Continuous Fermentation of Alcohol-Free Beer - Bioreactor Hydrodynamics and Yeast Physiology

UMinho|2012
Continuous fermentation of alcohol-free

Andr Manuel de Oliveira Mota beer - bioreactor hydrodynamics and
yeast physiology
Universidade do Minho
Escola de Engenharia
Andr Manuel de Oliveira Mota

beer - bioreactor hydrodynamics and
yeast physiology
June of 2012
Escola de Engenharia
Andr Manuel de Oliveira Mota

beer - bioreactor hydrodynamics and
yeast physiology
Doctoral Dissertation for PhD degree in Biological and

Chemical Engineering
Supervisor of the thesis:

Professor Doutor Jos Antnio Couto Teixeira
June of 2012
AUTORIZADA A REPRODUO PARCIAL DESTA TESE APENAS PARA EFEITOS

DE INVESTIGAO, MEDIANTE DECLARAO ESCRITA DO INTERESSADO, QUE A TAL SE
COMPROMETE;
Universidade do Minho, ___/___/______

Assinatura: ________________________________________________
If I have seen further it is by standing on ye sholders of Giants

Isaac Newton
a v M o ta
N u ria
III
T h e a c c o m p lis h m e n t o f th is th e s is w a s fin a n c ia lly s u p p o rte d b y a

P h D s c h o la rs h ip fro m F u n d a o p a ra a C i n c ia e T e c n o lo g ia (R e f.:
S F R H /B D /3 7 0 8 2 /2 0 0 7 ), in s e rte d in th e P ro g ra m a P o te n c ia l H u m a n o
Q u a d ro d e R e fe r n c ia E s tra t g ic o N a c io n a l (P O P H Q R E N ) - T ip o lo g ia
4 .1 - F o rm a o A v a n a d a . T h e P O P H -Q R E N is c o -fin a n c e d b y F u n d o
S o c ia l E u ro p e u (F S E ) a n d b y M in is t rio d a C i n c ia , T e c n o lo g ia e E n s in o
S u p e rio r (M C T E S ).
IV
Abstract/Resumo
Abstract
In th is s tu d y , th e c o n tin u o u s p ro d u c tio n w ith im m o b iliz e d c e lls o f
b e e r w ith o u t a lc o h o l w a s in v e s tig a te d . A p a rt fro m th e e v a lu a tio n o f th e
v a rio u s p a ra m e te rs a ffe c tin g th e q u a lity o f th e c o n tin u o u s p ro d u c tio n o f
a lc o h o l-fre e b e e r, a d e ta ile d s tu d y o n th e h y d ro d y n a m ic s o f th re e p h a s e b io re a c to rs w a s a ls o d o n e.
O n e o f th e m a jo r c o s ts a s s o c ia te d w ith c o n tin u o u s fe rm e n ta tio n s is
th e c a rrie r c o s t w h e re th e y e a s t is im m o b iliz e d . T w o c a rrie rs th a t a re
b y p ro d u c ts o f th e b re w in g in d u s try a n d a g ric u ltu re (c o rn c o b s a n d
s p e n t g ra in s ) w e re c o n s id e re d a n d a p p lie d in tw o d iffe re n t ty p e s o f
b io re a c to rs : g a s-lift a n d fix e d -b e d , u s in g s m a ll a n d la rg e p a rtic le s,
re s p e c tiv e ly .
T h e p re s e n c e o f s o lid p a rtic le s in th re e-p h a s e s y s te m s c a n in flu e n c e
th e c o u rs e o f th e fe rm e n ta tio n , a s th e h y d ro d y n a m ic s o f th e s y s te m
m a y b e m o d ifie d . T h u s , in th is w o rk th e in flu e n c e o f s p e n t g ra in s in
re g im e tra n s itio n o n a b u b b le c o lu m n w a s s tu d ie d . In th e p re s e n c e o f
s p e n t g ra in s , th e lim its w h e re th e h o m o g e n e o u s re g im e p re v a ils a re
s m a lle r th a n w ith o u t s o lid s . T h e e ffe c t is s im ila r to liq u id s w ith lo w
v is c o s ity . D iffe re n t c o n fig u ra tio n s o f g a s-lift b io re a c to rs w e re a ls o
e v a lu a te d
and
th e
c o n fig u ra tio n
w ith
b e tte r
g lo b a l
h y d ro d y n a m ic
p ro p e rtie s fo r th e c o n tin u o u s fe rm e n ta tio n o f a lc o h o l-fre e b e e r w a s

s e le c te d . H a v in g th is d o n e , th e lo c a l h y d ro d y n a m ic p ro pe rtie s o f th e
th re e -p h a s e s y s te m w e re c h a ra c te riz e d u s in g a d v a n c e d te c h n iq u e s
s u c h a s o p tic a l fib e rs , tra d itio n a lly a p p lie d in tw o -p h a s e s y s te m s . L o c a l
s tu d ie s
in d ic a te d
th a t
th e
c h a ra c te ris tic s
a re
m o re
a ffe c te d b y th e fe rm e n ta tio n m e ta b o lite s (e .g . e th a n o l) th a n th e s o lid s .

T h e g a s -lift re a c to r c o n fig u ra tio n w a s d e m o n s tra te d to b e th e o n e
th a t a llo w e d fo r th e o b te n tio n o f a b e tte r q u a lity b e e r. F o r th is s y s te m
w e re a ls o te s te d th e in flu e n c e o f th e c o m p o s itio n o f th e g a s -p h a s e in
th e fo rm a tio n o f a ro m a a n d fla v o u r c o m p o u n d s , a s w e ll a s th e in flu e n c e
o f a lo n g -te rm fe rm e n ta tio n o n th e p h y s io lo g y o f im m o b iliz e d y e a s t. T h e
re s u lts in d ic a te d th a t th e u s e o f n itro g e n in th e g a s p h a s e e x h ib its a n
Abstract/Resumo
in te re s tin g p o te n tia l in te rm s o f m ix in g a n d m a in te n a n c e o f a n a e ro b ic
c o n d itio n s e s s e n tia l in a c o n tin u o u s fe rm e n ta tio n . F in a lly s tra te g ie s fo r
m a in ta in in g a n d m o n ito rin g th e p h y s io lo g y o f th e im m o b iliz e d y e a s t
h a v e b e e n s u g g e s te d . A m o n g th e s e a re th e re g u la r a d d itio n o f fre s h
c a rrie r (s p e n t g ra in s ) a n d c o n tro l o f th e n u m b e r b u d s c a rs re s u ltin g
fro m th e y e a s t c e ll d iv is io n .
In th e c o n tin u o u s p ro d u c tio n o f a lc o h o l-fre e b e e r, s e v e ra l k n o w le d g e
a re a s a re in v o lv e d a n d its in te ra c tio n is o f fu n d a m e n ta l im p o rta n c e . It
s ta rts w ith th e s e le c tio n o f th e b e s t c a rrie r a nd y e a s t s tra in . T h e n , th e
b io re a c to r d e s ig n b a s e d o n th e in te ra c tio n b e tw e e n its h y d ro d y n a m ic
p ro p e rtie s a n d th e fe rm e n ta tio n p e rfo rm a n c e . In th is w o rk th e c o m p le x
re la tio n b e tw e e n th e s e d iffe re n t fie ld s a llo w e d th e a c q u is itio n o f n e w
k n o w le d g e th a t w ill b e u s e fu l in th e fu tu re .
K e y w o rd s : a lc o h o l-fre e b e e r, c o n tin u o u s fe rm e n ta tio n , b io re a c to rs ,

h y d ro d y n a m ic s , s p e n t g ra in s , im m o b iliz a tio n .
VI
Abstract/Resumo
Resumo
N e s te tra b a lh o , fo i e s tu d a d a a p ro d u o d e c e rv e ja s e m lc o o l e m
c o n tn u o ,
u tiliz a n d o
le v e d u ra s
im o b iliz a d a s .
tra b a lh o
e n g lo b o u
d ife re n te s re a s , c o m o : o e s tu d o d a h id ro d in m ic a d e b io re a c to re s
trif s ic o s , e o e s tu d o d e d iv e rs o s p a r m e tro s q u e a fe c ta m a q u a lid a d e
d a fe rm e n ta o c o n tn u a d e c e rv e ja s e m lc o o l.
U m d o s p rin c ip a is c u s to s a s s o c ia d o s fe rm e n ta o c o n tin u a o
c u s to d o s s u p o rte s o n d e s e im o b iliz a m a s le v e d u ra s . A s s im fo ra m
e s tu d a d o s d o is s u p o rte s q u e s o s u b p ro d u to s d a in d u s tria a g rc o la e
c e rv e je ira (e s p ig a s d e m ilh o e s p e n t g ra in s ). D e a c o rd o c o m o s
re s u lta d o s
o b tid o s ,
os
s u p o rte s ,
de
d ife re n te s
d ime n s e s ,
fo ra m
a p lic a d o s a d o is tip o s d e b iore a c to re s - " g s -lift" (p a rtc u la s m e n o re s )

e le ito fix o (p a rtc u la s m a io re s ).
A
p re s e n a
de
p a rtc u la s
s lid a s
em
s is te m a s
trif s ic o s
pode
in flu e n c ia r o d e c u rs o d e u m a fe rm e n ta o . A s s im n e s te tra b a lh o fo i
e s tu d a d a a in flu n c ia d o s s p e n t g ra in s n a tra n s i o d e re g im e e m
c o lu n a d e b o lh a s . O e s tu d o te v e c o m o o b je c tiv o s im u la r u m a p a rte d o
re a c to r g a s -lift (tu b o a s c e n d e n te ) e e n te n d e r o s m e c a n is m o s fs ic o s
q u e a i o c o rre m . N a p re s e n a d o s s p e n t g ra in s o s c a u d a is ao s q u a is o
re g im e h o m o g n e o p re v a le c e s o m e n o re s , e fe ito s im ila r a lq u id o s
c o m b a ix a v is c o s id a d e . S e g u id a m e n te a in flu n c ia d o s s p e n t g ra in s
fo i e s tu d a d a p a ra d ife re n te s c o n fig u ra e s d e b io re a c to re s g a s-lift. N o
fim fo i s e le c c io n a d a a c o n fig u ra o q u e a p re s e n ta v a p ro p rie d a d e s
h id ro d in m ic a s g lo b a is q u e m e lh o r s e a d a p ta m a s c a ra c te rs tic a s d e
u m a fe rm e n ta o c o n tin u a d e c e rv e ja s e m lc o o l. F e ita a s e le c o d o
b io re a c to r
e s tu d o u -s e
as
p ro p rie d a d e s
h id ro d in m ic a s
lo c a is
do
s is te m a trif s ic o . P a ra ta l re c orre u -s e a t c n ic a s m a is re fin a d a s (fib ra s

p tic a s ), n o rm a lm e n te u tiliz a d a s e m s is te m a s b if s ic o s . A s s im fo i
n e c e s s rio d e s e n v o lv e r u m m to d o p a ra a u tiliz a o d e s ta s t c n ic a s
e m s is te m a s trif s ic o s q u e p o d e s e r ta m b m a p lic a d a a s is te m a s c o m
e le v a d a
d e n s ida d e
c e lu la r.
Os
e s tu d o s
lo c a is
in d ic a ra m
que
as
c a ra c te rs tic a s h id ro d in m ic a s s o m a is a fe c ta d a s p e lo s s u b p ro d u to s
d a fe rm e n ta o d o q u e p e lo s s lid o s .
VII
Abstract/Resumo
F o ra m ta m b m re a liz a d a s e x p e ri n c ia s q u e p e rm itira m e s tu d a r a
in flu n c ia
de
d iv e rs o s
le v e d u ra ,
s u p o rte )
na
p a r m e tro s
(tip o
fe rm e n ta o
de
b io re a c to r,
c o n tin u a .
E n tre
os
e s tirp e
de
p a r m e tro s
e s tu d a d o s o p ro d u to fin a l q u e a p re s e n ta v a m e lh o r q u a lid a d e fo i o b tid o

u tiliz a n d o u m b io re a c to r g s -lift c o m le v e d u ra s im o b iliz a d a s e m s p e n t
g ra in s . N e s te s is te m a fo ra m a in d a te s ta d o s a in flu n c ia d o tip o d a
fa s e g a s o s a n a fo rm a o d e c o m p o s to s d o a ro m a e s a b o r, a s s im c o m o
a
in flu n c ia
de
uma
fe rm e n ta o
c o n tin u a
de
lo n g a
d u ra o
na
fis io lo g ia d a s le v e d u ra s im o b iliz a d a s . O s re s u lta d o s in d ic a ra m q u e o

a z o to
a p re s e n ta
um
p o te n c ia l
in te re s s a n te
na
m a n u te n o
das
c o n d i e s d e m is tu ra e d e a n a e ro b io s e e s s en c ia is n u m a fe rm e n ta o
c o n tn u a . F in a lm e n te e s tra t g ia s p a ra a m a n u te n o e c o n tro lo d a
fis io lo g ia
das
le v e d u ra s
im o b iliz a d a s
fo ra m
s u g e rid a s .
E n tre
e la s
e n c o n tra m -s e : a a d i o re g u la r d e s u p o rte fre s c o ( s p e n t g ra in s ) e o

c o n tro lo d a s c ic a triz e s d a s le v e d u ra s re s u lta n te s d a s u a d iv is o c e lu la r.
N a p ro d u o c o n tin u a d e c e rv e ja s e m lc o o l in te ra g e m d iv e rs a s
a re a s d o c o n h e c im e n to . E s te e s tu d o in ic ia-s e p e la s e le o d o m e lh or
s u p o rte
e s tirp e
de
le v e d u ra .
P o s te rio rm e n te
p a s s a-s e
ao
d e s e n v o lv im e n to d e u m b io re a c to r q u e b a s e a d o n a in te ra o e n tre a s
s u a s p ro p rie d a d e s h id ro d in m ic a s e a q u a lid a d e d a fe rm e n ta o . A o
lo n g o d e s te tra b a lh o a c o m p le x a re la o e n tre d ife re n te s re a s d o
c o n h e c im e n to
p e rm itiu
a q u is i o
de
novos
sem
lc o o l,
c o n h e c im e n to s
que
p o d e r o s e r te is n o fu tu ro .
P a la v ra s-c h a v e :
c e rv e ja
fe rm e n ta o
b io re a c to re s , h id ro d in m ic a , s p e n t g ra in s , im o b iliz a o
VIII
c o n tn u a ,
Ackowledgments
Ackowledgments
M y fi rs t a c k o w le d g m e n t a re to m y tw o s u p e rv is o rs p ro fe s s o rs J o s
A n t n io T e ix e ira a n d A n t n io A u g u s to V ic e n te , w h o b e lie v e d in m y
s c ie n tific a b ilitie s a n d w h o m in v ite d m e to th is p ro je c t. I a ls o th a n k to
F u n d a o p a ra a C i n c ia e T e c n o lo g ia th a t fi n a n c ia lly s u p p o rte d th is
s tu d y
d u rin g
48
m o n th s
th ro u g h
PhD
s c h o la rs h ip
(S F R H /B D /3 7 0 8 2 /2 0 0 7 ). I a ls o h a v e to a c k o w led g e a ll m y fo re in g
s u p e rv is o rs :
P ra g u e ,
Tomas
Czech
(L a b o ra to ire
B r n iy k
R e p u b lic ),
des
(In s titu te
P h ilip p e
E c o u le m e n ts
of
C h e m ic a l
Sechet
and
G o p h y s iq u e s
T e c h n o lo g y
A la in
et
in
C a rte llie r
In d u s trie ls
de
lU n iv e rs it d e G re n o b le in G re n o b le , F ra n c e ); fo r th e ir u n iq u e s c ie n tific
s u p p o rt th a t w a s e s s e n c ia l to m e .
M y s e c o n d a k o w le d g e m e n ts a re to a ll m y la b o ra to ry c o le g u e s (fro m
P o rtu g a l,
to
Czech
R e p u b lic
and
F ra n c e ),
who
had,
d u rin g
my
P h D s tu d y in g y e a rs , s u p p o rte d m e . A ls o fo r s o lv in g a lo t o f m y
te c h n ic a l p ro b e m s d u rin g th e las t y e a rs I s in c e re ly th a n k to th e
w o n d e rfu l
e n g in e e rin g -te c h n ic ia n s
(R u d o lf
Young,
Manuel
S a n to s ,
M a d a le n a V ie ira , J e a n-M a rc ) in a ll th re e in s titu tio n s I h a v e w o rk e d .

I a ls o h a v e to a c k o w le d g e m y frie n d s fo r th e ir frie n d s h ip a n d s u p p o rt.
And
th e
la s t
but
not
th e
le a s t,
th a n k
to
my
d e a re s t
fa m ily
(g ra n d p a re n ts , o n c le s , a u n ts a n d c o u s in s ). E s p e c ia lly to m y p a re n ts
(T e re s a a n d P a u lo ), m y s is te r a n d m y g ra n d m o th e r (N ilz a M o ta ), w h o
ta u g h t m e to lo v e s c ie n c e .
T o y o u a ll, th is th e s is is in p a rt, fro m y o u . T h a n k y o u a ll.
IX
List of Publications
List of Publications
C o n fe re n c e p ro ce e d d in g s
Immobilization of Yeast Cells on Corn Cob Particles: A Preliminary Study.
Andr Mota, Daniel P. Silva, Tom! Brnyik, Antnio A. Vicente, Jos A.
Teixeira. VII Brazilian Meeting on Chemistry of Food and Beverages. So
Paulo, 6th December 2008
Influence of Different Pre-Treatment Methods on The Immobilization Of
Brewers Yeast on Corn Cob Particles. Mota, A., Teixeira, J., SIPAL VII
International Symposium on the production of Alcohol and Yeasts, Mxico,
25th June 2009
Measurement of local gas-phase properties using an optical probe in a
three-phase systems. Mota, A., Vicente, A. A., Teixeira, J. A., Sechet, P.,
Cartellier, A. Poster in conference GLS10, Braga, June (2011)
A rtic le s in J o u rn a ls
Formation of flavour active compounds during continuous alcohol-free beer
production: The influence of yeast strain, reactor configuration and carrier
type. Mota A., Novak P., Macieira F., Vicente A., Teixeira J., Smogrovicov
D., Brnyik T. Journal of the American Society of Brewing Chemists, 69(1), 17, 2011.
Effect of spent grains on flow regime transition in bubble column. A.
Mota., Antnio A. Vicente., Jos A. Teixeira. Chemical Engineering Science,
66, 3350-3357, 2011.
CFD simulation and experimental measurement of gas holdup and liquid
interstitial velocity in internal loop airlift reactor. Simcik, M., Mota, A.,
Ruzicka, M. C., Vicente, A. A., Teixeira, J. A. Chemical Engineering Science.
66: pp. 3268-3279, 2011.
Hydrodynamic Properties of a Three-Phase Internal Gas-Lift Reactor with
an Enlarged Degasing Zone Using Spent Grains as solid-phase. Mota, A.,
Simcik, M., Ruzicka, M. C.,Vicente, A. A., Teixeira, J. A.. (Work accepted for
oral presentation in GLS11 Seul 2013. It is going to be submitted in a special
issue of Chemical Engineering Science).
Table of Contents
Table of Contents
I.Chapter I Motivation and Outline ........................................................ 1!
I.1! Thesis Motivation .......................................................................2!
I.1.1!
Alcohol-free beer: definition, history and market .............................. 2!
I.2! Research Aims ...........................................................................5!

I.3! Thesis Outline ............................................................................6!
I.4! References ................................................................................7!
II.Chapter II Alcohol-free Beer.............................................................. 8!
II.1! Methods to Produce Alcohol-free Beer .........................................9!
II.1.1!
II.1.2!
Techniques to remove alcohol from regular beer ............................. 9!

Traditional methods to restrict ethanol formation .......................... 11!
II.2.1!
II.2.2!
II.2.3!
Continuous beer fermentation using cell immobilization systems ..... 15!

Immobilization and physiological changes on yeast ....................... 17!
Industrial applications ................................................................ 22!
II.3.1!
II.3.2!
Industrial applications ................................................................ 25!

Future trends and challenges ...................................................... 26!
II.4.1!
II.4.2!
II.4.3!
II.4.4!
II.4.5!
Higher alcohols .........................................................................

Esters ......................................................................................
Organic acids ............................................................................
Aldehydes .................................................................................
Vicinal diketones .......................................................................
II.2! Continuous Beer Production ...................................................... 14!
II.3! Continuous Alcohol-free Beer Production ................................... 24!

II.4! Beer Quality and Flavour: Origin and Influence ............................ 26!
30!
32!
34!
35!
37!
II.5! References .............................................................................. 40!

III.! Chapter III Selection of the Best Immobilization Carrier for
Brewing Yeast Cells .............................................................................. 43!
III.1! Objectives ............................................................................... 44!
III.2! Introduction ............................................................................. 44!
III.2.1!
III.2.2!
III.2.3!
III.2.4!
III.2.5!
III.2.6!
Immobilization of biocatalysts: history and application..................

Continuous high cell density systems ..........................................
Immobilization methods .............................................................
Carrier properties and specific characteristics .............................
Spent grains and corncobs: cellulose-based carriers ....................
Chemical modification of carriers ...............................................
44!
45!
46!
48!
49!
51!
III.3.1!
III.3.2!
III.3.3!
III.3.4!
III.3.5!
III.3.6!
III.3.7!
III.3.8!
Determination of corncobs chemical composition .........................

Carrier particles for immobilization .............................................
Derivatization method ...............................................................
Pre-treatment of carriers ...........................................................
Conditions tested .....................................................................
Medium and microorganisms ......................................................
Adsorption tests .......................................................................
Immobilization with yeast growth test .........................................
53!
53!
54!
54!
55!
56!
56!
57!
III.4.1!
III.4.2!
III.4.3!
III.4.4!
III.4.5!
General considerations ..............................................................

Corncob composition ................................................................
Selection of best DEC concentration...........................................
The effect of Cc pre-treatment in carrier adsortion capacity ..........
Effect of yeast cell growth on Cc immobilization capacity .............
58!
59!
60!
63!
64!
III.3! Material and Methods ............................................................... 53!
III.4! Results and Discussion ............................................................. 58!
III.5! Conclusions ............................................................................. 67!

III.6! References .............................................................................. 68!
XI
Table of Contents
IV.!
Chapter IV Effect of Spent Grains on Flow Regime Transition in a
Bubble Column ..................................................................................... 70!
IV.1! Objectives ............................................................................... 71!
IV.2! Introduction............................................................................. 71!
IV.2.1!
IV.2.2!
Bubble columns: flow regime transition ....................................... 72!

Solids effect on three-phase bubble column ................................ 74!
IV.3.1!
IV.3.2!
IV.3.3!
IV.3.4!
IV.3.5!
IV.3.6!
Apparatus and measurements .....................................................

Solid-phase characterization: spent grains ..................................
Gas hold-up .............................................................................
Solids hold-up ..........................................................................
Measurements errors .................................................................
Evaluation of critical gas hold-up and critical gas velocity .............
77!
77!
78!
79!
80!
80!
IV.4.1! Spent grains characterization .....................................................

IV.4.2! Bed expansion versus water columns differential pressure for gas
hold-up determination ...............................................................................
IV.4.3! Gas-hold up vs gas-flow: solids influence ....................................
IV.4.4! Gas-hold up vs gas-flow: drift-flux ..............................................
83!
IV.3! Material and Methods ............................................................... 77!
IV.4! Results and Discussion ............................................................. 83!

84!
86!
90!
IV.5! Conclusions ............................................................................ 96!

IV.6! References .............................................................................. 96!
V.Chapter V Global Hydrodynamic Properties of Three-phase iGLR ..... 99!

V.1! Objectives ..............................................................................100!
V.2! Introduction............................................................................100!
V.2.1!
V.2.2!
V.2.3!
V.2.4!
Gas-lift reactor design and configurations ................................... 101!

Gas-phase global properties and methods ................................... 103!
Solid-phase global properties and methods ................................. 106!
Liquid-phase global properties and methods ................................ 108!
V.3.1!
V.3.2!
V.3.3!
V.3.4!
V.3.5!
V.3.6!
Gas-lift reactor: dimensions and characteristics ........................... 110!

Solids distribution and hold-up .................................................. 111!
Gas hold-up ............................................................................. 112!
Mixing time determination .......................................................... 112!
Circulation time determination ................................................... 113!
Liquid velocity determination ..................................................... 113!
V.4.1!
V.4.2!
V.4.3!
V.4.4!
Solid particles distribution ......................................................... 114!

Gas hold-up ............................................................................. 122!
Mixing and circulation time ........................................................ 128!
Liquid velocity .......................................................................... 132!
V.3! Material and Methods ..............................................................110!
V.4! Results and Discussion ............................................................114!
V.5! Conclusions ...........................................................................134!

V.6! References .............................................................................135!
VI.!
Chapter VI CFD Simulation of Gas Hold-up and Liquid Interstitial
Velocity in Two-phase iGLR ................................................................. 138!
VI.1! Objectives ..............................................................................139!
VI.2! Introduction............................................................................139!
VI.2.1!
CFD simulations in iGLR: advantages and limitations ................... 142!
VI.3.1!
VI.3.2!
Experimental setup .................................................................. 144!

Simulation setup ...................................................................... 145!
VI.4.1!
VI.4.2!
Auxiliary simulation results ....................................................... 153!

Comparison with experiments .................................................... 157!
VI.3! Material and Methods ..............................................................144!

VI.4! Results and Discussion ............................................................153!
VI.5! Conclusions ...........................................................................165!
VI.6! References .............................................................................166!
XII
Table of Contents
VII.! Chapter VII Local Hydrodynamic Properties of Three-phase iGLR

168!
V I I . 1 !O b j e c t i v e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 9 !
V I I . 2 !I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 9 !
VII.2.1!
VII.2.2!
VII.2.3!
VII.2.4!
Measurement of local gas-phase properties ............................... 170!

Solid-phase properties and distribution ..................................... 179!
Liquid-phase .......................................................................... 180!
Hydrodynamic model: local hydrodynamics ................................ 182!
VII.3.1!
VII.3.2!
VII.3.3!
VII.3.4!
VII.3.5!
VII.3.6!
VII.3.7!
VII.3.8!
VII.3.9!
VII.3.10!
VII.3.11!
Experimental apparatus for gas hold-up measurements ............... 186!

New signal processing and validation ........................................ 188!
Experimental conditions .......................................................... 191!
Solids properties .................................................................... 192!
High speed camera measurements and image analysis ................ 192!
Statistical method................................................................... 193!
PIV measurements .................................................................. 193!
Microorganism and medium ..................................................... 194!
Free biomass determination ..................................................... 194!
Immobilized biomass determination ......................................... 194!
Cell viability flow cytometry ................................................. 195!
VII.4.1!
VII.4.2!
VII.4.3!
VII.4.4!
VII.4.5!
VII.4.6!
Calibration of new system ........................................................ 196!

Gas-phase measurements ........................................................ 198!
Liquid-phase measurements ..................................................... 209!
Solids-phase measurements ..................................................... 211!
Model optimization results ....................................................... 215!
Fermentation results ............................................................... 216!
V I I . 3 !M a t e r i a l a n d M e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 6 !
V I I . 4 !R e s u l t s a n d D i s c u s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 6 !
V I I . 5 !C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 8 !
V I I . 6 !R e f e r e n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 9 !
VIII.!
Chapter
VIII
Continuous
Primary
Alcohol-free
Beer
Fermentation: Influence of Yeast Strain, Reactor Conditions, Carrier Type
and Ageing ......................................................................................... 222!
VIII.1! Objectives ........................................................................... 223!
VIII.2! Introduction ......................................................................... 223!
VIII.2.1! Gas-lift reactor ..................................................................... 224!
VIII.2.2! Packed-bed reactor ............................................................... 226!
VIII.3! Material and Methods ........................................................... 227!

VIII.3.1!
VIII.3.2!
VIII.3.3!
VIII.3.4!
VIII.3.5!
VIII.3.6!
VIII.3.7!
VIII.3.8!
VIII.3.9!
Microorganisms and medium ................................................... 227!

Carrier preparation ................................................................ 228!
Packed-bed reactor ............................................................... 229!
Gas-lift reactor characteristics and startup ............................... 230!
Batch fermentations ............................................................... 231!
Yeast cells analysis ................................................................ 231!
Out-flow analytical methods .................................................... 235!
Determination of residence time distribution ............................. 238!
Fermentations ....................................................................... 239!
VIII.4.1!
VIII.4.2!
VIII.4.3!
VIII.4.4!
VIII.4.5!
VIII.4.6!
General considerations ........................................................... 240!

Fermentations performance and flavour compounds................... 243!
Effect of yeast strain .............................................................. 248!
Effect of reactor design and carrier type .................................. 251!
Effect of gas-phase in the final product.................................... 253!
Effect of ageing: preliminary studies ........................................ 261!
VIII.4! Results and Discussion ......................................................... 240!
VIII.5! Conclusions ......................................................................... 267!

VIII.6! References........................................................................... 268!
IX.!
Chapter IX Conclusions and Future Work ................................. 271!
X.Apendix I ......................................................................................... 274!
XIII
List of Figures
List of Figures
Figure I-1. Beer market in Portugal in the last Five Years. Legend: !: Total Beer
Consumption; ": Regular Beer Consumption; -#$% Alcohol-free Beer Consume.
(Data from http://www.apcv.pt/asp/ in 15 December 2011) ............................. 4!

t h
Figure III-1. Evolution of optical density of free cells during immobilization time,
obtained for Cc2 particles derivatized with 50% (w/w) DEC solution. .............. 57!
Figure III-2. A: Ion exchange capacities (IEC) of different carrier particles at
different concentrations of DEC solution. B: Influence of derivatization on the
immobilized biomass (iBio) for different carriers. Legend: ! - Cc1; ! - Cc2; ! Cc3; ! - SG. ............................................................................................ 61!
Figure III-3. Ion Exchange Capacity for: A Cc pre-treatment A; B Cc pretreatment B. Legend: ! - Cc1; ! - Cc2; ! - Cc3; ! - SG. ............................ 63!
Figure III-4.Yeas cells adsorption for: A Cc pre-treatment A; B Cc pre-treatment
B. Legend: ! - Cc1; ! - Cc2; ! - Cc3; ! - SG. .......................................... 64!
Figure III-5. Immobilized biomass (iBio) expressed in amount of biomass per gram of
dry Carrier (mg /g ) versus DEC solution concentration for: A Cc pre-treatment
A; B Cc pre-treatment B. Legend: ! - Cc1; ! - Cc2; ! - Cc3; ! - SG. ....... 66!
B I O
D C
Figure IV-1. Definition of flow regime transition in BC and identification of flow

regimes and critical values. Legend: Ho Homogeneous regime; Tr Transition
regime; He Heterogeneous regime; q Beginning of Ho regime; q Critical
point (End of Ho regime); q End of Transition regime. Adapted from Ruzick et
al. (2003). ................................................................................................ 73!
S
H e
Figure IV-2. Comparison between gas hold-up obtained by Water Columns (Gas
hold-up ) and Bed expansion (Gas hold-up ): A Solids load up to
20% (wt.WET BASIS/vol.); B Solids load up to 20% (wt. WET BASIS /vol.). Legend: !
water;
!4% (wt.WET BASI /vol.);
!
8%
(wt.WET BASI /vol.);
+
12% (wt.WET BASIS/vol.); " - 20% (wt.WET BASIS/vol.). ........................................ 85!
W C
B E
Figure IV-3.Primary data obtained from different gas hold-up methods: A Bed
Expansion; B water Columns. Legend: !water; !-4% (wt.WET BASIS/vol.); !8% (wt. WET BASIS /vol.); +-12% (wt. WET BASIS /vol.); "-20% (wt.WET BASIS /vol.). ... 86!
Figure IV-4. Exemple of small bubbles near the BC wall. .................................... 88!
Figure IV-5. A - Solids influence at low gas flow rates in gas hold-up (Bed
Expansion). Legend: # q = 0,2 cm/s; ! - 0,4 cm/s; B Solids influence at high
gas flow rates in gas hold-up (Bed Expansion). Legend: # q = 2,5 cm/s; ! 2,7 cm/s .................................................................................................. 89!
Figure IV-6. Drift-flux plot: Drift-flux j (m/s) vs. Gas hold-up (vol./vol.). Legend: "
(smooth line): j by Eq. IV-4; $ (Data points): j by Eq. IV-7; ! (Blank Data):
Critical point. A, B, C, D, E: Different solid loads (0, 4, 8,12 % (wt.WET BASIS/vol.))
............................................................................................................... 91!
t
E X P
Figure IV-7. Evaluation of critical gas hold-up and critical gas flow with solid load.
Legend: % - Bed Expansion; ! - Water Columns........................................... 93!
Figure V-1. Different configurations used in GLR. Legend: a) Internal-loop GLR
(iGLR); b) Split GLR; c) External-loop GLR. ................................................. 101!
Figure V-2. Inverted tube manometer arrangement. (Adapted from Chisti, 1989)... 106!
Figure V-3. Internal Gas-lift reactor design used in this chapter. Legend: A - Riser
Hold-up measuring points; B - Downcomer Hold-up measuring points; C - Riser
pH probes; D - Downcomer pH probes; E - Conductivity probe point ............. 111!
Figure V-4. Solids sampling locations for solid distribution determination. .......... 112!
Figure V-5. Solids hold-up at different GLR positions (A,B,C,D,E) for riser A
configuration at different gas superficial velocity (1 cm/s; 2.5 cm/s; 5 cm/s and
7.5 cm/s) in riser. .................................................................................... 116!
XIV
List of Figures
Figure V-6. Solids hold-up at different GLR positions (A,B,C,D,E) for riser B
7.5 cm/s) in riser. .................................................................................... 120!
Figure V-7. Solids hold-up at different GLR positions (A,B,C,D,E) for riser C
7.5 cm/s) in riser. .................................................................................... 121!
Figure V-8. Gas hold-up (vol./vol.) vs. Gas superficial velocity in riser (U / (cm/s))
for different risers configurations. Legend: &-0% (wt.WET BASIS/vol.); !4% (wt.WET BASIS /vol.); "-8% (wt. WET BASIS /vol.); '-12% (wt. WET BASIS /vol.); (
20% (wt. WET BASIS /vol.). ............................................................................ 123!
G r
Figure V-9. Driving force (! -! ) vs. Gas superficial velocity (U ) for different risers
configurations. Legend: & - 0% (wt. WET BASIS /vol.); ! - 4% (wt.WET BASIS /vol.);
"- 8% (wt.WET BASIS /vol.); '-12% (wt.WET BASIS /vol.); ( 20% (wt.WET BASIS /vol.).
.............................................................................................................. 126!
G R
G r
G d
Figure V-10. Mixing time vs. Gas superficial velocity (U /(cm/s)) at different solid
load.
Legend:
&-0% (wt.WET BASIS
/vol.);
!-4% (wt.WET BASIS
/vol.);
"8% (wt.WET BASIS /vol.); '-12% (wt.WET BASIS /vol.); (20% (wt.WET BASIS /vol.).... 129!
Figure V-11. Liquid velocity (m/s) in riser and downcomer vs. Solid load
(% (wt.WET BASIS
/vol.))
for
different
risers
configurations.
Legend:
! - 0% (wt. WET BASIS /vol.); ) -4% (wt.WET BASIS /vol.); ) - 8% (wt. WET BASIS /vol.);
)- 12% (wt. WET BASIS /vol.); ) 20% (wt. WET BASIS /vol.)............................... 133!
S
Figure VI-1. Relationships between hydrodynamic parameters and other bioreactor

performance indices. Legend: U Overall Heat Transfer coefficient (W/(m2 C));
k a Overall volumetric mass transfer coefficient (s ). Adapted from Chisti,
1989. ...................................................................................................... 141!
H
- 1
Figure VI-2. Internal Gas-lift Reactor geometry (not at scale). Legend: D1

Distributor diameter; D2 Riser internal diameter; D3 - Riser external diameter;
D4 Column internal diameter; D5 Top degassing zone internal diameter; H1
Distance between distributor and beginning of riser; H2 Riser length; H3
Distance between riser end and beginning of top part; H4 Height of conical
section from top part; H5 Height of cylindrical section of top part. ............. 145!
Figure VI-3. Computational grid for the iGLR cross-section at Risers A, B (left) and
C (right). ................................................................................................. 146!
Figure VI-4. Riser A simulation results (section VI. 4.1). Comparison of two drag
closures for 5mm bubbles. (a) Riser liquid interstitial velocity V (m/s), (b) riser
gas holdup #, and (c) downcomer gas holdup # vs. riser gas superficial velocity
U (m/s). Legend: *-Tomiyama (Eq. VI-13); '-Schiller-Naumann (Eq. VI-15). . 155!
L
Figure VI-5. Case B simulation results (section VII.4.1). Tomiyama drag closure (Eq.
VI-13) results for 3 mm (') and 5mm bubble (*). Legend: (a) - Riser liquid
interstitial velocity V (m/s), (b) - riser gas holdup vs. riser gas superficial
velocity U (m/s)....................................................................................... 156!
L
Figure VI-6. Riser A simulations vs. experiments (Section VII-4.2): Fluent simulations
(*), CFX simulations (!), experiments ('). (a) Riser liquid interstitial velocity V
(m/s), (b) downcomer liquid interstitial velocity V , (c) riser gas holdup (#), and
(d) downcomer gas holdup # vs. riser gas superficial velocity U (m/s). ........... 158!
L
Figure VI-7. Riser A simulations: Gas holdup fields for gas flow rates 1.9, 3.6, 4.5,
9.1 and 13.6 L/min (riser gas superficial velocity ~ 1.0, 2.0, 2.5, 5.0, 7.5 cm/s).
.............................................................................................................. 159!
Figure VI-8. Riser B simulations vs. experiments. Legend: *-Fluent simulations
(black marks), !-CFX simulations (stars), '-experiments (white marks). (a) Riser
liquid interstitial velocity V , (b) downcomer liquid interstitial velocity V (m/s),
and (c) riser gas holdup # vs. riser gas superficial velocity U (m/s). ............... 160!
L
Figure VI-9. Riser B simulations: Gas holdup fields for gas flow rates 1.9, 3.6, 4.5,
9.1 and 13.6 l/min (riser gas superficial velocity ~ 1.0, 2.0, 2.5, 5.0, 7.5 cm/s)
.............................................................................................................. 161!
XV
List of Figures
Figure VI-10. Riser C simulations vs. experiments Legend: * - Fluent simulations

(black marks), !- CFX simulations (stars), ' - experiments (white marks). (a)
Riser liquid interstitial velocity V (m/s), (b) downcomer liquid interstitial velocity
V (m/s), (c) riser gas holdup #, and (d) downcomer gas holdup # vs. riser gas
superficial velocity U (m/s). ...................................................................... 163!
L
Figure VI-11. Riser C simulations: Instantaneous gas holdup fields for gas flow
rates 1.9, 3.6, 4.5, 9.1, 13.6, 17.9 and 26.8 l/min (riser gas superficial velocity ~
0.5, 1.0, 1.3, 2.6, 3.8, 5.0, 7.5 cm/s). ......................................................... 164!
Figure VII-1. Techniques used for local hydrodynamics characterization in g-l and gl-s systems. ............................................................................................ 171!
Figure VII-2. Optical fiber typical operation method and indicator function obtained
during bubble piercing. From Vejrazka et al. (2010). .................................... 173!
Figure VII-3.Bubble detection and determination of bubble caracteristic points.
Legend: A Beginning of Bubble; B End of Bubbles; C Begin rising; D End
of Rising. (in Cartellier, 1992) ................................................................... 175!
Figure VII-4. Bubble interface/probe interaction parameters. In Cartellier, 1992. . 176!
Figure VII-5. Signal amplitude distribution (Left Graph) and time series amplitude
(Right Graph) obtained by Mena et al (2008) for g-l-s system using alginate
beads. Adapted from Mena et al. (2008). .................................................... 178!
Figure VII-6. Photo of injection tip developed around the optical fiber (left). Diagram
of Injection system operating (right). Legend: A liquid injection tubes; B
Optical probe output signal; C Optical probe tip (optical fiber); D Liquid
injected arount optical fiber (dashed line) ................................................... 179!
Figure VII-7.Flow diagram for MATLAB program used in this model. Starting from
initial model parameters and experimental data, new parameters are calculated
in order to achieved values of U , e and U that are similar (5%) to the
experimental data. ................................................................................... 185!
G r
G r
L r
Figure VII-8. Experimental setup for gas hold-up experiments. Legend: 1. Air-Lift
Reactor; 2. Optical Probe; 3. Electro valve; 4. Light source+ Photo detector; 5.
Oscilloscope; 6. Acquisition Board ............................................................ 186!
Figure VII-9. MATLAB program layout (see Appendix I)...................................... 188!
Figure VII-10. Raw signal obtain for riser radial central position at Q =400mL/min.
Top graph shows optical probe signal. Bottom Graph shows electovalve signal.
(The total time where corresponds to 7 seconds) ......................................... 189!
S
Figure VII-11. Graphical explanation of cutting zones. Legend: Add1 area removed
before injection (from 0% to 50% of Injection peak time). Add2 area removed
after injection (from 0% to 100% of Injection peak time) .............................. 190!
Figure VII-12.Optical probe measurement location in the GL reactor .................. 191!
Figure VII-13. Experimental setup for PIV measurements. Legend: 1. Air Source; 2.
Rotameter; 3. Air-Lift Reactor; 4. Laser + Laser sheet maker; 5. ................... 193!
Figure VII-14. Error obtained at different Add1 and Add 2 for different gas-phase
characteristics: Gas hold-up, Bubble Velocity and Bubble Chord. Legend: A
Q = 250 mL/min; B Q = 400 mL/min; ! - 0.0% Add1; ) - 12.5% Add1;
) - 25.0% Add1; ) - 12.5% Add1; )- 50.0% Add1 ..................................... 197!
A I R
A I R
Figure
VII-15.
Solids
influence
in
local
gas-phase
properties.
Legend: A Q = 250 ml/min; B Q = 400 ml/min; ! - Gas Hold-up (% (vol./vol.));
& - Bubble Velocity (cm/s); ' - Bubble Chord (mm). ................................... 200!
R
Figure VII-16. HSC Images from GL reactors riser. White lines are the simulated
theoriacally piercing of a bubble and each length was used to determined chord.
Legend. A Air-Water; B Air-Water-SG (0.8% (wt. WET BASIS /vol.)); BAir-WaterSG (2% (wt.WET BASIS/vol.)); ........................................................................ 202!
Figure VII-17. Expected gas flow (&) vs. Experimental gas flow ('and!). ........... 204!
Figure VII-18. Cell concentration influence in gas-phase properties (voidage, bubble
velocity and chord) on water-air system at 250 mL/min. ............................... 207!
XVI
List of Figures
Figure VII-19. The evolution of Sauter (D ), equivalent (D ) diameter and sphericity

with the yeast cells concentrations. Values obtained from the HSC images.
Legend: ! - D (mm); & - D (mm); ' - Sphericity (-). ................................. 208!
3 2
E Q
E Q
3 2
Figure VII-20. Study of liquid velocity in the downcomer. vs radial profile (A

QAIR=250 mL/min; B QAIR=400 mL/min) and the influence of SG concentration in
average liquid velocity (C) and liquid flow (D) in the downcomer. Legend: ! 0% (wt.WET BASIS
/vol.) SG;
&-2% (wt.WET BASIS/vol.) SG;
"4% (wt.WET BASIS/vol.) SG; '-6% (wt.WET BASIS /vol.) SG; (-QAIR=250 mL/min; + QAIR=400 mL/min. .................................................................................... 210!
Figure VII-21. Solids Velocity (cm/s) in downcomer versus the air flow rate. The
values were obtained by HSC videos and determined manually. The SG load was
2% (wt.WET BASIS/vol.). ............................................................................... 212!
Figure VII-22. Modelization Values versus Experimental results. Legend: &250 mL/min; ! - 400 mL/min. .................................................................... 215!
Figure VIII-1. Experimental layout of the continuous packed-bed reactor: 1 = cold
room, 2 = wort barrel, 3 = peristaltic pump, 4 = PBR, 5 = cooling system; I, II
and III = sampling points, IV = beer outflow, V = gas outlet. ......................... 230!
Figure VIII-2. Laboratory scale installation for primary beer fermentation: 1-air
supply; 2-mass flow controller; 3-gas sterilization filter; 4-CO2 bottles; 5rotameters; 6-peristaltic pump; 7-thermostated cold room; 8-wort barrel; 9-gas
outlet; 10-gas-lift reactor; I. beer sampling; II. carrier sampling.................... 231!
Figure VIII-3. Experimental residence time distribution (RTD) curves (&) for PBR (A)
and GLR (B) vs. mathematical simulations (models) of RTD curves: - CSTR in
series model; - - - dispersion model; ! ! dispersion model with dead zones. . 243!
Figure VIII-4. Ethanol (A) and free cells (B) concentration troughout the PBR length
(from 0 cm to 41 cm) for S. pastorianus strain W96 ( ) and S. cerevisiae BY4743
( ) .......................................................................................................... 250!
Figure VIII-5.A pH evolution vs gas composition during primary AFB fermentation;
B Free and Immobilized biomass evolution vs gas-phase composition; " - pH;
# - iBIO (gBIOMASS/gDRY CARRIER); & - Concenration of free cells (g/L); -- : CO /Air
mixture; -- : CO /N mixture; : Wort barrel pH. ........................................... 255!
2
Figure VIII-6. A Sugars evolution vs gas composition during primary AFB

fermentation; B Ethanol evolution vs gas-phase composition; " - Glucose; # Maltose; & - Maltotriose;" - Ethanol ; -- : CO /Air mixture; -- : CO /N mixture.
.............................................................................................................. 256!
2
Figure VIII-7. Variation of immobilized biomass concentration (!) and carrier amount
inside the reactor (!) during the experiment. Black arrows identify carrier
addition at fermentation days (left to right): 18 (6.9 g); 20 (2.4 g); 22 (3.8 g);
26 (3.8 g); 33 (2.9 g) and 56 (12.9 g). ...................................................... 262!
t h
t h
r d
t h
n d
t h
Figure VIII-8. Relationships between immobilized dead/non viable cells (") and
intracellular glycogen (#), neutral lipids ($), and bud scars (&) during the
course of the fermentation experiment. ...................................................... 265!
Figure VIII-9. MATS analysis results for free cells (Top) and immobilized cells
(Bottom). Results are expressed as a percentage of cells adhered to a given
solvent. Legend: Period A: up to 30 day; Period B: from 30 to 56 day; ! Cloroform; & - Hexane; ' - Decane; " - Ethyl Acetate................................ 266!
t h
t h
XVII
t h
List of Tables
List of Tables
Table II-1. Examples of carrier types and operation systems used for continuous
beer fermentation and/or maturation by immobilized brewing yeast ................ 16!
Table II-2. Cellular changes accompanying senescence (adapted from Powell et al.,
2000) ....................................................................................................... 20!
Table II-3. Genes associated with longevity in Saccharomyces cerevisiae (adapted
from Powell, 2000) .................................................................................... 21!
Table II-4. Beer major Flavour compounds in continuous and batch systems for
regular (A and B) and AFB (C) ..................................................................... 28!
Table III-1. Continuous biotechnology processes using ICT: main characteristics . 46!
Table III-2. Chemical composition of SG. (from Mussatto et al., 2006) ................. 50!
Table III-3. Main size characteristics of the corn cob and SG particles used for
immobilization experiments ........................................................................ 53!
Table III-4. All conditions (Derivatizations/Pretreatments) used for adhesion tests 55!
Table III-5. Chemical composition of the Cc particles ........................................ 60!
Table III-6. Immobilization time (min) for different types of carrier....................... 62!
Table IV-1. Spent grains properties in wet basis at T=25 C and P=1 atm ............. 84!
Table V-1. Riser configurations used in this work. ............................................ 110!
Table V-2. Resume of all conditions used in this work ....................................... 110!
Table V-3. Average of the experimental values of solids hold-up and error* in iGLR
sections. ................................................................................................. 118!
Table V-4. Parameters obtained for the linear relation between downcomers and
risers gas hold-up (e =a e +b ) ................................................................ 128!
1 0
G d
G r
Table V-5. Parameters for equation t =a u ....................................................... 130!

.
G r
Table VI-1. Riser configurations used in this work. ........................................... 144!

Table VII-1.Results evaluation by OP and HSC for QAIR=250 mL/min. .................. 202!
Table VII-2. D (mm) and Sphericity determined by Manual Image Analysis (MIA) and
using the Automatic Statistics Tool (AST). Both were obtained from the same
images by the HSC. .................................................................................. 203!
3 2
Table VII-3. Values of riser linear and superficial velocities calculated from
equations 9 and 10 for all studied situations. .............................................. 211!
Table VII-4. Velocity values of different phases in different zones of GL reactor for
2%(vol./vol.) of solids. ............................................................................. 214!
Table VII-5. Parameters obtained after optimization. ......................................... 216!
Table VII-6. Fermentation and Gas-phase properties obtained at last day of
fermentation. ........................................................................................... 218!
Table VIII-1. Wort used in this Chapter (VIII) for alcohol free-beer production ..... 227!
Table VIII-2. Composition of the SMM and respective concentrations. ................ 228!
Table VIII-3. Conditions used for optimizing continuous primary AFBs fermentation.
.............................................................................................................. 239!
Table VIII-4. Hydrodynamic properties of packed-bed (PBR) and gas-lift (GLR)
reactors. ................................................................................................. 242!
Table VIII-5. Production conditions and basic parameters of real alcohol-free beers
produced in continuous PBR and iGLR with SG and Cc as carriers, and in batch
fermentations. ......................................................................................... 245!
Table VIII-6. Higher alcohol (HA), Esters (ES) and Organic Acids (OA) content (mg/L)
of real AFBs produced in continuous PBR and iGLR with SG and Cc as carriers,
and in batch fermentations. ...................................................................... 247!
Table VIII-7. Mean concentration and associated error (p=0.05) of volatile
compounds during different stages of continuous fermentations. .................. 260!
Table VIII-8. Ageing Fermentation performance parameters at different stages. ... 263!
XVIII
Nomenclature
Nomenclature
Symbol
Description
Unit
C o e ffic ie n t o f D a rw in ia n d rift
C ro s s s e c tio n a re a
m2
A c c e le ra tio n (v e c to r)
m /s 2
Alb
L a te ra l a re a o f re a c to r b o tto m s
m2
A600
A b s o rv a n c e a t 6 0 0 n m
CD
D ra g c o e ffic ie n t
ch
C h o rd le n g th
mm
P h a s e -d is trib u tio n p a ra m e te r
d12
D is ta n c e b e tw e e n tw o p o in ts
mm
D is ta n c e /d ia m e te r
deq
E q u iv a le n t d ia m e te r
A x ia l d is p e rs io n c o e ffic ie n t
m 2 /s
B u b b le (e q u iv a le n t) d ia m e te r
$PT
T o ta l F ric tio n lo s s
Pa
$Pf
F ric tio n lo s s
Pa
P h a s e h o ld -u p
Eo
E o tw o s n u m b e r
D ra g fu n c tio n
A c c e le ra tio n d u e to g ra v ity
m /s 2
T u rb u le n t k in e tic e n e rg y p ro d u c tio n
J /(m 3 s )
D is ta n c e /h e ig h t
m or mm
H1
C o lu m n h e ig h t (W a te r C o lu m n n r 1 )
mm
H2
C o lu m n h e ig h t (W a te r C o lu m n n r 2 )
mm
hD
D is p e rs io n h e ig h t
D rift flu x
m 3 /(m 2 s )
M o m e n tu m tra n s fe r c o e ffic ie n t
k g /(m 3 s 1 )
Kf
F ric tio n lo s s c o e ffic ie n t
T u rb u le n t k in e tic e n e rg y
m 2 /s 2
C h a ra c te ris tic le n g th
F o rc e o n e p h a s e is a c tin g o n o th e r p h a s e
N /m 3
N u m b e r o f ta n k s
XIX
Nomenclature
Nomenclature
Symbol
Description
Unit
OTRTOTAL
T o ta l O x y g e n T ra n s fe r R a te
m g /(L .h )
OTROS
S u p p ly O x y g e n T ra n s fe r R a te
m g /(L .h )
OTRDO
D is s o lv e d O x y g e n T ra n s fe r R a te
m g /(L .h )
P re s s u re
Pa
PL
P re s s u re d iffe re n c e
Pa
Pe
P e c le t n u m b e r
QG
G a s F lo w ra te
L /m in
QL
L iq u id flo w ra te
L /h
Re
R e y n o ld s n u m b e r
T im e
m in o r s
TG
G a s re s id e n c e tim e in o p tic a l p ro b e
TM
G a s m o u n tin g tim e in o p tic a l p ro b e
tm
M ix in g tim e
M e a n b u b b le ris e s p e e d
m /s
u0
T e rm in a l b u b b le s p e e d
m /s
S u p e rfic ia l v e lo c ity
m /s
In s ta n ta n e o u s v e lo c ity (v e c to r)
m /s
Ubt
B u b b le te rm in a l v e lo c ity
m /s
V o lu m e
Vslp
V o lu m e o f s a m p le
L in e a r v e lo c ity
m /s
V e lo c ity flu c tu a tin g p a rt
m /s
vdr
D rift v e lo c ity
m /s
In te rs titia l v e lo c ity
m /s
VR
R e a c to r w o rk in g v o lu m e
VD
D e a d z o n e s v o lu m e
XTGV
V a lu e s fro m T G V m e th o d
XINJ
V a lu e s fro m n e w in je c tio n m e th o d
XX
Nomenclature
Greek symbols
Symbol
Description
Unit
% /#
P h a s e v o lu m e fra c tio n (H o ld -u p )
&
F lu id d e n s ity
k g /m 3
'
S o lid lo a d
% (v o l./v o l.)
M o le c u la r d y n a m ic v is c o s ity
mPa.s
A d im e n s io n a l tim e
M e a n re s id e n c e tim e
m in
(D
A d im e n s io n a l tim e w ith d e a d v o lu m e s
)D
M e a n re s id e n c e tim e w ith d e a d v o lu m e s m in
T u rb u le n t d y n a m ic v is c o s ity
Pa.s
*t
T u rb u le n t k in e m a tic v is c o s ity
m 2 /s
S u rfa c e te n s io n
N /m
P a rtic le re la x a tio n tim e
Sub/superscript
DC
G
L
S
H
th e o
exp
c
C
bb
r
d
c
D
i,k
m
TD
B
D ry C a rrie r
G a s -p h a s e
L iq u id -p h a s e
S o lid -p h a s e
P s e u d o h o m o g e n e o u s -p h a s e
T h e o ric a l
E x p e rim e n ta l
C ritic a l
C o lu m n s
B o tto m
R is e r
D o w n c o m e r/D is p e rs e p h a s e
C o n tin u o u s p h a s e
D ra g (fo rc e )
In d e x o f a p h a s e
M ix tu re p ro p e rty
T u rb u le n t d is p e rs io n (fo rc e )
B u b b le
XXI
Abbreviations
Abbreviations
AFB
Alcohol-free beer
AATases
Acyltransferases
AL
Aldehydes
AST
Automatic statistics tool
BC
Bubble columns
CFD
Computional fluid dynamics
CCP
Cold contact process
Cc
Corncobs
CSTR
Continuous stirred tank reactor
dSG
Dry spent grains
ES
Esters
FAN
Free amino nitrogen
g-l
gas liquid
g-l-s
gas liquid solid
HA
Higher alcohols
HoR
Homogeneous regime
HeR
Heterogeneous regime
HSC
High speed camera
iGLR
Internal-loop gas-lift reactor
ICT
Immobilized cell technology
ILV
Isoleucine, leucine and valine
iBio
Immobilized biomass (gBiomass/gDry
IEC
Ion exchange capacity (meq/g)
LAB
Low-alcohol beer
MIA
Manual image analysis
O.D.
Optical density
OA
Organic acids
OTR
Oxygen transfer rate
OP
Optical probe
PBR
Packed-bed reactor
PFR
Plug Flow Reactor
Rpm
Rotations per minute
SG
Spent grains
SMM
Synthetic mineral medium
TGV
Treatment for gaseous velocity
WAI
Water adsortion index
wSG
Wet spent grains
XXII
Carrier
Chapter I
I. Chapter I Motivation and Outline
I.1 Thesis Motivation
I.2 Research Aims
I.3 Thesis Outline
I.4 References
Chapter I
I.1 Thesis Motivation

I.1.1
Alcohol-free beer: definition, history and market
T h e e x c e s s o f a lc o h o l c o n s u m p tio n c o n trib u te d m o re th a n a n y o th e r
fa c to r to th e o c c u rre n c e o f d o m e s tic , la b o u r an d d riv in g a c c id e n ts ,
v io le n c e ,
abuses,
m e n ta l
in c a p a c ity
and
even
d e a th .
E th a n o l
is
a b s o rb e d b y d iffu s io n a n d d is trib u te d th ro u g h a ll h u m a n b o d y tis s u e s

by
th e
b lo o d .
(F e rre ira
and
W illo u g h b y ,
2 0 0 8 ).
It
has
been
a ls o
m e n tio n e d th a t m o d e ra te a lc o h o l c o n s u m p tio n h a s b e tte r lo n g-te rm

e ffe c ts o n h u m a n h e a lth , e v e n th a n n o c o n s u m p tio n (B r n y ik e t a l.,
2 0 1 2 ; F e rre ira a n d W illo u g h b y , 2 0 0 8 ). T h e m a in re a s o n s a re d u e to th e
p re s e n c e o f p h e n o lic c o m p o u n d s (h ig h a n tio x id a n t c a p a c ity ), e s s e n tia l
v ita m in s a n d m in e ra ls (m a g n e s iu m ) (B a m fo rth , 2 0 0 2 ; B r n y ik e t a l.,
2 0 1 2 ).
These
compounds
a re
a ls o
p re s e n t
in
a lc o h o l-fre e
b e e rs,
a lth o u g h a t a lo w e r q u a n tity (B a rto lo m e t a l., 2 0 0 0 ), w ith th e m a in

a d v a n ta g e o f n o t h a v in g th e p e rv e rs e e ffe c ts o f e x c e s s iv e a lc o h o l
c o n s u m p tio n (B r n y ik e t a l., 2 0 1 2 ; F e rre ira a n d W illo u g h b y , 2 0 0 8 ).
F ro m a s im p le p o in t o f v ie w , b e e r c a n b e c la s s ifie d a c c o rd in g to its
a lc o h o l c o n te n t, b u t th e re is n o c o n s e n s u s o n th is . A c c o rd in g to th e
E u ro p e a n U n io n (E U ) d ire c tiv e 9 8 /3 3 /C E E b e e r is a b e v e ra g e d e s crib e d
by
th e
EU
code
NC 2203
w ith
an
a lc o h o l
c o n te n t
s u p e rio r
to
0 .5 % (v o l./v o l.) (C C E , 1 9 9 2 ; C E , 2 0 0 5 ). in m o s t o f th e c o u n trie s ,

g e n e ra lly
a lc o h o l-fre e
beer
(A F B )
or
n o
a lc o h o l
b e e r
(o r
d e a lc o h o liz e d b e e r ) c o n ta in s le s s th a n 0 .5 % (v o l./v o l.) o f a lc o h o l,

w h ile lo w a lc o h o l b e e rs (L A B ) m a y c o n ta in u p to 1 .2 % (v o l./v o l.) a n d
b e e rs
c o n ta in in g
b e tw e e n
1 .2 % (v o l./v o l.)
and
3 .0 % (v o l./v o l.)
a re
c o n s id e re d to b e re d u c e d in a lc o h o l (L e w is a n d Y o u n g , 1 9 9 5 ). In
s o m e c o u n trie s , d u e to c u ltu ra l a n d re lig io u s rule s , A F B s c a n n o t e x c e e d
0 .0 5 % (v o l./v o l.) (B r n y ik , e t a l., 2 0 1 2 ). T h e te rm in o lo g y u s e d in th is
th e s is w ill b e th e o n e u s e d in E U le g is la tio n , w h e re a n A F B c o n ta in s
le s s th a n 0 .5 % (v o l./v o l.) o f e th a n o l (C C E , 1 9 9 2 ).
Chapter I
H is to ric a lly , A F B p ro d u c tio n in c re a s e d du rin g a p e rio d (1 9 2 0-1 9 3 3 )

b e tw e e n th e tw o W o rld W a rs , e s p e c ia lly in th e U n ite d S ta te s d u e to th e
N a tio n a l P ro h ib itio n A c t, a ls o c a lle d : D ry L a w . T h is la w fo rb id th e
p ro d u c tio n , im p o rta tio n a n d e x p o rta tio n o f in to x ic a n t liq u o rs (b e v e ra g e s
w ith m o re th a n 0 .5 % (v o l./v o l.) o f e th a n o l) to a n d fro m th e U n ite d
S ta te s o f A m e ric a . F o r th e b re w in g in d u s try in th e U n ite d S ta te s o f
A m e ric a , th is la w w a s th e d riv in g fo rc e th a t le d to th e in c re a s e o f A F B s
p ro d u c tio n a n d th e d e v e lo p m e n t o f n e w p ro d u c tio n m e th o d s . N a tu ra lly ,
w h e n th e D ry L a w w a s a b o lis h e d in 1 9 3 3 , A F B s p ro d u c tio n d e c lin e d .
In th e p a s t tw o d e c a d e s th e h ig h ly c o m p e titiv e m a rk e ts in b re w in g
in d u s try , a llie d to th e g lo b a l tre n d fo r a h e a lth ie r life s ty le w ith o u t
a lc o h o l, re s tric tin g d riv in g a n d la b o u r la w s a n d re lig io u s is s u e s le d to
th e d e v e lo p m e n t a n d in c re a s e d c o n s u m p tio n o f A F B (B r n y ik e t a l.,
2 0 1 2 ; C . M A R M , 2 0 1 1 ). T h e p ro d u c tio n o f A F B s is s till a g ra in in th e
d e s e rt o f th e g lo b a l b e e r p ro d u c tio n . O v e r th e la s t fiv e y e a rs th e
a v e ra g e s a le s o f A F B s in c re a s e d b y 5 0 % o n ly in E u ro p e . A c tu a lly S p a in ,
th e la rg e s t c o n s u m e r c o u n try o f lo w a lc o h o l b e v e ra g e s , s o ld 9 .5 % o f
A F B s in 2 0 1 0 (C . M A R M , 2 0 1 1 ). O n th e o th e r h a n d in G e rm a n y , w h ic h
h a s th e b ig g e s t b e e r m a rk e t, th e a v e ra g e s a le s o f A F B s a re b e tw e e n
4 % a n d 5 % o f to ta l b e e r c o n s u m p tio n (B r n y ik e t a l., 2 0 1 2 ). T h is h a s
b e e n a ttrib u te d to E U le g is la tio n th a t re s tric ts a lc o h o l c o n s u m p tio n in
m a n y d a ily a c tiv itie s (s c h o o ls , w o rk in g , d riv in g ) o r in s o m e c o u n trie s in
s p o rts e v e n ts (e g . S ta d iu m s o f fo o tb a ll).
In
2006
th e
beer
P o rtu g u e s e
m a rk e t
re p re s e n te d
1 .5 %
of
th e
P o rtu g u e s e g ro s s d o m e s tic p ro d u c t g e n e ra tin g o v e r 1 .5 th o u s a n d s o f

m illio n s E u ro s (A P V C , 2 0 0 6 ). P o rtu g u e s e b e e r c o n s u m p tio n h a s b e e n
d e c re a s in g o v e r th e la s t fiv e y e a rs (F ig u r e I- 1 ). In a v e ra g e a P o rtu g u e s e
in h a b ita n t c o n s u m e s p e r y e a r 6 0 .6 L o f b e e r (A P V C , 2 0 1 1 ). In th e la s t
fiv e y e a rs , th e c o n s u m p tio n o f A F B s in P o rtu g a l fo llo w s a tre n d o f
c o n s u m p tio n s im ila r to re g u la r b e e rs . A F B s m a rk e t s h a re is v e ry lo w
and
v a rie s
b e tw e e n
2 .5 %
to
3 .3 %
of
g lo b a l
P o rtu g u e s e
beer
c o n s u m p tio n . T h is m a rk e t s h a re is s im ila r to th e o n e fo u n d in o th e r E U
Chapter I
c o u n trie s , w ith th e e x c e p tio n o f S p a in (A P V C , 2 0 1 1 ; B r n y ik e t a l.,
6500"
240"
180"
6000"
120"
5500"
60"
5000"
0"
Consumption of AFB (1E5 L)"
Consumption of regular
beer (1E5 L)"
2 0 1 2 ; C . M A R M , 2 0 1 1 ).
Time / Years"
Figure I-1. Beer market in Portugal in the last Five Years. Legend: !: Total
Beer Consumption; ": Regular Beer Consumption; -#$% Alcohol-free Beer
Consume. (Data from http://www.apcv.pt/asp/ in 15th December 2011)
A fe w y e a rs a g o th e re w e re h ig h e x p e c ta tio n s in th e in c re a s in g
m a rk e t o f A F B s , b u t th e s a le s d id n o t in c re a s e a s m u c h a s in itia lly
p re d ic te d . It w a s e x p e c te d th a t th e n e e d o f a h e a lth ie r life s ty le a n d
re s tric tin g c iv il la w s w o u ld d riv e th e c o n s u m p tio n fro m re g u la r b e e r to
A F B . A lth o u g h th e A F B m a rk e t s h a re in P o rtu g a l h a s b e en c o n s ta n t, th e
d e c re a s e o n A F B c o n s u m p tio n in d ic a te s a s h iftin g o n d rin k in g h a b its b y
th e P o rtu g u e s e p o p u la tio n . In F ig u r e I- 1 it is s u g g e s te d th a t th e
c o n s u m e r o f A F B in P o rtu g a l h a s a s im ila r p ro file th a n a c o n s u m e r o f
re g u la r
b e e r.
Reasons
fo r
th is
a re
v a ria b le
and
in c lu d e :
m e d ic a l
in d ic a tio n s , o b lig a tio n d u e to d riv in g a n d o p e ra tin g h e a v y m a c h in e s ,

even
m o ra l
p o s itio n s
about
a lc o h o l
abuse
(w w w .Ib e s a .p t,
access
2 0 /0 4 /2 0 1 0 ).
In o rd e r to c o n trib u te fo r a h e a lth ie r life s ty le in EU c o u n trie s a n d
c o n s id e rin g th e c o m p e titiv e b e e r m a rk e t n e w A F B p ro d u c tio n m e th o d s
th a t re d u c e p ro d u c tio n c o s ts a n d in c re a s e A F B q u a lity a re w o rth o f
re s e a rc h a n d d e v e lo p m e n t.
Chapter I
I.2 Research Aims

The
m a in
o b je c tiv e
of
th is
th e s is
was
to
d e v e lo p
la b-s c a le
te c h n o lo g y fo r c o n tin u o u s A F B p ro d u c tio n th a t w o u ld o v e rc o m e th e
e x is tin g m e th o d s in te rm s o f v o lu m e tric p ro d u c tiv ity a n d w o u ld b e
c o m p a ra b le in te rm s o f p ro d u c t q u a lity . T h e p a rtia l g o a ls to b e
a c h ie v e d a re :
$ S e le c tio n o f a n e c o n o m ic a lly a ffo rd a b le c a rrie r m a te ria l fo r y e a s t
c e lls im m o b iliz a tio n .
$ E v a lu a te th e im p o rta n c e o f re a c to r ty p e , y e a s t s tra in a n d c a rrie r
ty p e in a c o n tin u o u s lim ite d p rim a ry fe rm e n ta tio n o f A F B s .
$ S tu d y th e c o n s e q u e n c e s o f th e im m o b iliz e d b io m a s s a g e in g o n
p ro d u c t q u a lity a n d p ro p o se a s tra te g y to m in im is e its a d v e rs e e ffe c ts
$ S tu d y th e in flu e n c e o f c a rrie r p ro p e rtie s in th re e-p h a s e s y s te m
(g a s -liq u id -s o lid ) h y d ro d y n a m ic s a n d u n d e rs ta n d h o w th a t c a n in flu e n c e
b io re a c to r p e rfo rm a n c e .
$ D e v e lo p , b a s e d o n g lo b a l h y d ro d y n a m ic s tu d ie s , th e b io re a c to r
d e s ig n fo r th e s p e c ific re q u ire m e n ts o f th e lim ite d p rim a ry fe rm e n ta tio n
p ro c e s s w ith c e ll im m o b iliz e d o n a c e llu lo s e -b a s e c a rrie r.
$ U n d e rs ta n d th e lo c a l h y d ro d y n a m ic s o f th e d e s ig n e d b io re a c to r
in d u c e d b y th e c a rrie r p a rtic le s a n d s tu d y th e in flu e n c e o f th e lim ite d
fe rm e n ta tio n in lo c a l h y d ro d y n a m ic s .
Chapter I
I.3 Thesis Outline

T h is th e s is is s u b d iv id e d in e ig h t (8 ) c h a p te rs w e re th e w o rk is
p re s e n t a n d d is c u s s e d . E a c h c h a p te r, w ith th e e x c e p tio n o f C h a p te r II
a n d c h a p te r IX , is d iv id e d in: In tro d u c tio n , M a te ria l a n d M e th o d s ,
R e s u lts a n d D is c u s s io n a n d C o n c lu s io n s . In th e firs t p a rt o f th is th e s is
is re la te d w ith c a rrie r s e le c tio n (C h a p te r III), th e s e c o n d p a rt w ith th e
th re e -p h a s e b io re a c to r h y d ro d y n a m ic s (C h a p te r IV to V II), w h ile a th ird
p a rt (C h a p te r V III) w ith c o n tin u o u s fe rm e n ta tio n o f A F B .
In C h a p te r II s e v e ra l m e th o d s o f A F B p ro d u c tio n a re d e s c rib e d , b o th
in b a tc h a n d c o n tin u o u s m o d e , a t la b o ra to ry a n d in d u s tria l s c a le a s w e ll
a s im p o rta n t in fo rm a tio n a b o u t b e e r q u a lity a n d fla v o u r.
In C h a p te r III th e s e le c tio n b e tw e e n tw o c e llu lo s e-b a s e d c a rrie rs fo r
y e a s t c e lls im m o b iliz a tio n is in v e s tig a te d . In a d d itio n th e m e th o d s fo r
im p ro v in g im m o b iliz a tio n a re s tu d ie d .
In C h a p te r IV th e fu n d a m e n ta l s tu d y o f s p e n t g ra in s p a rtic le s
in flu e n c e in th re e-p h a s e B u b b le C o lu m n s (B C ) is re p o rte d .
In C h a p te r V th e g lo b a l th re e-p h a s e in te rn a l-lo o p g a s -lift re a c to r
(iG L R ) h y d ro d y n a m ic s a re s tu d ie d a n d th e b e s t re a c to r c o n fig u ra tio n fo r
c o n tin u o u s A F B is s e le c te d .
In c h a p te r V I C F D s im u la tio n fo r g -l s y s te m in iG L R is p e rfo rm e d in
o rd e r to e x p la in b e tte r th e p h e n o m e n a th a t o c c u rs in th is ty p e o f
re a c to r.
In C h a p te r V II th e lo c a l th re e -p h a s e G L R h y d ro d y n a m ic s a re s tu d ie d
u n d e r fe rm e n ta tio n c o n d itio n s a n d a m e th o d fo r m e a s u rin g lo c a l g a s
p ro p e rtie s in th re e-p h a s e sy s te m s is p ro p o s e d .
In C h a p te r V III th e s tu d y o f d iffe re n t s y s te m s fo r c o n tin u o u s lim ite d
A F B p rim a ry fe rm e n ta tio n a re d e s c rib e d . B e e r q u a lity a n d fla v o u r
c o m p o u n d s fo rm a tio n a t d iffe re n t c o n d itio n s , a s w e ll a s s tra te g ie s fo r
lo n g -te rm fe rm e n ta tio n a re p rop o s e d .
In C h a p te r IX th e g e n e ra l c o n c lu s io n s a n d s u g g e s tio n s fo r fu tu re
w o rk a re d e s c rib e d .
Chapter I
I.4 References
APVC, Associao Portuguesa de Produtores de Cerveja (2006). Indstria cervejeira
nacional contribui para 1,5% do PIB. Orient, pp. 1-2.
APVC, Associao Portuguesa de Produtores de Cerveja (2011). Relatrios Anuais
do Mercado cervejeiro em Protugal (in portuguese). Retrieved December 15 ,
2011, from http://www.apcv.pt/asp/
Bamforth, C. W. (2002). Nutritional aspects of beer a review. Nutrition Research,
22(1), 227237.
Brnyik, T., Silva, D.P., Baszczy"ski, M., Lehnert, R., and Almeida e Silva, J.B.
(2012). A review of methods of low alcohol and alcohol-free beer production.
Journal of Food Engineering, 108(4), 493-506.
MARM, Convenio MARM - Cerveceros de Espaa (2011). Informe socioeconomico
del sector de la cerveza en Espaa (in Spanish). Report (p. 36).
CCE, C. C. E. (1992). Directiva 92/83/CEE do Conselho relativa a harmonizao da
estrutura dos impostos especiais sobre o consumo de lcool e bebidas lcolicas
(in Protuguese). Directiva 92/83/CEE do Conselho.
CE, C. E. (2005). Regulamento (CE) N.o 1967/2005 Da Comisso relativo
classificao de certas mercadorias na Nomenclatura Combinada. (in Portuguese).
Jornal Oficial da Comisso Europeia, 10-12.
Ferreira, M. P. and Willoughby, D. (2008). Alcohol consumption: the good, the bad,
and the indifferent. Applied Physiology, Nutrition and Metabolism, 33(1), 12-20.
Lewis, M. J. and Young, T. W. (1995). Brewing. (M. J. Lewis and T. W. Young, Eds.).
London: Chapman and Hall.
www.Ibesa.pt. (2010). Cerveja sem lcool. Retrieved April 20, 2010, from
www.ibesa.pt
t h
Chapter II
II. Chapter II Alcohol-free Beer
II.1 Methods to Produce Alcohol-free Beer
II.2 Continuous Beer Production
II.3 Continuous Alcohol-free Beer Production
II.4 Beer Quality and Flavour: Origin and Influence
II.5 References
Chapter II
II.1 Methods to Produce Alcohol-free Beer

A F B h a s b e e n in d u s tria l p ro d u c e d s in c e th e b e g in n in g o f X X c e n tu ry
a n d d iffe re n t te c h n o lo g ie s h a v e b e e n d e v e lo p e d o v e r th e la s t c e n tu ry ,
p a rtic u la rly o v e r th e la s t th irty y e a rs . B e e r w ith lo w a lc o h o l c o n te n t c a n
b e p ro d u c e d b y : (1 ) u s in g a d d itio n a l te c h n iq u e s to re m o v e th e a lc o h o l;
(2 ) tra d itio n a l m e th o d s fo r re s tric te d e th a n o l fo rm a tio n ; (3 ) c o n tin u o u s
fe rm e n ta tio n (B r n y ik e t a l., 2 0 0 5 ; B r n y ik e t a l., 2 0 1 2 ; L e h n e rt e t a l.,
2 0 0 9 ; L e w is a n d Y o u n g , 1 9 9 5 ).
II.1.1
These
Techniques to remove alcohol from regular beer

m e th o d s
re q u ire
an
in v e s tm e n t
in
a d d itio n a l
b re w in g
e q u ip m e n t. T h e te c h n iq u e s th a t a re b e in g u s e d to re m o v e th e a lc o h o l
fro m re g u la r b e e rs a re e ith e r th e rm a l-b a s e d (v a c u u m e v a p o ra tio n o r
d is tilla tio n ) o r m e m b ra n e -b a s e d (d ia ly s is ; re v e rs e o s m o s is ) (L e w is a n d
Y o u n g , 1 9 9 5 ).
II.1.1.1
Thermal-based process
V a c u u m e v a p o ra tio n is ty p ic a lly a th re e-s ta g e p ro c e s s . In th e firs t

s ta g e , p re -h e a te d b e e r (3 5 C ) p a s s e s u n d e r v a c u u m tro u g h a p la te
e v a p o ra to r h e a te d a t 5 0 C w h e re th e b e e r is d e g a s s e d a n d th e v o la tile
c o m p o u n d s a re lib e ra te d . T h e b e e r s te a m g o e s to a v a c u u m c o lu m n
w h e re is s e p a ra te d in tw o : A F B a n d a n a lc o h o l-ric h c o n d e n s a te . T h e
a ro m a c o m p o u n d s a re re c o v e re d fro m d e g a s s e d C O 2 (b y w a te r o r A F B )
a n d re -a d d e d to th e p re v io u s ly o b ta in e d A F B . T h e d e v ic e s u s e d fo r
v a c u u m e v a p o ra tio n s h o u ld b e a b le to re m o v e e ffic ie n tly e th a n o l a t
s h o rt re s id e n c e tim e s . T h e m a in v a c u u m e v a p o ra to rs u s e d a re th in film
e v a p o ra to rs , w h ic h c a n o p e ra te b y g ra v ity o r u s in g a m e c h a n ic a l d e v ic e
p ro m o tin g a ro ta tio n a l m o v e m e n t. T h e m a in a d v a n ta g e o f u s in g v a c u u m
e v a p o ra tio n m e th o d s o v e r a tm o s p h e ric e v a p o ra tio n is th e b e tte r b e e r
ta s te p re s e rv a tio n . T h e th e rm a l p ro c e s s a llo w : (1 ) to re m o v e c o m p le te ly
th e a lc o h o l fro m b e e r (# 0 .0 5% (v o l./v o l.)); (2 ) th e c o m m e rc ia liz a tio n o f
b y -p ro d u c ts
Chapter II
(a lc o h o l
fo r
v in e g a r
p ro d u c tio n );
(3 )
c o n tin u o u s
and
a u to m a tic o p e ra tio n w ith s h o rt s ta rt-u p p e rio d s ; a n d (4 ) fle x ib ility

(a d a p te d to s e v e ra l b e e r c o m p o s itio n s a n d v o lu m e s fro m 2 0 0 L to
4 0 0 0 0 L ).
H o w e v e r,
th e
p u rc h a s e
of
th e s e
s y s te m s
re q u ire s
(1 )
s ig n ific a n t in v e s tm e n t; (2 ) h ig h ru n n in g c o s ts (e n e rg y c o n s u m p tio n ); (3 )
th e rm a l d a m a g e (lo s s o f v o la tile s ) o f b e e r. B e s id e s th e s e re m o v a l s te p s ,
a t th e e n d , th e re s u ltin g L A B h a s to b e d ilu te d w ith o x y g e n -fre e w a te r
a n d c a rb o n a te d (B r n y ik e t a l., 2 0 1 2 ; L e w is a n d Y o u n g , 1 9 9 5 ).
II.1.1.2
Membrane processes
A ll th e m e m b ra n e p ro c e s s e s h a v e le s s th e rm a l im p a c t o n A F B
q u a lity . T h e y c a n o p e ra te a u to m a tic a lly a n d a re fle x ib le , b u t a t th e
s a m e tim e th e y re q u ire s ig n ific a nt c a p ita l a n d ru n n in g c o s ts .
T h e d riv in g fo rc e in d ia ly s is is th e c o n c e n tra tio n g ra d ie n t b e tw e e n
b e e r a n d d ia ly s in g m e d iu m , w h ic h n o rm a lly o p e ra te s in c o u n te r-flo w .
B rie fly , b e e r (1 C to 6 C ) p a s s e s tro u g h a d ia ly s e r w h e re s m a ll
m o le c u le s (a s e th a n o l) p a s s tro u g h a s e le c tiv e m e m b ra n e in to th e
d ia ly s a te . H o w e v e r, s o m e a s p e c ts m u s t b e ta k e n in a c c o u n t a s th e
d ia ly s a te
p re s s u re
to
a v o id
CO2
in flu e n c e .
The
d ia ly s a te
is
th e n
d e a lc o h o liz e d in a v a c u u m d is tilla tio n c o lu m n . T h e p ro c e s s e ffic ie n c y

d e p e n d s m a in ly o n th e m e m b ra n e s c o m p o s itio n (c e llu lo s e o r s y n th e tic )
a n d a rra n g e m e n t (h o llo w fib e rs ), b e e r flo w ra te a n d c o m p o s itio n o f th e
d ia ly s a te . A s th e m e m b ra n e s a re n o t e th a n o l s e le c tiv e , o th e r im p o rta n t
fla v o u r b e e r c o m p o u n d s a re re m o v e d , w h ic h c a n b e o v e rc o m e by th e
in tro d u c tio n o f th e s e in th e d ia ly s a te to re d u c e th e fla v o u r c o m p o u n d s
lo s s e s .
In re v e rs e o s m o s is , th e u s e d m e m b ra n e is m o re s e le c tiv e th a n th e
o n e n o rm a lly u s e d in d ia ly s is . R e s e rv e o s m o s is p rin c ip le ta k e s in
account
th a t
w a te r
and
e th a n o l
p e rm e a te
m o re
th a n
o th e r
beer
c o m p o u n d s . B e e r p a s s e s tro u g h th e s u rfa c e o f a m e m b ra n e a t h ig h
p re s s u re
(4 0 b a r)
and
th e
most
p e rm e a b le
s u b s ta n c e s
c ro s s
th e
m e m b ra n e . T h is m e th o d c o n c e n tra te s b e e r a n d th e tra n s fe r o f e th a n o l
a ls o in c re a s e s . T h e n p u re o r d e m in e ra lise d w a te r is a d d e d (d ia filtra tio n )
10
w h ile
e th a n o l
Chapter II
s till
passes
th e
m e m b ra n e
u n til
th e
fin a l
e th a n o l
c o n c e n tra tio n is a c h ie v e d . In re v e rs e o s m o s is n o h e a t is a p p lie d ,

h o w e v e r m e m b ra n e c o s t a n d th e h ig h o p e ra tio n p re s s u re s re q u ire d a re
m a in d is a d v a n ta g e s . G e n era lly , re s e rv e o s m o s is A F B s h a v e s ig n ific a n t
lo s s e s o f v o la tile c o m p o u n d s (7 0 % to 8 0 % o f h ig h e r a lc o h o ls , 8 0 % to
9 0 % o f e s te rs ) (B r n y ik e t a l., 2 0 1 2 ; L e w is a n d Y o u n g , 1 9 9 5 ). T h e
m a jo rity
of
in d u s tria l
a p p lic a tio n s
a re
th e rm a l/m e m b ra n e
based,
s o m e tim e s e v e n a m ix o f b o th , b u t u s u a lly b re w in g in d u s trie s h id e o r

p a te n t
th e ir
p ro c e s s
to
a v o id
b e in g
used
by
o th e r
c o m p a n ie s
(B a rto lo m e t a l., 2 0 0 0 ).
II.1.2
Traditional methods to restrict ethanol formation
T ra d itio n a l m e th o d s to re s tric t e th a n o l fo rm a tio n m a in ly u s e s m a lle r

b re w in g o p e ra tio n s th a t a llo w p ro d u c in g A F B . T h e s e o p e ra tio n s in c lu d e
c h a n g e s in m a s h in g , a rre s te d o r lim ite d fe rm e n ta tio n a n d u tiliz a tio n o f
s p e c ia l y e a s t (B r n y ik e t a l., 2 0 1 2 ; L e w is a n d Y o u n g , 1 9 9 5 ).
II.1.2.1
Change in mashing
M a s h in g is o n e s ta g e o f b e e r w o rt p ro d u c tio n , w h e re th e d e g ra d a tio n
o f s ta rc h to fe rm e n ta b le s u g a rs a n d s o lu b le d e x trin s o c c u rs . S o th e
m a s h in g s te p h a s a h u g e in flu e n c e in th e fin a l c o n c e n tra tio n o f s u g a rs
in th e w o rt, th u s in fin a l b e e r e th a n o l c o n te n t. If c h a n g e s a re o p e ra te d
in th is p ro c e s s th e fe rm e n ta b le s u g a r c o n c e n tra tio n c a n b e a lte re d . T h e
c h a n g e s th a t c a n b e o p e ra te d d u rin g m a s h in g to re d u c e w o rt s u g a rs
c o n c e n tra tio n a re : th e a p p lic a tio n o f h ig h te m p e ra tu re s (7 5 C -8 0 C )
th a t d e a c tiv a te s # -a m y la s e ; th e c o ld w a te r m a lt e x tra c tio n (< 6 0 C ),
w h ic h e x tra c t m a lt fla v o u r c o m p o u n d s w ith o u t in c re a s in g w o rt g ra v ity ;
spent
g ra in s
re -m a s h in g
(e x tru s io n
or
a c id
h y d ro ly s is ;
and
th e
u tiliz a tio n o f g ra in s d e fic ie n t in # a m y la s e (B r n y ik e t a l., 2 0 1 2 ; L ew is

a n d Y o u n g , 1 9 9 5 ).
11
II.1.2.2
Chapter II
Limited or arrested fermentation
M o d ific a tio n s in n o rm a l p rim a ry b e e r fe rm e n ta tio n to p ro d u c e A F B s

a re p ro b a b ly th e m o s t u s u a lly m e th o d s a p p lie d in in d u s try . T h e s e
a lte ra tio n s a re m a d e b y a rre s t o r lim it o f th e fe rm e n ta tio n a n d a llo w
b re w in g
in d u s trie s
to
w o rk
w ith
tra d itio n a l
e q u ip m e n t.
The
m a in
o b je c tiv e is to k e e p a lo w e th a n o l c o n te n t. A rre s te d fe rm e n ta tio n is

o b ta in e d b y re m o v in g y e a s t b e fo re e x c e s s iv e a tte n u a tio n o c c u rs (ra p id
c o o lin g
or
filtra tio n ),
te m p e ra tu re s
(0 C
fo llo w e d
to
1 C)
by
p rio r
m a tu ra tio n
to
new
fo r
10 days
filtra tio n ,
at
lo w
c a rb o n a tio n ,
s ta b iliz a tio n a n d s te riliz a tio n . L im ite d fe rm e n ta tio n m e th o d s a re d o n e

b y c re a tin g c o n d itio n s th a t lim it th e y e a s t m e ta b o lis m . A m o n g th e s e ,
th e c o ld c o n ta c t p ro c e s s (C C P ), w h ich is d o n e u s in g a lo w g ra v ity w o rt
fe rm e n te d a t lo w te m p e ra tu re s (0 C to 1 C ), h a s a g o o d p o te n tia l a n d
h ig h v o la tile c o m p o u n d s a re p ro d u c e d . L o w a ld e h y d e re d u c tio n is
a c h ie v e d le a d in g to s tro n g in flu e n c e o f w o rty o ff-fla v o u rs in th e
p ro d u c e d A F B (P e rp te a n d C o llin , 1 9 9 9a , 1 9 9 9 b ).
W h e n A F B s a re p ro d u c e d b y th e s e m e th o d s , to im p ro v e its q u a lity
s o m e o p e ra tio n s s h o u ld b e ta k e n in a c c o u n t: (1 ) a d d in g o f d a rk /p a le
c a ra m e l m a lt (c o n trib u te s to fin a l fla v o u r); (2 ) d ilu tio n o f w o rt b e fo re
b o ilin g (re d u c e s b ittern e s s , in c re a s e s e s te rs a n d h ig h e r a lc o h o ls ); (3 )
d e c re a s e w o rt p H (lo w te m p e ra tu re a tte n u a tio n p e r s e is n o t c a p a b le to
d o th a t) (N a rz is s e t a l., 1 9 9 2 ).
II.1.2.3
The
use
Special yeasts utilization

of
s p e c ia l
y e a s t,
c a p a b le
of
p e rfo rm in g
lim ite d
fe rm e n ta tio n c o n s is ts o n u s in g th e ir lo w o r z e ro a b ility to p ro d u c e
e th a n o l. S p e c ia l y e a s t c a n b e o b ta in e d b y th e s e le c tio n o f a p ro p e r
m ic ro b ia l s tra in a s S . lu g w ig ii u n a b le to fe rm e n t m a lto s e - th e m a in
w o rt
sugar
(N a rz is s
et
a l.,
1 9 9 2 ).
O th e r
m e th o d s
in c lu d e
th e
d e v e lo p m e n t o f n e w y e a s t s tra in s b y fo rc in g m o d ific a tio n o f b re w in g

y e a s t b y ra n d o m m u ta tio n u s in g u ltra v io le t irra d ia tio n (N a rv til e t a l.,
2 0 0 1 ) o r b y u s in g g e n e tic e n g in e e rin g te c h n iq u e s th a t in te n tio n a lly
d e le te g e n e s fro m y e a s t c e lls (S e le c k y e t a l., 2 0 0 8 ). N o rm a lly th e
12
Chapter II
in te n tio n a lly d e le te d g e n e s a re in v o lv e d in th e s y n th e s is o f e n z y m e s
p re s e n t in th e tric a rb o x y lic a c id 's (T C A ) c y c le . A m o n g th e s e , th e u s u a l
d e le te d g e n e s a re : C IT 1 , A C O 1 , F U M 1 , M D H 1 , ID H 1 ID H 2 , K G D 1 , K G D 2 ,
a n d L P D 1 (P rz y b y la-Z a w is la k e t a l., 1 9 9 9 ; R e p e tto a n d T z a g o lo ff, 1 9 9 0 ;
S e le c k y e t a l., 2 0 0 8 ). T h e d e v e lo p m e n t o f n e w y e a s t s tra in s is n o rm a lly
d o n e in h a p lo id /d ip lo id la b o ra to ry s tra in s a n d n o t in in d u s tria l y e a s t
s tra in s , w h ic h n o t o n ly a re a llo p lo id s a s a ls o h a v e im p o rta n t in d u s tria l
c h a ra c te ris tic s (fe rm e n ta tio n ra te , fla v o u r fo rm a tio n , flo c c u la tio n ). T h e
id e a l y e a s t s tra in is a h y b rid b e tw e e n th is g e n e tic a lly m o d ifie d s tra in s
and
in d u s tria l
s tra in s ,
c a rry in g
a ll-im p o rta n t
fo r
in d u s tria l A F B p ro d u c tio n . In a d d itio n , th e g e ne tic d e v e lo p m e n t a n d

a p p lic a tio n o f y e a s t s tra in s h a s le g a l o b s ta c le s a n d g e n e ra te s c o n c e rn s
in th e c o n s u m e rs . N o w a d a y s , n o b re w in g in d u s trie s w ill ta k e th e ris k o f
u s in g g e n e tic a lly m o d ifie d y e a s t s tra in s to p ro d u c e a n A F B .
II.1.2.4
Continuous fermentation
C o n tin u o u s fe rm e n ta tio n o f b e e r a n d A F B h a s b e e n , s in c e th e la s t
d e c a d e s , o n e o f th e m o s t s tu d ie d a re a s in s e v e ra l b re w in g re s e a rc h
g ro u p s fro m d iffe re n t u n iv e rs itie s a c ro s s th e g lo b e (L e h n e rt e t a l., 2 0 0 9 ;
M e n s o u r e t a l., 1 9 9 7 ; P e rp te a n d C o llin , 1 9 9 9a ; S e le c k y e t a l., 2 0 0 8 ;
Van
Ie rs e l
c o n tin u o u s
et
a l,
1995;
m e th o d s
in
V irk a ja rv i,
b re w in g
2 0 0 1 ).
in d u s try
The
has
im p le m e n ta tio n
been
d riv e n
by
of
th e
p o te n tia l e c o n o m ic a l a d v a n ta g e s . S o m e e x a m p le s a t in d u s tria l s c a le
a re k n o w n (s e e S e c tio n II.2 .7 ). H o w e v e r, u n b a lan c e d fla v o u r is s u e s
h a v e n o t y e t b e e n to ta lly o v e rc o m e , le a d in g to th e n e c e s s ity o f fu rth e r
in v e s tig a tio n fo r th e a p p lic a tio n o f c o n tin u o u s p ro c e s s e s (B r n y ik , e t
a l., 2 0 0 8 ; M e n s o u r e t a l., 1 9 9 7 ; V irk a ja rv i a n d L in k o , 1 9 9 9 ; W illa e rt a n d
N e d o v ic , 2 0 0 6 ).
13
Chapter II
II.2 Continuous Beer Production

N o w a d a y s in b e e r in d u s try th e tra d itio n a l b a tc h p ro c e s s o f b e e r
fe rm e n ta tio n
and
m a tu ra tio n
s till
p re v a ils
over
th e
c o n tin u o u s
p ro c e s s e s . C o n s id e rin g th a t a re s e v e ra l d iffe re n t ty p e s o f b e e rs , in
tra d itio n a l b a tc h s y s te m s th e p rim a ry b e e r fe rm e n ta tio n ta k e s p la c e fo r
5 to 1 0 d a y s w h ile m a tu ra tio n ta k e s 1 to 3 w e e k s (B r n y ik e t a l., 2 0 0 8 ;
M e n s o u r e t a l., 1 9 9 7 ). T h e m o s t s u c c e s s fu l c o n tin u o u s fe rm e n ta tio n
s y s te m s d e v e lo p e d a re b a s e d o n im m o b iliz e d c e ll te c h n o lo g y (IC T ), b u t
d e s p ite its a d v a n ta g e s th e y a re s till n o t b e in g fu lly a p p lie d a t in d u s tria l
s c a le (B r n y ik e t a l., 2 0 0 5 ; M e n s o u re t a l., 1 9 9 7 ; P ilk in g to n e t a l., 1 9 9 8 ).
T h e m a in a d v a n ta g e o ffe re d b y th e s e s y s te m s w h e n c o m p a re d w ith
tra d itio n a l fe rm e n ta tio n a n d m a tu ra tio n , is th e p ote n tia l tim e s a v in g s b y
re d u c in g
th e
p ro d u c tio n
tim e ,
w h ic h
is
re fle c te d
in
e c o n o m ic a l
a d v a n ta g e s . B y u s in g c o n tin u o u s s y s te m s th e b e e r p ro d u c tio n tim e h a s

b e e n re d u c e d fro m a w e e k-b a s e (2 to 4 w e e k s ) to a d a ily -b a s e (2 to
5 d a y s ) (B r n y ik e t a l., 2 0 0 6 , 2 0 0 5 ; D m n y e t a l., 1 9 9 8 ; V a n Ie rs e l e t
a l., 1 9 9 5 ; V irk a ja rv i, 2 0 0 1 ). C o n tin u o u s b e e r p ro d u c tio n g iv e s th e
p o s s ib ility o f n o t o n ly u s in g s h o rt fe rm e n ta tio n tim e s a n d re d u c e
d o w n tim e s
(fillin g ,
c le a n in g
and
s ta n d
b y ),
but
a ls o
re d u c e
th e
in v e s tm e n t c a p ita l c o s ts , o p e ra tio n c o s ts a n d s p a c e re q u ire m e n ts

(B r n y ik e t a l., 2 0 0 5 ; M e n s o u r e t a l., 1 9 9 7 ; P ilk in g to n e t a l., 1 9 9 8 ). T h e
in v e s tm e n t c o s ts w e re e s tim a te d to v a ry b e tw e e n 1 5 % a n d 1 0 0 % o f th e
b a tc h p ro c e s s (B r n y ik e t a l., 2 0 0 5 ; M e n s o u r e t a l., 1 9 9 7 ; P a ju n e n et
a l., 1 9 9 1 ; V irk a ja rv i, 2 0 0 1 ) a n d d e p e n d m o s tly o n th e c a rrie r c o s t.
O p e ra tin g c o s t c a n b e b o th 7 0 % lo w e r a n d 8 0 % h ig h e r th a n in b a tc h
s y s te m ,
d e p e n d in g
on
th e
a p p lie d
te c h n o lo g y ,
e s tim a te d
annual
p ro d u c tio n a n d c o m p re h e n s iv e n e s s o f th e e c o n o m ic s tu d y . M ore o v e r,
th e a re a re q u ire m e n ts a re e x p e c te d to b e s m a lle r th a n fo r tra d itio n a l
te c h n o lo g y (P a ju n e n e t a l., 1 9 9 1 ). O n th e o th e r s id e , th e re a re m a n y
re a s o n s th a t s e n te n c e d c o n tin u o u s fe rm e n ta tio n to fa ilu re (B r n y ik e t
a l., 2 0 0 5 ; M e n s o u r e t a l., 1 9 9 7 ; P ilk ing to n e t a l., 1 9 9 8 ; W illa e rt a n d
N e d o v ic , 2 0 0 6 ): e n g in e e rin g p ro b le m s (o p e ra tio n c o n d itio n s , p ro c e s s
14
Chapter II
h y g ie n e ); u n b a la n c e d fla v o u r (c e ll p h y s io lo g y , a g e in g ); c o n ta m in a tio n ;
tra d itio n .
Nowadays
th e
m a in
o b je c tiv e
in
th e
a re a
of
c o n tin u o u s
beer
fe rm e n ta tio n in v e s tig a tio n is to re d u c e s ig n ific a n tly th e p ro d u c tio n

tim e s a n d , a t th e s a m e tim e , e n s u re a u n ifo rm h ig h q u a lity b e e r. It is
u s e le s s fo r b re w in g in d u s try to p ro d u c e b e e r in c o n tin u o u s w ith o u t th e
s a m e c h a ra c te ris tic s o f th e tra d itio n a l b e e r d rin k e d b y th e re g u la r
c o n s u m e rs . B e e r m a k in g is o n e o f th e o ld e s t b io te c h n o lo g y p ro c e s s
k n o w n b y m e n , a n d c o n s e q u e n tly tra d itio n a s p e c ts h a v e to b e ta k e n
in to a c c o u n t (L e w is a n d Y o u n g , 1 9 9 5 ). T ra d itio n is a n a d d itio n a l
o b s ta c le
to
be
s o lv e d
by
s c ie n tific
c o m m u n ity
and
co n s is ts
in
d e m o n s tra tin g th e a d v a n ta g e s o f c o n tin u o u s ly b e e r te c h n o lo g y to th e

b re w in g e n g in e e rs /m a s te rs . T h e s e a d v a n ta g e s m u s t b e in itia lly b a s e d in
e c o n o m ic
a s p e c t,
p ro d u c tio n
of
but
s h o u ld
d iffe re n t
beer
go
beyond.
ty p e s
o n ly
They
by
s h o u ld
a llo w
c ha n g in g
th e
th e
w o rt
c o m p o s itio n a n d o p e ra tio n p a ra m e te rs w ith o u t th e n e c e s s ity o f n e w

s ta rt-u p o f th e b io re a c to r.
II.2.1
Continuous beer fermentation using cell immobilization

systems
A c c o rd in g to M e n s o u r e t a l. (1 9 9 7 ), s y s te m s u s in g im m o b iliz e d y e a s t
c e lls fo r c o n tin u o u s ly b e e r p ro d u c tio n h a v e b e e n p ro p o s e d fo r m o re
th a n 3 0 y e a rs (M e n s o u r e t a l., 1 9 9 7 ). N a rz is s a n d H e llic h (1 9 7 1 , 1 9 7 2 )
c ite d b y V irk a ja rv i a n d L in k o (1 9 9 9 ) w e re p io n e e rs in th e u s e o f
im m o b iliz in g
c o n s is ts
in
yeast
a
fo r
beer
p a c k e d -b e d
fe rm e n ta tio n .
fo rm e d
by
T h e ir
k ie s e lg u h r
s im p le
and
b io re a c to r
yeast
in
k ie s e lg u h r filte r th ro u g h w h ic h w o rt flo w s . H o w e v e r th e b io re a c to rs
life tim e w a s o n ly 7 1 0 d a y s b e fo re c lo g g in g .
S in c e th e n , IC T h a v e b e e n a p p lie d a n d s u c c e s s fu lly d e v e lo p e d fo r
c o n tin u o u s b e e r a n d A F B p ro d u c tio n . S o m e o f th e n h a v e b e e n a p p lie d
a t in d u s tria l s c a le b o th o n m a tu ra tio n a n d p rim a ry fe rm e n ta tio n (B r n y ik
e t a l., 2 0 0 5 ; M e n s o u r e t a l., 1 9 9 7 ; P ilk in g to n e t a l., 1 9 9 8 ). S o it is n o t
15
Chapter II
s u rp ris in g th a t o v e r th e la s t d e c a d e s , d iffe re n t re a c to rs de s ig n s , a s w e ll
a s s e v e ra l im m o b iliz e d m e th o d s , w e re p ro p o s e d fo r c o n tin u o u s p rim a ry
fe rm e n ta tio n a n d m a tu ra tio n (o r s e c o n d a ry fe rm e n ta tio n ) b y s e v e ra l
s c ie n tific g ro u p s a n d b re w in g in d u s trie s .
Table II-1. Examples of carrier types and operation systems used for
continuous beer fermentation and/or maturation by immobilized brewing yeast
Carrier material
Reactor type
Process
References
Ca-alginate
Packed-bed
Maturation
(Dmny et al., 1998;

Smogrovicov and
Domny, 1999)
,-carrageenan
Packed-bed
Maturation
(Dmny et al., 1998),
, -carrageenan
Gas-lift
Primary
Fermentation
(Smogrovicov and
Domny, 1999)
Porous glass (Siran)
Packed-bed ;
Gluten pellets
Gas-lift
Maturation
($mogrovi$ov et al.,
1999)
Spent grains
Gas-lift
Primary
Fermentation
(Brnyik et al., 2006)
Corncobs
Packed-bed
Maturation
(Brnyik et al., 2006)
PVA particles
Gas-lift
Primary
fermentation
(Bezbradica et al.,
2007)
Spent grains
Gas-lift
Primary
fermentation
(Lehnert et al., 2009)
(Virkajrvi and Kronlf,

Primary
Fermentation
1998)
C a rrie r c o s t is o n e o f th e m a in in v e s tm e n t c o s ts w h e n b e e r is
p ro d u c e d in a c o n tin u o u s m o d e . S e v e ra l ty p e s o f c a rrie rs h a v e b e e n
te s te d to im m o b iliz e b re w in g y e a s t (B r n y ik e t a l., 2 0 0 5 ; P ilk in g to n e t
a l., 1 9 9 8 ). T h e c a rrie r ty p e d e p e n d s o n th e p ro c e s s to b e a p p lie d :
p rim a ry fe rm e n ta tio n o r m a tu ra tio n . N o t o n ly d iffe re n t im m o b iliz a tio n
m e th o d s a re im p lie d , b u t a ls o d iffe re n t re a c to rs d e s ig ns o p e ra tin g a t
d iffe re n t c o n d itio n s (s e e T a b le II- 1 ). In th e la s t 1 5 y e a rs , th e c e llu lo s ebase
c a rrie rs
have
g a in e d
im p o rta n c e
m a in ly
due
to
th e ir
good
im m o b iliz a tio n c a p a c itie s , h ig h a v a ila b ility a n d lo w c o s t (B r n y ik e t a l.,

2 0 0 1 ). M o re in fo rm a tio n a b o u t th is s u b je c t is a v a ila b le in C h a p te r III.
16
II.2.2
Chapter II
Immobilization and physiological changes on yeast
S in c e th e e a rly a tte m p ts to d e v e lo p a c o n tin u o u s p ro c e s s fo r b e e r

p ro d u c tio n th e c h a n g e s in im m o b iliz e d c e lls m e ta b o lis m h a v e b e e n
p o s tu la te d a s th e m a in re a s o n fo r u n b a la n c e d fla v o u r o f fin a l b e e r
(B r n y ik e t a l., 2 0 0 5 ; M e n s o u r e t a l., 1 9 9 7 ; V irk a ja rv i a n d L in k o , 1 9 9 9 ).
It
has
been
re p o rte d
th a t
im m o b iliz e d
c e lls
in d u c e
d iffe re n t
p h y s io lo g ic a l re s p o n s e s w h e n c o m p a re d w ith fre e c e ll s y s te ms . T h e s e
c h a n g e s a re m a in ly in m e ta b o lic fu n c tio n s (s u b s tra te u p ta k e , p ro d u c ts
fo rm a tio n , e n z y m e s e x p re s s io n a n d /o r a c tiv ity ). M o s t p ro b a b ly th e y
o c c u r d u e to th e d iffe re n t m ic ro -e n v iro n m e n ta l c o n d itio n s th a t re s u lt
fro m c e ll-im m o b iliz a tio n (J u n te r e t a l., 2 0 0 2 ; S h e n e t a l., 2 0 0 3 ).
T h e s e c h a n g e s c a n b e d u e to : in c re a s e d le v e ls o f D N A , p ro d u c tio n o f
s tru c tu ra l c a rb o h y d ra te s a n d g ly c o g e n (B r n y ik e t a l., 2 0 0 5 ; D o ra n a n d
B a ile y , 1 9 8 6 ), in c re a s e s a tu ra tio n o f fa tty a c id s c o n te n t, m o d ific a tio n s
o f g e n e e x p re s s io n le v e ls , c e ll w a ll a n d m e m b ra n e c o m p o s itio n (S h e n e t
a l., 2 0 0 3 ; V e rb e le n e t a l., 2 0 0 6 ). T h is la s t o n e h a s im p o rta n t im p a c t o n
s e v e ra l e n z y m e s , S E N S O p ro te in s , tra n s p o rte rs a n d m e m b ra n e flu id ity
th a t p la y a n im p o rta n t ro le o n e th a n o l to le ra n c e (Q u n e t a l., 2 0 0 2 ) s u g a r
u p ta k e
and
a m in o
a c id s
m e ta b o lis m .
It
is
e x p e c te d
th a t
yeast
p h y s io lo g ic a l c h a n g e s a ffe c t fin a l b e e r fla v o u r. H o w e v e r, it is d iffic u lt to

d e s c rib e th e d ire c t e ffe c ts o f im m o b iliz a tio n d u e to th e c o m p le x ity o f
th e d iffe re n t im m o b iliz a tio n s s y s te m s , w h ic h c a n in d u c e o n e o r m o re
c h a n g e in y e a s t m e ta b o lis m . N e v e rth e le s s , s e v e ra l re a s o n s h a v e b e e n
p ro p o s e d
fo r
consequence
th e
of
m o d ific a tio n s
th a t
occur
im m o b iliz a tio n .
The
m a in
in
yeast
re a s o n s
c e ll
a re
(1 )
as
a
th e
c o n tin u o u s m o d e o f re a c to r o p e ra tio n ; (2 ) in te rn a l a n d e x te rn a l m a s s
tra n s fe r lim ita tio n ; (3 ) s p e c ific e n v iro n m e n t; (4 ) a g e in g o f im m o b iliz e d
c e lls (B r n y ik e t a l., 2 0 0 5 ; M a s s c h e le in e t a l., 1 9 9 4 ; V e rb e le n e t a l.,
2 0 0 6 ).
17
II.2.2.1
Chapter II
Continuous mode of reactor operation
W h e n th e tra d itio n a l b e e r fe rm en ta tio n s a re c a rrie d o u t in b a tc h

p ro c e s s , th e y e a s t re g u la te s a n d a d ju s ts its m e ta b o lis m d u rin g th e
fe rm e n ta tio n w ith re s p e c t to th e c h a n g e s in th e m e d iu m c o m p o s itio n .
T h is d o e s n o t h a p p e n in s te a d y-s ta te im m o b iliz e d c e lls s y s te m s w h e re
th e
c e lls
a re
not
exposed
to
s ig n ific a n t
a lte ra tio n s
in
re a c tio n
e n v iro n m e n t (M a s s c h e le in e t a l., 1 9 9 4 ). In a c o n tin u o u s p ro c e s s , th e

y e a s t c e lls d o n o t m a k e a n y d is tin c tio n b e tw e e n la g , e x p o n e n tia l a n d
s ta tio n a ry g ro w th p h a s e s , b e c a u s e th e m e d iu m c o m p o s itio n is a lm o s t
c o n s ta n t
over
th e
tim e .
T h is
is
th e
b ig g e s t
d iffe re n c e
b e tw e e n
c o n tin u o u s a n d b a tc h p ro c e s s e s . In o rd e r to m in im iz e it, c o n tin u o u s

beer
fe rm e n ta tio n
s y s te m s
u s u a lly
c o n s is t
in
tw o
(o r
m o re )
fe rm e n ta tio n s v e s s e ls (c o m b in in g a g ita te d v e s s e ls a n d p lu g-flo w lik e

re a c to rs ) to a c h ie v e th e c o rre c t b e e r b a la n c e a n d fla v o u r (B e z b ra d ic a e t
a l., 2 0 0 7 ; B r n y ik e t a l., 2 0 0 6 ; M e n s o u r e t a l., 1 9 9 7 ; S m o g ro v ic o v a n d
D o m n y , 1 9 9 9 ; V irk a j rv i a n d K ro n l f, 1 9 9 9 ; Y a m a u c h i e t a l., 1 9 9 5 ).
II.2.2.2
Internal and external mass transfer limitations
T h e re a re tw o ty p e s o f m a s s tra n s fe r m e c h a n is m s th a t c a n lim it th e
c o n tin u o u s b e e r fe rm e n ta tio n w h e re IC T is a p p lie d : th e in te rn a l a n d
e x te rn a l m a s s tra n s fe r. T h e e x te rn a l m a s s tra n s fe r d e p e n d s la rg e ly o n
th e a p p lie d re a c to r, its d e s ig n a n d re g im e o f op e ra tio n .
T h e in te rn a l m a s s tra n s fe r lim ita tio n s a re re la te d m a in ly w ith m a te ria l
d iffu s io n in s id e th e b io film a n d /o r in s id e th e c a rrie r (e .g . a lg in a te )
w h e re c e lls a re e n tra p p e d . T h e s e lim ita tio n s a re , w h e n c o m p a re d w ith
fre e -c e lls s y s te m s , th e m o s t u s u a l ju s tific a tio n to th e o fte n -o b s e rv e d
d e c re a s e in im m o b iliz e d c e lls g ro w th ra te (M a s s c h e le in e t a l., 1 9 9 4 ) o r
th e v a ria tio n in s p e c ific c o m p o u n d p ro d u c tiv itie s (T a ip a e t a l., 1 9 9 3 ).
M o re o v e r th e s e lim ita tio n s c re a te a s p e c ific m ic ro-e n v iro n m e n t o n th e
im m o b iliz e d c e lls . E x te rn a l a n d in te rn a l m a s s tra n s fe r b a rrie rs d o n o t
h a v e a d e e p im p a c t in th e s u rfa c e o f p o ro u s a n d n o n-p o ro u s c a rrie rs . If
th e re is a lo w b io film th ic k n e s s , a n a lm o s t d ire c t c o n ta c t o f im m o b iliz e d
c e lls w ith th e b u lk liq u id o c c u rs a n d th e re fo re n o s ig n ific a n t m a s s
18
Chapter II
tra n s fe r lim ita tio n s o c c u r a n d c o n s e q u e n tly th e re is n o t a d e v e lo p e m e n t

o f a s p e c ific m ic ro e n v iro n m e n t. (B r n y ik e t a l., 2 0 0 5 ).
II.2.2.3
Specific environment
T h e a c tiv ity o f th e im m o b iliz e d c e lls a ro u n d /in s id e th e s o lid m a trix is

in flu e n c e d b y th e d e v e lo p e d m ic ro e n v iro n m e n t. T h e re a re n u m e ro u s
e ffe c ts o f im m o b iliz a tio n o n c e ll p h y s io lo g y re p o rte d in lite ra tu re
(B r n y ik
et
a l.,
im m o b iliz e d
yeast
2 0 0 5 ).
The
in c lu d e
re p o rte d
changed
p h y s io lo g ic a l
m e ta b o lic
changes
fu n c tio n s
such
of
as
s u b s tra te u p ta k e (S h e n e t a l., 2 0 0 4 ), p ro d u c t fo rm a tio n a n d a lte re d

e n z y m e e x p re s s io n a n d a c tiv ity (Ie rs e l e t a l., 2 0 0 0 ). S o m e a u th o rs a g re e
th a t th e e n h a n c e d m e ta b o lic a c tiv ity c a n b e a ttrib u te d a ls o to th e
s u rfa c e s e n s in g re s p o n s e s in im m o b iliz e d c e lls b u t th e re as o n s a re n o t
y e t c le a rly e x p la in e d . A m o n g a ll c h a n g e s in im m o b iliz e d y e a s t, th e
in c re a s e o f e th a n o l c o n te n t (Q u n e t a l., 2 0 0 2 ), m o d ific a tio n s a t D N A
(D o ra n a n d B a ile y , 1 9 8 6 ) le v e l a n d c h a n g e s in c e lls p ro p e rtie s a re th e
m o s t im p o rta n t.
II.2.2.4
Ageing
A g e in g is th e p re d e te rm in e d tra n s itio n o f a n in d iv id u a l c e ll fro m th e

y o u th to o ld a g e th a t fin a lly c u lm in a te s in d e a th . T h e H a y flic k lim it is
th e m a x im u m life s p a n p o te n tia l o f a c e ll (a s S a c c h a ro m y c e s c e re v is ia e ).
E m p iric a lly , it c o rre s p o n d s to th e n u m b e r o f d iv is io ns w h ic h o n e c e ll is
c a p a b le o f (b e tw e e n 1 0 to 3 0 d iv is io n s ). D e a th in y e a s t c e lls m a y o c c u r
v ia tw o d iffe re n t w a y s : n e c ro s is a n d s e n e s c e n c e (P o w e ll e t a l., 2 0 0 0 ).
A c c o rd in g to M o n c h e t a l. (1 9 9 5 ) c ite d b y P o w e l e t a l. (2 0 0 0 ),
n e c ro s is is d e fin e d a s th e a c cu m u la tio n o f irre p a ra b le d a m a g e in
in tra c e llu la r c o m p o n e n ts c o m p ro m is in g c e ll in te g rity a n d le a d in g to
a u to ly s is . O n th e o th e r s id e , s e n e s c e n c e is th e p re d e te rm in e d c e s s a tio n
o f life a s a re s u lt o f a g e n e tic a lly c o n tro lle d p ro g re s s io n fro m y o u th to
o ld a g e (P o w e ll e t a l., 2 0 0 0 ). T h e o rig in o f n e c ro s is is d iv e rs e a n d
depends
on
e n v iro n m e n ta l
fa c to rs
(s tre s s
c o n d itio n s ;
d e le tio n
or
d is ru p tio n o f g e n e s ; a c c u m u la tio n o f D N A d a m a g e ) (M a s k e ll e tl a l.,
19
Chapter II
2 0 0 3 ), w h ile s e n e s c e n c e s ta rts w h e n n o m o re c e ll d iv is io n o c c urs

(P o w e ll e t a l., 2 0 0 0 ; S in c la ir e t a l., 1 9 9 8 ). S e n e s c e n c e c a n b e re la te d
w ith th e y e a s t c e ll c y c le /d iv is io n . T h e k n o w le d g e a b o u t y e a s t c e ll c y c le
is
v e ry
im p o rta n t o n
b re w in g
in d u s try
because
a fte r th e
p rim a ry
fe rm e n ta tio n th e y e a s t c e lls a re c o lle c te d an d re -u s e d . T h is k n o w le d g e

a llo w s th e o p tim iz a tio n o f th e n u m b e r o f re u s in g b y p re d ic tin g th e
fo rm a tio n o f b io m a s s o n th e n e x t s te p .
Table II-2. Cellular changes accompanying senescence (adapted from Powell
et al., 2000)
Characteristic
Change
Cell size
Surface wrinkle
Bud scar number
Cell wall chitin
Specific gene expression
Protein synthesis
Generation time
Increase
Increase
Increase
Increase
Altered
Decrease
Increase
D u rin g a n o rm a l fe rm e n ta tio n th e p e rc e n ta g e o f o ld c e lls , w h ic h h a v e

lo w e r
m e ta b o lic
a c tiv ity ,
in c re a s e s ,
re s u ltin g
in
irre v e rs ib le
m o d ific a tio n s o n c e ll a p p e a ra n c e (B a rk e r e t S m a rt, 1 9 9 6 ; M a s k e ll e t a l.,

2 0 0 3 ). T h e s e m o rp h o lo g ic a l a n d p h y s io lo g ic a l c h a n g e s c a n a c t a s
b io m a rk e rs in th e d e te rm in a tio n o f c e ll a g e o r fo r fe rm e n ta tio n c o n tro l,
e s p e c ia lly in c o n tin u o u s fe rm e n ta tio n s w ith im m o b iliz e d y e a s t c e lls
(B a rk e r a n d S m a rt, 1 9 9 6 ; M a s k e ll e t a l., 2 0 0 3 ).
T h e re a re s e v e ra l a g e in g a n d s e n e s c e n c e m e c h a n is m s th a t h a v e b e e n
p ro p o s e d , s u c h a s (P o w e ll e t a l., 2 0 0 0 ; S in c la ir e t a l., 1 9 9 8a , 1 9 9 8 b ):
- th e re la tio n b e tw e e n s u rfa c e- to -v o lu m e ra tio a n d th e in c re a s in g
n u m b e rs o f b u d s c a rs (re d u c tio n o f th e s u rfa c e a re a fo r b u d d in g a n d
n u trie n t e x c h a n g e ),
- th e g ra d u a l lo s s o f te lo m e ric s e q u e n c e s (re s p on s ib le fo r e n s u rin g
th a t a c o m p le te re p lic a tio n o c c u rs ),
- c y to p la s m s e n e s c e n t fa c to rs (a c c u m u la tio n o f to x ic c o m p u n d s ),
- g e n e tic fa c to rs .
20
Chapter II
A m o n g a ll o f th e s e , g e n e tic c o n tro l o f a g e in g h a s g a in e d th e b ig g e s t
im p o rta n c e o v e r th e la s t te n y e a rs (K e n n e d y e t a l., 1 9 9 5 ; P o w e ll e t a l.,
2 0 0 0 ). C o n s e q u e n tly , a n u m b e r o f g e n e s th a t m a y in flu e n c e lo n g e v ity in
y e a s t h a v e a lre a d y b e e n id e n tifie d a n d a re lis te d b e lo w (T a b le II- 3 ).
Table II-3. Genes associated with longevity in Saccharomyces cerevisiae
(adapted from Powell, 2000)
Gene
Function
Observations
LAG1
Unknown
LAG2
Unknown
RAS
Nutritional status
SIR4
Gene silencing
AGE
Stress response
a
Kenedy et al, 1995
Expressed in young cells

Related with longevity
Silence AGE stress responsea
In b re w in g in d u s try , th e te rm a g e in g h a s b e e n in c o rre c tly u s e d ,

b e c a u s e y e a s t a g e is m e a s u re d c h ro n o lo g ic a lly b y th e n u m b e r o f tim e s
a p o p u la tio n is re u s e d in s u c c e s s iv e fe rm e n ta tio n s . A fte r fin is h in g th e
p ry m a ry
beer
fe rm e n ta tio n
th e
c y lin d ro -c o n ic a l
ta n k
is
c o o le d
to
p ro m o te s e d im e n ta tio n . C e lls a re d e p o s ite d in th e c o n ic a l p a rt o f th e

ta n k , th e o ld e s t fra c tio n b e in g lo c a te d in th e b o tto m a n d th e y o u n g e s t
c e lls in th e to p . T h e flo c c u la tio n p o te n tia l o f ye a s t c e lls is re g u la te d b y
c e ll w a ll p ro p e rtie s th a t c h a n g e th ro u g h o u t th e c e ll life s p a n (P o w e ll e t
a l.,
2 0 0 3 ).
ro u g h
c e ll
s u rfa c e
to p o g ra p h y
p ro m o te s
c e ll-to -c e ll
a d h e s io n d u rin g th e o n s e t o f flo c c u la tio n . A lth o u g h s e d im e n ta tio n

c a p a c ity is s tra in-s p e c ific , c e ll s iz e m a y trig g e r a n im p o rta n t ro ll in
flo c c u la tio n a c tin g a s a n u c le a tio n p o in t (B a rk e r a n d S m a rt, 1 9 9 6 ;
P o w e ll e t a l., 2 0 0 3 ).
In th e o ry , c o n tin u o u s fe rm e n ta tio n p ro c e s s e s w ith im m o b iliz e d y e a s t
th a t
has
lo w
H a y flic k
lim it
(m a x im u m
number
of
po s s ib le
c e ll
d iv is io n s ) is n o t re c o m m e n d e d (B r n y ik e t a l., 2 0 0 5 ). T h e m a in re a s o n is
th a t lo n g p e rio d s o f c o n tin u o u s fe rm e n ta tio n a re d e s ira b le fro m th e
e c o n o m ic p o in t o f v ie w a n d y e a s t w ith lo w H a y flic k lim it w o u ld n o t
s u p p o rt lo n g fe rm e n ta tio n ru n s (V irk a j rv i a n d K ro n l f, 1 9 9 9 ; V irk a ja rv i,
2 0 0 1 ). It is k n o w n th a t a g e d b re w in g y e a s t c h a n g e s th e ir flo c c u la tio n
c h a ra c te ris tic s (S o a re s a n d M o ta , 1 9 9 6 ) a n d fe rm e n ta tio n p e rfo rm a n c e
(P o w e ll e t a l., 2 0 0 3 ). In a d d itio n , m u ta tio n s in b re w in g y e a s t le a d in g to
21
Chapter II
a lte ra tio n s a t m o rp h o lo g ic a l/b io c h e m ic a l le v e l d u rin g fe rm e n ta tio n c a n

o c c u r. In a p ra tic a l p o in t o f v ie w it w o u ld im p o rta n t to k n o w : (1 ) th e
in flu e n c e o f th e d iffe re n t im m o b iliz a tio n m e th o d s o n a g e in g o f b re w in g
y e a s t a n d (2 ) th e e ffe c t o f s e n e s c e n c e o n c e ll v ita lity a n d fe rm e n ta tio n
p e rfo rm a n c e (B r n y ik e t a l., 2 0 0 5 ).
II.2.3
Industrial applications
C o n tin u o u s p rim a ry fe rm e n ta tio n a n d m a tu ra tio n o f b e e r h a s b e e n a

fie ld
of
in te re s t
fo r
b re w in g
s in c e
th e
1970s.
A p p lic a tio n s
fo r
c o n tin u o u s b e e r p rim a ry fe rm e n ta tio n a n d/o r m a tu ra tio n h a v e b e e n

im p le m e n te d a t la rg e s c a le C u lto r L td a n d T u c h e n h a g e n ; K irin
B re w e ry ; M e u ra D e lta ; L a b a tt B re w in g C o m p a n y (M e n s o u r e t a l., 1 9 9 7 ).
T h e C u lto r L td a n d T u c h e n h a g e n s y s te m p ro c e s s w a s d e v e lo p e d in
th e e a rly 1 9 9 0 s b y a c o n s o rtiu m . T h e y d e v e lo p e d tw o p ro c e s s e s : (1 )
a c c e le ra te d m a tu ra tio n (d ia c e ty l re d u c tio n ) a n d (2 ) c o n tin u o u s A F B . T h e
c a rrie r u s e d w a s D E A E-c e llu lo s e (S p e z y m e G D C ) in a d o w n flo w
P a c k e d -b e d re a c to r (P B R ). T h e m a tu ra tio n s y s te m h a s b e e n o p e ra tio n a l
s in c e 1 9 9 2 a t in d u s tria l s c a le (1 E 8 L ) in th e S in e b ry c h o ff B re w e ry in
K e ra v a (F in la n d ), w h e re it is u s e d a lo n g s id e w ith tra d itio n a l m a tu ra tio n
p ro c e s s
(G E A
P ro c e s s
E n g in e e rin g ,
2008;
Mensour
et
a l.,
1 9 9 7 ).
N o w a d a y s , th is s y s te m (Im m o c o n m a tu ra tio n ) is a v a ila b le a n d s o ld b y

G E A d iv is o n fro m T u c h e n h a g e n .
T h e K irin B re w e ry C o ., L td (J a p a n ) s y s te m w a s e v e n tu a lly th e firs t
in d u s tria l s y s te m in v o lv in g c o n tin u o u s p rim a ry b e e r fe rm e n ta tio n a n d
m a tu ra tio n . It is a m u lti-s ta g e s y s te m c o m p o s e d b y B C re a c to r a n d tw o
P B R (m a in fe rm e n ta tio n ) a n d a th ird P B R (m a tu ra tio n ). O v e ra ll, th e
c o n tin u o u s p ro d u c tio n o f b e e r ta k e s p la c e w ith in th re e to fiv e d a y s . T h e
in itia l 2 0 L s y s te m w a s s c a le d -u p to 5 0 0 L a n d th e n to 1 0 0 0 0 L .
H o w e v e r s o m e u n e x p e c te d d ra w b a c k s (h ig h e r c a p ita l a n d o p e ra tin g
c o s ts ) c o n d e m n e d to fa ilu re th e 1 0 0 0 0 L in s ta lla tio n (M e n s o u r e t a l.,
1 9 9 7 ).
22
Chapter II
M e u ra D e lta h a s d e v e lo p e d a m u lti-s ta g e b io re a c to r s y s te m fo r th e
p ro d u c tio n o f a lc o h o l-fre e a n d re g u la r b e e r. Y e a s t c e lls a re im m o b iliz e d
b y a d s o rp tio n o n th e in te rn a l s u rfa c e o f th e s ilic o n c a rb id e ro d s . B a s e d
o n th e s u c c e s s , M e u ra D e lta is try in g to im p le m e n t th e ir d e s ig n to
in d u s tria l s c a le . L ittle s c a le-u p p ro b le m s a re e x p e c te d , b e c a u s e th e
s y s te m h a s a m o d u la r d e s ig n (M e n s o u r e t a l., 1 9 9 7 ; M e u ra , 2 0 0 7 ).
T h e L a b a tt B re w e rie s (C a n a d a ) in a s s o c ia tio n w ith th e D e p a rtm e n t o f
C h e m ic a l a n d B io c h e m ic a l E n g in e e rin g a t th e U n iv e rs ity o f W e s te rn
O n ta rio ,
p ro p o s e d
c o n tin u o u s
beer
fe rm e n ta tio n
s y s te m
u s in g
im m o b iliz e d y e a s t c e lls in , -c a rra g e e n a n . A d d itio n a lly th e y d e v e lo p e d a

c o n tin u o u s b e a d p ro d u c tio n p ro c e s s (D e c a m p s e t a l., 2 0 0 4 ) b a s e d o n
s ta tic m ix e rs . A d ra ft tu b e g a s-lift b io re a c to r w ith a d e g a s s in g z o n e w a s
a p p lie d fo r th e p rim a ry fe rm e n ta tio n o f b e e r. L a b b a t B re w e ry w a s th e
firs t in u s in g a g a s -L ift b io re a c to r fo r c o n tin u o u s b e e r p ro d u c tio n .
M o re o v e r th e e x p e rim e n ta l w o rk a llo w s n o t o n ly re s e a rc h g ro u p s to b e
fa m ilia ris e d w ith th e IC T b u t a ls o h o w it c o u ld b e a p p lie d in c o n tin u o u s
b e e r p ro d u c tio n . B e fo re th is a p p lic a tio n th e m a jo r p ro b le m w a s re la te d
w ith th e p ro d u c tio n o f p o o r fla v o u r p ro file m a in ly d u e to lo w fre e a m in o
n itro g e n (F A N ) u p ta k e fro m im m o b iliz e d c e lls . L a b a tt B re w e ry , w ith th e
im p le m e n ta tio n o f a g a s -lift b io re a c to r (g o o d m ix in g p ro file s ) w ith a
d ra ft tu b e a n d s m a ll s iz e d im m o b ilis a tio n b e a d s ( 1 m m in d ia m e te r)
managed
to
re d u c e
s ig n ific a n tly
mass
tra n s fe rs
lim ita tio n s .
C o n s e q u e n tly th e F A N u p ta k e w a s b e tte r a n d a fin a l p ro d u c t o f

a c c e p ta b le
q u a lity
was
p ro d u c e d .
The
v a lu a b le
e x p e rie n c e
and
k n o w le d g e g a in e d th ro u g h th e p re lim in a ry im m o b iliz e d c e ll w o rk a t
L a b a tt, p ro v id e d a s o lid fo u n d a tio n w h ic h is n o w u s e d to d e v e lo p th is
te c h n o lo g y in to a n in d u s tria lly fe a s ib le v e n tu re (M e n s o u r e t a l., 1 9 9 7 ).
23
Chapter II
II.3 Continuous Alcohol-free Beer Production

A s re p o rte d b e fo re , A F B s a re m a in ly p ro d u c e d , a t in d u s tria l s c a le , b y
a rre s te d o r lim ite d fe rm e n ta tio n (a s C C P ). F e rm e n tin g a n A F B is
te c h n ic a lly
s im p le
and
re s e m b le s
s e c o n d a ry
The
c o n tin u o u s p ro d u c tio n o f A F B is b a s e d o n th e lim ite d fe rm e n ta tio n

s tra te g y . T h e p ro p o s e d A F B c o n tin u o u s fe rm e n ta tio n s y s te m s g e n e ra lly
c o n s is t o f o n e -s ta g e b io re a c to r a n d a re s im ila r to th e p re v io u s ly
re p o rte d c o n tin u o u s m a tu ra tio n s y s te m s (lo w te m p e ra tu re , lo w w o rt
g ra v ity a n d s h o rt c o n ta c t tim e ). T h e m a in a im is to re m o v e th e w o rty
fla v o u r w ith o u t a h ig h a lc o h o l c o n c e n tra tio n (o r o n ly v e ry lo w a m o u n ts
o f a lc o h o l) in th e b e e r (B r n y ik e t a l., 2 0 1 2 ; V irk a ja rv i a n d L in k o , 1 9 9 9 ).
C o n s id e rin g th a t th e m o s t s u c c e s s fu lly a p p lie d s y s te m s in in d u s try
a re
fo r
m a tu ra tio n
p ro d u c tio n
of
AFB
of
beer
has
(s e e
b ig
s e c tio n
p o te n tia l
II.2 .3 ),
th e
a p p lic a tio n
c o n tin u o u s
due
its
h ig h
p ro d u c tiv itie s w h e n c o m p a re d w ith th e e x is tin g m e th o d s . In fa c t, th e

s till lo w n u m b e r o f A F B s c o n s u m e rs a n d a b s e n c e o f a lo n g tra d itio n in
its p ro d u c tio n /c o n s u m p tio n (B ra n y ik e t a l., 2 0 1 2 ) c a n le a d to a n e a s ie r
im p le m e n ta tio n o f c o n tin u o u s A F B s p ro d u c tio n s y s te m s a t in d u s tria l
s c a le . In a d d itio n , s o m e a p p lic a tio n s m a n a g e d a g o o d re d u c tio n o f
a ld e h y d e s b y th e b io re a c to r d e s ig n o n ly (p h y s ic a l m e c h a n is m ). A F B s
a re k n o w n b y th e ir w o rty o ff-fla v o u rs a n d a ld e h y d e s h a v e b e e n re p o rte d
a s th e re a s o n fo r th is c h a ra c te r (P e rp te a n d C o llin , 1 9 9 9a ).
M a n y re s e a rc h g ro u p s h a v e b e e n d e v e lo p in g a n d o p tim iz in g d iffe re n t
c o n tin u o u s A F B s s y s te m s . T h e m a in d iffe re n c e s a re in re a c to r d e s ig n ,
im m o b iliz a tio n m e th o d a n d c a rrie r u s e d . T h e m a in re a c to rs u s e d a re
P a c k e d -b e d
lik e
re a c to rs
(V a n
Ie rs e l
et
a l.,
1 9 9 5 ).
However
and
c o n s id e rin g th e g o o d m ix in g a n d a ld e h y d e re d u c tio n p ro p e rtie s (b y

s trip p in g )
of
iG L R ,
th is
re a c to r
has
a ls o
g a in e d
c o n tin u o u s A F B s p ro d u c tio n (L e h n e rt e t a l., 2 0 0 9 ).
24
im p o rta n c e
fo r
II.3.1
Chapter II
Industrial applications
A lth o u g h s e v e ra l s y s te m s h a v e b e e n p ro p o s e d a n d im p le m e n te d o v e r
th e la s t y e a rs fo r c o n tin u o u s A F B s p ro d u c tio n th e m e th o d s u s e d in
in d u s try a re n o t k n o w n b y th e m a jo rity o f th e s c ie n tific c o m m u n ity . T h e
m a in
c o n c lu s io n
fe rm e n ta tio n
to
s y s te m s
in d u s tria l c o n tin u o u s
p o in te d
a re
be
th e
ta k e n
have
AFB
need
of
not
is
yet
th a t
th e
been
p ro d u c tio n . T h e
in d u s try
to
p ro p o s ed
s u c c e s s fu lly
m a in
a c q u ire
c o n tin u o u s
a p p lie d
re a s o n s
s p e c ia l
th a t
in
a re
e q u ip m e n t
(b io re a c to r a n d a d d itio n a l e q u ip m e n t fo r ru n n in g it c on tin u o u s ly ) a n d to
a p p ly a d d itio n a l te c h n iq u e s (im m o b iliz a tio n m e th o d s ) a n d m a te ria ls
(c a rrie r) n o t c o m m o n in b re w in g in d u s try (B r n y ik e t a l., 2 0 1 2 ; M e n s o u r
e t a l., 1 9 9 7 ; P ilk in g to n e t a l., 1 9 9 8 ). A n o th e r p o s s ib le c o n c lu s io n m a y
be
re la te d
w ith
th e
im p o rta n c e
of
c o n tin u o u s
s y s te m s
u s in g
im m o b iliz e d c e lls o n c o n tin u o u s A F B s p ro d u c tio n th a t m a y le a d b re w in g

c o m p a n ie s to h id e th e ir p ro g re s s in th is p a rtic u la r fie ld . A s re fe rre d
b e fo re o n ly o n e in d u s tria l a p p lic a tio n fo r c o n tin u o u s A F B s is k n o w n th e C u lto r L td a n d T u c h e n h a g e n s y s te m . In fa c t, it is n o t a p u re A F B
b u t a L A B . C o n s id e rin g th e s im ila ritie s b e tw e e n A F B a n d L A B , s o m e
d e ta ils o f th is in d u tria l a p p lic a tio n w ill b e d e s c rib e d b e lo w .
The
s y s te m
d e v e lo p e d
by
th e
C u lto r
L td
and
Tuchenhagen
c o n s o rtiu m to p ro d u c e c o n tin u o u s ly A F B u s in g im m o b iliz e d c e lls in a

P B R , w h e n c o m p a re d to th e c la s s ic a l a rre s te d b a tc h fe rm e n ta tio n , w a s
fo u n d to p ro d u c e a b e tte r ta s tin g L A B w ith a n im p ro v e d p ro d u c t
c o n s is te n c y . T h e B a v a ria B V (N e th e rla n d s ) is u s in g th is s y s te m fo r th e
c o n tin u o u s p ro d u c tio n o f 1 5 0 0 0 m 3 o f L A B p e r y e a r. It is a lre a d y o n e o f
B a v a ria 's b e s t s e llin g L A B . A p e rs o n a l c o m m u n ic a tio n fro m H e ik k i
Lommi
(b u s in e s s m a n a g e r o f C u lto r T e c h n o lo g y ) c ite d b y M e n s o u r e t
a l. (1 9 9 7 ) in d ic a te d th a t o th e r c o m p a n ie s (F a x e in De n m a rk , O tta k rin g e r
in A u s tria ), h a v e a ls o p u rc h a s e d th is te c h n o lo g y a n d a re p re s e n tly
p ro d u c in g c o n tin u o u s ly A F B . In re c e n t y e a rs th e G E A g ro u p to g h e te r
w ith T u c h e n h a g e n d e v e lo p e d th e Im m o c o n s y s te m fo r c o n tin u o u s ly A F B
p ro d u c tio n . T h e y c la im th a t m a n y b re w e rie s a re u s in g th e ir m e th o d ,
25
Chapter II
s u c h a s S in e b ry c h o ff B re w e ry in K e ra v a F in la n d (G E A P ro c e s s
E n g in e e rin g , 2 0 0 8 ; M e n s o u r e t a l., 1 9 9 7 ).
II.3.2
Future trends and challenges
R e g u la r A F B s a re k n o w n b y th e w o rty o ff-fla v o u r a n d la c k o f b o d y .
C o n s id e rin g th a t e v e n th e m o s t c o m m o n in d u s tria l a p p ro a c h e s c a n n o t
s o lv e th e s e is s u e s , a n d th a t c o n tin u o u s fe rm e n ta tio n s te c h n o lo g y
d e v e lo p m e n t h a s b e e n ra is in g fa s t th is c a n b e a p ro m is in g fie ld o f
s tu d y in th e fu tu re . M a n y s tu d ie s h a v e to b e m a d e to a c h ie v e n o t o n ly
th e p ro d u c tio n o f a w e ll-b a la n c e d A F B , b u t a ls o in th e s c a le-u p is s u e s
o f th e u s e d b io re a c to rs . T h u s , it is im p o rta n t to :
- u n d e rs ta n d b e tte r th e p h y s io lo g ic a l c h a n g e s o f im m o b iliz e d y e a s t;
- c o n tro l a g e in g is s u e s in a lo n g-te rm fe rm e n ta tio n ;
- u n d e rs ta n d th e in te ra ctio n b e tw e e n h y d ro d y n a m ic a s p e c ts (k L a ;
m ix in g p ro file s , c h a n n e llin g ) a n d y e a s t m e ta b o lis m fo r e a c h p ro p o s e d
s y s te m .
II.4 Beer Quality and Flavour: Origin and Influence

T h e tw o d is tin c t a s p e c ts to b e e r q u a lity a re th e n a tu re o r k in d a n d
th e d e g re e o f e x c e lle n c e. T h e re a re m a n y q u a lity p a ra m e te rs in b e e r
fla v o u r, c o lo u r, c la rity , d e g re e o f d e g ra d a tio n , p re s e n c e (o r n o t) o f
fo a m , p H a n d a lc o h o l c o n te n t a re th e m o s t im p o rta n t.
F la v o u r, a c c o rd in g to th e E B C -A S B C d e fin itio n , is th e c o m b in a tio n o f
o lfa c to ry a n d g u sta to ry a ttrib u te s p e rc e iv e d d u rin g ta s tin g . It in c lu d e s
ta c tile , th e rm a l, p a in a n d k in e s th e tic e ffe c ts . T h e m o s t im p o rta n t
a s p e c ts o f fla v o u r a re m o u th -fe e l, ta s te a n d o d o r (B rig g s e t a l, 2 0 0 4 ).
M o u th -fe e l is d e fin e d a s th e ta c tile p e rc e p tio n a t th e lin in g o f th e
m o u th
(to n g u e
s m o o th n e s s ,
and
te e th )
a s trin g e n c y
and
(d ry in g ),
is
in v o lv e d
te m p e ra tu re
in
th e
and
p e rc e p tio n
CO2
of
s e n s a tio n .
T a s te is s e n s e d b y to n g u e w h e re fo u r (4 ) fla v o u rs (s w e e t, s a lt, s o u r a n d
b itte r) a re re c o g n iz e d , w h ile th e a ro m a is s e n s ed b y th e o lfa c to ry
26
Chapter II
s y s te m (B rig g s e t a l., 2 0 0 4 ; L e w is a n d Y o u n g , 1 9 9 5 ). T h e p re d o m in a n t
in flu e n c e s o n b e e r fla v o u r a re d e riv e d fro m h o p b itte rn e s s a n d a ro m a ,
m a lt c o m p o n e n ts a n d fe rm e n ta tio n (L e w is a n d Y o u n g , 1 9 9 5 )
T h e re a re s e v e ra l c o m p o u n d s th a t c o n tribu te to b e e r fla v o u r. E a c h
c o m p o u n d h a s d iffe re n t th re s h o ld s , i.e ., p e rc e p tio n o f a s u b s ta n c e th a t
is p re s e n t in a c o n c e n tra tio n th a t c a n ju s t b e id e n tifie d . In th e n e x t
ta b le (T a b le II- 4 ) a re s u m m a riz e d th e m a in b e e r fla v o u r c o m p ou n d s .
T a b le II- 4 c o m p a re s th e v a lu e s p re s e n te d in lite ra tu re fo r re g u la r b e e rs
a n d A F B s to c o n tin u o u s a n d b a tc h p ro d u c tio n m o d e s .
F ro m T a b le II- 4 , it is p o s s ib le to o b s e rv e th a t c o n tin u o u s p ro c e s s
h a s s im ila r v a lu e s to th e tra d itio n a l b a tc h fo r h ig h e r a lc o h o ls a n d
e s te rs . T h e m a in p ro b le m o c c u rs in to ta l V D K s c o n c e n tra tio n . V D K s
a m o u n t fo r re g u la r b e e rs p ro d u c e d in c o n tin u o u s is u s u a lly h ig h e r th a n
b y b a tc h p ro c e s s a n d th a n th e th re s h o ld v a lu e s (B r n y ik e t a l., 2 0 0 6 ;
P a ju n e n e t a l. (2 0 0 1 ) in W illa e rt a n d N e d o v ic , 2 0 0 6 ). H o w e v e r th is d o e s
n o t o c c u r in c o n tin u o u s A F B m a in ly d u e to th e o p e ra tio n a l p a ra m e te rs
(8 C ) th a t lim it th e fe rm e n ta tio n a n d y e a s t m e ta b o lis m . C o n s e q u e n tly
th e fo rm a tio n o f a ll m a in fla vo u r c o m p o u n d s is lo w e r. A m o n g th e m
V D K s c o n c e n tra tio n in c o n tin u o u s A F B is s im ila r to th e b a tc h p ro c e s s
(L e h n e rt e t a l., 2 0 0 8 ). S o m e a u th o rs (P e rp te a n d C o llin , 1 9 9 9b )
c o n s id e re d
th a t,
due
to
AFB
lo w
a lc o h o l
c o n te n t,
th e ir
fla v o u r
c o m p o u n d s th re s h o ld s h o u ld b e c o m p a re d w ith th e fa lv o u r th re s h o ld
c o m p o u n d in w a te r a n d n o t in b e e r a s is n o rm a lly d o n e . B e in g s o , in
T a b le II- 4 th e th re s h o ld in w a te r a n d b e e r a re d is p la y e d fo r d iffe re n t
fla v o u r c o m p o u n d s .
27
Chapter II
Table II-4. Beer major Flavour compounds in continuous and batch systems for regular (A and B) and AFB (C)
Class
Compound / mg/L or
g/L*
Continuous system
A
Batch process
Threshold
in water
(E) /
(mg/L)
990
Threshold in
beer (D) /
(mg/L)
Flavour in Beer (D)
14000
Alcohol, strong
Alcohol
Alcohol
Alcohol, Banana,
Sweetish, Aromatic
Alcohol, Banana,
Sweetish, Aromatic
Solvent, Fruity,
Sweetish
Banana, Apple,
Solvent, Estery
Apple, Fruty,
Sweetish, Aniseed,
Estery
Apple, Sweetish,
Fruty
-
Alcohol
Ethanol
38500
3900
44200
< 3900
Higher
Alcohols
(HA)
Propanol
IsoButanol
Amyl-Alcohol
(2-Methyl-butanol)
Isoamyl alcohol
(3-Methyl-butanol)
TOTAL HA
16.1
8.3
24.9
8.4
1.6
1.6
17.5
12.5
10
8.2
2
1.8
0.59
800
200
55.8
8.6
70
7.65
1.2
65
47.0
51
0.22
70
80.2
80.3
11.8
100
69.2
11.45
Ethyl acetate
11.3
26.4
0.58
17.2
21.5
0.78
33
Isoamyl acetate
1.00
1.5
1.2-1.6
Ethyl Caproate
0.2
0.01
0.4
0.01
0.21-0.23
Ethyl Caprylate
0.6
0.22
1.4
0.01
0.9
Amyl Acetates
Total ES
0.2
11.5
28.2
0.02
0.83
1.2
18.4
24.8
0.06
0.86
1.2 **
-
Esters
(ES)
Legend: A Brnyik et al. (2006): Spent Grains (Regular beer); B Pajunen et al (2001) in Willaert and Nedovic (2006):
Aspen wood chips (average values of Regular beer); C Lehnert et al (2008): Spent Grains (AFB); D Meilgaard (1975); E Czerny et al. (2008).
* Values in g/L ** Value from Lehnert et al (2008). *** from Perpte and Collin (1999b)
28
Chapter II
Table II-4. (continuation)

Class
Vicinal
Diketones
(VDKs)
Continuous system
Compound / (mg/L or
g/L*)
Diacetyl
(2,3
Butanedione)
2,3-Pentanedione
Total VDKs
Batch process
Threshold
in water (E)
/ (mg/L)
Threshold
in beer (D)
/ (mg/L)
Flavour in Beer (D)
0.25
0.15
Diacetyl, butter scotch
0.29
0.15
0.4
0.06
0.03
0.05
0.9
-
Diacetyl, Fruity
-
Green leaves, Fruty,

Worty
Bitter, Vinous,
Hexanal*
0.2
0.2
2.4
0.35
aldehyde
Aldehydes 2-Methyl propanal*
1.7
0.3
0.49
1***
Malty, Chocolate,
3-Methyl butanal *
8.7
1.6
0.5
0.6***
Almond***
Methional
Furfural*
1.5
2.7
8000
150
Paper, husk
Legend: A Brnyik et al. (2006): Spent Grains (Regular beer); B Pajunen et al (2001) in Willaert and Nedovic (2006):
Aspen wood chips (average values of Regular beer); C Lehnert et al (2008): Spent Grains (AFB); D Meilgaard (1975); E Czerny et al. (2008).
* Values in g/L ** Value from Lehnert et al (2008). *** from Perpte and Collin (1999b)
Acetaldehyde
13.1
8.5
29
6.9
0.025
10
II.4.1
Chapter II
Higher alcohols
H ig h e r a lc o h o ls (H A ) a re a lc o h o ls th a t h a v e m o re c a rb o n a to m s th a n
e th a n o l (L e w is a n d Y o u n g , 1 9 9 5 ) a n d c o n trib u te to b e e r fla v o u r d u e to
th e ir a lc o h o lic o r s o lv e n t-lik e a ro m a c a u s in g a w a rm m o u th-fe e l. T h e y
a re fo rm e d d u rin g m a in fe rm e n ta tio n a n d c a n b e d iv id e d in a lip h a tic
and
a ro m a tic .
The
m a in
a lip h a tic
HA
a re :
n-p ro p a n o l,
is o b u ta n o l
(2 m e th y lp ro p a n o l), a m y l a n d is o a m y l a lc o h o ls , w h ile th e m a in a ro m a tic

HA
a re :
2 -p h e n y le th a n o l,
u n d e s ira b le
(B r n y ik
et
ty ro s o l
a l.,
and
2008;
try p to p h o l,
W illa e rt
and
b e in g
th e
N e d o v ic ,
la tte r
2006;
M e ilg a a rd , 1 9 7 5 ).
T h e H A a re fo rm e d fro m th e ! -k e to -a c id s p o o l. T h e s e a c id s a re
d e c a rb o x y la te d
to
a ld e h y d e s
and
fu rth e r
re d u c e d
(b y
a lc o h o l
d e h y d ro g e n a s e ) to H A . T h e ! -k e to -a c id s a re s y n th e s iz e d tro u g h tw o
p a th w a y s : th e a n a b o lic a n d c a ta b o lic ro u te s . In th e a n a b o lic ro u te , th e
! -k e to -a c id s
fo rm a tio n
a ris e s
c a rb o h y d ra te
m e ta b o lis m .
In
in
he
th e
a m in o
c a ta b o lic
a c id
ro u te ,
s y n th e s is
! -k e to -a c id s
fro m
a re
s y n th e s iz e d b y th e u p ta k e o f w o rt a m in o a c id s fro m y e a s t c e lls (B r n y ik
e t a l., 2 0 0 8 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). T he fin a l c o n c e n tra tio n o f H A
is d e te rm in e d b y th e u p ta k e o f th e c o rre s p o n d in g a m in o a c id a n d
c a rb o h y d ra te u tiliz a tio n ra te a n d its fo rm a tio n is re la te d w ith F A N
u p ta k e (B r n y ik e t a l., 2 0 0 8 ; M a s s c h e le in e t a l., 1 9 9 4 ; W illa e rt a n d
N e d o v ic , 2 0 0 6 ). T h e c o n tribu tio n o f a n a b o lic o r c a ta b o lic p a th w a y is
u n c le a r a n d v a rie s fo r e a c h a lc o h o l. It is re p o rte d th a t th is c o n trib u tio n
is in flu e n c e d b y w o rt a m in o a c id c o m p o s itio n , fe rm e n ta tio n s ta g e a n d
y e a s t s tra in . M o re o v e r, H A c a n a ls o b e o rig in a te d fro m th e re d u c tio n of
k e to n e s a n d a ld e h y d e s p re s e n t in w o rt. (B r n y ik e t a l., 2 0 0 2 ).
R e g a rd in g T a b le II- 4 a n d h a v in g in m in d th e h ig h ta s te th re s h o ld o f
th e s e c o m p o u n d s , th e g a p b e tw e e n H A fo rm a tio n d u rin g c o n tin u o u s
a n d tra d itio n a l b e e r fe rm e n ta tion s d o e s n o t re p re s e n t th e m o s t s e rio u s
fla v o u r p ro b le m (B r n y ik e t a l., 2 0 0 8 ; S m o g ro v ic o v a n d D o m n y ,
1 9 9 9 ). Y e t, a n in c re a s e d p ro p a n o l y ie ld fo llo w e d b y d e c re a s e d i-b u ta n o l
30
Chapter II
a n d is o a m y l a lc o h o l fo rm a tio n w a s n o tic e a b le in s o m e o f th e c o n tin u o u s

s y s te m s (B r n y ik e t a l., 2 0 0 8 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ).
G e n e ra lly , im m o b iliz e d c e lls s y s te m s b y e n tra p m e n t a re re la te d w ith
lo w H A fo rm a tio n , m a in ly d u e to m a s s tra n s fe r lim ita tio n s th a t le a d s to
p o o r im m o b iliz e d c e ll g ro w th a n d lo w F A N u p ta k e b y th e im m o b iliz e d
c e lls . O n th e o th e r h a n d , c o n tin u o u s fe rm e n ta tio n u s in g im m o b iliz e d
c e lls s y s te m s b y a d s o rp tio n (s p e n t g ra in s , w o o d c h ip s ) h a v e h ig h F A N
u p ta k e , a s w e ll a s g o o d im m o b iliz e d c e ll g ro w th , w h ic h , n a tu ra lly , le a d s
to g o o d fo rm a tio n o f H A (B r n y ik e t a l., 2 0 0 8 ; M a s s c h e le in e t a l., 1 9 9 4 ;
S m o g ro v ic o v a n d D o m n y , 1 9 9 9 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ).
In c o n tin u o u s s y s te m s , it is p o s s ib le to c o n tro l th e fo rm a tio n o f H A
by
c h o o s in g
fe rm e n ta tio n
an
a p p ro p ria te
c o n d itio n s
yeast
(C O 2 ,
s tra in ,
w o rt
T e m p e ra tu re ,
c o m p o s itio n ,
w o rt
v e lo c ity ),
im m o b iliz a tio n m e th o d (a d s o rp tio n o r e n tra p m e n t) a n d re a c to r d e s ig n

(Y a m a u c h i e t a l., 1 9 9 5 ). H A fo rm a tio n is re la te d w ith y e a s t g ro w th
in te n s ity . T h e re fo re s o lu tio n s to in c re a s e th e ir fo rm a tio n a re m a in ly
s o lu tio n s th a t in c re a s e y e a s t c e lls g ro w th , s u c h a s : th e in c re a s e fo
a e ra tio n a n d te m p e ra tu re a n d th e re d u c e o f d is s o lv e d C O 2 . T h e H A
p ro d u c tio n s
is
e x tre m e ly
im p o rta n t
as
th e y
a re
re la te d
w ith
th e
p ro d u c tio n o f e s te rs (B r n y ik e t a l., 2 0 0 8 ; S m o g ro v ic o v a n d D o m n y ,
1 9 9 9 ; W illa e rt a n d N ed o v ic , 2 0 0 6 ).
II.4.1.1
Higher alcohols production in continuous AFBs
It h a s b e e n re p o rte d th a t in A F B s d u e to lim ite d fe rm e n ta tio n o r

a lc o h o l re m o v a l te c h n iq u e s th e c o n c e n tra tio n o f H A is lo w (B ra n y ik e t
a l., 2 0 1 2 ). M o re o v e r, th e c o n c e n tra tio n o f u n d e s ira b le H A s u ch a s
ty ro s o l a n d try p to p h o l is a ls o lo w (B a rto lo m e t a l., 2 0 0 0 ). A s to ld
b e fo re (s e c tio n II.1 ), c o n tin u o u s A F B p ro d u c tio n is n o rm a lly o b ta in e d b y
lim itin g y e a s t g ro w th in te n s ity . C o n s e q u e n tly , th e c o n c e n tra tio n o f H A
is n o rm a lly v e ry lo w c o m p a re d w ith c o n tin u o u s re g u la r b e e rs p ro d u c tio n
(s e e T a b le II- 4 ). H o w e v e r, w h e n c o m p a re d w ith c o m m e rc ia l A F B s th e
v a lu e s a re s im ila r. L e h n e rt e t a l (2 0 0 8 ) p ro v e d th a t th e in c re a s in g o f
d is s o lv e d o x y g e n in th e re a c to r in c re a s e s th e H A p ro d u c tio n . It w a s
31
Chapter II
s u s ta in e d th a t in c re a s in g d is s o lv e d o x y g e n , it in c re a s e s c a rb o h y d ra te
a n d a m in o a c id m e ta b o lis m a n d c o n s e q u e n tly th e ! -k e to -a c id s a n d H A
p ro d u c tio n . O n ly a t h ig h o x y g e n tra n s fe r ra te s (! 1 0 m g /L /h ) th e a m o u n t
o f H A w a s h ig h e r th a n in th e c o m m e rc ia l A F B s (L e h n e rt e t a l., 2 0 0 8 ).
II.4.2
Esters
E s te rs (E S ) re p re s e n t th e la rg e s t g ro u p o f fla v o u r a c tiv e c o m p o u n d s
in b e e rs . T h e ir g re a t im p o rta n c e in fla v o u r is a ttrib u te d to its lo w
th re s h o ld th a t c o n fe rs a fru ity a ro m a in b e e rs .
E s te rs a re s y n th e s iz e d
b y th e re a c tio n b e tw e e n a n a lc o h o l a n d a n a c y l-C o A m o le c u le , w h ic h is
c a ta ly z e d
by
a lc o h o l
a c y ltra n s fe ra s e s
(A A T a s e s ).
S e v e ra l
o th e r
e s te ra s e s h a v e b e e n re p o rte d . A lth o u g h e s te r fo rm a tio n in y e a s t h a s

b e e n v e ry s tu d ie d o v e r th e p a s t y e a rs (V e rs tre p e n e t a l., 2 0 0 3 ), th e
k n o w le d g e a b o u t th e p h y s io lo g ic a l ro le o f th e s e c o m p o u n d s is ra th e r
u n c le a r (B r n y ik e t a l., 2 0 0 8 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). T h e E S c a n
b e d iv id e d in to tw o m a in g ro u p s : (1 ) a c e ta te e s te rs s u c h a s e th y l
a c e ta te (m o s t c o m m o n b e c a u s e is d e riv e d fro m e tha n o l), is o a m y l
a c e ta te a n d p h e n y le th y l a c e ta te ; a n d (2 ) th e s o-c a lle d e th y l o r m e d iu m
c h a in fa tty a c id e s te rs e .g . e th y l c a p ro a te a n d e th y l c a p ry la te (B r n y ik
e t a l., 2 0 0 8 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). E S p ro d u c tio n in b a tc h
s y s te m s o c c u rs d u rin g y e a s t g ro w th (6 0 % ) a n d s ta tio n a ry p h a s e (4 0 % )
(W illa e rt a n d N e d o v ic , 2 0 0 6 ). T w o fa c to rs a re im p o rta n t o n th e ra te o f
e s te r fo rm a tio n : th e a v a ila b ility o f th e tw o s u b s tra te s (a c e ty l/a c y l-C o A
a n d a lc o h o ls ), a n d th e a c tiv ity o f e n z y m e s (A A T a s e s ). E s te r fo rm a tio n is
h ig h ly d e p e n d e n t o n th e y e a s t s tra in u s e d a n d o n c e rta in fe rm e n ta tio n
p a ra m e te rs s u c h a s te m p e ra tu re , p itc h in g ra te , to p p re s s u re a n d
a e ra tio n (C a ld e rb a n k a n d H a m m o n d , 1 9 9 4 ; V a n Ie rs e l e t a l., 1 9 9 9 ;
V e rs tre p e n a n d K lis , 2 0 0 6 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). H igh e r w o rt
a e ra tio n a ffe c ts e s te rs s y n th e s is , b e c a u s e fa tty a c id s fro m w o rt re d u c e
th e a v a ila b ility o f a c y l-C o A (u s e d fo r y e a s t g ro w th a n d m e m b ra n e lip id
s y n th e s is ) (B r n y ik e t a l., 2 0 0 8 ; L e h n e rt e t a l., 2 0 0 8 ; L e w is a n d Y o u n g ,
1 9 9 5 ). S o m e s tu d ie s re p o rte d tha t th e m a in fa c to r c o n tro llin g e s te r
b io s y n th e s is is th e e x p re s s io n le v e l o f th e A T F 1 g e n e , w h ic h c o d e s fo r
32
Chapter II
A A T a s e . T h e e x p re s s io n o f A T F 1 g e n e is re p re s s e d b o th b y o x y g e n a n d
u n s a tu ra te d fa tty a c id s (V a n Ie rs e l e t a l., 1 9 9 9 ; V e rs tre p e n e t a l., 2 0 0 3 ;
W illa e rt a n d N e d o v ic , 2 0 0 6 ).
When
s tu d in g
th e
ES
p ro d u c tio n
in
c o n tin u o u s
and
tra d itio n a l
s y s te m , th e o v e ra ll te n d e n c y in d ic a te s re d u c e d e s te r fo rm a tio n in
c o n tin u o u s s y s te m s (B r n y ik e t a l., 2 0 0 8 ). T h e la c k o f E S in c o n tin u o u s
s y s te m s is b e lie v e d to h a p p e n d u e to th e lo w c e llu la r m e ta b o lic
a c tiv itie s in th e s e s y s te m s (W illa e rt a n d N e d o v ic , 2 0 0 6 ). T h is te n d e n c y
m ig h t b e d u e to in c o rre c t a e ra tio n (la c k o f k n o w le d g e o f k L a ), y e a s t
s tra in a n d c e ll p h y s io lo g y , p ro c e s s d e s ig n (e .g . ty p e o f im m o b iliz a tio n )
(B r n y ik
et
a l.,
2 0 0 8 ),
a g e in g
a n d /o r
g e n e tic
d rift
(e s p e c ia lly
in
im m o b iliz e d s y s te m s ) (S a to e t a l., 2 0 0 1 ), w o rt c o m p o s itio n (s p e c ific

g ra v ity , lip id a n d F A N c o n te n t) a n d p ro c e s s c o n d itio n s (te m p e ra tu re ,
p H , a g ita tio n ) (D u fo u r e t a l., 2 0 0 3 ). H o w e v e r, s o m e re p o rts s h o w e d
h ig h e r E S p ro d u c tio n in c o n tin u o u s im m o b iliz e d c e lls s y s te m s (V a n
Ie rs e l
et
a l.,
1999;
W illa e rt
and
N e d o v ic ,
2 0 0 6 ).
The
th e o re tic a l
e x p la n a tio n re la te s th e E S in c re a s e w ith th e lo w m a s s tra n s fe r a n d

d is s o lv e d o x y g e n . T h is d e c re a s e re d u c e s th e im m o b iliz e d y e a s t c e lls
g ro w th in te n s ity a n d a n in c re a s e in th e a v a ila b ility o f a c y l-C o A fo r E S
s y n th e s is in s te a d o f fa tty -a c id s fo rm a tio n o c c u rs (W illa e rt a n d N e d o v ic ,
2 0 0 6 ). S h e n e t a l, (2 0 0 3 ) fo u n d a s ig n ific a n t re d u c tio n (3 5 % ) o f th e
a m o u n t o f to ta l fa tty a c id s in im m o b iliz e d y e a s t c e lls , w h ic h a llo w e d th e
a c y l-C o A s to b e c h a n n e lle d fo r e s te rs p ro d u c tio n (S h e n e t a l., 2 0 0 3 ).
M o re o v e r lo w fa tty a c id s w ill h a v e a le s s re p re s s iv e e ffe c t o n A T F 1
g e n e c o n s id e re d o n e o f m a in g e n e s in c o d in g E S fo rm a tio n (S h e n e t a l.,
2 0 0 3 ).
II.4.2.1
Esters production in continuous AFBs
B y a n a ly s in g th e c o m m e rc ia l b e e rs p re s e n t in T a b le II- 4 , it is
p o s s ib le to o b s e rv e th a t E S p ro d u c tio n in A F B s c a n b e u p to 3 0 tim e s
le s s th a n in re g u la r b e e rs . M o re o v e r, to p ro d u c e c o n tin u o u s ly A F B s lo w
te m p e ra tu re s a re n o rm a lly a p p lie d (0 C to 1 2 C ), w h ic h lim it y e a s t
g ro w th , a n d a ls o E S p ro d u c tio n . V a n Ie rs e l a n d h is c o lla b o ra to rs (1 9 9 9 )
33
Chapter II
o b s e rv e d b e tte r E S p ro d u c tio n a t h ig h te m p e ra tu re s (1 2 C ) a n d lo w
o x y g e n c o n te n t. T h e y a ls o o b s e rv e d th a t th e in c re a s e in E S c o n te n t
w a s re la te d w ith a d e c re a s e d o f fa tty a c id c o n te n t a s s o c ia te d w ith a n
in c re a s e o f e n z y m e a c tiv ity (V a n Ie rs e l e t a l., 1 9 9 9 ). L e h n e rt e t a l (2 0 0 8 )
re p o rte d a d u a l e ffe c t fro m o x y g e n in E S p ro d u c tio n . T h e y s h o w e d th a t
in c re a s e in o x y g e n c o n c e n tra tio n le a d s to in c re a s e o f H A c o n te n t in
A F B s , w h ic h is a s u b s tra te o n E S p ro d u c tio n . H o w e v e r, p e r s e , th is
in c re a s e o n s u b s tra te c o n c e n tra tio n w a s n o t e n o u g h a n d w a s c o u n te rb a la n c e d b y lo w e n z y m e a c tiv ity d u e to o x y g e n c o n c e n tra tio n , o v e ra ll
le a d in g to n o E S in c re a s e (L e h n e rt e t a l., 2 0 0 8 ). In c re a s in g o x y g e n
tra n s fe r ra te , th e y e a s t c e ll u s e s d iffe re n t m e ta b o lic p a th w a y s th a t a re
re la te d w ith c e ll d iv is io n a n d n o t w ith E S p ro d u c tio n . Es te rs s y n th e s is
is a c o m p le x p ro c e s s in v o lv in g m a n y fa c to rs a n d is d iffic u lt to c o n tro l.
In o rd e r to s o lv e th is p ro b le m , m u ta n ts a n d g e n e tic a lly m o d ifie d y e a s t
m ig h t b e a p ro m is in g in s tru m e n t to c o n tro l fla v o u r is s u e s in c o n tin u o u s
p ro c e s s (V e rs tre p e n e t a l., 2 0 0 1, 2 0 0 3 ).
R e a c to r d e s ig n m a y in flu e n c e ES c o n c e n tra tio n in th e fin a l p ro d u c t,
m a in ly in G L R w h e re s trip p in g o f E S b y th e g a s-p h a s e m a y o c c u r a n d
le a d to lo w E S c o n c e n tra tio n . E x p e rim e n ts p ro v e d th a t th e p h y s ic a l
re m o v a l o f E S b y s trip p in g is re d u c e d (b e lo w 3 0 % ) a t d ilu tio n ra te s u p
to 0 .3 h - 1 . T h e e x c e p tio n w a s th e e th y l c a p ry la te (lo s s e s b e tw e e n 3 0 %
a n d 8 0 % ) (M a c ie ira , 2 0 0 8 ).
II.4.3
O rg a n ic
Organic acids
a c id s
(O A )
in
beer
a re
d e riv e d
fro m
w o rt
and
yeast
m e ta b o lis m . T h e s h o rt c a rb o n s k e le to n o rg a n ic a c id s s e c re te d b y
y e a s ts a re d e riv e d fro m : (1 ) th e inc o m p le te tu rn o v e r o f T C A c y c le a n d
(2 ) th e a m in o a c id s c a ta b o lis m . O A re d u c e th e p H d u rin g fe rm e n ta tio n
c o n fe rrin g a s o u r ta s te . In a d d itio n th e y p ro v id e a n in c re a s e o f
m ic ro b ia l s ta b ility o n th e fin a l p ro d u c t (B r n y ik e t a l., 2 0 0 8 ; S e le c k y e t
a l., 2 0 0 8 ). It c a n b e s ta te d th a t th e O A c o n c e n tra tio n in c o n tin u o u s ly
s y s te m s s h o u ld b e s u c h th a t d o e s n o t in flu e n c e b e e r fla v o u r b u t k e e p s
34
Chapter II
a g o o d m ic ro b ia l p ro te c tio n . T h e re a re fe w s tu d ie s a b o u t O A fo rm a tio n
d u rin g c o n tin u o u s b e e r p ro d u c tio n . T h e o n e s a v a ila b le re p o rt little
d iffe re n c e in th e to ta l o rg a n ic a c id c o n c e n tra tio n . T h e c o n tro l o n O A
c o n c e n tra tio n in c o n tin u o u s b e e r fe rm e n ta tio n is s im ila r to H A a n d is
b a s e d o n th e re g u la tio n o f c e ll g ro w th a n d s u b trate c o n s u m p tio n ra te .
(B r n y ik e t a l., 2 0 0 8 , S m o g ro v ic o v a n d D o m n y , 1 9 9 9 ; Y a m a u c h i e t
a l., 1 9 9 5 .)
II.4.3.1
Organic acids production in continuous AFBs
W o rk in g w ith T C A g e n e d e fic ie n t y e a s t s tra in s , S e le ck y e t a l. (2 0 0 8 )

w e re a b le to p ro d u c e A F B s w ith h ig h s a c c h a rid e c o n c e n tra tio n w ith lo w
n itro g e n u tiliz a tio n . B u t th e lo w e th a n o l w a s c o m p e n s a te d b y a h ig h O A
fo rm a tio n m a in ly s u c c in a te , fu m a ra te , la c tic a n d m a la te . T h e o b s e rv e d
h ig h le v e ls o f la c tic a c id (1 4 4 m g /L to 4 9 5 m g /L ) w e re c o n s id e re d
im p o rta n t b e c a u s e it c o n fe rs g o o d p ro te c tio n a g a in s t c o n ta m in a tio n
a n d m a s k s u n a c c e p ta b le w o rty o ff-fla v o u rs ty p ic a l fro m A F B s (S e le c k y
e t a l, 2 0 0 8 ).
II.4.4
Beer
Aldehydes
a ld e h y d e s
a ris e
m a in ly
d u rin g
w o rt
p ro d u c tio n
(m a s h in g ,
b o ilin g ) a n d p a rtia lly a re fo rm e d d u rin g fe rm e n ta tio n b y y e a s t(B a rto lo m

e t a l., 2 0 0 0 ). A ld e h y d e s a re v e ry im p orta n t d u e to th e ir lo w th re s h o ld ,
w h ic h is h u n d re d tim e s le s s th a n th e o th e r c o m p o u n d s (s e e T a b le II- 4 ).
T h e y c o n trib u te la rg e ly to th e w o rty o ff-fla v o u r d e te c te d p a rtic u la rly in
lo w -a lc o h o l b e e r p ro d u c e d b y lim ite d fe rm e n ta tio n (P e rp te a n d C o llin ,
1 9 9 9 a , 1 9 9 9 b ). S e v e ra l a ld e h y d e s p re s e n t in w o rt h a v e b e e n id e n tifie d
a s re s p o n s ib le fo r w o rty o ff fla v o u r in b e e rs (3 m e th y l b u ta n a l, 2 m e th y l b u ta n a l, h e x a n a l, h e p ta n a l).
B e e rs m a d e b y c o n tin u o u s fe rm e n ta tio n s h o w a s lig h tly in c re a s e d
a c e ta ld e h y d e c o n te n t w h en c o m p a rin g to c o n v e n tio n a l b e e rs (B r n y ik e t
a l., 2 0 0 4 ; M e n s o u r e t a l., 1 9 9 7 ). U s u a lly th is is d u e to th e fa c t th a t th e
m a jo rity o f c o n tin u o u s s y s te m s s h o w e x c e s s c e ll g ro w th a n d /o r o v e r-
35
Chapter II
a e ra tio n . A c e ta ld e h y d e c o n te n t h a s to b e c o n tro lle d e ith e r b y p ro p e r

o x y g e n s u p p ly (B r n y ik e t a l., 2 0 0 6 ) o r b y p ro lo n g e d m a tu ra tio n
(K ro n l f a n d L in k o , 1 9 9 2 ). H ig h e r ra te s o f a ld e h y d e re d u c tio n w e re
o b s e rv e d a t h ig h e r te m p e ra tu re s , b u t re s id e n c e tim e a p p e a re d to b e a
m a jo r fa c to r in d e te rm in in g re s id u a l a ld e h y d e le v e ls (De b o u rg e t a l.,
1 9 9 4 ; V a n Ie rs e l e t a l., 1 9 9 5 ). It is a ls o re p o rte d th a t th e e n z y m a tic
m e c h a n is m s
in v o lv e d
in
a ld e h y d e
re d u c tio n
c a p a c ity
w e re
not
n e g a tiv e ly a ffe c te d (D e b o u rg e t a l., 1 9 9 5 ) o r im p ro v e d (V a n Ie rs e l e t a l.,

2 0 0 0 ) b y in flu e n c e o f th e im m o b iliza tio n m e th o d . Im m o b iliz e d c e lls , d u e
to h ig h e r g lu c o s e flu x s h o w e d b o th h ig h e r a ld e h y d e s re d u c tio n a n d
h ig h e r
a c tiv ity
of
a lc o h o l
d e h y d ro g e n a s e ,
c o m p a rin g
to
fre e
c e ll
s y s te m s (V a n Ie rs e l e t a l., 2 0 0 0 ).
II.4.4.1
Aldehydes production in continuous AFBs
A s in d ic a te d b e fo re , in A F B s th e p re s e n c e o f a ld e h y d e s is c ru c ia l fo r
a b a la n c e d fin a l b e e r fla v o u r. In A F B s th e fla v o u r th re s h o ld s a re
b e lie v e d to b e m o re n e a r w a te r th re s h o ld s th a n in b e e r, m a in ly d u e to
th e ir lo w a lc o h o l c o n te n t (P e rp te a n d C o llin , 1 9 9 9b ). W o rt a ld e h y d e s
d u rin g
b a tc h
fe rm e n ta tio n s
a re
re d u c e d
re la tiv e ly
s w iftly ,
but
as
c o n tin u o u s A F B s a re n o rm a lly lim ite d fe rm e n ta tio n s s o m e c o n c e rn s

a ris e . A m o n g th e s e , 2 - a n d 3 -m e th y lb u ta n a l, h y d ro x y m e th y lfu rfu ra l
(B a rto lo m a n d a l., 2 0 0 0 ) a n d m e th io n a l (P e rp te a n d C o llin , 1 9 9 9 ) a ris e
m a in ly fro m w o rt p re p a ra tio n (m a s h in g ).
P e rp te a n d C o llin (1 9 9 9 b ) s tu d ie d th e a ld e h y d e re d u c tio n in C C P
u s in g a P B R . A fte r 8 h th e y fo u n d th a t a ld e h y d e re d u c tio n is a lm o s t
c o m p le te a n d it is in flu e n c e d b y te m p e ra tu re (8 5 % a t 2 8 C a n d 6 0 % a t
0 C ), p itc h in g ra te a n d w o rt c o m p o s itio n (b in d in g e ffe c t o f p ro te in s ). In
a
la te r
a rtic le ,
th e
same
a u th o rs
fo u n d
th a t
m e th io n a l
(3-
m e th y lth io p ro p io n a ld e h y d e ) w a s th e k e y c o m p o u n d re s p o n s ib le fo r th e
w o rty , u n p le a s a n t, fla v o u r o f A F B s (P e rp te a n d C o llin , 1 9 9 9 b ). T h is
a ld e h y d e d e riv e s fro m th e re a c tio n b e tw e e n m e th io n in e w ith g lu c o s e o r
m a lto s e a n d h a s a h ig h b o ilin g p o in t. S o th is c o m p o u n d h a s lo w lo s s e s
d u rin g b re w in g p ro c e s s . M o re o v e r, it is v e ry h a rd to d e te c t a n d h a s a
36
Chapter II
v e ry lo w th re s h o ld : 1 .7 p p b in w a te r (P e rp te a n d C o llin , 1 9 9 9 b ) a n d
2 5 0 p p b in b e e r (M e ilg a a rd , 1 9 7 5 ).
A c e ta ld e h y d e is a ls o o n e o f th e im p o rta n t a ld e h y d e s fo u n d in b e e rs
b e c a u s e it is a p re c u rs o r o f m a n y o th e r c o m p o u n d s (H A , e th a n o l e tc ).
Its fo rm a tio n is re la te d w ith fe rm e n ta tio n in te n s ity . T h e re fo re th e
a c e ta ld e h y d e c o n c e n tra tio n in c re a s e s w ith th e in c re a s e o f th e o x y g e n
tra n s fe r ra te . A s its fo rm a tio n is e s s e n tia l, a c e ta ld e h y d e re d u c tio n is
lo w e r, h o w e v e r its re d u c tio n is g e n e ra lly h ig h e r th a n 8 5 % a n d th e fin a l
c o n c e n tra tio n is s im ila r to th e th re s h o ld in re g u la r b e e rs (1 0 m g /L , s e e
T a b le II- 4 ) (L e h n e rt e t a l., 2 0 0 8 ). N e v e rth e le s s , if th e fin a l a c e ta ld e h y d e
c o n c e n tra tio n s o b ta in e d in c o n tin u o u s A F B s a re c o m p a re d w ith th e
th re s h o ld in w a te r (0 .0 2 5 m g /L ) a s s u g g e s te d b y s o m e a u th o rs , a n d
th e n its fla v o u r in flu e n c e c a n b e re le v a n t. S o m e s tu d ie s h a v e b e e n
made
to
m e a s u re
th e
lo s s
of
a ld e h y d e s
by
s trip p in g
(p h y s ic a l
m e c h a n is m )) in a n iG L R a n d it w a s fo u n d th a t in th is ty p e o f re a c to rs , a t
lo w e r d ilu tio n ra tes (b e lo w 0 .2 h - 1 ), th e a ld e h y d e re m o v a l (b y s trip p in g )
was
good
and
h ig h e r
th a n
65%
(M a c ie ira ,
2 0 0 8 ).
D u rin g
s e v e ra l
c o n tin u o u s ly A F B fe rm e n ta tio n s u s in g s p e n t g ra in s a s c a rrie r a n d

d iffe re n t s tra in s , it w a s a lw a y s o b ta in e d a n a ld e h y d e re d u c tio n a b o v e
th e b a tc h p ro c e s s (8 5 % v s 6 5 % ) a n d n o in flu e n c e o n th e g a s-p h a s e
(p u re C O 2 v s A ir/C O 2 m ix tu re ) w a s n o te d (M a c ie ira , 2 0 0 8 ).
II.4.5
Vicinal diketones
A m o n g th e k e to n e s p re s e n t in b e e r, th e tw o v ic in a l d ik e to n e s
(V D K s ): d ia c e ty l (2 ,3 -b u ta n e d io n e ) a n d 2 ,3-p e n ta n e d io n e a re th o s e th a t
a re o f e x tre m e im p o rta n c e o n b e e r o ff-fla v o u r. D ia c e ty l is th e m o s t
fla v o u r-a c tiv e ,
because
it
has
v e ry
lo w
ta s te
th re s h o ld
of
a p p ro x im a te ly 0 .1 5 m g /L (M e ilg a a rd , 1 9 7 5 ). T h e y a re re s p o n s ib le fo r a n
u n c le a n , s w e e tis h ta s te in b e e r, w h ic h turn s in to a b u tte ry o ff-fla v o u r if
in h ig h e r c o n c e n tra tio n (W illa e rt a n d N e d o v ic , 2 0 0 6 ).
V D K s a re p ro d u c e d d u rin g m a in fe rm e n ta tio n fro m y e a s t m e ta b o lite s
(! -a c e to la c ta te a n d ! -k e to b u ty ra te ) th a t a re s e c re te d in to th e g re e n -
37
Chapter II
b e e r. T h e s e a re p ro d u c e d a s in term e d ia te s in th e b io s y n th e s is o f th e
a m in o a c id s (is o le u c in e , le u c in e a n d v a lin e IL V p a th w a y ) (L e w is a n d
Y o u n g , 1 9 9 5 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). D u rin g m a tu ra tio n p h a s e ,
d ia c e ty l is re -a s s im ila te d a n d re d u c e d b y y e a s t to a c e to in a n d 2 ,3b u ta n e d io l,
compounds
w ith
re la tiv e ly
h ig h
fla v o u r
th re s h o ld s
(M e ilg a a rd , 1 9 7 5 ). T h e a m o u n t o f d ia c e ty l fo rm e d is re la te d w ith th e
in te rc e llu la r v a lin e s c o n c e n tra tio n , b u t a t h ig h c o n c e n tra tio n s o f v a lin e
th e re
is
an
in h ib ito ry
e ffe c t
on
th e
enzyme
re s p o n s ib le
fo r
!-
a c e to la c ta te p ro d u c tio n (P e te rs e n e t a l, 2 0 0 4 ). G e n e ra lly fe rm e n ta tio n

c o n d itio n s th a t p ro m o te y e a s t g ro w th a re re la te d w ith h ig h p ro d u c tio n
of
! -a c e to la c ta te ,
th u s
h ig h
d ia c e ty l
c o n c e n tra tio n s
(W illa e rt
and
N e d o v ic , 2 0 0 6 ).
In m o s t c o n tin u o u s b e e r fe rm e n ta tio n p ro d u c e d u s in g IC T , th e
a m o u n t o f to ta l d ia c e ty l te n d s to b e h ig h e r w h e n c o m p a rin g w ith
tra d itio n a l s y s te m s (B r n y ik e t a l, 2 0 0 5 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ).
S e v e ra l e x p la n a tio n s fo r th is tre n d a re : (1 ) c h a n g e o f c e ll p h y s io lo g y
in d u c e d
by
th e
im m o b iliz a tio n
(in c re a s in g
th e
e x p re s s io n
of
a c e to h y d ro x y a c id s y n th e ta s e g e n e ); (2 ) a lte ra tio n o f a m in o a c id (IL V

p a th w a y
changes)
m e ta b o lis m
in
im m o b iliz e d
c e lls ;
(3 )
enhanced
a n a b o lic fo rm a tio n o f a m in o a c id p re c u rs o rs d u e to ra p id y e a s t g ro w th
(B r n y ik e t a l, 2 0 0 5 ).
In o rd e r to c o n tro l a n d /o r a v o id th e e x c e s s iv e V D K s fo rm a tio n d u rin g
im m o b iliz e d c o n tin u o u s fe rm e n ta tio n , th e c o n tro l s tra te g ie s in c lu d e : (1 )
a d d itio n
of
! -a c e to la c ta te
d e c a rb o x y la s e ;
(2 )
u s in g
of
g e n e tic a lly
m o d ifie d b re w e rs y e a s t (e n c o d in g ! -a c e to la c ta te d e c a rb o x y la s e ); (3 )
c o n tro l o f o p e ra tio n c o n d itio n s (B r n y ik e t a l, 2 0 0 6 ; G o d tfre d s e n e t a l,
1 9 8 4 ; K ro n lo f a n d L in k o , 1 9 9 2 ; Y a m a u c h i e t a l., 1 9 9 5 ). C o n s id e rin g
e n z y m e c o s ts a n d th e u s u a l re lu c ta n c e o f c o n s u m e rs to th e u s e o f
g e n e tic a lly m o d ifie d o rg a n is m s in fo o d in d u s try , o th e r o p tio n s s h o u ld
b e u s e d . T h e s e c o n s is t m a in ly in lim itin g th e g ro w th o f im m o b iliz e d
y e a s t, p ro c e s s c h a n g e s , a n d s e le c tio n o f a n a p p ro p ria te y e a s t s tra in .
H o w e v e r c o n tro l th ro u g h g ro w th re g u la tio n is d iffic u lt a n d w a s fou n d to
b e in e ffe c tiv e (B r n y ik e t a l, 2 0 0 6 ; Y a m a u c h i e t a l., 1 9 9 5 ). T h e m a in
38
re a s o n
because
Chapter II
h ig h
d ia c e ty l
a m o u n ts
a re
fo u n d
in
c o n tin u o u s
m a tu ra te d b e e r is d u e to th e s h o rt tim e s n o rm a lly u s e d in c o n tin u o u s

m a tu ra tio n . Y a m a u c h i e t a l (1 9 9 5 ) s u g g e s te d a s tra te g y to re d u c e
d ia c e ty l e ffe c tiv e ly . T h is w a s o b ta in e d b y a p p ly in g a n in te rm e d ia te h e a t
tre a tm e n t
b e tw e e n
th e
p rim a ry
and
s e c o n d a ry
fe rm e n ta tio n
(m a tu ra tio n ). T h e h e a tin g w a s m a d e u n d e r a n a e ro b ic c o n d itio n s a t

9 0 C fo r 1 0 m in p rio r to m a tu ra tio n . T h e m a in d ra w b a c k w a s th e
n e c e s s ity o f im p le m e n tin g a n a d itio n a l c e n trifu g a tio n s te p (to re m o v e
b io m a s s ) b e fo re h e a tin g tre a tm e n t (Y a m a u c h i e t a l., 1 9 9 5 ).
II.4.5.1
Vicinal diketones production in continuous AFBs
A s th e te m p e ra tu re s u s e d in c o n tin u o u s A F B p ro d u c tio n a re n orm a lly

lo w a n d th e V D K s c o n c e n tra tio n d e p e n d s o n th e y e a s t g ro w th in te n s ity
n o rm a lly th e v a lu e s o f V D K s a re b e lo w th re s h o ld fo r re g u la r b e e rs
(T a b le II- 4 ). A t lo w te m p e ra tu re s th e ! -a c e to la c ta te p ro d u c tio n is lo w e r
a n d m o s t o f it is b a la n c e d to IL V p a th w a y . W o rk in g a t lo w te m p e ra tu re
a n d lo w d is s o lv e d o x y g e n c o n c e n tra tio n s w ill d e c re a s e th e V D K s
p ro d u c tio n (V a n Ie rs e l, 1 9 9 9 ).
39
Chapter II
II.5 References
Barker, M. G., and Smart, K. A. (1996). Morphological changes associated with the
cellular aging of a brewing yeast strain. Journal of the American Society of
Brewing Chemists, 54(2), 121126.
Bartolom, B., Pena-Neira, A. and Gmez-Cordovs, C. (2000). Phenolics and
related substances in alcohol-free beers. European Food Research and
Technology, 210(6), 419423.
Bezbradica, D., Obradovic, B., Leskosek-Cukalovic, I., Bugarski, B., and Nedovic, V.
(2007). Immobilization of yeast cells in PVA particles for beer fermentation.
Process Biochemistry, 42(9), 1348-1351.
Briggs, D. E., Boulton, C. A., Brookes, P. A., and Stevens, R. (2004). Brewing
Science and practice (p. 2547). Cambridge: Woodhead Publishing Limited.
Brnyik, Toms, Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2001).
Spent grainsa new support for brewing yeast immobilisation. Biotechnology
Letters, 23(13), 10731078. Springer.
Brnyik, T., Vicente, A. A., Dostlek, P., and Teixeira, J. A. (2005). Continuous beer
fermentation using immobilized yeast cell bioreactor systems. Biotechnology
progress, 21(3), 653-63.
Brnyik, T., Silva, D. P., Vicente, A. A., Lehnert, R., Silva, J. B. A., Dostlek, P., and
Teixeira, J. A. (2006). Continuous immobilized yeast reactor system for complete
beer fermentation using spent grains and corncobs as carrier materials. Journal of
industrial microbiology and biotechnology, 33(12), 1010-8.
Brnyik, T., Vicente, A. A., Dostlek, P., and Teixeira, J. A. (2008). A review of
flavour formation in continuous beer fermentations. Journal of the Institute of
Brewing, 114(1), 3-13.
Brnyik, T., Silva, D. P., Baszczy!ski, M., Lehnert, R., and Almeida e Silva, J. B.
(2012). A review of methods of low alcohol and alcohol-free beer production.
Journal of Food Engineering, 108(4), 493-506.
Calderbank, J., and Hammond, J. (1994). Influence of higher alcohol availability on
ester formation by yeast. Journal of the American Society of Brewing Chemists,
52(2), 84-90.
Czerny, M., Christlbauer, M., Christlbauer, M., Fischer, A., Granvogl, M., Hammer,
M., Hartl, C., Hernandez, N. M. and Schieberle, P. (2008). Re-investigation on
odour thresholds of key food aroma compounds and development of an aroma
language based on odour qualities of defined aqueous odorant solutions.
European Food Research and Technology, 228(2), 265-273.
Dufour, J.-P., Malcorps, P., And Silcock, P. (2003). Control of Ester Synthesis
During Brewery Fermentation. In K. Smart (Ed.), Brewing yeast fermentation
performance (2nd ed., pp. 213-233). Oxford: Blackwell Science.
Decamps, C., Norton, S., Poncelet, D., and Neufeld, R. J. (2004). Continuous pilot
plant-scale immobilization of yeast in"!-carrageenan gel beads. AIChE Journal,
50(7), 1599-1605.
Doran, P. M., and Bailey, J. E. (1986). Effects of immobilization on growth,
fermentation properties, and macromolecular composition of Saccharomyces
cerevisiae attached to gelatin. Biotechnology and Bioengineering, 28, 73-87.
Dmny, Zoltan, Smogrovicov, D., Gemeiner, P., Sturdk, E., Ptkov, J., and
Malovkov, A. (1998). Continuous secondary fermentation using immobilized
yeast. Biotechnology Letters, 20(11), 10411045.
GEA Process Engineering. (2008). Immocon - Rapid and continuous maturation of
beer by means of immobilized yeast.
Junter, G. A., Coquet, L., Vilain, S., and Jouenne, T. (2002). Immobilized-cell
physiology: current data and the potentialities of proteomics. Enzyme and
Microbial Technology, 31(3), 201212.
Kennedy, B. K., Austriaco, N. R., Zhang, J., and Guarente, L. (1995). Mutation in the
Silencing Gene SIR4 Can Delay Aging in S. cerevisiae. Cell, 80, 485-496.
Lehnert, R., Kurec, M., Brnyik, T., and Teixeira, J. A. (2008). Effect of Oxygen
Supply on Flavour Formation During Continuous Alcohol-free Beer Production: A
Model Study. Journal of American Society of Brewing Chemist, 66(4), 233-238.
40
Chapter II
Lehnert, R., Novk, P., Macieira, F., Kurec, M., Teixeira, J. A., and Brnyik, T.
(2009). Optimisation of Lab-Scale Continuous Alcohol-Free Beer Production.
Czech. Journal of Food Science, 27(4), 267-275.
Lewis, M. J., and Young, T. W. (1995). Brewing. (M. J. Lewis and T. W. Young, Eds.).
London: Chapman and Hall.
Macieira, F. F. (2008). Continuous fermentation of real alcohol-free beer. Master
Thesis, University of Minho, Braga
Maskell, D., Kennedy, A., Hodgson, J., and Smart, K. (2003). Chronological and
replicative lifespan of polyploid (syn. S. pastorianus). FEMS Yeast Research, 3(2),
201-209.
Masschelein, C. A., Ryder, D. S., and Simon, J.-P. (1994). Immobilized Cell
Technology in Beer Production. Critical Reviews in Biotechnology, 14(2), 155-177.
Meilgaard, M. C. (1975). Flavour chemestry of beer: Part II: Flavour and Threshold of
239 aroma volatiles. MBAA Technical Quarterly, 12(3), 151-168.
Mensour, N. A., Margaritis, A., Briens, C. L., Pilkington, P. H., and Russell, I. (1997).
New developemnts in the Brewing Industry using immobilized yeast cell biorector
systems. Journal of Institute of Brewing, 103, 363-370.
Meura. (2007). The Meurabrew!: the brewhouse of the future. Brochure. Retrieved at
12
th
of
December
in
2010,
from
the
website:
http://www.meura.com/uploads/pdf/Meurabrew Paper.pdf
Monch, D., Krugwer, E., and Stahl, U. (1995). Effects of stress on brewery yeasts.
Monatsschr Brau, 48, 288-299.
Narvtil, M., Dmny, Z., !turdk, E., !mogrovicov, D., and Gemeiner, P. (2001).
Production of non-alcoholic beer using free and immobilized cells of
Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle. Biotechnology
and Applied Biochemistry, 35(2), 133-140.
Narziss, L., Miedaner, H., Kern, E., and Leibhard, M. (1992). Technology and
composition of non-alcoholic beers. Processes using arrested fermentation.
Brauwelt International, 4, 396-410.
Nelson, J., and Griffin, E. G. (1916). Adsortion of invertase. Journal of the American
Chemical Society, 38(5), 11091115.
Pajunen, E., Gronqvist, A., and Ranta, B. (1991). Immobilized Yeast Reactor
Application in Continuous Secondary Fermentation in Industrial Scale Operation.
Proceedings of the European Brewing Convention Congress, Lisbon (pp. 361-368).
Lisbon: IRL Press: Oxford.
Pajunen, E., Tapani, K., Berg, H., Ranta, B., Bergin, J., and Lommi, H. (2001).
Controlled beer fermentation with continuous on-stage immobilized yeast reactor.
28 EBC Congress, Chapter 49 (pp. 1-12).
Perpte, Philippe, and Collin, S. (1999a). Fate of the worty favours in a cold contact
fermentation. Food Chemistry, 66, 359-363.
Perpte, P, and Collin, S. (1999b). Contribution of 3-methylthiopropionaldehyde to
the worty flavour of alcohol-free beers. Journal of agricultural and food chemistry,
47(6), 2374-8.
Pilkington, P., Margaritis, A., Mensour, N., and Russell, I. (1998). Fundamentals of
immobilized yeast cells for continuous beer fermentation: a review. Journal of the
Institute of Brewing, 104(1), 1931. London: Harrison and Sons, 1904-.
Powell, C. D., Zandycke, S. M. V., Quain, D. E., and Smart, K. A. (2000). Replicative
ageing and senescence in Saccharomyces cerevisiae and the impact on brewing
fermentations. Microbiology, 146, 1023-1034.
Powell, C., Quain, D., and Smart, K. (2003). The impact of brewing yeast cell age on
fermentation performance, attenuation and flocculation. FEMS Yeast Research,
3(2), 149-157.
Przybyla-Zawislak, B., Gadde, D. M., Ducharme, K., and McCammon, M. T. (1999).
Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA)
function using a collection of mutants defective in all TCA cycle genes. Genetics,
152(1), 153.
Qun, J., Shanjing, Y., and Lehe, M. (2002). Tolerance of immobilized baker s yeast
in organic solvents. Enzyme and Microbial Technology, 30, 721-725.
Repetto, B., and Tzagoloff, a. (1990). Structure and regulation of KGD2, the
structural gene for yeast dihydrolipoyl transsuccinylase. Molecular and cellular
biology, 10(8), 4221-32.
t h
41
Chapter II
Sato, M., Watari, J., and Shinotsuka, K. (2001). Genetic instability in flocculation of
bottom-fermenting yeast. Journal of American Society of Brewing Chemist, 59(3),
130-134.
Sato, T., and Tosa, T. (1999). Enzymes, Immobilization methods. In M. C. Flickinger
and S. W. Drew (Eds.), Encyclopedia of Bioprocess Technology: Fermentation,
Biocatalysis and Bioseparation (1st ed., pp. 1062-1064). New York: New York:
John Wiley and Sons, Inc.
Selecky, R., Smogrovicova, D., and Sulo, P. (2008). Beer with Reduced Ethanol
Content Produced Using Saccharomyces cerevisiae Yeasts Deficient in Various
Tricarboxylic Acid Cycle Enzymes. Journal Of The Institute Of Brewing, 114(2), 97101.
Shen, H., Moonjai, N., Verstrepen, K., and Delvaux, F. (2003). Impact of attachment
immobilization on yeast physiology and fermentation performance. Journal of the
American Society of Brewing Chemists, 61(2), 7987.
Sinclair, D a, Mills, K., and Guarente, L. (1998a). Molecular mechanisms of yeast
aging. Trends in biochemical sciences, 23(4), 131-4.
Sinclair, David, Mills, K., and Guarente, L. (1998b). Aging in Saccharomyces. Annual
Reviews in Mibrobiology, 52, 533-60.
Smogrovicov, D., and Domny, Z. (1999). Beer volatile by-product formation at
different
fermentation
temperature
using
immobilized
yeasts.
Process
Biochemistry, 34(8), 785-794.
Soares, E. V., and Mota, M. (1996). Flocculation onset, growth phase, and
genealogical age in Saccharomyces cerevisiae. Canadian Journal of Microbiology,
42(6), 539-547.
Taipa, M. A., Cabral, J. M. S., and Santos, H. (1993). Comparison of glucose
fermentation by suspended and gel-entrapped yeast cells: An in vivo nuclear
magnetic resonance study. Biotechnology and Bioengineering, 41, 647-653.
Virkajrvi, I., And Kronlf, J. (1999). Long-term stability of immobilized yeast
columns in primary fermentation. Journal of the American Society of Brewing
Chemists, 56(2), 70-75.
Van Iersel, M. F. M., Meersman, E., Swinkels, W., Abee, T., and Rombouts, F. M.
(1995). Continuous production of non-alcohol beer by immobilized yeast at low
temperature. Journal of Industrial Microbiology and Biotechnology, 14(6), 495
501.
Van Iersel, M. F. M., Van Dieren, B., Rombouts, F. M., and Abee, T. (1999). Flavour
formation and cell physiology during the production of alcohol-free beer with
immobilized Saccharomyces cerevisiae. Enzyme and Microbial Technology, 24(7),
407411.
Verbelen, P. J., De Schutter, D. P., Delvaux, F., Verstrepen, K. J., and Delvaux, F. R.
(2006). Immobilized yeast cell systems for continuous fermentation applications.
Biotechnology letters, 28(19), 15151525.
Verstrepen, K J, Derdelinckx, G., Delvaux, F. R., Winderickx, J., Thevelein, J. M.,
Bauer, F. F., and Pretorius, I. S. (2001). Late fermentation expression of FLO1 in
Saccharomyces cerevisiae. Journal of American Society of Brewing Chemists,
59(2), 69-76.
Verstrepen, Kevin J, and Klis, F. M. (2006). Flocculation, adhesion and biofilm
formation in yeasts. Molecular microbiology, 60(1), 5-15.
Verstrepen, Kevin J, Derdelinckx, G., Dufour, J.-P., Winderickx, J., Thevelein, J. M.,
Pretorius, I. S., and Delvaux, F. R. (2003). Flavour-active esters: adding fruitiness
to beer. Journal of bioscience and bioengineering, 96(2), 110-8.
Virkajarvi, I. (2001). Feasibility of continuous main fermentation of beer using
immobilized yeast. Vtt Publications. Technical Research Centre Of Finland Espoo.
Virkajarvi, I., and Linko, M. (1999). Immobilization: A revolution in traditional
brewing. Naturwissenschaften, 86(3), 112122.
Willaert, R., and Nedovic, V. (2006). Review Primary beer fermentation by
immobilized yeast a review on flavour formation and control strategies. Journal
of Chemical Technology and Biotechnology, 81, 1353-1367.
Yamauchi, Y., Okamoto, T., Murayama, H., Nagara, A., Kashihara, T., Yoshida, M.,
Yasui, T., et al. (1995). Rapid Fermentation of Beer Using an Immobilized Yeast
Multistage Bioreactor System. Applied Biochemistry And Biotechnology, 53.
42
Chapter III
III. Chapter III Selection of the Best

Immobilization Carrier for Brewing Yeast
Cells
III.1 Objectives
III.2 Introduction
III.3 Material and Methods
III.4 Results and Discussion
III.5 Conclusions
III.6 References
43
Chapter III
III.1 Objectives
W h e n im p le m e n tin g a c o n tin u o u s fe rm e n ta tio n s y s te m us in g IC T it is
n e c e s s a ry
to
c o n s id e r
some
a s p e c ts
such
as:
c a rrie r
p ro p e rtie s ,
im m o b iliz a tio n m e c h a n is m a n d th e re a c to r c o n fig u ra tio n . O n e o f th e

m a in
p ro b le m s
when
c o n tin u o u s
p rim a ry
beer
fe rm e n ta tio n s
a re
p e rfo rm e d is th e tim e th a t ta k e s fo r b io re a c to r sta rt-u p (7 to 1 5 d a y s )

i.e ., th e tim e th a t is n e c e s s a ry fo r th e b io re a c to r to h a v e e n o u g h
im m o b iliz e d b io m a s s (iB io ) th a t a llo w s w o rk in g a t h ig h d ilu tio n ra te s
(B r n y ik e t a l., 2 0 0 1 ). If th e in itia l a m o u n t o f c e lls a d s o rb e d to th e
c a rrie r is in c re a s e d , th e s ta rt-u p tim e w ill b e re d u c e d . A d s o rp tio n
d e p e n d s o n m a n y fa c to rs a n d o c c u rs n a tu ra lly in y e a s t c e lls a n d is
m o re th a n a s im p le a n d p h y s ic a l m e c h a n is m . It is a d y n a m ic pro c e s s
u n til th e e q u ilib riu m b e tw e e n c e ll-c a rrie r a tta c h m e n t a n d d e ta c h m e n t
p ro c e s s e s is re a c h e d .
In th is c h a p te r th e m a in o b je c tiv e w a s to s tu d y th e in flu e n c e o f
c a rrie r
ty p e
(s p e n t
g ra in s
and
c o rn c o b s )
and
of
th e ir
d e g re e
of
d e riv a tiz a tio n o n th e in itia l a d s o rp tio n o f y e a s t c e lls . In a d d itio n , fo r

c o rn c o b s it w a s s tu d ie d th e in flu e n c e o f th e ir s iz e , a s w e ll a s th e
in flu e n c e
of
d e riv a tiz a tio n
d iffe re n t
on:
(1 )
p re-tre a tm e n t
m e th o d s
and
io n
and
c o rn c o b s
exchange
(2 )
th e
d e g re e
of
a d s o rp tio n
c a p a c ity .
III.2 Introduction
III.2.1 Immobilization of biocatalysts: history and application
B io p ro c e s s e s u s in g im m o b iliz e d b io c a ta ly s ts (e n z y m e s /c e lls ) h a v e
b e e n re c e iv in g in c re a s in g w o rld w id e a tte n tio n b e c a u s e o f th e ir b e n e fits
o f lo w e n v iro n m e n ta l p o llu tio n a n d e c o n o m ic a l u tiliz a tio n o f n a tu ra l
re s o u rc e s a n d e n e rg y (S a to a n d T o s a , 1 9 9 9 ). T h e p io n e e r w o rk o f
N e ls o n
and
G riffin
(1 9 1 6 )
in
th e
e a rly
XX
c e n tu ry
was
th e
firs t
d e m o n s tra tio n o f im m o b iliz e d b io c a ta ly s ts (N e ls o n a n d G riffin , 1 9 1 6 ). A t

th e p re s e n t, im m o b iliz e d b io c a ta ly s ts h a v e c o n trib u te d to a w id e v a rie ty
44
Chapter III
o f fie ld s in in d u s try lik e th e p ro d u c tio n o f e n e rg y b y b io lo g ic a l p ro c e s s

o r p ro d u c tio n o f u s e fu l c o m p o u n d s (S a to a n d T o s a , 1 9 9 9 ). O v e r th e la s t
fo rty y e a rs , m a n y b io te c h n o lo g y p ro c e s s e s b a s e d in im m o b iliz e d c e lls
s y s te m s h a v e b e e n s tu d ie d . P a rtic u la rly a n in c re a s e in th e d e v e lo p m e n t
o f im m o b iliz e d c e lls s y s te m s o c c u rre d s in c e th e e a rly 1 9 8 0 s (J u n te r a n d
J o u e n n e , 2 0 0 4 ). T h e s e s y s te m s c a n o c c u r n a tu ra lly b y s p o n ta n e o u s
a d s o rp tio n o r a rtific ia lly b y e n tra p m e n t a n d a tta c h m e n t te c h n iq u e s .
C e ll im m o b iliz a tio n c a n b e d e fin e d a s th e ph y s ic a l c o n fin e m e n t o f a
m ic ro o rg a n is m in a c e rta in d e fin e d re g io n o f s p a c e w ith re te n tio n o f its
c a ta ly tic a c tiv itie s , w h ic h c a n b e u s e d in re p e titiv e o r c o n tin u o u s
p ro c e s s e s (J u n te r a n d
w e re
o rig in a lly
J o u e n n e , 2 0 0 4 ). C e ll im m o b iliz a tio n m e th o d s
a d a p te d
The
fro m
m a in
th o s e
a d v a n ta g e
e a rlie r
fro m
a p p lie d
u s in g
in to
c e lls
enzyme
in s te a d
of
e n z y m e s is to a v o id th e e x tra c tio n /p u rific a tio n s te p s . T h e s e s te p s

re d u c e e n z y m e a c tiv ity , s ta b ility a n d c o s t. A c tu a lly im m o b iliz e d c e lls
s y s te m s a re a p p lie d in m a n y d iffe re n t fie ld s , s u c h a s th e b io s y n th e s is
a n d b io c o n v e rs io n s o f s e v e ra l p ro d u c ts (e n z y m e s, a n tib io tic s , a m in o
a c id s , O A , a lc o h o ls a n d p o ly s a c c h a rid e s ); e n v iro n m e n ta l p ro c e s s e s
(b io fe rtiliz a tio n , b io re m e d ia tio n ); fo o d p ro c e s s in g (b e e r, w in e , la c to s e
h y d ro ly s is ); b io s e n s o rs (a m in o a c id s c h o le s te ro l, to x ic ity te s ts , m e ta ls )
(J u n te r a n d J o u e n n e , 2 0 0 4 ).
III.2.2 Continuous high cell density systems

The
m a jo r
a d v a n ta g e
of
u s in g
im m o b iliz e d
c e lls
s y s te m s
over
c o n v e n tio n a l (s u s p e n d e d c e lls ) c u ltu re s is th a t it a llo w s w o rk in g w ith

h ig h c e ll d e n s ity s y s te m s a n d tu rn s e a s y th e a p p lic a tio n o f c o n tin u o u s
p ro c e s s e s . T h e m a in a d v a n ta g e s a n d d is a d v a n ta g e s o f c o n tin u o u s
p ro c e s s e s u s in g IC T o v e r s u s p e n d e d c e lls c u ltu re s are s u m m a riz e d in
T a b le III- 1 (G a m a e t a l., 2 0 0 3 ; K o u rk o u ta s e t a l., 2 0 0 4 ; S a to a n d T o s a ,
1 9 9 9 ; V e rb e le n e t a l., 2 0 0 6 ; V irk a ja rv i a n d L in k o , 1 9 9 9 ).
T h e m o s t im p o rta n t a d v a n ta g e in d ic a te d in T a b le III- 1 is th e h ig h
re a c tio n s ra te s o b ta in e d (d u e to h ig h d ilu tio n ra te s u s e d ) in c o n tin u o u s
45
Chapter III
p ro c e s s e s u s in g IC T . T h e e c o n o m ic b e n e fits a re th e d riv in g fo rc e fo r
th e re c e n t in c re a s e o f c o n tin u o u s b io te c h n o lo g y p ro c e s s e s . A lth o u g h
s o m e d is a d v a n ta g e s c a n b e o b s e rv e d , im m o b iliz e d c e lls s y s te m s a re a
k e y te c h n o lo g y u s e d to p e rfo rm e ffic ie n t c o n tin u o u s b io p ro c e s s e s
(G a m a e t a l., 2 0 0 3 ; S a to a n d T o s a , 1 9 9 9 ; V e rb e le n e t a l., 2 0 0 6 ).
Table III-1. Continuous biotechnology processes using ICT: main
characteristics
Advantages
Disadvantages
High cell concentration in bioreactor
By-products formation
Higher reaction rates
Low permeation to substrates
Biocatalytic regeneragion activity
Higher nutrients and energy requirement
Utilization of high dilution rates without washout

Improve product consistency
Increased cell activity during storage
Reusability of the biocatalyst
Risk of product contamination by cells

leaked from carriers
Low production due to mass transfer
limitation
Ageing of cells in a long-term
fermentation (months)
Hygienization and contamination
Higher specific product yields

Controlled microenvironment
Maintaining of catalytic activity
Easier downstream processing
III.2.3 Immobilization methods

T h e s e le c tio n o f th e a p p ro p ria te im m o b iliz a tio n m e th o d is n e c e s s a ry
a n d h a s a c ru c ia l ro le in th e fin a l p e rfo rm a n c e o f th e im m o b iliz e d c e lls
s y s te m .
T h e re fo re
it
is
v e ry
im p o rta n t
to
know
th e
d iffe re n t
im m o b iliz a tio n m e th o d s . T h e y c a n b e d iv id e d in to fiv e c a te g o rie s :

c a rrie r b in d in g (e g . C e lls a tta c h e d to a s u rfa c e ); c ro s s b in d in g (e g .:
a g e n ts th a t in d u c e c e ll flo c c u la tio n ); e n tra p m e n t (e g .: C e lls in s id e a
p o ro u s
m a trix );
m e c h a n ic a l
c o n ta in m e n t
b e h in d
b a rrie r
(e g .:
m ic ro e n c a p s u la tio n) a n d c o m b in e d m e th o d s . E a c h m e th o d h a s its
m e rits a n d fa u lts . T h is is w h y th e c h o ic e o f a n a p p ro p ria te m e th o d is s o
im p o rta n t a n d s h o u ld ta k e in to a c c o u n t th e p u rp os e o f im m o b iliz a tio n ,
46
Chapter III
c e lls c h a ra c te ris tic s , ty p e o f re a c tio n a n d re a c to r d e s ig n (B r n y ik e t a l.,

2 0 0 4 ; S a to a n d T o s a , 1 9 9 9 ; V e rb e le n e t a l., 2 0 0 6 ; K o b a y a s h i e t a l.,
1998)
The
c a rrie r-b in d in g
m e th o d
is
based
on
th e
a tta c h m e n t
of
b io c a ta ly s ts to w a te r-in s o lu b le c a rrie r s u rfa c e th ro u g h a c o v a le n t/io n ic

bond,
p h y s ic a l
a d s o rp tio n
and
b io s p e c ific
b in d in g .
The
c o v a le n t-
b in d in g m e th o d is u s u a lly a p p lie d to im m o b iliz e d e n z y m e s , d u e to th e

to x ic ity o f th e a g e n ts n e e d e d fo r fo rm a tio n o f c o v a le n t b o n d s , a n d th e
d iffic u lty in fin d in g th e id e a l c o n d itio n s fo r c e ll im m o b iliz a tio n . T h e c e ll
s u rfa c e h a s s o m e re a c tiv e c o m p o n e n ts (p ro te in s , s a c c h a rid e s ), w h ic h
c a n b e u s e d fo r c o v a le n t b o n d in g b e tw e e n c e lls a n d a m o d ifie d o r
a c tiv a te d c a rrie r. P h y s ic a l a d s o rp tio n is u s u a lly p re fe rre d in IC T fo r th e
p ro d u c tio n o f a lc o h o l b e v e ra g e s o v e r th e u s e o f in d u c e rs th a t m a y
in te rfe re w ith th e p ro c e s s . T h e a d s o rp tio n o c c u rs n a tu ra lly b y th e
a tta c h e m e n t o f c e lls to c a rrie rs a n d it is s e n s itiv e to e n v iro n m e n ta l
fa c to rs s u c h a s : p H , io n ic s tre n g th , c o n c e n tra tio n o f re a c ta n ts a n d
te m p e ra tu re . T h e p h e n o m e n a in v o lv e d a re p rim a rily b a s e d o n V a n d e r
W a a ls fo rc e s , io n ic a n d h y d ro g e n b o n d s , h y d ro p h o b ic in te ra c tio n s
b e tw e e n c e ll s u rfa c e a n d th e c a rrie r (B r n y ik e t a l., 2 0 0 4 ; S a to a n d
T o s a , 1 9 9 9 ; V e rb e le n e t a l., 2 0 0 6 ; K o b a y a s h i e t a l., 1 9 9 8 ).
T h e c ro s s -lin k e d m e th o d u s e s b i- o r m u ltifu n c tio n a l re a g e n ts b u t n o t
c a rrie rs a s th e p re v io u s m e th o d . R e a g e n ts a re u s e d to lin k c e lls w ith
each
o th e r
and
c e ll
p e lle ts
a re
fo rm e d .
Among
th e s e
rea g e n ts ,
g lu ta ra ld e h y d e , to lu e n e d iis o c y a n a te a n d h e x a m e th y le n e d iis o c y a n a te
a re c o m m o n ly u s e d . A p a rtic u la rly c a s e o f c ro s s-lin k e d m a y o c c u rs in
y e a s t c e lls : flo c c u la tio n . Y e a s t flo c c u la tio n is c h a ra c te riz e d b y c e ll-c e ll
a g g re g a tio n . It is a re v e rs ib le a n d c a lc iu m d e p e n d e n t p ro c e s s in w h ic h
c e lls a d h e re to fo rm flo c s /p e lle ts . It in v o lv e s p ro te in s th a t s e le c tiv e ly
b in d m a n n o s e re s id u e s p re s e n t o n th e c e ll w a lls o f y e a s t c e lls (B r n y ik
e t a l., 2 0 0 5 ; K o b a y a s h i e t a l., 1 9 9 8 ; K o u rk o u ta s e t a l., 2 0 0 4 ; V e rb e le n
e t a l., 2 0 0 6 ).
In th e e n tra p m e n t m e th o d , th e c e lls a re p la c e d w ith in a c o n fin e d
m a trix . T h e e n tra p m e n t o f c e lls c a n b e d o n e b y n a tu ra l d iffu s io n o f c e lls
47
Chapter III
in to a p o ro u s m a trix o r th e p o ro u s m a trix is s y n th e s iz e d a ro u n d c e lls

(C a -a lg in a te ,
agar
e tc ).
T h is
is
ve ry
p o p u la r
m e th o d
fo r
c e ll
im m o b iliz a tio n b u t h a s s o m e d is a d v a n ta g e s: (1 ) lo w m a s s tra n s fe r ra te s

a n d (2 ) lo w re n e w a b le c a p a c ity o f c a rrie r (G a m a e t a l., 2 0 0 3 ; S a to a n d
T o s a , 1 9 9 9 ; V e rb e le n e t a l., 2 0 0 6 ).
M e c h a n ic a l c o n ta in m e n t o f c e lls b e h in d a b a rrie r is a c h ie v e d b y
u s in g m ic ro p o ro u s m e m b ra n e filte rs , c e ll e n tra p m e n t in m ic ro c a p s u le ,
o r b y c e ll im m o b iliz a tio n in th e in te ra c tio n s u rfa c e o f tw o im m is c ib le
liq u id s . C o n ta in m e n t o f c e lls u s in g m ic ro p o ro u s m e m b ra n e m ig h t b e
u s e d in c o n tin u o u s p roc e s s e s , b u t m e m b ra n e-c lo g g in g is s u e s c a n
a ris e .
S o m e tim e s d iffe re n t m e c h a n is m s o f c e ll im m o b iliz a tio n in s id e a
re a c to r m a y o c c u r. F o r in s ta n c e , y e a s t c e lls im m o b iliz a tio n c a n o c c u r
b y n a tu ra l a d h e s io n in s id e c a rrie r c a v itie s a n d a t s a m e tim e y e a s t
flo c c u la tio n /s e d im e n ta tio n o f c e lls c a n b e a ls o p re s e n t (B r n y ik e t a l.,
2 0 0 4 ; V e rb e le n e t a l., 2 0 0 6 ). T h e re fo re , it is v e ry im p o rta nt n o t o n ly to
s e le c t b u t a ls o to u n d e rs ta n d a ll im m o b iliz a tio n m e c h a n is m s in v o lv e d in
th e s tu d ie d s y s te m . T h u s , to p e rfo rm a c o n tin u o u s fe rm e n ta tio n w h e re
h ig h c e ll d e n s ity s y s te m is p re s e n t a ll th e s e c o n c e rn s a b o u t c e ll
im m o b iliz a tio n ty p e , te c h n iq u e s a n d m e th o d s s h o u ld b e c o n s id e re d .
III.2.4 Carrier properties and specific characteristics

W h e n im m o b iliz e d c e lls s y s te m s a re a p p lie d is v e ry im p o rta n t to
c h o o s e th e c o rre c t c a rrie r/s u rfa c e w h e re c e lls c a n b e im m o b iliz e d . T h e
c a rrie r s h o u ld b e : c h e a p ; in e rt, i.e ., s h ou ld n o t h a v e a n y in flu e n c e o n
th e p ro c e s s ; re g e n e ra b le i.e ., a b le to b e u s e d s e v e ra l tim e s ; a p p lic a b le
a t la rg e s c a le ; s ta b le (m e c h a n ic a l, th e rm a l, c h e m ic a l a n d b io lo g ic a l);
a n d fo o d g ra d e (o n fo o d in d u s try a p p lic a tio n s ). In a d d itio n , th e c a rrie r
s h o u ld h a v e a b ig s u rfa c e a re a , w ith fu n c tio n a l g ro u p s a llo w in g h ig h
c e lls lo a d s w ith o u t a ffe c tin g c e ll a c tiv ity a n d w ith m in im a l m a s s tra n s fe r
lim ita tio n s (B r n y ik e t a l., 2 0 0 5 ; K o b a y a s h i e t a l., 1 9 9 8 ; K o u rk o u ta s e t
a l., 2 0 0 4 ; V e rb e le n e t a l., 2 0 0 6 ).
48
Chapter III
T h e re a re d iffe re n t c a rrie rs th a t c a n b e us e d fo r im m o b iliz a tio n o f

c e lls s u c h a s : p o ro u s g la s s (V irk a ja rv i e t P o h ja la , 2 0 0 0 ); C a -a lg in a te
(D m n y e t a l., 1 9 9 8 ); P V A p a rtic le s (B e z b ra d ic a e t a l., 2 0 0 7 ); s p e n t
g ra in s (B r n y ik e t a l., 2 0 0 1 ); a n d w o o d c h ip s (V irk a ja rv i e t L in k o , 1 9 9 9 ).
R e c e n tly
c e llu lo s e-b a s e d
c a rrie rs
have
been
used
fo r
c e ll
im m o b iliz a tio n th a n k s to th e ir a d v a n ta g e o u s p ro p e rtie s a s : h ig h c e ll

lo a d in g , h ig h a v a ila b ility a n d th e rm a l s ta b ility (F u jii e t a l., 1 9 9 9 ). In th e
case
of
spent
g ra in s ,
th e
m e c h a n is m
c o n s is ts
of
m e c h a n ic a l re te n tio n in p o re s a n d c a v itie s , h y d ro p h o b ic fo rc e s th a t
le a d to a s ta b le c e ll-c a rrie r a d h e s io n , a n d flo c c u la tio n-lik e c e ll-c e ll
in te ra c tio n s (B r n y ik e t a l., 2 0 0 5 ) a re in v o lv e d a s w e ll.
III.2.5 Spent grains and corncobs: cellulose-based carriers

In th is th e s is o n ly tw o c a rrie rs w e re u s e d : s p e n t g ra in s (S G ) a n d
c o rn c o b s (C c ). In th is p a rtic u la rly c h a p te r w e e v a lu a te th e c a p a c ity o f
C c a n d S G a s a c a rrie r fo r y e a s t c e ll im m o b iliz a tio n .
III.2.5.1
Corncobs
C o rn c o b s a re c e llulo s e -b a s e d m a te ria l th a t m a k e s th e m a s u ita b le

m a te ria l fo r im m o b iliz a tio n . It is c h e a p a n d a v a ila b le in a la rg e a m o u n t.
A c c o rd in g to th e F o o d a n d A g ric u ltu re O rg a n iz a tio n o f th e U n ite d
N a tio n s (F A O , 2 0 0 8 ), th e a n n u a l p ro d u c tio n o f c o rn c o b s w o rld w id e is
about
695x109 kg,
m e a n in g
th a t
fo r
e v e ry
100 kg
of
c o rn
g ra in
a p p ro x im a te ly 1 8 k g o f c o rn c o b s a re p ro d u c e d (T o rre e t a l., 2 0 0 8 ).
III.2.5.2
Spent
Spent grains
g ra in s
re p re s e n ts
a re
about
th e
most
8 5 % (w t./w t.)
p ro d u c e d
of
to ta l
b re w in g
b y-p ro d u c t
b y -p ro d u c ts
g e n e ra te d
and
by
b re w in g in d u s try . O n a v e ra g e S G re p re s e n ts 3 1 % (w t./w t.) o f th e

o rig in a l m a lt w e ig h t, a n d a re p ro d u c e a p p ro x im a te ly 2 0 k g p e r 1 0 0 L o f
b e e r. S G a re a v a ila b le a t lo w o r n o c o s t th ro u g h o u t th e y e a r, b e c a u s e
beer
is
not
seasonal
p ro d u c t
lik e
49
w in e .
So
SG
a re
p ro d u c e d
Chapter III
c o n tin u o u s ly in la rg e q u a n titie s . S G c o n s is t o f th e h u s k p e ric a rp s e e d

c o a t la y e rs th a t c o v e r th e o rig in a l b a rle y g ra in , b e in g ric h in c e llu lo s e
a n d n o n -c e llu lo s ic p o ly s a c c h a rid e s , lig n in , s o m e p ro te in s a n d lip id s .
T h e c h e m ic a l c o m p o s itio n o f S G v a rie s a c c o rd in g to : b a rle y v a rie ty ,
h a rv e s t, m a ltin g a n d m a s h in g c o n d itio n s , re g io n , q u a lity a n d ty p e o f
a d ju n c ts a d d e d . In g e ne ra l, S G a re c o n s id e re d a s a lig n o c e llu lo s e
m a te ria l ric h in p ro te in a n d fib re . (M u s s a tto e t a l., 2 0 0 6 )
W h e n S G a re d is c a rd e d fro m b re w in g in d u s try (a ls o c a lle d b a rle y
s p e n t g ra in s ) th e y h a v e a n 8 0 % (w t./w t.) c o n te n t in w a te r. A s a re s u lt it
h a s a lo w b io lo g ic a l s ta b ility (! 3 0 d a y s s to ra g e ). A m o n g th e s e v e ra l
p ro c e s s e s
to
in c re a s e
th e
s h e lf-life
of
SG,
d ry in g
is
th e
most
s u c c e e d e d o n e . D ry in g n o t o n ly in c re a s e s S G s h e lf-life , b u t a ls o
re d u c e s th e ir w e ig h t a n d c o n s e q u e n tly tra n s p o rta tio n s c o s ts (M u s s a tto
e t a l., 2 0 0 6 ). D u e to S G p ro p e rtie s (p ro te in a n d fib e r c o n te n t), it c a n b e
u s e d fo r s e v e ra l a p p lic a tio n s s u c h a s fo o d in g re d ie n t fo r h u m a n (h ig h
p ro te in flo u r); a n im a l n u tritio n (c a ttle fe ed ); e n e rg y p ro d u c tio n (b io g a s );
c h a rc o a l p ro d u c tio n ; b ric k c o m p o n e n t; p a p e r m a n u fa c tu re ; a d s o rb e n t
(v o la tile o rg a n ic c o m p o u n d s , c a d m iu m , le a d , c h ro m iu m ). S G h a v e a ls o
a p p lic a tio n s in b io te c h n o lo g y p ro c e s s e s a s : s u b s tra te fo r c u ltiv a tio n o f
m ic ro o rg a n is m s
and
enzyme
p ro d u c tio n ;
c a rrie r
in
b re w in g
(p re-
tre a te d ); s o u rc e o f a d d e d-v a lu e p ro d u c ts .
Table III-2. Chemical composition of SG. (from Mussatto et al., 2006)
Component
% (dry weight)
Cellulose
Arabinoxylan
Lignin
Protein
Lipid
Ash
16.8 25.4
21.8 28.4
11.9 27.8
15.2 24.0
0.0 10.6
2.4 4.6
A m o n g a ll d e s c rib e d a p p lic a tio n s , S G c a n b e u s e d a s c a rrie r in

c o n tin u o u s b re w in g p rim a ry fe rm e n ta tio n (B r n y ik , e t a l., 2 0 0 1 ; B r n y ik
e t a l., 2 0 0 2 ). S G o b ta in e d b y B r n y ik e t a l (2 0 0 1 ) a fte r p re -tre a te d w ith
H C l a n d N a O H (a cid /b a s e tre a tm e n t) a re c o n s id e re d to h a v e id e a l
50
Chapter III
c o n d itio n s fo r y e a s t c e lls im m o b iliz a tio n . T h e a d v a n ta g e s o f S G a s

c a rrie r a re : h ig h y e a s t lo a d in g c a p a c ity w ith o u t re q u irin g c h e m ic a l
m o d ific a tio n ,
re g e n e ra b le
fe rm e n ta tio n
c o n d itio n s ,
(u s in g
h ig h ly
NaOH
s o lu tio n ),
a v a ila b le ,
cheap
in e rt
(n o t
under
re q u irin g
s ig n ific a n t tre a tm e n ts ) (B r n y ik e t a l., 2 0 0 1 ). T h e p ro c e s s is v e ry

la b o rio u s a n d h a s a lo w y ie ld (1 0 % fro m ra w d ry S G ), w h ic h is
c o n s id e re d a m ain d is a d v a n ta g e o f p re-tre a te d S G (B r n y ik e t a l.,
2 0 0 1 ). C o n s id e rin g ra w S G w a te r c o n te n t a n d a c id /b a s e m e th o d y ie ld ,
fo r e a c h 1 0 0 k g o f ra w S G le a v in g b re w in g in d u s try o n ly 2 k g o f tre a te d
S G c a n b e o b ta in e d . H o w e v e r it is s u g g e s te d th a t fo r in d u s tria l
a p p lic a tio n s o n ly N a O H tre a tm e n t (b a s e tre a tm e n t) c a n b e a p p lie d to
c le a n th e ra w S G (B r n y ik e t a l., 2 0 0 1 ).
B r n y ik
et
a l.
p h y s ic o c h e m ic a l
h y d ro p h o b ic
(2 0 0 4 )
p e rfo rm e d
p ro p e rtie s .
p a rtic le s ,
w ith
It
an
showed
s u rfa c e
e x te n s iv e
s tu d y
th a t
a re
n e g a tiv e
SG
c h a rg e d .
of
SG
w e tta b le ,
The
same
a u th o rs fo u n d th a t b a s e -tre a te d S G w e re le s s w e tta b le a n d th e n m o re
h y d ro p h o b ic th a n a c id /b a s e d-tre a te d S G (B r n y ik e t a l., 2 0 0 4 ).
III.2.6 Chemical modification of carriers

T h e c h e m ic a l m o d ific a tio n o f th e c a rrie r s u rfa c e c a n in c re a s e c e ll
im m o b iliz a tio n , th ro u g h in te n s ific a tio n o f th e b in d in g b e tw e e n c e ll
s u rfa c e a n d c a rrie r (B r n y ik e t a l., 2 0 0 1 ; F u jii e t a l., 1 9 9 9 ). S e v e ra l
p ro d u c ts c a n b e u s e d to in c re a s e d c e ll im m o b iliz a tio n in s u rfa c e s , a s :
d ie th y la m in o e th y l (D E A E ) (A l-a d h a m i e t a l, 2 0 0 2 ; B r n y ik e t a l., 2 0 0 1 ;
F u jii e t a l., 1 9 9 9 ), 2 -d ie th y la m in o -e th y lc h lo rid e h y d ro c h lo rid e (D E C )
(A n trim a n d H a rris , 1 9 9 1 ), p o ly e th y le n im in e (D S o u z a a n d M e lo , 2 0 0 1 ;
D S o u z a e t a l, 1 9 8 6 ; M e lo e t D S o u z a , 1 9 9 9 ), p o ly (s ty re n e -ra n -s u lfo n ic
a c id )(F u jii e t a l., 1 9 9 9 ), p o ly s ty re n e fib e rs (Y o s h io k a e t S h im a m u tra ,
1 9 8 6 ) a n d p o ly e th y le n e im in e c e llu lo s e (P E IC ) (F u jii e t a l., 1 9 9 9 ). S G a n d
C c a re c e llu lo s e-b a s e d m a te ria ls th a t c a n b e d e riv a tiz e d in o rd e r to
in c re a s e
th e ir
io n
exchange
c a p a c ity
(IE C ),
a ttra c tiv e n e s s o f its s u rfa c e fo r c e ll a d h e s io n .
51
i.e .,
to
in c re a s e
th e
Chapter III
D E C s o lu tio n h a s b e e n s u c c e s s fu lly te s te d b y A n trim a n d H a rris fo r

d e riv a tiz a tio n o f c e llu lo s e (1 9 9 1 ). T h e d e riv a tiz a tio n m e th o d u s e d h a s
b e e n o rig in a lly p ro p o s e d in th e fie ld o f e n z y m e im m o b iliz a tio n . It is a
p ro c e s s fo r in c re a s in g a d s o rp tio n c a p a c ity o f g ra n u la r c e llu lo s ic io n
e x c h a n g e c o m p o s ite s in o rd e r to im p ro v e th e p ro d u c t c o n c e n tra tio n
(A n trim a n d H a rris , 1 9 9 1 ). C o n s id e rin g th a t th e o u te r m e m b ra n e o f
y e a s t c e lls is fu ll o f p ro te in s , it is b e lie v e d th a t th is m e tho d c a n b e
a p p lie d
fo r
c e ll
im m o b iliz a tio n ,
as
o th e r
m e th o d s
im m o b iliz a tio n w e re b e fo re (J u n te r a n d J o u e n n e , 2 0 0 4 ).
52
fo r
enzyme
Chapter III
III.3 Material and Methods

III.3.1 Determination of corncobs chemical composition
A fte r b e in g d rie d a t 4 0 C in a n o v e n fo r 1 2 h , th e C c w e re c u t in to
s m a ll p ie c e s (1 -3 c m in le n g th ), m ille d in a k n iv e s m ill to p a s s th ro u g h
a 0 .4 m m s c re e n fo r th e d e te rm in a tio n o f th e ir c h e m ic a l c o m p o s itio n .
T w o g ra m s o f m ille d C c w e re tre a te d w ith 1 0 m L o f 7 2 % (v o l./v o l.)
H 2 S O 4 u n d e r s tirrin g a t 4 5 C fo r 7 m in . T h e m ix tu re w a s a u to c la v e d fo r
3 0 m in a t 1 .0 5 b a r fo r th e c o m p le te h y d ro ly s is o f o lig o m e rs . A fte r
filtra tio n
th ro u g h
S e p -P a k
C18
c a rtrid g e
(a ro m a tic
compounds
re m o v a l), th e h y d ro ly s a te w a s a n a lyz e d in a M e ta C a rb 6 7 H c o lu m n a t
4 5 C u s in g a S h im a d z u c h ro m a to g ra p h w ith re fra c tiv e-in d e x d e te c to r.
T h e m o b ile p h a s e w a s 0 .0 0 5 m o l/L H 2 S O 4 a t 0 .6 m L /m in flo w ra te .
S u g a r c o n c e n tra tio n s , re p o rte d a s g lu c a n a n d x y la n , w e re d e te rm in e d
fro m c a lib ra tio n c u rv e s o b ta in e d w ith p u re c o m p o u n d s . L ig n in w a s
d e te rm in e d b y g ra v im e tric a n a ly s is (R o c h a , 2 0 0 0 ).
III.3.2 Carrier particles for immobilization

F o r im m o b iliz a tio n e x p e rim e n ts th e C c w e re c u t in to th re e d iffe re n t
s iz e s : (1 ) c y lin d e rs ( 0 .6 c m h ig h ), (2 ) th e s a m e c y lin d e rs w e re c u t in to
! o f c y lin d e rs a n d (3 ) m ille d m a te ria l (d < 0 .0 5 c m ). T h e m a in
c h a ra c te ris tic s o f th e p a rtic le s a re d e p ic te d in th e T a b le III- 3 .
Table III-3. Main size characteristics of the corn cob and SG particles used
for immobilization experiments
Carrier Particle
Diameter / cm
Height / cm
Volume / cm3
Cylinders (Cc1)
2.22 1.8
0.60 0.02
2.34 0.4
1/4 Cylinder (Cc2)
1.13 0.1
0.60 0.02
0.60 0.1
Milled (Cc3)
SG
0.1 and 0.05

0.3 and 0.1
-----
5.24x10-4 and 6.54x10-5

---
53
Chapter III
III.3.3 Derivatization method

T e n g ra m s o f c a rrie r (C c o r S G ) w e re p la c e d in to a n E rle n m e y e r fla s k
w ith 2 8 5 m L o f d e io n iz e d w a te r. T h e s u s p e n s io n w a s s tirre d a n d h e a te d
a t 4 0 C a n d th e n 8 6 g o f N a 2 S O 4 a n d 1 2 g o f N a O H w e re a d d e d .
S u b s e q u e n tly 2 0 g o f D E C s o lu tio n (2 5 , 5 0 , a n d 7 5 % (w t./v o l.)) w e re
a d d e d a t 4 0 C d u rin g tw o h o u rs (2 h ) p e rio d . T h e m ix tu re w a s th e n
s tirre d fo r 3 0 m in a t 4 0 C . It w a s fo llo w e d b y n e w a d d itio n o f 1 0 g o f
N a O H a n d 2 0 g o f D E C s o lu tio n (2 5 , 5 0 , a n d 7 5 % (w t./v o l.)) a t 4 0 C fo r
a tw o h o u rs (2 h ) p e rio d . T h e fin a l m ix tu re w a s s tirre d fo r 3 0 m in a t
6 0 C . T h e m ix tu re w a s c o o le d a n d th e p H a d ju s te d to 6 .5 u s in g
3 0 % (v o l./v o l.) H 2 S O 4 s o lu tio n . T h e d e riv a tiz e d c a rrie r w a s w a s h e d w ith
d is tille d w a te r a n d d rie d a t 6 0 C .
III.3.4 Pre-treatment of carriers

III.3.4.1
SG preparation
T h e h y d ro ly s is o f re s id u a l s ta rc h y e n d o s p e rm a n d e m b ry o o f th e
b a rle y k e rn e l p re s e n t in th e ra w S G w a s d o n e a c c o rd in g to th e m e th o d
p ro p o s e d b y B r n y ik e t a l (2 0 0 1 ). R a w S G (1 0 0 g ) w e re m ix e d in
1 5 0 0 m L o f 0 .3 5 M (3 % (v o l./v o l.)) H C l s o lu tio n fo r 2 .5 h a t 6 0 C . T h e
m ix tu re w a s c o o le d a n d w a s h e d w ith w a te r. T h e re m a in in g s o lid s
(m a in ly th e h u s k s o f th e b a rle y g ra in ), w e re p a rtia lly d e lig n ifi e d b y
s h a k in g (1 2 0 rp m ) in 5 0 0 m L o f 0 .5 M N a O H a t 3 0 C fo r 2 4 h . A fte r
b e in g w a s h e d s e v e ra l tim e s w ith w a te r u n til n e u tra l p H a n d d rie d , th e
c a rrie r ( 1 0 g D R Y
BASE
) w a s re a d y to b e u s e d . T h e p re p a ra tio n p ro c e d u re
g iv e s 1 0 % (w t./w t.) y ie ld fro m d ry S G . T h e d ry in g s te p s a p p lie d in th e

p re p a ra tio n p ro ce d u re w e re n e c e s s a ry o n ly in o rd e r to q u a n tify th e
y ie ld s (B r n y ik e t a l., 2 0 0 1 ).
III.3.4.2
Cc pre-treatment A
D ry C c (2 0 g ) w e re a d d e d to 3 0 0 m L o f 1 % (v /v ) o f H C l fo r 1 h a t
6 0 C . T h e n th e C c w e re w a s h e d w ith d is tille d w a te r a n d w a s a d d e d
54
Chapter III
1 % (w /v ) o f N a O H . T h e m ix tu re w a s s tirre d fo r 5 h a t 1 2 0 rp m . T h e
c a rrie r w a s th e n c le a n e d a g a in w ith d is tille d w a te r u n til n e u tra l p H a n d
d rie d
at
40 C.
T h is
p re -tre a tm e n t
was
p e rfo rm e d
to
th re e
Cc
c o n fo rm a tio n s (s e e T a b le III- 3 ). T h is m e th o d p ro v id e s a y ie ld o f 8 0 % ,
5 0 % a n d 2 0 % o f C c 1 , C c 2 , a n d C c 3 p a rtic le s re s p e c tiv e ly .
III.3.4.3
Cc pre-treatment B
D ry C c (2 0 g ) w e re a d d e d to 2 0 0 0 m L o f d is tille d w a te r a n d a u to c la v e
fo r 1 5 m in a t 1 2 1 C (1 .0 5 b a r). A fte r c o o lin g th e c a rrie r w a s w a s h e d
w ith d is tille d w a te r a n d a d d e d to 3 0 0 m L o f 1 % (w t./v o l.) o f N a O H
(0 .2 5 M ) fo r 1 h a t 1 2 0 rp m . T h e n C c w e re c le a n e d w ith d is tille d w a te r
u n til
n e u tra l
pH.
T h is
p re-tre a tm e n t
was
p e rfo rm e d
to
th re e
Cc
c o n fo rm a tio n s (s e e T a b le III- 3 ). T h is m e th o d p ro v id e s a y ie ld o f 8 0 % ,
5 0 % a n d 2 0 % o f C c 1 , C c 2 , a n d C c 3 p a rtic le s re s p e c tiv e ly .
III.3.5 Conditions tested

T a b le III- 4 p re s e n ts a ll th e d iffe re n t c o n d itio n s m a d e to s e le c t th e
b e s t c a rrie r fo r y e a s t c e lls im m o b iliz a tio n .
Table III-4. All conditions (Derivatizations/Pretreatments) used for adhesion
tests
Method/Carrier
Cc1
Cc2
Cc3
SG*
0% (wt./vol.) DEC
!!
25% (wt./vol.) DEC
50% (wt./vol.) DEC
!!
75% (wt./vol.) DEC
Cc Pre-Treatment: A
!!
!!
!!
Cc Pre-Treatment: B
!!
!!
!!
Cc Method: A + 50% (wt./vol.) DEC
!!
!!
!!
Cc Method: B + 50% (wt./vol.) DEC
!!
!!
!!
! - Adhesion test only.

!! - Adhesion test and Immobilization test with yeast growth.
* All SG used were pre-treated accordingly Brnyik et al. (2001).
55
Chapter III
III.3.6 Medium and microorganisms

Y e a s t c e lls u s e d in th is c h a p te r w e re S a c c h a ro m y c e s c e re v is ia e
C C M I 8 9 0 (C u ltu re c o lle c tio n o f in d u s tria l m ic ro o rg a n is m o f IN E T I,
L is b o n ,
P o rtu g a l).
The
m e d iu m
c o m p o s itio n
used
th ro u g h o u t
th is
c h a p te r is p re s e n te d in T a b le III- 5 .
Table III-5. Medium Composition
Component
C o n c e n tra tio n / (g /L )
G lu c o s e
20
Y e a s t E x tra c t
M g S O 4 .7 H 2 O ; 5
0 .4
KH2PO4
NH4SO4
III.3.7 Adsorption tests

G ro w th m e d iu m w a s in o c u la te d a n d in c u b a te d fo r 1 2 h a t 3 0 C a n d
1 5 0 rp m . T h e m e d iu m w a s th e n c e n trifu g e d a n d th e s u p e rn a ta n t w a s
d is c a rd e d . T h e p e lle t w a s re-s u s p e n d e d in 2 0 0 m L o f d is tille d w a te r
a n d th e o p tic a l d e n s ity (O .D .) w a s m e a s u re d a t 6 0 0 n m (O .D . 0 ) u s in g a
Tecan
S u n ris e
96
w e ll
M ic ro p la te
Reader
(T e c a n
G ro u p
L td .,
M n n e d o rf, S w itz e rla n d ). T h e c e llu la r s u s p e n s io n w a s a d d e d to 3 g o f

c a rrie r a n d s a m p le s o f c e ll s u s p e n s io n w e re ta k e n e v e ry 5 m in (O .D . i )
u n til O .D . s ta b iliz a tio n M e th o d A (F ig u r e III- 1 ). A fte r O .D . s ta b iliz a tio n
th e c a rrie r w a s g e n tly s e p a ra te d fro m s o lu tio n , w a s h e d a n d th e a m o u n t
o f a tta c h e d b io m a s s w a s d e te rm in e d b y d ry w e ig h t m e th o d p ro p o s e d b y
B r n y ik e t a l (2 0 0 2 b ) M e th o d B . T h e a m o u n t o f im m o b iliz e d b io m a s s
(iB io in m g B I O /g D C ) b y m e th o d A w a s d e te rm in e d fro m E q . III- 1 :
iBio =
W Free Bio (i) ! W Free Bio (0)
W h e re W F r e e
E q . III- 1
WCarrier (Dry Base )

Bio
(i)
is
th e
w e ig h t o f fre e
b io m a s s
d e te rm in e d fro m O .D . v s C B i o m a s s (g /L ) c a lib ra tio n c u rv e .
56
a t tim e = 0 ,..,I
Chapter III
Figure III-1. Evolution of optical density of free cells during immobilization

time, obtained for Cc2 particles derivatized with 50% (w/w) DEC solution.
III.3.8 Immobilization with yeast growth test

A n E rle n m e y e r fla s k c o n ta in in g 1 8 0 m L m e d iu m a n d 1 .5 g o f c a rrie r
was
in o c u la te d
s u s p e n s io n .
A fte r
w ith
12 h
20 mL
of
of
p re v io u s ly
in o c u la tio n
tim e ,
p re p a re d
O .D .
at
yeast
c e lls
600 nm
was
d e te rm in e d . T h e m e d iu m w a s re m o v e d b y filtra tio n (W h a tm a n n r 1 ,
M a id s to n e , U K ) a n d th e b io c a ta ly s t (c a rrie r + im m o b iliz e d c e lls ) w a s
c o lle c te d . T h e b io c a ta ly s t w a s th e n w a s h e d w ith 4 0 0 m L o f w a te r a n d
filte re d . A fte r filtra tio n th e b io c a ta ly s t w a s p a lc e d in a n E rle n m e y e r
fla s k w ith 1 0 0 m L o f m e d iu m a n d s tirre d s tro n g ly (6 0 0 rp m ). S a m p le s o f
O .D . a re ta k e n u n til s ta b iliz a tio n M e th o d C . A fte r s ta b iliz a tio n , th e
liq u id w a s re m o v e d a n d th e c a rrie r wa s h e d . F in a lly , th e c a rrie r w a s
d rie d (4 0 C ) a n d w e ig h te d to c a lc u la te c a rrie r lo s s e s .
57
Chapter III
III.4 Results and Discussion

III.4.1 General considerations
In th is s e c tio n th e re s u lts fro m th e p e rfo rm e d te s ts (T a b le III- 4 ) a re
p re s e n te d a n d d is c u s s ed . In o rd e r to a c h ie v e th e m a in a im o f c a rrie r
s e le c tio n s o m e c o n s id e ra tio n s w e re ta k e n in to a c c o u n t a n d a re th e
b a s e o f fu tu re d is c u s s io n . T h e s e c o n s id e ra tio n s in c lu d e th e m a in
c h a ra c te ris tic s th a t a c a rrie r p a rtic le s h o u ld h a v e to b e s u c c e s s fu lly
a p p lie d in a c o n tin u o u s p rim a ry fe rm e n ta tio n o f A F B . B o th S G a n d C c
a re c e llu lo s e-b a s e d , c h e a p , a n d a v a ila b le in la rg e s c a le . T h e n e e d to
p re -tre a te d S G p a rtic le s is c o n s id e re d to b e a d ra w b a c k in h e re n t to th is
c a rrie r.
C h e m ic a l m o d ific a tio n o f c e llu lo s e-b a s e d fib re s h a s b e e n d e v e lo p e d
to in c re a s e in itia l y e a s t c e lls a d h e s io n to th e c a rrie rs . In o u r w o rk w e
u s e d th e m e th o d p ro p o s e d b y A n trim a n d H a rris (1 9 9 1 ) . In itia lly th e
m in im u m
c o n c e n tra tio n
of
DEC
s o lu tio n
th a t
in c re a s e s
c e ll
im m o b iliz a tio n w a s d e te rm in e d (T a b le III- 4 ) in n o n -tre a te d C c c a rrie r

p a rtic le s .
A fte r th is , C c p re-tre a tm e n ts w e re p e rfo rm e d (s e e s e c tio n s 0 a n d
III.4 .5 ). T h e m a in o b je c tiv e o f u s in g th e s e tw o p re-tre a tm e n ts w a s to
in c re a s e b o th th e c e llu lo s e c o n te n t a n d p o ro s ity (c a v itie s ) to a c h ie v e a
m o re e ffic ie n t in itia l y e a s t c e lls a d s o rp tio n . T h e p re-tre a tm e n t A is a
m o d ific a tio n o f S G s p re -tre a tm e n t. In itia lly th e h e m ic e llu lo s e a n d
s ta rtc h a tta c h e d to C c p a rtic le s w a s re m o v e d w ith H C l fo llo w e d b y a
d e lig n ific a tio n w ith N a O H . T h e c o n d itio n s a p p lie d to S G c o u ld n o t b e
u s e d in C c b e c a u s e th e y w e re to o a g g re s s iv e a n d d e s tro y e d a ll C c
p a rtic le s . O n th e o th e r h a n d , th e p re -tre a tm e n t B is b a s e d in a u to h y d ro ly s is
and
d e lig n ific a tio n
(N a O H )
m e th o d s
to
h y d ro ly z e d
h e m ic e llu lo s e s (R u iz e t a l., 2 0 1 1 ). U s u a lly th e c o n d itio n s u s e d in a u toh y d ro ly s is a n d d e lig n ifica tio n m e th o d s a re m o re a g g re s s iv e (h ig h e r

te m p e ra tu re s )
th a n
th e
c o n d itio n s
58
used
in
th is
w o rk .
The
a u to -
Chapter III
h y d ro ly s is m a in a im is to p ro d u c e a s u b s tra te to b e u s e d in b o th
s a c c h a rific a tio n p ro c e s s e s a n d c e llu la s e p ro d u c tio n (R u iz e t a l., 2 0 1 1 ).
In o u r c a s e , w e w a n te d o n ly to u s e it fo r y e a s t c e lls im m o b iliz a tio n s o
th e o b je c tiv e w a s to d o a le s s e x te n s iv e a n d le s s c o s tly p re-tre a tm e n t
to in c re a s e th e im m o b iliz a tio n c a p a c ity w ith o u t re d u c in g s ig n ific a n tly
th e s h a p e a n d m e c h a n ic a l re s is ta n c e o f C c pa rtic le s . T h e re fo re le s s
a g g re s s iv e c o n d itio n s th a n th o s e n o rm a lly a p p lie d in a u to-h y d ro ly s is
m e th o d s w e re c h o s e n .
F in a lly , in th e a d h e s io n te s ts , th e m e th o d u s e d to m e a s u re c e ll
a d h e s io n w a s th e re d u c tio n o f O .D . (a t 6 0 0 n m ) o f fre e c e lls in d is tille d
w a te r d u rin g tim e M e th o d A (F ig u r e III- 1 a n d E q . III- 1 ). T h is m e th o d
is b a s e d o n th e a s s u m p tio n th a t a ll c e lls th a t a re n o t fre e a re a d s o rb e d
o n th e c a rrie r. T h is is s u p p o rte d b y th e s h o rt tim e o f th is k in d o f te s ts
(m a x im u m o f 3 0 m in ), w h ic h a v o id s th e in flu e n c e s o f c e llu la r g ro w th
a n d d e a th ra te s . T h e m e th o d p ro p o s e d b y B r n y ik e t a l (2 0 0 4 ) M e th o d
B w a s n o t u s e d in th is c a s e b e c a u s e th e e rro r p re s e n t in C c a n d S G
p a rtic le s d u e to c a rrie r lo s s e s (a ro u n d 1 0 % ) w a s to o b ig c o n s id erin g
th e lo w a m o u n t o f y e a s t c e lls th a t a re in itia lly a tta c h e d (B r n y ik e t a l.,
2 0 0 4 ). F o r th e s a m e re a s o n , w h e n im m o b iliz a tio n te s ts w ith y e a s t c e lls
g ro w in g w e re m a d e th e m e th o d u s e d w a s a m e c h a nic a l m e th o d in itia lly
p ro p o s e d b y B r n y ik e t a l (2 0 0 1 ) M e th o d C .
III.4.2 Corncob composition

B e fo re
te s ts ,
Cc
c h e m ic a l
c h a ra c te riz a tio n
was
p e rfo rm e d . T h e c o m p o s itio n o f C c p a rtic le s is g iv e n in T a b le III- 5 .

T h e re s u lts a re g iv e n a s th e c o n te n t o f g lu c a n (c o rre s p o n d in g to
c e llu lo s e ),
x y la n
and
a c e ty l
g ro u p s
(b o th
c o rre s p o n d in g
to
h e m ic e llu lo s e s ), a n d s o lu b le lig n in a n d K la s o n lig n in (c o rre s p o n d in g to

to ta l lig n in ). T h e s e d a ta is in go o d a g re e m e n t w ith o th e r re s u lts
re p o rte d e a rlie r (G a rro te e t a l., 2 0 0 2 ).
59
Chapter III
Table III-5. Chemical composition of the Cc particles

Compounds
Corn Cobs ( % )
Cellulose
Glucan
Hemicelluloses
Xylan
Acetyl Group
Total Lignin
Soluble Lignin
Klason Lignin
Total weight
31.8 0.8
31.8 0.8
33.6 1.1
29.4 0.2
3.1 0.4
27.9 0.4
11.3 0.2
16.6 0.4
92.2 0.9
III.4.3 Selection of best DEC concentration

T h e d e riv a tiz a tio n m e th o d p ro p o s e d b y A n trim a n d H a rris (1 9 9 9 ) w a s
u s e d in o rd e r to in c re a s e th e C c a n d S G a d s o rp tio n c a p a c itie s b y
in c re a s in g th e ir io n e x c h a n g e c a p a c ity (IE C ). In F ig u r e III- 2 A th e v a lu e s
o f IE C o b ta in e d fo r th e th re e d iffe re n t s h a p e s o f C c a n d S G a t d iffe re n t
c o n c e n tra tio n s o f th e d e riv a tiz a tio n a g e n t (D E C ) a re s h o w n .
F ig u r e III- 2 A s h o w s th a t, g e n e ra lly , th e d e riv a tiz a tio n in c re a s e d
s lig h tly th e IE C o f C c p a rtic le s . It a ls o s h o w s th a t th e re is a s iz e e ffe c t
o f th e C c p a rtic le s o n IE C , b e in g th is e ffe c t h ig h e r fo r s m a lle r p a rtic le s
(C c 3 a n d S G ). T h is re s u lt w a s e x p e c te d in th e s e p a rtic le s d u e to th e ir
h ig h e r s u p e rfic ia l a re a . T h e h ig h e s t IE C w a s o b ta in e d fo r S G p a rtic le s .
T o o b ta in a m o re e ffe c tiv e d e riv a tiz a tio n , i.e ., to a c h ie v e a g o o d IE C b y
d e riv a tiz a tio n
of
c e llu lo s e-b a s e d
c a rrie rs ,
th e
c e llu lo s e
must
be
a v a ila b le . In th e c a s e o f C c , c e llu lo s e re p re s e n ts c a 3 0 % o f its

c o m p o s itio n
(T a b le
III- 5 )
w h ile
in
p re-tre a te d
SG
it
re p re s e n ts
9 0 % (w t./w t.) (B r n y ik e t a l., 2 0 0 4 ). B e in g s o , is n o t s tra n g e th a t fo r S G

th e in c re a s e o f IE C w a s m o re e ffe c tiv e th a n fo r C c . F o r c o n c e n tra tio n s
a b o v e 5 0 % o f D E C s o lu tio n s , th e IE C in C c d id n o t in c re a s e a s m u c h a s
e x p e c te d . T h is m ig h t b e d u e to th e c e llu lo s e a m o u n t in C c , w h ic h is
lim ite d . S o th e h ig h e r D E C c o n c e n tra tio n h a s n o e ffe c t o n th e d e g re e o f
d e riv a tiz a tio n o f C c p a rtic le s .
60
Chapter III
IEC /
(meq/
gDC)!
A!
2.0!
1.5!
1.0!
0.5!
0.0!
0!
25!
50!
75!
% DEC solution (wt./vol.)!
iBio. /
(mgBIO/
gDC)! 50!
40!
30!
20!
10!
0!
B!
0!
25!
50!
75!
Figure III-2. A: Ion exchange capacities (IEC) of different carrier particles at

different concentrations of DEC solution. B: Influence of derivatization on the
immobilized biomass (iBio) for different carriers. Legend: ! - Cc1; ! - Cc2;
! - Cc3; ! - SG.
B r n y ik
et
a l.
(2 0 0 1 )
used
SG
as
c a rrie r
fo r
fe rm e n ta tio n in b u b b le c o lu m n s a n d o b ta in e d v a lu e s o f IE C b e tw e e n
0 .4 5 a n d 0 .8 m e q /g D C . C c 1 a n d C c 2 p a rtic le s s h o w e d lo w e r IE C w h e n
c o m p a re d w ith S G , w h ic h is a n in d ic a tio n th a t th e s e p a rtic le s h a v e
lo w e r a d s o rp tio n c a p a c ity th a n S G a n d C c 3 . U p to 5 0 % (w t./v o l.) o f
D E C s o lu tio n th e v a lu e s o f IE C fo r C c 3 (IE C : 5 0 % D E C = 0 .7 3 m e q /g D C )
a n d S G (IE C : 5 0 % D E C = 0 .7 9 m e q /g D C ) a re s im ila r to th o s e re p o rte d in
lite ra tu re . F o r S G th e m a x im u m IE C (1 .8 m e q /g D C ) w a s o b ta in e d w h e n
7 5 % (w t./v o l.) w e re u s e d . In o rd e r to a n a ly z e c e llu lo s e-b a s e d c a rrie rs
w ith s im ila r IE C a n d s iz e s , a n d b a s e d in th e re s u lts p re s e n te d in F ig u re
III- 2 A , th e c o n c e n tra tio n o f 5 0 % (w t./v o l.) o f D E C s o lu tio n w a s s e le c te d
a s th e b e s t c o m p ro m is e .
In F ig u r e III- 2 B it is p o s s ib le to v e rify th a t fo r C c 2 , C c 3 a n d S G th e
d e riv a tiz a tio n in c re a s e d th e c a rrie rs lo a d c a p a c ity fo r c o n c e n tra tio n o f
D E C s o lu tio n s a b o v e 5 0 % (w t./v o l.). O n th e o th e r h a n d fo r C c 1 p a rtic le s
61
Chapter III
th e re w a s n o re a l diffe re n c e o f im m o b iliz a tio n fo r th e n o n d e riv a tiz e d

and
d e riv a tiz e d
c a rrie r.
The
lo w
v a lu e s
of
yeast
c e lls
a d h e s io n
(iB io M A X = 2 0 m g B I O /g D C ) in th e s e p a rtic le s c a n b e a ls o e x p la in e d b y th e ir
s iz e . T h e y a re b ig g e r a n d th e a re a in c o n ta c t w ith th e c e ll s u s p e n sio n is
lo w e r th e fo re le s s im m o b iliz a tio n is e x p e c te d to o c c u r.
T h e v a lu e s o f im m o b iliz a tio n , ta k in g in a c c o u n t th e te c h n iq u e u s e d
in th is w o rk , a re c o n s id e re d g o o d . It m u s t b e p o in te d o u t th a t th is
m e th o d is u s e d o n ly fo r a d s o rp tio n o f y e a s t c e lls a n d d o es n o t a llo w
th e c o m p le te fo rm a tio n o f y e a s t b io film d u e to th e a b s e n c e o f c e ll
g ro w th .
T h e re s u lts o b ta in e d w h e n th e a d s o rp tio n tim e is e v a lu a te d a ls o h e lp
s u s ta in in g th is s ta te m e n t. T h e a d s o rp tio n tim e is th e tim e th a t is
n e e d e d to a c h ie v e th e e q u ilib riu m b e tw e e n c e lls a d s o rp tio n /d e s o rp tio n
in e a c h a s s a y . T h e tim e o f a d s o rp tio n is d e te rm in e d w h e n th e re s u lts o f
O .D . a re s ta b iliz e d , i.e ., w h e n th e v a lu e s o f O .D . o f fre e c e lls a re le s s
th a n 1 0 % o f th e O .D . fin a l v a lu e (s e e F ig u re III- 1 ). T h e a d s o rp tio n tim e
is a q u a lita tiv e d a ta , w h ic h c a n b e u s e d to e v a lu a te th e tim e o f
a d s o rp tio n in a re a l fe rm e n ta tio n . T a b le III- 6 p re s e n ts th e a d s o rp tio n
tim e s fo r e a c h a s s a y .
In
g e n e ra l,
th e
a d s o rtio n
tim e
in c re a s e s
w ith
th e
in c re a s e d
c o n c e n tra tio n o f D E C s o lu tio n d u e to h ig h lo a d c a p a c ity o f d e riv a tiz e d

p a rtic le s , w h ic h n e e d m o re tim e to a c h ie v e th e e q u ilib riu m b e tw e e n
a d s o rp tio n a n d d e s o rp tio n .
Table III-6. Immobilization time (min) for different types of carrier.
% (wt./vol.)
DEC solution
0
25
50
75
Immobilization time (min)

SG
10
20
25
25
Cc1
15
20
30
20
Cc2
15
20
10
15
Cc3
15
15
20
25
F ro m a ll th e s e re s u lts (F ig u r e III- 2 a n d T a b le III- 6 ) it is p o s s ib le to

d e te rm in e th a t 5 0 % (w t./v o l.) o f D E C s o lu tio n is th e b e s t c o n c e n tra tio n
to p e rfo rm C c a n d S G d e riv a tiz a tio n a c c o rd in g to th e p ro p o s e d m e th o d .
62
Chapter III
T h is c o n c e n tra tio n is s e le c te d b e c a u s e w e c a n a c h ie v e g o o d IE C a n d
b io m a s s a d s o rp tio n lo a d w ith o u t in c re a s in g to o m u c h th e a d s o rp tio n
tim e .
III.4.4 The effect of Cc pre-treatment in carrier adsortion capacity

H a v in g in m in d th a t b e tte r re s u lts w e re o b ta in e d w ith S G in th e
p re v io u s s e c tio n , a n d c o n s id e rin g th a t d e riv a tiz a tio n h a d m o re e ffe c t o n
th is c a rrie r d u e to th e h ig h e r c e llu lo s e c o n c e n tra tio n , tw o d iffe re n t p retre a tm e n ts w e re te s te d o n C c p a rtic le s (w ith a n d w ith o u t d e riv a tiz a tio n
u s in g
DEC
s o lu tio n
of
5 0 % (w t./v o l.)).
The
re s u lts
of
IE C
and
a d s o rb e d b io m a s s a re p re s e n te d in F ig u r e III- 3 a n d F ig u r e III- 4 ,
IEC /
(meq/
gDC)!
1.5!
A!
1.0!
0.5!
0.0!
0%!
50%!

B!
IEC /
(meq/
gDC)! 1.2!
1.0!
0.8!
0.6!
0.4!
0.2!
0.0!
0%!
50%!
Figure III-3. Ion Exchange Capacity for: A Cc pre-treatment A; B Cc pretreatment B. Legend: ! - Cc1; ! - Cc2; ! - Cc3; ! - SG.
F ig u r e III- 3 in d ic a te s th a t IE C in c re a s e d o n ly fo r th e p re-tre a tm e n t B
in C c 3 p a rtic le s . T h is s u g g e s ts th a t th e p re-tre a tm e n t d id n o t in c re a s e
63
s ig n ific a n tly
th e
Chapter III
c e llu lo s e
ra tio
in
Cc
p a rtic le s ,
as
e x p e c ted .
C o n s e q u e n tly th e v a lu e o f IE C d id n o t in c re a s e .
F ig u r e III- 4 c le a rly s h o w s th a t th e c e ll a d s o rp tio n w a s h ig h e r fo r C c
p re -tre a te d w ith H C l a n d N a O H . N e v e rth e le s s th e re s u lts w e re s im ila r to
th e o n e s o b ta in e d fo r C c p a rtic le s w ith o u t a n y p re -tre a tm e n t (F ig u r e
III- 2 ). P re -tre a tm e n t o f c a rrie r p a rtic le s a t in d u s tria l s c a le is o n ly
ju s tifie d
if
th e
im p a c t
on
a d s o rp tio n
(a n d
c o n s e q u e n tly
on
im m o b iliz a tio n ) is h ig h . T h e re s u lts s u g g e s te d th a t a p re-tre a tm e n t in

C c p a rtic le s is n o t ju s tifie d , b e c a u s e n o re a l im p ro v e m e n t o f th e
a d s o rp tio n p ro p e rtie s o f th e c a rrie r w a s o b s e rv e d .
iBio. /
(mgBIO/
gDC)!
A!
50!
40!
30!
20!
10!
0!
0%!
50%!

iBio. /
(mgBIO/
gDC)!
B!
50!
40!
30!
20!
10!
0!
0%!
50%!

Figure III-4.Yeas cells adsorption for: A Cc pre-treatment A; B Cc pretreatment B. Legend: ! - Cc1; ! - Cc2; ! - Cc3; ! - SG.
III.4.5 Effect of yeast cell growth on Cc immobilization capacity

T h e p re v io u s te s ts in d ic a te th a t D E C s o lu tio n c a n p la y a n im p o rta n t
ro le o n a d s o rp tio n c a p a c ity o f c e llu lo s e-b a s e d c a rrie rs . H o w e v e r it
d o e s n o t p ro v id e fu th e r in fo rm a tio n o n h o w it c a n re a lly in c re a s e
64
Chapter III
C e ll
in v o lv e s
in itia l
a d s o rp tio n
and
b io film fo rm a tio n in th e c a rrie r. It is k n o w n th a t y e a s t c e lls , w h e n

im m o b iliz e d , s u ffe r c h a n g e s in p h y s io lo g y a n d th e ir g ro w th ra te c a n b e
a ffe c te d w h e n c o m p a re d w ith fre e c e lls g ro w th (J u n te r e t a l, 2 0 0 2 ). In
o rd e r to c o n firm th is , im m o b iliz a tio n te s ts d u rin g y e a s t c e lls g ro w th
w e re p e rfo rm e d . In F ig u r e III- 5 th e re s u lts o f y e a s t im m o b iliz a tio n
d u rin g y e a s t g ro w th a re p re s e n te d fo r th e s itu a tio n s in d ic a te d in T a b le
III- 4 . A s e x p la in e d b e fo re , th e m e th o d u s e d in th is s itu a tio n w a s a b it
d iffe re n t d u e to th e s ig n ific a n t p a rtic le s d e g ra d a tio n c a u s e d b y N a O H
M e th o d C .
In F ig u r e III- 5 th e re s u lts s u g g e s t th a t n o im m o b iliz a tio n o c c u rs in
c a rrie r p a rtic le s S G a n d C c 3 . F o r o th e r C c p a rtic le s th e re a re c e lls
a tta c h e d , a n d y e a s t c e ll im m o b iliz a tio n o n to S G is d e s c rib e d e ith e r in
b u b b le c o lu m n s o r a ir-lift re a c to rs (B r n y ik e t a l., 2 0 0 1 ; B r n y ik e t a l.,
2 0 0 2 b ). T h e e x p la n a tio n fo r lo w im m o b iliz a tio n re lie s o n th e m e th o d
u s e d . In te rm s o f s iz e , C c 3 a n d S G a re s im ila r. S o it is e x p e c te d th e y
h a v e s im ila r b e h a v io u r u n d e r s im ila r im m o b iliz a tio n c o n d itio n s . T h e la c k
o f im m o b iliz e d c e lls is d u e to th e te n s io n fo rc e s p re s e n t d u rin g y e a s t
c e lls g ro w th . T h e h ig h liq u id v e lo c ity a n d tu rb u le n c e inc re a s e s s h e a r
fo rc e s in th e liq u id -p h a s e a n d th is in flu e n c e s b io film fo rm a tio n . T h u s
th e
is
re d u c e d
and
s im ila r
a d s o rp tio n
re s u lts
a re
o b ta in e d . S e v e ra l w o rk s re la tin g th e s e tw o fa c to rs h a v e b e e n re p o rte d
a n d th e re la tio n b e tw e e n h ig h s h e a r s tre s s fo rc e s a n d b io film fo rm a tio n
in d ic a te s th e a d v e rs e e ffe c t o f s h e a r o n im m o b iliz e d b io m a s s (v a n
L o o s d re c h t e t a l., 2 0 0 2 ). A s p a rtic le s C c 1 a n d C c 2 a re b ig g e r, it is
p o s s ib ly th a t th e y h a v e a h ig h e r n u m b e r o f b ig g e r c a v itie s fo r c e lls to
c o lo n iz e a n d in th e s e reg io n s th e s h e a r s tre s s e ffe c t is re d u c e d . T h u s
th e y e a s t b io film h a s b e tte r c o n d itio n s to g ro w th a n d fo rm a b io film .
A n a ly s in g o n ly th e s e tw o la s t C c p a rtic le s (C c 1 a n d C c 2 ), fro m
F ig u re III- 5 . it c a n b e c o n c lu d e d th a t p re-tre a tm e n t A s h o w e d to b e
s lig h tly b e tte r fo r y e a s t im m o b iliz a tio n a n d b io film fo rm a tio n . It h a s
b e e n d e s c rib e d th a t a c id /b a s e p re-tre a tm e n ts a re q u ite e ffe c tiv e in
re d u c in g th e h e m ic e llu lo s e a n d lig n in fro m lig n o c e llu lo s ic c o m p o u n d s .
65
Chapter III
T h is w ill a llo w m o re c e llu lo se to b e p re s e n t e ith e r fo r e n z y m a tic

d e g ra d a tio n o f c e llu lo s e (Z h a n g a n d L y n d , 2 0 0 4 ) o r c e ll im m o b iliz a tio n .
T h e p re -tre a tm e n t A is b a s e d o n ty p ic a lly a c id /b a s e p re-tre a tm e n ts .
C o n s id e rin g th a t in p re -tre a tm e n t B th e c o n d itio n s u s e d a re le s s
a g g re s s iv e th a n u s u a l (R u iz e t a l., 2 0 1 1 ), it is e x p e c te d th a t a fte r
a p p ly in g
th e
p re-tre a tm e n t
to
Cc,
m o re
c e llu lo s e
is
a v a ila b le .
T h e re fo re a h ig h e r im m o b iliz a tio n is e x p e c te d fo r th is p re-tre a te m n t,

w h ic h is c o n firm e d b y th e re s u lts in F ig u re III- 5 . In b o th p re -tre a tm e n ts
th e im m o b iliz a tio n re s u lts in d e riv a tiz e d p a rtic le s (C c 1 a n d C c 2 ) w e re
h ig h e r. H o w e v e r fro m F ig u r e III- 5 o n ly fo r C c 1 th e d e riv a tiz a tio n w a s
s h o w n to h a v e a n in flu e n c e o n y e a s t im m o b iliz a tio n b e in g m o re th a n th e
d o u b le fo r b o th p re-tre a tm e n ts (A a n d B ).
A!
iBio. /
(mgBIO/
gDC)! 80!
60!
40!
20!
0!
0!
50!
B!
iBio. /
(mgBIO/
gDC)! 80!
60!
40!
20!
0!
0!
50!
Figure III-5. Immobilized biomass (iBio) expressed in amount of biomass per

gram of dry Carrier (mgBIO/gDC) versus DEC solution concentration for: A Cc
pre-treatment A; B Cc pre-treatment B. Legend: ! - Cc1; ! - Cc2; ! Cc3; ! - SG.
T h e d e riv a tiz a tio n p ro c e s s is la b o rio u s a n d c o s tly if a p p lie d a t

in d u s tria l s c a le . T h e s e re s u lts in d ic a te th a t d e riv a tiz e d C c p a rtic le s d id
n o t in c re a s e s ig n ific a n tly b io film g ro w th , a s e x p e c te d . S im ila r re s u lts
66
Chapter III
w e re o b ta in e d b y B r n y ik e t a l (2 0 0 1 ) u s in g D E A E m o d ifie d S G in
b u b b le
c o lu m n s .
The
re s u lts
suggest
th a t
th e
in itia l
a d h e s io n
im p ro v e m e n t o b ta in e d p re v io u s ly in th e d e riv a tiz e d c a rrie rs , h a s n o re a l

im p a c t o n b io film fo rm a tio n .
T h e re s u lts o b ta in e d fo r C c a n d S G p a rtic le s d e riv a tiz e d w ith D E C
s o lu tio n a s w e ll a s th e re s u lts o b ta in e d b y B r n y ik a n d c o-w o rk e rs
(2 0 0 1 ), in d ic a te th a t d e riv a tiz a tio n o f c e llu lo s e -b a s e d c a rrie rs w ith
th e s e c o m p o u n d s (D E A E a n d D E C ) d o e s n o t h a v e a p o s itiv e im p a c t in
y e a s t c e lls im m o b iliz a tio n .
III.5 Conclusions
In th is c h a p te r th e c a p a c ity o f tw o c e llu lo s e-b a s e d c a rrie rs (C c
a n d S G ) fo r c e ll im m o b iliz a tio n w a s e v a lu a te d a n d th e influ e n c e o f
s e v e ra l fa c to rs w a s s tu d ie d .
It w a s c o n c lu d e d th a t n o n p re -tre a te d C c p a rtic le s a n d S G
d e riv a tiz e d b y th e p ro p o s e d m e th o d h a v e b e tte r a d s o rp tio n p ro p e rtie s ;
th e c o n c e n tra tio n o f 5 0 % (w t./v o l.) in D E C w a s th e b e s t c o m p ro m is e
b e tw e e n IE C , a d s o rp tio n tim e a n d c e ll a d s o rp tio n . A m o n g a ll te s te d
p a rtic le s th e s m a lle r (S G a n d C c 3 ) w e re c o n s id e re d to b e th e b e s t. T h e
p re -tre a tm e n t m e th o d s u s e d in C c p a rtic le s d id n o t in c re a s e th e
a d s o rp tio n p ro p e rtie s o f C c .
It
was
p ro v e d
th a t
s iz e
e ffe c t
c o u ld
p la y
ke y
ro le
in
im m o b iliz a tio n w h e re b ig g e r C c p a rtic le s s h o w e d b e tte r re s u lts fo r

im m o b iliz a tio n . H o w e v e r th e p a rtic le s iz e s h o u ld d e p e n d o n re a c to r
ty p e a n d c o n fig u ra tio n . It is s u g g e s te d th a t b ig g e r C c p a rtic le s c a n b e
u s e d in p a c k e d b e d-lik e re a c to rs , w h ile C c 3 a n d S G p a rtic le s c a n b e
u s e d in g a s lift-lik e re a c to rs , m a in ly d u e to m in im u m flu id iz a tio n o f g a s
n e c e s s a ry to o b ta in a h o m o g e n e o u s s o lid flo w in s id e th is re a c to r.
T h e m a in c o n c lu s io n is th a t th e d e riv a tiz e d c a rrie rs s h o w e d
b e tte r a d s o rp tio n p ro p e rties , b u t th a t d id n o t m e a n b e tte r fin a l c e ll
im m o b iliz a tio n (b io film fo rm a tio n ) p ro p e rtie s . B e in g s o , C c p a rtic le s a n d
67
Chapter III
S G s h o u ld b e a p p lie d in c o n tin u o u s fe rm e n ta tio n w ith o u t a n y a d d itio n a l

tre a tm e n t, a s th e y a lre a d y h a v e a g o o d c e ll im m o b iliz a tio n c a p a c ity .
III.6 References
Al-Adhami, A. J. H., Bryjak, J., Greb-markiewicz, B., and Peczyn, W. (2002).
Immobilization of wood-rotting fungi laccases on modifed cellulose and acrylic
carriers. Process Biochemistry, 37, 1387-1394.
Antrim, R. L., and Harris, D. W. (1991). Treatment of cellulosic ion exchange
composites in hot aqueous medium to increase adsortion of macromolecules. US
Patent nr. 5001063.
Bezbradica, D., Obradovic, B., Leskosek-Cukalovic, I., Bugarski, B., and Nedovic, V.
Brnyik, T., Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2001). Spent
grainsa new support for brewing yeast immobilisation. Biotechnology Letters,
23(13), 10731078.
Brnyik, Toms, Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2002a).
Continuous primary beer fermentation with brewing yeast immobilized on spent
grains. Journal of the Institute of Brewing, 108(4), 410-415.
Brnyik, Toms, Vicente, A. A., Oliveira, R., and Teixeira, J. A. (2004a).
Physicochemical
surface
properties
of
brewing
yeast
influencing
their
immobilization onto spent grains in a continuous reactor. Biotechnology and
bioengineering, 88(1), 84-93.
Brnyik, Toms, Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2004b).
Continuous primary fermentation of beer with yeast immobilized on spent grains The effect of operational conditions. Journal of The American Society of Brewing
Chemists, 62(1), 29-34.
Brnyik, Toms, Vicente, A. A., Dostlek, P., and Teixeira, J. A. (2005). Continuous
beer fermentation using immobilized yeast cell bioreactor systems. Biotechnology
progress, 21(3), 653-663.
Dmny, Z., Smogrovicov, D., Gemeiner, P., Sturdk, E., Ptkov, J., and
DSouza, S. F., and Melo, J. S. (2001). Immobilization of bakers yeast on jute fabric
through adhesion using polyethylenimine: application in an annular column reactor
for the inversion of sucrose. Process Biochemistry, 36, 677-681.
DSouza, S. F., Melo, J. S., Deshpande, A., and Nadkarni, G. B. (1986).
Immobilization of yeast cells by adhesion to glass surface using Plyethylenimine.
Biotechnology Letters, 8(9), 643-648.
FAO, F. and A. O. of the U. N. (2008). FAOSTAT statistics database. Retrieved July
8 , 2008, from: http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567
Fujii, N., Sakurai, A., Onjoh, K., and Sakakibara, M. (1999). Influence of surface
characteristics of cellulose carriers on ethanol production by immobilized yeast
cells. Process Biochemistry, 34(2), 147152.
Gama, F. M., Aires-Barros, M. R., and Cabral, J. (2003). Engenharia Enzimatica (in
portuguese) (p. 272). Lidel Edies Tcnicas, Lisboa.
Junter, G. A., Coquet, L., Vilain, S., and Jouenne, T. (2002). Immobilized-cell
physiology: current data and the potentialities of proteomics. Enzyme and
Microbial Technology, 31(3), 201212.
Junter, G.-A., and Jouenne, T. (2004). Immobilized viable microbial cells: from the
process to the proteome em leader or the cart before the horse. Biotechnology
Advances, 22(8), 633-658.
Kang, S., and Choi, H. (2005). Effect of surface hydrophobicity on the adhesion of S.
cerevisiae onto modified surfaces by poly(styrene-ran-sulfonic acid) random
copolymers. Colloids and surfaces. B, Biointerfaces, 46(2), 70-77.
t h
68
Chapter III
Kobayashi, O., Hayashi, N., Kuroki, R., and Sone, H. (1998). Region of FLO1 Proteins
Responsible for Sugar Recognition. Journal of Bacteriology, 180(24), 6503-6510.
Kourkoutas, Y., Bekatorou, A., Banat, I. M., Marchant, R., and Koutinas, A. A.
(2004). Immobilization technologies and support materials suitable in alcohol
beverages production: a review. Food Microbiology, 21(4), 377397.
Melo, J. S., and DSouza, S. F. (1999). Simultaneous filtration and immobilization of
cells from a flowing suspension using a bioreactor containing polyethylenimine
coated cotton threads: Application in the continuous inversion of concentrated
sucrose syrups. World Journal of Microbiology and Biotechnology, 15, 17-21.
Mussatto, S., Dragone, G., and Roberto, I. (2006). Brewers spent grain: generation,
characteristics and potential applications. Journal of Cereal Science, 43(1), 114.
Rocha, G.J.M. (2000). PhD. Thesis, So Carlos/Universidade de So Paulo, Brazil
Ruiz, H., Ruzene, D. S., Silva, D. P., da Silva, F. F. M., Vicente, A. A, and Teixeira,
J. A. (2011). Development and characterization of an environmentally friendly
process sequence (autohydrolysis and organosolv) for wheat straw delignification.
Applied biochemistry and biotechnology, 164(5), 629-641.
Ruzene, D. S., Silva, D. P., Vicente, A. a, Teixeira, J. a, de Amorim, M. T. P., and
Gonalves, A. R. (2009). Cellulosic films obtained from the treatment of sugarcane
bagasse fibers with N-methylmorpholine-N-oxide (NMMO). Applied biochemistry
and biotechnology, 154(1-3), 38-47.
Sato, T., and Tosa, T. (1999). Enzymes, Immobilization methods. In M. C. Flickinger
and S. W. Drew (Eds.), Encyclopedia of Bioprocess Technology: Fermentation,
Biocatalysis and Bioseparation (1st ed., pp. 1062-1064). New York: John Wiley
and Sons, Inc.
Torre, P., Aliakbarian, B., Rivas, B., Dominguez, J. M., and Converti, A. (2008).
Release of ferulic acid from corn cobs by alkaline hydrolysis. Biochemical
Engineering Journal, 40(3), 500-506.
immobilized yeast a review on flavour formation and control strategies. Jounal
Yoshioka, T., and Shimamutra, M. (1986). Studies of Plystyrene-based ion-exchange
fiber. V. Immobilization of microorgnism cells by adsorption on a novel fiber-form
anion exchanger. Bull. Chem. Soc. Jpn, 59, 77-81.
Zhang, Y. H. P., and Lynd, L. R. (2004). Toward an aggregated understanding of
enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnology
and bioengineering, 88(7), 797-824.
69
Chapter IV
IV. Chapter IV Effect of Spent Grains on

Flow Regime Transition in a Bubble
Column
IV.1 Objectives
IV.2 Introduction
IV.3 Material and Methods
IV.4 Results and Discussion
IV.5 Conclusions
IV.6 References
70
Chapter IV
IV.1 Objectives
T h e a im o f th is w o rk is to c o n trib u te to th e s tu d y o f th e e ffe c t o f S G
o n th e flo w re g im e tra n s itio n in a b u b b le c o lu m n . S G a re s o lid s w ith
u n iq u e c h a ra c te ris tic s -fla t, c e llu lo s e -b a s e d , c o m p le te ly w e tta b le , lo w
s iz e a n d lo w -d e n s ity p a rtic le s (B r n y ik e t a l., 2 0 0 1 ) a n d th e re is a n
a b s e n c e o f s tu d ie s o n th e in flu e n c e o f th is ty p e o f p a rtic le s in th re ep h a s e b u b b le c o lu m n s . T h is w a s th e d riv in g fo rc e to p e rfo rm th is w o rk .
C o n s id e rin g
th e
p re v io u s
im p o rta n c e
of
SG
fo r
c o n tin u o u s
AFB
p ro d u c tio n in a n iG L R th e s tu d y o f th e in flu e n c e o f th e s o lid s -p h a s e in

b u b b le c o lu m n s re g im e flo w tra n s itio n w ill a llo w : (1 ) m im ic th e ris e r
c o n d itio n s ; (2 ) u n d e rs ta n d th e p h y s ic a l m e c h a n is m b y w h ic h S G m a y , o r
m a y n o t in flu e n c e th e iG L R h y d ro d y n a m ic s .
O th e r o b je c tiv e s a ris e a s th e d e te rm in a tio n o f s p e c ific S G p ro p e rtie s
a n d th e d e v e lo p m e n t o f a c a lib ra tio n m e th o d to d e te rm in e g a s h o ld-u p
in th re e -p h a s e iG L R .
IV.2 Introduction
O v e r th e la s t d e c a d e s h y d ro d y n a m ic s o f g a s-liq u id -s o lid (g -l-s )
s y s te m s h a s b e e n in te n s iv e ly s tu d y d u e to th e ir a p p lic a tio n s in s e v e ral
in d u s tria l fie ld s s u c h a s p e tro c h e m ic a l, c h e m ic a l, b io c h e m ic a l a n d
b io te c h n o lo g y p ro c e s s e s . T h e s tu d y o f th e h y d ro d y n a m ic s o n th re ep h a s e s y s te m s p re s e n ts a c h a lle n g e to s e v e ra l re s e a rc h c o m m u n itie s ,
w h ic h
a re
d e a lin g
w ith
b u b b le
c o lu m n s ,
a irlift
re a c to rs ,
flo ta tio n
c o lu m n s , b u b b ly flo w a n d flu id iz e d b e d s . M o s t o f th e re c e n t w o rk
d e a lin g w ith b u b b le c o lu m n s (B C ) is fo c u s e d o n th e s ta b ility o f its flo w
re g im e (M e n a e t a l., 2 0 0 5 a ; R u z ic k a , e t a l.,2 0 0 1 a , 2 0 0 3 ; Z a h ra d n ik e t
a l., 1 9 9 7 )
71
Chapter IV
IV.2.1 Bubble columns: flow regime transition

In B C tw o m a in flo w re g im e s o c c u r: th e h o m o g e n e o u s re g im e a n d th e
h e te ro g e n e o u s re g im e , th a t m a y b e id e n tifie d b y v a ry in g g a s in p u t
(R u z ic k a e t a l., 2 0 0 1 a ; Z a h ra d n ik e t a l., 1 9 9 7 ). T h e h o m o g e n e o u s
re g im e (H o R ) is c h a ra c te riz e d b y a u n ifo rm b u b b le ris e th ro u g h th e
c o lu m n . B u b b le s u s u a lly h a v e s im ila r s iz e , a re s p h e ric a l, s m a ll a n d ris e
a lm o s t v e rtic a l. T h e re is a n y la rg e-s c a le liq u id c irc u la tio n a n d o th e r
p h e n o m e n a a s c o a le s c e n c e a n d b re a k-u p a re n e g lig ib le (R u z ic k a , e t a l.,
2 0 0 1 a ). In c o n tra s t, th e h e te ro g e n e o u s re g im e (H e R ) is c h a ra c te riz e d b y
a
la rg e
b u b b le
s iz e
d is trib u tio n .
M a c ro-s c a le
liq u id
c irc u la tio n ,
c o a le s c e n c e a n d a p a ra b o lic /n o n-u n ifo rm ra d ia l p ro file o f h o ld -u p w ith

a m a x im u m a t c e n tre a re ty p ic a l o n th is re g im e (M e n a e t a l., 2 0 0 5 a ).
T h e tra n s itio n s ta rts w h e n th e H o R lo s e s its s ta b ility a n d g ra d u a l
p ro c e s s o c c u rs w h e re th e re a re a n in c re a s in g n u m b e r o f c o h e re n t
s tru c tu re s (c irc u la tio n s ) w ith in c re a s in g s iz e a n d in te n s ity (M e n a e t a l.,
2 0 0 5 a ; R u z ic k a e t a l., 2 0 0 1a ; Z a h ra d n ik e t a l., 1 9 9 7 ).
Due
to
th e ir
c h a ra c te ris tic s ,
HoR
and
HeR
have
d iffe re n t
h y d ro d y n a m ic b e h a v io u r. T h is re su lts in d iffe re n t m a s s , h e a t, a n d
m o m e n tu m tra n s fe r p ro p e rtie s . C o n s e q u e n tly , it is im p o rta n t to c la rify
h o w o p e ra tin g p a ra m e te rs (re a c to r g e o m e try , g a s a n d liq u id flo w ra te s ,
p ro p e rtie s o f th e c o n ta c tin g p h a s e s ) a c t o n flo w re g im e p ro p e rtie s a n d
tra n s itio n (Z a h ra d n ik e t a l., 1 9 9 7 ).
P a ra m e te rs s u c h a s s u p e rfic ia l g a s v e lo c ity , c o lu m n d ia m e te r, liq u id
a n d g a s p h a s e p ro p e rtie s a n d d is trib u to r g e o m e try a c t s im u lta n e o u s ly
o n re g im e tra n s itio n (S h a ik h a n d A l-D a h h a n , 2 0 0 7 ). T h e s e le c tio n o f th e
c o rre c t
d is trib u to r
is
re q u ire d
to
s tu d y
re g im e
tra n s itio n ,
b e in g
p e rfo ra te d o r p o ro u s p la te s th e m o s t c o m m o n ly a p p lie d a t la b o ra to ry
a n d in d u s tria l s c a le (Z a h ra d n ik e t a l., 1 9 9 7 ). Z a h ra d n ik e t a l. (1 9 9 7 )
d e m o n s tra te d th a t p e rfo ra te d p la te s w ith h o le s in fe rio r to 1 m m a n d
p o ro u s p la te s a re a d e q u a te to c h a ra c te riz e re g im e tra n s itio n . V ija y a n e t
a l (2 0 0 7 ) s tu d ie d th e in flu e n c e o f s p a rg e r g e om e try in th e re g im e
tra n s itio n , e v a lu a tin g th e in flu e n c e o f th e ra tio b e tw e e n a re a o f s p a rg e r
72
Chapter IV
a n d a re a o f c o lu m n c ro s s s e c tio n . T h e y fo u n d th a t if th is ra tio is
in c re a s e d th e c ritic a l a n d g lo b a l v a lu e s o f g a s h o ld-u p a ls o in c re a s e
(V ija y a n e t a l., 2 0 0 7 ).
G e n e ra lly , re g im e e v o lu tio n is o b s e rv e d b y th e in c re a s in g o f g a s flo w
ra te in p u t a n d b y d e te rm in e d th e c o rre s p o n d e n t g a s h o ld-u p . T h e re a re
s e v e ra l te c h n iq u e s to d e te rm in e lo c a l o r g lo b a l g a s h o ld-u p s u c h a s :
b e d e x p a n s io n (M e n a e t a l., 2 0 0 5 a ; R u z ic k a e t a l., 2 0 0 1a , 2 0 0 3 ;
Z a h ra d n ik e t a l., 1 9 9 7 ), p re s s u re d ro p , d y n a m ic g a s d is e n g a g e m e nt
(S c h u m p e a n d G ru n d , 1 9 8 6 ; Y a n g e t a l., 2 0 1 0 ), c o n d u c tiv ity (W a rs ito e t
a l., 1 9 9 7 ) a n d o p tic a l fib e rs (C a rte llie r, 1 9 9 0 ). T h e a n a ly s is o f th e g a s
h o ld -u p v e rs u s s u p e rfic ia l g a s v e lo c ity (F ig u re IV - 1 ) s h o w s th a t H o R
a p p e a rs a s a c o n v e x lin e w h ile H e R fo llo w s a ra tio n a l fu n c tio n (c o n c a v e
lin e ). T h e s e lin e s a re c o n n e c te d b y a tra n s itio n z o n e fo r in te rm e d ia te
g a sM.C.
v eRuzicka
lo c ity
v a lu eEngineering
s (R uJournal
z ic k96a (2003)
e t 1522
a l., 2 0 0 1 a ).
et al. / Chemical
16
Nomenclature
a
c
d
D
e
g
H
j
k1, k2
q
R
t
u
u0
v
We
gas holdup e [-]
bubble drift coefficient ()

Tr
He
Ho
parameter of Eq. (8) ()
bubble diameter (m)
column diameter (m)
c
gas holdup (porosity, voidage, volumetric
bubble concentration) ()
gravity (m/s2 )
column height (clear liquid height,
unaerated liquid level) (m)
drift-flux (m/s)
parameters of Eq. (8) ()
gas flow rate (linear gas velocity, specific
gas flow, superficial gas velocity) (m/s)
0
qs
qc
qhe
correlation coefficient ()
gas flow rate q [m/s]
bubble retention time (s)
Fig. 1. Definition sketch of flow regimes in bubble column. Full line:
mean gas phase rise velocity (bubble swarm
i g u r e(m/s)
I V - 1 . D e f i n i t i Hohomogeneous
o n o f f l o w regime;
r e g iTrtransition
m e t r a nregime;
s i t i oHeheterogeneous
n in BC and identification of
velocity, bubble slip F
velocity)
plate
f l o(single
w r e bubble
g i m e s a n d c r regime;
i t i c aqls stable
value
s . operation
L e g eregime,
n d : beginning
H o ofH homogeneous
omogeneous regime; Tr
terminal bubble velocity
regime; qc critical point, end of homogeneous regime, beginning of
T r a nvelocity)
s i t i o n(m/s)
r e g i m e ; H eregime
Htransition;
e t e r oqghe end
e n e of
o uregime
s r etransition,
g i m ebeginning
; q S ofBheteroeginning of Ho regime;
velocity, isolated bubble
gas velocity insideqorifice
(m/s)
r i t i c a l p o i n t ( E n dgeneous
of H
o r eec critical
g i m e )voidage.
; q H eBroken
Eline:
n d pure
o f heterogeneous
Transition regime. Adapted
regime;
C C
regime. f r o m R u z i c k e t a l . ( 2 0 0 3 ) .
orifice Weber number ()
Greek letters
There are numerous results scattered in literature about
liquid kinematic viscosity

(m2 /s) Identification
the effect
of the liquid
on the transition
gas holdup in the hetIV.2.1.1
of the
flowviscosity
regime
hydrodynamic bubble diffusivity (m2 /s)

erogeneous regime. Generally, it is reported that the holdup
liquid dynamic viscosity (mPa s)

decreases with increasing viscosity. This is attributed to the
R e g im e tra n s itio n presence
id e n tific
n is ofpbig
o sand
s ibfast
lebubbles
b y awith
p p ly in g th e d rift-flu x
density (kg/m3 )
of largea tio
population
interfacial surface tension

short retention time in the bed [1,2,4,6,7]. Not only the visc o n c(N/m)
e p t in itia lly p rocous
p omedia
s e d arebfavourable
y W a llis
(1 9 6 of
9 ).big bubbles
In thdiis m e th o d , th e d rift
(liquid surface tension)
for formation
relative free plate area ()

rectly at the gas distributor [1,2,8], but also they promote
flu x , j G L (th e v o lu m e tric
x of e
ith e r and
p hsuppress
a s e re
la tiv
e to a s u rfa c e m o v in g
bubbleflu
coalescence
[2,4,810]
bubble
breakup
Subscripts
in the bed [9,11]. The decrease of holdup is reflected by
a t th e v o lu m e tric a vvarious
e ragcorrelations
e v e lo ccontaining
ity ) is thep lo
tte d a g a in s t th e g a s h o ld-u p .
c
critical value (end of homogeneous regime,
viscosity effect. They
n
beginning of regime
form
different
of n:th e tra n s itio n fro m
T htransition)
e c h a n g e in th e are
s lousually
p e ofo fthe th
e e curvwith
e re
p re values
s e n ts
g
gas
0.053 and 0.16 [2], 0.05 [1], 0.22 and others [12].
l
liquid
On the other hand, there are studies reporting controvers
stable plate operation regime (beginning of
sial effects of the viscosity [1,2,11,13]. Indeed, both increase
homogeneous regime)
and decrease of the heterogeneous gas holdup have been ob73 was found at low viscosity
served [8,12,1417]. An increase
< 3, a decrease at moderate viscosity = 311, and a
the circulations progressively enhance the bubble velocity
roughly constant holdup at higher viscosity > 11 [14]. To
(e.g. u = u0 + const. q), namely in the central core of the
reconcile this ambiguity, the viscosity was supposed to play
Chapter IV
h o m o g e n e o u s to h e te ro g e n e o u s flo w . T h is c o n c e p t h a s b e e n u s e d a n d
m o d ifie d b y s e v e ra l a u th o rs fo r d e te rm in in g re g im e tra n s itio n (K ris h n a ,
e t a l., 1 9 9 9 ; M e n a e t a l., 2 0 0 5 a ; R u z ic k a , e t a l., 2 0 0 1a , 2 0 0 1 b ; V ia l e t
a l., 2 0 0 0 ) S e v e ra l m o d e ls fo r re g im e tra n s itio n h a v e b e e n p ro p o s e d
b a s e d o n : (1 ) b u b b le d ra g fo rc e , (2 ) g a s p h a s e s lip v e lo c ity , (3 ) e n e rg y
b a la n c e o f th e tw o flo w , (4 ) b u b b le s iz e , (5 ) c o u p lin g b e tw e e n p h a s e s
(G h a ra t e t J o s h i, 1 9 9 2 ; H y n d m a n e t a l., 1 9 9 7 ; J o s h i e t L a li, 1 9 8 4 ;
K ris h n a , 1 9 9 1 ; R iq u a rts , 1 9 7 9 ; R u z ic k a e t a l., 2 0 0 1a ). T h e m o d e l
p ro p o s e d b y R u z ic k a e t a l. (2 0 0 1a ) b a s e d o n th e c o n c e p t o f th e
D a rw in ia n d rift o f b u b b le s w a s a b le to d e s c rib e w ith g o o d a c c u ra c y th e
tra n s itio n b e tw e e n H o R a n d H e R in a tw o-p h a s e s y s te m . H o w e v e r s o m e
fa c to rs th a t a ffe c t re g im e s ta b ility a s c o lu m n d im e n s io n s , liq u id p ha s e
p ro p e rtie s a n d
s o lid s
p re s e n c e
a re
n o t e x p lic itly
in v o lv e d
in
th e
p ro p o s e d m o d e l a n d e x p e rim e n ts h a v e b e e n d o n e o v e r th e la s t y e a rs to
v a lid a te p a rtic u la r a s p e c ts o f th e s ta b ility c rite ria (M e n a e t a l., 2 0 0 5 a ;
R u z ic k a e t a l., 2 0 0 1 a , 2 0 0 8 ).
IV.2.2 Solids effect on three-phase bubble column

In th re e -p h a s e B C re a c to r, th e e ffe c t o f s o lid s o n g a s h o ld -u p h a s
b e e n th e fo c u s o f s e v e ra l s tu d ie s (B a n is i e t a l., 1 9 9 5 ; G a n d h i e t a l.,
1999; Mena
e t a l., 2 0 0 5a , 2 0 5 5 b , 2 0 0 8 ). T h e s e
s y s te m s
can
be
c la s s ifie d a s liq u id-g a s flo w w ith th e p re s e n c e o f s o lid s o r a s liq u id

s o lid
flu id iz e d
beds
w ith
th e
p re s e n c e
of
gas
b u b b le s .
However
c o m p a ris o n b e tw e e n th e d iffe re n t s tu d ie s is d iffic u lt a n d re s u lts a re

o fte n c o n tra d ic to ry , d u e to d iffe re n c e s in c o lu m n d e s ig n , o p e ra tin g
p a ra m e te rs (m a in ly liq u id th ro u g h p u t) a n d s o lid s p ro p e rtie s . P ro p e rtie s
o f th e s o lid p a rtic le s c a n b e q u ite d iffe re n t d e p e n d in g o n th e siz e ,
s h a p e , d e n s ity , w e tta b ility , h y d ro p h o b ic a n d s u rfa c e p ro p e rtie s . H a v in g
th is in m in d , th e ir e ffe c t in g a s h o ld-u p a n d flo w re g im e tra n s itio n is fa r
fro m b e in g to ta lly e x p la in e d d e s p ite s e v e ra l a tte m p ts . B a n is i e t a l.
(1 9 9 5 ) re p o rte d th a t th e p re s e n c e o f s o lid s d e c re a s e d g a s h o ld-u p
w h ile a d u a l e ffe c t w a s o b s e rv e d b y o th e r a u th o rs (M e n a , e t a l., 2 0 0 5a ;
X ie e t a l., 2 0 0 3 ). T h e p re s e n c e o f s o lid s o n b u b b le c o lu m n s a ffe c ts th e
74
Chapter IV
g a s -liq u id m ix tu re in s e v e ra l w a y s : b u b b le fo rm a tio n (Y o o e t a l., 1 9 9 7 ),

b u b b le ris e , a x ia l a n d ra d ia l p ro file s (G a n d h i e t a l., 1 9 9 9; W a rs ito e t a l.,
1 9 9 7 ), m ix in g a n d d is p e rs io n , m a s s tra n s fe r (M e n a e t a l., 2 0 0 5 b ), g a s
h o ld -u p a n d flo w re g im e s (M e n a e t a l., 2 0 0 5 a ).
G e n e ra lly g a s h o ld -u p d e c re a s e s w ith s o lid s c o n c e n tra tio n . T h e re a re
s e v e ra l p o s s ib le e x p la n a tio n s fo r th is k in d o f e ffe c t s u c h a s in cre a s e d
c o a le s c e n c e (G a n d h i e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ) a n d re d u c tio n o f
b u b b le b re a k u p (G a n d h i e t a l., 1 9 9 9 ), in c re a s e d a p p a re n t v is c o s ity ,
s te ric e ffe c t.
In w h a t c o n c e rn s th e e ffe c t o f a p p a re n t v is c o s ity , s o m e a u th o rs
c o n s id e r th e s o lid a n d liq u id p h a s e a s a p s e u d o-h o m o g e n e o u s p h a s e ,
th is re q u irin g th e n e e d to d e fin e a n a p p a re n t v is c o s ity d u e to s o lid
p re s e n c e in liq u id (F re ita s e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ). In a s im ila r w a y
to w h a t h a s b e e n re p o rte d fo r tw o -p h a s e flo w s w h e re a h ig h e r v is c o s ity
d e c re a s e s g a s h o ld -u p , w h ile a t lo w v is c o s itie s th e o p p o s ite o c c u rs
(E is s a a n d S c h u g e rl, 1 9 7 5 ; R u z ic k a e t a l., 2 0 0 3 ), a n id e n tic a l b e h a v io u r
m ig h t b e e x p e c te d fo r th e e ffe c t o f s o lid s c o n c e n tra tio n o n g a s-h o ld u p . N e v e rth e le s s , re c e n t s tu d ie s in re g im e tra n s itio n s tu d y in g th e e ffe c t
o f liq u id v is c o s ity re p o rte d th a t e v e n a t lo w v is c o s ity v a lu e s th e g lo b a l
h o ld -u p d e c re a s e s (Y a n g e t a l., 2 0 1 0 ). In g e n e ra l, a n in c re a s e o f
v is c o s ity is re la te d w ith a d e c re a s e o n g a s h o ld-u p . W h e n s o lid s a re
p re s e n t, s o m e a u th o rs c o n s id e r th e s o lid a n d liq u id p h a s e a s a
p s e u d o -h o m o g e n e o u s p h a s e . T h is le a d s to th e n e e d o f d e fin in g a n
a p p a re n t v is c o s ity d u e to th e s o lid p re s e n c e in th e liq u id th a t w o u ld
a ffe c t liq u id p h a s e p ro p e rtie s a s d e n s ity a n d v is c o s ity (F re ita s e t a l.,
1 9 9 9 ; L u e t a l., 1 9 9 5 ). A s fa r a s w e a re a w a re s o m e re la tio n s to
d e te rm in e d v is c o s ity a re re p o rte d in lite ra tu re b e in g th e m o s t c o m m o n
th e o n e s b y O liv e r e t a l. (1 9 6 1 ) (c ite d b y L u e t a l. (1 9 9 5 )); T h o m as
(1 9 6 5 ) (c ite d b y Y o o e t a l. 1 9 9 7 ); B a rn e a a n d M iz ra h i (1 9 7 3 ) (c ite d b y
G a n d h i e t a l., (1 9 9 9 )) a n d M e tz n e r (1 9 8 5 ) (c ite d b y Y o o e t a l., (1 9 9 7 )).
T h e c h a n g e in v is c o s ity p ro m o te d b y s o lid s in th e liq u id-p h a s e
re v e a ls th a t p o s s ib le re la tio n s /in te ra c tio n s be tw e e n s o lid s a n d v is c o s ity
a re lik e ly to o c c u r o n th e ir e ffe c t o n flo w re g im e d e s ta b iliz a tio n o f g -l-s
75
Chapter IV
s y s te m s . M e n a e t a l. (2 0 0 5 a ) n o tic e d th a t o n ly fo r a h ig h s o lid c o n te n t
th e v is c o s ity h a d a n im p o rta n t c o n trib u tio n fo r th is ; h o w e v e r, th is
c a n n o t b e g e n e ra liz e d a s th e s o lid e ffe c t o n th e a p p a re n t v is c o s ity o f
th e m ix tu re d e p e n d s o n th e p ro p e rtie s o f th e s o lid s a p p lie d . It s e e m s
th a t th e B C d e s ig n a n d s iz e a ls o p la y a n im p o rta n t ro le w h e n a ll th e s e
a s p e c ts a re c o n s id e re d (R u z ic k a , e t a l., 2 0 0 1 b ,; R u z ic k a e t a l., 2 0 0 3 ;
Y a n g e t a l., 2 0 1 0 ).
IV.2.2.1
Solids dual effect on regime transition
T h e d u a l e ffe c t th a t h a s b e e n re p o rte d in th e re c e n t y e a rs le a d to a
d is c u s s io n o f w h a t is th e re a l e ffe c t o f s o lid s in B C . It s e e m s th a t s iz e ,
c o n c e n tra tio n a n d w e tta b ility p la y a n im p o rta n t ro le o n th is e ffe c t.
A c c o rd in g ly to B a n is i e t a l. (1 9 9 5 ) fin e p a rtic le s in s m a ll a m o u n t
(s u p p re s s in g
(p ro m o tin g
c o a le s c e n c e )
b re a k u p )
te n d
and
to
la rg e
in c re a s e
p a rtic le s
in
h o ld-u p ,
h ig h
w h ile
amount
m o d e ra te
c o n c e n tra tio n s a n d s iz e s s e e m to d e c re a s e g a s h o ld-u p . C o n c e rn in g

th e in flu e n c e o f th e w e ta b ility , its re a l e ffe c t o n g a s h o ld-u p re m a in s
u n c le a r.
J a m ia la h m a d i
and
M u lle r-S te in h a g e n
(1 9 9 1 )
re p o rt
th a t
w e tta b le p a rtic le s in c re a s e d h o ld-u p w h ile n o n -w e tta b le p a rtic le s h a v e

th e o p p o s ite e ffe c t. H o w e v e r, M e n a e t a l. (2 0 0 5 a ), w o rk e d w ith a lg in a te
b e a d s (lo w d e n s ity a n d c o m p le te ly w e tta b le s o lid s ) a n d fo u n d th a t a
d u a l e ffe c t is p re s e n t - fo r lo w s o lid c o n te n t (< 3 .0 % ) s o lid s e n h a n c e d
th e H o R re g im e s ta b iliz a tio n a n d g lo b a l h o ld -u p in c re a s e d w h ile fo r
h ig h e r s o lid s c o n te n t (> 3 .0 % ) th e o p p o s ite e ffe c t w a s o b s e rv e d
(B a n is i e t a l., 1 9 9 5 ; J a m ia la h m a d i a n d M u lle r-S te in h a g e n , 1 9 9 1 ; M e n a ,
e t a l., 2 0 0 5 a ).
F ro m a c ritic a l p o in t o f v ie w th e a c tu a l k n o w le d g e o f th e flo w re g im e
tra n s itio n in th re e -p h a s e s y s te m re m a in s s c a rc e . T h e m a in re a s o n is
re la te d n o t o n ly w ith th e c o rre c t d a ta in te rp re ta tio n in te rm s o f p h y s ic a l
m e c h a n is m b u t a ls o w ith th e d iffic u lty o f re la tin g a ll re s u lts re p o rte d in
lite ra tu re
76
Chapter IV
IV.3 Material and Methods

IV.3.1 Apparatus and measurements
M e a s u re m e n ts w e re p e rfo rm e d in a c y lin d ric a l P le x ig la s b u b b le
c o lu m n w ith in te rn a l d ia m e te r o f 0 .1 4 2 m . T h e d is trib u to r w a s a c e ra m ic
p o ro u s p la te w ith 0 .0 9 m o f d ia m e te r a n d a n a p p ro x im a te p o ro s ity o f
3 8 % (v o l.). It e n s u re s th e th re e re g im e s : h o m o g e n e o u s , tra n s itio n a n d
h e te ro g e n e o u s . C o m p re s s e d filte re d a ir w a s th e g a s-p h a s e (T = 2 5 C ,
P = 1 .0 1 b a r) a n d w a te r th e liq u id-p h a s e (T = 2 0 C ). T h e c le a r liq u id
h e ig h t w a s H L 0 = 1 .0 9 m fo r a ll e x p e rim e n ts (n o liq u id th ro u g h p u t). T h e
d e p e n d e n c e o f th e g a s h o ld -u p (! ) o n th e g a s flo w ra te (Q G ) w a s
m e a s u re d th re e tim e s a n d th e n a v e ra g e d . T h e g a s s u p e rfic ia l v e lo c ity
v a rie d in th e ra n g e U G = 0 m /s (0 d m 3 /s ) to U G = 0 .0 2 7 m /s (0 .4 3 d m 3 /s ),
c o v e rin g th e h o m o g e n e o u s a n d p a rt o f th e tra n s itio n re g im e . T h e g a s
flo w w a s m e a s u re d w ith a M a s s F lo w C o n tro lle r (A lic a t S c ie n tific , In c .,
T u c s o n A Z , U S A ) a n d v a ria tio n s in g a s s u p e rfic ia l v e lo c ity c lo s e to th e
tra n s itio n p o in t w e re w ith in 2 m m /s (0 .0 3 3 d m 3 /s ).
IV.3.2 Solid-phase characterization: spent grains

S G , a lm o s t fla t p a rtic le s , w ith eq u iv a le n t d ia m e te r d E Q < 2 .1 m m w e re
th e s o lid p h a s e . T h e s iz e d is trib u tio n o f th e p a rtic le s w a s d e te rm in e d
by
s ie v in g
in to
fra c tio n s
u s in g
p o rta b le
s ie v e
shaker
(M o d e l
A n a ly s e tte , F rits c h , G e rm a n y ).
IV.3.2.1
Preparation of spent grains
S p e n t G ra in s w e re p re p a re d ac c o rd in g ly s e c tio n III.3 .4 .1 w ith o u t th e

a c id h y d ro ly s is s te p (B r n y ik e t a l., 2 0 0 4 ).
77
IV.3.2.2
Chapter IV
Water absorption index (WAI) determination
W A I w a s d e te rm in e d a c c o rd in g ly M u s s a to e t a l (2 0 0 9 ). M a in ly a
s a m p le o f 2 .5 g o f s p e n t g ra in s (d ry b a s e ) w a s s u s p e n d e d in 3 0 m L o f
d is tille d w a te r in a 5 0 m L c e n trifu g e d tu b e . T h e s lu rry w a s s tirre d fo r
o n e m in u te a t ro o m te m p e ra tu re a n d th e n c e n trifu g e a t 3 0 0 0 g a n d
2 5 C
fo r
1 0 m in .
The
s u p e rn a ta n t
was
d is c a rd e d
and
c a lc u la te d fro m w e ig h t re m a in in g g e l a n d e x p re s s a s g W E T
IV.3.2.3
SG
WAI
/g D R Y
SG
was
.
Specific weight of wet spent grains (wSG)
S G a re a c e llu lo s e-b a s e m a te ria l a n c o n s e q u e n tly
is a b le to a b s o rb
w a te r. T h u s s p e c ific w e ig h t d e te rm in a tio n b e c a m e d iffic u lt. B e in g s o

w S G s p e c ific w e ig h t w a s d e te rm in e d b y vo lu m e in c re a s e d m e th o d . A
la rg e a m o u n t o f d ry S G (d S G ) 1 5 g w a s s u s p e n d e d in 3 0 m L o f
d is tille d w a te r a n d c e n trifu g e d (3 0 0 0 g a n d 2 5 C fo r 1 0 m in ). T h e
s u p e rn a ta n t w a s d is c a rd e d a n d th e re m a in in g w S G w e re a d d e d to
5 0 0 m L o f d is tille d w a te r. V o lu m e o f w S G w a s d e te rm in e d b y w a te r
v o lu m e
in c re a s e
and
s p e c ific
w e ig h t
(V w S G /W w S G )
d e te rm in e d .
The
re s u lts w e re p e rfo rm e d in trip lic a te .
IV.3.3 Gas hold-up

G a s h o ld -u p w a s m e a s u re d u s in g tw o te c h n iq u e s d e s c rib e d b e lo w .
IV.3.3.1
Bed expansion
In e a c h e x p e rim e n ta l ru n , th e g a s flo w w a s s e t, th e b e d h e ig h t w a s
re c o rd e d a fte r th e tim e re q u ire d to re a c h a s te a d y v a lu e w a s a c h ie v e d
(n e v e r le s s th a n fiv e m in u te s ). E a c h o f th e e ig h t ru n s w a s re p e a te d
th re e tim e s a n d th e v o id a g e v a lu e s w e re a v e ra g e d . G a s h o ld-u p w a s
d e te rm in e d a c c o rd in g to (D e c k w e r, 1 9 9 2 ; M e n a e t a l., 2 0 0 5 a ; R u z ic k a
e t a l.,2 0 0 a , 2 0 0 3 , 2 0 0 8 ; Y a n g e t a l., 2 0 1 0 ):
!G =
HGL + H L
HL
E q . IV - 1
78
IV.3.3.2
Chapter IV
Water columns differential pressure
T h e g a s h o ld -u p w a s d e te rm in e d b y m e a s u rin g s ta tic p re s s u re
d iffe re n c e b e tw e e n tw o h e ig h ts in th e B C . O u r in te re s t in u s in g th is
te c h n iq u e is re la te d w ith fu tu re te s ts to b e p e rfo rm e d in a iG L R .
M o re o v e r, it is re p o rte d th a t p re s s u re d iffe re n c e p e r s e is n o t e n o u g h
w h e n th re e p h a s e s y s te m s a re a p p lie d (B o y e r e t a l., 2 0 0 2 ) a n d a n
a d d itio n a l te c h n iq u e s h o u ld b e a p p lie d to d e te rm in e s o lid s h o ld-u p .
D iffe re n tia l p re s s u re w a s m e a s u re d b y th e d iffe re n c e in w a te r c o lu m n s .
For
each
set
of
e x p e rim e n ts ,
p re s s u re
d iffe re n c e s
(H 1 -H 2 )
w e re
m e a s u re d a t le a s t th re e tim e s d u rin g 5 m in (g a s s in g tim e b ig g e r th a n

5 m in ). T h e m e a n v a lu e w a s th e n u s e d to d e te rm in e g a s h o ld-u p b y th e
fo llo w in g e q u a tio n (F re ita s a n d T e ix e ira , 1 9 9 8 a , 1 9 9 8 b ):
!G =
H1 " H 2 #L " #S
"
$ !S
d12
#L
E q . IV - 2
IV.3.4 Solids hold-up

S o lid s h o ld -u p a n d d is trib u tio n w e re d e te rm in e d b y u s in g th e m e th o d
d e v e lo p e d b y F re ita s e t a l. (1 9 9 7 ). B rie fly , a s a m p le r a d a p te d to re ta in
s p e n t g ra in s w ith 6 0 m L o f v o lu m e is u s e d fo r c o lle c tin g s o lid s . T h e
s o lid s s a m p le is c o lle c te d b e tw e e n th e tw o p o in ts w h e re th e ! G is
m e a s u re d . T h e s o lid s w e re filtra te d a n d d rie d a t 1 0 5 C fo r 1 2 h . T h e
s o lid s v o lu m e w a s d e te rm in e d (c o n s id e rin g th e ir W A I a n d s p e c ific
w e ig th ) a n d s o lid s h o ld -u p ( ! S ) c a lc u la te d a c c o rd in g ly (F re ita s e t a l.,
1 9 9 7 ):
!S =
VS
Vspl
E q . IV - 3
79
Chapter IV
IV.3.5 Measurements errors

T h e re la tiv e e rro r fo r b e d e x p a n s io n m e th o d is c o n s id e re d to b e le s s
th a n 5 % . O n th e h o m o g e n e o u s a n d in th e b e g in n in g o f tra n s itio n re g im e
th e la y e r is u n ifo rm a n d th e in te rfa c e is e a s y to lo c a te w ith a 1 m m
p re c is io n (p re c is io n o f s c ale m illim e tre p a p e r). T h is re s o lu tio n w a s
c o n s id e re d a d e q u a te d u e to th e h e ig h t o f th e c o lu m n u s e d (1 0 9 0 m m ).
H o w e v e r,
when
tra n s itio n
s ta rts
to
occur
and
waves
appear
th e
d e te rm in a tio n o f th e h e ig h t o f th e B C is d iffic u lt (H G + L ). T o m in im iz e th is
e ffe c t, th e o b ta in e d v a lu e c o rre s p o n d s to th e m e a n o f th e v a lu e s
m e a s u re d d u rin g s e v e ra l o s c illa tio n s . A t th e e n d o f tra n s itio n a n d in th e
b e g in n in g
of
h e te ro g e n e o u s
re g im e
o s c illa tio n s
w e re
at
m a x im u m
3 0 m m a ro u n d th e m e a n v a lu e . H a v in g in m in d th e in c re a s e in h e ig h t
c o lu m n fo r th e s e s u p e rfic ia l g a s v e lo c itie s , it w a s p o s s ib le to h a v e a n
e x p e rim e n ta l e rro r n o t e x c e e d in g 5 % .
T h e re s o lu tio n o b ta in e d o n th e w a te r c o lu m n m e th o d w a s th e s a m e
a s fo r b e d e x p a n s io n , fo r th e h ig h e s t flo w ra te s . H o w e v e r, fo r th e lo w
g a s flo w ra te s , th e e rro r is la rg e r (u p to 1 0 % ), c o n s id e rin g th e
m e a s u re d d iffe re n c e s o f th e h e ig h t in w a te r c o lu m n s (1 0 to 1 2 0 m m ).
T h e m e a s u re m e n t e rro r a s s o c ia te d w ith s o lid s h o ld-u p d e te rm in a tio n is
c o n s id e re d to b e n o m o re th a n 1 0 % (F re ita s a n d T e ix e ira , 1 9 9 8 ).
O v e ra ll, th e c o m b in e d e rro r fo r th e d e te rm in a tio n o f g a s h o ld-u p u s in g
is a t its m a x im u m 1 5 % .
IV.3.6 Evaluation of critical gas hold-up and critical gas velocity

C o n s id e rin g th e p rim a ry d a ta o b ta in e d (! v s U G ), th e c ritic a l p o in t
c o u ld b e d e te rm in e d a s th e in fle x io n p o in t o f th e d a ta g ra p h . H o w e v e r,
its
d ire c t
d e te rm in a tio n
in
th e
g ra p h
is
d iffic u lt
and
in a c c u ra te .
C o n s e q u e n tly , th e d a ta w e re re-p lo tte d a c c o rd in g to th e d rift-flu x

m o d e l a n d th e in fle x io n p o in t d e te rm in e d fro m th e d e v ia tio n o f th e d a ta
fro m th e th e o re tic a l lin e o f th e u n ifo rm re g im e . T h is is a s ta n d a rd
p ro c e d u re .
80
Chapter IV
T h e th e o re tic a l lin e j= j( ! G ) is d e fin e d a s :
j theo = !G " (1# !G ) " u
E q . IV - 4
W h e re u is th e m e a n s lip s p e e d in c a s e o f n o liq u id flu x th ro u g h th e

c o lu m n . T h e c o n c e p t o f D a rw in ia n d rift w a s u s e d to d e te rm in e th e
b u b b le m e a n s lip s p e e d (R u z ic k a e t a l., 2 0 0 1 a ). T h u s :
$ a ! #G '
utheo = u0 ! &1"
)
% 1" #G (
E q . IV - 5
W h e re u 0 is th e b u b b le te rm in a l s p e e d , a n d a th e b u b b le d rift
c o e ffic ie n t.
For
each
d a ta
lin e
! G e x p (Q E X P ),
th e y
a re
o b ta in e d
by
lin e a riz a tio n o f E q . V -5 , u s in g th e b a s ic re la tio n :
!G exp =
Qexp
E q . IV - 6
U exp
T h e e x p e rim e n ta l d rift-flu x is o b ta in e d fro m E q . IV - 4 to g e th e r w ith

E q . IV - 6 :
j exp = (1! "G ) # Qexp
E q . IV - 7
T h e tra n s itio n b e g in s w h e re E q . IV - 7 s e p a ra te s fro m E q . IV - 4 : it is

th e c ritic a l p o in t [U C ,! C ], th e in s ta b ility th re s h o ld . T h e v a lu e s o f U C a n d
! C a re th e q u a n tita tiv e m e a s u re s o f th e h o m o g e n e o u s re g im e s ta b ility .
T h e e v a lu a tio n p ro c e d u re is a n ite ra tiv e p ro c e s s . T h e h o m o g e n e o u s
d a ta ra n g e is in itia lly a s s e s s e d , th e n is u s e d fo r th e lin e a riz a tio n , till
th e c o rre la tio n c o e ffic ie n t o f th e lin e a riz a tio n is s u ffic ie n tly c lo s e to
u n ity .
T h e re g im e tra n s itio n w a s a ls o fo u n d u s in g th e s lip-s p e e d c o n c e p t,
w h e re , a t th e c ritic a l p o in t, th e s lip s p e e d d a ta u e x p , d e p a rts fro m th e
u t h e o o b ta in e d fro m th e m o d e l lin e .
T h e firs t c rite ria o f th e d rift flu x m o d e l is b a s e d o n th e c o u p lin g o f
p h a s e s , i e ., o n th e m a s s c o n s e rv a tio n o f th e p h a s e s . T h e s lip s p e e d
c o n c e p t is b a s e d o n th e fa c t th a t, in H o R , th e b u b b le s p e e d d e c re a s e s
w ith th e in c re a s e o f h in d ra n c e c a u s e d b y th e in c re a s e o f b u b b le
c o n c e n tra tio n .
81
Chapter IV
T h e o b ta in e d re s u lts a re th e a v e ra g e o f th e s e tw o m e th o d s . S in c e
th e s e tw o m e th o d s a re e q u iv a le n t, o n ly d iffe re n t c o-o rd in a te s a re u s e d ,
th e re s u lts s h o u ld b e s im ila r. T h is w a s th e te s t o f c o rre c tn e s s (M e n a e t
a l., 2 0 0 5 a ; R u z ic k a e t a l., 2 0 0 8 ).
In th e lite ra tu re , th e s ta b ility c rite ria n o rm a lly u s e d o n g -l-s s y s te m s
a re s c a rc e a n d s e v e ra l p rin c ip le s h a v e b e e n a p p lie d . In itia lly , th e y h a v e
b e e n b a s e d o n c o rre la tio n s o b ta in e d fro m e x p e rim e n ta l d a ta b u t th e y
la c k in te rm s o f u n iv e rs a l a p p lic a tio n d u e to th e ir s p e c ific ity (K ris h n a ,
1 9 9 1 ; R e illy e t a l., 1 9 9 4 ; W ilk in s o n e t a l., 1 9 9 2 ). T h e o re tic a l c rite ria
b a s e d o n th e o re tic a l c o n c e p ts a re m o re a c c ura te a n d m a y b e a p p lie d a
p rio ri ( R u z ic k a a n d T h o m a s , 2 0 0 3 ; S h n ip e t a l., 1 9 9 2 ) o r a p o s te rio r a s
th e s lip s p e e d c o n c e p t a n d th e d rift-flu x m o d e l (u s e d in th is w o rk ).
82
Chapter IV
IV.4 Results and Discussion

In th is c h a p te r it w a s e v a lu a te d th e in flu e n c e o f s o lid s in B C re g im e
flo w
tra n s itio n .
The
s o lid s
used
w e re
SG
and
p o s s ib le
p h y s ic a l
m e c h a n is m s th a t in flu e n c e th e re g im e flo w tra n s itio n in B C is d is c u s s e d

b e llo w .
IV.4.1 Spent grains characterization

T h e s o lid p a rtic le s (S G ) u s e d in th is c h a p te r a re a little d iffe re n t fro m
th e o n e s u s e d in th e p re v io u s c h a p te rs (III a n d V III). T h e m a in re a s o n is
th e la c k o f th e a c id p re-tre a tm e n t s te p (III.3 .4 .1 ). C o n s id e rin g th e n e e d
o f h ig h a m o u n t o f S G (a b o u t 5 k g o f d S G ) to w o rk in th e B C d e s c rib e d ,
th e lo w y ie ld (2 k g d S G /1 0 0 k g B G S ), th e re a g e n t a n d e n e rg y c o s ts , a n d th e
tim e n e c e s s a ry to o b ta in s u c h a m o u n t it w a s d e c id ed to re m o v e th e
a c id
s te p
fro m
th e
SG
p re p a ra tio n .
M o re o v e r
d u rin g
th e
SG
c h a ra c te riz a tio n n o re le v a n t d iffe re n c e s w e re fo u n d b o th in W A I a n d

d e n s ity b e tw e e n b a s e-tre a te d a n d a c id /b a s e-tre a te d S G .
S G a fte r p re-tre a tm e n t a re m a in ly c e llu lo s e (B r n y ik e t a l., 2 0 0 1 ).
T h e y a re c o m p le te ly w e tta b le p a rtic le s a n d in c re a s e s iz e w h e n in
c o n ta c t w ith w a te r. In o rd e r to d e te rm in e th e ir d e n s ity is im p o rta n t to
d is tin g u is h b e tw e e n d e n s ity o f d S G a n d w S G . In itia lly it w a s trie d to
d e te rm in e th e d e n s ity o f d S G . H o w e v e r e ith e r u s in g th e p ro p o s e d
m e th o d (s e c tio n IV .3 .2 .3 ) o r o th e r m e th o d s p ro p o s e d in lite ra tu re
(p ic n o m e te r m e th o d ) it w a s im p o s s ib le to d e te rm in e d w ith a c c u ra c y th e
d S G d e n s ity . T h is w a s m a in ly d u e to th e ir u n iq u e c h a ra c te ris tic s o f
w e tta b ility a n d s iz e c h a n g e . H a v in g th is in m in d it w a s o n ly d e te rm in e d
th e s p e c ific d e n s ity o f w S G p a rtic le s . T h is w a s im p o rta n t n o t o n ly
b e c a u s e d e n s ity o f p a rtic le s is c ru c ia l w h e n h y d ro d y n a m ic s tu d ie s a re
p e rfo rm e d b u t a ls o b e c a u s e th e re a l e ffe c t o n re a c to r h y d ro d y n a m ic w ill
b e d u e to th e w e t p a rtic le s in a q u e o u s s o lu tio n s . In o rd e r to
d e te rm in e th e d e n s ity o f w S G p a rtic le s it is a ls o n e c e s s a ry to d e te rm in e
th e W A I. O n T a b le IV - 1 th e v a lu e s o f w S G p ro p e rtie s (d e n s ity a n d W A I)
d e te rm in e d a re p re s e n t.
83
Chapter IV
Table IV-1. Spent grains properties in wet basis at T=25 C and P=1 atm
P ro p e r ty
A v e ra g e
S td e v
Qv
W A I / (g W E T /g D R Y )
! W E T S G / (g /m L )
8 .1 2 1
1 .0 3 7
0 .1 1 2
0 .0 1 0
1 .3 7 %
0 .9 7 %
! W A T E R / (g /m L )
0 .9 9 8
0 .0 0 0
0 :0 2 %
M u s s a to e t a l. (2 0 0 9 b ) o b ta in a W A I fo r S G o f 9 .0 3 b e in g th e
d iffe re n c e e x p la in e d e ith e r b y th e d iffe re n t p re-tre a tm e n t u s e d (b a s e tre a t o n ly ) o r b y d iffe re n t b a rle y v a rie ty u s e d in this w o rk . T h e h ig h W A I
is a m e a s u re o f th e a b ility to a d s o rb w a te r, w h ic h is v e ry im p o rta n t
a s p e c t fo r im m o b iliz a tio n o f m ic ro o rg a n is m s . T h is a b ility a llo w th a t
w a te r is a lw a y s p re s e n t fo r c e lls to g ro w th a n d d e v e lo p e d (M u s s a tto e t
a l., 2 0 0 9 b ). B e s id e s th is h ig h w a te r a d s o rp tio n , w h ic h p ro v e s th e ir
w e tta b ility c a p a c ity , S G h a v e h y d ro p h o b ic c h a ra c te r (B r n y ik e t a l.,
2 0 0 4 ) a n d h ig h c ritic a l h u m id ity p o in t, w h ic h in d ic a te s th a t n o rm a lly lo w
a m o u n ts o f w a te r a re b o u n d e d to the p a rtic le s (M u s s a tto e t a l., 2 0 0 9 b ).
T h is a m b ig u o u s c a p a c ity o f S G a re id e a l fo r m ic ro o rg a n is m s g ro w th a n d
im m o b iliz a tio n b e c a u s e m a n a g e to k e e p h ig h w a te r co n te n t a n d a t th e
s a m e tim e c re a te a h y d ro p h o b ic e n v iro n m e n ta l th a t d e c re a s e s th e
a b ility o f im m o b iliz e d y e a s t (m o re h y d ro p h o b ic ) to b e re le a s e in th e
b u lk (B r n y ik e t a l., 2 0 0 4 ).
IV.4.2 Bed expansion versus water columns differential pressure

for gas hold-up determination
F o r th e s y s te m c o n s id e re d , th e d e te rm in a tio n o f g a s h o ld -u p u s in g
b o th te c h n iq u e s s h o w e d s im ila r re s u lts . T h e m e a s u rin g o f g a s h o ld-u p
b y w a te r c o lu m n s c o m b in e d w ith th e m o d ifie d m e th o d fo r s o lid h o ld-u p
d e te rm in a tio n u s e d b y F re ita s e t. a l (1 9 9 7 ) a p p e a rs to b e a s u ita b le
m e th o d to d e te rm in e d g a s h o ld -u p in th re e -p h a s e s y s te m s w h e re S G
a re p re s e n t. In fa c t a n d c o n s id e rin g th e s o lid d is trib u tio n in th e e n tire
B C th e d iffe re n c e b e tw e e n th e e x p e rim e n ta l a n d th e o re tic a l v a lu e s o f
s o lid lo a d d e te rm in e d b y th is te c h n iq u e w a s 2 .9 7 % ; 3 .3 2 % ; 3 .9 8 % a n d
6 .9 4 % fo r 4 % ; 8 % ; 1 2 % ; 2 0 % (w t. W E T
84
BASIS
/v o l.) re s p e c tiv e ly . T h e
Chapter IV
o b ta in e d e rro rs a re in th e s a m e ra n g e o f th e o n e s o b ta in e d b y o th e r
a u th o rs (F re ita s a n d T e ix e ira , 1 9 9 8 a , 1 9 9 8 b ; F re ita s e t a l., 1 9 9 7 ).
H a v in g in m in d th a t th e s e v a lu e s h a v e b e e n c a lc u la te d w ith th e
e x p e rim e n ta l v a lu e s fo r g a s h o ld-u p , it is c le a r th a t th e a p p lie d
te c h n iq u e m a y b e a p p lie d w ith a g o o d a c c u ra c y.
M o re o v e r,
th e
re s u lts
p re s e n te d
in
F ig u re
IV - 2
th a t
show
th e
e x p e rim e n ta l e rro rs fo r th e tw o te c h n iq u e s c o n firm th is c o n c lu s io n . In

F ig u re IV - 2 A , th e d e v ia tio n s fo r a ll ra n g e o f s o lid s lo a d s u s e d a re
s h o w n w h ile in F ig u r e IV - 2 B o n ly th e firs t th re e s o lid s lo a d s a re
c o n s id e re d (4 % 8 % a n d 1 2 % (w t. W E T
BASIS
0.160!
eWC (% / (vol./vol))!
20%!
0.120!
0.080!
0.040!
Gas hold-upWC / (vol./vol.) !
0.160!
0.000!
0.000!
/v o l.)).
15%!
0.120!
0.080!
0.040!
0.000!
0.040! 0.080! 0.120!
Gas hold-upWC / (vol./vol.)!
0.000!
0.040! 0.080! 0.120!

Gas hold-upBE / (vol./vol.) !
Figure IV-2. Comparison between gas hold-up obtained by Water Columns

(Gas hold-upWC) and Bed expansion (Gas hold-upBE): A Solids load up to
20% (wt.WET BASIS/vol.); B Solids load up to 20% (wt.WET BASIS/vol.). Legend:
! water; !- 4% (wt.WET BASIS/vol.); ! - 8% (wt.WET BASIS/vol.);
+ - 12% (wt.WET BASIS/vol.); ! - 20% (wt.WET BASIS/vol.).
T h e m a x im u m a n d m e a n d e v ia tio n s b e tw e e n th e re s u lts o b ta in e d w ith

th e tw o d iffe re n t te c h n iq u e s a re , re s p e c tiv e ly , 2 6 % a n d 7 % , w ith m o s t
o f th e m e a s u re d v a lu e s w ith e rro rs in th e ra n g e 8 % a n a c c e p ta b le
re s u lt h a v in g in m in d th e a p p lie d te c h n iq u e s . N e v e rth e le s s th e h ig h e s t
e rro rs o c c u r a t th e lo w e s t g a s flo w ra te s a n d h ig h s o lid s c o n te n t. In
fa c t, fo r th e m a x im u m s o lid s c o n te n t a n d d u e to th e n o n -h o m o g e n e ity
o f s o lid s in th e c o lu m n , e s p e c ia lly a t lo w g a s flo w s , th e c o m b in e d
m e th o d fo r s o lid s a n d g a s h o ld -u p d e te rm in a tio n is n o t s u ita b le . It
85
Chapter IV
s e e m s th a t, fo r th e s e ty p e o f s o lid s , o n ly s o lid s lo a d v a lu e s b e lo w
1 2 % (w t. W E T
BASIS
e v a lu a tio n
of
2 0 % (w t. W E T
BASIS
/v o l.)
th e
a re
d a ta
a c c u ra te
in
te rm s
enough
of
to
re g im e
p e rfo rm
tra n s itio n .
c o rre c t
Thus,
fo r
/v o l.) s o lid s lo a d th e re s u lts w e re n o t u s e d fo r th e
d e te rm in a tio n o f re g im e tra n s itio n . A n y w a y n o re g im e tra n s itio n w a s

o b s e rv e d a t th is s o lid s lo a d . F o r h ig h e r g a s flo w ra te s th e a g re e m e n t
b e tw e e n th e d a ta w a s a lw a y s b e lo w 4 % . T h e re fo re b o th te c h n iq u e s
w e re c o n s id e re d fo r th e d e te rm in a tio n o f c ritic a l v a lu e s (! C a n d U C ) fo r
re g im e tra n s itio n .
IV.4.3 Gas-hold up vs gas-flow: solids influence

In th e g ra p h o f th e F ig u r e IV - 3 a re p lo tte d th e c u rv e ! (q ) o b ta in e d fo r
e a c h c o n c e n tra tio n o f s o lid s (0 2 0 % (w t. W E T
Gas Hold-up (% / (vol./vol.))!
Gas Hold-up (% / (vol./vol.))!
A!
12%!
9%!
6%!
3%!
0%!
0.000!
0.010!
0.020!
BASIS
B!
12%!
9%!
6%!
3%!
0%!
0.000!
0.030!
/v o l.)).
0.010!
0.020!
0.030!
UG / (m/s)!
UG / (m/s)!
Figure IV-3.Primary data obtained from different gas hold-up methods: A

Bed Expansion; B water Columns. Legend: ! water;
!- 4% (wt.WET BASIS/vol.); ! - 8% (wt.WET BASIS/vol.); + - 12% (wt.WET BASIS/vol.);
! - 20% (wt.WET BASIS/vol.).
F ig u r e IV - 3 s h o w s th a t, in th is c o lu m n , re g im e tra n s itio n o c c u rs a t
lo w e r g a s flo w s th a n th e o n e s o b ta in e d b y o th e r a u th o rs in B C (M e n a e t
a l., 2 0 0 5 a ; R u z ic k a e t a l., 2 0 0 1 a , 2 0 0 1 b ). H o w e v e r th e v a lu e s o f g a s
h o ld -u p a re in th e s a m e ra n g e . T h e re a re tw o p o s s ib le e x p la n a tio n s fo r
th is : b u b b le c o lu m n s iz e a n d th e e ffe c t o f th e d is trib u to r.
86
Chapter IV
It is re p o rte d th a t c o lu m n s iz e (d ia m e te r a n d h e ig h t) in flu e n c e s th e
g lo b a l h o ld -u p . A ls o , s p a rg e r in flu e n c e o v e r th e c o lu m n a x is c a n g o u p
to fo u r tim e s th e c o lu m n d ia m e te r (G a n d h i e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ).
F o r th e s a m e g a s flo w , R u z ic k e t a l. (2 0 0 1 b ) o b s e rv e d th a t g a s h o ld-u p
v a lu e s a re u s u a lly lo w e r w h e n B C d ia m e te r in c re a s e s a n d /o r c o lu m n
h e ig h t
in c re a s e s .
Thus
th e
c ritic a l
v a lu e s
(! C
vs.
UC)
fo r
re g im e
tra n s itio n a ls o d im in is h (R u z ic k a e t a l., 2 0 0 1 b ). G e n e ra lly , g a s s p a rg e rs

in B C h a v e a d ia m e te r th a t o c c u p ie s a ll c ro s s-s e c tio n o f th e B C . A s in
o u r c a s e th e s p a rg e r c o rre s p o n d s to 2 /3 o f th e b u b b le d ia m e te r, th is
c h a ra c te ris tic is re s p o n s ible fo r a h ig h e r h e ig h t u n til g a s flo w s ta b iliz e s
a n d fo r lo c a l liq u id c irc u la tio n n e a r th e s p a rg e r. T h is m e a n s th a t th e
h e ig h t o f th e c o lu m n n e c e s s a ry fo r th e g a s to a c h ie v e a fla t p ro file is
h ig h e r a s th e re is a re g io n a t th e b o tto m o f th e c o lu m n w h e re liq u id
c irc u la tio n o c c u rs (le s s v is ib le a t lo w flo w s ), w h ic h d iffic u lt a ra p id
s ta b iliz a tio n o f th e g a s p ro file s a s ty p ic a lly o c c u rs in H o R . T h e liq u id
c irc u la tio n is a c o n s e q u e n c e o f th e h ig h e r a m o u n t o f b u b b le s in th e
c e n te r o f th e c o lu m n im m e d ia te ly a b o v e th e d is trib u to r, re s u ltin g in
lo c a l lo w e r h o ld -u p v a lu e s . T h e in flu e n c e o f s p a rg e r g e o m e try o n g a s
h o ld -u p v a lu e s h a s a ls o b e e n s tu d ie d b y V ija y a n e t a l. (2 0 0 7 ). A s th e
g a s h o ld -u p v a lu e s o b ta in e d in th is w o rk a re in th e s a m e ra n g e v a lu e s
a s th o s e re p o rte d b y th e s e a u th o rs w h e n s im ila r H /D a n d A s p a r g e r /A C
ra tio s a re c o n s id e re d , o u r e x p e rim e n ta l v a lu e s m a y b e c o n s id e re d to b e
w ith in th e e x p e c te d ra n g e (V ija y a n e t a l., 2 0 0 7 ).
T h e s e le c tio n o f th is d is trib u to r is re la te d w ith o u r in te re s t o n th e
s tu d y o f th e th re e -p h a s e h y d ro d y n a m ic s in a n in te rn a l a irlift re a c to r, a s
in th e s e s y s te m s th e d is trib u to r c ro s s s e c tio n is o f th e s a m e o rd e r o f
m a g n itu d e o f th e a irlift ris e r c ro s s s e c tio n a n d a lw a y s s m a lle r th a n th e
to ta l c o lu m n c ro s s s e c tio n . T h e a p p lie d d is trib u to r a llo w e d to re a c h th e
o b je c tiv e o f s o lid s flu id iz a tio n a t lo w g a s flo w ra te s (e x c e p t fo r the
m a x im u m s o lid s c o n c e n tra tio n ).
E v e n if th is s y s te m w o u ld b e h a v e d iffe re n tly fro m th o s e re p o rte d in
lite ra tu re , w h ic h is n o t th e c a s e , it w o u ld s till p o s s ib le to c o n c lu d e o n
th e e ffe c t o f th e s o lid p h a s e o n flo w re g im e tra n s itio n , a s e x p e rie n c e s
87
Chapter IV
w e re d o n e fo r a ll c a s e s in th e s a m e B C . In a d d itio n , th e p re s e n c e o f
b o th re g im e flo w (H o R a n d H e R ) in a ir-w a te r s y s te m s w a s c le a rly
id e n tifie d .
F ro m F ig u re IV - 3 , it is a ls o p o s s ib le to o b s e rv e , a t lo w g a s flo w s ,
th a t th e p re s e n c e o f th e s o lid p h a s e c a u s e s a n in c re a s e in g a s h o ld-u p .
T h is m a y b e a ttrib u te d to a s ta b iliz a tio n e ffe c t o f th e b u b b le s a t lo w
g a s flo w s , b e in g th e o p p o s ite e ffe c t o b s e rv e d a s g a s flo w is in c re a s e d .
T h is s ta b iliz a tio n e ffe c t c a u s e s a n in c re a s e in g a s re s id e n c e tim e a n d
c o n s e q u e n tly h o ld -u p is h ig h e r. T h is is a ls o re la te d w ith a d e c re a s e o f
th e h e ig h t th a t th e g a s n e e d s to a c h ie v e th e fla t p ro file , ty p ic a l fo r H o R .
T h e e ffe c ts a s s o c ia te d w ith th e u s e o f a s p a rg e r n o t o c c u p y in g th e
e n tire c ro s s s e c tio n o f th e c o lu m n m a y a ls o c o n trib u te to th e o b s e rv e d
in c re a s e in g a s h o ld-u p . A s d u e to th e ir s e d im e n ta tio n p ro p e rtie s a n
in c re a s e in s o lid s c o n c e n tra tio n n e a r th e d is trib u to r o c c u rs (s p e c ia lly a t
lo w g a s flo w a n d h ig h s o lid s lo a d ), a h ig h e r in te ra c tio n b e tw e e n
b u b b le s a nd p a rtic le s o c c u rs . T h e re is a ls o th e s te ric e ffe c t (p re s e n c e
o f s o lid s ) o f s p e n t g ra in s a s w e ll a s its s u rfa c e p ro p e rtie s th a t c a n h a v e
a n im p o rta n t e ffe c t o n th e in te ra c tio n s b e tw e e n s o lid s a n d b u b b le s .
T h is e ffe c t re d u c e s b u b b le ris e v e lo c ity le a d in g to a s lig h t in c re a s e o n
h o ld -u p (L u e t a l., 1 9 9 5 ; M e n a e t a l., 2 0 0 5 a ). In fa c t, w h e n s o lid s w e re
p re s e n t, a la rg e r a m o u n t o f s m a lle r b u b b le s n e a r th e w a ll wa s o b s e rv e d
in c o m p a ris o n w ith a ir-w a te r s y s te m s a s o b s e rv e d in n e x t fig u re .
Figure IV-4. Exemple of small bubbles near the BC wall.
88
Chapter IV
S p e n t g ra in s w e tta b ility is a n o th e r fa c to r th a t m a y c o n trib u te to th e

o b ta in e d re s u lts . A ls o , it h a s b e e n re p o rte d th a t th e in flu e n c e o f th e
p h y s ic a l p ro p e rtie s o f s o lid s is h ig h e r w h e n s o lid s h a v e a s m a ll s iz e
c o m p a re d w ith b u b b le s iz e , a s is th e c a s e o f S G (M e n a e t a l., 2 0 0 5 a ). A
h ig h e r d ra g fo rc e o n th e b u b b le s u rfa c e (g a s-liq u id in te rfa c e ) is c re a te d
b y th e p re s e n c e o f s m a ll s iz e s o lid s w ith a c o n s e q u e n t re d u c tio n o n
b u b b le ris e v e lo c ity a n d g a s h o ld-u p in c re a s e (M e n a e t a l., 2 0 0 5 a ). T h e
c o m b in a tio n
of
th e
a b o v e -m e n tio n e d
e ffe c ts
c o n trib u te s
fo r
th e
o b s e rv e d in c re a s e in g a s h o ld-u p a t lo w e r g a s flo w s w h e n s o lid s a re

p re s e n t. T h is is in a g re e m e n t w ith J a m ia la h m a d i a n d M u lle r-S te in h a g e n
(1 9 9 1 ) th a t c o n c lu d e d th a t w e tta b le p a rtic le s in c re a s e d h o ld-u p b y
s u p p re s s in g c o a le s c e n c e w h ile n o n-w e tta b le p a rtic le s h a d th e o p p o s ite
e ffe c t. It m a y a ls o b e n o te d th a t th e re s is ta n c e pro m o te d b y s o lid s
s e d im e n ta tio n
was
e v id e n t
c o n c e n tra tio n w a s te s te d (2 0 % (w t. W E T
BASIS
when
m a x im u m
s o lid
/v o l.)), a s n o flu id iz a tio n o f
s o lid p a rtic le s w a s o b s e rv e d a t th e lo w e s t g a s flo w ra te s .

12.5%!
Gas Hold-up (% (vol./vol.))!
Gas Hold-up (% (vol./vol.))!
3.8%!
3.0%!
2.3%!
1.5%!
0.8%!
12.0%!
11.5%!
11.0%!
10.5%!
0.0%!
0!
5!
10!
% Solids (vol./vol.)!
0!
15!
5!
10!
% Solids (vol./vol.)!
15!
Figure IV-5. A - Solids influence at low gas flow rates in gas hold-up (Bed
Expansion). Legend: ! q = 0,2 cm/s; " - 0,4 cm/s; B Solids influence at
high gas flow rates in gas hold-up (Bed Expansion). Legend: ! q = 2,5
cm/s; " - 2,7 cm/s
O n F ig u r e IV - 5 A , it is p o s s ib le to v e rify th a t w h e n s o lid s a re p re s e n t,
a t lo w g a s flo w s , g lo b a l g a s h o ld-u p te n d s to in c re a s e w ith a m a x im u m
89
Chapter IV
a ro u n d 8 % (v o l./v o l.) o f s o lid s . In H o R , th is re s u lt is a c o n s e q u e n c e o f

p a rtic le -b u b b le
in te ra c tio n s
th a t
re s u lt
in
b u b b le
b re a k-u p
and
d is p e rs io n , re d u c e d b u b b le ris e v e lo c ity a n d c o n s e q u e n t in c re a s e in
h o ld -u p .
O n th e o th e r h a n d , F ig u r e IV - 5 B s h o w s th a t in H e R a t h ig h flo w s th e
o p p o s ite e ffe c t is p re s e n t re v e a lin g a du a l e ffe c t o f th e p a rtic le s . A t
th e s e v a lu e s o f g a s flo w (H e R ) liq u id c irc u la tio n is h ig h e r a n d m o re
p ro n o u n c e d , th e e ffe c t o f p a rtic le-b u b b le in te ra c tio n b e in g lo w e r.
B u b b le -b u b b le in te ra c tio n s in c re a s e d a n d c o a le s c e n c e o c c u rre d . A t
th is s ta g e , c o a le s c e n ce d o e s n o t s e e m to b e s ig n ific a n tly a ffe c te d b y
th e p re s e n c e o f th e s o lid p h a s e . H o w e v e r, w ith th e o b ta in e d d a ta , it is
im p o s s ib le
to
id e n tify /e v a lu a te
th e
im p o rta n c e
of
th e
p h y s ic a l
m e c h a n is m s re la te d w ith s o lid p ro p e rtie s (w e tta b ility , s iz e , d e n s ity ). At

h ig h
gas
flo w s ,
c o a le s c e n c e
2 0 % (w t. W E T
la rg e r
phenomena
BASIS
b u b b le s
at
w e re
th e s e
v is u a lly
o b s e rv e d
s ta g e s .
The
re v e a lin g
absence
of
/v o l.) s o lid s d a ta is re la te d w ith th e fa c t th a t H o R w a s
n o t a c h ie v e d u n d e r th is s o lid s lo a d c o n d itio n (s e e F ig u r e IV - 6 ).
IV.4.4 Gas-hold up vs gas-flow: drift-flux

In F ig u re IV - 6 a re p lo tte d th e D rift-flu x re s u lts fo r th e d a ta o b ta in e d
b y th e B e d E x p a n s io n m e th o d . T h is m e th o d a llo w s fo r a n a c c u ra te
d e te rm in a tio n
of
th e
c ritic a l
p o in t
(p o in t
at
w h ic h
HoR
s ta b ility
d is a p p e a rs ). T h e c ritic a l p o in t, w h ic h c o rre s p o n d s to th e in fle x io n

p o in ts o n F ig u r e IV - 3 , is n o w c le a rly id e n tifie d o n F ig u re IV - 6 (o p e n
s y m b o ls ) fo r th e d iffe re n t e xp e rim e n ta l c o n d itio n s . A s p re v io u s ly s a id ,
th is p o in t c o rre s p o n d s to th e b e g in n in g o f s e p a ra tio n o f th e p lo ts o f th e
e x p e rim e n ta l a n d c a lc u la te d d a ta .
90
Chapter IV
A!
0.04!
B!
0.03!
j / (m/s)!
j / (m/s)!
0.03!
0.02!
0.02!
0.01!
0.01!
0!
0!
0!
0.04!
0.08!
0.12!
0!
Gas hold-up / (vol./vol.)!
0.02!
0.01!
0.01!
0!
0!
0.04!
0.08!
0.12!
0!
0.04!
0.08!
0.12!
E!
0.03!
j / (m/s)!
0.12!
D!
0.03!
j / (m/s)!
j / (m/s)!
0.02!
0!
0.08!
C!
0.03!
0.04!
0.02!
0.01!
0!
0!
0.04!
0.08!
0.12!

Figure IV-6. Drift-flux plot: Drift-flux j (m/s) vs. Gas hold-up (vol./vol.).
Legend: ! (smooth line): jt by Eq. IV-4; ! (Data points): jEXP by Eq. IV-7; "
(Blank Data): Critical point. A, B, C, D, E: Different solid loads (0, 4, 8,12 %
(wt.WET BASIS /vol.))
91
Chapter IV
F ro m th e re s u lts p lo tte d in F ig u r e IV - 6 , a d e c re a s e o f c ritic a l g a s

h o ld -u p a n d c ritic a l g a s flo w w ith s o lid s lo a d is c le a rly o b s e rv e d .
C ritic a l g a s h o ld -u p d e c re a s e p re s e n ts a lin e a r b e h a v io u r, w h ile c ritic a l
gas
flo w
has
1 2 % (w t. W E T
an
BASIS
e x p o n e n tia l
tre n d .
T h is
s u g g e s ts
th a t
above
/v o l.) o f S G lo a d in g th e c ritic a l g a s flo w is s im ila r to th e
m in im u m g a s flu id iz a tio n flo w fo r th is s y s te m . In fa c t, a s s a id b e fo re ,

w h e n th e h ig h e s t s o lid s lo a d w a s a p p lie d , th e v a lu e fo r m in im u m
flu id iz a tio n g a s v e lo c ity w a s o n ly 2 0 % in fe rior to th e c ritic a l g a s flo w
fo r 1 2 % (w t. W E T
s o lid s
lo a d ,
BASIS
/v o l.) lo a d in g . T h is in d ic a te s th a t, u n d e r th e te s te d
1 2 % (w t. W E T
BASIS
/v o l.)
s o lid s
lo a d
was
th e
m in im u m
c o n c e n tra tio n w h e re H o R c o u ld b e e s ta b lis h e d a n d o n ly fo r a s m a ll

ra n g e
of
gas
v e lo c itie s
(0 .2 c m /s < U G < 0 .5 c m /s ).
C o n c e rn in g
th e
c ritic a l g a s h o ld -u p , th e fo llo w in g c o rre la tio n c a n b e fo u n d to d e s c rib e

its d e p e n d e n c e o n s o lid s lo a d (E q .IV - 8 ):
! C = 0.10 " 0.56 # $ S R 2 = 0.94; Rxy = 0.97

T h is
e q u a tio n
in d ic a te s
th e
way
E q . IV - 8
spent
g ra in s
in flu e n c e
re g im e
tra n s itio n o n th e s tu d ie d b u b b le c o lu m n . E q .IV - 8 in te rc e p ts y -a x is a t a

v a lu e lo w e r th a n th e o n e s fo u n d in th e lite ra tu re (M e n a e t a l., 2 0 0 5a ),
th is b e in g re la te d w ith th e a b o v e a d d re s s e d s p e c ific is s u e s o f th is B C .
W h e n c o m p a rin g th e s lo p e o f E q . IV - 8 w ith th e o n e re p o rte d b y M e n a e t
a l. (2 0 0 5 a ) fo r a lg in a te b e a d s a s th e s o lid p h a s e , th e o b ta in e d v a lu e
w ith S G is tw o tim e s la rg e r. T h is a llo w s c o n c lu d in g th a t S G h a v e a m o re
p ro n o u n c e d e ffe c t o n re g im e tra n s itio n th a n a lg in a te b e a d s . T h is is n o t
u n e x p e c te d a s th e p ro p e rtie s o f b o th s o lid s a re d iffere n t, m a in ly s iz e ,
s h a p e a n d w e tta b ility .
P re s e n te d re s u lts in d ic a te th a t a re d u c tio n in H o R re g im e s ta b ility is
o b s e rv e d w h e n s o lid s a re p re s e n t (F ig u re IV - 6 a n d F ig u r e IV - 7 ). It w a s
v is u a lly v e rifie d tha t w h e n g a s flo w w a s in c re a s e d b ig b u b b le s s ta rt to
a p p e a r, e s p e c ia lly in th e c o lu m n c e n tre , d u e to c o a le s c e n c e . In fa c t, a t
m a x im u m
s o lid s
lo a d
w h e re
no
HoR
was
e s ta b lis h e d ,
b u b b le
c o a le s c e n c e w a s o b s e rv e d e v e n a t th e lo w e s t flo w s . A t th e h ig h e s t
flo w ,
s lu g
re g im e
was
p re s e n t,
ty p ic a l
s itu a tio n
fo r
BCs
w ith
d ia m e te rs in fe rio r to 2 0 c m (D e c k w e r, 1 9 9 2 ). T h is re s u lt c o n tra d ic ts th e
92
one
o b ta in e d
by
Chapter IV
J a m ia la h m a d i
and
M u lle r-S te in h a g e n
(1 9 9 1 )
th a t
o b s e rv e d th a t w e tta b le p a rtic le s in c re a s e g a s h o ld-u p b y s u p p re ss in g

c o a le s c e n c e . In fa c t th is o c c u rs a t lo w g a s flo w s (F ig u re IV - 5 A ), b u t
n o t a t h ig h e r o n e s (F ig u r e IV - 5 B ). A s a b o v e s a id , th is d u a l e ffe c t o f S G
is a ttrib u te d to th e B C d e s ig n . T h e in c re a s e in g a s h o ld-u p b y th e
a c tio n o f S G m e a n s th a t m o re b u b b le s a re p re s e n t a n d in te ra c tio n s
b e tw e e n b u b b le s a re h ig h e r. T h is in c re a s e d ra te o f c o llis io n s p ro m o te s
c o a le s c e n c e . W ith th e g a s h o ld-u p in c re a s e in th e H o R b y th e p re s e n c e
o f th e s o lid p h a s e , n o n-u n ifo rm itie s a re fo rm e d in th e g a s p h a s e a n d
th e s ta b ility o f H o R is re d u c e d . T h u s th e H e R is a c h ie v e d e a rlie r a n d
th e c ritic a l v a lu e s (! C v s . U C ) a re lo w e r w h e n s o lid s a re p re s e n t.
0.03!
0.09!
UC / (m/s)!
0.12!
0.06!
0.02!
0.01!
0.03!
0!
0!
0%!
4%!
8%! 12%! 16%!
0%!
% Solid Load (wWET BASIS/vol.)!
4%!
8%!
12%! 16%!
% Solid Load (wWET BASIS/vol.)!
Figure IV-7. Evaluation of critical gas hold-up and critical gas flow with solid
load. Legend: ! - Bed Expansion; " - Water Columns
It is a ls o p o s s ib le to o b s e rv e th a t e ith e r b e d e x p a n s io n a n d w a te r
c o lu m n s te c h n iq u e s s h o w s im ila r re s u lts (F ig u re IV - 7 ). T h is in d ic a te s o r
c o n firm s th a t, fo r s o lid s lo a d s in fe rio r to 1 2 % (w t. W E T
BASIS
/v o l.), th e
c o m b in a tio n o f s o lid h o ld -u p m e th o d a n d w a te r c o lu m n s a p p e a r to b e a
s u ita b le
te c h n iq u e
to
e v a lu a te
gas
h o ld-u p
in
d iffe re n t
re a c to rs
c o n fig u ra tio n s .
It w a s s u g g e s te d th a t s o lid s e ffe c ts c a n b e s im ila r to w h a t o c c u rs in
a lo w v is c o s ity liq u id o n b u b b le c o lu m n s (M e n a e t a l., 2 0 0 5 ). In o u r
93
Chapter IV
c a s e , c ritic a l v a lu e s (! C v s . U C ) d e c re a s e w ith s o lid s lo a d , w h ic h is in

a g re e m e n t w ith p u b lis h e d re s u lts o n v is c o s ity e ffe c t o n flo w re g im e
tra n s itio n (Y a n g e t a l., 2 0 1 0 ). R u z ic k e t a l., (2 0 0 3 ) re p o rte d a d u a l
e ffe c t o f liq u id v is c o s ity w h e n lo w v is c o s ity (< 2 ,1 m P a .s ) a n d h ig h
v is c o s ity liq u id s a re p re s e n t. M e a n w h ile , re c e n t s tu d ie s p u b lis h e d b y
Y a n g e t a l. (2 0 1 0 ) id e n tifie d th a t a t lo w v is c o s ity liq u id s th e re is a fa s t
d e c re a s e o f c ritic a l v a lu e s (g a s h o ld-u p a n d g a s flo w ) fo llo w e d b y a
s m a ll d e c re a s e . T h e c ritic a l v is c o s ity w h e re th is c h a n g e o c c u rs w a s
a ro u n d 3 m P a .s . T h e b e h a v io u r o f c ritic a l g a s flo w v s . v is c o s ity w a s
(Y a n g e t a l., 2 0 1 0 ):
qC1 = !0.019ln( L ) + 0.025; qC 2 = !0.018ln( L ) + 0.056 for L < 2.8 mPa " s E q . IV - 9
T h e s e a u th o rs c o n s id e re d th a t th e firs t v a lu e fo r th e c ritic a l g a s flo w
- q C 1 c o rre s p o n d s to th e e n d o f th e h o m o g e n e o u s re g im e w h e n th e
firs t b ig b u b b le s a re fo rm e d a n d th e s e c o n d o n e -
q C 2 is o b ta in e d
w h e n fu ll h e te ro g e n e o u s re g im e is e s ta b lis h e d (Y a n g e t a l., 2 0 1 0 ).
A c c o rd in g ly , th e y s u g g e s t th a t th e v a lu e s o b ta in e d fo r c ritic a l v a lue s o f
tra n s itio n c o rre s p o n d to th e v a lu e w h e n h o m o g e n e o u s re g im e s lo s e s
s ta b ility (q C 1 ).
O liv e r
et
a l.
(1 9 6 1 )
c ite d
by
Lu
et
a l.
(1 9 9 5 )
s u g g e s te d
th e
d e te rm in a tio n o f a p s e u d o-h o m o g e n e o u s v is c o s ity fo r a s o lid -liq u id

m ix tu re a c c o rd in g to th e fo llo w in g e q ua tio n :
S !L = L f (" S )
!1
E q . IV - 1 0
W h e re ,
(1" 0.75 # ! )(1" 2.15 # ! )

f (! ) =
1/ 3
S
(1" ! S )
E q . IV - 1 1
T h e a p p lic a tio n o f th is e q u a tio n to o u r e x p e rim e n ta l d a ta w a s b a s e d

o n th e fa c t th a t th e s o lid p h a s e u s e d a lg in a te b e a d s - b y L u e t a l.
(1 9 9 5 ) h a d s im ila r p ro p e rtie s d e n s ity a n d s iz e - to th e s o lid s u s e d in
th is w o rk . In o u r c a s e , th e fo llo w in g e m p iric a l c u rv e re la tin g c ritic a l g a s
flo w w ith th e p s e u d o -h o m o g e n e o u s v is c o s ity (1 m P a . s < S - L < 1 .7 m P a . s )
w a s o b ta in e d :
94
Chapter IV
qC1 = !0.032ln( L ) + 0.02 ( R 2 = 0.98)

T h is
re la tio n
1 2 % (w t. W E T
BASIS
was
o b ta in e d
fo r
s o lid s
E q . IV - 1 2
lo a d
0%,
4%,
8%
and
/v o l.) a s o n ly in th e s e s itu a tio n s tra n s itio n re g im e w a s
o b s e rv e d . T h e o b ta in e d v a lu e s a re in th e s a m e ra n g e a s th o s e o f
E q . IV - 9 (Y a n g e t a l., 2 0 1 0 ).
T h e s e re s u lts d e m o n s tra te th a t th e in flu e n c e o f S G lo a d in th e c ritic a l
v a lu e c o rre s p o n d in g to th e e n d o f H o R (q C ) fo llo w s th e s a m e p a tte rn o f
lo w v is c o s ity liq u id s . It s e e m s th a t, d e p e n d in g o n th e c o lu m n s iz e
(d ia m e te r a n d h e ig h t), ty p e o f d is trib u to r a n d ty p e o f s o lid s , th e d u a l
e ffe c t o f s o lid s a n d v is c o s ity s u g g e s te d b y s o m e a u th o rs (M e n a e t a l.,
2 0 0 5 a ; R u z ic k a , e t a l., 2 0 0 1 ) m a y o r m a y n o t o c c u r (Y a n g e t a l., 2 0 1 0 )
a s in th is c a s e . It is im p o rta n t to n o tic e th a t E q . IV - 1 2 is lim ite d to th is
p a rtic u la r s y s te m (a ir-w a te r-S G ), b e in g s im ila r e v a lu a tio n s p o s s ib le a n d
d e s ira b le fo r o th e r g a s-liq u id -s o lid s y s te m s in o rd e r to h a v e a b e tte r
u n d e rs ta n d in g o f its h y d ro d y n a m ic s
A lth o u g h th e p re s e n te d in fo rm a tio n a im s a t c o n trib u tin g to th e e ffe c t
o f a s o lid p h a s e o n th e h y d ro d y n a m ic s o f g -l-s s y s te m s , fu rth e r w o rk is
re q u ire d to c la rify h o w s o lid s in flu e n c e re g im e tra n s itio n a n d th e
m a g n itu d e o f th is in flu e n c e . O b ta in e d re s u lts a llo w e d to c o n firm th a t
th a t s o lid in flu e n c e is re la te d w ith th e lo w v is c o s ity o f th e p s e u d oh o m o g e n e o u s liq u id p h a s e fo rm e d a n d th a t o th e r p a ra m e te rs s u c h a s
s te ric e ffe c t, w e tta b ility , B C d e s ig n a n d d is trib u to r g e o m e try p la y a ls o
a n im p o rta n t ro le in flo w re g im e tra n s itio n .
95
Chapter IV
IV.5 Conclusions
T h e e ffe c t o f S G p a rtic le s o n h o m o g e n e o u s re g im e s ta b ility a n d
re g im e tra n s itio n in a th re e -p h a s e B C w a s in v e s tig a te d e x p e rim e n ta lly .
T h e s ta b ility w a s e x p re s s e d b y th e c ritic a l v a lu e s o f g a s h o ld u p a n d g a s
flo w
ra te .
s ta b iliz a tio n
The
on
e x p e rim e n ts
HoR
fo r
lo w
showed
gas
th a t
flo w
th e
ra te .
s o lid s
p ro m o te d
M o re o v e r,
it
was
d e m o n s tra te d th a t s p e n t g ra in s d e c re a s e th e c ritic a l v a lu e s w h e re th e
H o R p re v a ils . T h is in flu e n c e w a s d e m o n s tra te d to h a v e s im ila r e ffe c t to
th e o n e fo u n d fo r lo w v is c o s ity liq u id s o n re g im e tra n s itio n . In a d d itio n ,
it w a s p o s s ib le to c o n c lu d e o n th e im p o rta n c e o f th e s te ric e ffe c t o f
s o lid s a s w e ll a s th e ir s p e c ific p ro p e rtie s (w e tta b ility ) o n re g im e
tra n s itio n .
D e s p ite th is , th e m e c h a n is m s b y w h ic h s o lid s a ffe c t th e re g im e
tra n s itio n is fa r fro m b e in g u n d e rs to o d . F u rth e rm o re th e in flu e n c e o f
c o lu m n s iz e a n d ty p e o f s o lid s m a k e s d iffic u lt a re a l a s s e s s m e n t w h e n
a ll re s u lts a v a ila b le in lite ra tu re a re c o m p a re d
IV.6 References
Banisi, S., Finch, J. A., Laplante, A. R. and Weber, M. E. (1995). Effect of solid
particles on gas holdup in flotation columns - I. measurement. Chemical
Engineering Science, 50(14), 23292334.
Brnyik, T., Vicente, A. A., Machado Cruz, J. M. and Teixeira, J. A. (2001). Spent
grains a new support for brewing yeast immobilisation. Biotechnology Letters,
23(13), 10731078.
Brnyik, T., Vicente, A. A., Oliveira, R. and Teixeira, J. A. (2004). Physicochemical
surface properties of brewing yeast influencing their immobilization onto spent
grains in a continuous reactor. Biotechnology and bioengineering, 88(1), 84-93.
Cartellier, A. (1990). Optical probes for local void fraction measurements:
Characterization of performance. Review of Scientific Instruments, 61(2), 874-886.
Deckwer, W. D. (1992). Bubble Column Reactors. (Wiley, Ed.). Chichester.
Eissa, S. H. and Schugerl, K. (1975). Holdup and backmixing investigation in co- and
counter-current bubble columns. Chemical Engineering Science, 30, 1251-1256.
Freitas, C., Vicente, A. A., Mota, M. and Teixeira, J. A. (1997). A new sampling
device for measuring solids hold-up in a three-phase system. Biotechnology
Techniques, 11(7), 489-492.
Freitas, C. and Teixeira, J. A. (1998a). Solid-phase distribution in an airlift reactor
with an enlarged degassing zone. Biotechnology Techniques, 12(3), 219-224.
Freitas, C. and Teixeira, J. A. (1998b). Hydrodynamic studies in an airlift reactor
with an enlarged degassing zone. Bioprocess and Biosystems Engineering, 18(4),
267279.
96
Chapter IV
Freitas, Carla, Fialov, M., Zahradnik, J., and Teixeira, J. A. (1999). Hydrodynamic
model for three-phase internal-and external-loop airlift reactors. Chemical
Engineering Science, 54, 5253-5258.
Gandhi, B., Prakash, A. and Bergougnou, M. A. (1999). Hydrodynamic behavior of
slurry bubble column at high solids concentrations. Powder Technology, 103(2),
8094.
Gharat, S. D. and Joshi, J. B. (1992). Transport phenomena in bubble column
reactors I: flow pattern. The Chemical Engineering Journal, 48, 141-151.
Hyndman, C. L., Larachf, F. and Guy, C. (1997). Understanding gas-phase
hydrodynamics in bubble columns!: a convective model based on kinetic theory.
Chemical Engineering Science, 52(1), 63-77.
Jamialahmadi, M. and Muller-Steinhagen, H. (1991). Effect of solid particles on gas
hold-up in bubble columns. Canadian Journal of Chemical Engineering, 69, 390393.
Joshi, J.B. and Lali, A. M. (1984). Velocity-hold up relationship in multiphase
contactorsA unifed approach. Frontier in chemical reaction engineering. New
Delhi: Wiley.
Krishna, R. (1991). A Model For Gas Holdup In Bubble Columns Incorporating The
Influence Of Gas Density On Flow Regime Transitions, 46, 2491-2496.
Krishna, R., Ellenberger, J. and Maretto, C. (1999). Flow regime transition in bubble
columns. International Communications Heat Mass Transfer, 26(4), 467-475.
Lu, W-J., Hwang, S-J. and Chang, C-M. (1995). Liquid velocity and gas holdup in
three-phase internal loop airlift reactors with low-density particles. Chemical
Mena, P. C., Ruzicka, M. C., Rocha, F. A., Teixeira, J. A. and Drahos, J. (2005a).
Effect of solids on homogeneousheterogeneous flow regime transition in bubble
columns. Chemical Engineering Science, 60(22), 6013-6026.
Mena, P. C., Pons, M. N., Teixeira, J. A. and Rocha, F. A. (2005b). Effect of Solids
on Gas-Liquid Mass Transfer and Bubble Characteristics in Three-Phase Systems.
7th World Congress of Chemical Engineering.
Mena, P. C., Rocha, F. A., Teixeira, J. A., Sechet, P., and Cartellier, A. (2008).
Measurement of gas phase characteristics using a monofibre optical probe in a
three-phase flow. Chemical Engineering Science, 63(16), 4100-4115.
Mussatto, S. I., Aguilar, C. N., Rodrigues, L. R. and Teixeira, J. a. (2009a).
Colonization of Aspergillus japonicus on synthetic materials and application to the
production of fructooligosaccharides. Carbohydrate research, 344(6), 795-800.
Mussatto, S. I., Aguilar, C. N., Rodrigues, L. R. and Teixeira, J. A. (2009b).
Fructooligosaccharides and B-fructofuranosidase production by Aspergillus
japonicus immobilized on lignocellulosic materials. Journal of Molecular Catalysis
B: Enzymatic, 59, 76-81.
Reilly, I. G., Scott, D. S., de Bruijn, T. J. W. and MacIntyre, D. (1994). The role of
gas phase momentum in determining gas holdup and hydrodynamic flow regimes
in bubble column. Canadian Journal of Chemical Engineering, 72, 3-12.
Riquarts, H. P. (1979). Model representation of homogeneous and heterogeneous
two-phase flow in fluidized beds and bubble columns. German Chemical
Engineering, (2), 268274.
Ruzicka, M. C., Zahradnik, J., Draho!, J., and Thomas, N. H. (2001a). Homogeneousheterogeneous regime transition in bubble columns. Chemical Engineering
Science, 56(15), 46094626
Ruzicka, M. C., Draho!, J., Fialov, M. and Thomas, N. H. (2001b). Effect of bubble
column dimensions on flow regime transition. Chemical Engineering Science, 56,
6117-6124.
Ruzicka, M. C. and Thomas, N. H. (2003a). Buoyancy-driven instability of bubbly
layers: analogy with thermal convection. International Journal of Multiphase Flow,
29, 249-270.
Ruzicka, M. C., Draho!, J., Mena, P. C. and Teixeira, J. A. (2003b). Effect of
viscosity on homogeneousheterogeneous flow regime transition in bubble
columns. Chemical Engineering Journal, 96(1-3), 1522.
Ruzicka, M. C., Vecer, M. M., Orvalho, S. and Drahos, J. (2008). Effect of surfactant
on homogeneous regime stability in bubble column. Chemical Engineering
Science, 63(4), 951-967.
97
Chapter IV
Schumpe, A. and Grund, G. (1986). The Gas Disengagement Technique for Studying
Gas Holdup Structure in Bubble Columns. The Canadian Journal of Chemical
Engineering, 64, 891-896.
Shaikh, A. and Al-Dahhan, M. H. (2007). A Review on Flow Regime Transition in
Bubble Columns. International Journal of Chemical Reactor Engineering, 5, R1.
Shnip, A. I., Kolhatkar, R. V., Swamy, D. and Joshi, J. B. (1992). Criteria for the
transition from the homogeneous to the heterogeneous regime in two-dimensional
bubble column reactors. International Journal of Multiphase Flow, 18, 705-726.
Vial, C., Camarasa, E., Poncin, S., Wild, G., Midoux, N. and Bouillard, J. (2000).
Study of hydrodynamic behaviour in bubble columns and external loop airlift
reactors through analysis of pressure fluctuations. Chemical Engineering Science,
55, 2957-2973.
Vijayan, M., Schlaberg, H. I. and Wang, M. (2007). Effects of sparger geometry on
the mechanism of flow pattern transition in a bubble column. Chemical
Engineering Journal, 130, 171-178.
Wallis, G. B. (1969). One-dimensional Two-phase Flow. New York: McGraw-Hill.
Warsito, Ohkawa, M., Maezawa, A. and Uchida, S. (1997). Flow structure and phase
distributions in a slurry bubble column. Chemical Engineering Science, 52(21-22),
3941-3947.
Wilkinson, P. M., Spek, A. P. and van Dierendonk, L. L. (1992). Design parameters
estimation for scale-up of high-pressure bubble columns. A.I.Ch.E. Journal, 38,
544-554.
Xie, T., Ghiaasiaan, S. M., Karrila, S. and Mcdonough, T. (2003). Flow regimes and
gas holdup in paper pulp water gas three-phase slurry flow. Chemical
Engineering Science, 58, 1417 - 1430.
Yang, J. H., Yang, J-I., Kim, H-J., Chun, D. H., Lee, H-T. and Jung, H. (2010). Two
regime transitions to pseudo-homogeneous and heterogeneous bubble flow for
various liquid viscosities. Chemical Engineering and Processing: Process
Intensification, 49, 1044-1050.
Yoo, D. H., Tsuge, H., Terasaka, K. and Mizutani, K. (1997). Behavior of bubble
formation in suspended solution for an elevated pressure system. Chemical
Engineering Science, 52(21-22), 37013707.
Zahradnik, J., Fialova, M., Ru!i!ka, M., Draho", J., Ka"tnek, F. and Thomas, N. H.
(1997). Duality of the gas-liquid flow regimes in bubble column reactors. Chemical
98
Chapter V
V. Chapter V Global Hydrodynamic

Properties of Three-phase iGLR
V.1 Objectives
V.2 Introduction
V.3 Material and Methods
V.4 Results and Discussion
V.5 Conclusions
V.6 References
99
Chapter V
V.1 Objectives
T h e m a in o b je c tiv e o f th is c h a p te r w a s to d e te rm in e th e in flu e n c e o f
S G lo a d in g o n h y d ro d y n a m ic p a ra m e te rs (s o lid s a n d g a s h o ld-u p s ,
liq u id
v e lo c ity ,
c irc u la tio n /m ix in g
tim e s )
fo r
th re e
d iffe re n t
c o n fig u ra tio n s o f a n iG L R w ith e n la rg e d to p d e g a s s in g z o n e . T h e m a in

g o a l w a s to d e te rm in e th e b e s t iG L R d e s ig n th a t c o n fe rs s u ita b le
h y d ro d y n a m ic c o n d itio n s fo r a fe rm e n ta tio n o f A F B in c o n tin u o u s m o d e .
V.2 Introduction
G a s -lift re a c to r h y d ro d y n a m ic s h a v e b e e n s tu d ie d o v e r th e p a s t y e a rs
d u e to th e ir a b ility to b e u s e d in s e v e ra l c h e m ic a l a n d b io te c h n o lo g y
p ro c e s s e s .
The
im p o rta n c e
of
s tu d y in g
th e
g a s-lift
re a c to r
h y d ro d y n a m ic s o n b io te c h n o lo g y p ro c e s s e s h a s b e e n a lre a d y re fe rre d
e s p e c ia lly in fe rm e n ta tio n p ro ce s s e s , w h e re s o lid p h a s e w ith s im ila r
d e n s ity to fe rm e n ta tio n b ro th s is p re s e n t (F re ita s a n d T e ix e ira , 1 9 9 8 ;
K le in e t a l., 2 0 0 3 ).
In th is th e s is (C h a p te r III a n d V III) tw o s o lid s h a v e b e e n s tu d ie d fo r
c o n tin u o u s A F B p ro d u c tio n in an iG L R . T h e re s u lts p re s e n te d in
C h a p te r V III (s e c tio n V III.3 .1 ) in d ic a te th a t b o tto m -fe rm e n tin g s tra in s
u s in g S G a s c a rrie r a re s u ita b le fo r th e c o n tin u o u s p ro d u c tio n o f A F B in
a n iG L R . In C h a p te r IV th e in flu e n c e o f S G a s s o lid-p h a s e o n th e re g im e
flo w
tra n s itio n
in
BC,
as
w e ll
as
th e
p o s s ib le
in v o lv ed
p h y s ic a l
m e c h a n is m s a re s tu d ie d . T h e p re v io u s re s u lts (C h a p te r IV ) s h o w e d th a t
th e p ro p e rtie s o f S G a re v e ry im p o rta n t, b e c a u s e th e y re d u c e th e tim e
w h e re th e H o R is p re s e n t.
T h e b e h a v io u r o f S G in th e iG L R h y d ro d y n a m ic s is s till u n k n o w n .
M o re o v e r, it is a ls o im p o rta n t to w e ll d e fin e s o m e a s p e c ts o f th e iG L R
ris e r c o n fig u ra tio n : a c ru c ia l d e s ig n p a ra m e te r fo r th is ty p e o f re a c to rs
(K le in
et
a l.,
2 0 0 3 ).
In
o rd e r
to
e s ta b lis h
th e
lim its
w h e re
th e
b io te c h n o lo g y p ro c e s s e s fo r A F B u s in g S G in a n iG L R c a n o p e ra te , it is
im p o rta n t to u n d e rs ta n d h o w th e s e c e llu lo s e-b a s e d p a rtic le s c a n a ffe c t
th re e -p h a s e iG L R h y d ro d y n a m ic s .
100
Chapter V
V.2.1 Gas-lift reactor design and configurations

T h e g a s -lift re a c to r is a m o d ifie d b u b b le c o lu m n a n d th re e g e n e ra l
c o n fig u ra tio n s a re c o n s id e re d : in te rn a l, s p lit o r e x te rn a l (F ig u re V - 1 ).
Figure V-1. Different configurations used in GLR. Legend: a) Internal-loop

GLR (iGLR); b) Split GLR; c) External-loop GLR.
A m o n g th e G L R ty p e s , d iffe re n t s p e c ific c o n fig u ra tio n s a re p re s e n t:

(1 ) th e p o s itio n o f th e g a s d is trib u to r; (2 ) p re s e n c e o r a b s e n c e o f a n
e n la rg e d to p s e c tio n . T h e s e s p e c ific a tio n s d e p e n d m a in ly o n th e ir fin a l
a p p lic a tio n (F re ita s a n d T e ix e ira , 1 9 9 8 ; V a n B e n th u m e t a l., 2 0 0 0 ).
T h e g -l s e p a ra tio n in a n iG L R ta k e s p la c e a t th e to p p a rt o f th e
re a c to r. In te rm s o f iG L R te rm in o lo g y , th e p re s e n c e o f a s e p a ra to r is
c o n s id e re d
when:
DSEP>DC
(C h is ti,
1989;
K le in
et
a l.,
2 0 0 3 ).
The
p re s e n c e o f a n e n la rg e d to p s e c tio n is u s u a lly a p p lie d to e a s ie r th e g a s

re le a s e . In a d d itio n it a llo w s th e s e d im e n ta tio n o f p a rtic le s th a t m a y b e
p re s e n t in th e liq u id m e d iu m th a t a re e x p e c te d to b e k e p t in d e fin ite ly
in s id e th e iG L R (fo r e x a m p le w h e n c o n tin u o u s m o d e o f o p e ra tio n is
a p p lie d ). T h is is a c h ie v e d b e c a u s e th e c ro s s-s e c tio n a l a re a in c re a s e
re d u c e s th e d o w n w a rd s liq u id (a n d s o lid s ) v e lo c ity in to th e d o w n c o m e r
a n d th u s a b e tte r g a s d is e n g a g e m e n t a n d s o lid s s e d im e n ta tio n o c c u rrs
(D o lg o s e t a l., 2 0 0 1 ). D u e to th e s e re a s o n s , th e iG L R u s e d in p re v io u s
e x p e rim e n ts w a s e q u ip p e d w ith a d e g a s s in g s e c tio n in th e to p p a rt
(F ig u r e V - 3 ). In F ig u r e V - 3 it is p o s s ib le to o b s e rv e th e p re s e n c e o f a
101
Chapter V
d o u b le -b a rrie r in th e re a c to r o u tle t. T h is d o u b le-b a rrie r h e lp s in th e

re d u c tio n o f th e b io c a ta ly s t lo s s e s w h e n w e a re o p e ra tin g th e iG L R a t
c o n tin u o u s m o d e . T h e o u te r b a rrie r is o p e n e d a t b o tto m a n d to p a n d
th e in n e r b a rrie r is o p e n e d o n ly a t th e to p . T h u s , it is th e in n e r b a rrie r
th a t d e fin e s th e re a c to r c o n te n ts le v e l. T h is a rra n g e m e n t o b lig e s liq u id
p h a s e to a s p e c ific m o v e m e n t: in itia lly th e liq u id p h a s e g o e s d o w n to
p a s s th e firs t b a rrie r a n d th e n u p w a rd s to le a v e th e re a c to r. T h e firs t
b a rrie r a v o id s n o t o n ly th e p re s e n c e o f g a s-p h a s e d u rin g th e liq u ids o lid s e p a ratio n b u t a ls o re d u c e s th e liq u id flu c tu a tio n s n e a r th e in n e r
b a rrie r. W h e n th e liq u id is g o in g u p w a rd s in th e s p a c e b e tw e e n th e tw o
b a rrie rs , it is th e n e a s ie r to c o m p le te th e s e p a ra tio n o f b ig g e r p a rtic le s
(c a rrie r, y e a s t flo c s , e tc ), w h ic h th e o p e ra to r w a n ts to m a in ta in in s id e
th e re a c to r. In o u r c a s e , w e w a n t to k e e p th e b io c a ta ly s t (S G + y e a s t
c e lls )
in s id e
th e
iG L R
d u rin g
lo n g-te rm
fe rm e n ta tio n
and
th is
c o n fig u ra tio n w ith th e e n la rg e d to p s e c tio n p lu s th e d o u b le-b a rrie r in

o u tle t, p la y s a n im p o rta n t ro le to a v o id b ig lo s s e s o f b io c a ta ly s t.
T h e m a jo rity o f p u b lis h e d p a p e rs a b o u t th e in flu e n c e o f re a c to r
c o n fig u ra tio n o n its h y d ro d y n a m ic s s tu d ie s th e e ffe c t o f: ra tio b e tw e e n
d o w n c o m e r a n d ris e r a re a s (A d /A r ), ris e r le n g th (H r ), g a s s p a rg e r d e s ig n
a n d lo c a tio n , liq u id h e ig h t a n d th e p re s e n c e /a b s e n c e o f a n e n la rg e to p
d e g a s s in g s e c tio n (K le in e t a l., 2 0 0 3 ; L u e t a l., 1 9 9 5 ; O liv ie ri e t a l.,
2 0 0 7 ). K le in e t a l. (2 0 0 3 ) in d ic a te d th a t u s in g th e s a m e e n la rg e d to p
d e g a s s in g z o n e , s im p ly b y c h a n g in g th e ris e r le n g th d iffe re n t iG L R
c o n fig u ra tio n s c a n b e a c h ie v e d . It is s u g g e s te d th a t if th e ris e r to p e n d
m a tc h e s
th e
o p e n in g
of
to p
s e c tio n ,
it
is
o n ly
c o n s id e re d
one
s e p a ra to r. T h e s e p a ra tio n o f g a s a n d s o lid p h a s e s fro m th e liq u id th e n

ta k e s p la c e o n ly in th e e n la rg e d to p d e g a s s in g s e c tio n . O n th e o th e r
h a n d , if th e ris e r to p e n d is b e lo w th e to p s e c tio n o p e n in g th e n it is
c a lle d a d u a l s e p a ra to r (T a b le V - 1 a n d F ig u r e V - 3 ). T h is d u a l s e p a ra to r
c o n s is ts o f tw o p a rts : th e lo w e r p a rt a c tin g a s a b u b b le s e p a ra to r a n d a
to p p a rt (e n la rg e d z o n e ) a c tin g a s a p a rtic le s e p a ra to r (K le in , e t a l.,
2 0 0 3 ).
102
Chapter V
T h e ty p e s o f s p a rg e rs c o m m o n ly u s e d in b io te c h n o lo g y p ro c e s s e s
a re th e p e rfo ra te d p la te a n d in a le s s e r d e g re e th e p o ro u s p la te s .
D iffe re n t s p a rg e rs o rig in a te d iffe re n t b u b b le s s iz e s , w h ic h c a n h a v e a
s ig n ific a n t e ffe c t o n re a c to r h y d ro d y n a m ic s . G e n e ra lly s in te re d p la te s
w ith p o ro s ity 1 a n d p e rfo ra te d p la te s w ith 0 .5 m m o rific e s a n d 0 .2 %
o f fre e p la te a re a ra tio a re id e a l to o b ta in b o th flo w re g im e s (H o R a n d
H e R ) (Z a h ra d n ik e t a l., 1 9 9 7 ). In a n iG L R it is b e lie v e d th a t s p a rg e r
lo c a tio n h a s a h ig h e r e ffe c t o n b u b b le s d istrib u tio n a n d o n th e re a c to r's
h y d ro d y n a m ic s (C h is ti, 1 9 8 9 ) th a n its ty p e (p o ro u s o r p e rfo ra te d p la te ).
F o r s in te re d p la te s w ith p o ro s ity 1 a n d p e rfo ra te d p la te s w ith 0 .5 m m
o rific e s a n d 0 .2 % th is is v a lid s in c e th e b u b b le s fo rm e d a re s im ila r in
s iz e a n d s h a p e .
T h e th re e -p h a s e iG L R h y d ro d y n a m ic b e h a v io u r in v o lv e s th e s tu d y o f
s e v e ra l p a ra m e te rs a s : g a s a n d s o lid h o ld-u p , m ix in g tim e , c irc u la tio n
tim e a n d liq u id v e lo c ity in ris e r a n d d o w n c o m e r (F re ita s a n d T e ix e ira ,
1 9 9 8 a ; K le in e t a l., 2 0 0 3 ; K o rp ija rv i e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ;
M e rc h u k e t a l., 1 9 9 8 ; O liv ie ri e t a l., 2 0 0 7 ).
T h e in te ra c tio n b e tw e e n a ll th e s e p a ra m e te rs w ith th e o p e ra tio n
p a ra m e te rs (g a s flo w , s o lid c o n te n t, liq u id a n d s o lid s p ro p e rtie s ) w ill
d e te rm in e th e b e s t iG L R c o n fig u ra tio n to b e u s e d o n e a c h p ro c e s s . A
c o m p ro m is e b e tw e e n re a c to r c o n fig u ra tio n , o p e ra tio n p a ra m e te rs a n d
h y d ro d y n a m ic p ro p e rtie s is e s s e n tia l to e s ta b lis h th e b e s t c o n d itio n s to
a c h ie v e b e tte r fe rm e n ta tio n p e rfo rm a n c e s .
V.2.2 Gas-phase global properties and methods

In iG L R th e g a s-p h a s e is in je c te d in th e ris e r (d ra ft tu b e ) p ro m o tin g
th e m ix in g a n d liq u id c irc u la tio n in s id e th e re a c to r (F ig u re V - 1 .a ). It is
k n o w n th a t s o lid s in flu e n c e th e b e h a v io u r o f g a s-liq u id (g -l) m ix tu re .
T h is in flu e n c e c a n b e e ith e r in g a s-p h a s e a n d /o r in liq u id -p h a s e . In
o rd e r
to
u n d e rs ta n d
th e
re a l
s o lid
in flu e n c e ,
it
is
im p o rta n t
to
u n d e rs ta n d a n d c h a ra c te riz e liq u id a n d g a s p h a s e s e ith e r in th e g -l a n d

in g a s -liq u id -s o lid (g -l-s ) s y s te m s .
103
Chapter V
T h e in flu e n c e o f s o lid s in th e g a s-p h a s e is c h a ra c te riz e d b y : (1 )

c h a n g in g b u b b le s p ro p e rtie s s u c h a s : s h a p e , ris e a n d fo rm a tio n v e lo c ity
(F a n e t a l., 1 9 9 9 ; Y a n g e t a l., 2 0 0 7 ; Y o o e t a l., 1 9 9 7 ); (2 ) a lte re d ra d ia l
a n d a x ia l g a s-p h a s e (h o ld -u p ) p ro file s (G a n d h i e t a l., 1 9 9 9 ; W a rs ito e t
a l., 1 9 9 7 ); (3 ) in flu e n c e m ix in g a n d d is p e rs io n (M a ts u m o to e t a l., 1 9 8 9 );
(4 ) m o d ifie d g a s h o ld -u p (B a n is i e t a l., 1 9 9 5 a , 1 9 9 5 b ; G a n d h i e t a l.,
1 9 9 9 ; J a m ia la h m a d i e t M u lle r-S te in h a g e n , 1 9 9 1 ) a n d flo w re g im e s
p ro file (M e n a e t a l., 2 0 0 5 ; W a rs ito e t a l., 1 9 9 7 ; X ie e t a l., 2 0 0 3 ).
G e n e ra lly , in iG L R th e p re s e n c e o f s o lid s d e c re a s e s th e g a s h o ld -u p
in th e ris e r. T h is d e c re a s e is b ig g e r a t g a s s u p e rfic ia l v e lo c itie s h ig h e r
th a n 0 .2 m /s . D e p e n d in g o n th e iG L R c o n fig u ra tio n th e g a s-p h a s e m a y
o r n o t b e p re s e n t in th e d o w n c o m e r. G e n e ra lly b u b b le s e n te r in th e
d o w n c o m e r w h e n g a s s u p e rfic ia l v e lo c itie s a re b e tw e e n 0 .1 m /s a n d
0 .3 m /s (F re ita s e t a l., 1 9 9 8 a ; K le in e t a l. 2 0 0 3 a ). A t th e s e c o n d itio n s ,
liq u id v e lo c ity in d o w n c o m e r is s u c h th a t it d ra g s b u b b le s to th is p a rt
o f th e re a c to r. T h e s o lid s a ls o in flu e n c e th e p re s e n c e o f g a s-p h a s e in
th e d o w n c o m e r o f iG L R , e s p e c ia lly w h e n in th e p re s e n c e o f a d u a l
s e p a ra to r. F re ita s e t a l (1 9 9 8a ) a n d K le in e t a l (2 0 0 3a ) re s u lts in d ic a te d
th a t th e p re s e n c e o f s o lid s (u p to 2 0 % (v o l./v o l.)) o n th e s a m e iG L R
c o n fig u ra tio n (A d /A r = 3 .6 7 ; d u a l s e p a ra to r to p s e c tio n ) in c re a s e s th e
d o w n c o m e r g a s h o ld -u p a t lo w e r g a s s u p e rfic ia l v e lo c itie s (0 .1 m /s to
0 .2 m /s ). T h e re is a d u a l e ffe c t o f s o lid s in d o w c o m e rs g a s h o ld -u p : a t
lo w g a s s u p e rfic ia l v e lo c itie s (u p to 0 .3 m /s ) s o lid s in c re a s e g a s h o ldu p in d o w n c o m e r, a t h ig h e r v e lo c itie s (> 0 .4 m /s ) s o lid s p re s e n c e
d e c re a s e s th e d o w n c o m e r g a s h o ld-u p (F re ita s a n d T e ix e ira , 1 9 9 8a ;
K le in e t a l., 2 0 0 3a ). A p o s s ib le e x p la n a tio n fo r th is d u a l e ffe c t lie s in
th e c o m b in e d e ffe c t o f c o a le s c e n c e a n d th e b u b b le s lip v e lo c ity . In
d o w n c o m e r, b u b b le s lip v e lo c ity h a s a d irec t in flu e n c e o n g a s h o ld -u p .
If b u b b le s s lip v e lo c ity is s m a lle r th a n th e d o w n c o m e rs liq u id lin e a r
v e lo c ity , b u b b le s a re e n tra in e d b y th e liq u id . T h e s p a c e o c c u p ie d b y
th e s o lid s re d u c e s th e s p a c e fo r b u b b le s to flo w a n d th e h in d ra n c e
cause
by
it
d e c re a s es
th e ir
s lip
v e lo c ity .
M o re o v e r
at
lo w
gas
s u p e rfic ia l v e lo c itie s , th e c o a le s c e n c e p h e n o m e n a is re d u c e d , th u s
b u b b le s a re s m a ll a n d w ith lo w s lip v e lo c ity . B u b b le s w ith th e s e
104
Chapter V
c h a ra c te ris tic s a re e a s ily a rre s te d to d o w n c o m e r in c re a s in g th e g a s

h o ld -u p in th is s e c tio n . O n th e o th e r h a n d , a t h ig h g a s s u p e rfic ia l
v e lo c itie s th e in te ra c tio n s b e tw e e n b u b b le s in c re a s e d u e to th e ir
a m o u n t a n d le s s s p a c e a v a ila b le (o c c u p ie d b y s o lid s ), w h ic h p ro m o te s
c o a le s c e n c e . A t th is p o in t th e c o a le s c e n c e im p a c t is h ig h e r th a n th e
h in d ra n c e c a u s e d b y s o lid s p re s e n c e . C o n s e q u e n tly th e b u b b le s a re
b ig g e r w ith h ig h s lip v e lo c ity a n d th e n fe w e r b u b b le s a re e n tra in e d to
th e d o w n c o m e r re d u c in g th e g a s h o ld-u p in th is p a rt o f th e iG L R . It is
im p o rta n t to re fe r th a t th is is u s e d to e x p lain th e d o w n c o m e rs g a s
h o ld -u p d iffe re n c e b e tw e e n th e g -l-s a n d g -l s y s te m s . M o re o v e r, th is
m a y n o t b e a s s im p le a s it s e e m s , b e c a u s e in G L R s th e re a re a lo t o f
d iffe re n t m e c h a n is m s th a t m a y in flu e n c e g a s h o ld-u p in d o w n c o m e r a n d
th e im p a c t o f e a c h o n e o f th e m is n o t y e t c le a r (C h is ti, 1 9 9 8 ).
T h e m e c h a n is m b y w h ic h s o lid s in flu e n c e th e g lo b a l g a s h o ld-u p h a s
b e e n s tu d ie d a n d s o m e a tte m p ts h a v e b e e n m a d e to e x p la in it (B a n is i
e t a l, 1 9 9 5 a , 1 9 9 5 b ; M e n a e t a l., 2 0 0 5 ). T h e s o lid s in flu e n c e h a s b e e n
id e n tifie d m a in ly fo r B C . T h e s o lid p h y s ic a l m e c h a n is m in flu e n c e in G L R
s h o u ld b e id e n tic a l to B C , e s p e c ia lly in ris e r w h e re th re e-p h a s e s a re
a lw a y s p re s e n t. T h e o re tic a lly a n d d u e to th e d iffe re n c e s b e tw e e n th e s e
re a c to rs (liq u id flo w , d e s ig n , m ix in g p ro file ), th e in te n s ity o f e a c h
m e c h a n is m m ig h t b e d iffe re n t.
To
d e te rm in e
g e n e ra lly
g lo b a l
n o n -in v a s iv e
gas
h o ld -u p
te c h n iq u e s
in
a re
th e
ris e r
used.
In
and
iG L R
downcomer
d e s ig n
th e
p re s s u re d ro p b e tw e e n tw o p o in ts in th e ris e r/d o w n c o m e r, a n d th e
D y n a m ic G a s D is e n g a g e m e n t te c h n iq u e s a re c o m m o n ly a p p lie d (B o y e r
e t a l., 2 0 0 2 ; D e c k w e r, 1 9 9 2 ). P re s s u re d ro p b e tw e e n tw o p o in ts a llo w s
to m e a s u re th e liq u id o r g a s h o ld -u p a n d c a n b e o b tain e d b y d iffe re n tia l
p re s s u re s e n s o rs (B o y e r e t a l., 2 0 0 2 ; F re ita s e t a l., 2 0 0 0 ; K le in , e t a l.,
2 0 0 3 ) o r b y a in v e rte d m a n o m e te r tu b e (C h is ti, 1 9 8 9 ; M e rc h u k e t a l.,
1 9 9 8 ) u s e d in th is w o rk F ig u re V - 2 .
105
Chapter V
Figure V-2. Inverted tube manometer arrangement. (Adapted from Chisti,

1989)
In tw o p h a s e -flo w , th e g a s h o ld -u p is d e te rm in e d a c c o rd in g to :
!G =
"L
d
$ h
"L # "G d12
Eq. V-1
W h e re d h is th e d is ta n c e b e tw e e n liq u id c o lu m n s (F ig u r e V - 2 ) a n d d 1 2
is th e d is ta n c e b e tw e e n th e m e a s u rin g p o in ts (1 a n d 2 in F ig u r e V - 2 ).
Is im p o rta n t to n o te th a t in g -l-s s y s te m s th e p re s s u re d ro p p e r s e is
n o t e n o u g h to m e a s u re a ll p h a s e s h o ld-u p a n d a n a d d itio n a l te c h n iq u e
m u s t b e u s e d . G e n e ra lly , it is p re fe rre d to u s e a d iffe re n t te c h niq u e to
m e a s u re th e s o lid -p h a s e h o ld -u p (B o y e r e t a l., 2 0 0 2 ; F re ita s a n d
T e ix e ira , 1 9 9 8 a , 1 9 9 8 b ; K le in , e t a l., 2 0 0 3 ).
V.2.3 Solid-phase global properties and methods

In iG L R n o t o n ly s o lid in flu e n c e o n g a s h o ld -u p is im p o rta n t, b u t a ls o
s o lid d is trib u tio n th ro u g h o u t th e re a c to r. F re ita s a n d T e x e ira (1 9 9 8 b )
fo u n d th a t fo r lo w -d e n s ity p a rtic le s (a lg in a te b e d s ) in a n iG L R th e re is
a n a x ia l a n d ra d ia l s o lid s d is trib u tio n a t lo w g a s flo w s (F re ita s a n d
T e ix e ira , 1 9 9 8 b ). U s in g th e s a m e s o lid s , K le in e t a l (2 0 0 3a , 2 0 0 3 b )
106
Chapter V
fo u n d th a t s o lid d is trib u tio n a re a ls o a ffe c te d b y ris e r c o n fig u ra tio n

w h e re u n ifo rm s o lid d is trib u tion is a c h ie v e d a t lo w e r ra tio b e tw e e n
d o w n c o m e r a n d ris e r (K le in e t a l., 2 0 0 3 a ). T h e w o rk o f K le in e t a l.
(2 0 0 3 a ) s h o w e d th a t fo r a s m a ll s o lid lo a d g a s v e lo c ity d o e s n o t h a v e a
b ig in flu e n c e o n s o lid d is trib u tio n a n d th e s e a re m a in ly lo c a te d in th e
b o tto m p a rt o f b o th ris e r a n d d o w n c o m e r fo r d iffe re n t in te rn a l d ra ft
c o n fig u ra tio n s (K le in e t a l., 2 0 0 3a ). T h e re fo re , it is e x p e c te d to h a v e
s im ila r s o lid s d is trib u tio n fo r o u r re a c to r c o n fig u ra tio n , d u e to s im ila r
p ro p e rtie s b e tw e e n S G a n d a lg in a te b e d s . M o re o v e r, in a fe rm e n ta tio n
u s in g S G a s ca rrie r fo r b io m a s s im m o b iliz a tio n th e s o lid c o n c e n tra tio n s
u s e d a re n o rm a lly u p to 1 2 % (w t. W E T
BASIS
/v o l.) (K le in e t a l., 2 0 0 3a ;
B r n y ik e t a l., 2 0 0 2 ). F ro m K le in e t a l. (2 0 0 3 ) w o rk it is n o t c le a r if ris e r
le n g th h a s a n y in flu e n c e o n s o lid s d is trib u tio n fo r h ig h e r ra tio s b e tw e e n
ris e r a n d d o w n c o m e r c ro s s-s e c tio n (A d /A r = 3 .6 7 ), n o rm a lly fo u n d in iG L R
fo r c o n tin u o u s A F B p rim a ry fe rm e n ta tio n (L e h n e rt e t a l. 2 0 0 8 , 2 0 0 9 ) a n d
th e s a m e u s e d in C h a p te r V III.
S e v e ra l
m e th o d s
can
be
used
to
m e a s u re
s o lid s
h o ld-u p
and
d is trib u tio n . T h e s e m e th o d s c a n b e e ith e r (1 ) n o n-in v a s iv e u s in g a

tra c e r (B o y e r e t a l., 2 0 0 2 ) o r p re s s u re d ro p (K le in e t a l., 2 0 0 3 a ;
R o d rg u e z e t a l., 1 9 9 9 ); a n d /o r (2 ) in v a s iv e u s in g s a m p le rs (F re ita s a n d
a l., 1 9 9 9 ). T ra c e r m e th o d s a re n o rm a lly a p p lie d in flu id iz a tio n c o lu m n s .
T h e s o lid tra c e r is a p a rtic le th a t s h o u ld h a v e th e s a m e s h a p e s iz e a n d
d e n s ity
of
s tu d y
p a rtic le s .
T h e re
a re
d iffe re n t
k in d s
of
tra c e rs :
c o lo u re d , m a g n e tic a n d flu o re s c e n t (B o y e r e t a l., 2 0 0 2 ). W e n g e a t a l

(1 9 9 9 ) c ite d b y K le in e t a l (2 0 0 3 a ) in itia lly p ro p o s e d th e p re s s u re d ro p
m e th o d . T h is m e th o d s ta rts b y th e m e a s u re m e n t o f h y d ro s ta tic p re s s u re
in th e g -l-s d is p e rs io n fo llo w e d b y in te rru p tio n o f g a s flo w (c o m p le te
g a s d is e n g a g e m e n t). T h e n a s e c o n d m e a s u re m e n t in th e re s u ltin g tw op h a s e (s o lid liq u id ) d is p e rs io n is p e rfo rm e d . T h e m e a s u re m e n t p e riod
h a s to b e s h o rt e n o u g h to a v o id s ig n ific a n t s e d im e n ta tio n o f th e s o lid
p a rtic le s . K le in e t a l. (2 0 0 3a ) u s e d th is m e th o d fo r a lg in a te b e a d s a n d
th e m e a s u rin g tim e w a s a ro u n d 2 5 0 s (K le in e t a l., 2 0 0 3 a , 2 0 0 3 b ).
H o w e v e r th e S G s s e d im e n ta tio n is v e ry fa s t a n d fo rm e d a c o m p a c t
la y e r in le s s th a n a m in u te , w h ic h is v e ry h a rd to re m o v e w ith o u t
107
Chapter V
p h y s ic a l in te rfe ren c e . M o re o v e r, th is m e th o d la c k s in fo rm a tio n a b o u t

s o lid s d is trib u tio n , w h ic h is a ls o a v e ry im p o rta n t p a ra m e te r in iG L R .
S a m p le r d e v ic e s a s th e d e v e lo p e d b y F re ita s e t a l. (1 9 9 7 ) c a n b e v e ry
u s e fu l fo r s o lid s h o ld -u p d e te rm in a tio n a n d a llo w a ls o to s tu d y th e
s o lid s d is trib u tio n in th e iG L R w ith re la tiv e e rro rs le s s th a n 1 0 %
(F re ita s a n d T e ix e ira , 1 9 9 7 , 1 9 9 8 b ).
V.2.4 Liquid-phase global properties and methods

It is a ls o im p o rta n t to ta k e in to a c c o u n t th e liq u id -p h a s e p ro p e rtie s
in th re e -p h a s e s y s te m s , m a in ly th e c h a n g e s th a t o c c u r in th is p h a s e .
T h e s e m a y o r m a y n o t b e c a u s e d b y th e p re s e n c e o f s o lid s . U s u a lly ,
c irc u la tio n a n d m ix in g tim e s a re im p o rta n t p a ra m e te rs to s tu d y th e
m ix in g a n d d is p e rs io n o f th e iG L R . A s s o c ia te d to th is , liq u id v elo c itie s
in d o w n c o m e r a n d ris e r a re n o rm a lly d e te rm in e d . L iq u id v e lo c ity is a
c ru c ia l p a ra m e te r th a t is a p p lie d in s e v e ra l m o d e ls fo r d e s c rib in g th e
iG L R h y d ro d y n a m ic s (C h is ti, 1 9 8 9 ; F re ita s e t a l., 1 9 9 9 ; G a rc ia-C a lv o e t
a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ) a n d a ls o u s e d w h e n s c a le-u p is p e rfo rm e d
(C h is ti, 1 9 8 9 ; F re ita s e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ; R o d rg u e z e t a l.,
1 9 9 9 ).
T o m e a s u re th e p a ra m e te rs lis te d a b o v e , tra c e r te c h n iq u e s a re
a p p lie d . D iffe re n t k in d s o f tra c e rs c a n b e u s e d : th e rm a l (G a rc ia -C a lv o
e t a l., 1 9 9 9 ), a c id (F re ita s a n d T e ix e ira , 1 9 9 8 a ; L u e t a l., 1 9 9 5 ; M e rc h u k
e t a l., 1 9 9 8 ; C h is ti, 1 9 8 9 ) a n d m a g n e tic (B la e t a l., 2 0 0 4 ; K le in e t a l.,
2 0 0 4 ; V a n B e n th u m e t a l., 2 0 0 0 ) tra c e rs . A c id tra c e rs a re u s e d m a in ly
to d e te rm in e th e d o w n c o m e r liq u id lin e a r v e lo c ity . In s u m m a ry , tw o p H
e le c tro d e s (s a m e re s p o n s e tim e ) a re p lac e d in d o w n c o m e r a n d a p u ls e
o f s u lp h u ric a c id (4 M to 8 M ) is in je c te d a t th e to p o f th e d o w n c o m e r.
T h e liq u id lin e a r v e lo c ity is th e n c a lc u la te d fro m th e e q u a tio n :
v Lr =
delectrodes
!t probes
Eq. V-2
T h e n b y a m a s s b a la n c e (E q . V - 3 ), it is p o s s ib le to d e te rm in e th e
d o w n c o m e r liq u id lin e a r v e lo c ity a n d a ls o th e ris e r a n d d o w n c o m e r
108
Chapter V
liq u id s u p e rfic ia l v e lo c itie s E q . V - 4 (C h is ti, 1 9 8 9 ; F re ita s a n d T e ix e ira ,

1 9 9 8 a ):
Eq. V-3
Eq. V-4
K n o w in g th e le n g th o f ris e r a n d d o w n c o m e r, it is th e n p o s s ib le to
d e te rm in e th e c irc ula tio n tim e b y (C h is ti, 1 9 8 9 ):
Eq. V-5
C irc u la tio n tim e is a ls o re la te d w ith m ix in g tim e , w h ic h is d e fin e d a s
th e tim e (t M ) n e c e s s a ry to o b ta in a re q u ire d d e g re e o f m ix in g (! M ):
Eq. V-6
S e v e ra l m e th o d s a re u s e d to m e a s u re th e m ix in g tim e . T h e s e a re
s im ila r to th e m e th o d s u s e d to m e a s u re liq u id lin e a r v e lo c ity : (1 )
c o n d u c tim e tric ;
(2 )
th e rm a l;
(3 )
a c id ;
(4 )
ra d io a c tiv e ;
and
(5 )
c o lo rim e tric . M ix in g tim e in d ic a te s th e m ix in g q u a lity o f th e re a c to r a n d

d e p e n d s o n iG L R c o n fig u ra tio n a n d d e s ig n (F o n s e c a a n d T e ix e ira ,
2 0 0 7 ). G e n e ra lly , in iG L R , m ix in g tim e d e c re a s e s ra p id ly a t lo w e r g a s
flo w s (0 .0 1 m /s to 0 .3 m /s ), a fte r th a t th e re is a s ta b iliz a tio n a n d o n ly a
b ig in c re a s e o f g a s flo w c a n re d u c e s ig n ific a n tly th e m ixin g tim e
(F re ita s a n d T e ix e ira , 1 9 9 8 a ; K le in e t a l., 2 0 0 3 ; M e rc h u k e t a l., 1 9 9 8 ).
S im ila r b e h a v io u r c a n b e fo u n d in c irc u la tio n tim e (t C ). C o n s id e rin g th a t
c irc u la tio n a n d m ix in g tim e s a re v e ry im p o rta n t p a ra m e te rs o n th e
c h a ra c te riz a tio n o f th e m ix in g p e rfo rm a n c e o f a iG L R , th e m e th o d s u s e d
s h o u ld h a v e e n o u g h tim e re s o lu tio n to c a p tu re th e liq u id flu c tu a tio n s
b e tw e e n th e tw o p o in ts w h e re th e m e a s u re m e n t ta k e s p la c e .
109
Chapter V
V.3 Material and Methods

V.3.1 Gas-lift reactor: dimensions and characteristics
T h e e x p e rim e n ts w e re d o n e in a P e rs p e x iG L R w ith a to p e n la rg e d
d e g a s s in g z o n e (F ig u r e V - 3 ). T h e iG L R h a s to ta l v o lu m e o f 6 0 L a n d th e
re le v a n t d im e n s io n s a re in d ic a te d . M e a s u re m e n ts w e re d o n e fo r th re e
d iffe re n t ris e r tu b e s d e s c rib e d in T a b le V - 1 . T h e ris e rs w e re lo c a te d 2 .3
c m a b o v e th e d is trib u to r.
Table V-1. Riser configurations used in this work.
R is e r L a b e l
R is e r A
R is e r B
R is e r C
Dri / cm
6 .2
6 .2
8 .7
Hr / cm
120
140
120
Dci / cm
1 4 .2
1 4 .2
1 4 .2
A r i /A d
3 .9 7
3 .9 7
1 .7 9
Legend: Dri Riser internal diameter; Hr height of riser; Dci Column

internal diameter; Ari/Ad Ratio between internal riser area and downcomer
area.
T h e g a s e n te re d th e a irlift th ro u g h th e 1 0 c m d iam e te r p o ro u s p la te
s p a rg e r. T h e d ia m e te r o f th e a c tiv e z o n e o f th e s p a rg e r, th ro u g h
w h ic h th e g a s flo w w a s p a s s in g , w a s a ro u n d 8 c m . A ir (2 0 2 5 C )
a n d ta p w a te r (1 8 C 2 2 C ) w e re u s e d a s th e g a s a n d liq u id p h a s e .
T h e n e t w a te r flo w th ro u g h th e a irlift w a s z e ro .
Table V-2. Resume of all conditions used in this work
UAIR/
(c m /s )
1
2 .5
5
7 .5
QG(A/B) /
(L /m in )
1 .9
4 .5
9 .1
1 3 .6
QG(C) /
(L /m in )
3 .6
8 .9
1 7 .8
2 6 .8
% S o lid s
(w t. W E T B A S I S /V o l.)
0; 4; 8; 12; 20
0; 4; 8; 12; 20
0; 4; 8; 12; 20
0; 4; 8; 12; 20
F o u r g a s s u p e rfic ia l v e lo c itie s in th e ris e r ra n g in g fro m 1 to 7 .5 c m /s

w e re s tu d ie d a n d fiv e s o lid lo a d s w e re te s te d (T a b le V - 2 ). T h e a ir flo w
w a s c o n tro lle d b y a 1 0 0 S L P M m a s s flo w c o n tro lle r (A lic a t S c ie n tific s ,
T u c s o n , U S A ).
110
Chapter V
5 cm
42 cm
1 cm
23 cm
35.5 cm
E
6.5 cm
OUT FLOW
17 cm
14.2 cm
25 cm
6.2
cm
34 cm
8.4
cm
LEGENDA:
A - Riser Hold-up measuring points
B - Downcomer Hold-up measuring points
C - Riser pH probes
D - Downcomer pH probes
E - Conductivity probe area
141 cm
120
cm
A
17
cm
C
7.5
cm
Figure V-3. Internal Gas-lift reactor design used in this chapter. Legend: A Riser Hold-up measuring points; B - Downcomer Hold-up measuring points; C
- Riser pH probes; D - Downcomer pH probes; E - Conductivity probe point
V.3.2 Solids distribution and hold-up

T h e s a m e s o lid s u s e d in p re v io u s c h a p te r (s e c tio n IV .3 .2 .1 ) w e re
u s e d in th e p re s e n t c h a p te r. S o lid s h o ld-u p a n d d is trib u tio n w e re
d e te rm in e d b y u s in g th e m e th o d d e v e lo p e d b y F re ita s e t a l (1 9 9 7 ) a s
d e s c rib e d in s e c tio n IV .3 .4 . T h e s a m p le r w a s in tro d u c e d in to th e
re a c to r in s e v e ra l p o in ts : A ,B ,C ,D ,E (F ig u re V - 4 ). T h e n s o lid s v o lu m e
w a s d e te rm in e d a n d s o lid s h o ld-u p c a lc u la te d a c c o rd in g ly E q . IV - 3 :
111
Chapter V
Figure V-4. Solids sampling locations for solid distribution determination.
V.3.3 Gas hold-up

G a s h o ld -u p w a s d e te rm in e d u s in g F re ita s a n d T e ix e ira (1 9 9 8 a )
p ro c e d u re b y m e a s u rin g p re s s u re d iffe re n c e b e tw e e n T o p a n d B o tto m
o f b o th ris e r a n d d o w n c o m e r (d = 1 .0 9 m ). D iffe re n tia l p re s s u re w a s
m e a s u re d
by
w a te r
c o lu m n s
w e ig h t
d iffe re n c e .
For
each
set
of
e x p e rim e n ts , p re s s u re d iffe re n c e s (d h i ) in ris e r a n d d o w n c o m e r w e re

m e a s u re d a t le a s t th re e tim e s d u rin g n o le s s th a n 5 m in u te s . T h e m e a n
v a lu e w a s th e n u s e d to d e te rm in e d g a s h o ld-u p (ris e r/d o w c o m e r) u s in g
th e E q . IV - 2 . E a c h m e a s u re m e n t w a s d o n e in trip lic a te (C h is ti, 1 9 8 9 ;
F re ita s a n d T e ix e ira , 1 9 9 8 a )
V.3.4 Mixing time determination

M ix in g tim e w a s d e te rm in e d a s th e tim e fo r v aria tio n o f N a C l
c o n c e n tra tio n (5 0 m L o f 3 0 0 g /L N a C l s o lu tio n w e re in je c te d a t b o tto m
o f re a c to r (E p o in t in F ig u r e V - 3 )), to b e w ith in 5 % o f fin a l v a lu e
(F re ita s a n d T e ix e ira, 1 9 9 8 a ). E a c h e x p e rim e n t w a s re p e a te d th re e
tim e s .
112
Chapter V
V.3.5 Circulation time determination

C irc u la tio n tim e w a s d e te rm in e d b y in je c tin g 1 0 m L o f H 2 S O 4 s o lu tio n
(4 .5 M ). T h e p H e v a lu a tio n in ris e r a n d d o w n c o m e r w a s re c o rd e d b y 4
p H m e te rs : tw o M e th ro m m o d e l 6 2 0 p ro b e s in th e ris e r a n d tw o m o d e l
691
p ro b e s
c o n n e c te d
to
in
a
th e
PC
d o w n c om e r
w ith
NI
(M e th ro m ,H e ris a u ! ,S w itz e rla n d )
d a ta
a c q u is itio n
b o a rd
(N a tio n a l
In s tru m e n ts , A u s tin , T e x a s , U S A ) a n d re c o rd e d to file s u s in g L a b v ie w

(N a tio n a l In s tru m e n ts , A u s tin , T e x a s , U S A ). B y a v e ra g in g c o n s e c u tiv e
p e a k s in e a c h p H re s p o n s e c u rv e , c irc u la tio n tim e w a s d e te rm in e d
a c c o rd in g to E q . V - 5 (C h is ti, 1 9 8 9 ; F re ita s a n d T e ix e ira , 1 9 9 8 a ).
V.3.6 Liquid velocity determination

T h e liq u id v e lo c ity in ris e r a n d d o w n c o m e r w a s d e te rm in e d fro m th e
c u rv e s
o b ta in e d
on
tw o
pH
m e te rs
(d 1 2 = 1 .0 9 m )
lo c a te d
in
ris e r/d o w n c o m e r. B y k n o w in g th e tim e th e tra c e r ta k e s to tra v e l fro m

o n e e le c tro d e to th e o th e r, th e liq uid v e lo c ity w a s d e te rm in e d u s in g th e
E q . V - 2 . In o rd e r to a v o id m e a s u rin g e rro rs d u e to a c id d is s o c ia tio n
o n ly
th e
in itia l
peaks
in
pH
re s p o n s e
c u rv e s
w e re
used.
Each
e x p e rim e n ta l a s s a y w a s re p e a te d in trip lic a te (C h is ti, 1 9 8 9 ; F re ita s a n d

T e ix e ira , 1 9 9 8 a ).
113
Chapter V
V.4 Results and Discussion

In th is c h a p te r th e m a in h y d ro d y n a m ic p a ra m e te rs fo r th re e d iffe re n t
ris e rs c o n fig u ra tio n in a n iG L R w ith a n e n la rg e d to p s e c tio n w e re
s tu d ie d . T h e m a in o b je c tiv e w a s to c h o o s e o n e o f th e s e c o n fig u ra tio n s
fo r c o n tin u o u s p rim a ry A F B fe rm e n ta tio n . K le in e t a l (2 0 0 3 b ) d e te rm in e d
th a t fo r g -l a n d g -l-s s y s te m s th e m a in h y d ro d y n a m ic p a ra m e te rs (g a s
h o ld -u p , liq u id v e lo c ity , s o lid s h o ld-u p a n d c irc u la tio n tim e ) d o n o t
s u ffe r re le v a n t c h a n g e s w h e n th e re a c to r is o p e ra tin g in b a tc h o r in
c o n tin u o u s m o d e (K le in e t a l., 2 0 0 3b ). T h e m a in re a s o n w a s th e
m a x im u m d ilu tio n ra te u s e d in b io te c h n o lo g y p ro c e s s e s . N o rm a lly it is
b e llo w 0 .6 h - 1 (in A F B p ro d u c tio n b e llo w 0 .4 h - 1 ), s o th e liq u id flo w ra te
th a t p a s s e s th ro u g h o u t th e G L R b io re a c to r is le s s th a n 1 % o f th e liq u id
flo w ra te in s id e th e iG L R . T h e re fo re th e te s ts w e re m a d e u s in g th e
s a m e s o lid s a s in p re v io u s c h a p te r a n d in b a tc h s y s te m . T a p w a te r
in s te a d o f s y n th e tic m in e ra l m e d iu m (S M M ) o r re a l w o rt w a s u s e d in th e
h y d ro d y n a m ic s tu d ie s . T h e m a in w o rk w a s to d e te rm in e th e b e s t re a c to r
c o n fig u ra tio n fo r c o m p le te m ix in g a n d d is trib u tio n o f S G in th e iG L R .
T h e u s e o f S M M o r re a l w o rt w o u ld in c re a s e th e ris k o f c o n ta m in a tio n
b e tw e e n th e d iffe re n t e x p e rim e n ts , w h ic h w o u ld u n d o u b te d ly a lte r th e
liq u id m e d iu m c o m p o s itio n a n d in flu e n c e th e fin a l re s u lts . F re s h ta p
w a te r fro m m u n ic ip a l w a te r c o m p a n y w a s u s e d . E v e ry tw o /th re e d a y s ,
b e fo re te s tin g n e w c o n d itio n s th e e x p e rim e n ts in g -l s y s te m s w e re
re p e a te d in o rd e r to c o n tro l if s o m e re le v a n t c h a n g e s in w a te r (liq u idp h a s e ) c o u ld b e fo u n d , h o w e v e r n o re le v a n t v a ria tio n s w e re d ete c te d .
V.4.1 Solid particles distribution

A s re fe rre d th e s o lid s d is trib u tio n a lo n g th e iG L R is re a lly im p o rta n t,
e s p e c ia lly w h e n fe rm e n ta tio n p ro c e s s e s a re in v o lv e d , b e c a u s e it w ill
re d u c e th e p ro b a b ility o f h a v in g d e a d z o n e s in th e iG L R , w h ic h m a y
le a d to in a p p ro p ria te fe rm e n ta tio n p e rfo rm a n c e s . O n th e fig u re s b e lo w
(F ig u re V - 5 , F ig u r e V - 6 , a n d F ig u re V - 7 ) th e v a lu e s o f s o lid h o ld-u p fo r
a ll c o n fig u ra tio n s a t d iffe re n t g a s flo w a n d s o lid s lo a d a re d is p la y e d .
114
Chapter V
T h e a v e ra g e s o lid lo a d s o b ta in e d a m o n g th e fiv e s e c tio n s u s e d a re

p re s e n te d in T a b le V - 3 . T h e e rro r b e tw e e n th e m e a s u re d v a lu e s a n d th e
e x p e c te d v a lu e s is o n a v e ra g e 7 % w ith a m a x im u m a n d m in im u m e rro r
o f 1 4 % a n d 0 % re s p e c tiv e ly . T h e v a lu e s a re in a g re e m e n t w ith F re ita s
a n d T e ix e ira (1 9 9 8 b ) w h o u s e d th e s a m e m e th o d a n d o b ta in e d a ro u n d
1 0 % e rro r b e tw e e n e x p e rim e n ta l a n d e x p e c te d v a lu e o f s o lid lo a d
(F re ita s a n d T e ix e ira , 1 9 9 8 b ). T a b le V - 3 a ls o in d ic a te s th e v a ria tio n
c o e ffic ie n t, w h ic h m a y b e u s e d a s a n in d ic a to r o f s o lid s d is trib u tio n
in s id e th e iG L R . S ta tis tic a lly th e v a ria tio n c o e ffic ie n t (V C ) in d ic a te s th e
re p ro d u c ib ility o f th e e x p e rim e n ta l p o in ts . T h is c o e ffic ie n t w a s o b ta in e d
fro m th e s o lid s h o ld -u p m e a s u re m e n ts in th e d iffe re n t iG L R a re a s
(A ,B ,C ,D ,E in F ig u r e V - 4 ). In o u r c a s e it c a n b e u s e d to o b ta in a
q u a lita tiv e e v a lu a tio n o f S G s p a rtic le s d is trib u tio n in s id e th e iG L R . In
te rm s o f v a lu e if V C is n e a r 1 0 0 % , it m e a n s th a t th e s o lid s h o ld -u p in
d iffe re n t re a c to r p o s itio n s a re s im ila r, th u s a g o o d s o lid s d is trib u tio n is
o b ta in e d . T h e m a in d ra w b a c k o f u s in g th is c rite rio n fo r e v a lu a tio n o f
s o lid s d is trib u tio n is th a t a t lo w e r s o lid lo a d s th e m e th o d s u ffe rs th e
m a x im u m v a ria tio n s , th u s th e V C c rite ria c a n o n ly b e u s e d to c o m p a re
th e s a m e s o lid lo a d c o n d itio n s .
V.4.1.1
Riser A
R e s u lts o b ta in e d fo r R is e r A p re s e n te d in F ig u r e V - 5 a n d T a b le V - 3
in d ic a te d , a s re fe rre d in th e lite ra tu re , th a t fo r th is c o n fig u ra tio n th e re
is a n a x ia l a n d ra d ia l d istrib u tio n o f s o lid s , w h ic h is m o re p ro n o u n c e d
a t th e tw o lo w e r g a s s u p e rfic ia l v e lo c itie s (1 c m /s a n d 2 .5 c m /s ) (F re ita s
a n d T e ix e ira , 1 9 9 8 b ; K le in e t a l., 2 0 0 3a , 2 0 3 3 b ). M o re s o lid p a rtic le s
a re p re s e n t in th e b o tto m p a rt o f th e re a c to r (s e c tio n s B , C a n d D in
F ig u re V - 4 ), w h ile it is th e o p p o s ite in th e to p p a rt (s e c tio n s A a n d E in
F ig u re V - 4 ). D o lg o s e t a l (2 0 0 1 ) d e fin e th re e m a in re g im e s th a t m a y
o c c u r in th is (ris e r A in a w o rk in g v o lu m e o f 5 0 L ) iG L R c o n fig u ra tio n :
(A ) liq u id flo w s d ire c tly to th e d o w n c o m e r (p a rtic le re s id e n c e tim e fro m
5 s to 1 0 s ); (B ) liq u id p re v a ils in th e e d d ie s b e tw e e n th e to p o f th e ris e r
a n d th e b e g in n in g o f th e e n la rg e d to p s e c tio n (re s id e n c e tim e 1 0 s to
115
20
Chapter V
s ) a n d (C ) th e liq u id th a t flo w s to th e to p e n la rg e d s e c tio n
(re s id e n c e tim e a b o v e 3 0 s ).
Figure V-5. Solids hold-up at different GLR positions (A,B,C,D,E) for riser A
configuration at different gas superficial velocity (1 cm/s; 2.5 cm/s; 5 cm/s
and 7.5 cm/s) in riser.
T h e y fo u n d th a t th e flo w p a tte rn A a n d B o c c u rs m o re o fte n th a n th e

flo w p a tte rn C a t U G r = 4 .6 c m /s . T h e re fo re a t lo w g a s flo w ra te s th e
liq u id flo w s m a in ly in th e ris e r a n d th e d o w n c o m e r, w h ich m a y e x p la in
th e s o lid s d is trib u tio n o b ta in e d fo r th e lo w e r g a s flo w ra te s . T h e s o lid s
d is trib u tio n in o u r c a s e is in flu e n c e d n o t o n ly b y th e liq u id e x c h a n g e
b e tw e e n th e b o tto m a n d th e to p p a rt o f th e re a c to r, b u t a ls o b y th e
s e ttlin g p ro p e rtie s o f th e p artic le s u s e d . T h e s e tw o p h e n o m e n a a re
o p p o s ite . T h e liq u id e x c h a n g e a c ts to e q u a liz e th e s o lid p a rtic le
116
Chapter V
c o n c e n tra tio n s , w h ile th e s e ttlin g p a rtic le s a c t to in c re a s e th e p a rtic le

c o n c e n tra tio n in th e b o tto m p a rt o f th e iG L R . W h e n a lo w ris e r le n g th is
s e t (ris e r A ) th e re is a d u a l s e p a ra to r, w h ic h h a s a n e ffe c t o n th e
re a c to r flo w fie ld o f th e to p p a rt. A t lo w g a s s u p e rfic ia l v e lo c itie s th e
liq u id e x c h a n g e is w e a k e r a n d re g im e A a n d B a re fa v o u re d (D o lg o s e t
a l., 2 0 0 1 ), w h ic h to g e th e r w ith th e p a rtic le s s e ttlin g te n d e n c y le a d s to a
p o o re r s o lid s d is trib u tio n in o u r iG L R . A s th e ris e r tu b e is s h o rte r, th e
e n tra n c e in th e to p e n la rg e d z o n e is fu rth e r a w a y a n d in te rm e d ia te d b y
a tu rb u le n t re g io n (u n d e r th e to p o f th e ris e r tu b e ). L iq u id c irc u la tio n
e d d ie s a re p re s e n te d in th is re g io n , w h ic h a c c o rd in g ly to D o lg o s e t a l
(2 0 0 1 ) in d u c e s a ty p ic a l liq u id b e h a v io u r: m o s t o f th e liq u id th a t re a c h
th is re g io n is e n tra in e d in th e e d d ie s a n d a fte r a s h o rt c irc u la tio n in th is
z o n e g o e s b a c k to th e d o w n c o m e r, w ith o u t re a c h in g th e to p e n la rg e d
s e c tio n .
T h e re fo re
at
lo w e r
gas
s u p e rfic ia l
v e lo c itie s
th e
liq u id
c irc u la tio n fo llo w s th e p a tte rn s A a n d B , c o n s id e rin g th a t S G p a rtic le s

m o v e d in th e s a m e w a y a s th e liq u id flo w s a n d th e ir s e ttlin g p ro p e rtie s
m a k e m o re lik e ly th e e x is te n c e o f a x ia l a n d ra d ia l s o lid s d is trib u tio n .
M o re o v e r, th e a x ia l s o lid d is trib u tio n re s u lts a re in a c c o rd a n c e w ith th e
o b ta in e d b y K le in e t a l (2 0 0 3a ) th a t u s e d a lg in a te b e a d s a n d s im ila r
re a c to r d e s ig n (e x c e p t s p a rg e r c o n fig u ra tio n ).
V.4.1.2
Riser B
In F ig u r e V - 6 it is in d ic a te d th a t n o a x ia l a n d ra d ia l d is trib u tio n s a re
p re s e n t, w ith th e e x c e p tio n o f lo w e s t g a s flo w a n d h ig h s o lid lo a d s
w h e re a lo w e r s o lid c o n te n t in th e c o n ic a l p a rt (s e c tio n E ) m a y b e
d e te c te d . In a d d itio n , th e re s u lts p re s e n te d in T a b le V - 3 fo r th is ris e r
in d ic a te th a t th e m a x im u m v a ria tio n c o e ffic ie n t (1 5 % ) is o b ta in e d a t
m in im u m g a s s u p e rfic ia l v e lo c ity (1 c m /s ) a n d m in im u m s o lid lo a d
(4 % (w t. W E T
BASIS
/v o l.)). C o m p a rin g b e tw e e n ris e r B a n d A it s e e m s th a t
in c re a s in g ris e r le n g th h a s a s ig n ific a n t e ffe c t o n im p ro v in g s o lid

d is trib u tio n in s id e th e re c to r. R is e r B w a s a b le to in c re a s e th e s o lid s
h o m o g e n e ity e v e n a t lo w g a s flo w ra te s .
117
Chapter V
Table V-3. Average of the experimental values of solids hold-up and error* in
iGLR sections.
Riser % SolidsTEO (vol./vol.)
4%
8%
A
12 %
20 %
4%
8%
B
12 %
20 %
4%
8%
C
12 %
20 %
UG /
(cm/s)
% SolidsEXP (vol./vol.)
AVG STDEV
VC
Error*
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
4%
4%
4%
4%
9%
9%
9%
8%
13%
13%
13%
13%
22%
20%
19%
19%
0.017
0.012
0.006
0.008
0.032
0.018
0.014
0.009
0.035
0.012
0.008
0.005
0.027
0.013
0.009
0.012
38.80%
27.69%
14.41%
18.91%
36.84%
21.19%
16.47%
10.14%
26.44%
9.04%
5.79%
3.78%
12.17%
6.69%
4.59%
6.14%
7%
4%
8%
4%
8%
8%
9%
6%
9%
10%
8%
6%
11%
0%
3%
4%
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
4%
4%
4%
4%
9%
9%
9%
9%
13%
13%
13%
13%
21%
20%
20%
20%
0.007
0.006
0.005
0.005
0.008
0.003
0.004
0.003
0.011
0.008
0.010
0.007
0.017
0.007
0.009
0.006
15.65%
13.87%
11.56%
11.17%
8.83%
3.87%
4.22%
3.49%
8.50%
6.22%
7.59%
5.63%
8.27%
3.33%
4.52%
2.95%
5%
9%
8%
10%
9%
9%
12%
12%
10%
7%
6%
5%
6%
2%
1%
1%
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
1
2.5
5
7.5
4%
5%
4%
4%
9%
9%
9%
9%
12%
11%
11%
11%
20%
19%
19%
19%
0.012
0.007
0.005
0.005
0.014
0.015
0.009
0.005
0.023
0.019
0.009
0.004
0.049
0.028
0.013
0.013
27.79%
15.77%
11.12%
12.07%
15.27%
16.17%
10.38%
5.61%
20.28%
17.10%
8.34%
4.08%
24.87%
14.46%
7.12%
7.05%
12%
13%
11%
11%
14%
13%
10%
7%
4%
7%
9%
9%
1%
4%
6%
5%
*Error is the difference between the expected (theoretical value) and the
measured value (experimental value). Legend: AVG Average; STDEV
Standard derivation; VC Variance coeficienc
118
Chapter V
K le in e t a l (2 0 0 3a ) fo u n d th a t ris e r d ia m e te r in flu e n c e w a s c ru c ia l fo r
b e tte r s o lid s d is trib u tio n th a n ris e r le n g th , e s p e c ia lly a t h ig h g a s flo w s
(K le in
et
a l.,
2 0 0 3 a ).
In
our
case,
if
a ll
ris e r
c o n fig u ra tio n s
a re
c o m p a re d , th e ris e r le n g th p ro v e s to h a v e b e tte r in flu e n c e o n s o lid s

d is trib u tio n th a n th e d ia m e te r in c re a s e . T h e p o s s ib le e x p la n a tio n lie s in
th e a m o u n t o f liq u id (c o n s e q u e n tly S G ) e x c h a n g e d b e tw e e n th e to p a n d
b o tto m p a rt o f th e iG L R . In th is c a s e , th e liq u id e x c h a n g e c o u n te ra c ts
th e s e ttlin g te n d e n c y o f th e S G p a rtic le s a n d h o m o g e n iz e s th e s o lid
d is trib u tio n A t th is c o n fig u ra tio n th e liq u id flo w p a tte rn s d e s c rib e d b y
D o lg o s e t a l. (2 0 0 1 ) a re n o t p re s e n t b e c a u s e th e liq u id le a v in g th e ris e r
d ire c tly e n te rs in th e e n la rg e d to p s e c tio n . T h is p ro v id e s a b e tte r liq u id
e x c h a n g e in th e a x ia l d ire c tio n a n d b e tter s o lid s m ix in g . It is e x p e c te d
to h a v e a s h a rp e r v e lo c ity p ro file a t th e lo w e r b o u n d a ry o f th e iG L R to p
p a rt w ith u p w a rd liq u id flu x in th e iG L R c e n te r a n d d o w n w a rd flu x n e a r
th e iG L R e x te rio r w a lls . If th e ris e r is s h o rte r (ris e r A ), th e n th e liq u id
le a v in g th e ris e r w o u ld n e e d to p a s s th e b u b b le s e p a ra to r s e c tio n
(tu rb u le n t re g io n ) w ith its flo w c irc u la tio n s e d d ie s b e fo re e n te rin g th e
a irlift to p s e c tio n . T h is w o u ld re s u lt in a fla tte r v e lo c ity p ro file a n d th u s
w e a k e r liq u id e x c h a n g e . M o re o v e r, a t th is co n fig u ra tio n (ris e r B ), a s
th is tu rb u le n t re g io n is s u p p re s s e d , th e in flu e n c e o f s o lid s p a rtic le s
s e ttlin g is lo w e r b e c a u s e th e m ix in g b e tw e e n th e u p w a rd a n d d o w n w a rd
liq u id is re d u c e d . T h e a b o v e m e n tio n e d re a s o n s a llo w a b e tte r liq u id
exchange
th ro u g h o u t
th e
a x ia l
d ire c tio n
re s u ltin g
in
m o re
h o m o g e n e o u s s o lid p a rtic le d is trib u tio n b e tw e e n th e b o tto m a n d th e

to p p a rt o f th e a irlift (F ig u r e V - 6 ).
N e v e rth e le s s , th is h o m o g e n e ity c a n h a v e a n a d v e rs e e ffe c t, w h e n
c o n tin u o u s s y s te m s a re u s e d , b e c a u s e th e p ro b a b ility o f c a rrie r (S G )
p a rtic le to w a s h -o u t is h ig h e r. T h a t is w h y in th e to p e n la rg e d s e c tio n
th e re is a d o u b le-b a rrie r a t th e o u tle t (s e e s e c tio n V .2 .1 ), w ith th e
o b je c tiv e to d e c re a s e th e s o lid s lo s e s w h e n th e iG L R o p e ra te s in
c o n tin u o u s m o d e (F ig u r e V - 3 )
119
Chapter V
Figure V-6. Solids hold-up at different GLR positions (A,B,C,D,E) for riser B
V.4.1.1
Riser C
In ris e r C (F ig u r e V - 7 ) a s o n ris e r A (F ig u r e V - 5 ), it is a ls o p re s e n t a n
a x ia l a n d ra d ia l s o lid s d is trib u tio n a t lo w e r g a s s u p e rfic ia l v e lo c itie s . B y
c o m p a ris o n w ith ris e r A a t th e s e c o n d itio n s , in ris e r C th e a x ia l
d is trib u tio n is m o re p ro n o u c e d w ith th e h ig h e s t s o lid c o n c e n tra tio n in
th e ris e r a n d d o w n c o m e r s e c tio n s (C a n d D re s p e c tiv e ly ). T h is c a n b e
e x p la in e d b y th e d e c re a s e o f th e ra tio A r /A d (T a b le V - 3 ), m e a n in g th a t
th e d o w n c o m e r c ro s s-s e c tio n is a ls o s m a lle r a n d th e liq u id v e lo c ity in
th a t s e c tio n is h ig h e r (F ig u r e V - 1 1 ). T h is w ill in c re a s e th e m ix in g a n d
th e c irc u la tio n e d d ie s in th e tu rb u le n c e re g io n , w h ic h fa v o u rs th e flo w
120
Chapter V
p a tte rn s A a n d B , p re v io u s ly e x p la in e d . B e in g s o , th e s o lid s a re
p re fe ra b ly m o v e d to th e d o w n c o m e r in s te a d o f g o in g u p to th e to p
s e c tio n a n d th e n th e y c irc u la te m a in ly in th e iG L R b o tto m p a rt. T h e s e
re s u lts s u g g e s te d th a t a n in c re a s e in ris e r d ia m e te r c a u s e s a n in c re a s e
in th e s o lid s n o n -h o m o g e n e itie s a n d th e ra d ia l a n d a x ia l d is trib u tio n
w a s m o re p ro n o u n c e d . C o n s id e rin g th a t K le in e t a l. (2 0 0 3 ) o b ta in e d
b e tte r s o lid s d is trib u tio n a t h ig h e r A r /A d th e p o s s ib le e x p la n a tio n fo r
th is lie s in th e d iffe re n t p ro p e rtie s o f th e s o lid s u s e d , a lg in a te b e a d s
a n d S G , u s e d in K le in e t a l (2 0 0 3 ) w o rk a n d in th is w o rk , re s p e c tiv e ly .
Figure V-7. Solids hold-up at different GLR positions (A,B,C,D,E) for riser C
121
Chapter V
T h e re s u lts p re s e n te d in T a b le V - 3 s h o w e d th a t fo r th e s a m e s o lid
lo a d th e h ig h e s t v a ria tio n c o e ffic ie n ts a re a lw a y s fo r th e s h o rte s t ris e rs
(A a n d C ). C o m p a rin g a ll c o n fig u ra tio n s u s e d (R is e r A , B a n d C ), it c a n
b e o b s e rv e d th a t th e e ffe c t o f ris e r le n g th h a s m o re im p a c t o n s o lid s
d is trib u tio n th a n th e e ffe c t o f ris e r d ia m e te r. T h e s e re s u lts s u g g e s t th a t
ris e r B is b e tte r s u ite d to a c h ie v e b e tte r h o m o g e n e ity o f S G d is trib u tio n
a lo n g a ll iG L R s e c tio n s .
V.4.2 Gas hold-up

In F ig u r e V - 8 it c a n b e o b s e rv e d th a t in th e iG L R th e ris e r g a s h o ldu p in c re a s e s w ith g a s s u p e rfic ia l v e lo c ity . G e n e ra lly th e re is a d e c re a s e
o f g lo b a l g a s h o ld -u p w h e n s o lid s a re p re s e n t (F ig u re V - 8 ). T h e b ig g e r
in flu e n c e w a s d e te c te d fo r ris e r A a t U G r = 7 .5 c m /s .
T h e lo w d iffe re n c e s fo u n d c a n b e e x p la in e d b y tw o fa c to rs : (1 ) th e
m e th o d a c c u ra c y , w h ic h h a s b e e n p ro v e d to h a v e a n a s s o c ia te d e rro r
b e tw e e n 1 5 % a n d 2 0 % (s e c tio n IV .4 .2 ); a n d (2 ) th e p ro p e rtie s (d e n s ity )
o f S G th a t a re v e ry s im ila r to w a te r a n d d o e s n o t in flu e n c e s ig n ific a n tly
th e liq u id -p h a s e p ro p e rtie s .
T h e g a s h o ld -u p in ris e r d e c re a s e s b o th w ith in te rn a l ris e r d ia m e te r
d e c re a s e (R is e r C to R is e r A ) a n d w ith th e in c re a s e o f ris e r le n g th (R is e r
A a n d R is e r B ).
T h e in c re a s e o f d ia m e te r in c re a s e s ris e r c ro s s-s e c tio n a re a a n d
c o n s e q u e n tly s e v e ra l p h e n o m e n a c a n o c c u r. T h e g a s h o ld-u p in ris e r
w h e n th e b u b b le c irc u la tio n re g im e is n o t p re s e n t is re la te d w ith liq u id
lin e a r v e lo c ity b y :
!Gr =
UGr
v Lr + U slip
Eq. V-7
W h e n th e b u b b le c irc u la tio n re g im e is p re s e n t (v a lid fo r ! G d ! 0 .0 8 ):
!Gr =
UGr 0 + !Gd v Ld " U slip # Ad / Ar

v Lr + U slip
122
Eq. V-8
Chapter V
W h e re v L / d r + /-U s l i p is th e to ta l g a s v e lo c ity in th e ris e r a n d in th e

d o w n c o m e r. F o r th e s a m e g a s s u p e rfic ia l v e lo c itie s , g a s h o ld-u p
Gas Hold-up / (vol./vol.)!
Riser - A!
0.15!
0.10!
0.05!
0.00!
0!
2!
4!
6!
8!
depends on vLr and Uslip.
Downcomer - A!
0.12!
0.09!
0.06!
0.03!
0.00!
0!
2!
0.15!
0.10!
0.05!
0.00!
2!
4!
6!
UGr / (cm/s)!
8!
Riser - B!
0!
0.15!
0.10!
0.05!
0.00!
2!
4!
6!
8!
Downcomer - B!
0.12!
0.09!
0.06!
0.03!
0.00!
0!
2!
Riser - C!
0!
6!
UGr / (cm/s)!
8!
UGr / (cm/s)!
4!
UGr / (cm/s)!
4!
6!
UGr / (cm/s)!
8!
Downcomer - C!
0.12!
0.09!
0.06!
0.03!
0.00!
0!
2!
4!
6!
UGr / (cm/s)!
8!
Figure V-8. Gas hold-up (vol./vol.) vs. Gas superficial velocity in riser (UGr /
(cm/s)) for different risers configurations. Legend: ! - 0% (wt.WET BASIS/vol.);
" - 4% (wt.WET BASIS/vol.); ! - 8% (wt.WET BASIS/vol.); # - 12% (wt.WET BASIS/vol.);
$ 20% (wt.WET BASIS/vol.).
C o n s id e rin g th a t v L r > U s l i p . c h a n g e s in U s l i p in th e ris e r w o u ld h a v e a

re d u c e d e ffe c t in ris ers g a s h o ld -u p . T h e h ig h e r g a s h o ld -u p d e te c te d
in ris e r C (c o m p a re d w ith ris e r A ) w h e n n o b u b b le re g im e c irc u la tio n is
p re s e n t (U G r < 5 c m /s ) is m a in ly d u e to v L r d e c re a s e . F ro m F ig u r e V - 1 1 ,
123
th e
vLr
d e c re a s e
Chapter V
is
a ro u n d
U G r = 7 .5 c m /s fo r 2 0 % (w t. W E T
10%
BASIS
le s s
in
ris e r
(v L r = 0 .9 m /s
at
/v o l) o f S G ) th a n in th e o th e r ris e rs
(R is e r B : v L r = 1 .0 m /s a t U G r = 7 .5 c m /s fo r 2 0 % (w t. W E T
BASIS
/v o l) o f S G ).
C o n s id e rin g th e v L r d e c re a s e o b s e rv e d in ris e r C (F ig u re V - 1 1 ) a n d b y
th e E q . V - 7 th e m a x im u m ! G r in c re a s e w a s e x p e c te d to b e 1 0 % .
G e n e ra lly fo r U G r = 7 .5 c m /s th e g a s h o ld -u p in ris e r C is a ro u n d 2 0 %
m o re th a n in ris e r A w h ic h c a n b e e x p la in e d b y th e b u b b le c irc u la tio n
re g im e p re s e n t in ris e r C a n d w h ic h c a n b e e x p la in e d b y E q . V - 7 .
A p p ly in g E q . V - 8 to th e b u b b le c irc u la tio n re g im e p re s e n t in ris e r C
when
U G r = 7 .5 c m /s ,
it
is
p o s s ib le
to
o b s e rv e
th a t,
u s in g
th e
e x p e rim e n ta l v a lu e s o f v L r / d (F ig u r e V - 1 1 ) a n d ! G d (F ig u r e V - 8 ), th e U s l i p
o b ta in e d a re v e ry lo w . T h is is in a c c o rd a n c e w ith v is u a l in d ic a tio n s th a t
s h o w e d th e p re s e n c e o f s m a ll b u b b le s (lo w U s l i p ) in th e d o w n c o m e r
s e c tio n .
In F ig u re V - 8 it is s h o w n th a t g a s h o ld -u p d e c re a s e s w ith th e
in c re a s e o f ris e r le n g th (R is e r A to B ), th e e x c e p tio n o c c u rrin g a t h ig h
s o lid s lo a d (2 0 % (w t. W E T
BASIS
/v o l.)), w h e re th e g a s h o ld -u p fo r ris e r A
(0 .0 6 2 ) a n d ris e r B (0 .0 5 9 ) a re s im ila r (F ig u r e V - 8 ). A s it w a s s h o w n
p re v io u s ly , th e g a s h o ld -u p in ris e r c a n b e e x p la in e d b y fa c to rs s u c h a s
th e liq u id lin e a r v e lo c ity a n d th e d riv in g fo rc e . In th is c a s e (ris e r A
c o m p a re d w ith ris e r B ) th e in te rp la y b e tw e e n th e s e tw o p h e n o m e n a m a y
a ls o b e u s e d to e x p la in th e o b s e rv e d re s u lts . G e n e ra lly , th e g a s h o ld u p in ris e r A is h ig h e r th a n in ris e r B b e c a u s e w h e n b u b b le s e n te r in th e
d o w n c o m e r th e y re d u c e th e liq u id lin e a r v e lo c ity in th e d o w n c o m e r
(v L d ). T h is re d u c tio n o c c u rs b e c a u s e th e d riv in g fo rc e ( ! G r - ! G d ) is
re d u c e d (F ig u r e V - 9 ) w h e n b u b b le s a re e n tra in e d in th e d o w n c o m e r
(U G r = 5 c m /s a n d U G r = 7 .5 c m /s ). T h u s , b y th e m a s s c o n s e rv a tio n la w th e
liq u id lin e a r v e lo c ity in th e ris e r (v L r ) is a ls o re d u c e d . F ro m E q . V - 7 th e
re d u c tio n o f liq u id lin e a r v e lo c ity in th e ris e r (ris e r A ) in c re a s e s th e g a s
h o ld -u p (c o n s id e rin g U G r a n d U s l i p c o n s ta n t), w h ic h c a n e x p la in th e
h ig h e r g a s h o ld -u p s fo u n d in th e g -l s y s te m (F ig u re V - 8 ) fo r ris e r A .
H o w e v e r,
re g a rd in g
the
F ig u r e
V-11
th e
ris e r
lin e a r
v e lo c ity
at
U G r = 7 .5 c m /s is 1 .0 2 c m /s a n d 0 .9 c m /s re s p e c tiv e ly fo r ris e r A a n d B .
124
Chapter V
T h is c o n tra d ic ts E q . V - 7 w h ic h b y th e v a lu e s fro m F ig u re V - 1 1 s h o u ld
p re s e n t h ig h e r g a s h o ld -u p s fo r ris e r B a n d n o t A a s o b s e rv e d (F ig u re
V - 8 ). T h is d is c re p a n c y (m a in ly fo r th e g -l s y s te m ) c a n b e d u e to s e v e ra l
fa c to rs th a t in te ra c t a m o n g th e m a n d s h o u ld b e ta k e n in a c c o u n t.
A m o n g th e m a re : th e c h a n g e s in U s l i p , liq u id flo w p a tte rn s (d u e to th e
p re s e n c e /a b s e n c e o f a tu rb u le n t re g io n a b o v e th e ris e r to p s e n d ) a n d
c o a le s c e n c e p h e n o m e n a in te n s ity a t d iffe re n t ris e r c o n fig u ra tio n s .
W h e n h ig h e r s o lid lo a d is p re s e n t a t U G r ! 5 c m /s , th e g a s h o ld -u p in
ris e r A a n d ris e r B a re s im ila r. T h is c a n h a p p e n d u e to b u b b le
c o a le s c e n c e , w h ic h c a n b e e n h a n c e d b y th e S G p a rtic le s . C o a le s c e n c e
w ill in c re a s e b u b b le s iz e a n d th u s th e b u b b le s lip v e lo c ity , w h ic h w ill
d e c re a s e th e d o w n c o m e r g as h o ld -u p . If d u e to s o lid s p re s e n c e th e g a s
h o ld -u p in d o w n c o m e r d e c re a s e s , th e d riv in g fo rc e ( ! G r ! G d ) in c re a s e s .
B y a n a ly s in g F ig u r e V - 8 a n d F ig u r e V - 9 (a t U G r = 7 .5 c m /s ), it c a n b e
o b s e rv e d th e d e c rea s e o f g a s h o ld -u p in d o w n c o m e r fo r a ll s o lid lo a d
(F ig u r e V - 8 ) a n d th e c o n s e q u e n t d riv in g fo rc e in c re a s e (F ig u r e V - 9 ).
C o n s e q u e n tly th e ris e r liq u id v e lo c ity is h ig h e r a n d th e g a s h o ld-u p in
ris e r w ill d e c re a s e .
It c a n b e a rg u e d th a t th e d e c re a s e o f g a s h o ld-u p in th e ris e r w ill
d e c re a s e th e d riv in g fo rc e . C o n s e q u e n tly th e p re v io u s e x p la n a tio n (fo r
g -l-s s y s te m ) is n o t v a lid . H o w e v e r, fro m F ig u re V - 9 fo r ris e r A , th e
d riv in g fo rc e re m a in s c o n s ta n t fo r a ll s o lid lo a d s a t U G r = 7 .5 c m /s . In th e
p re s e n c e o f s o lid s , th e g a s h o ld -u p is n o t o n ly in flu e n c e d b y th e ris e r
c o n fig u ra tio n (liq u id flo w p a tte rn s , U s l i p , c o a le s c e n c e ) b u t a ls o b y th e
s o lid -p h a s e in flu e n c e in th e g a s-p h a s e . T h e g -l-s is a c o m p le x s y s te m
w h e re th e s o lid s in flu e n c e m a y a lte r th e g a s-p h a s e p ro p e rtie s , w h ic h
m a y o r n o t h a v e in flu e n c e o n th e flo w p a tte rn o f th e re a c to r. T h is
in flu e n c e o f s o lid s in th e iG L R flo w p a tte rn s h a s d iffe re n t in te n s itie s a t
d iffe re n t iG L R c o n fig ura tio n s .
.
125
Chapter V
Riser - B!
Riser - A!
0.08!
0.06!
0.06!
eGr-eGd!
eGr-eGd!
0.08!
0.04!
0.04!
0.02!
0.02!
0.00!
0.00!
0!
2!
4!
6!
0!
8!
2!
4!
6!
8!
UGr / (cm/s)!
UGr / (cm/s)!
Riser - C!
0.08!
eGr-eGd!
0.06!
0.04!
0.02!
0.00!
0!
2!
4!
6!
8!
UGr / (cm/s)!
Figure V-9. Driving force (!GR-!Gd) vs. Gas superficial velocity (UGr) for
different risers configurations. Legend: ! - 0% (wt.WET BASIS/vol.);
" - 4% (wt.WET BASIS/vol.); ! - 8% (wt.WET BASIS/vol.); # - 12% (wt.WET BASIS/vol.);
T h e in c re a s e o f ris e r le n g th in th is re a c to r c o n fig u ra tio n s u p p re s s e s

th e b u b b le s e p a ra to r (lo w e r p a rt o f d u a l s e p a ra to r) p re s e n t in th e o th e r
tw o c o n fig u ra tio n s . T h is re a c to r p a rt is s m a ll (a b o u t 2 0 c m in le n g th )
a n d c re a te s re s is ta n c e to b u b b le ris in g (tu rb u le n c e re g io n ), a n d m ig h t
le a d to m o re b u b b le s b e in g d ra g g e d to th e d o w n c o m e r.
F o r th e s a m e g a s s u p e rfic ia l v e lo c itie s in th e ris e r A , th e g a s h o ld-u p
v a lu e s o b ta in e d w ith S G p a rtic le s , a re in th e s a m e ra n g e o f th e re s u lts
o b ta in e d b y F re ita s a n d T e ix e ira (1 9 9 8 a ) w ith a lg in a te b e a d s (a n d s a m e
ris e r c o n fig u ra tio n ). In th e ir w o rk th e ris e r g a s h o ld -u p d e c re a s e s
126
Chapter V
s lig h tly w h e n s o lid s a re a p p lie d a t lo w g a s flo w s u p e rfic ia l v e lo c itie s ,

s im ila r re s u lts a re o b ta in e d in o u r c a s e(F re ita s a n d T e ix e ira , 1 9 9 8 a ).
M o re o v e r th e re s u lts p re s e n te d in F ig u re V - 8 in d ic a te th a t fo r ris e r B
th e in flu e n c e o f S G w a s e v e n le s s th a n in o th e r c o n fig u ra tio n s .
T h e d o w n c o m e r re s u lts s h o w th a t, o n ly a t lo w ris e rs le n g th , b u b b le s
a re g o in g in s id e th e d o w n c o m e r b e in g th is m o re s ig n ific a n t a t lo w e r
A r i /A d re la tio n (ris e r C ) a s d e s c rib e d in lite ra tu re (C h is ti, 1 9 8 9 ; K le in e t
a l., 2 0 0 3 ). O n F ig u r e V - 8 it c a n b e o b s e rv e d th a t d o w n c o m e r g a s h o ldu p in ris e r C is s im ila r a t h ig h g a s s u p e rfic ia l v e lo c itie s (U G r = 5 c m /s a n d
U G r = 7 .5 c m /s ). T h e m a in re a s o n is th a t a t U G r = 7 .5 c m /s c o m p le te b u b b le
c irc u la tio n o c c u rre d , e v e n in th e p re s e n c e o f S G p a rtic le s .
If A r i /A d ra tio is lo w , a ls o is th e d o w n c o m e r c ro s s-s e c tio n a re a , a n d
th e
liq u id
v e lo c ity
(> 0 .2 5
m /s )
th a t
goes
in to
th e
d o w n c o m e r is h ig h e r, w h ic h d ra g s m o re b u b b le s (U s l i p < 0 .2 5 m /s ) to th is
p a rt o f th e re a c to r. H o w e v e r th is d o e s n o t h a p p e n w h e n ris e r le n g th is
in c re a s e d d u e to th e s u p p re s s in g o f th e in te rm e d ia te p a rt b e tw e e n ris e r
a n d to p s e c tio n , w h ic h im p ro v e s th e d e g a s ific a tio n o f th e g a s a n d
re d u c e s th e p ro b a b ility o f b u b b le s to g o in s id e th e d o w n c o m e r. S im ila r
re s u lts w e re o b ta in e d b y K le in e t a l (2 0 0 3 ) w h e re a lm o s t n o b u b b le s
w e re p re s e n t in d o w n c o m e r w h e n th e ris e r e n d s m a tc h e d th e b e g in n in g
o f to p s e c tio n (K le in e t a l., 2 0 0 3 ).
P re v io u s s tu d ie s in d ic a te d th a t in g -l s y s te m s b u b b le s e n te r in th e
d o w n c o m e r fo r g a s s u p e rfic ia l v e lo c itie s b e tw e e n 1 0 c m /s (F re ita s a n d
T e ix e ira , 1 9 9 8 a ) a n d 1 5 c m /s . In o u r c a s e g a s h o ld -u p in d o w n c o m e r
s ta rts
to
in c re a s e
at
2 .5 c m /s
and
5 c m /s
fo r
ris e r
and
A,
re s p e c tiv e ly . T h e m a in re a s o n fo r th is d is c re p a n c y is th e ty p e o f
d is trib u to r u s e d . In o u r w o rk it w a s u s e d a p o ro u s p la te , w h ic h fo rm s
s m a lle r b u b b le s th a n th e d is trib u te r u s e d b y th e o th e r a u th o rs .
F ro m th e v a lu e s p re s e n t in F ig u r e V - 8 it w a s d e te rm in e d th e re la tio n
b e tw e e n ris e r a n d d o w n c o m e r g a s h o ld-u p . T h is re la tio n is lin e a r a n d
th e re s u lts fro m th e p a ra m e te rs e s tim a tio n a re p re s e n t in T a b le V - 4 .
127
Chapter V
T h e v a lu e s o f c o e ffic ie n ts a G a n d b G a re p re s e n te d in T a b le V - 4 a n d
re s u lt fro m th e lin e a r re la tio n o f ris e r a n d d o w n c o m e r g a s h o ld -u p to
d iffe re n t s o lid lo a d a n d ris e r c o n fig u ra tio n . A s in ris e r B n o g a s w a s
p re s e n t in th e d o w n c o m e r th is re la tio n w a s n o t p o s s ib le to o b ta in , b u t
th e v a lu e s fro m o th e r tw o ris e rs in d ic a te th a t th e v a lu e s fo u n d fo r a G
(! G d /! G r ) in g -l-s s y s te m s a re s im ila r to th e o n e s fo u n d in lite ra tu re a n d
s h o w a s im ila r b e h a v io u r: a d e c re a s e w ith s o lid lo a d (F re ita s a n d
T e ix e ira , 1 9 9 8 a ). O n th e o th e r h a n d , th e a G v a lu e s o b ta in e d fo r g -l
s y s te m (0 .9 2 a n d 0 .9 3 ) a re s lig h tly h ig h e r th a n th e o n e s p re s e n te d in
lite ra tu re : 0 .8 0 (L u e t a l., 1 9 9 5 ); 0 .8 8 (F re ita s a n d T e ix e ira , 1 9 9 8 a ) a n d
0 .8 9 (B la e t a l., 2 0 0 4 ). T h e d iffe re n c e s c a n b e e x p la in e d b y th e d iffe re n t
re a c to r d e s ig n a n d d is trib u to r u s e d . In o u r c a s e th e fo rm e d b u b b le s a re
s m a lle r,
th u s
m o re
p ro n e
to
e n tra in
in to
th e
d o w n c o m e r,
w h ic h
in c re a s e s th e d o w n c o m e r g a s h o ld-u p in th is s e c tio n a n d c o n s e q u e n tly

th e ra tio (! G d /! G r ).
Table V-4. Parameters obtained for the linear relation between downcomers
and risers gas hold-up (eGd=aG10eGr+bG)
% Solids
(wt.WET BASIS/vol.)
aG
Riser A
bG
R2
aG
Riser B
bG
R2
aG
Riser C
bG
R2
0.92 -0.03 0.95
---
---
---
0.93
-0.02
0.96
0.72 -0.02 0.92
---
---
---
0.94
-0.01
0.99
0.73 -0.02 0.99
---
---
---
0.74
0.00
0.93
12
0.75 -0.02 0.99
---
---
---
0.61
-0.02
1*
20
0.52 -0.01 0.99
---
---
---
0.54
-0.02
1*
*C o rre la tio n b a s e d o n ly in tw o e x p e rim e n ta l p o in ts
V.4.3 Mixing and circulation time

M ix in g
tim e
is
d e s c rib e d
as
th e
tim e
n e c e s s a ry
fo r
a c h ie v ing
h o m o g e n e ity in s id e th e re a c to r. G e n e ra lly in iG L R th e m ix in g tim e is

re la te d w ith th e g a s flo w s u p e rfic ia l v e lo c ity b y th e e q u a tio n :
b
t M = a ! uGr
Eq. V-9
It is re la te d w ith th e c irc u la tio n tim e , w h ic h is th e tim e n e c e s s a ry fo r

a s in g le e le m e n t to c o m p le te a lo o p in a n iG L R . T h e c o rre la tio n fo r
m ix in g tim e w a s u s e d to a p p ro x im a te th e e x p e rim e n ta l d a ta o b ta in e d
128
Chapter V
(F ig u r e V - 1 0 ) a n d th e v a lu e s fro m th e c o rre s p o n d in g p a ra m e te rs a re
p re s e n te d in T a b le V - 5 .
In iG L R th e m ix in g tim e d e c re a s e s ra p id ly a t lo w g a s s u p e rfic ia l
v e lo c itie s a n d s ta b iliz e s a t h ig h e r v e lo c itie s . F ro m F ig u r e V - 1 0 , it is
p o s s ib le to o b s e rv e a s im ila r tre n d in a ll re a c to r c o n fig u ra tio n s . It is
v e rifie d th a t R is e r B c o n fig u ra tio n s h o w e d th e lo w e r v a lu e s o f m ix in g
tim e (t M ) in d ic a tin g a g o o d m ix in g p ro file w h e n c o m p a re d w ith o th e r tw o
c o n fig u ra tio n s . T h is is p ro b a b ly d u e to th e a b s e n c e o f th e b u b b le
s e p a ra to r s e c tio n , w h ic h le a d s to a fa s t a x ia l m ix in g th ro u g h o u t a ll
iG L R . T h e a x ia l m ix in g is p ro m o te d b y th e liq u id c irc u la tio n in te n s ity
b e tw e e n ris e r a n d d o w n c o m e r (M o lin a e t a l., 1 9 9 9 ).
Riser A!
100!
Riser B!
50!
50!
tM / s!
tM / s!
75!
25!
0!
25!
0!
0.0! 2.5! 5.0! 7.5!
0.0!
UGr / (cm/s)!
2.5! 5.0! 7.5!

UGr / (cm/s)!
Riser C!
100!
tM / s!
75!
50!
25!
0!
0.0!
2.5!
5.0!
7.5!
UGr / (cm/s)!
Figure V-10. Mixing time vs. Gas superficial velocity (U /(cm/s)) at different
solid load. Legend: ! - 0% (wt.WET BASIS/vol.); " - 4% (wt.WET BASIS/vol.);
! - 8% (wt.WET BASIS/vol.); # - 12% (wt.WET BASIS/vol.);
A s in c o n fig u ra tio n B n o g a s is p re s e n t in th e d o w n c o m e r, th e d riv in g

fo rc e is h ig h e r e s p e c ia lly a t h ig h g a s flo w s (F ig u re V - 9 ), le a d in g to a
g o o d m ix in g p ro file . M o re o v e r in T a b le V - 5 th e v a lu e s o f a a re s m a lle r
129
Chapter V
fo r th is re a c to r c o n fig u ra tio n in d ic a tin g th e g o o d m ix in g p ro file o f it.

B e llo (1 9 8 1 ) c ite d b y M o lin a e t a l (1 9 9 9 ) p ro p o s e d a re la tio n b e tw e e n
th e d im e n s io n le s s m ix in g tim e ( ! = t M /t C ) a n d th e ra tio A d /A r i to b e
in d e p e n d e n t fro m g a s v e lo c ity :
Eq. V-10
W h e re , a c c o rd in g ly to B e llo (1 9 8 1 ), ! v a rie s b e tw e e n 5 .2 a n d 3 .5 fo r
e x te rn a l a n d in te rn a l G L R s , re s p e c tiv e ly . In o u r c a s e th e ! v a lu e s
o b ta in e d fo r g -l s y s te m in ris e r A a re s im ila r to th e v a lu e s re p o rte d in
lite ra tu re (B e llo , 1 9 8 1 ; M o lin a e t a l., 1 9 9 9 ).
Table V-5. Parameters for equation tM=a.uGrb
Configuration
%Solids (wt. WET BASIS/vol.)
R2 tM/tC
60.78 -0.7 0.98 2 . 5
3.57
41.87 -0.48 0.99 2 . 3
3.31
67.3 -0.62 0.96 2 . 8

48.76 -0.48 0.91 2 . 6
3.96
12
20
39.13 -0.43 0.74 2 . 1
3.06
34.64 -0.35 0.82 1 . 6
2.28
33.24 -0.64 0.86 1 . 1

30.94 -0.22 0.98 1 . 7
1.56
2.38
20
35.06 -0.82 0.92 1 . 7

28.45 -0.79 0.81 2 . 0
82.13 -0.46 0.92 5 . 8
8.00
83.84 -0.76 0.94 4 . 2

88.65 -0.76 0.9 4 . 6
5.79
41.05 -0.39 0.62 3 . 2

51.11 -0.4 0.81 4 . 3
4.45
8
12
8
12
20
3.75
2.46
2.85
6.36
5.96
In c a s e o f ris e r B th e lo w d im e n s io n le s s m ix in g tim e is d u e to th e
g o o d m ix in g p ro file s th u s lo w ! v a lu e s a re o b ta in e d . In o p p o s itio n , th e
ris e r C c o n fig u ra tio n h a s ! v a lu e s s im ila r to e x te rn a l G L R , w h ic h
in d ic a te s th a t th is c o n fig u ra tio n m ig h t w o rk a s tw o s e p a ra te d re a c to rs .
T h e h ig h m ix in g tim e s o b ta in e d in ris e r C c a n b e a re s u lt o f s e v e ra l
fa c to rs a n d h o w th e y in te ra c t to g e th e r. T h e s e fa c to rs a re th e ris e r
d ia m e te r, th e p re s e n c e o f a d u a l to p s e c tio n s e p a ra to r a n d c o n d u c tiv ity
130
Chapter V
p ro b e p o s itio n (F ig u r e V - 3 ). F ro m th e p o in t o f v ie w o f th e liq u id p h a s e ,
th e in c re a s e o f ris e r d ia m e te r le a d s to lo w e r liq u id v e lo c ity in th e ris e r
(F ig u r e V - 1 1 ). T h e re fo re th e a m o u n t o f liq u id th a t le a v e s ris e r C c a rrie s
le s s e n e rg y , a n d d o e s n o t re a c h th e to p e n la rg e d s e c tio n a s e a s ily a s in
ris e r A o r B . T h e re fo re , in th is c o n fig u ra tio n (ris e r C ), th e liq u id
e x c h a n g e b e tw e e n th e ris e r a n d th e d o w n c o m e r m a y b e re d u c e d w h ic h
le a d s to lo w e r m ix in g tim e s . In a d d itio n , th e p o s itio n o f th e c o n d u c tiv ity
p ro b e , u s e d to re a d th e m ix in g tim e , is lo c a te d a t e n la rg e d to p s e c tio n .
O n th e o th e r h a n d , th e in c re a s e in th e liq u id lin e a r v e lo c ity in th e
d o w n c o m e r le a d s to a n in c re a s e o f g a s h o ld-u p in th e d o w n c o m e r. A s
p re s e n te d in F ig u r e V - 9 in ris e r C th e d riv in g fo rc e ( ! r - ! d ) is s m a ll w h e n
c o m p a re d w ith ris e r A /B m a in ly d u e to th e p re s e n c e o f g a s in th e
d o w n c o m e r. In th e o ry th e lo w d riv in g fo rc e w ill re d u c e th e m ix in g tim e .
It is th e n im p o rta n t to u n d e rs ta n d b e tte r w h ic h e ffe c t is s tro n g e r in th e
m ix in g p ro file o f iG L R . T h e in te rp la y b e tw e e n liq u id v e lo c ity , d riv in g
fo rc e , b u b b le s lip v e lo c ity is c o m p le x a n d s e n s itiv e . In F ig u re V - 1 0 C it
c a n b e a ls o v e rifie d th a t a t h ig h s o lid c o n te n t th e m ix in g tim e is s m a ll.
A p o s s ib le e x p la n a tio n is th e o c c u rre n c e o f c o a le s c e n c e th a t c h a n g e s
th e c irc u la tio n p ro file o f th e re a c to r b e in g m o re liq u id tra n s p o rte d to
th e to p d e g a s s in g s e c tio n , p ro m o tin g a b e tte r m ix in g .
T h e d im e n s io n le s s m ix in g tim e in d ic a te s h o w m u c h b ig g e r is th e
n u m b e r o f lo o p s n e c e s s a ry fo r a co m p le te m ix in g . A s p re d ic te d , b e tte r
re s u lts w e re o b ta in e d fo r ris e r B b e in g th e o n ly c o n fig u ra tio n th a t n e e d s
a t m o s t 2 lo o p s fo r a c o m p le te m ix in g . In g e n e ra l, it is p o s s ib le to
o b s e rv e (F ig u r e V - 1 0 ) th a t S G p a rtic le s d e c re a s e th e m ix in g . T h e
e x c e p tio n is a t 8 % (w t. W E T
BASIS
/v o l.) in ris e r B w h e re th e m ix in g w a s
h ig h e r th a n th e re m a in in g s o lid s lo a d s u s e d (F ig u re V - 1 0 ). T h is m a y b e
d u e to c o a le s c e n c e p ro m o te d b y S G w h ic h le a d s to b ig g e r b u b b le s th a t
d ra g m o re liq u id to th e to p s e c tio n im p ro v in g th e a x ia l m ix in g . T h e
re la tiv e v e lo c ity b e tw e e n g a s- a n d liq u id -p h a s e s p ro m o te s th e a x ia l
m ix in g , th u s if b ig g e r b u b b le s a re p re s e n t, a x ia l m ix in g is in c re a s e d ,
w h ic h re d u c e s th e m ix in g tim e .
131
Chapter V
V.4.4 Liquid velocity

A s th e lin e a r liq u id v e lo c ity c a n in flu e n c e m ix in g , m a s s tra n s fe r a n d
s o lid s
s u s p e n s io n
p ro p e rtie s ,
it
is
v e ry
im p o rta n t
to
s tu d y
th is
p a ra m e te r in re a c to rs h y d ro d y n a m ic s , e s p e c ia lly fo r d e s ig n a n d s c a leu p p u rp o s e s (B la e t a l., 2 0 0 4 ; F re ita s , 2 0 0 2 ). T h e re s u lts p re s e n te d in

F ig u r e V - 1 1 a re in th e s a m e ran g e o f lin e a r liq u id v e lo c ite s fo u n d in
lite ra tu re (B la e t a l., 2 0 0 4 ; F re ita s , 2 0 0 2 ) a n d , a s e x p e c te d in d ic a te th a t
liq u id v e lo c ity in c re a s e s w ith g a s flo w . N o re le v a n t in flu e n c e o f s o lid s is
o b s e rv e d ,
w ith
2 0 % (w t. W E T
BASIS
th e
e x c e p tio n
of
some
d e c re a s e
fo u n d
when
/v o l.) o f S G p a rtic le s w e re a d d e d . E v e n in th e lite ra tu re
c o n s u lte d , th e e ffe c t o f s o lid s in lin e a r liq u id v e lo c ity re m a in s u n c le a r,

a s it c a n in c re a s e th e liq u id v e lo c ity (F re ita s a n d T e ix e ira , 1 9 9 8 a ) o r
d e c re a s e (F re ita s a n d T e ix e ira , 1 9 9 8 a ; L u e t a l., 1 9 9 5 ; M e rc h u k e t a l.,
1 9 9 8 ). T h e m a in re a s o n s fo r th is a re th e d iffe re n t G L R c o n fig u ra tio n s
a n d c o n d itio n s (ty p e o f s o lid s ) u s e d .
T h e re s u lts fo r th e d iffe re n t ris e rs a re in a c c o rd a n c e w ith th e o n e s
o b ta in e d b y K le in e t a l (2 0 0 3 ) a n d s u g g e s t th a t th e d ia m e te r in c re a se
(ris e r A to C ) h a s m o re in flu e n c e o n liq u id v e lo c ity th a n th e ris e r le n g th
in c re a s e (ris e r A to B ). W h e n th e d ia m e te r is in c re a s e d th e g a s h o ld-u p
a ls o
in c re a s e s
(F ig u r e
V-8)
but
fo r
ris e r
th e
gas
h o ld -u p
at
U G r = 7 .5 c m /s is s im ila r to th e g a s h o ld -u p a t U G r = 5 c m /s , w h ic h m ig h t
b e d u e to b u b b le c irc u la tio n p re s e n t a t th e h ig h e s t g a s s u p e rfic ia l
v e lo c ity (7 .5 c m /s ). T h e b u b b le c irc u la tio n re g im e o c c u rs e ith e r in th e
p re s e n c e o r a b s e n c e o f S G p a rtic le s . In th is re g im e , th e h ig h e r b u b b le s
flo w in a x ia l p o s itio n fro m th e b o tto m to th e to p , w h ile th e s m a lle r
b u b b le s (w ith lo w e r s lip v e lo c ity ) e n te r in th e d o w n c o m e r. D u e to th e
h ig h e r
downcomer
liq u id
v e lo c ity ,
w h ic h
re s u lts
fro m
th e
s m a ll
d o w n c o m e r c ro s s -s e c tio n a re a , th e s e s m a ll b u b b le s a re d ra g g e d to th e
b o tto m p a rt o f th e iG L R a n d e n te r a g a in in th e ris e r.
132
Chapter V
A!
1.00!
A!
0.50!
vd / (m/s)!
vr / (m/s)!
1.50!
0.25!
0.50!
0.00!
0.00!
1!
B!
1.50!
1.00!
2.5!
5.0!
7.5!
% Solid Load (wt/vol)!
B!
0.50!
vd / (m/s)!
vr / (m/s)!
1.0!
2.5!
5!
7.5!
0.25!
0.50!
0.00!
0.00!
1!
1!
2.5!
5!
7.5!
7.5!
C!
0.50!
vd / (m/s)!
vr / (m/s)!
1.00!
5!
C!
1.50!
2.5!
0.25!
0.50!
0.00!
0.00!
1!
2.5!
1!
5!
7.5!
2.5!
5!
7.5!
Figure V-11. Liquid velocity (m/s) in riser and downcomer vs. Solid load
(% (wt.WET BASIS/vol.)) for different risers configurations. Legend:
! - 0% (wt.WET BASIS/vol.); " - 4% (wt. WET BASIS/vol.); " - 8% (wt. WET BASIS/vol.);
" - 12% (wt. WET BASIS/vol.); " 20% (wt. WET BASIS/vol.).
133
Chapter V
W ith th e e x c e p tio n o f ris e r C , F ig u re V - 1 1 in d ic a te s th a t s o lid s

in flu e n c e a ffe c ts m o re th e liq u id v e lo c ity in th e d o w n c o m e r th a n in th e
ris e r, e s p e c ia lly a t h ig h g a s flo w s . T h e m a in re a s o n is th e a b s e n c e o f
h ig h a m o u n ts o f b u b b le s in th e d o w n c o m e rs A a n d B . T h e re is o n ly
c o n ta c t b e tw e e n th e liq u id - a n d s o lid -p h a s e a n d th e in flu e n c e o f s o lid s
in liq u id v e lo c ity is h ig h e r, p a rtic u la rly w h e n h ig h a m o u n ts o f S G a re
p re s e n t.
A fin a l n o te h a s to b e m a d e c o n c e rn in g th e c o n d itio n s te s te d . In
m o s t o f th e h y d ro d y n a m ic s tu d ie s fo u n d in lite ra tu re , th e u s ed g a s
s u p e rfic ia l v e lo c itie s a re th e s a m e a n d h ig h e r m a g n itu d e th a n th e o n e s
in th is w o rk . H o w e v e r, in c o n tin u o u s A F B p ro d u c tio n th e s u p e rfic ia l g a s
v e lo c ity is n o rm a lly 0 .5 c m /s (L e h n e rt e t a l., 2 0 0 8 ) a n d c a n g o u p to 3
c m /s (B r n y ik e t a l., 2 0 0 4 ) w ith a n a c c e p ta b le a m o u n t o f im m o b iliz e d
b io m a s s . B e in g s o , th e p re s e n te d h y d ro d y n a m ic e x p e rim e n ts w e re
p e rfo rm e d b o th in th e h y d ro d y n a m ic a n d fe rm e n ta tio n ra n g e . T h e
m e th o d s u s e d , m a in ly fo r g a s h o ld -u p d e te rm in a tio n , d u e to th e
o b s e rv e d e rro rs (1 5 % to 2 0 % ), d id n o t s h o w e d e n o u g h a c c u ra c y a t g a s
s u p e rfic ia l v e lo c ity lo w e r th a n 1 c m /s (s e e s e c tio n V .3 .1 ), th e re fo re n o
re s u lts w e re te s te d a t th is g a s flo w . H o w e v e r, it is b e lie v e d th a t the
fin a l c o n c lu s io n s o f th is c h a p te r a b o u t th e b e s t ris e r c o n fig u ra tio n
w o u ld n o t b e a ffe c te d . M o re o v e r in th e n e x t c h a p te r lo c a l h y d ro d y n a m ic
s tu d ie s , w ith m o re re fin e d te c h n iq u e s w ill b e p e rfo rm e d a t s u p e rfic ia l
g a s v e lo c itie s b e tw e e n 0 .5 c m /s a n d 1 c m /s .
V.5 Conclusions
T h e m a in o b je c tiv e o f th is c h a p te r w a s to u n d e rs ta n d th e e ffe c t o f S G
in th re e d iffe re n t c o n fig u ra tio n s o f iG L R w ith a n d e n la rg e d to p s e c tio n .
T h e o b ta in e d re s u lts o f s o lid s d is trib u tio n , g a s h o ld -u p , liq u id lin e a r
v e lo c ity c irc u la tio n a n d m ix in g tim e w e re u s e d to s e le c t b e tw e e n th e
c o n fig u ra tio n s te s te d th e m o s t s u ita b le to p e rfo rm a c o n tin u o u s p rim a ry
A F B fe rm e n ta tio n .
134
Chapter V
B e fo re th e fin a l c o n c lu s io n , it is im p o rta n t to h a v e in m in d th e id e a l
c o n d itio n s fo r c o n tin u o u s A F B fe rm e n ta tio n : (1 ) g o o d s o lid p a rtic le s
d is trib u tio n , (2 ) m in im u m g a s flo w ra te to flu id iz e a ll s o lid p a rtic le s , (3 )
a n a e ro b ic c o n d itio n s , (4 ) lo w g a s h o ld -u p in ris e r to a v o id h ig h
s trip p in g lo s e s o f fla v o u r c o m p o u n d s , (5 ) a b s e n c e o f b u b b le s in
d o w n c o m e r b e c a u s e n o rm a lly C O 2 is u s e d a n d it is k n o w n to h a v e
a d v e rs e e ffe c t o n y e a s t c e ll m e ta b o lis m , (6 ) g o o d m ix in g p ro p e rtie s , i
e ., if m ix in g tim e is s m a lle r th e in flu e n c e o f liq u id flo w w ill b e e v e n
s m a lle r.
A c lo s e lo o k to th e re s u lts s h o w e d th e ris e r B is th e m o s t s u ita b le
c o n fig u ra tio n te s te d to p e rfo rm th e c o n tin u o u s fe rm e n ta tio n o f A F B .
C o m p a rin g o n ly th e lo w e r g a s s u p e rfic ia l v e lo c ity (1 c m /s ) ris e r B w a s
th e o n e w h e re it w a s fo u n d : b e tte r h o m o g e n iz a tio n o f S G p a rtic le s ;
lo w e r g a s h o ld -u p s in ris e r a n d a b s e n c e o f g a s h o ld-u p in d o w n c o m e r;
lo w e r m ix in g a n d c irc u la tio n tim e s , a s w e ll a s m in im u m t M /t C .
T h e re fo re ris e r B w a s c o n s id e re d a s th e c o n fig u ra tio n th a t in c lu d e s
a ll
th e
to
p e rfo rm
c o n tin u o u s
AFB
p rim a ry
In o rd e r to h a v e a b e tte r u n d e rs ta n d in g o f th e re a l h y d ro d y n a m ic s in
th is re a c to r c o n fig u ra tio n lo c a l m e a s u re m e n ts w e re m a d e in a 6 L G L R .
T h is is d is c u s s e d in C h a p te r V II.
V.6 References
Banisi, S., Finch, J. A., Laplante, A. R. and Weber, M. E. (1995a). Effect of solid
particles on gas holdup in flotation columns-I. measurement. Chemical
Banisi, S., Finch, J. A., Weber, M. E. and Laplante, A. R. (1995b). Effect of solid
particles on gas holdup in flotation columns-II. Investigation of mechanisms of
gas holdup reduction in presence of solids. Chemical Engineering Science, 50(14),
2335-2342.
Bello, R. A. (1981). A characterization study of airlift contactors for application to
fermentations. PhD thesis. University of Waterloo, Ontario, Canada.
Bla, M., Ki, M. and Marko, J. (2004). Scale influence on the hydrodynamics of an
internal loop airlift reactor. Chemical Engineering and Processing, 43(12), 15191527.
Boyer, C., Duquenne, A.-M. and Wild, G. (2002). Measuring techniques in gas-liquid
and gas-liquid-solid reactors. Chemical Engineering Science, 57, 3185-3185.
135
Chapter V
Brnyik, T., Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2002).
grains. Journal Of The Institute Of Brewing, 108(4), 410-415.
Chisti, M. Y. (1989). Airlift Bioreactors (1st ed.). Essex: Elsevier Science Publishers
LTD.
Clift, R., Grace, J. R. and Weber, M. E. (1978). Bubbles, drops, and particles (Vol.
380). Academic press New York.
Deckwer, W. D. (1992). Bubble Column Reactors. (Wiley, Ed.). Chichester.
Dolgos, O., Klein, J., Vicente, A. A. and Teixeira, J. A. (2001). Behaviour of dual gasliquid separator in an internal-loop airlift reactor effect of top clearance. 28th
Conference SSCHE (pp. 21 - 25). Tatranks Matliare.
Fan, L-S., Yang, G. Q., Lee, D. J., Tsuchiya, K. and Luo, X. (1999). Some aspects of
high-pressure phenomena of bubbles in liquids and liquidsolid suspensions.
Chemical Engineering Science, 54(21), 46814709.
Fonseca, M. M. and Teixeira, J. A. (2007). Reactores Biolgicos (in Portuguese). L.
Lidel-edies tcnicas, 482.
Freitas, C., Vicente, A. A., Mota, M. and Teixeira, J. A. (1997). A new sampling
device for measuring solids hold-up in a three-phase system. Biotechnology
Techniques, 11(7), 489-492.
Freitas, C. and Teixeira, J. A. (1998a). Hydrodynamic studies in an airlift reactor
with an enlarged degassing zone. Bioprocess and Biosystems Engineering, 18(4),
267279. Springer.
Freitas, C. and Teixeira, J. A. (1998b). Solid-phase distribution in an airlift reactor
with an enlarged degassing zone. Biotechnology Techniques, 12(3), 219-224.
Freitas, C., Fialov, M., Zahradnik, J. and Teixeira, J. A. (1999). Hydrodynamic
Freitas, C., Fialov, M., Zahradnik, J. and Teixeira, J. A. (2000). Hydrodynamics of a
three-phase external-loop airlift bioreactor. Chemical Engineering Science, 55(21),
49614972.
Freitas, Carla. (2002). Bioreactores Multifsicos - Caracterizao Hidrodinmica e
de Transferncia de Massa (in Portuguese). PhD thesis, University of Minho.
8094.
Jamialahmadi, M. and Muller-Steinhagen, H. (1991). Effect of solid particles on gas
hold-up in bubble columns. Canadian Journal of Chemical Engineering, 69, 390393.
Klein, J., Vicente, A. A. and Teixeira, J. A. (2003a). Hydrodynamic considerations on
optimal design of a three-phase airlift bioreactor with high solids loading. Journal
Klein, J., Vicente, A. A. and Teixeira, J. A. (2003b). Hydrodynamics of a Three-phase
Airlift Reactor with an Enlarged Separator Application to High Cell Density
Systems. The Canadian Journal of Chemical Engineering, 81, 1-11.
Klein, J., Vicente, A. A. and Teixeira, J. A. (2004). Study of hydrodynamics and
mixing in an airlift reactor with an enlarged separator using magnetic. 3rd
International Symposium on Two-Phase Flow Modelling and Experiemntation (Vol.
5, pp. 22-24). Pisa.
Korpijarvi, J., Oinas, P. and Reunanen, J. (1999). Hydrodynamics and mass transfer
in an airlift reactor. Chemical Engineering Science, 54(13-14), 22552262.
Lehnert, R., Kurec, M., Brnyik, T. and Teixeira, J. A. (2008). Effect of Oxygen
Lehnert, R., Novk, P., Macieira, F., Kurec, M., Teixeira, J. A. and Brnyik, T. (2009).
Optimisation of Lab-Scale Continuous Alcohol-Free Beer Production. Czech
Journal of Food Science, 27(4), 267-275.
Engineering Science, 50(8), 13011310. Elsevier.
Matsumoto, T., Hidaka, N. and Morooka, S. (1989). Axial distribution of solid holdup
in bubble column for gasliquidsolid system. A.I.Ch.E. Journal, 31, 1701-1709.
136
Chapter V
Mena, P. C., Ruzicka, M. C., Rocha, F. A., Teixeira, J. A. and Drahos, J. (2005).
columns.
Chemical
Engineering
Science,
60(22),
6013-6026.
doi:10.1016/j.ces.2005.04.020
Merchuk, J., Contreras, A., Garcia, F. and Molina, E. (1998). Studies of mixing in a
concentric tube airlift bioreactor with different spargers. Chemical Engineering
Science, 53(4), 709-719.
Molina, E., Contreras, A. and Chisti, Y. (1999). Gas holdup, liquid circulation and
mixing behaviour of viscous newtonian media in a split-cylinder airlift bioreactor.
Trans
Olivieri, G., Marzocchella, a, Vanommen, J. and Salatino, P. (2007). Local and global
hydrodynamics in a two-phase internal loop airlift. Chemical Engineering Science,
62(24), 7068-7077.
Rodrguez, A., Garcia-Calvo, E., Prados, A. and Klein, J. (1999). A fluid dynamic
model for three-phase airlift reactors. Chemical Engineering Science, 54, 23592370.
Van Benthum, W., Van der Lans, R., Van Loosdrecht, M. and Heijnen, J. (2000). The
biofilm airlift suspension extension reactor-II: Three-phase hydrodynamics.
Chemical Engineering Science, 55(3), 699711.
Warsito, Ohkawa, M., Maezawa, A. and Uchida, S. (1997). Flow structure and phase
distributions in a slurry bubble column. Chemical Engineering Science, 52(21-22),
3941-3947.
Xie, T., Ghiaasiaan, S. M., Karrila, S. and Mcdonough, T. (2003). Flow regimes and
Yang, G. Q., Du, B. and Fan, L-S. (2007). Bubble formation and dynamics in gas
liquidsolid fluidizationA review. Chemical Engineering Science, 62, 2 - 27.
Yoo, D. H., Tsuge, H., Terasaka, K. and Mizutani, K. (1997). Behavior of bubble
formation in suspended solution for an elevated pressure system. Chemical
Engineering Science, 52(21-22), 37013707
Zahradnik, J., Fialova, M., Ru!i!ka, M., Draho", J., Ka"tnek, F. and Thomas, N. H.
(1997). Duality of the gas-liquid flow regimes in bubble column reactors. Chemical
137
Chapter VI
VI. Chapter VI CFD Simulation of Gas Holdup and Liquid Interstitial Velocity in Twophase iGLR
VI.1 Objectives
VI.2 Introduction
VI.3 Material and Methods
VI.4 Results and Discussion
VI.5 Conclusions
VI.6 References
138
Chapter VI
VI.1 Objectives
T h e m a in o b je c tiv e o f th is c h a p te r w a s to p e rfo rm c o m p u tin g flu id
d y n a m ic (C F D ) s im u la tio n s fo r th e tw o -p h a s e iG L R in o rd e r to e xp la in
a n d c o m p le m e n t th e p ra c tic a l re s u lts o b ta in e d in th e p re v io u s c h a p te r.
T h e m a in g o a l w a s to te s t th e a b ility o f o u r C F D s im u la tio n s e tu p to
c a p tu re g lo b a l c h a ra c te ris tic s o f th e flo w (m e a n liq u id in te rs titia l
v e lo c ity a n d g a s h o ld-u p b o th in ris e r a nd d o w n c o m e r s e c tio n s ) in o u r
iG L R w ith a n e n la rg e d d e g a s s in g z o n e . B e s id e s th is it w a s a ls o
im p o rta n t
to
c a p tu re
th e
th re e
e x p e rim e n ta lly
o b s e rv e d
b u b b le
c irc u la tio n re g im e s a n d s o v e rify if th e s im u la tio n s e tu p c o u ld b e la te r

u s e d to o b ta in th e g lo b a l c h ara c te ris tic fo r: (1 ) m o d ifie d g e o m e trie s ; (2 )
d iffe re n t flu id s . T h e c o m p a ris o n w a s d o n e fo r th e th re e d iffe re n t ris e r
tu b e s u s e d b e fo re (A , B , C s e e T a b le V I- 1 ) b u t o n ly fo r g -l s y s te m (a irw a te r). T o a p p ly th e C F D s im u la tio n s it w a s u s e d th e c o m m e rc ia l c o d e
F lu e n t 6 .3 u s in g a lg e b ra ic s lip m ix tu re m u ltip h a s e m o d e l. S im u la tio n s
re s u lts u s in g d iffe re n t m o d e l (fu ll E u le r E u le r) in d iffe re n t c o d e (C F X
1 2 .1 ) a re a ls o p re s e n te d in th e p a p e r. T h e s e c o n d a ry g o a l w a s to te s t
th e s e n s itiv ity o f th e s im u la tio n s to th e d iffe re n t b u b b le s lip v e lo c ity ,
a n d to th e d iffe re n t tu rb u le n c e c lo s u re m o d e ls (v a ria n ts o f ! " , ! # ).
T h is w a s d o n e u s in g th e m ix tu re m o d e l in F lu e n t 6 .3 c o d e .
VI.2 Introduction
The
k n o w le d g e
of
th e
iG L R
h y d ro d y n a m ic s
is
needed
fo r
th e
re a c to rs d e s ig n . B a s ic g lo b a l q u a n titie s s u c h a s g a s h o ld-u p a n d liq u id

v e lo c itie s in th e ris e r a n d in th e d o w n c o m e r, to ta l in te rfa c ia l a re a
a m o n g o th e rs n e e d to b e k n o w n . T h e h y d ro d y n a m ic a n d o th e r re le v a n t
p a ra m e te rs s u c h a s th e a irlift g e o m e try a re in te rre la te d a n d th e ir
re la tio n s h ip c a n b e q u ite c o m p le x b e c a u s e th e y , d ire c tly o r in d ire c tly ,
in flu e n c e e a c h o th e r a n d s o m e tim e s in c o m p le x in te ra c tio n s (C h is ti,
1 9 9 8 ). A s a n e x a m p le : th e d riv in g fo rc e fo r th e liq u id c irc u la tio n is th e
d iffe re n c e b e tw e e n th e ris e r a n d th e d o w n c o m e rs g a s h o ld-u p s . T h is is
b a la n c e d b y fric tio n lo s s e s in th e ris e r, d o w n c o m e r a n d in s e v e ra l p a rts
o f G L R g e o m e try . T h e fric tio n lo s s e s in th e c a s e o f iG L R a ls o o c c u r a n d
139
Chapter VI
a re in flu e n c e d b y b o tto m a n d to p c le a ra n c e s p a rts , w h ile in e x te rn a l

G L R th e s e lo s s e s a re in c o n n e c tin g p ip e s (F re ita s e t a l., 1 9 9 9 ).
H o w e v e r,
th e
re s u ltin g
liq u id
c irc u la tio n
a ffe c ts
th e
ris e r
and
d o w n c o m e r g a s h o ld -u p a n d th u s th e d riv in g fo rc e . T h e g a s h o ld-u p
d e p e n d s a ls o o n b u b b le s lip v e lo c ity , w h ic h d e p e n d s o n th e b u b b le
s iz e . B u b b le s iz e is in flu e n c e d b y : th e g a s d is trib u to r, c o a le s c e n c e
p ro p e rtie s
of
th e
in v o lv e d
flu id s
and
tu rb u le n c e .
T u rb u le n c e
is
in flu e n c e d b y liq u id c irc u la tio n . T h e re le v a n t h y d ro d y n a m ic p a ra m e te rs

n e e d to b e e ith e r o b ta in e d e x p e rim e n ta lly o r p re d ic te d b y m o d e ls o f
v a rio u s ty p e s . A lo t o f e x p e rim e n ta l d a ta a n d c o rre la tio n s h a v e b e e n
p u b lis h e d o n g lo b a l q u a n titie s (h o ld-u p , liq u id v e lo c itie s ) fo r iG L R . A
la rg e n u m b e r o f c o rre la tio n s fo r th e s e p a ra m e te rs a re c o m p ile d (C h is ti,
1 9 8 9 , 1 9 9 8 ; F re ita s e t a l., 1 9 9 9 ; G a rc ia-C a lv o e t a l., 1 9 9 9 ; L u e t a l.,
1 9 9 5 ). N e v e rth e le s s th e m a jo rity o f th e c o rre la tio n s p re s e n te d in th e
lite ra tu re a re re s tric te d in th e ir v a lid ity to th e s a m e re a c to r s iz e , ty p e
a n d g -l s y s te m u s e d (Y o u n g e t a l., 1 9 9 1 ). N o rm a lly , th e s e c o rre la tio n s
a re s y s te m s p e c ific , b e in g o f little u s e in d e s ig n o r s c a le-u p . T h e u s u a l
re q u ire m e n t is fo r e s tim a tio n o f e x p e c te d p e rfo rm a n c e in la rg e r o r
g e o m e tric a lly d iffe re n t re a c to rs o r flu id s (C h is ti, 1 9 9 8 ). S o m e a u th o rs
e m p lo y e d m o d e ls b a s e d o n m e c h a n ic a l e n e rg y b a la n c e in th e a irlift
re a c to r (C h is ti, 1 9 8 9 ; H e ijn e n e t a l., 1 9 9 7 ; V e rla a n e t a l., 1 9 8 6 ), b u t th e
in fo rm a tio n a b o u t fric tio n lo s s e s (fric tio n c o e ffic ie n ts ) m u s t b e p ro v id e d
a s a n in p u t p a ra m e te r. M o re re c e n t e x p e rim e n ta l m e a s u re m e n ts o f
a irlift g lo b a l h y d ro d y n a m ic c h a ra c te ris tic s (ris e r a n d d o w n c o m e r h o ldu p a n d v e lo c itie s ) c a n b e fo u n d fo r iG L R (M e rc h u k e t a l., 1 9 9 8 ; v a n
B a te n e t a l., 2 0 0 3 ) a n d e x te rn a l g a s -lift re a c to r (F re ita s e t a l., 1 9 9 9 ; V ia l
e t a l., 2 0 0 2 ).
140
Chapter VI
Solid-Liquid Mass
Transfer
Solids Suspension
Liquid Velocity
Heat Transfer
Microbial
Survival
UH
Mixing
Turbulence
Shear Rates
Gas-Liquid Mass
Transfer
Bubble Size
Bubble Rise
Velocity
kLaL
Gas Hold-up
Figure VI-1. Relationships between hydrodynamic parameters and other

bioreactor performance indices. Legend: UH Overall Heat Transfer
coefficient (W/(m2 C)); kLaL Overall volumetric mass transfer coefficient (s1
). Adapted from Chisti, 1989.
L u o a n d A l-D a h h a n (2 0 0 8 , 2 0 1 0 ) m e a s u re d lo c a l liq u id v e lo c ity

p ro file s , tu rb u le n t q u a n titie s u s in g th e c o m p u te r-a u to m a te d ra d io a c tiv e
p a rtic le tra c k in g (C A R P T ) te c h n iq u e a n d lo c a l g a s h o ld-u p p ro file s
u s in g c o m p u te d to m o g ra p h y in a n iG L R . T h e y o b s e rv e d s ig n ific a n t
e ffe c t o f to p a n d b o tto m c le a ra n c e s o n th e flo w . T h e y a ls o o b s e rv e d
th a t th e b u b b le s a re p ro n e to c o n c e n tra te in th e ris e r c e n te r in ra d ia l
d ire c tio n a n d th e c h a n g e fro m b u b b ly to c h u rn-tu rb u le n t flo w a t
s u p e rfic ia l g a s v e lo c ity o f 2 c m /s . A p a rt fro m e x p e rim e n ts , e m p iric a l
c o rre la tio n s o r th e o re tic a l m o d e ls
such
as
th e
m o d e ls
based
on
m e c h a n ic a l e n e rg y b a la n c e , C F D s im u la tio n s a re a to o l, w h ic h c a n b e
u s e d to s tu d y g a s-lift re a c to r h y d ro d y n a m ic s .
141
Chapter VI
T h e re a re tw o m a in g ro u p s o f m u ltip h a s e flo w m o d e ls u s a b le fo r
s im u la tio n s o n b u b b le c o llu m n a n d g a s-lift re a c to rs s c a le . In E u le r
E u le r m o d e ls a ll p h a s e s a re tre a te d a s in te rp e n e tra tin g c o n tin u a , w h ile
in E u le r L a g ra n g e m o d e ls th e m o tio n o f in d iv id u a l p a rtic le s is tra c k e d
th ro u g h th e c o n tin u o u s flu id . T h e E u le r L a g ra n g e m o d e ls , w h ic h tra c k
th e m o tio n o f e v e ry s in g le p a rtic le (a p p ro x im a te d a s a m a s s p o in t w ith
c lo s u re e q u a tio n fo r th e in te rp h a s e fo rc e s ), c a n b e u s e d fo r s m a lle r
s c a le p ro b le m s w ith lo w g a s h o ld -u p . O n ly E u le r E u le r m o d e ls (m ix tu re
m o d e l a n d fu ll E u le r E u le r m o d e l) w e re u s e d in th e p re s e nte d w o rk ,
th u s o n ly E u le r E u le r m o d e ls a re c o n s id e re d in th e fo llo w in g c h a p te r.
VI.2.1 CFD simulations in iGLR: advantages and limitations

T h e m a in a d v a n ta g e o f C F D s im u la tio n s if c o m p a re d to e x p e rim e n ts
is th a t n o e x p e rim e n ta l a p p a ra tu s h a s to b e b u ilt; th e re fore e q u ip m e n t
d im e n s io n s a n d w o rk in g flu id s c a n b e e a s ily c h a n g e d in s im u la tio n s .
H o w e v e r, th e q u a lity o f th e C F D s im u la tio n p re d ic tio n s , g re a tly d e p e n d s
o n h o w w e ll o r h o w b a d ly a re e m p lo y e d th e C F D m o d e ls , s u b-m o d e ls
a n d c lo s u re e q u a tio n s th a t d e s c rib e flo w p h e n o m e n a o c c u rrin g in th e
re a c to r. S in c e th e g -l flo w s a re v e ry c o m p le x w ith flo w p h e n o m e n a
o c c u rrin g o n a w id e ra n g e o f s p a c e a n d tim e s c a le s , m o d e lin g o f g -l
flo w s is s till a n o p e n s u b je c t a n d fa r fro m b e in g c o m p le te . M o re o v e r it
is s till n e c e s s a ry to v a lid a te s im u la tio n re s u lts a g a in s t e x p e rim e n ts . T h e
E u le r E u le r m o d e ls n e e d c lo s u re s fo r a ll re le v a n t in te rp h a s e fo rc e
(d ra g , lift, a d d e d m a s s , e tc .), fo r th e tu rb u le n c e (d u e to s in g le p h a s e
flo w a n d d u e to b u b b le s ) a s w e ll a s m o d e ls fo r b u b b le c o a le s c en c e a n d
b re a k -u p , b e c a u s e th e b u b b le s iz e is p re s e n t in m o s t o f th e c lo s u re
e q u a tio n s .
S o k o lic h in
et
a l.
(2 0 0 4 )
d is c u s s e d
th e
re le v a n c e
of
in d iv id u a l
in te rp h a s e fo rc e s fo r th e s im u la tio n a n d a ls o tu rb u le n c e m o d e llin g
is s u e s . T h e y o b s e rv e d a w e a k d e p e n d e n c y o f s im u la tio n re s u lts (in
p a rtia lly a e ra te d re c ta n g u la r b u b b le c o lu m n ) o n th e e m p lo y e d v a lu e o f
th e b u b b le s lip v e lo c ity . T h e y e x p la in e d th is w e a k d e p e n d e n c e b y th e
142
Chapter VI
fa c t th a t th e b u b b le to ta l v e lo c ity w a s th e s u m o f th e b u b b le s lip
v e lo c ity a n d th e liq u id v e lo c ity , w h ic h c a n b e re la tiv e ly h ig h , s o th e
c h a n g e in th e b u b b le s lip v e lo c ity h a d lo w e r im p a c t o n th e c a lc u la te d
g a s h o ld -u p (S o k o lic h in e t a l., 2 0 0 4 ). It c o u ld b e e x p e c te d fro m th e
s a m e re a s o n th a t th e s im ila r b e h a v io u r (w e a k d e p e n d e n c e o f ga s
h o ld u p o n th e b u b b le s lip v e lo c ity ) c o u ld b e fo u n d in a irlift s im u la tio n s ,
if b u b b le s a re p re s e n t o n ly in th e ris e r a n d th e d o w n c o m e r h o ld-u p is
z e ro , m a y b e th e d e p e n d e n c e c o u ld b e e v e n w e a k e r d u e to th e m o re
o rd e re d flo w in th e a irlift if c o m p a re d to th e b u b b le c o lu m n . H o w e v e r, if
th e g a s h o ld -u p in th e d o w n c o m e r is p o s itiv e , th e n th e e ffe c t o f th e
b u b b le s lip v e lo c ity c o u ld b e m u c h s tro n g e r d u e to c o u n te r c u rre n t flo w
o f b o th p h a s e s in th is g a s-lift re a c to r s e c tio n . It is th e n n o t s u rp ris in g
th a t s o m e a u th o rs d id s im u la tio n s o f g a s-lift re a c to r a n d o b ta in e d a
g o o d a g re e m e n t w ith e x p e rim e n ts e v e n w h e n in a p p ro p ria te c lo s u re fo r
d ra g fo rc e w a s u s e d (e .g . S c h ille r N a u m a n n c o rre la tio n fo r rig id s p h e re
d ra g u s e d fo r 5 m m e q u iv a le n t d ia m e te r a ir b u b b le in w a te r) in c a s e s
w ith z e ro d o w n c o m e r g a s h o ld -u p .
T h e re a re a n u m b e r o f p a p e rs d e d ic a te d to th e E u le r E u le r C F D
s im u la tio n s
of
a irlift
re a c to rs .
E x te rn a l-lo o p
a irlift
s im u la tio n
c o m p a ris o n s o f ra d ia l p ro file s o f g a s h o ld-u p a n d liq u id v e lo c ity c a n b e

fo u n d (C a o e t a l., 2 0 0 7 ; R o y e t a l., 2 0 0 6 ; V ia l e t a l., 2 0 0 2 ). T h e
c o m p a ris o n
of
a v e ra g e
gas
h o ld -u p
and
liq u id
v e lo c ities
w ith
e x p e rim e n ts fo r iG L R a n d fo r z e ro d o w n c o m e r g a s h o ld-u p c a n b e
fo u n d , (H u a n g e t a l., 2 0 0 8 ; M u d d e a n d v a n d e n A k k e r, 2 0 0 1 ; V a n B a te n
e t a l., 2 0 0 3 ). H u a n g e t a l. (2 0 0 8 ) a ls o re p o rte d w e a k d e p e n d e n c e o f th e
s im u la tio n
re s u lts
on
th e
b u b b le
v e lo c ity
p re s c rib e d
on
th e
to p
b o u n d a ry c o n d itio n . S im u la tio n s fo r c a s e s w ith n o n z e ro d o w n c o m e r

h o ld -u p w e re d o n e (H u a n g e t a l., 2 0 1 0 ; J ia e t a l., 2 0 0 7 ; O e y e t a l.,
2 0 0 1 ; T a lv y e t a l., 2 0 0 7 ). T a lv y e t a l. (2 0 0 7 ) c o m p a re d v e rtic a l a n d
h o riz o n ta l p ro file s o f g a s h o ld u p in ris e r a n d d o w n c o m e r, a n d h o riz o n ta l
liq u id v e lo c ity p ro file s in d o w n c o m e r w ith e x p e rim e n ts in a re c ta n g u la r
a irlift. A ls o J ia e t a l. (2 0 0 7 ) c o m p a re d s im u la te d a n d e x p e rim e n ta l
h o riz o n ta l p ro file s o f g a s h o ld -u p a n d liq u id v e lo c itie s in a re c ta n g u la r
a irlift a n d fo u n d a g o o d a g re e m e n t.
143
Chapter VI
VI.3 Material and Methods

VI.3.1 Experimental setup
T h e e x p e rim e n ts w e re d o n e in th e s a m e iG L R w ith a to p e n la rg e d
d e g a s s in g z o n e u s e d in p re v io u s c h a p te r (F ig u r e V - 3 a n d T a b le V - 1
a n d T a b le V - 2 in C h a p te r V s e c tio n V .3 .1 ) b u t o n ly d a ta u s in g a ir-w a te r
w a s c o n s id e re d fo r C F D s im u la tio n s .
In F ig u r e V I- 3 a n d T a b le V I- 1 a re d is p la y e d th e re a c to r d im e n s io n s
a n d g rid c e lls u s e d in th is w o rk
Table VI-1. Riser configurations used in this work.
Riser A
Riser B
Riser C
nr of grid
nr of grid
nr of grid
(mm)
(mm)
(mm)
cells
cells
cells
100
12
100
12
100
12
D1
62
12
62
12
87
12
D2
70
14
70
14
92
14
D3
142
28
142
28
142
28
D4
420
28
420
28
420
28
D5
23
5
23
5
23
5
H1
1200
60
1400
70
1200
60
H2
200
16
0
0
200
20
H3
170
14
170
14
170
12
H4
120
6
120
6
120
6
H5
Legend: D1 Distributor diameter; D2 Riser internal diameter; D3 - Riser
external diameter; D4 Column internal diameter; D5 Top degassing zone
internal diamter; H1 Distance between distributor and beginning of riser; H2
Riser length; H3 Distance between riser end and beginning of top part; H4
Height of conical section from top part; H5 Height of cylindrical section of
top part.
T h e liq u id in te rs titia l v e lo c ity in th e ris e r a n d in th e d o w n c o m e r w a s

m e a s u re d a s d e s c rib e d in s e c tio n V .3 .6 . T h e g a s h o ld-u p in th e ris e r
a n d d o w n c o m e r w a s e s tim a te d u s in g U -tu b e m a n o m e te rs (s e c tio n
V .3 .3 )
w ith o u t
th e
s o lid s
c o n s id e ra tio n
(!Si=0
in
Eq.
IV - 2 ).
The
a g re e m e n t o f th is e s tim a te w ith th e re a l g a s h o ld u p in th e a p p a ra tu s
m a y b e a ffe c te d b y a p re s s u re d ro p d u e to flo w w ith a m a x im u m e rro r
o f 1 5 % (s e c tio n IV .4 .2 ).
144
Chapter VI
Figure VI-2. Internal Gas-lift Reactor geometry (not at scale). Legend: D1

Distributor diameter; D2 Riser internal diameter; D3 - Riser external
diameter; D4 Column internal diameter; D5 Top degassing zone internal
diameter; H1 Distance between distributor and beginning of riser; H2
Riser length; H3 Distance between riser end and beginning of top part; H4
Height of conical section from top part; H5 Height of cylindrical section of
top part.
VI.3.2 Simulation setup

T h e m a in p a rt o f th e s im u la tio n s w a s d o n e in C F D c od e F lu e n t 6 .3
u s in g th e a lg e b ric s lip m ix tu re m o d e l. A p a rt o f th e flu e n t s im u la tio n s
w a s la te r re c a lc u la te d in th e C F X 1 2 .1 c o d e u s in g th e fu ll E u le r E u le r
m o d e l a n d th e ir re s u lts a re a ls o p re s e n te d , a lth o u g h fo r th e ris e r C
c o n fig u ra tio n C F X s im u la tio n s w e re n o t fin is h e d d u e to c o n v e rg e n c e
p ro b le m s a n d th e re s u lts a re n o t s h o w n .
T h e a irlift g e o m e try is s h o w n in F ig u re V I- 2 T h e g a s d is trib u to r in th e
s im u la tio n d o m a in g e o m e try h a d th e s a m e d ia m e te r a s th e ris e r. T h e
c o m p u ta tio n a l g rid w a s th e s a m e fo r b o th s im u la tio n s (F lu e n t a n d C F X )
a n d c o n ta in e d 4 8 1 0 0 (R is e r A ), 4 4 7 0 0 (R is e r B ) a n d 4 9 1 0 0 (R is e r C )
145
Chapter VI
g rid c e lls . T h e c o m p u ta tio n a l g rid (a t th e a irlift c ro s s-s e c tio n ) is s h o w n

in F ig u r e V I- 3 a n d n u m b e r o f g rid c e lls p e r e a c h a irlift d im e n s io n c a n
be
fo u n d
in
T a b le
V I- 1
The
g rid
was
s u ffic ie n t
to
o b ta in
g rid
in d e p e n d e n t re s u lts (s e e S e c tio n V I.4 .1 fo r th e g rid in d e p e n d e n c e te s t

re s u lt). T h e tim e s te p in a ll s im u la tio n s w a s 0 .0 0 5 s .
Figure VI-3. Computational grid for the iGLR cross-section at Risers A, B

(left) and C (right).
VI.3.2.1
Fluent simulations
T h e m ix tu re m o d e l u s e d in F lu e n t 6 .3 c o d e a s s u m e s th a t tw o o r m o re
p h a s e s a re in te rp e n etra tin g . It c a n s o lv e c a s e s w h e re tw o (o r m o re )
p h a s e s m o v e a t d iffe re n t v e lo c itie s a n d it a s s u m e s lo c a l e q u ilib riu m
o v e r s h o rt s p a tia l le n g th s c a le s , i.e . d is p e rs e d p h a s e s m o v e a t th e ir
te rm in a l
v e lo c itie s .
T h is
model
is
s im p le r
th a n
fu ll
E u le r-E u le r
m u ltip h a s e m o d e l. T h e m ix tu re m o d e l s o lv e s th e c o n tin u ity a n d th e

m o m e n tu m e q u a tio n fo r a m ix tu re w ith a v e ra g e d p ro p e rtie s (d e n s ity ,
v e lo c ity a n d v is c o s ity ), a n a lg e b ra ic e q u a tio n fo r th e d is p e rs e d p h a s e (s )
s lip v e lo c ity a n d a v o lu m e fra c tio n e q u a tio n fo r th e s e c o n d a ry p h a s e (s ).
T h e m ix tu re m o d e l s h o u ld re q u ire le s s c o m p u ta tio n a l re s o u rc e s th a n
th e E u le r-E u le r m o d e l, s in c e it s o lv e s fe w e r e q u a tio n s . H o w e v e r, it c a n
b e s o m e tim e s m o re d iffic u lt to c o n v e rg e w ith m o re ite ra tio n s re q u ire d
to b e d o n e , th u s d im in is h in g th e m e n tio n e d a d v a n ta g e o v e r th e E u le rE u le r m o d e l. M o d e l e q u a tio n s , b o u n d a ry c o n d itio n s a n d o th e r s o lv e r
p a ra m e te rs
a re
e x p la in e d
b e lo w .
The
m ix tu re
model
s o lv e s
th e
c o n tin u ity a n d m o m e n tu m e q u a tio n fo r th e m ix tu re , th e a lg e b ra ic

e q u a tio n fo r d is p e rs e d p h a s e s lip v e lo c ity a n d v o lu m e fra c tio n e q u a tio n
146
fo r
th e
Chapter VI
s e c o n d a ry
phase
(M a n n in e n
et
a l.,
1 9 9 6 ).
The
c o n tin u ity
e q u a tio n is
!"m /!t + # $ ( "m v m ) = 0
E q . V I 1
W h e re :
n
v m = "# 1 ! $% ' & k !k v k

m k =1
E q . V I 2
Is th e m a s s a v e ra g e d v e lo c ity , a n d
n
!m = # " k !k
Eq. VI 3
k =1
Is th e m ix tu re d e n s ity . T h e s u m s a re o v e r a ll (n ) p h a s e s (k ). T h e
m o m e n tu m e q u a tio n in th e m ix tu re m o d e l c a n b e o b ta in e d b y s u m m in g
m o m e n tu m e q u a tio n s o f a ll p h a s e s in th e E u le r-E u le r m o d e l.
The
m o m e n tu m e q u a tio n is :
! ( "m v m ) /!t + #.( "m v m v m ) = $#p + #.[ % (#v m + #T v m )] + "m g + ...

E q . V I 4
( n
+
... + #.* ' & k "k v dr, k v dr, k ) k =1
,
W h e re n is th e n u m b e r o f p h a s e s (tw o in o u r c a s e : a ir, w a te r), a n d

n
! = # " kk + t
E q . V I 5
k =1
Is th e m ix tu re e ffe c tiv e v is c o s ity , t is th e tu rb u le n t v is c o s ity a n d k

is th e m o le c u la r v is c o s ity o f p h a s e k , a n d
v dr,k = v k ! v m = v kc ! #$ 1 " %& .( ' i "i v ic

m
i=1
E q . V I 6
Is th e d rift v e lo c ity o f p h a s e k , w h e re
v kc = v k ! v c
E q . V I 7
Is th e s lip v e lo c ity b e in g th e c s u b s c rip t fo r th e c o n tin u o u s p h a s e .

T h e s lip v e lo c ity is c a lc u la te d fro m th e fo llo w in g a lg e b ra ic e q u a tio n :
&'
&' c *
v kc = ! k ( "k # "c ) a / f"k # ($t /% t )( k ' #
'c+
)
k
147
E q . V I 8
Chapter VI
T h e firs t te rm o n th e rig h t h a n d s id e o f E q . V I 8 is d u e to a d ra g
fo rc e , w h e re
!k =
"k dk2
18 c
E q . V I 9
Is k -p h a s e p a rtic le re la x a tio n tim e , a n d f is a d ra g fu n c tio n :
f = CD ! Re/24
E q . V I 1 0
B e in g R e e x p re s s e d b y :
Re =
dk v kc !c
c
E q . V I 1 1
T h e a c c e le ra tio n (a ):
a = g ! ( v m .") v m !
#v m
E q . V I 1 2
#t
A n d C D is d ra g c o e ffic ie n t. T h e d ra g c o e ffic ie n t (C D ) w a s c a lc u la te d
fro m T o m ia y a m a s c o rre la tio n (T o m iy a m a e t a l., 2 0 0 2 ) fo r a s in g le
b u b b le :
CD = max"min 24 /Re(1 + 0.15Re 0.687 ),72 /Re ,(8 /3)

$#
E!
%
E 0 + 4 '&
E q .V I 1 3
W h e re ,
E! =
"#gd 2
!
E q . V I 1 4
T h is m e a n s th a t th e d ra g c o e ffic ie n t is C D = 1 .2 1 5 fo r s in g le 5 m m
b u b b le , w ith a s lip v e lo c ity ~ 0 .2 3 m /s . In o rd e r to te s t th e e ffe c t o f
b u b b le s lip v e lo c ity o n s im u la tio n re s u lts S c h ille r-N a u m a n n s c lo s u re
w a s a ls o e m p lo y e d :
CD = 24 /Re(1 + 0.15Re 0.687 ) for Re < 1000 and C D = 0.44 for Re > 1000 E q . V I 1 5
E q . V I 1 3 w a s u s e d in a ll s im u la tio n s u n le s s s a id o th e rw is e . A t th is
p o in t is im p o rta n t to n o tic e a p o s s ib le b u g e ith e r in F lu e n t 6 .3 a n d
F lu e n t1 2 m ix tu re m o d e ls . If a firs t o rd e r tim e d is c re tiz a tio n s c h e m e is
u s e d th e n th e s lip v e lo c ity is c a lc u la te d c o rre c tly . If a s e c o n d o rd e r
s c h e m e is u s e d th e n it is c a lc u la te d in c o rre c tly (1 s t o rd e r ~ 0 .2 3 m /s ,
2 n d o rd e r ~ 0 .4 7 m /s ). T h is w a s te s te d fo r b o th u s e r d e fin e d c lo s u re a n d
148
Chapter VI
c lo s u re s th a t a re a lrea d y p re s e n t in th e s o lv e r. T h e s e c o n d te rm in E q .
V I 8 a p p e a rs d u e to a d is p e rs io n o f s e c o n d a ry p h a s e b y tu rb u le n c e ,
w h e re ! t is a tu rb u le n t P ra n d tl n u m b e r a n d " t is a tu rb u le n t d iffu s iv ity :
!k =
C k2
# # /(1 + # # )(1 + C$ #2 ) %1/ 2
"k
E q . V I 1 6
W h e re :
! " = v kc /(2 / 3k)1/ 2
E q . V I 1 7
C = 1.8 ! 1.35cos2 "
E q . V I 1 8
! = v kc v k /( v kc v k )
E q . V I 1 9
C w a s c o n s id e re d to b e e q u a l to 0 .0 9 . T h e # # is a tim e ra tio b e tw e e n
th e tim e s c a le o f th e e n e rg e tic tu rb u le n t e d d ie s a ffe c te d b y th e
c ro s s in g -tra je c to rie s e ffe c t a n d th e p a rtic le re la x a tio n tim e
! ! = 0.165(k /" )(
1+C # !2 /$ k
E q . V I 2 0
T h e v a lu e o f ! t w a s s e t to 0 .0 1 . T h e d e fa u lt v a lu e w a s 0 .7 5 . T h e
lo w e r v a lu e , w h ic h w a s u s e d in F lu e n t s im u la tio n s , e n h a n c e s a tu rb u le n t
d is p e rs io n . T h e lo w er v a lu e w a s c h o s e n , b e c a u s e fo r R is e r B b u b b le s
flo w in g in to th e ris e r d id n o t d is p e rs e fa s t e n o u g h a c ro s s th e w h o le
ris e r c ro s s s e c tio n a s it w a s v is u a lly o b s e rv e d in e x p e rim e n ts . T h e
m ix tu re c o m p o s itio n is u p d a te d b y s o lv in g th e tra n s p o rt e q u a tio n fo r
th e d is p e rs e d p h a s e s (o n ly o n e d is p e rs e d p h a s e in o u r c a s e )
! (" k #k ) /!t + $.(" k #k v m ) = %$.(" k #k v dr, k )
E q . V I 2 1
T h e s ta n d a rd $ % & m o d e l w a s u s e d to m o d e l tu rb u le n c e . T h is m o d e l
b e lo n g s to a g ro u p o f R e y n o ld s a v e ra g e d N a v ie r-S to k e s e q u a tio n
m o d e ls . T h e in s ta n ta n e o u s v e lo c ity in th e m o m e n tu m e q u a tio n is
d iv id e d in to th e a v e ra g e a n d flu c tu a tin g p a rt
u = v m + v'
E q . V I 2 2
T h e n th e m o m e n tu m e q u a tio n is e n s e m b le a v e ra g e d a n d th e re s u ltin g
te rm
!".( #v'v')
E q . V I 2 3
Is m o d e le d a s
149
Chapter VI
!. t (!v m + !v Tm )
E q . V I 2 4
W h e re t is tu rb u le n c e v is c o s ity , w h ic h a g a in re q u ire s a c lo s u re
e q u a tio n . T h e c lo s u re is :
t = !m C k 2 /"
E q . V I 2 5
T h e tu rb u le n t k in e tic e n e rg y , k , a n d tu rb u le n t d is s ip a tio n ra te , ! , a re
o b ta in e d b y s o lv in g th e ir tra n s p o rt e q u a tio n s :
! ( "m k) /!t + #.( "m v m k) = #.[ t,m /$ k % (#k)] + Gk,m + "m&
E q . V I 2 6
! ( "m# ) /!t + $.( "m v m# ) = $.[ t,m /% k & ($# )] + # /k(C1# Gk,m ' C2# "(# )
E q . V I 2 7
T h u s u n lik e th e m o le c u la r v is c o s ity , th e tu rb u le n t v is c o s ity is n o t a

c o n s ta n t, b u t it d e p e n d s o n a lo c a l flo w fie ld . T h e te rm G k , m is a
tu rb u le n c e p ro d uc tio n d u e to m e a n v e lo c ity s h e a r.
Gk,m = t !v m + (!v m )T : !v m
E q . V I 2 8
T h e tu rb u le n t m o d e l c o n s ta n ts w e re C = 0 .0 9 , " k = 1 , " ! = 1 .3 , C 1 ! =
1 .4 4 , C 2 ! = 1 .9 2 . T h e s e a re e m p iric a lly d e te rm in e d c o n s ta n ts fo u n d to
w o rk w e ll fo r a w id e ra n g e o f w a ll-b o u n d e d a n d fre e s h e a r flo w s (F lu e n t
In c ., 2 0 0 6 ). T h e b o u n d a ry c o n d itio n s w e re n o-s lip c o n d itio n o n a ll a irlift
w a lls . S ta n d a rd w a ll fu n c tio n w a s u s e d to m o d e l a v e lo c ity p ro file in th e
w a lls v ic in ity a n d to p ro v id e in n e r b o u n d a ry c o n d itio n fo r R e y n o ld s
a v e ra g e d v e lo c ity fie ld . Z e ro liq u id v e lo city a n d 0 .2 5 m /s g a s v e rtic a l
v e lo c ity w a s p re s c rib e d a t th e in le t fo r a ll p h a s e s . G a s v o lu m e fra c tio n
w a s th e n s e t to a c o n s ta n t v a lu e to o b ta in th e d e s ire d g a s flo w ra te in to
th e a irlift. Z e ro liq u id v e lo c ity w a s s e t a t th e o u tle t b o u n d a ry a n d n o
g ra d ie n ts w e re c o n s id e re d e ith e r fo r g a s v e lo c ity a n d g a s v o lu m e
fra c tio n . In itia l c o n d itio n w a s z e ro liq u id v e lo c ity a n d g a s v o lu m e
fra c tio n ,
in itia l
tu rb u le n c e
k in e tic
e n e rg y
was
# = 0 .0 0 1 m 2 /s 2 ,
and
d is s ip a tio n ra te ! = 0 .0 0 0 1 m 2 /s 3 . T h e tim e in te rv a l o f 3 0 s w a s th e n
s im u la te d to le t th e flo w re a c h a s te a d y o r p s e u d o-s te a d y s ta te . T h e n
a n o th e r 1 2 0 s o r m o re o f a flo w tim e w a s s im u la te d to o b ta in tim ea v e ra g e d q u a n titie s fo r e v a lu a tion .
150
Chapter VI
T h e s e g re g a te d p re s s u re-b a s e d s o lv e r in F lu e n t w a s u s e d to s o lv e
th e m o d e l e q u a tio n s , n o d e b a s e d g ra d ie n t o p tio n w a s u s e d to e v a lu a te
v a ria b le s
g ra d ie n ts .
P re s s u re-Im p lic it
w ith
S p littin g
of
O p e ra to rs
(P IS O ) s c h e m e w a s u s e d a s a p re s s u re-v e lo c ity c o u p lin g a lg o rith m ,

P R E S T O ! (P R E s s u re S T a g g e rin g O p tio n ) s c h e m e w a s u s e d fo r p re s s u re
d is c re tiz a tio n a n d th e Q U IC K s c h e m e fo r v e lo c ity (F lu e n t In c ., 2 0 0 6 ),
g a s v o lu m e fra c tio n , tu rb u le n c e k in e tic e n e rg y a n d d is s ip a tio n ra te . A
firs t o rd e r im p lic it s c h e m e w a s e m p lo y e d fo r th e tim e d is c re tiz a tio n .
VI.3.2.2
CFX simulation
E u le r-E u le r tw o -flu id m o d e l w a s u s e d in o u r s im u la tio n s d o n e in C F X

1 2 .1 . In o p p o s itio n w ith th e m ix tu re m o d e l, th is m o d e l s o lv e s c o n tin u ity
a n d m o m e n tu m e q u a tio n fo r e a c h p h a s e . T h u s fo r a tw o p h a s e flo w
th e re a re tw o c o n tin u ity a n d tw o m o m e n tu m e q u a tio n s , o n e s e t fo r
c o n tin u o u s
phase
(c )
and
one
fo r
d is p e rs e d
phase
(d ).
The
tw o
m o m e n tu m e q u a tio n s a re c o u p le d v ia p re s s u re (p re s s u re fie ld is s h a re d
b y b o th p h a s e s ), a n d v ia in te rp h a s e fo rc e te rm s , w h ic h a c c o u n ts fo r
v a rio u s fo rc e s (d ra g , lift, a d d e d m a s s , tu rb u le n t d is p e rs io n fo rc e a n d
o th e rs ) a n d c a n c e l e a c h o th e r o u t w h e n m o m e n tu m e q u a tio n s o f
in d iv id u a l p h a s e s a re a d d e d
to g e th e r. D ra g fo rc e
and
tu rb u le n c e
d is p e rs io n fo rc e w e re a c c o u n te d fo r in o u r C F X s im u la tio n s . T he
c o n tin u ity e q u a tio n s o f p h a s e c a n d d a re :
! (" c #c ) /!t + $.(!" c #c v c ) = 0
E q . V I 2 9
! (" d #d ) /!t + $.(!" d #d v d ) = 0
E q . V I 3 0
T h e m o m e n tu m e q u a tio n s o f p h a s e c a n d d a re :
! (" c #c v c ) /!t + $.(!" c #c v c ) = %" c $p + $. " c c,eff ($v c + $v c T ) + " c #c g + M cd

E q . V I 3 1
! (" d #d v d ) /!t + $.(!" d #d v d ) = %" d $p + $. " d d ,eff ($v d + $v d T ) + " d #d g + M dc

E q . V I 3 2
W h e re :
M cd = !M dc
E q . V I 3 3
151
Chapter VI
Is a fo rc e a c tin g o n th e p h a s e c d u e to p h a s e d (d ra g , lift, ), a n d
c , e f f a n d d , e f f a re e ffe c tiv e v is c o s itie s c o n s is tin g o f a m o le c u la r a n d a

tu rb u le n t v is c o s ity :
c,eff = c + tcand d ,eff = d + td
E q . V I 3 4
O n ly d ra g a n d tu rb u le n c e d is p e rs io n fo rc e w e re c o n s id e re d in o u r
s im u la tio n s :
M cd = M cd, D + M cd, TD
E q . V I 3 5
M cd,D = K cd (v d ! v c ) = (3/4)(CD /d)" d #d v d ! v c (v d ! v c )
E q . V I 3 6
W h e re d is d is p e rs e d p h a s e p a rtic le (b u b b le ) e qu iv a le n t d ia m e te r.
D ra g c o e ffic ie n t w a s s e t to C D = 1 .2 1 5 re s u ltin g in a b u b b le s lip v e lo c ity
~ 0 .2 3 m /s (~ 5 m m b u b b le in w a te r).
M cd,TD = CTD K cd v tc /! tc " (#$ d /$ d % #$ c /$ c )
E q . V I 3 7
W h e re C T D = 1 , K c d is m o m e n tu m tra n s fe r c o e ffic ie n t fo r d ra g fo rc e , ! t c
is tu rb u le n t k in e m a tic v is c o s ity o f th e c o n tin u o u s p h a s e , " t c = 0 .9 . T w o
e q u a tio n s #$% m o d e l w e re u s e d to m o d e l tu rb u le n c e a n d to o b ta in a
c lo s u re fo r tu rb u le n t v is c o s ity in th e c o n tin u o u s p h a s e:
tc = C !c k c2 /" c
E q . V I 3 8
! (" c #c kc ) /!t + $.(" c #c v c k c ) = $.[" c ( c + tc /% k )($kc )] + " c Gc & " c #c' c E q . V I 3 9
! (" c #c$ c ) /!t + %.(" c #c v c$ c ) = %.[" c ( c + tc /& k )(%$ c )] + " c$ c /k c (C1$ Gc ' C2$ #c$ c )
Eq. VI40
W h e re
Gc = tc !v c + (!v c ) T : !v c
E q . V I 4 1
T h e tu rb u le n t m o d e l c o n s ta n ts w e re C = 0 .0 9 , " k = 1 , " ! = 1 .3 , C 1 ! = 1 .4 4 ,
C 2 ! = 1 .9 2 . T u rb u le n c e v is c o s ity in th e d is p e rs e d p h a s e w a s c a lc u la te d
as:
td = ( !d / !c ) tc /"
E q . V I 4 2
W h e re " = 1 . T h e b o u n d a ry c o n d itio n s w e re n o-s lip c o n d itio n fo r liq u id

a n d fre e -s lip c o n d itio n fo r g a s p h a s e o n w a lls . Z e ro v e lo c ity fo r liq u id
a n d 0 .2 5 m /s fo r g a s p h a s e w e re u s e d . T h e v o lu m e fra c tio n o f g a s
152
Chapter VI
p h a s e w a s s e t to o b ta in th e d e s ire d v a lu e o f g a s flo w ra te . T h e
d e g a s s in g c o n d itio n w a s u s e d a t th e o u tle t liq u id v e lo c ity is s e t to
z e ro , w h ile th e g a s p h a s e is a llo w e d to e s c a p e .
VI.4 Results and Discussion

T h e re s u lts s e c tio n is d iv id e d in tw o p a rts . A u x ilia ry s im u la tio n
re s u lts a re p re s e n te d in s e c tio n V I.4 .1 . T h is s e c tio n c o n s is ts o f g rid
in d e p e n d e n c e te s t re s u lt, b rie f c o m p a ris o n o f fe w s im u la tio n s w ith
d iffe re n t tu rb u le n c e m o d e ls fo r a s e le c te d iG L R ris e r c o n fig u ra tio n , a n d
fin a lly a c o m p a ris o n o f tw o d iffe re n t c lo s u re s fo r th e d ra g fo rc e . T h e
m a in re s u lts a n d th e ir c o m p a ris o n w ith e x p e rim e n ta lly m e a s u re d d a ta
(liq u id in te rfa c ia l v e lo c ity a n d g a s h o ld -u p in th e ris e r a n d d o w n c o m e r)
a re p re s e n te d in s e c tio n V I.4 .2 .
T h e re s u lts fo r b o th s im u la tio n s e tu p s (F lu e n t, C F X ) a re p re s e n te d . In
e x p e rim e n ts , th e re w e re h ig h e r liq u id v e lo c itie s in th e d o w n c o m e r fo r
th e la rg e r d ia m e te r ris e r (R is e r C ), d u e to th e lo w e r d o w n c o m e r c ro s s s e c tio n a re a , w h ic h le a d th e b u b b le s (g a s-p h a s e ) to e n tra in in to th is
s e c tio n . If R is e r A (s h o rte r d ra ft tu b e ) a n d B (lo n g e r d ra ft tu b e ) a re
c o m p a re d , th e n fo r th e lo n g e r tu b e th e g a s s e p a ra tio n w a s b e tte r
re s u ltin g in n o g a s in th e d o w n c o m e r, w h ile fo r th e s h o rte r d ra ft tu b e
(c a s e A ) b u b b le s w e re e n tra in e d in to th e d o w n c o m e r fo r th e h ig h e s t g a s
flo w ra te . T h e s e re s u lts a re in a g re e m e n t w ith th e re s u lts o b ta in e d in
th e fo rm e r c h a p te r (s e e s e c tio n V .4 .2 ).
VI.4.1 Auxiliary simulation results

T h e g rid in d e p e n d e n c e te s t w a s d o n e fo r th e iG L R w ith ris e r B fo r a n
a irflo w ra te o f 3 .6 L /m in (U G = 1 c m /s s e e T a b le V - 2 in s e c tio n V -3 .1 ).
O n e s im u la tio n (F lu e n t) w a s ru n o n a re fin e d fin e g rid w ith 8 x m o re
g rid c e lls th a n in th e s ta n d a rd c o a rs e g rid c a s e (3 5 7 6 0 0 v s 4 4 7 0 0
c e lls ). T h e d iffe re n c e in th e liq u id in te rs titia l v e lo c ity a n d g a s h o ld-u p in
153
Chapter VI
th e ris e r w a s w ith in 1 % . T h e re fo re it c o u ld b e v e rifie d th a t th e s ta n d a rd

g rid w a s e n o u g h to o b ta in g rid in d e p e n d e n t re s u lts .
F o u r a d d itio n a l tu rb u le n c e m o d e ls in a d d itio n to s ta n d a rd !"# m o d e l
w e re te s te d (in F lu e n t) a t s a m e c o n d itio n s (ris e r B , 3 .6 L /m in ), p rio r to
th e m a in s im u la tio n c a m p a ig n . T h e s e fo u r m o d e ls w e re R N G !"# m o d e l,
RNG
!"#
model
w ith
s w irl
m o d ific a tio n
fo r
tu rb u le n c e
v is c o s ity ,
re a liz a b le !"# m o d e l a n d s ta n d a rd (n o t S S T ) !"$ m o d e l. F o r a d d itio n a l

d e ta ils a b o u t th e s e m o d e ls (s im u la tio n s w e re ru n w ith d e fa u lt s o lv e r
s e ttin g s ) a c o m p le te d e s c rip tio n is p re s e n t in F lu e n ts 6 .3 U s e r G u id e
(F lu e n t In c ., 2 0 0 6 ). U s in g th e m e n tio n e d tu rb u le n c e m o d e ls in s te a d o f
s ta n d a rd
!"# m o d e l h a d o n ly a m in o r im p a c t o n re s u lts o f th e
s im u la tio n s . T h e la rg e s t d iffe re n c e b e tw e e n s ta n d a rd !"# m o d e l re s u lts

a n d th e o th e r m o d e l re s u lts w a s 1 .4 % fo r in te rs titia l liq u id v e lo c ity a n d
4 .9 % fo r g a s h o ld -u p in ris e r o n c o a rs e g rid a n d 1 .7 % a n d 4 .9 % o n
th e fin e g rid re s p e c tiv e ly . S ta n d a rd !"# m o d e l w a s th e n k e p t fo r a ll
s u b s e q u e n t s im u la tio n s .
D iffe re n t c lo s u re s fo r d ra g c o e ffic ie n t w e re te s te d o n R is e rs A a n d B
to te s t th e e ffe c t o f c h a n g in g th e b u b b le s lip v e lo c ity o n s im u la tio n
re s u lts . T o m iy a m a e t a l. (2 0 0 2 ) c lo s u re fo r d ra g (E q . V I- 1 3 ) fo r a s in g le
b u b b le (5 m m e q u iv a le n t d ia m e te r) a n d S c h ille r-N a u m a n n c lo s u re (E q .
V I- 1 5 ) fo r 5 m m b u b b le w e re c o m p a re d fo r c a s e A , a n d T o m iy a m a
c lo s u re
fo r
3mm
and
5mm
b u b b le
w e re
c o m p a re d
fo r
case
B.
S im u la tio n s w e re d o n e fo r fiv e g a s flo w ra te s in ra n g e 1 .9 1 3 .6 L /m in

(1 .0 7 .5 c m /s ) fo r b o th ris e rs c o n fig u ra tio n s (A a n d B ).
R is e r A c o m p a ris o n b e tw e e n th e d ra g c lo s u re s is s h o w n in F ig u r e
V I- 4 . W e a re a w a re th a t th e S c h ille r-N a u m a n n c lo s u re is v a lid o n ly fo r
rig id s p h e re s , w h ic h is c e rta in ly n o t th e c a s e o f 5 m m a ir b u b b le in
w a te r. H o w e v e r, th e a im o f th is c o m p a ris o n w a s o n ly to e x a m in e a n
in flu e n c e o f c h a n g in g a s lip v e lo c ity o f b u b b le s o n s im u la tio n re s u lts .
T h e T o m iy a m a c lo s u re s re su lte d in b u b b le s lip v e lo c itie s (ris e r v o lu m e
a v e ra g e ) in ra n g e 2 2 .2 to 2 2 .9 c m /s d e p e n d in g o n th e g a s flo w ra te
(w a s lo w e r fo r h ig h e r g a s flo w ra te s ). T h e S c h ille r-N a u m a n n s lip
154
Chapter VI
v e lo c ity w a s a ro u n d 7 0 % h ig h e r, fro m 3 7 .6 to 3 8 .6 c m /s . N e v e rth e le s s ,

th is d iffe re n c e in s lip v e lo c itie s d id n o t c a u s e v e ry s ig n ific a n t c h a n g e s
in s im u la tio n o u tp u ts . T h e re la tiv e d iffe re n c e b e tw e e n th e g a s h o ld-u p
in ris e r w a s fro m 1 1 .2 to 1 5 .8 % a n d w a s d e c re a s in g w ith in c re a s in g g a s
flo w ra te (m a x im u m a b s o lu te h o ld -u p d iffe re n c e b e tw e e n th e tw o
c lo s u re s w a s 0 .8 % ).
b!
a!
1!
Gas Hold-up (-)!
0.08!
0.06!
0.8!
0.04!
VL(m/s)!
0.6!
0.4!
0.02!
0.2!
0!
0!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
UG (m/s)!
0.08!
c!
Gas Hold-up (-)!
0.008!
0.006!
0.004!
0.002!
0!
0!
0.05!
UG (m/s)!
0.1!
Figure VI-4. Riser A simulation results (section VI. 4.1). Comparison of two
drag closures for 5mm bubbles. (a) Riser liquid interstitial velocity VL (m/s),
(b) riser gas holdup !, and (c) downcomer gas holdup ! vs. riser gas
superficial velocity UG (m/s). Legend: ! - Tomiyama (Eq. VI-13);
" - Schiller-Naumann (Eq. VI-15).
T h e re a s o n fo r th is is th e c o -c u rre n t flo w o f liq u id a n d g a s p h a s e s in

th e ris e r. T h e g a s h o ld u p in th e ris e r is g iv e n b y
E q . V I 4 3
W h e re U G is g a s s u p e rfic ia l v e lo c ity g iv e n b y a irflo w ra te in to th e
a irlift a n d V G is g a s in te rs titia l v e lo c ity in th e ris e r, w h ic h is
E q . V I 4 4
155
Chapter VI
A s re fe rre d in th e lite ra tu re th e e ffe c t o f c ha n g in g th e b u b b le s lip

v e lo c ity V s l i p o n th e h o ld u p in th e a irlift ris e r is th e n s m a lle r th e n it
w o u ld b e in a c a s e o f b u b b le s ris in g th ro u g h a s ta g n a n t liq u id (V L = 0
c m /s ), b e c a u s e th e liq u id v e lo c ity V L in th e ris e r is re la tiv e ly h ig h if
c o m p a re d to b u b b le s lip v e lo c ity V s l i p (S o k o lic h in e t a l., 2 0 0 4 ). T h e
s itu a tio n w o u ld b e d iffe re n t in th e d o w n c o m e r, w h e re th e p h a s e s flo w
c o u n te r-c u rre n tly a n d th e s lip v e lo c ity v a lu e c o u ld h a v e a d e e p im p a c t
on
th e
downcomer
h y d ro d y n a m ic s .
T h e re
and
was
e ffe c tiv e ly
on
z e ro
th e
gas
w h o le
h o ld u p
a irlift
in
th e
d o w n c o m e r fo r a ll g a s flo w ra te s w h e re ris e r A w a s u s e d , e x c e p t fo r th e
h ig h e s t o n e w ith T o m iy a m a d ra g c o rre la tio n , w h e n th e g a s ju s t s ta rte d
to b e e n tra in e d in to th e d o w n c o m e r, a n d th e d o w n c o m e r h o ld u p w a s
~ 8 % o f th e ris e r h o ld u p . T h e d iffe re n c e b e tw e e n liq u id in te rs titia l
v e lo c itie s fo r th e tw o d ra g c o e ffic ie n t c o rre la tio n s w a s fro m 1 .6 to 8 .5 %
a n d w a s d e c re a s in g w ith in c re a s in g o f th e g a s flo w ra te w ith a s h a rp e r
d ro p o f th is d iffe re n c e fo r th e h ig h e s t g a s flo w ra te , w h e re th e d riv in g
fo rc e fo r liq u id c irc u la tio n w a s d e c re a s e d b y th e s m a ll a m o u n t o f g a s
e n tra in e d in to th e d o w n c o m e r (T o m ia y a m a c o rre la tio n c a s e ).
For
R is e r
c o n fig u ra tio n ,
th e
c o m p a ris o n
b e tw e e n
s im u la tio n s
a s s u m in g 3 m m a n d 5 m m d ia m e te r b u b b le s is s h o w n in F ig u r e V I- 5 .
T o m iy a m a d ra g c o rre la tio n w a s u s e d fo r b o th .
a!
1!
Gas Hold-up (-)!
VL / (m/s)!
0.8!
b!
0.08!
0.06!
0.6!
0.04!
0.4!
0.02!
0.2!
0!
0!
0!
0.02!
0.04!
0.06!
0.08!
0!
UG / (m/s)!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
Figure VI-5. Case B simulation results (section VII.4.1). Tomiyama drag

closure (Eq. VI-13) results for 3 mm (!) and 5mm bubble ("). Legend: (a) Riser liquid interstitial velocity VL (m/s), (b) - riser gas holdup vs. riser gas
superficial velocity UG (m/s).
156
Chapter VI
T h e re s u ltin g v o lu m e a v e ra g e d b u b b le s lip v e lo c itie s in th e ris e r w e re

fro m 1 9 .4 to 2 0 .0 c m /s (3 m m b u b b le ) a n d fro m 2 2 .3 to 2 3 .0 c m /s
(5 m m b u b b le ). T h e re w a s on ly a v e ry s m a ll d iffe re n c e b e tw e e n 3 m m
and
5 mm
b u b b le
s im u la tio n s ,
b e in g
re a s o n s
fo r
th is
th e
same
p re v io u s ly d e s c rib e d . T h e re la tiv e d iffe re n c e b e tw e e n g a s h o ld-u p in

th e ris e r w a s fro m 2 .2 % to 3 .5 % , a n d fro m 0 .6 % to 1 .8 % fo r th e ris e r
liq u id in te rs titia l v e lo c ity . O n c e m o re th is d iffe re n c e w a s d e c re a s in g
w ith in c re a s in g o f g a s flo w ra te a n d n o g a s w a s p re s e n t in th e
d o w n c o m e r a t a ll g a s flo w ra te s .
It c o u ld b e s ta te d th a t if th e g a s p h a s e is p re s e n t o n ly in th e ris e r
a n d n o t in th e d o w n c o m e r, th e n th e im p a c t o f a b u b b le s lip v e lo c ity
c h a n g e is n o t s o s ig n ific a n t d u e to c o c u rre n t flo w o f b o th p h a s e s .
H o w e v e r, if th e g a s p h a s e is ris in g th ro u g h a s ta g n a n t liq u id o r e v e n if
th e p h a s e s a re flo w in g c o u n te r c u rre n tly (d o w n c o m e r), th e n , o f c o u rs e ,
th is im p a c t c a n b e m u c h m o re s ig n ific a n t.
VI.4.2 Comparison with experiments

S im u la tio n re s u lts fo r th e th re e a irlift c o n fig u ra tio n s (A , B a n d C ) a n d
th e ir c o m p a ris o n w ith e x p e rim e n ta lly m e a s u re d d a ta a re p re s e n te d in
th is s e c tio n . B o th F lu e n t a n d C F X s im u la tio n re s u lts a re s h o w n fo r
ris e rs A a n d B , s im u la tio n s fo r ris e r C w e re d o n e o n ly in F lu e n t. G a s
flo w ra te s in th e s im u la tio n s w e re fro m 1 .9 to 1 3 .6 L /m in (1 .0 5
7 .5 1 c m /s g a s s u p e rfic ia l v e lo c ity b a s e d o n ris e r c ro s s s e c tio n ) fo r
ris e rs A a n d B , a n d fro m 1 .9 to 2 6 .8 L /m in (0 .5 3 7 .5 1 c m /s ) fo r ris e r
C.
VI.4.2.1
Case A
L iq u id in te rs titia l v e lo c itie s a n d g a s h o ld-u p in th e ris e r a n d in th e

d o w n c o m e r o b ta in e d fro m s im u la tio n s (F lu e n t a n d C F X ) a n d fro m
e x p e rim e n ts a s a fu n c tio n o f g a s flo w ra te a re s h o w n in F ig u r e V I- 6 .
G a s h o ld u p fie ld s fo r d iffe re n t g a s flo w ra te s c a n b e s e e n in F ig u re
V I- 7 . A v e ry g o o d a g re e m e n t w a s o b ta in e d fo r liq u id in te rs titia l v e lo c ity .
157
Chapter VI
T h e re la tiv e d iffe re n c e b e tw e e n s im u la tio n s (F lu e n t) a n d e x p e rim e n ts

w a s fro m 0 .7 to 4 .4 % a n d it w a s in c re a s in g w ith g a s flo w ra te .
T h e F lu e n t a n d C F X s im u la tio n s a ls o a g re e d v e ry w e ll w ith e a c h o th e r
w ith C F X p re d ic tin g s lig h tly lo w e r v a lu e s . T h e s itu a tio n w a s w o rs e in
th e c a s e o f g a s h o ld -u p .
a!
1.0!
0.6!
0.15!
VL (m/s)!
0.20!
VL (m/s)!
0.8!
0.4!
0.2!
0.10!
0.05!
0.00!
0.0!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
c!
0.09!
0.06!
0.03!
d!
0.08!
Gas Hold-up (-)!
0.12!
Gas Hold-up (-)!
b!
0.25!
0.06!
0.04!
0.02!
0.00!
0.00!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
Figure VI-6. Riser A simulations vs. experiments (Section VII-4.2): Fluent

simulations (!), CFX simulations ("), experiments (#). (a) Riser liquid
interstitial velocity VL (m/s), (b) downcomer liquid interstitial velocity VL, (c)
riser gas holdup (!), and (d) downcomer gas holdup ! vs. riser gas superficial
velocity UG (m/s).
S im u la tio n s s tro n g ly u n d e rp re d ic te d b o th g a s h o ld-u p in th e ris e r

(3 0 % re la tiv e d iffe re n c e a t th e h ig h e s t g a s flo w ra te u s e d ) a n d in th e
d o w n c o m e r, a t th e m o m e n t w h e n b u b b le s w e re s ta rtin g to b e e n tra in e d
in to
th e
d o w n c o m e r.
The
gas
h o ld -u p
fro m
s im u la tio n s
in
th e
d o w n c o m e r fo r th e h ig h e s t g a s flo w ra te w a s le s s th a n 1 /1 0 o f th e
e x p e rim e n ta lly o b ta in e d v a lu e . If a g a s in te rs titia l v e lo c ity is e s tim a te d
fro m V G = U G /! G , w h e re U G is th e g a s s u p e rfic ia l v e lo c ity g iv e n b y g a s
flo w ra te a n d ! G is th e e x p e rim e n ta lly m e a s u re d g a s h o ld u p , th e n fo r
158
Chapter VI
th e h ig h e s t g a s flo w ra te th e V G w o u ld b e ~ 7 4 c m /s . T h is V G v a lu e is
a c tu a lly lo w e r th a n th e m e a s u re d a n d s im u la te d liq u id v e lo c ity (V L e x p =
8 6 .5 c m /s a t m a x im u m g a s flo w ra te : in F ig u r e V I- 6 ) a n d , c o n s id e rin g
th e E q . V I- 4 4 w o u ld re s u lt in a n e g a tiv e b u b b le s lip v e lo c ity , w h ic h is o f
Riser A
c o u rs e n o t p o s s ib le .
Figure VI-7. Riser A simulations: Gas holdup fields for gas flow rates 1.9,
3.6, 4.5, 9.1 and 13.6 L/min (riser gas superficial velocity ~ 1.0, 2.0, 2.5, 5.0,
7.5 cm/s).
T h e re a s o n o f th e d is c re p a n c y b e tw e e n s im u la tio n a n d e x p e rim e n t is
th e n p ro b a b ly e ith e r d u e to th e e x p e rim e n ta l m e th o d u s e d to o b ta in
e s tim a te s o f g a s h o ld -u p in th e d o w n c o m e r o r d u e to im p e rfe c t m o d e l
c o n s id e ra tio n s (b u b b le s h a p e , ris e v e lo c ity , e tc ). A p re s s u re d ro p d u e
to liq u id flo w c a n n e g a tiv e ly in flu e n c e h o ld-u p m e a s u re d w ith U -tu b e
m a n o m e te rs . N e v e rth e le s s , it w a s v is u a lly o b s e rv e d g a s b u b b le s in th e
d o w n c o m e r a t h ig h e r g a s s u p e rfic ia l v e lo c itie s in ris e r A c o n fig u ra tio n .
B e in g s o th e d is c re p a n c y o b ta in e d fo r d o w n c o m e r g a s h o ld-u p c a n b e
in flu e n c e d
e ith e r
by
th e
m e a s u rin g
c o n s id e ra tio n s .
159
m e th o d
and
by
s im u la tio n
Chapter VI
Case B
VI.4.2.2
S im u la tio n s
(F lu e n t
and
CFX)
and
e x p e rim e n ts
of
ris e r
a re
c o m p a re d in F ig u r e V I- 8 . G a s h o ld u p fie ld s fo r d iffe re n t g a s flo w ra te s

c a n b e s e e n in F ig u r e V I- 9 .
A g o o d a g re e m e n t w a s o b ta in e d fo r liq u id in te rs titia l v e lo c ity . T h e
re la tiv e d iffe re n c e b e tw e e n s im u la tio n s (F lu e n t) a n d e x p e rim e n ts v a rie d
fro m 4 .6 to 8 .3 % . A s fo r th e c a s e A , th e F lu e n t a n d C F X s im u la tio n s
g a v e v e ry s im ila r re s u lts w ith C F X p re d ic tin g s lig h tly lo w e r v a lu e s o f
liq u id in te rs titia l v e lo c ity . T h e s im u la te d a n d e x p e rim e n ta lly m e a s u re d
g a s h o ld -u p a g re e m e n t in th e ris e r w a s p o o r. T h e re la tiv e d iffe re n c e in
th e ris e r h o ld -u p fo r th e lo w e s t g a s flo w ra te w a s 4 0 .4 % a n d c o n tin u a lly
d e c re a s e d to 1 5 .5 % fo r th e h ig h e s t g a s flo w ra te .
a!
1.0!
0.20!
0.6!
0.15!
VL (m/s)!
0.8!
VL (m/s)!
b!
0.25!
0.10!
0.4!
0.05!
0.2!
0.00!
0.0!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
c!
0.08!
0.06!
Gas hold-up (-)!
0.04!
0.02!
0.00!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0.08!
Figure VI-8. Riser B simulations vs. experiments. Legend: ! - Fluent

simulations (black marks), "- CFX simulations (stars), # - experiments (white
marks). (a) Riser liquid interstitial velocity VL, (b) downcomer liquid interstitial
velocity VL(m/s), and (c) riser gas holdup ! vs. riser gas superficial velocity
UG(m/s).
160
Chapter VI
T h e g a s h o ld -u p w a s o v e rp re d ic te d b y s im u la tio n s in th is s itu a tio n

(ris e r B ) c o n tra ry to th e p re vio u s ris e r c o n fig u ra tio n (A ). If ris e r B
s im u la tio n s a re c o m p a re d w ith ris e r A s im u la tio n s (R is e r B h a s 2 0 c m
lo n g e r d ra ft tu b e ), th e n it c a n b e n o tic e d th a t th e p re d ic te d liq u id
in te rs titia l v e lo c itie s a re v e ry s im ila r, w ith ris e r B v e lo c itie s 2 .7 %
2 .9 % h ig h e r if c o m p a re d to ris e r A e x c e p t fo r th e h ig h e s t g a s flo w ra te ,
w h e re th e R is e r B v e lo c ity is 6 .1 % h ig h e r, b e c a u s e o f s ta rtin g b u b b le
p e n e tra tio n in to th e d o w n c o m e r in ris e r A . E x p e rim e n ta lly m e a s u re d
ris e r liq u id in te rs titia l v e lo c itie s fo r ris e r B we re 6 .3 -1 1 .6 % h ig h e r th e n
in ris e r A d e p e n d in g o n th e g a s flo w ra te . If th e s a m e c o m p a ris o n is
d o n e fo r g a s h o ld -u p (R is e r A v s . R is e r B ) th e n fo r s im u la tio n s th e
re la tiv e d iffe re n c e is a g a in lo w (R is e r B ris e r h o ld u p 1 .7-4 .6 re la tiv e %
lo w e r). H o w e v e r, th e e x p e rim e n ta lly m e a s u re d h o ld-u p in R is e r B w a s
2 4 .7 % - 3 3 .2 % re la tiv e lo w e r th a n in ris e r A (e x c e p t fo r th e lo w e s t g a s
flo w ra te ).
T h e d iffe re n c e b e tw e e n ris e r A a n d R is e r B in e x p e rim e n ts
Riser B
w a s m u c h h ig h e r th a n in s im u la tio n s .
Figure VI-9. Riser B simulations: Gas holdup fields for gas flow rates 1.9,
3.6, 4.5, 9.1 and 13.6 l/min (riser gas superficial velocity ~ 1.0, 2.0, 2.5, 5.0,
7.5 cm/s)
161
T h is
suggest
Chapter VI
th a t
th e
re a s o n
fo r
b ig g e r
d is c re p a n c y
b e tw e e n
e x p e rim e n ta l a n d s im u la tio n d a ta in R is e r B is p ro b a b ly a c o n s e q u e n c e
o f th e e x p e rim e n ta l m e th o d u s e d to e s tim a te g a s h o ld u p s (U -tu b e
m a n o m e te rs ).
VI.4.2.3
Case C
In th e R is e r C th re e d iffe re n t re g im e s w e re e x p e rim e n ta lly o b s e rv e d

w ith th e c h a n g e o f g a s flo w ra te . A t firs t, fo r th e lo w e s t g a s flo w ra te ,
th e re w e re n o g a s b u b b le s in s id e th e d o w n c o m e r. T h e n fo r h ig h e r ra te s
th e b u b b le s s ta rte d to b e e n tra in e d in to th e d o w n c o m e r, b u t th e b u b b le
fro n t d id n o t re a c h th e d o w n c o m e r b o tto m y e t. A n d fin a lly , fo r th e
h ig h e s t g a s flo w ra te , th e fro n t re a c h e d th e a irlift b o tto m a n d b u b b le s
s ta rte d to c irc u la te w ith a c o rre s p o n d in g c h a n g e o f in te rs titia l liq u id
v e lo c ity a n d g a s h o ld u p v s . g a s flo w ra te c u rv e tre n d s .
T h e re s u lts a re s h o w n in F ig u re V I- 1 0 (g a s h o ld -u p s a n d liq u id
in te rs titia l v e lo c itie s ) a n d in F ig u r e V I- 1 1 (s im u la te d g a s h o ld -u p
fie ld s ).T h e m a in q u e s tio n w a s if th e s im u la tio n s w e re a b le to c a p tu re
th is b e h a v io u r. T h e a n s w e r w a s th a t th e y c o u ld , b u t o n ly p a rtia lly .
T h e re w a s a n in itia lly g o o d a g re e m e n t in liq u id in te rs titia l v e lo c itie s .
T h e d iffe re n c e b e tw e e n th e s im u la tio n a n d th e e x p e rim e n t fo r th e
lo w e s t e x p e rim e n ta lly m e a s u re d g a s flo w ra te w a s < 3 .5 % a n d it w a s
c o n tin u a lly in c re a s in g w ith g a s flo w ra te . It w a s 8 .5 % fo r th e s e c o n d
h ig h e s t flo w ra te , w h ic h is s till o k , b u t fo r th e h ig h e s t flo w ra te , w h e re
th e re w a s a s te e p e r ris e o f th e e x p e rim e n ta lly m e a s u re d liq u id v e lo c ity
d u e to th e re g im e c h a n g e , th e e x p e rim e n t/s im u la tio n d iffe re n c e w a s
46%.
G a s h o ld -u p in th e ris e r a n d in th e d o w n c o m e r a n d a ls o th e p o in t a t
w h ic h b u b b le s s ta rt to e n te r th e d o w n c o m e r w a s c a p tu re d v e ry w e ll.
H o w e v e r, th e a g re e m e n t fo r th e h ig h e s t g a s flo w ra te w a s ra th e r b a d .
T h e b u b b le fro n t d id n o t re a c h th e a irlift b o tto m in th e s im u la tio n s a n d
th e re a p p e a re d n o b u b b le c irc u la tio n re g im e e v e n fo r th e m a x im a l g a s
flo w ra te .
162
Chapter VI
a!
0.8!
0.40!
0.30!
VL (m/s)!
VL (m/s)!
0.6!
0.4!
0.2!
0.20!
0.10!
0.00!
0.0!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
c!
d!
0.15!
Gas hold-up (-)!
0.15!
Gas hold-up (-)!
b!
0.50!
0.10!
0.10!
0.05!
0.05!
0.00!
0.00!
0!
0.02!
0.04!
0.06!
UG (m/s)!
0!
0.08!
0.02!
0.04!
0.06!
0.08!
UG (m/s)!
Figure VI-10. Riser C simulations vs. experiments Legend: ! - Fluent

simulations (black marks), "- CFX simulations (stars), # - experiments (white
marks). (a) Riser liquid interstitial velocity VL(m/s), (b) downcomer liquid
interstitial velocity VL(m/s), (c) riser gas holdup !, and (d) downcomer gas
holdup ! vs. riser gas superficial velocity UG (m/s).
T h e liq u id in te rs titia l v e lo c ity d id n o t c h a n g e m u c h w ith a n in c re a s e

in g a s flo w ra te in th e s im u la tio n s , e x c e p t fo r th e lo w e r ra te s (o n ly 6 .2 %
in c re a s e
fro m
gas
flo w
ra te
4 .5 L /m in
to
2 6 .8 L /m in ).
The
liq u id
s u p e rfic ia l v e lo c ity in s im u la tio n s (a n d th u s a to ta l c irc u la tin g liq u id

flo w ra te ), w a s e v e n d e c re a s in g w ith g a s flo w ra te . T h e re a s o n w a s th a t
th e d riv in g fo rc e fo r th e liq u id c irc u la tio n , th e d iffe re n c e b e tw e e n
a v e ra g e h o ld -u p in th e ris e r a n d in th e d o w n c o m e r, w a s s lo w ly
d e c re a s in g w ith g a s flo w ra te fro m a c e rta in p o in t (Q G = 4 .5 L /m in ,
U G = 1 .2 6 c m /s ), w h ile th e g a s h o ld -u p in th e ris e r a n d th e d o w n c o m e r
w e re
ris in g
lin e a rly .
The
a b s o lu te
d iffe re n c e
b e tw e e n
th e m
(ris e r/d o w n c o m e r) w a s 1 .7 % a t Q G = 4 .5 L /m in (U G = 1 .2 6 c m /s ) a n d
1 .2 % a t 2 6 .8 L /m in (U G = 7 .5 1 c m /s ). T h e liq u id s u p e rfic ia l v e lo c ity in th e
ris e r d ro p p e d fro m 3 4 .6 c m /s to 3 2 .3 c m /s in th is g a s flo w ra te ra n g e in
s im u la tio n s .
163
Chapter VI
Riser C
Figure VI-11. Riser C simulations: Instantaneous gas holdup fields for gas
flow rates 1.9, 3.6, 4.5, 9.1, 13.6, 17.9 and 26.8 l/min (riser gas superficial
velocity ~ 0.5, 1.0, 1.3, 2.6, 3.8, 5.0, 7.5 cm/s).
F o r ris e r C s im u la tio n s , th e re w a s a v e ry g o o d a g re e m e n t w ith

e x p e rim e n ts fo r lo w g a s flo w ra te s . T h e d e v ia tio n fro m e x p e rim e n ts
th e n s ta rte d to in c re a s e s lo w ly w ith g a s flo w ra te a n d th e n it ro s e
s u d d e n ly fo r th e h ig h e s t g a s flo w ra te w ith th e o n s e t o f a b u b b le
c irc u la tio n re g im e , w h ic h w a s n o t c a p tu re d . A s it w a s p o in te d o u t
e a rlie r, flo w in a d o w n c o m e r (c o u n te r-c u rre n t flo w ) is in flu e n c e d b y a
b u b b le s lip v e lo c ity m u c h m o re th a n a flo w in a ris e r (c o-c u rre n t).
D e m a n d s o n th e a c c u ra c y o f d ra g fo rc e c lo s u re a re th e n q u ite h ig h if
b u b b le s a re p re s e n t in th e d o w n c o m e r. D ra g c o e ffic ie n t a n d b u b b le s lip
v e lo c ity fo r g iv e n flu id s d e p e n d o n b u b b le s iz e , s h a p e , lo c a l g a s h o ld u p
a n d o th e r fa c to rs (p re s e n c e o f s u rfa c ta n ts ). T o o b ta in a c o rre c t b u b b le
s lip v e lo c ity (a ) a re liab le c lo s u re e q u a tio n fo r it is n e e d e d a n d (b )
c o rre c t in p u ts in to th is c lo s u re a re re q u ire d . O u r c lo s u re w a s E q . V I- 8 .
A b u b b le s iz e c a n b e e ith e r p re s c rib e d d ire c tly o r it c a n b e a re s u lt
fro m a b u b b le c o a le s c e n c e /b re a k-u p m o d e l s o lu tio n . S o lv in g g o v e rn ing
e q u a tio n s o f th e flo w p ro v id e s th e lo c a l g a s h o ld-u p . In o u r s im u la tio n s
th e b u b b le s iz e w a s p re s c rib e d d ire c tly (5 m m fo r a ll flo w ra te s , a ll
b u b b le s
assumed
to
have
same
s iz e ). T h e
weak
p o in t o f o u r
s im u la tio n s a n d th e c a u s e o f d is a g re e m e n t b e tw e e n sim u la tio n s a n d

e x p e rim e n ts c a n b e a n y o f th e a b o v e-m e n tio n e d is s u e s , p ro b a b ly th e
s p e c ific a tio n o f b u b b le s iz e a n d /o r th e s lip v e lo c ity a d ju s tm e n t o n th e
164
Chapter VI
lo c a l g a s h o ld u p . A lth o u g h m e a s u re m e n ts o f b u b b le s iz e w e re d o n e
w ith a h ig h -s p e e d c a m e ra , th e y c o u ld b e d o n e o n ly fo r v e ry lo w g a s
flo w ra te s n o t re le v a n t fo r o u r s tu d y . T h e b u b b ly la y e r s o o n b e c a m e
o p a q u e a s th e h o ld u p in c re a s e d .
T h e u s e o f lo c a l te c h n iq u e s (e g . o p tic a l p ro b e s ) to m e a s u re th e
b u b b le s p ro p e rtie s (v e lo c ity , s iz e ) is n o w v e ry im p o rta n t a n d it s e e m s
th e n e x t s te p in th is p ro c e s s . L o c a l a x ia l m e a s u re m e n ts w ill a ls o a llo w
s tu d y in g th e o c c u rre n c e o f c o a le s c e n c e a n d b re a k-u p a s w e ll a s
m e a s u rin g its in te n s ity , w h ic h c a n b e c o m p u te d la tte r u s in g s im ila r
C F D s s e tu p s . T h is in fo rm a tio n s e e m s c ru c ial in o rd e r to g e t c lo s e r th e
s im u la tio n s
and
th e
e x p e rim e n ta l
v a lu e s .
F in a lly
th is
can
be
c o m p le m e n te d b y m e a s u re m e n ts a n d s im u la tio n u s in g S G a s s o lid
p h a s e in a g -l-s s y s te m .
VI.5 Conclusions
T h e p rim a ry g o a l o f th is w o rk w a s to m a k e a c o m p a ris o n b e tw e e n
s im u la tio n s a n d e x p e rim e n ts to s e e h o w w e ll th e s im u la tio n s a re a b le to
p re d ic t liq u id in te rs titia l v e lo c ity a n d g a s h o ld-u p b o th in th e ris e r a n d
in th e d o w n c o m e r fo r a g -l s y s te m . T h re e d iffe re n t a irlift ris e r tu b e s fo r
a ra n g e o f g a s flo w ra te s w e re te s te d. It w a s s h o w n th a t th e re w a s a
g o o d a g re e m e n t re g a rd in g th e liq u id in te rs titia l v e lo c itie s in th e ris e r
and
th e
downcomer
w ith
e rro rs
m o s tly
w e ll b e lo w
10%
w ith
an
e x c e p tio n o f h ig h e r g a s flo w ra te s (a n d e s p e c ia lly th e h ig h e s t o n e ) in

la rg e r d ia m e te r ris er (R is e r C ). T h e a g re e m e n t o f th e g a s h o ld -u p (ris e r
a n d d o w n c o m e r) b e tw e e n s im u la tio n s a n d e x p e rim e n ts w a s p o o re r. T h e
s im u la tio n s o v e r-p re d ic te d th e ris e r g a s h o ld u p in ris e r A a n d u n d e rp re d ic te d it in ris e r B . H o w e v e r, a s it w a s d is c u s s e d , it c o u ld b e a n
e rro r in e x p e rim e n ta l m e a s u re m e n ts ra th e r th a n a s im u la tio n e rro r. T h e
a g re e m e n t o f th e g a s h o ld -u p (ris e r a n d d o w n c o m e r) fo r R is e r C w a s
g o o d , e x c e p t fo r th e h ig h e s t g a s flo w ra te , w h e re a flo w re g im e h a d
a lre a d y
changed
to
b u b b le
c irc u la tio n
re g im e ,
w h ic h
was
not
c a p tu re d b y s im u la tio n s . A m o re a c c u ra te m o d e llin g o f th e d ra g
165
Chapter VI
fo rc e /b u b b le s lip v e lo c ity w o u ld b e n e e d e d to o b ta in b e tte r a g re e m e n t

w ith e x p e rim e n ts in th e c a s e s , w h e n th e re is a g a s p h a s e p re s e n t in th e
d o w n c o m e r. T h e s im u la tio n s e tu p is co n s id e re d to b e g o o d e n o u g h to
m o d e l c a s e s w ith n o g a s p h a s e b e in g e n tra in e d in to th e d o w n c o m e r.
T h e s e c o n d a ry g o a l w a s to te s t a n e ffe c t o f v a rio u s m o d e ls a n d
p a ra m e te rs o n s im u la tio n re s u lts .
T h is in c lu d e d c o m p a ris o n b e tw e e n
tw o c o m m e rc ia l c o d e s w ith tw o d iffe re n t m o d e ls , m ix tu re m o d e l (F lu e n t)
a n d E u le r-E u le r m o d e l (C F X ), te s tin g d iffe re n t d ra g fo rc e c lo s u re s a n d
tu rb u le n c e m o d e ls (v a ria n ts o f !"# , !"$ ). T h e F lu e n t s im u la tio n s w ith
m ix tu re m o d e l a n d C F X s im u la tio n s w ith E u le r-E u le r m o d e l g a v e th e
s a m e re s u lts w ith o n ly m in o r d iffe re n c e s b e tw e e n th e m . T h e te s tin g o f
d ra g
c lo s u re s
s u g g e s te d
th a t
an
a c c u ra te
m o d e llin g
of
d ra g
fo rc e /b u b b le s lip v e loc ity is re q u ire d , if th e re a re b u b b le s p re s e n t in a

d o w n c o m e r d u e to c o u n te r-c u rre n t flo w o f g a s- a n d liq u id -p h a s e . N o t
s o m u c h a c c u ra c y is n e e d e d fo r a ris e r flo w m o d e llin g , b e c a u s e th e c oc u rre n t flo w o f g a s - a n d liq u id -p h a s e a n d th e re la tiv e ly h ig h liq uid
v e lo c ity if c o m p a re d to th e b u b b le s lip v e lo c ity . T h re e v e rs io n s o f a !"#
tu rb u le n c e m o d e l a n d a !"$ m o d e l g a v e v e ry s im ila r re s u lts .
VI.6 References
Cao, C., Dong, S. and Guo, Q. (2007). Experimental and numerical simulation for
gas liquid phases flow structure in an external-loop airlift reactor. Industrial and
Engineering Chemistry Research, 46, 73177327.
Chisti, M. Y. (1989). Airlift Bioreactors (1st ed.). Essex: Elsevier Science Pub. LTD.
Chisti, Y. (1998). Pneumatically agitated bioreactors in industrial and enviromental
bioprocessing: Hydrodynamics, hydraulics and transport phenomena. Applied
Mechanic Reviews, 51(1), 33-112.
Fluent Inc. (2006). Fluent 6.3 User s Guide. Lebanon, NH: Fluent Inc.
Garcia-Calvo,E., Rodrguez,A., Prados,A. and Klein,J. (1999). A fluid dynamic model
for three-phase airlift reactors. Chemical Engineering Science, 54, 2359-2370.
Heijnen, J., Hols, J., van Der Lans, R. M. van Leeuwen, H. M., Mulder, A., and
Weltevrede, R. (1997). A simple hydrodynamic model for the liquid circulation
velocity in a full-scale two- and three-phase internal airlift reactor operating in the
gas recirculation regime. Chemical Engineering Science, 52(15), 2527-2540.
Huang, Q., Yang, C., Yu, G. and Mao, Z. S. (2008). Sensitivity study on modeling an
internal airlift loop reactor using a steady 2D two-fluid model. Chemical
Engineering and Technology, 31, 17901798.
166
Chapter VI
Huang, Q., Yang, C., Yu, G. and Mao, Z. S. (2010). CFD simulation of hydrodynamics
and mass transfer in an internal airlift loop reactor using a steady two-fluid model.
Chemical Engineering Science, 65, 5527-5536.
Jia, X., Wen, J., Wei, F. and Yuan, Q. (2007). Local hydrodynamics modeling of gas
liquidsolid three-phase airlift loop reactor. Industrial and Engineering Chemistry
Research, 46, 5210-5220.
Manninen,M., Taivassalo,V. and Kallio,S. (1996). On the mixture model for
multiphase flow. (p. 288).
Merchuk, J., Contreras, A., Garcia, F. and Molina, E. (1998). Studies of mixing in a
concentric tube airlift bioreactor with different spargers. Chemical Engineering
Science, 53(4), 709-719.
Mudde, R. F. and van den Akker, H. E. A. (2001). 2D and 3D simulations of an
internal airlift loop reactor on the basis of a two-fluid model. Chemical
Oey, R. S., Mudde, R. F., Portela, L. M. and van den Akker, H. E. A. (2001).
Simulation of a slurry airlift using a two-fluid model. Chemical Engineering
Science, 56, 673-681.
Roy, S., Dhotre, M. T. and Joshi, J. B. (2006). CFD Simulation of Flow and Axial
Dispersion in External Loop Airlift Reactor. Chemical Engineering Research and
Design, 84, 677-690.
Sokolichin,A., Eigenberger,G. and Lapin,A. (2004). Simulation of buoyancy driven
bubbly flow: established simplifications and open questions. AIChE Journal, 50(1),
2445.
Talvy, S., Cockx, A. and Line, A. (2007). Modeling hydrodynamics of gasliquid airlift
reactor. AIChE Journal, 53, 335353.
Tomiyama,A., Tamai,H., Zun,I. and Hosokawa,S. (2002). Transverse migration of
single bubbles in simple shear flows. Chemical Engineering Science, 57, 18491858.
Van Baten, J. M., Ellenberger, J. and Krishna, R. (2003). Hydrodynamics of internal
air-lift reactors: experiments versus CFD simulations. Chemical Engineering and
Processing, 42(10), 733742.
Verlaan, P., Tramper, J., VanT Reit, K. and Luyben, K. C. H. A. M. (1986). A
hydrodynamic model for an airlift-loop bioreactor with external loop. Chemical
Engineering Journal, 33, B43-B53.
Vial, C., Poncin, S., Wild, G. and Midoux, N. (2002). Experimental and theoretical
analysis of the hydrodynamics in the riser of an external loop airlift reactor.
Chemical Engineering Science, 57, 4745-4762.
Young, M. A., Carbonell, R. G. and Ollis, D. F. (1991). Airlift bioreactors: analysis of
local two-phase hydrodynamics. AIChE Journal, 37(3), 403-428.
van Baten, J. M., Ellenberger, J. and Krishna, R. (2003). Hydrodynamics of internal
air-lift reactors: experiments versus CFD simulations. Chemical Engineering and
Processing, 42, 733-742.
167
Chapter VII
VII. Chapter VII Local Hydrodynamic

Properties of Three-phase iGLR
VII.1 Objectives
VII.2 Introduction
VII.3 Material and Methods
VII.4 Results and Discussion
VII.5 Conclusions
VII.6 References
168
Chapter VII
VII.1 Objectives
T h e m a in o b je c tiv e in th is c h a p te r w a s to s tu d y th e lo c a l in flu e n c e o f
S G in th e G L R h y d ro d y n a m ic s , in o rd e r to u n d e rs ta n d b e tte r th e
in flu e n c e o f s u c h p a rtic u la rly p a rtic le s . In a d d itio n a n d d u e to in itia lly
te c h n ic a l p ro b le m s o th e r o b je c tiv e s lie u n d e r th e m a in o b je c tiv e , s u c h
as:
- D e v e lo p m e n t a n d c a lib ra tio n o f a m e th o d to m e a s u re g a s-p h a s e
p ro p e rtie s u s in g a n O P ,
- E v a lu a tio n o f th e e ffe c t o f lo w s o lid c o n c e n tra tio n in iG L R lo c a l
h y d ro d y n a m ic s a t s im ila r g a s flo w c o n d itio n s u s e d in c o n tin u o u s A F B
p rim a ry fe rm e n ta tio n ,
- S tu d y th e in flu e n c e o f y e a s t ce lls in o p tic a l p ro b e m e a s u re m e n ts a s
w e ll a s th e in flu e n c e o f m ic ro o rg a n is m s g ro w th o n th e g a s-p h a s e a fte r
s ta r-u p a n d im m o b iliz a tio n s ta g e (s e e c h a p te r IV ).
VII.2 Introduction
In c h a p te r V . th e g lo b a l h y d ro d y n a m ic p a ra m e te rs o f th re e-p h a s e
iG L R w e re s tu d ie d a n d d is c u s s e d . T h e m a in c o n c lu s io n in d ic a te d th e
a d e q u a te iG L R c o n fig u ra tio n fo r c o n tin u o u s A F B p rim a ry fe rm e n ta tio n .
A m o n g th e o p tio n s te s te d , th e id e a l c o n fig u ra tio n s h o u ld h a v e a n A d /A r
ra tio e q u a l to 3 .6 7 a n d th e le n g th o f ris e r s h o u ld g o fro m tw o
c e n tim e tre s a b o v e s p a rg e r (s h o u ld e n s u re b u b b le s d ia m e te r b e tw e e n 2
a n d 4 m m ) to th e b e g in n in g o f c o n ic a l p a rt fro m e n la rg e d to p s e c tio n .
T h e in flu e n c e o f S G p a rtic le s o n g a s h o ld-u p fo r th is c o n fig u ra tio n w a s
n o t e n tire ly c le a r (s e e s e c tio n V .4 .2 ). T h e re fo re in th is c h a p te r it w ill b e
s tu d ie d th e in flu e n c e o n lo c a l iG L R h y d ro d y n a m ic s o f S G p a rtic le s fo r a
s im ila r c o n fig u ra tio n iG L R .
A s re fe rre d b e fo re lo c a l m e a s u re m e n ts o f g a s-, s o lid - a n d liq u id p h a s e re q u ire th e u s e o f d iffe re n t a n d re fin e d te c h n iq u e s to b e tte r
u n d e rs ta n d th e e ffe c t o f S G h a v e in th e g -l m ix tu re .
169
Chapter VII
VII.2.1 Measurement of local gas-phase properties

In c h a p te r V g lo b a l g a s h o ld -u p a n d liq u id v e lo c ity fo r th e s e le c te d
ris e r c o n fig u ra tio n w e re n o t s ig n ific a n tly a ffe c te d b y th e p re s e n c e o f
s o lid s . H o w e v e r it w a s m e n tio n e d th a t it w a s d u e to th e lo w s e n s ib ility
o f th e m e th o d s u s e d . T h e re fo re it w a s im p o rta n t to s tu d y th e lo c a l
h y d ro d y n a m ic s o f th e th re e-p h a s e iG L R u s in g S G a s s o lid-p h a s e .
T h e re a re s e v e ra l te c h n iq u e s to s tu d y lo c a l flo w s tru c tu re s (B o y e r e t
a l., 2 0 0 2 ) a n d a re re p re s e n te d in F ig u r e V II- 1 . b e lo w .
A m o n g th e s e te c h n iq u e s , th e m a in n o n-in v a s iv e a re to m o g ra p h y (! o r X -ra y , c a p a c ita n c e o r re s is tiv ity , u ltra s o u n d ) a n d v is u a liz a tio n (H ig h
S p e e d C a m e ra (H S C ), P IV , e tc ) te c h n iq u e s , w h ile a m o n g in v a s iv e
te c h n iq u e s a re p h a s e d e te c tio n p ro b e s : n e e d le , h e a t tra n s fe r a n d
u ltra s o u n d p ro b e s (B o y e r e t a l., 2 0 0 2 ; A la in C a rte llie r, 1 9 9 0 ).
In v a s iv e te c h n iq u e s b e s id e s m o re e x p e n s iv e a n d h a rd to a p p ly a re
o fte n u s e d a t in d u s tria l s c a le . T h e re a s o n s fo r th is a re : (1 ) th e ty p ic a l
tu rb u le n t flo w re g im e s fo u n d in in d u s tria l re a c to rs ; (2 ) o p a q u e re a c to rs ;
a n d (3 ) a b ility to c o lle c t d a ta o n lin e a n d c o n tin u o u s ly (B o y e r e t a l.,
2 0 0 2 ). A lth o u g h , in th e la te r y e a rs , to m o g ra p h y b e g in s to b e a p p lie d in
some
in d u s tria l
p ro c e s s ,
th e
m a in
d ra w b a c k
of
th is
te c h n iq u e
a p p lic a tio n in g -l-s s y s te m s re m a in s th e e q u ip m e n t c o s t. O n th e o th e r

h a n d , p h a s e d e te c tio n p ro b e s a re c h e a p e r a n d e a s ie r to a p p ly in
in d u s tria l
p ro c e s s e s .
S e n s o rs
based
on
te m p e ra tu re ,
c a p a c ita nc e ,
o p tic a l p ro p e rtie s a n d c o n d u c tiv ity a re c o m m o n ly u s e d . F re q u e n tly th e y

a re a p p lie d fo r tw o-p h a s e flo w (O liv ie ri e t a l. 2 0 0 7 ; J h a w a r a n d P ra k a s h
2 0 0 7 ) a n d m o re re c e n tly to th re e p h a s e flo w s y s te m s (M e n a e t a l.
2 0 0 8 ), u s u a lly a t lo w s o lid s h o ld -u p (B o y e r e t a l. 2 0 0 2 ). In o u r w o rk , to
s tu d y th e g a s -p h a s e tw o te c h n iq u e s w e re u s e d : p h o to g ra p h ic m e th o d
(h ig h s p e e d c a m e ra ) a n d o p tic a l p ro b e (O P ). T h e c h o ic e fo r th e s e tw o
m e th o d s w a s to m e a s u re w ith m a x im u m a c c u ra c y th e lo c a l g a s-p h a s e
p ro p e rtie s in th e g -l-s s y s te m p re s e n t in th e ris e r o f a n iG L R .
170
Chapter VII
Local Hydrodynamic Techniques!
Invasive!
Non-Invasive!
Tomagraphic
Techniques!
Pitot tubes!
Laser Doppler
Anemometry!
Heat Transfer
Probes!
Radioactive
Techniques!
Ultrasound!
Polarographic
Techniques!
Needle Probes!
Visualization
Techniques!
1.! Photographic (photo and video)!

2.! Radiographic!
3.! Particle Image Velocimetry (PIV)!
1.! Optical
fiber!
2.! Impedance
probes!
Figure VII-1. Techniques used for local hydrodynamics characterization in g-l

and g-l-s systems.
VII.2.1.1
Photographic technique: high speed camera
H ig h s p e e d c a m e ra (H S C ) is th e to o l fo r th e p h o to g ra p h ic te c h n iq u e .
T h is is a m e th o d u s e d in v is u a liz a tio n te c h n iq u e s m a in ly a p p lie d fo r
q u a lita tiv e flo w a n a ly s is a n d q u a n tita tiv e b u b b le p ro p e rtie s s u c h a s :
s h a p e , s iz e , a re a a n d v o lu m e . B a s ic a lly , it c o n s is ts in film in g th e
s y s te m (g -l o r g -l-s ) flo w , w h ic h is v e ry im p o rta n t b e c a u s e d u e to th e ir
s m a ll s c a le a n d fa s t b u b b le ra is in g h u m a n e y e is lim ite d to c a p tu re a ll
in fo rm a tio n . H o w e v e r s o m e lim ita tio n s a re k n o w n a s th e in a b ility to u s e
th is te c h n iq u e a t h ig h g a s flo w s , re a c to r w a ll tra n s p a re n c y , o u t o f
171
Chapter VII
fo c u s , illu m in a tio n a n d im a g e a n a ly s is (B o y e r e t a l., 2 0 0 2 ; F e rre ira e t

a l., 2 0 1 1 ). T h e re a c to r w a ll, a s w e ll a s fo c u s a n d illu m in a tio n c a n b e
s o lv e d , o r re d u c e d e x p e rim e nta lly , e s p e c ia lly b y u s in g a b a c k lig h t w e re
lig h t p a s s e s tro u g h a d iffu s io n la y e r b e fo re illu m in a tin g th e re a c to r.
(M e n a e t a l, 2 0 0 5 a , 2 0 0 5 c , 2 0 0 8 ). N e v e rth e le s s , tre a tm e n t o f th e
o b ta in e d im a g e s is n o t a n e a s y ta s k to p e rfo rm . M o s t o f th e c o m m e rc ia l
s y s te m s fo r b u b b le p o p u la tio n c h a ra c te riz a tio n , i e ., th e id e n tific a tio n
o f s in g le a n d o v e rla p p in g b u b b le s a re d o n e b y m a n u a l o r s e m i-m a n u a l
m e th o d s (F e rre ira e t a l., 2 0 1 1 ). T h is id e n tific a tio n c a n b e d o n e b y a
c rite riu m a s th e c o n c a v ity in d e x (C I) b u t it is n o rm a lly u s e d a t g a s
s u p e rfic ia l v e lo c itie s b e lo w 3 c m /s . W h e n b u b b le s a re s e p a ra te d a n d
c la s s ifie d
th e n
th e
c o m m e rc ia l
p ro g ra m s
a re
a b le
to
d e te rm in e
s u c c e s s fu lly th e b u b b le s p ro p e rtie s a s : th e p ro je c te d a re a , e q u iv a le n t
d ia m e te r, th e F e re t d ia m e te rs d is trib u tio n , e lo n g a tio n a n d s p h e ric ity
(F e rre ira e t a l., 2 0 1 1 ; M e n a e t a l., 2 0 0 5 c ).
R e c e n tly F e rre ira e t a l. (2 0 1 1 ) d e v e lo p e d a n a u to m a tic m e th o d b a s e d
o n a n o n lin e te c h n iq u e fo r s u c ro s e c ry s ta l m o rp h o lo g y to id e n tify
b e tw e e n
s in g le
and
g ro u p
(tw o
or
m o re )
of
b u b b le s .
T h is
c la s s ific a tio n is b a s e d o n a s e rie s o f p ro b a b ilitie s a n d a ls o a llo w s th e

c h a ra c te riz a tio n o f s y s te m c o m p le x ity /tu rb u le n c e (F e rre ira e t a l., 2 0 1 1 ).
U s u a lly w h e n th re e-p h a s e s y s te m s a re a p p lie d , th e u s e o f a u to m a tic
im a g e
a n a ly s is
of
b u b b le s
is
v e ry
d iffic u lt,
e s p e c ia lly
when
th e
d iffe re n c e o f g re y le v e l b e tw e e n s o lid s a n d bu b b le s is n o t s ig n ific a n t.

H S C v id e o s c a n b e a ls o u s e d to d e te rm in e p a rtic le s (b u b b le s o r s o lid s )
v e lo c ity , h o w e v e r e v e n in c o m m e rc ia l p ro g ra m s th e tre a tm e n t is d o n e
m a n u a lly .
VII.2.1.2
Optical probes characteristics: the LEGI optical probe
O v e r th e la s t tw o d e c a d e s o p tic a l p ro b e s h a v e b e e n d e v e lo p e d a n d
s u c c e s s fu lly a p p lie d in B C re a c to r lo c a l g a s -p h a s e h y d ro d y n a m ic s (D ia s
e t a l., 2 0 0 0 ; H a m a d a n d H e , 2 0 1 0 ; H o n g e t a l., 2 0 0 4 ; P o u p o t a n d
C a rte llie r, 1 9 9 9 ; S h e n e t a l., 2 0 0 8 ). T h e y o p e ra tio n is re la tiv e ly s im p le
a n d c o n s e q u e n tly th e ir u tiliz a tio n h a v e b e e n in c re a s e d . T h e y a llo w
172
Chapter VII
m e a s u rin g n o t o n ly g a s -p h a s e c o n c e n tra tio n s (h o ld -u p ) b u t a ls o b u b b le

v e lo c itie s ,
s iz e
d is trib u tio n s ,
mean
in te rfa c ia l
a re a ,
mean
S a u te r
d ia m e te r a n d a re a ls o a b le to id e n tify flo w re g im e s . H o w e v e r, th is is
o n ly p o s s ib le if s o m e flo w s tru c tu re s o r b u b b le s h a p e a s s u m p tio n s a re
ta k e n in a c c o u n t (C a rte llie r, 1 9 9 2; M e n a e t a l., 2 0 0 8 ).
D o u b le (o r m u lti) tip p ro b e s a re a ls o a p p lie d in o rd e r to m e a s u re
p a ra m e te rs s u c h a s b u b b le v e lo c itie s , d ia m e te r a n d in te rfa c ia l a re a
(B o y e r e t a l., 2 0 0 2 ; C h a u m a t e t a l., 2 0 0 5 ; D ia s e t a l., 2 0 0 0 ). T h e m a jo r
d ra w b a c k o n th is is th e re d u c tio n o f s p a tia l re s o lu tio n . In a d d itio n ,
w h e n d o u b le tip p ro b e s a re a p p lie d o n g -l-s s y s te m s , th e re is a h ig h e r
p ro b a b ility to p ro m o te a n in c re a s e o f s o lid s n e a r the tip , w h ic h c a n le a d
to in a c c u ra te m e a s u re s .
Figure VII-2. Optical fiber typical operation method and indicator function
obtained during bubble piercing. From Vejrazka et al. (2010).
C a rte llie r (1 9 9 0 ) d e ve lo p e d a s in g le tip p ro b e , w h ic h a llo w s a re fin e d

d e s c rip tio n o f th e flo w . T h is p ro b e is a b le to m e a s u re v o id fra c tio n a n d
b u b b le v e lo c ity (C a rte llie r, 1 9 9 0 ). D u e to th e ir s m a ll s iz e th e s p a tia l
re s o lu tio n
is
v e ry
h ig h ,
w h ic h
a ls o
a llo w s
th e
d e s c rip tio n
of
m ic ro s tru c tu ra tio n . H o w e v e r s o m e c o m p le x in te ra c tio n s (tip -b u b b le
173
Chapter VII
in te ra c tio n ) m u s t b e ta k e n in a c c o u n t o n th is s y s te m , p a rtic u la rly w h e n

b u b b le v e lo c ity is m e a s u re d (C a rte llie r a n d R iv i re , 2 0 0 1 ).
O p tic a l p ro b e p rin c ip le is v e ry s im p le (F ig u r e V II- 2 ). T h e lig h t is
g e n e ra te d in a L E D o r L a s e r d io d e (6 0 0 to 8 0 0 m m ) a n d p a s s e s tro u g h a
fib e r c o n n e c ted to a c o u p le r. T h is d e v ic e c o n n e c ts th e p ro b e a n d
re c e iv e s its re s p o n s e s ig n a l th a t is tra n s m itte d to a n o p tic a l/e le c tric
c o n v e rto r. T h e o p tic a l s ig n a l is th e n c o n v e rt in to e le c tric a l s ig n a l
(O liv ie r 1 9 9 9 ).
The
e le c tric a l
s ig n a l
is
fra g m e n te d
in
o rd e r
to
e x tra c t
a ll
c h a ra c te ris tic p o in ts (fra g m e n ts ) a n d c a rrie d o u t a s a m p lin g o rd e rin g

b a s e d o n its a m p litu d e .
In F ig u re V II- 3 a re p re s e n te d th e m o s t im p o rta n t p o in ts to b e
d e te c te d fro m th e s ig n a l w h e n a b u b b le is p ie rc e d b y th e fib er. T h e
e le c tric a l
s ig n a l
tre a tm e n t
m e th o d ,
c a lle d
T re a tm e n t
fo r
Gaseous
V e lo c ity (T V G ), w a s p ro p o s e d b y C a rte llie r (1 9 9 2 ), a n d c a n b e d iv id e d in

fo u r s ta g e s :
1 . D e te c tio n o f ty p ic a l a m p litu d e s (V L a n d V G );
2 . D e te c tio n o f th e c h a ra c te ris tic p o in ts A a n d B ;
3 . D e te c tio n o f th e c h a ra c te ris tic s p o in ts C a n d D ;
4 . E x p lo ita tio n o f th e re d u c e d d a ta .
In itia lly th e k n o w le d g e o f th re e a m p litu d e s is im p o rta n t: liq u id (V L ),
g a s (V G ) a n d n o is e (V B ) v o lta g e le v e ls , w h ic h re p re s e n t th e s u rro u n d in g
p h a s e a ro u n d th e p ro b e tip . V G is n o t e a s y to d e fin e m a in ly d u e to
d iffe re n c e b e tw e e n V G in a d ry p ro b e a n d V G o f a b u b b le . V B , th e
e le c tric n o is e , is c o n s id e re d s im ila r b o th in liq u id- o r g a s -p h a s e . T h e s e
p a ra m e te rs c a n b e m a n u a lly in tro d u c e d o r c o m p u te d . T h e V B c a n e a s ily
b e d e te rm in e d fro m a s c o p e re a d in g w h ile th e V L a n d V G d e te rm in a tio n
im p lie s th e a s s u ra n c e o f tw o c rite ria : (1 ) V G -V L > 1 a n d (2 ) V L /V B > 3 . T o
a v o id p o s s ib le flu c tu a tio n s a s a fe ty c o e ffic ie n t (S C ) is ta k e n in a c c o u n t
a n d fo r c o m p u ta tio n p u rp o s e s V B is d e fin e a s V B C = S C x V B /2 , w h e re S C is
u s u a lly lo w e r th a n 2 . T h e n , s ig n a l fra g m e n ta tio n is d o n e b y is o la tin g a ll
tim e in te rv a ls fro m ra w s ig n a l fro m w h ic h v o lta g e e x c e e d s V L + V B C .
174
Chapter VII
Figure VII-3.Bubble detection and determination of bubble caracteristic

points. Legend: A Beginning of Bubble; B End of Bubbles; C Begin
rising; D End of Rising. (in Cartellier, 1992)
F o r e a c h in te rv a l, A a n d B a re d e te rm in e d , th e u s e d c rite ria fo r A a n d
B b e in g th e v e ry b e g in n in g o f th e a s c e n d in g a n d th e v e ry b e g in n in g o f
th e d e s c e n d in g ra m p, re s p e c tiv e ly . T o re d u c e e rro rs th a t m a y o c c u r,
c rite ria p a ra m e te rs a re in tro d u c e d to e n s u re th e c o rre c t d e te rm in a tio n
o f A a n d B . D e te rm in a tio n o f C a n d D p o in ts d e p e n d s o n a c o rre c t
b u b b le p ie rcin g a n d s e v e ra l c rite ria a re u s e d to e n s u re th a t o n ly
b u b b le s s ig n a l w ith th e w e ll d e fin e p la te a u a re u s e d . F o r e v e ry in te rv a l,
th e d iffe re n c e in a m p litu d e (D V ) b e tw e e n liq u id a n d g a s is c o m p u te d .
T h e n , u s in g th e le v e ls 1 0 % a n d 9 0 % o f D V , p o in ts C a n d D a re
o b ta in e d b y in te rp o la tio n b e tw e e n th e c lo s e s t p o in ts c o rre s p o n d in g to
s u c h le v e ls . T h e T G V s o ftw a re d e te rm in e s th e tim e s s e rie s o f t A , t B , t C
a n d t D . T h e la tte r tw o a re z e ro if th e p re v io u s re fe rre d c rite ria a re n o t
a c h ie v e d . G a s re s id e n c e tim e (T G ) is g iv e n b y t B -t A w h ile ris in g tim e (T U )
is c a lc u la te d b y t D -t C . If T U v a lu e s a re le s s th a n th e s a m p lin g ra te th e y
a re e lim in a te d . U s in g T G a n d T U is p o s s ib le to d e te rm in e d lo c a l g a s
p h a s e a n d b u b b le p ro p e rtie s a c c o rd in g th e fo llo w in g e q u a tio n s :
G a s h o ld -u p
!G =
"T
E q . V II 1
t acq
175
Chapter VII
B u b b le V e lo c ity :
v B = A ! TMB
E q . V II 2
B u b b le C h o rd :
chB = v B ! TG
E q . V II 3
T h e a n a ly s is o f a ll m e a s u re m e n ts in a T G -T U o r c h B -|V 0 | p la n e c a n b e
u s e fu l to d is tin g u is h s e v e ra l b u b b le p o p u la tio n s a n d g e t in fo rm a tio n
a b o u t th e flo w re g im e .
Some
e rro rs
h a ve
been
re p o rte d
fo r
lo c a l
c h a ra c te ris tic
m e a s u re m e n ts u s in g o p tic a l p ro b e s a n d a re re la te d m a in ly w ith th e
p ie rc in g b e tw e e n o p tic a l fib e r a n d b u b b le a s e x e m p lifie d in F ig u r e
V II- 4 (C a rte llie r 1 9 9 2 , 1 9 9 8 ; C a rte llie r a n d B a rra u , 1 9 9 8 ). T h e m o s t
u s u a l a re e ffe c ts o f: (1 ) b lin d in g ; (2 ) d riftin g a n d (3 ) c ra w lin g . In
a d d itio n , a v o id in g e rro rs in T U a n d v B m e a s u re m e n t a s w e ll a s in p ro b e
c le a n lin e s s is e s s e n tia l to a c h ie v e a n a c c u ra te m e a s u re m e n t.
interfaceat t
-Active
lengthof theprobe
2.bParameters
F i g u r e V I I - 4FIG.
. Bu
b l e i n t e rfor
f a cthe
e /analysis
p r o b e ofi nattwo-dimensional
e r a c t i o n p a rprobe/interface
ameters. In Cartellier,
interaction.
1992.
stic events on a bubble signature.
T,=f( I vol ,R,B) for a probe and a liquid/gas pair.t d e te c te d d u e

T h e b lin d in g e ffe c t o c c u rs w h e n s m a ll c h o rd s a re n o(2)
of a probe to the passage
to im p etorfe c t tip d e w e ttin g . T h e d rifting e ffe c t h a p p e n s w h e n th e
ovides an opportunity
Let us recall that the previous experiments lead to a speerface characteristics.
In
a
case
theg form
b u b b le tra je ccial
to ry
c hof
a nEq.
g e s(2)
le aofd in
to a s m a ll c h o rd d e te c tin g o r e v e n to
between T, and Vi has
n o d e te c tio n a tT,=L*/I
a ll. F in
th e Rc raCO,
w linfl=O
g eor
ffe90,
c t is d(3)
u e to b u b b le
V,la lly for
s either parallel to or perThese ideal cases
d emay
fo rmnot
a tio n where
a n d dL*e sisa ca econstant
le ra tio nwhen
o f th
e bisugreater
b b le wthan
h e n10iscm/s.
p ie rc e d b y th e
1V,l
ing conditions, and addipresented
influence
tip . B lin d in g Ina the
n d tests
d riftin
g le a dhereafter,
to a n the
und
e re s timofa both
tio n Roand
f g a s h o ld-u p ,
nterface curvature for exfi is also investigated. The rise time is still defined as the
wever, owing to the comduration between the point C at 10% and the point D at
enomena occurring during
90% of the voltage difference DV between liquid and gas
he present analysis will be
levels. More precisely, DV is defined by the static liquid
scopic parameters. For a
level VL on one hand, and by the asymptotic value of the
, these are: the geometric
plateau VG, even if this level176
deviates from the static gas
erface Vi, the radii of curlevel. The uncertainty on T, is determined for every test; it
n of the probe axis with
never exceeds 7% including some subjectivity of the opernterface p1 and /3*. These
ator in the determination of DV. The latency length L is
Chapter VII
w h ile th e c ra w lin g le a d s to a n o v e re s tim a tio n a s th e b u b b le s ta y s

m o re tim e p ie rc e d b y tip .
E rro rs re la te d w ith p ie rc in g a re v e ry im p o rta n t b e c a u s e ris in g tim e
(T U ) d e p e n d o n ! (a n g le b e tw e e n th e p ro b e a nd th e n o rm a l to in te rfa c e )
a n d " (a n g le b e tw e e n th e p ro b e a n d th e b u b b le v e lo c ity v e c to r)
(C a rte llie r a n d B a rra u , 1 9 9 8 ). It w a s s h o w n th a t fo r ! v a lu e s in fe rio r to
1 0 T U is w e a k ly s e n s ib le h o w e v e r w h e n ! in c re a s e s th e ris in g tim e T U
s ta rts to b e im p o s s ib le to d e te rm in e (T U *v B ! c te = L ). T h u s , it is d iffic u lt
to a rra n g e s o m e c o rre la tio n b e tw e e n a ll th e s e p a ra m e te rs e s p e c ia lly , in
re a l s itu a tio n s , w h e re it is d iffic u lt to p re d ic t ! . In o rd e r to h a v e lo w
v a lu e s o f ! , a n d b e a b le to d e te rm in e v e lo c ity fro m T U , th e p ro b e n e e d s
to b e a lm o s t in p e rp e n d ic u la r p o s itio n c o m p a re d to th e flo w d ire c tio n
(C a rte llie r a n d B a rra u , 1 9 9 8 ; M e n a e t a l., 2 0 0 8 ).
V e jra z k a e t a l (2 0 1 0 ) fo u n d th a t g a s h o ld -u p m e a s u re m e n t e rro rs a re
d u e to th e in tru s iv e p ro p e rty o f p ro b e s . T h e y s tu d ie d th e in te ra c tio n
b e tw e e n a n o p tic a l p ro b e a n d a b u b b le w h e re b o th o p tic a l p ro b e
d is p la c e m e n t a n d b u b b le d e fo rm a tio n w e re fo u n d to b e th e c a u s e s fo r
th e m e a s u re m e n t e rro rs d is p la y e d , e s p e c ia lly w h e n c h o rd s d is trib u tio n
is c a lc u la te d (V e jra z k a e t a l., 2 0 1 0 ). H o w e v e r it is b e lie v e d th a t th e
a b ility to e v a lu a te g a s h o ld -u p is n o t s o ro u g h ly a ffe c te d b y th e s e
causes.
W h e n o p tic a l p ro b e s a re a p p lie d in g -l-s s y s te m s th e s o lid s s h o u ld
n o t: (1 ) h a v e s u c h rig id ity s o th a t th e y c o u ld b re a k th e o p tic a l fib e r
w h e n in te ra c tio n o c c u rs ; (2 ) c o n ta m in a te th e o ptic a l fib e r. O p tic a l fib e r
c o n ta m in a tio n d e p e n d s m a in ly fro m th e s o lid s p ro p e rtie s a n d s iz e . In
g e n e ra l s o lid p ro p e rtie s a re s u c h th a t h a v e n o in flu e n c e in o p tic a l p ro b e
s ig n a l, b u t th e re a re e x c e p tio n s . F o r e x a m p le it w a s fo u n d b y M e n a e t
a l (2 0 0 8 ) th a t a lg in a te b e d s , a s e m i-rig id s o lid , le t s o m e re s id u e s a fte r
tip -s o lid in te ra c tio n a n d w ro n g s ig n a ls in p ro b e v o lta g e w e re re p o rte d
a s it c a n b e o b s e rv e d in F ig u re V II- 5 (M e n a e t a l., 2 0 0 8 ).
O n th e o th e r h a n d s m a ll p a rtic le s m a y fo rm a n a g g lo m e ra te a ro u n d
th e o p tic a l fib e r a v o id in g th e in te ra c tio n b e tw e e n tip a n d b u b b le s . T h is
177
a g g lo m e ra tio n
Chapter VII
of
s m a ll
p a rtic le s
depends
on
th e ir
p ro p e rtie s
(h y d ro p h o b ic ity ; s u rfa c e c h a rg e ; e tc ) le a d in g to th e fo rm a tio n o f a

s o lid s c lu s te r a ro u n d th e tip .
Figure VII-5. Signal amplitude distribution (Left Graph) and time series
amplitude (Right Graph) obtained by Mena et al (2008) for g-l-s system using
alginate beads. Adapted from Mena et al. (2008).
S o lid -tip
in te ra c tio n
may
in d u c e
in a d e q u a te
s ig n a ls
due
to
c o n ta m in a tio n o f th e o p tic a l fib e r. M e n a e t a l (2 0 0 8 ) fo u n d th a t a lg in a te

b e d s c o n ta m in a tio n in d u c e s a s ig n ific a n t s ig n a l in c re a s in g (v o lta g e
p e a k ) a ffe c tin g th e s ig n a l p ro c e s s in g (F ig u r e V II- 5 ). H a v in g in m in d th a t
a ll m e th o d s d e v e lo p e d in s ig n a l p ro c e s s in g a re o p tim iz e d fo r tw o-p h a s e
flo w , it is n e c e s s a ry to a d a p t th e m w h e n th re e-p h a s e s y s te m s a re u s e d .
T h e re fo re a n e w p ro b le m a ris e s d u e to tip m e c h a n ic a l s e n s itiv ity a n d
th e ir re s is ta n c e to s o lid p a rtic le s im p a c t. In th e ir w o rk o th e r s o lid s
(g la s s a n d p o ly s ty re n e ) w e re te s te d in o rd e r to e v a lu a te th e e ffe c ts o f
s o lid s o n m e a s u re m e n t a c c u ra c y . A m o n g a ll s tu d ie d s o lid o n ly a lg in a te
b e d s in d u c e s e rro rs in o p tic a l p ro b e re s p o n s e s ig n a l. C o n s id e rin g th e
c o m p le x
and
s ig n a l
a n a ly s is
p ro c e d u re s
used,
th e
s u c c e s s fu ll a p p lic a tio n o f this s p e c ific p ro b e o n th re e p h a s e s y s te m s

w a s a c h ie v e d (M e n a e t a l., 2 0 0 8 ).
T h e c o n ta m in a tio n c a u s e d b y c lu s te r fo rm a tio n a ro u n d th e tip is a
v e ry d iffe re n t ty p e o f c o n ta m in a tio n a n d d is tin c t fro m th e o n e th a t
o c c u rre d in M e n a e t a l (2 0 0 8 ) w o rk . T h e p a rtic le a g g lo m e ra tio n a v o id s
b u b b le -tip in te ra c tio n a n d it is n o t p o s s ib le to a c c u ra te ly m e a s u re th e
lo c a l p ro p e rtie s o f th e s y s te m .
178
Chapter VII
VII.2.2 Solid-phase properties and distribution

T h e p a rtic le s te s te d o n th is w o rk w e re th e c e llu lo s e-b a s e d (S G ). T h e
S G a re g e n e ra lly h yd ro p h o b ic a n d h a v e a n e g a tiv e c h a rg e a t lo w p H ,
w h ic h a lo n g w ith th e ir ru g o s ity a n d n o n-u n ifo rm s u rfa c e fu ll o f c a v itie s
a n d h o le s a llo w s th e m to im m o b iliz e c e lls v e ry e ffe c tiv e ly . T h e a b ility o f
S G , w h e n in th e p re s e n c e o f w a te r, to fo rm a g g re g a te s e s p e c ia lly in th e
s e c tio n s o f th e re a c to r (c o n ic a l p a rt, s e e S e c tio n V .4 .1 ) w h e re th e liq u id
v e lo c ity is lo w e r (d e a d z o n e s ) c a n re s u lt fro m
th e ir h y d ro p h o b ic
s u rfa c e . P a rtic le s a n d b u b b le s s iz e a re in th e s a m e ra n g e s o th e tip c a n

p ie rc e o n e o r m o re S G p a rtic le s th a t a re flo w in g u p w a rd s in th e ris e r.
T h e s e p ie rc e d p a rtic le s w ill th e n in te ra c t w ith th e m o v in g p a rtic le s a n d
a g g lo m e ra tio n o f S G is o b s e rv e d a ro u n d th e p ro b e tip u n til n o b u b b le
s ig n a l is d e te c te d .
T o o v e rc o m e th is p ro b le m it w a s im p le m e n te d a n in je c tio n s y s te m
(F ig u r e V II- 6 ) th a t a c ts p e rio d ic a lly n e a r th e p ro b e tip . T h e liq u id-p h a s e
is in je c te d p e rio d ic a lly a n d th e tu rb u le n c e th a t it p ro m o te s c le a n s th e
tip o f a ll c lu s te r fo rm e d .
Figure VII-6. Photo of injection tip developed around the optical fiber (left).
Diagram of Injection system operating (right). Legend: A liquid injection
tubes; B Optical probe output signal; C Optical probe tip (optical fiber); D
Liquid injected arount optical fiber (dashed line)
179
Chapter VII
H o w e v e r th e re a re s o m e d ra w b a c k s w h e n in je c tio n n e a r th e p ro b e is
a p p lie d : b e c a u s e o f th e in d u c e d p e rtu rb a tio n s , p a rts o f th e s ig n a l,
c o rre s p o n d in g to th e tim e s w h e n in je c tio n is d o n e , m u s t b e re m o v e d
fro m th e o p tic a l p ro b e ra w s ig n a l in o rd e r to a c h ie v e a n a c c u ra te s ig n a l
e v a lu a tio n . In p ra c tic a l te rm s , th e in je c tio n s y s te m w a s n o t a b le to b e
u s e d a t m o re th a n 6 .1 % s o lid s (w t
WET BASIS
./v o l.).
A s v e rifie d in C h a p te r V , in a n iG L R n o t o n ly s o lid in flu e n c e o n g a s

h o ld -u p is im p o rta n t b u t a ls o s o lid d is trib u tio n th ro u g h o u t th e re a c to r.
C o n s id e rin g th e re s u lts fro m p re v io u s c h a p te r, it is e x p e c te d to h a v e
s im ila r s o lid s d is trib u tio n in th e iG L R te s te d . B e in g s o a n d to e a s ie r
fu tu re e v a lu a tio n , th e s o lid s d is trib u tio n th ro u g h o u t th e iG L R w a s
c o n s id e re d to b e a lm o s t h o m o g e n e o u s (s e e s e c tio n V .4 .1 .2 ).
VII.2.3 Liquid-phase
It is a ls o im p o rta n t to ta k e in to a c c o u n t th e liq u id -p h a s e p ro p e rtie s
in th re e -p h a s e s y s te m s . T h e s o lid s m a y in d u c e c h a n g e s in th e liq u idp h a s e p ro p e rtie s , w h ic h w ill a ffe c t th e g -l m ix tu re . U s u a lly c irc u la tio n
and
m ix in g
tim e s
a re
im p o rta n t
p a ra m e te rs
to
s tu d y
th e
m ix in g ,
d is p e rs io n a n d te n s io n fie ld p ro p e rtie s o f th e G L R s . A s s o c ia te d to th is ,
liq u id v e lo c itie s in d o w n c o m e r a n d ris e r a re n o rm a lly d e te rm in e d , th e s e
v e lo c itie s b e in g im p o rta n t p a ra m e te rs to u s e in s e v e ra l m o d e ls th a t
d e s c rib e th e G L re a c to r h y d ro d y n a m ic s .
T ra c e r te c h n iq u e s u s e d in th e p re v io u s c h a p te r d o n o t a llo w u s to
k n o w th e lo c a l flo w c h a ra c te ris tic s in th e d o w n c o m e r. F o r th is , fin e
te c h n iq u e s m a y b e u s e d (F ig u r e V II- 1 . ) a s , fo r e x a m p le , L D A L a s e r
D o p p le r A n e m o m e try (n o n in v a s iv e ); P IV P a rtic le Im a g e V e lo c im e try
(n o n -in v a s iv e ); H e a t p ro b e s (in v a s iv e ). In th is w o rk , c o n s id e rin g th e
liq u id v e lo c ity a n d th e in flu e n c e o f S G , P IV w a s u s e d .
180
VII.2.3.1
P a rtic le
Chapter VII
Particle image velocimetry

im a g e
v e lo c im e try
(P IV )
is
te c h n iq u e
th a t
has
been
s u c c e s s fu lly u s e d to d e te rm in e v e lo c ity fie ld s in s in g le-p h a s e flo w a t

lo w g a s h o ld -u p v a lu e s . A la s e r s h e e t is u s e d to illu m in a te a d e fin e d
p a rt o f th e flu id th a t c o n ta in s s e e d in g p a rtic le s (d e n s ity s im ila r to th e
flu id ). A H S C is s y n c h ro n iz e d w ith th e la s e r b e a m a n d tw o o r m o re
p ic tu re s a re ta k e n in s h o rt tim e in te rv a ls (! t). T h e n th e s e im a g e s a re
tre a te d (filte re d a n d c le a n e d o f n o is e ) a n d th e d is ta n c e b e tw e e n th e
tw o s e e d in g p a rtic le s is c a lc u la te d . K n o w in g th e tim e in te rv a l b e tw e e n
th e p ic tu re s , it is th e n p o s s ib le to d e te rm in e th e in s ta n ta n e o u s v e lo c ity
fie ld o f th e liq u id . B y p ro c e s s in g th e in s ta n ta n e o u s v e lo c ity , th e
a v e ra g e v e lo c ity in d iffe re n t z o n e s o f th e re a c to r c a n b e o b ta in e d . T h e
m a in p ro b le m o f u s in g P IV is to id e n tify th e s e e d in g p a rtic le s b e tw e e n
th e fra m e s . U s u a lly to d is c rim in a te w e ll th e d iffe re n t p h a se s flu o re s c e n t
s e e d in g p a rtic le s a re u s e d fo r th e liq u id a n d th e filte re d s ig n a l is
a n a ly s e d (B o y e r e t a l., 2 0 0 2 ; U n a d k a t e t al., 2 0 0 9 ). P IV te c h n iq u e is a
re fin e d te c h n iq u e g e n e ra lly u s e d to o b ta in in s ta n ta n e o u s liq u id v e lo c ity
a n d flu id p ro p e rtie s . In o u r c a s e , w e u s e d it to d e te rm in e th e ty p e o f
flo w in th e d o w c o m e r w h e re o n ly liq u id a n d s o lid-p h a s e w e re p re s e n t.
In g a s -lift re a c to rs th e liq u id -p h a s e v e lo c ity a n d c irc u la tio n d e p e n d s o n
th e g a s flo w ra te a c c o rd in g to :
b
v Li = a ! UGr
for i = r or d
E q . V II 4
W h e re a d e p e n d s o n th e flu id p ro p e rtie s w h ile U G r is d e te rm in e d b y

th e flo w re g im e a n d b o th b y th e re a c to r g e o m e try (O n k e n a n d W e ila n d ,
1 9 8 3 ). L iq u id v e lo c ity in flu e n c e s c irc u la tio n a n d m ix in g tim e in th is k in d
o f re a c to rs , a s s h o w e d in a p re v io u s c h a p te r, d e p e n d s o n g e o m e try
p ro p e rtie s a s th e ra tio s : A d /A r a n d H r /H C , th e p re s e n c e o r a b s en c e o f a
to p s e c tio n a s w e ll a s its c o n fig u ra tio n (F o n s e c a e t T e ix e ira , 2 0 0 7 ).
K le in e t a l (2 0 0 3 ) w o rk in g w ith a s im ila r g a s-lift re a c to r fo u n d th a t liq u id
flo w th ro u g h th e re a c to r (to d ilu tio n ra te s u p to 0 .6 h -1 ) h a s n o re le v a n t
in flu e n c e in th e m a in h y dro d y n a m ic p a ra m e te rs (s e c tio n V I.3 ) (K le in e t
a l., 2 0 0 3 ). S o , a ll m e a s u re m e n ts w e re d o n e in b a tc h s y s te m .
181
Chapter VII
VII.2.4 Hydrodynamic model: local hydrodynamics

F o r th is w o rk a h y d ro d y n a m ic m o d e l w a s d e v e lo p e d b a s e d o n th e
h y d ro d y n a m ic m o d e ls p re s e n t in lite ra tu re (C h is ti 1 9 8 9 ; L u e t a l. 1 9 9 5 ;
F re ita s e t a l. 1 9 9 9 ). T h is a llo w s h a v in g a b e tte r u n d e rs ta n d in g o f th e
in flu e n c e
of
th e
s o lid
phase
on
s y s te m
h y d ro d y n a m ic s .
Some
a s s u m p tio n s w e re m a d e :
- re a c to r c o n ta in s fo u r s e c tio n s : B o tto m (b ), R is e r (r), D o w n c o m e r
(d ) a n d T o p (t).
- a p s e u d o -h o m o g e n e o u s p h a s e (H ) w a s c o n s id e re d .
- s o lid s d is trib u tio n is h o m o g e n e o u s w h ic h le a d s to th e fa c t th a t th e
p ro p e rtie s o f th e p s e u d o -h o m o g e n e o u s p h a s e a re c o n s ta n t in a ll p a rts
o f th e re a c to r.
- n o g a s is p re s e n t in d o w n c o m e r.
F ro m th e d rift-flu x m o d e l d e v e lo p e d b y Z u b e r e t F in d la y (1 9 6 5 ) th e
re la tio n b e tw e e n g a s lin e a r v e lo c ity a n d s u p e rfic ia l v e lo c ity o f g a s a n d
liq u id , a s w e ll a s b u b b le te rm in a l v e lo c ity c a n b e w ritte n a s :
vG = C ! (UG + U L ) + U bt
E q . V II 5
W h e re C is th e d is trib u tio n p a ra m e te r a n d in d ic a te s th e flu x p a tte rn .

W h e n C = 1 it m e a n s th a t th e flu x d is trib u tio n is fla t. C is re la te d to g a s
d is trib u tio n a n d n o t v e lo c ity p ro file . In th e a ir-lift re a c to r ris e r:
vGr = C ! (UGr + U Lr ) + U bt
E q . V II 6
T h e re la tio n b e tw e e n lin e r a n d s u p e rfic ia l v e lo c ity w ith g a s h o ld-u p

c a n b e d e fin e d a s :
!Gr =
UGr
vGr
E q . V II 7
C o m b in in g E q . V II 6 a n d E q . V II 7 o n e g e ts :
!Gr =
UGr
C " (UGr + U Lr ) + U bt
E q . V II 8
182
Chapter VII
E q . V II 8 w ill b e u s e d to o b ta in , b y fittin g to e x p e rim e n ta l d a ta , th e

p a ra m e te rs : C a n d U b t . T h e d e te rm in a tio n o f liq u id v e lo c ity in ris e r is
d o n e c o n s id e rin g th a t th e p re s s u re d iffe re n c e b e tw e e n d o n w c o m e r a n d
ris e r (P d ) is e q u a l to th e to ta l fric tio n lo s s (- ! P T )(L u e t a l., 1 9 9 5 ):
Pd = ( !H " Hd # !G" Gd ) $ ( !L" Lr + !G"Gr ) # g # H
E q . V II 9
K n o w in g th a t ! H > > ! G a n d c o n s id e rin g th a t " H r = 1 - " G r a n d " H d = 1 ,
" G d = 0 , th e P d e q u a tio n c a n b e re-w ritte n :
Pd = !H " # Gr " g " H
E q . V II 1 0
T h e to ta l fric tio n lo s s in e a c h re a c to r s e c tio n is g iv e n b y th e

e q u a tio n :
!"PT = # !"Pf
E q . V II 1 1
W h e re (- ! P f ) i is th e p re s s u re d ro p in s e c tio n i (i= ris e r, d o w n c o m e r,

to p , b o tto m ) a n d is d e te rm in e d b y :
(!"P ) = 1/2 # $
f
# k fi # v Li2
E q . V II 1 2
F o r R is e r a n d D o w n c o m e r k f i is d e te rm in e d a c c o rd in g ly :
k fr = 4 ! f r ! H r /dr and k fr = 4 ! f d ! H d /(dc " dr,ext )
E q . V II 1 3
W h e re f i (fo r i= r o r d ) is d e te rm in e d b y th e B la s iu s e q u a tio n :
f i = 0.0791Re !0.25
E q . V II 1 4
And
Re Hi =
!Hi " v Li " di

Hi
E q . V II 1 5
U s in g c o rre c tio n fa c to r p ro p o s e d
(a ) b y G a rc ia-C a lv o a n d L e t n
(F re ita s 2 0 0 2 ) a n d c o n s id e rin g # H i a n d H i c o n s ta n t th ro u g h o u t a ll
re a c to r, th e k f i e q u a tio n c a n b e re-w ritte n :
k fi =
0.25
4 ! " ! 0.0791! H i ! Hi
0.25
#Hi
! di0.25 ! v Li0.25
183
E q . V II 1 6
Chapter VII
A s ! , " H i a n d H i d e p e n d o n s o lid c o n c e n tra tio n a p a ra m e te r # w a s

c o n s id e re d (F re ita s e t a l. 1 9 9 9 ):
! = " # 4 # 0.0791# 0.25 # $ %0.25
E q . V II 1 7
A s k f t o p < < k f b o t t o m o n ly th e p re s s u re d ro p in th e b o tto m
w ill b e
c o n s id e re d a n d d e te rm in e d a c c o rd in g ly (C h is ti 1 9 8 9 ):
k fbb
"A %
= 11.4 ! $ d '
# Alb &
0.79
0.2 <
Ad
< 1.8
Alb
E q . V II 1 8
T h u s a t s te a d y-s ta te c o n s id e rin g th e re la tio n:
Pd = !"PT
C o m b in in g
Eq.
V II 1 0 ,
E q . V II 1 9
Eq.
V II 1 1 ,
Eq.
V II 1 2
and
Eq.
V II 1 3
c o n s id e rin g :
v Li =
U Li
1! "Gi ! " Si
E q . V II 2 0
And
Ai ! U Li = Ar ! U Lr
E q . V II 2 1
T h e fin a l e q u a tio n fo r liq u id s u p e rfic ia l v e lo c ity is

2
2 ! g ! H r,d
k fbb
$A '
2
! ("Gr # " Gd ) = & r ) !
2 U Lr + ...
% Ad (
(1# "Gd # " Sd )
1.75
*
$ Ar '
Dr#1.25
(Dc # Dr ) #1.25 1.75
... + ,
+
!
& )
1.75
1.75 / ! H r,d ! 0 ! U Lr
(1#
"
#
"
)
A
(1#
"
#
"
)
% d(
Gr
Sr
Gd
Sd
,+
/.
E q . V II 2 2
T h e s e e q u a tio n s a re s o lv e d a c c o rd in g to th e fo llo w in g d ia g ra m
(F ig u re V II- 7 ).
184
Chapter VII
Model Parameters:
C,!,Ubt
Experimental Data:
UGr, eGr, ULr
Initial estimations (literature):

C0=1;
b0=0.02,
ubt0=0.25.
Initial conditions:
Ugr00=Ugr;
egr00=eGr,
Ulr00=Ulr.
!Gr determination (eq.15)
ULr determination (eq. 29)

New parameters:
C; B; ubt0.
New conditions:
Ugr00=Ugr(i);
egr00=egr(i),
ulr00=ulr(i)Ulr.
Yes
Ulr(i)-ulr(i-1)"0.001
No
No
Error"[(ULrEXP-ULr)/ULREXP+(eGrEXP-eGr)/
eGREXP]
Yes
End
Figure VII-7.Flow diagram for MATLAB program used in this model. Starting
from initial model parameters and experimental data, new parameters are
calculated in order to achieved values of UGr, eGr and ULr that are similar
(5%) to the experimental data.
185
Chapter VII
VII.3 Material and Methods

VII.3.1 Experimental apparatus for gas hold-up measurements
Figure VII-8. Experimental setup for gas hold-up experiments. Legend: 1. AirLift Reactor; 2. Optical Probe; 3. Electro valve; 4. Light source+ Photo
detector; 5. Oscilloscope; 6. Acquisition Board
T h e iG L R u s e d in th is w o rk is o f th e c o n c e n tric d ra u g h t tu b e ty p e
w ith a n e n la rg e d to p s e c tio n fo r d e g a s s in g a n d a to ta l w o rk in g v o lu m e
o f 6 L (F ig u r e V II- 8 ). T h e d im e n s io n s o f th e re a c to r a re a s fo llo w s : to ta l
h e ig h t= 9 6 c m ; d o w n c o m e rs le n g th = 6 6 c m a n d in s id e d ia m e te r= 7 c m ;
d ra u g h t tu b e le n g th = 6 4 c m , d ia m e te r= 3 c m , a n d th ic k n e s s = 0 .5 c m ;
a n d c y lin d ric a l p a rts le n g th = 1 9 .5 c m a n d d ia m e te r= 1 9 c m . T h e a n g le
186
Chapter VII
b e tw e e n th e c o n ic a l s e c to r a n d th e m a in b o d y w a s 5 1 . G a s w a s
in je c te d th ro u g h a d is trib u to r (1-c m d ia m e te r) w ith fiv e n e e d le s , e a c h o f
0 .2 m m in d ia m e te r, a n d p lac e d 1 .7 c m b e lo w th e a n n u lu s o f th e ris e r.
T h e o u tflo w o f th e re a c to r w a s p la c e d b e h in d a s e d im e n ta tio n b a rrie r,
th u s m in im iz in g c a rrie r lo s s e s . T h e w a te r le v e l in th e re a c to r w a s
a lw a y s k e p t c o n s ta n t a t th e s a m e h e ig h t. T e m p e ra tu re a n d p re s s u re
w e re a m b ie n t (2 1 C a n d 1 a tm ). T h e d e s ire d g a s flo w w a s a d ju s te d
w ith a ro ta m e te r (M R 3 0 0 0 s e rie s F lo w m e te r, K e y In s t., T re v o s e , U S A )
o p e ra tin g a t 1 a tm a n d 2 1 C .
T h e c y c lic in je c tio n (In je c tio n tim e (s )/T o ta l c y c le tim e (s )= 0 .5 /2 .5 ) w a s
p e rfo rm e d b y u s in g o n e e le c tro -v a lv e (S ira i, M ila n , Ita ly ) c o n tro lle d b y
P C e q u ip p e d w ith L A B V IE W (N a tio n a l In s tru m e n ts , A u s tin , T e x a s , U S A ).
T h e s ig n a l a c q u is itio n o f th e e le c tro-v a lv e a n d o p tic a l p ro b e w e re
re c o rd e d
u s in g
an
a c q u is itio n
b o a rd
NI
B N C-2 1 1 0
(N a tio n a l
In s tru m e n ts , A u s tin , T e x a s , U S A ) a n d L A B V IE W p ro g ra m , w h ic h s a v e s
th e s ig n a l in a b in a ry file p e r a s s a y . T h e n e a c h file w a s tre a te d u s in g
M A T L A B 6 .1 (M a th W o rk s , N a tic k , M A , U S A ) d e v e lo p e d to re a d th e
b in a ry file s o b ta in e d . T h e s a m e p ro g ra m w a s u s e d to d e te rm in a te a ll
c h a ra c te ris tic p o in ts fro m o p tic a l p ro b e s ig n a l (t A , t B , t C a n d t D ), a s w e ll
a s to c a lc u la te a ll g a s p h a s e p ro p e rtie s d e te rm in e d fro m E q . V II 1 , V II2 a n d V II- 3 . T h e s a m p lin g ra te w a s 1 0 k H z a n d th e d u ra tio n o f e a c h
a s s a y w a s 8 0 0 s . E a c h e x p e rim e n ta l c o n d itio n w as re p e a te d 5 tim e s in
o rd e r to h a v e e n o u g h m e a s u re d b u b b le s (a t le a s t 1 0 0 0 ) th a t a s s u re
s ta tis tic a l e v a lu a tio n .
187
Chapter VII
Binary File from Optical Probe (OP) Experiment Reading!
Conversion of binary file in two arrays: OP voltage; Electro valve

voltage!
Removing Injection times in OP voltage array (Sections)!
Determination per section: VG; VL ; Nr of Bubbles (N Bub)!
If N Bub/Section > 1!
YES!
NO!
Determination of tG; tU;

tAQ; !SECTION;!
Section Rejected!
Figure VII-9. MATLAB program layout (see Appendix I).
VII.3.2 New signal processing and validation

In th e ra w d a ta tre a tm e n t th e in flu e n c e o f th e in je c tio n is re m o v e d
a n d o n ly th e d a ta w h e re n o in je c tio n s a re p e rfo rm e d is a n a ly s e d to
d e te rm in e d
g a s-p h a s e
p ro p e rtie s :
gas
h o ld -u p ,
b u b b le
c h o rd
and
v e lo c ity . F o r v a lid a tio n o f th e n e w s y s te m , te s ts w e re p e rfo rm e d o n ly in

g -l s y s te m in o rd e r to e v a lu a te if th e m o d ific a tio n (in je c tio n s y s te m p lu s
n e w m e th o d ) m a d e h a s a re a l in flu e n c e in th e m e a s u re m e n t.
T h e o b ta in e d s ig n a l is c o m p o s e d b y tw o c u rv e s : o n e w h e re th e s ig n a l
re c o rd e d b y th e O P is p re s e n t a n d o th e r w hic h c o rre s p o n d s to th e
s ig n a l o f th e in je c tio n s (F ig u r e V II- 1 0 ).
A t e a c h in je c tio n th e re is a p e rtu rb a tio n in th e p ro b e a n d in th e flo w
le a d in g
to
m e a s u re m e n t
e rro rs
due
to :
(1 )
p ro b e
m o v e m e n t;
(2 )
in c re a s e d b u b b le re s id e n c e tim e (h ig h e r t G ); (3 ) b u b b le d e fle c tio n

(d e te c tio n o f a b u b b le th a t n o rm a lly w a s n o t d e te c te d a n d v ic e-v e rs a ).
188
Chapter VII
Figure VII-10. Raw signal obtain for riser radial central position at
QGAS=400mL/min. Top graph shows optical probe signal. Bottom Graph shows
electovalve signal. (The total time where corresponds to 7 seconds)
In o rd e r to a v o id th e s e e rro rs th e s ig n a l is th e n c u t b e fo re a n d a fte r
th e in je c tio n a n d o n ly th e re s t o f th e s ig n a l is u s e d fo r g a s h o ld-u p
d e te rm in a tio n . T h e c u t s ig n a l is th e n a n a ly s e d a n d th e g a s h o ld-u p
c a lc u la te d . S o th e c u ttin g tim e (p e rc e n ta g e o f in je c tio n tim e ) b e c o m e s
o n e o f th e c ritic a l p a ra m e te rs fo r h o ld -u p d e te rm in a tio n . D iffe re n t
c u ttin g z o n e s a ro u n d th e in je c tio n w e re p e rfo rm e d a nd g a s h o ld -u p
d e te rm in e d fo r e a c h z o n e . T h e v a lid a tio n e x p e rim e n ts w e re p e rfo rm e d
in a ir-w a te r m ix tu re u s in g th e in je c tio n s y s te m a n d w ith o u t th e in je c tio n
s y s te m . T h e n th e re s u lts w e re c o m p a re d b e tw e e n th e n e w s y s te m a n d
th e o b ta in e d u s in g th e a lg o rith m d e v e lo p e d b y A la in C a rte llie r (1 9 9 2 )
T G V . T h e re s u lts fro m th e n e w d e v e lo p e d s y s te m w e re p e rfo rm e d a t
d iffe re n t
c u ttin g
p e rc e n ta g e s
b e fo re
and
a fte r
th e
in je c tio n
tim e
(d iffe re n t c u ttin g z o n e s ). T h e c u ttin g z o n e s (F ig u re V II- 1 1 ) in c lu d e n o t
189
Chapter VII
o n ly th e tim e w h e n th e in je c tio n w a s p e rfo rm e d b u t a ls o fra c tio n s o f

tim e b e fo re a n d a fte r th e re fe rre d in je c tio n . C o n s id e rin g th a t th e
in je c tio n in flu e n c e is b ig g e r a fte r it o c c u rs th a n b e fo re , th e z o n e s
e v a lu a te d w e re : A D D 1 - u p to 5 0 % le s s tim e b e fo re th e b e g in n in g o f
in je c tio n ; A D D 2 - u p to 1 0 0 % m o re a fte r th e in je c tio n tim e . T h is m e a n s
th a t, p e r e a c h in je c tio n p e rio d (2 .5 s ), th e m in im u m tim e re m o v e d w a s
0 .5 s c o rre s p o n d in g o n ly to th e in je c tio n a n d th e m a x im u m tim e w a s
1 .2 5 s c o rre s p o n d in g to a c u ttin g o f 5 0 % b e fo re a n d 1 0 0 % a fte r th e
in je c tio n (in c lu d e d ) a s e x p la in e d in F ig u r e V II- 1 1 .
Figure VII-11. Graphical explanation of cutting zones. Legend: Add1 area

removed before injection (from 0% to 50% of Injection peak time). Add2 area
removed after injection (from 0% to 100% of Injection peak time)
T h e e rro r b e tw e e n g a s h o ld -u p m e a s u re u s in g th e T G V p ro g ra m a n d
u s in g th e n e w m e th o d w a s c a lc u la te d u s in g th e e q u a tio n :
%Error =
abs(xTGV ! x INJ )
" 100
xTGV
E q . V II 2 3
W h e re x re p re s e n ts th e g a s -p h a s e p ro p e rtie s : h o ld -u p ( ! B ); b u b b le
v e lo c ity (v B ) o r b u b b le c h o rd le n g th (c h B ).
190
Chapter VII
VII.3.3 Experimental conditions
6L
Top view of Riser
r7
r15 r22
Legend:
B
0 <-> A : 10 cm
A <-> B : 20 cm
ri <-> rj : 7 mm
Figure VII-12.Optical probe measurement location in the GL reactor
T h e o p tic a l p ro b e w a s lo c a te d a t tw o h e ig h ts fro m th e to p o f th e
ris e r: h A = 1 0 c m
and hB=30 cm
a n d m e a s u re m e n ts w e re p e rfo rm e d
v a ry in g th e ra d ia l p o s itio n . T h e ra d ia l p o s itio n s w e re , re s p e c tiv e ly , r= 0 ,

7 m m . r= 0 c o rre s p o n d s to th e c o lu m n a x is . T h e fe w p o s itio n s s tu d ie d
in th e ra d ia l p o s itio n s w e re b e c a u s e o f (1 ) th e lo w e r s p a tia l re s o lu tio n
d u e to in je c tio n s y s te m u s e d , a n d (2 ) th e tim e /s to ra g e s p a c e th a t w a s
n e c e s s a ry to o b ta in e d e n o u g h d a ta fo r e a c h s tu d ie d p o s itio n .
191
Chapter VII
VII.3.4 Solids properties

S p e n t g ra in s w e re p re-tre a te d a c c o rd in g to th e a c id /b a s e m e th o d
p ro p o s e d b y B r n y ik e t a l. (2 0 0 1 ). S p e n t g ra in s a fte r p re -tre a tm e n t a re
a lm o s t fla t p a rtic le s , w ith e q u iv a le n t d ia m e te r d E Q < 2 .1 m m a n d d e n s ity
!=1037 kgWET
BASIS
/m 3 .
The
s iz e
d is trib u tio n
of
th e
p a rtic le s
was
d e te rm in e d b y s ie v in g in to fra c tio n s u s in g a p o rta b le s ie v e s h a k e r

(M o d e l A n a ly s e tte , F rits c h , G e rm a n y ). W ith th e o b ta in e d d a ta , th e
e q u iv a le n t d ia m e te r w a s c a lc u la te d . T h e s o lid s a re c o m p le te ly w e tta b le
w ith a w a te r a d s o rp tio n in d e x (W A I) o f 8 ,1 2 g W E T /g D R Y a n d s e d im e n ta tio n
v e lo c ity is a b o u t (0 .8 3 0 .1 6 ) c m /s . T h e fo llo w in g fiv e s o lid lo a d in g s
w e re u s e d 0 % (w a te r), 2 % , 4 % , 6 % (w t. W E T
BASIS
/v o l.).
VII.3.5 High speed camera measurements and image analysis

T o a n a ly s e th e s p e c ific b e h a v io u r a n d in flu e n c e o f th e S G in g -l
m ix tu re a H S C , re c o rd in g a t 1 0 0 0 fra m e s p e r s e c o n d , w a s u s e d . T h is
a llo w s v is u a l e v a lu a tio n a n d c o n firm a tio n o f th e in fo rm a tio n o b ta in e d b y
th e o p tic a l p ro b e u s in g th e n e w m e th o d . H o w e v e r th is w a s o n ly
a c h ie v e d
at
lo w
s o lid
(u p
to
2 % (w t. W E T
BASIS
/v o l.))
b e c a u s e a t h ig h e r v a lu e s n o in fo rm a tio n fro m im a g e s w a s p o s s ib le to
b e o b ta in e d . H S C v id e o s w e re re c o rd e d u s in g F lo w V ie w
TM
p ro g ra m a n d
th e b u b b le p ro p e rtie s (m e a n c h o rd a n d m in im u m a n d m a x im u m F e re t
d ia m e te rs ) a n a ly s is w a s p e rfo rm e d m a n u a lly . Im a g e s iz e s o b ta in e d h a v e
a 1 2 8 0 x 1 0 2 4 re s o lu tio n a n d th e m a n u a l m e a s u re m e n t e rro r w a s b e lo w
5 % . In o rd e r to c o n firm th e q u a lity o f th e m a n u a l a n a ly s is p ro c e s s ,
some
e x p e rim e n ts
w e re
a ls o
a n a ly s e d
u s in g
th e
s ta tis tic a l
to o l
p ro p o s e d b y F e rre ira e t a l. (2 0 1 1 ). T h is to o l w a s s u c c e s s fu lly a p p lie d in

th e a u to m a tic id e n tific a tio n o f s in g le b u b b le s a n d b u b b le g ro u p s w ith a
m in im u m a g re e m e n t o f 9 0 % (F e rre ira e t a l., 2 0 1 1 ).
192
Chapter VII
VII.3.6 Statistical method

The
s ta tis tic a l
a n a ly s is
of
th e
g a s-p h a s e
p ro p e rtie s
(h o ld-u p ,
v e lo c ity , c h o rd s iz e ) w a s c a rrie d o u t u s in g s in g le-fa c to r a n a ly s is o f

v a ria n c e
(A N O V A ),
w h ile
m u ltip le
c o m p a ris o n
te s ts
w e re
used
to
d e te rm in e th e s ta tis tic a l s ig n ific a n c e w ith a 9 5 % c o n fid e n c e le v e l. F o r

th e d a ta a n a ly s is , M A T L A B s o ftw a re w a s u s e d .
VII.3.7 PIV measurements

P IV m e a s u re m e n ts w e re m a d e to d e te rm in e th e liq u id v e lo c ity fie ld in
th e d o w n c o m e r. T h e P IV -L IF s y s te m w a s a L a V is io n D A V IS w ith a T w in s
U ltra Y a g 2 x 3 0 m J L a s e r (w a v e p e a k 5 3 2 n m ). T h e c a m e ra (P ro X 2 M )
w ith a m in im u m in te rfra m e o f 1 1 0 n s w a s u s e d to re c o rd e P IV -L IF
im a g e s . T h e la s e r w a s p a s s e d th ro u g h a le n s , w h ic h s h a p e d th e
re s u ltin g b e a m in to a lig h t s h e e t. T h e d e la y b e tw e e n tw o p ic tu re s w a s
1 2 0 s . T h e fie ld o f v ie w w a s 2 3 2 m m ! 1 5 0 m m a n d in c lu d e d th e
d o w c o m e r c e n te r. C o n s e q u e n tly , 1 5 0 d o u b le im a g e p a irs w e re o b ta in e d
in e a c h a s s a y , a n d w e re c o n s id e re d to b e s u ffic ie n t to o b ta in s ta tis tic s
re s u lts . E a c h a s s a y w a s p e rfo rm e d in trip lic a te
Figure VII-13. Experimental setup for PIV measurements. Legend: 1. Air

Source; 2. Rotameter; 3. Air-Lift Reactor; 4. Laser + Laser sheet maker; 5.
193
Chapter VII
Recording camera; 6. Acquisition and Synchronized System; 7. Laser sheet

measurement viewed from above
VII.3.8 Microorganism and medium

T h e m ic ro o rg a n is m u s e d d u rin g th e e x p e rim e n ta l w o rk w a s a b re w in g
y e a s t s tra in
(S a c c h a ro m y c e s
c e re v is ia e ) s u p p lie d
by
th e
company
U N IC E R (B e b id a s d e P o rtu g a l, S .A ., S . M a m e d e d e In fe s ta ).
T h e y e a s t w a s c u ltiv a te d in a s y n th e tic m o d e l m e d iu m (S M M ) w ith
s a m e c o m p o s itio n a s p re s e n te d in C h a p te r V III.2 . B a rre ls w ith 5 0 L o f
S M M w e re s te riliz e d b y a u to c la v in g a t 1 2 0 C , 1 0 0 k P a fo r 6 0 m in .
VII.3.9 Free biomass determination

A s a m p le o f 1 0 m L w a s re m o v e d fro m th e re a c to r o u tflo w a n d it w a s
c e n trifu g e d (H e ttic h , U n iv e rs a l 3 2 0 R ) fo r 5 m in a t 5 0 0 0 rp m a n d 4 C .
T h e s u p e rn a ta n t w a s re m o v e d a n d th e s e d im e n t w a s d ilu te d (1 :5 ra tio )
w ith is o to n ic s o lu tio n (0 .9 % (w t./v o l.) N a C l). T h e o p tic a l d e n s ity o f th e
s o lu tio n w a s m e a s u re d w ith a s p e c tro p h o to m e te r (W P A - B io w a v e II) a t
6 0 0 n m w a v e le n g th . E a c h s a m p le d e te rm in a tio n w a s d o n e in trip lic a te .
The
re s u lts
of
b io m a s s
w e re
c a lc u la te d
u s in g
c a lib ra tio n c u rv e th a t re la te d g B I O M A S S /L v s . O .D . (6 0 0 n m ).
VII.3.10 Immobilized biomass determination

A s a m p le c o n ta in in g a p p ro x im a te ly 1 g o f d ry b io c a ta ly s t (c a rrie r +
im m o b iliz e d c e lls ) w as ta k e n fro m th e re a c to r. T h e b u lk liq u id w a s
re m o v e d w ith a s y rin g e a n d th e c a rrie r w a s w a s h e d w ith d is tille d w a te r
in o rd e r to re m o v e th e fre e c e lls c a p tu re d b e tw e e n th e c a rrie r p a rtic le s
a n d d rie d a t 8 0 C . T h e n th e d ry s a m p le w a s w e ig h e d a n d d iv id e d in
th re e E rle n m e y e r fla s k s (p re v io u s w e ig h te d ) w ith 5 0 m L o f 3 % (w /v )
N a O H s o lu tio n a n d w a s s h a k e n a t 1 5 0 rp m fo r 2 4 h . D u rin g th is tim e th e
im m o b iliz e d c e lls w e re c o m p le te ly re m o v e d fro m
th e c a rrie r. T h e
re le a s e d c e lls w e re w a s h e d w ith d is tilla te w a te r a n d d rie d a t 8 0 C . T h e
194
Chapter VII
a m o u n t o f im m o b iliz e d y e a s t b io m a s s w a s d e te rm in e d fro m th e w e ig h t
d iffe re n c e
b e fo re
and
a fte r
th e
tre a tm e n t
w ith
NaOH
s o lu tio n .
C o rre c tio n s o f th e b io m a s s w e ig h t w e re c a rrie d o u t a d m ittin g 6 % o f

lo s s e s .
VII.3.11 Cell viability flow cytometry

T h e flo w c y to m e try m e a s u re m e n ts w e re ta k e n u s in g a A c c u ri C 6 flo w
c y to m e te r (A c c u ri, M ic h ig a n , U S A ). T h e F C M a n a ly z e r c o n ta in s a s o lid
s ta te 4 8 8 n m (F L 1 -H ) a n d a 6 4 0 n m (F L 2 -H ) d io d e la s e r a n d is a ls o
e q u ip p e d w ith a n a u to s a m p le r u n it. T h e c e ll v ia b ility w a s d e te rm in e d
b y s ta in in g y e a s t c e lls w ith P ro p id iu m io d id e (P I). F o r th a t, 1 m L o f
y e a s t c e lls s u s p e n s io n w a s m ix e d w ith 1 0 L o f P I in a d a rk ro o m fo r
1 0 m in .
10 L
Then
c y to m e try .
C e ll
th a t
of
yeast
s u s p e n s io n
s u s p e n s io n
was
c o rre s p o n d s
d ilu te d
to
was
in
a n a ly s e d
o rd e r
m in im u m
of
to
150
by
flo w
a c h ie v e
e v e n ts
a
(o r
c e lls )/ L d e te c te d in F L 1-H c h a n n e l. T h e d e a d c e lls s ta in e d in re d b y P I

w e re
d e te c te d
c a lib ra tio n
in
c u rv e
F L 2 -H
th a t
channel
re la te d
and
number
(6 0 0 n m ) w a s d e te rm in e d .
195
c o n s id e re d
of
T o ta l
non
c e lls /m L
v ia b le .
vs.
O .D .
Chapter VII
VII.4 Results and Discussion

VII.4.1 Calibration of new system
B e fo re m e a s u rin g th e g a s p h a s e p ro p e rtie s in g -l-s s y s te m s it w a s
n e c e s s a ry to k n o w th e ra n g e o f c u ttin g z o n e s w h e re th e n e w m e th o d is
v a lid , i e ., w h e re the e rro r b e tw e e n th e n e w a n d T G V m e th o d s is lo w e r.
A n in itia l o b s e rv a tio n in d ic a te d th a t th e n e w s y s te m g e n e ra lly o v e r
e s tim a te s th e g a s -p h a s e p ro p e rtie s fo r 2 5 0 m L /m in , w h ile fo r 4 0 0
m L /m in th e re s u lts a re u n d e re s tim a te d . T h e re la tiv e e rro r b e tw e e n th e
tw o m e th o d s is p re s e n te d in F ig u re V II- 1 4 . It is s h o w n th a t e ith e r
B u b b le V e lo c ity o r C h o rd S iz e a re n o t v e ry a ffe c te d b y th e d iffe re n t
c u ttin g z o n e s . H o w e v e r, th e s a m e d o e s n o t h a p p e n fo r g a s h o ld-u p . It
is
im p o rta n t
to
have
in
a c c ou n t
th a t
among
th e
g a s -p h a s e
c h a ra c te ris tic s d e te rm in e d u s in g th e O P , o n ly th e g a s h o ld-u p is

d e p e n d e n t o n th e a c q u is itio n tim e (E q . V II 1 ). S o it is u n d e rs ta n d a b le
w h y d iffe re n t c u ttin g z o n e s ra tio s a ffe c t th e fin a l a c q u is itio n tim e , a n d
th e re fo re in flu e n c e th e g a s h o ld -u p . T h u s th e b e s t (le s s e rro r) c u ttin g
z o n e s o b ta in e d w e re : fo r 2 5 0 m L /m in A d d 1 < 3 7 .5 % a n d A d d 2 < 3 7 .5 % ,
a n d fo r 4 0 0 m L /m in 2 5 % < A d d 1 < 5 0 % a n d 5 0 % < A d d 2 < 1 0 0 % . In s id e
th e s e v a lu e s th e e rro r fo r 2 5 0 m L /m in w a s a lw a y s b e lo w 1 0 % a n d fo r
4 0 0 m L /m in b e lo w 5 % . T h e e rro r fo r b u b b le v e lo c ity w a s 1 0 % a n d 2 0 %
fo r 2 5 0 m L /m in a n d 4 0 0 m L /m in re p e c tiv e ly , w h ile fo r b u b b le c h o rd w a s
1 5 % a n d 2 0 % . T h e b ig g e s t e rro r in b u b b le p ro p e rtie s (v e lo c ity a n d
c h o rd ) is m a in ly re la te d w ith lo w fre q u e n c y a c q u is itio n u s e d 1 0 k H z ,
w h e n c o m p a re d w ith th e u s e d in th e T G V m e th o d 5 0 k H z (C a rte llie r,
1 9 9 8 ). T h e lo w fre q u e n c y o f s ig n a l u s e d w a s n e c e s s a ry to a v o id h u g e
b in a ry file s th a t w e re n o t p o s s ib le to b e tre ate d b y a v a ila b le m e th o d s .
196
Chapter VII
A!
B!
20.0!
% Error (Gas Hold-up)!
% Error (Gas Hold-up)!
20.0!
15.0!
10.0!
5.0!
15.0!
10.0!
5.0!
0.0!
0.0!
% Add2!
20.0!
10.0!
% Error (Bubble Velocity)!
% Error (Bubble Velocity)!
% Add2!
5.0!
0.0!
15.0!
10.0!
5.0!
0.0!
% Add2!
% Add2!
20.0!
% Error (Bubble Chord)!
% Error (Bubble Chord)!
15.0!
10.0!
5.0!
0.0!
15.0!
10.0!
5.0!
0.0!
% Add2!
% Add2!
Figure VII-14. Error obtained at different Add1 and Add 2 for different gasphase characteristics: Gas hold-up, Bubble Velocity and Bubble Chord.
Legend: A QAIR= 250 mL/min; B QAIR= 400 mL/min; ! - 0.0% Add1; " 12.5% Add1;
" - 25.0% Add1; " - 12.5% Add1; "- 50.0% Add1
197
Chapter VII
VII.4.2 Gas-phase measurements

T h e m a in re s u lts o b ta in e d to d e te rm in e th e g a s-p h a s e p ro p e rtie s in
th e ris e r w e re d o n e u s in g a n o p tic a l p ro b e . H o w e v e r a n d du e to th e
im p le m e n ta tio n o f th e n e w m e th o d to u s e th is p ro b e fo r th is s y s te m
s o m e b u b b le c h a ra c te ris tic s w e re a ls o d e te rm in e d u s in g a H S C in o rd e r
to e v a lu a te a n d v a lid a te th e re s u lts . T h e m a x im u m s o lid c o n te n t th a t
th e n e w s y s te m w a s a b le to w o rk w a s 6 % S G (w t. W E T
BASIS
/v o l.). A b o v e
th is v a lu e , th e c le a n in g s y s te m w a s n o t a b le to d o a n e ffic ie n t tip
c le a n in g .
T o c h e c k th e s ig n ific a n t d iffe re n c e s b e tw e e n a ll m e a s u re m e n ts , th e
re s u lts w e re s ta tis tic a lly e v a lu a te d . A s in d ic a te d in s e c tio n V II.3 .3 , d a ta
fro m tw o d iffe re n t a x ia l p o s itio n s in th e ris e r (A a n d B in F ig u r e V II- 1 2 )
w e re a n a ly s e d . F o r e a c h s o lid s c o n c e n tra tio n , tw o d iffe re n t g a s flo w s
w e re te s te d : 2 5 0 a n d 4 0 0 m L /m in . B ra n y ik e t a l (2 0 0 5 ) fo u n d th a t
2 5 0 m L /m in w a s a g o o d c o m p ro m is e b e tw e e n m ix in g tim e , b io c a ta ly s t
s e d im e n ta tio n , a n d th e m a x im u m im m o b iliz a tio n fo r y e a s t c e lls (a ro u n d
0 .6 g B I O M A S S /g C A R R I E R ). A s a t lo w e rs g a s flo w s S G s e d im e n ta tio n w a s
o b s e rv e d , th e g a s flo w o f 4 0 0 m L /m in w a s a ls o te s te d in o rd e r to s tu d y
g a s flo w e ffe c t. B e tw e e n th e tw o a x ia l s tu d ie d p o in ts (A a n d B in F ig u r e
V II- 1 2 ), n o s ta tis tic d iffe re n c e w a s fo u n d . B e in g s o a n d c o n s id e rin g
th a t th e e x p e rim e n ts u s in g th e H S C w e re m a d e a t th e a x ia l p o s itio n in
th e m id d le o f th e ris e r (B in F ig u re V II- 1 2 ) o n ly th e v a lu e s o b ta in e d
u s in g th e o p tic a l p ro b e a t th is p o s itio n a re p re s e n te d in th e re s u lts .
R a d ia l g a s-p h a s e p ro p e rtie s w a s a ls o a n a ly s e d fo r e a c h c o n d itio n
(a x ia l p o s itio n s , d iffe re n t g a s flo w s a n d s o lid lo a d s ) a s in d ic a te d in
F ig u r e V II- 1 2 . H o w e v e r it w a s v e rifie d th a t n o s ig n ific a n t d iffe re n c e s in
th e re s u lts w e re d e te c te d b e tw e e n th e ra d ia l p o s itio n s s tu d ie d . T h e n ,
fo r th e g a s -p h a s e a fla t re g im e in ra d ia l p o s itio n c a n b e c o n s ide re d . In
a d d itio n , th is w a s v a lid n o t o n ly in g a s h o ld-u p m e a s u re m e n ts , b u t a ls o
fo r b u b b le v e lo c ity a n d b u b b le c h o rd .
F ro m
F ig u re
V II- 1 5 ,
it
is
p o s s ib le
to
v e rify
th a t
gas
h o ld -u p
d e c re a s e s w h e n s o lid s a re p re s e n t. B y th e s ta tis tic a l a n a ly s is a n d fo r
198
Chapter VII
th e lo w e s t g a s flo w te s te d (2 5 0 m L /m in ), th e g a s h o ld -u p d e c re a s e is
o n ly s ig n ific a n t fo r s o lid s lo a d a b o v e 4 % (w t. W E T
BASIS
/v o l.). O n th e o th e r
h a n d , fo r th e h ig h e s t g a s flo w (4 0 0 m L /m in ) th e g a s h o ld -u p d e c re a s e
w h e n s o lid s a re a d d ed . H o w e v e r n o s ig n ific a n t d iffe re n c e o n g a s h o ldu p d e c re a s e s w a s n o te d a m o n g th e d iffe re n t s o lid s c o n c e n tra tio n s . S G
a re k n o w n to g e n e ra lly d e c re a s e th e g a s h o ld-u p a t h ig h g a s v e lo c itie s
in
BC
m a in ly
due
to
o c c u rre n c e
of
c o a le s c e n c e
(C h a p te r
IV ).
C o a le s c e n c e in c re a s e s b u b b le s iz e a n d v e lo c ity . In o u r c a s e th e c h o rd
s iz e a n d v e lo c ity in c re a s e d w ith s o lid lo a d . C h o rd s iz e in c re a s e is n o t
d ire c tly re la te d w ith b u b b le s iz e a s b u b b le p ro p e rtie s (s h a p e , s h e ric ity )
c a n a ls o c h a n g e . F ro m s ta tis tic a l a n a ly s is , it is c le a r th a t b u b b le c h o rd
in c re a s e d , w h ic h is a n in d ic a tio n fo r b u b b le c o a le s c e n c e to o c c u r.
S im ila r re s u lts w e re fo u n d w h e n a n id e n tic a l o p tic a l fib e r w a s u s e d a n d
te s te d w ith a lg in a te b e a d s (M e n a e t a l., 2 0 0 8 ). T o a c c e s s th e re a l
b u b b le s iz e d is trib u tio n , it is n e c e s s a ry to a s s u m e a s p e c ific flo w
s tru c tu re (M e n a e t a l., 2 0 0 8 ). M o re o v e r, to tra n s fo rm c h o rd in a c tu a l
d ia m e te r is n o t a n e a s y ta s k e v e n a t a n a rro w b u b b le c h o rd d is trib u tio n
fo r a lm o s t s p h e ric a l b u b b le s in h o m o g e ne o u s re g im e .
In a d d itio n th e g a s h o ld -u p re s u lts a t 4 0 0 m L /m in (0 .9 4 c m /s )
p re s e n te d
(4 % (w t. W E T
F ig u r e
in
BASIS
V II- 1 5
fo r
g -l
and
g -l-s
s y s te m s
/v o l.)) a n d th e re s u lts o b ta in e d fo r th e lo w e s t g a s
s u p e rfic ia l v e lo c ity (1 c m /s ) in C h a p te r V a re s im ila r (ra n g e o f g a s h o ldu p b e tw e e n 1 % (v o l./v o l.) a n d to 2 % (v o l./v o l.)). T h e s e re s u lts s h o w
a c c o rd a n c e b e tw e e n g lo b a l a n d lo c a l h y d ro d y n a m ic m e th o d s u s e d in
th is th e s is . E v e n a t d iffe re n t re a c to r s c a le s 6 0 L a n d 6 L in C h a p te r V
a n d V II re s p e c tiv e ly th is s im ila rity b e tw e e n th e re s u lts is in a c c o rd a n c e
w ith th e s c a le re s u lts o b ta in e d b y B la a n d c o-w o rk e rs (2 0 0 4 ). T h e y
w o rk e d w ith th re e iG L R (1 0 L , 3 2 L a n d 2 0 0 L ) a n d th e ir re s u lts
in d ic a te d th a t fo r g a s s u p e rfic ia l v e lo c itie s b e lo w 0 .1 5 m /s th e g a s h o ld
u p in ris e r o f iG L R is n o t in flu e n c e d b y th e in c re a s e o f re a c to r s c a le
(B la e t a l., 2 0 0 4 ).
199
Chapter VII
T o g e t a b e tte r u n d e rs ta n d in g o f th e u n d e rly in g p h y sic a l m e c h a n is m s

in v o lv e d in th e c h o rd s iz e a n d v e lo c ity in c re a s e , d a ta w e re
o b ta in e d
u s in g th e H S C .
A!
B!
2.20!
Gas Hold-up / % (vol./vol.)!
Gas Hold-up / % (vol./vol.)!
1.20!
1.00!
0.80!
1.80!
1.40!
1.00!
0.60!
0!
2!
4!
6!
8!
0!
2!
% SG (wt.WET BASIS/vol.)!
65.0!
65.0!
60.0!
60.0!
vB / (cm/s)!
vB / (cm/s)!
8!
B!
70.0!
55.0!
50.0!
55.0!
50.0!
45.0!
45.0!
40.0!
40.0!
35.0!
35.0!
0!
2!
4!
6!
8!
0!
2!
4!
6!
8!
A!
2.8!
B!
2.50!
2.5!
2.20!
2.2!
chB / mm!
chB / mm!
6!
A!
70.0!
4!
1.9!
1.6!
1.90!
1.60!
1.30!
1.3!
1.0!
1.00!
0!
2!
4!
6!
8!
0!
2!
4!
6!
8!
Figure VII-15. Solids influence in local gas-phase properties.

Legend: A QAIR= 250 ml/min; B QAIR= 400 ml/min; ! - Gas Hold-up
(% (vol./vol.)); " - Bubble Velocity (cm/s); # - Bubble Chord (mm).
200
Chapter VII
V is u a liz a tio n te c h n iq u e s a re v e ry u s e fu l in g -l a n d g -l-s s y s te m s

(B o y e r e t a l. 2 0 0 2 ; M e n a e t a l., 2 0 0 5 ; M a c e ira s e t a l., 2 0 1 0 ; F e rre ira e t
a l., 2 0 1 1 ). T h e o b ta in e d in fo rm a tio n m a y b e e ith e r q u a lita tiv e o r
q u a n tita tiv e . D e s p ite th e c o m m e rc ia l m e th o d s d e v e lo p e d to p e rfo rm
a u to m a tic im a g e a n a ly s is th e re is s till th e n e e d fo r th e o p e ra to r to d o a
s e m i-m a n u a l b u b b le c h a ra c te riz a tio n . T h is is m o re re le v a n t w h e n im a g e
a n a ly s is is a p p lie d to d e te rm in e b u b b le p ro p e rtie s in g -l-s s y s te m s .
W h e n s o lid s a re p re s e n t, it is p o s s ib le to u s e a u to m a tic m e th o d s if th e
s o lid s g re y le v e l is d iffe re n t fro m b u b b le s g re y le v e l (M e n a e t a l.,
2 0 0 5 b ). O th e rw is e th e s e m e th o d s c a n lo o s e a c c u ra c y d u e to s o lid s
p re s e n c e . In o u r c a s e th e s o lid s u s e d lim ite d th e im a g e s o b ta in e d b y
H S C b e c a u s e th e g re y le ve l o f S G is s im ila r to b u b b le s g re y le v e l. T h a t
m a d e m o re d iffic u lt to d is tin g u is h b e tw e e n b u b b le a n d p a rtic le (s e e
F ig u re V II- 1 6 ). E v e n th e m a n u a l d e te rm in a tio n o f b u b b le p ro p e rtie s
a b o v e 2 % (v o l./v o l.) w a s n o t p o s s ib le b e lo w th e re p o rte d e rro r. T h e
s ta tis tic a l to o l p ro p o s e d b y F e rre ira e t a l. (2 0 1 1 ) w a s u s e d , b u t o n ly to
a n a ly s e th e g -l s y s te m s a n d g -l-s s y s te m im a g e s a t lo w e s t g a s flo w a n d
s o lid c o n te n t (0 .8 % (v o l./v o l.)). B e in g s o , th e re s u lts w e re o b ta in e d
m a n u a lly a n d th e n c o m p a re d w ith th e p o s s ib le re s u lts o b ta in e d b y th e
s ta tis tic s to o l m e th o d .
T h e m a in a im o f u s in g H S C in th is w o rk w a s to ob ta in m o re d a ta th a t
c o n firm s
and
v a lid a te s
th e
re s u lts
o b ta in e d
fro m
o p tic a l
p ro b e
m e a s u re m e n ts . T o d o th is , H S C im a g e s a n d v id e o s w e re u s e d to
d e te rm in e d
b u b b le
v e lo c ity
and
b u b b le
c h o rd
(m a n u a lly ).
B u b b le
v e lo c ity w a s d e te rm in e d b y m a n u a lly tra c k in g b u bb le s b e tw e e n 2 0

fra m e s th a t c o rre s p o n d to a tim e in te rv a l o f 2 0 m illi-s e c o n d s . B u b b le
C h o rd w a s d e te rm in e d b y c o n s id e rin g a v irtu a l o p tic a l p ro b e p ie rc in g a
b u b b le in a p e rp e n d ic u la r p o s itio n (s e e im a g e b e lo w ).
In T a b le V II- 1 a re p re s e n te d th e d iffe re n c e s b e tw e e n H S C re s u lts
a n d O p tic a l P ro b e fo r th e lo w e s t g a s flo w a t 0 a n d 2 % (v o l./v o l.) s o lid
lo a d .
201
Chapter VII
Figure VII-16. HSC Images from GL reactors riser. White lines are the
simulated theoriacally piercing of a bubble and each length was used to
determined chord. Legend. A Air-Water; B Air-Water-SG
(0.8% (wt.WET BASIS/vol.)); B Air-Water-SG (2% (wt.WET BASIS /vol.));
In T a b le V II- 1 it is p o s s ib le to o b s e rv e th a t th e re s u lts o b ta in e d w ith

th e o p tic a l p ro b e a re in a c c o rd a n c e w ith th e o n e s o b ta in e d b y th e H S C .
G e n e ra lly , fo r v e lo c ity v a lu e s th e H S C re s u lts a re h ig h e r, w h ile fo r
b u b b le c h o rd th e s e o n ly o c c u r in g -l (a ir-w a te r). W h e n S G p a rtic le s a re
p re s e n t th e a n a ly s is o f th e im a g e s is m o re d iffic u lt (F ig u r e V II- 1 6 ), e v e n
u s in g th e m a n u a l m e th o d s a n d m a in ta in in g th e s a m e c rite ria . F o r th e s e
c a s e s , th e s o lid s n o is e in th e im a g e a ffe c ts th e id e n tific a tio n a n d
d e te rm in a tio n o f b u b b le bo u n d a ry , i. e ., th e in te rfa c e b e tw e e n g a s a n d
liq u id p h a s e s . (F ig u r e V II- 1 6 ). T h is m a y e x p la in th e lo w e s t b u b b le
c h o rd o b ta in e d b y H S C re s u lts .
Table VII-1.Results evaluation by OP and HSC for QAIR=250 mL/min.
Gas-phase
properties
Bubble Velocity
(cm/s)
Bubble Chord (mm)
Air-Water
Air-Water-2 %
SG (wt.WET BASIS/vol.)
HSC
OP
HSC
OP
48.45.7
44.37.5
66.95.9
64.85.0
1.70.5
1.30.3
2.00.3
2.10.4
F o r th e v e lo c ity d e te rm in a tio n us in g th e H S C , th e b u b b le re fe re n c e
p o in t u s e d w a s th e b u b b le c e n tra l p o in t, w h ic h is e a s ie r (le s s e rro r) to
a c c e s s e v e n w h e n s o lid s a re p re s e n t.
T h e m e a n S a u te r d ia m e te r a n d b u b b le s s p h e ric ity w e re d e te rm in e d
fo r a ll te s te d c o n d itio n s u s in g th e H S C (s e e M ate ria l a n d M e th o d s ). T h is
202
Chapter VII
w a s th e n c o m p a re d w ith th e re s u lts o b ta in e d b y th e s ta tis tic s to o l

m e th o d p ro p o s e d b y F e rre ira e t. a l (2 0 1 1 ). In T a b le V II- 2 th e re s u lts
show
th a t
th e
e rro r
b e tw e e n
Manual
Im a g e
A n a ly s is
(M IA )
and
A u to m a tic S ta tis tic s T o o l (A S T ) a n a ly s is is n o m o re th a n 6 % a n d 7 % fo r

D 3 2 a n d S p h e ric ity re s p e c tiv e ly .
Table VII-2. D32 (mm) and Sphericity determined by Manual Image Analysis
(MIA) and using the Automatic Statistics Tool (AST). Both were obtained from
the same images by the HSC.
%
Solids
QAIR
D32 (mm)
Sphericity
(mL/min) MIA AST
MIA
AST
250
3.82 3.86 0.599 0.564
0
400
3.96 3.90 0.598 0.586
250
3.62 3.43 0.588 0.563
0.1
400
3.86
NP
0.576
NP
250
3.80
NP
0.625
NP
0.25
400
3.72
NP
0.620
NP
Legend: NP Not possible to evaluate by the program due to bad image
quality.
In T a b le V II- 2 it is p o s s ib le to v e rify th a t fo r 2 5 0 m L /m in th e v a lu e o f
D 3 2 (fo r b u b b le ) is lo w e r w h e n s o lid s a re pre s e n t w ith a m in im u m a t
0 .8 % (v o l./v o l.), w h ile a t 4 0 0 m L /m in th e v a lu e s d e c re a s e d w ith s o lid
c o n te n t. H o w e v e r in te rm s o f b u b b le s p h e ric ity th e re is a s m a ll
d e c re a s e fo r 0 .8 % (v o l./v o l.) s o lid s a n d fo r 2 % (v o l./v o l.) a n in c re a s e is
n o te d . A t th is s o lid co n te n t th e s p h e ric ity is e v e n h ig h e r th a n th e o n e
o b s e rv e d in a ir-w a te r s y s te m . T h is re s u lt is in a c c o rd a n c e w ith th e
p re v io u s w h e re a n in c re a s e in c h o rd s iz e o c c u rs in th e p re s e n c e o f
s o lid s .
If
b u b b le s
a re
c o n s id e re d
as
fla tte n e d
o b le te n o id ,
th e
s p h e ric ity is d e te rm in e d b y th e ra tio b e tw e e n th e m in o r a n d m a jo r a x e s

(M e n a e t a l., 2 0 0 8 ). T h e re fo re th e in c re a s e in s p h e ric ity c a n b e d u e to :
in c re a s e o f m in o r a x e (w h ic h c o rre s p o n d to th e c h o rd m e a s u re d w ith
th e o p tic a l p ro b e ) o r th e d e c re a s e o f m a jo r a x is . In fa c t th e re s u lts fro m
H S C s h o w e d a n in c re a s e in m in o r a x is a c c o m p lis h e d b y a d e c re a s e o f
m a jo r a x is . G e n e ra lly , b u b b le s p h e ric ity in c re a s e s w ith g a s flo w a n d fo r
h ig h s o lid s lo a d (M e n a e t a l., 2 0 0 8 ). In c re a s in g s o lid c o n te n t o r g a s
b lo w s p ro m o te s b u b b le-b u b b le in te ra c tio n a n d c o a le s c e n c e , w h ic h
le a d s to b ig g e r a n d ro u n d e d b u b b le s . In o u r c a s e , s p h e ric ity in c re a s e d
a t lo w s o lid c o n te n t, w h ic h is c o n s is te n t w ith th e p re v io u s re s u lts
203
Chapter VII
o b ta in e d . T h e o b ta in e d re s u lts s u g g e s te d th a t o u r p a rtic le s p ro m o te
c o a le s c e n c e . B u b b le c o a le s c e n c e o c c u rs in th re e s te p s : (1 ) in itia l film
fo rm a tio n , (2 ) film th in n in g a n d fin a lly , (3 ) film ru p tu re . Z o n e t a l (2 0 0 2 )
s h o w e d th a t th e in c re a s e o f h y d ro p h o b ic p a rtic le s c o n c e n tra tio n le a d s
to
b u b b le
p o p u la tio n
s w ift
fro m
s m a ll
to
b ig g e r.
H y d ro p h o b ic
p a rtic le s a c t in g -l in te rfa c e re d u c in g th e e n e rg y b a rrie r to film ru p tu re

a n d c o n s e q u e n tly c oa le s c e n c e is p ro m o te d (Z o n e t a l., 2 0 0 2 ). O n th e
o th e r h a n d c o m p le te ly w e tta b le p a rtic le s w e re fo u n d to in c re a s e g a s
h o ld -u p (Z o n e t a l., 2 0 0 2 ). S G a re c o m p le te ly w e tta b le p a rtic le s ,
h o w e v e r th e ir s u rfa c e is m a in ly h y d ro p h o b ic (J a m ia la h m a d i a n d M u lle rS te in h a g e n , 1 9 9 1 ). B e in g s o , a n d c o n s id e rin g th e o b s e rv e d g a s h o ld-u p
d e c re a s e , it m e a n s th a t h y d ro p h o b ic p ro p e rtie s o f S G h a v e m o re
in flu e n c e in b u b b le p ro p e rtie s th a n th e ir w e tta b ility . T h u s c o a le s c e n c e
c a n o c c u r in g -l-s w h e re S G a re p re s e n t.
F o r a ll e x p e rim e n ts (g a s flo w s a n d s o lid s lo a d s ) b y a n a ly s in g F ig u r e
V II- 1 5 a n d F ig u r e V II- 1 6 a s w e ll a s T a b le V II- 1 a n d T a b le V II- 2 , it is
p o s s ib le to c o n c lu d e th a t o p tic a l p ro b e s re s u lts a re c o n s is te n t a n d
fe a s ib le . N e v e rth e le s s , it w a s n e c e s s a ry to c e rtify th e re s u lts , e n s u rin g
th a t o u r d a ta o b ta in e d b y th e o p tic a l p ro b e w e re in d e e d a c c u ra te . T o
d o th is , th e th e o re tic a l g a s flo w a c ro s s th e ris e r c ro s s-s e c tio n w a s
c a lc u la te d fro m th e b u b b le v e lo c ity re s u lts . In th e n e x t fig u re (F ig u r e
V II- 1 7 ) it is s h o w n th a t th e g a s flo w w a s u n d e re s tim a te d a t 4 0 0 m L /m in
w h ile fo r 2 5 0 m L /m in it is s h o w n th e re s u lts a re a ro u n d th e th e o re tic a l
QAIR DET / (mL/min)!
v a lu e .
400.0!
325.0!
250.0!
175.0!
100.0!
200! 250! 300! 350! 400! 450!
QAIR EXP. /
(mL/min)!
Figure VII-17. Expected gas flow (!) vs. Experimental gas flow ("and#).
204
Chapter VII
A s re fe rre d b e fo re th e o p tic a l p ro b e u n d e re s tim a tio n is re la te d w ith

b lin d in g a n d d riftin g e ffe c ts . In th e c a s e o f o u r iG L R it w a s p o s s ib le to
o b s e rv e d u rin g th e H S C m e a s u re m e n ts th a t s o m e b u b b le s w e re n o t
ra is in g v e rtic a lly a n d th is w a s m o re fre q u e n t w h e n s o lid s w e re p re s e n t.
T h is b e h a v io u r fro m b u b b le s is d e fin e d b y th e liq u id p ro p e rtie s , a s w e ll
a s b y th e re a c to r c o n fig u ra tio n . In th is p a rtic u la r iG L R th e re is a
d e g a s s in g to p p a rt w h e re th e re is a s e d im e n ta tio n b a rrie r th a t re d u c e s
c a rrie r lo s s e s d u rin g a c o n tin u o u s fe rm e n ta tio n . A s th e to p p a rt is n o t
e x a c tly s y m m e tric a l th e liq u id th a t g o e s d o w n th e d o w n c o m e r is n o t
u n ifo rm ly d is trib u te d th ro u g h a ll th e c ro s s s e c tio n . It m e a n s th a t th e
liq u id flo w is n o t e x a c tly th e s a m e in s o m e p a rts o f th e b o tto m -ris e r
c o n n e c tio n w h e re a s th e a m o u n t o f liq u id th a t g o e s in to th e ris e r is
h ig h e r in s o m e z o n e s p ro m o tin g b u b b le s p in a n d d riftin g . T h is h a s a n
e ffe c t in th e o p tic a l p ro b e a c c u ra c y to m e a s u re th e ris in g tim e (T U ).
M o re o v e r th e a c q u is itio n ra te (1 0 k H z ) u s e d w a s fiv e tim e s in fe rio r to
th e n o rm a lly o n e u s e d b y c o m m e rc ia l p ro g ra m w h e re th e T G V m e th o d
(C a rte llie r, 1 9 9 2 ) is a p p lie d fo r tw o -p h a s e flo w . In a d d itio n , c alib ra tio n
s tu d ie s a lre a d y in d ic a te d th a t a t 4 0 0 m L /m in th e e rro rs o b ta in e d to
m e a s u re b u b b le v e lo c ity in tw o-p h a s e flo w a re u n d e re s tim a te d b y 2 0 %
w h e n c o m p a rin g th e n e w w ith th e fo rm e r T G V m e th o d . A t 2 5 0 m L /m in
g a s flo w , th e n e w s y s te m o v e r e s tim a te d b u b b le v e lo c ity b y 1 0 %
(C a rte llie r, 1 9 9 2 ). T h is 1 0 % d ife re n c e is in s id e th e o p tic a l p ro b e e rro r
fo r tw o -p h a s e flo w s (C a rte llie r, 1 9 9 2 ). It is th e n p o s s ib le to c o n c lu d e
th a t th e s y s te m u s e d w a s s u c c e s s fu lly a p p lie d a t th e lo w e r g a s flo w
ra te
used
and
th e
re s u lts
can
be
e v a lu a te d
w ith
a c c u ra c y .
The
m e th o d /te c h n iq u e a p p lie d w a s h a n d-m a d e ; e s p e c ia lly th e in je c tio n

s y s te m , a n d a ll th is s y s te m c a n b e la rg e ly im p ro v e d . T h is im p ro v e m e n t
w ill a llo w fo r e x a m p le a s tro n g e r in je c tio n flu x a n d c o n s e q u e n tly a m o re
a c c u ra te m e a s u re a t h ig h e r g a s flo w s .
VII.4.2.1
Cell influence in gas properties of two-phase flow
T h e d riv in g fo rc e o f th is w o rk w a s to u n d e rs ta n d n o t o n ly th e
in flu e n c e o f S G in G L re a c to rs h y d ro d y n a m ic s b u t a ls o h o w it is
205
Chapter VII
a ffe c te d b y a fe rm e n ta tio n u s in g y e a s t c e lls : S . c e re v is ia e . B e in g s o , it

w a s n e c e s s a ry to e v a lu a te h o w th e y e a s t b y its e lf in flu e n c e s g a s-p h a s e
p ro p e rtie s . S tu d ie s w h e re in c re a s in g a m o u n ts o f a c o n c e n tra te d c e ll
s o lu tio n (0 .1 0 5 g /m L ) w e re a d d e d to a ir-w a te r s y s te m w e re p e rfo rm e d ,
re s u lts fro m o p tic a l p ro b e a n d H S C w e re ta k e n a n d e v a lu a te d . In F ig u r e
V II- 1 8 a re p re s e n te d th e in flu e n c e o f c e ll c o n c e n tra tio n in g a s-p h a s e
p ro p e rtie s . F ig u r e V II- 1 8 s h o w s th a t little c e ll c o n c e n tra tio n h a s a lo t
o f in flu e n c e in th e g a s -p h a s e p ro p e rtie s . G e n e ra lly it is o b s e rv e d a n
in c re a s e o f g a s h o ld -u p , b u b b le v e lo c ity a n d c h o rd s iz e . T h is in d ic a te s
th a t a m in im u m v o lu m e o f c e lls w a s a b le to in d u c e c h a n g e s in th e g a sp h a s e . H o w e v e r, b u b b le s p ro p e rtie s d id n o t c h a n g e s ig n ific a n tly a b o v e
th e lo w e r c e ll c o n c e n tra tio n , i. e ., g a s h o ld-u p , b u b b le v e lo c ity /c h o rd
d iffe re n c e a t h ig h c e ll c o n c e n tra tio n is s im ila r to th e v a lu e s o b ta in e d a t
lo w c e ll c o n c e n tra tio n .
In F ig u re V II- 1 8 it is p o s s ib le to o b s e rv e th a t a n in c re a s e o f g a s
h o ld -u p is a c c o m p lis h e d b y a n in c re a s e o f b u b b le c h o rd a n d v e lo c ity .
T h e s e re s u lts a re o d d b e c a u s e if g a s h o ld-u p in c re a s e s th e b u b b le
v e lo c ity w a s e x p e c te d to d e c re a s e a s w e ll a s b u b b le c h o rd . S m a lle r
b u b b le s ris e s lo w e r th u s re s id e n c e tim e is h ig h e r, w h ic h in c re a s e s th e
g a s h o ld -u p . O n e p o s s ib le e x p la n a tio n fo r th is p h e n o m e n o n is th e c e ll
in flu e n c e o n th e o p tic a l fib e r m e a s ure m e n t m e c h a n is m . T h is in flu e n c e
c a n b e : (1 ) g e n e ric in flu e n c e in a ll p a ra m e te rs (g a s h o ld -u p ; b u b b le
v e lo c ity
and
c h o rd );
(2 )
s p e c ific
e ffe c t
by
a ffe c tin g
o n ly
th e
m e a s u re m e n t o f b u b b le v e lo c ity a n d b u b b le c h o rd . In th e firs t c a s e , th e
o v e re s tim a tio n w a s d u e to c ra w lin g e ffe c t c a u s e d b y th e p re s e n c e o f
c e lls . C e lls m a y a c t o n th e liq u id p ro p e rtie s in flu e n c in g a ls o th e
re fle c tiv e in d e x o f th e liq u id a n d c o n s e q u e n tly T G a n d T M m e a s u re m e n ts
a re a ffe c te d le a d in g to w ro n g v a lu e s . T h e s e c o n d e x p la n a tio n ta k e s into
a c c o u n t th e fa c t th a t T U m e a s u re m e n t is m o re s e n s ib le to e rro rs th a n
T G . T h is is ju s tifie d c o n s id e rin g th e te n d e n c y to u n d e re s tim a te b u b b le
v e lo c ity fo r th is p ro b e (s e e F ig u re V II- 1 6 ). In th is c a s e , c e lls w ill a c t o n
liq u id -g a s in te rfa c e le a d in g to w ro n g T M m e a s u re m e n ts . A c c o rd in g to
B r n y ik e t a l. (2 0 0 4 ), y e a s t c e lls h y d ro p h o b ic ity in c re a s e s b y 3 0 % in
c o n tin u o u s s y s te m s w h e n th e b io film s ta rts to b e d e v e lo p e d (B r n y ik ,
206
Chapter VII
e t a l. 2 0 0 4 ). M o re o v e r, it is k n o w n th a t in B C re a c to rs , h y d ro p h o b ic
Gas hold-up / (% (vol./vol.))!
p a rtic le s p ro m o te c o a le s c e n c e (Z o n e t a l. 2 0 0 2 ).
2.00!
1.75!
1.50!
1.25!
1.00!
0.00!
0.10!
0.20!
0.30!
0.40!
0.50!
0.40!
0.50!
0.40!
0.50!
[Cells] / (g/L)!
vB / (cm/s)!
80.00!
70.00!
60.00!
50.00!
0.00!
0.10!
0.20!
0.30!
[Cells] / (g/L)!
chB / (mm)!
2.25!
2.00!
1.75!
1.50!
0.00!
0.10!
0.20!
0.30!
[Cells] / (g/L)!
Figure VII-18. Cell concentration influence in gas-phase properties (voidage,

bubble velocity and chord) on water-air system at 250 mL/min.
A c c o rd in g to F ig u r e V II- 1 8 , c o a le s c e n c e d o e s n o t o c c u r in o u r
s y s te m b e c a u s e n o g a s h o ld -u p d e c re a s e is o b s e rv e d . C o n s id e rin g th is ,
it is m o re lik e ly th a t c e lls in d u c e s o m e e rro r o n th e b u b b le-tip
207
Chapter VII
in te ra c tio n a n d th e s ig n a l o v e r e s tim a tio n in th e ir p re s e n c e is a n

im p o rta n t a s p e c t to c o n s id e r w h e n o p tic a l p ro b e is u s e d to d e te rm in e
g a s -p h a s e c h a ra c te ris tic s in fe rm e n ta tio n s b ro th . B e in g s o th e in c re a s e
o f g a s h o ld -u p , b u b b le v e lo c ity a n d b u b b le c h o rd w h e n in th e p re s e n c e
o f th e s e y e a s t c e lls w a s fo u n d to b e : 4 2 .7 4 3 .3 1 % , 3 6 .7 4 4 .4 2 % a n d
3 0 .2 6 3 .5 7 % re s p e c tiv e ly . F u rth e r s tu d ie s a re n e e d e d to h a v e a b e tte r
o f th is ty p e o f p h e n o m e n a a n d h o w b io m a s s c a n re a lly a ffe c t b u b b le
p ro p e rtie s (s h a p e , v e lo c ity , fre q u e n c y ). F u rth e r s tu d ie s u s ing H S C w e re
m a d e w h e n c e lls w e re p re s e n t. A s w h e n S G w h e re u s e d , o n ly a s m a ll
c o n c e n tra tio n o f c e lls w e re te s te d , b e c a u s e th e in c re a s e o f c e lls le a d s
to a n in c re a s e o f th e tu rb id ity w h e n th e y a re a d d e d to g -l s y s te m s .
0.60!
0.58!
3.8!
0.55!
3.6!
0.53!
3.4!
Sphericity / (-)!
DEQ or D32 / (mm)!
4.0!
0.50!
0.000! 0.050! 0.100! 0.150! 0.200! 0.250!

[Cells] / (g/L)!
Figure VII-19. The evolution of Sauter (D32), equivalent (DEQ) diameter and
sphericity with the yeast cells concentrations. Values obtained from the HSC
images. Legend: ! - DEQ (mm); " - D32 (mm); # - Sphericity (-).
T h is tu rb id ity w a s m e a s u red in te rm s o f c e ll a b s o rp tio n a n d th e

re la tio n b e tw e e n a b s o rp tio n a n d c e ll c o n c e n tra tio n w a s p lo tte d . F ig u r e
V II- 1 9 s h o w s th a t a s m a ll a m o u n t o f c e lls d o e s n o t h a v e s ig n ific a n t
in flu e n c e e ith e r o n s p h e ric ity , e q u iv a le n t o r S a u te r d ia m e te r. E v e n
th o u g h a s m a ll d e c re a s e is d e te c te d , th e re s u lts in d ic a te d th a t u s e d
y e a s t c e lls d id n o t h a v e a re a l in flu e n c e o n g a s-p h a s e p ro p e rtie s .
T h e re fo re th e v a ria tio n o b ta in e d b y th e o p tic a l p ro b e is m o re lik e ly to
be
due
to
yeast
c e ll
in flu e n c e
in
m e c h a n is m .
208
o p tic a l
p ro b e
m e a s u re m e n t
Chapter VII
VII.4.3 Liquid-phase measurements

T h e P IV te c h n iq u e u s e d a llo w s u s n o t o n ly to d e te rm in e th e a v e ra g e
liq u id v e lo c ity in th e d o w n c o m e r fo r d iffe re n t s o lid lo a d , b u t a ls o to
o b ta in th e liq u id v e lo c ity p ro file .
In F ig u r e V II- 2 0 A /B it is p o s s ib le to o b s e rv e th a t th e liq u id v e lo c ity
p ro file in th e d o w n c o m e r is h ig h e r a t c e n tra l p o s itio n b e tw e e n ris e r a n d
d o w n c o m e r th a n n e a r th e c o lu m n w a ll (r= 1 .5 c m ). T h e s e re s u lts w e re
o n ly o b ta in e d in th e o u te r p a rt o f th e d o w n c o m e r, b u t th e p ro file in th e
in n e r p a rt s h o u ld h a v e s im ila r b e h a v io u r. If w e c o n s id e r th e d im e n s io n s
o f th e g a s -lift re a c to r, in tw o -p h a s e flo w (a ir-w a te r) th e th e o re tic a l flo w
re g im e is tra n s ie n t (R e < 2 3 0 0 ) fo r liq u id v e lo c itie s in d o w n c o m e r b e lo w
8 c m /s . A d d in g s o lid s , a c c o rd in g to th e e q u a tio n s p ro p o s e d b y L u e t a l.
(1 9 9 5 ) fo r p s e u d o -h o m o g e n e o u s p h a s e , th e a p p a re n t v is c o s ity a n d
d e n s ity w ill in c re a s e . F o r th e s e tw o p ro p e rtie s th e a p p a re n t v is c o s ity
in c re a s e is h ig h e r th a n th e a p p a re n t d e n s ity in c re a s e . A c c o rd in g to
R e y n o ld s (R e ) e q u a tio n (E q . V II- 1 5 ), if th e fo rm e r p h e n o m e n o n o c c u rs
th e n th e R e n u m b e r w ill d e c re a s e a n d th e o re tic a lly th e s o lid s a d d e d w ill
h e lp to s ta b iliz e th e la m in a r flo w o f th e p s e u d o-h o m o g e n e o u s p h a s e in
th e d o w n c o m e r s e c tio n . C o n s id e rin g th e la m in a r re g im e s ta b iliz a to n b y
SG,
it
is
fe a s ib le
to
a d m it
tra n s ie n t
p ro file
of
th e
p s e u d o-
h o m o g e n e o u s p h a s e in th e d o w n c o m e r. In F ig u r e V II- 2 0 C /D it is s h o w n
th a t fo r b o th g a s flo w s th e in flu e n c e o f S G in th e a v e ra g e liq u id v e lo c ity
in th e d o w n c o m e r is n e g lig ib le a t 4 0 0 m L /m in . N e v e rth e le s s a t 2 5 0
m L /m in , a s m a ll d e c re a s e in liq u id v e lo c ity is p re s e n t. T h e v a lu e s
p re s e n te d in F ig u r e V II- 2 0 C /D a re o b ta in e d b y a v e ra g in g th e tw o
c e n tra l
ra d ia l
p o in ts
(F ig u re
V II- 2 0 A /B ).
The
re s u lts
in
F ig u re
V II- 2 0 C /D a re a m e re in d ic a tio n th a t s o lid s in c re a s e s ta b iliz e d th e flo w

in d o w n c o m e r, w h ic h m e a n s th a t th e tu rb u le n c e d e c re a s e s . In a lim it
s itu a tio n , if s o lid s a m o u n t is s ig n ific a n tly in c re a s e d , n o c irc u la tio n
w o u ld b e o b s e rv e d b e c a u s e th e g a s flo w w o u ld n o t b e e n o u g h to
flu id iz e a ll s o lid s . T h is is in a c c o rd a n c e w ith re s u lts o b ta in e d b y o th e r
a u th o rs (F re ita s , 2 0 0 2 ).
209
Chapter VII
A!
B!
12.0!
12.0!
vL / (cm/s)!
15.0!
vL / (cm/s)!
15.0!
9.0!
6.0!
9.0!
6.0!
3.0!
3.0!
0.0!
0.0!
0.60!
0.85!
1.10!
1.35!
1.60!
0.60!
Downcomer Radial Position /

(mm)!
1.10!
1.35!
1.60!
Downcomer Radial Position /

(mm)!
C!
12.0!
0.85!
D!
300.0!
10.0!
QLd / (cm3/s)!
VLd / (cm/s)!
275.0!
8.0!
250.0!
225.0!
200.0!
175.0!
6.0!
0.00!
5.00!
0.00!
10.00!
5.00!
10.00!
Figure VII-20. Study of liquid velocity in the downcomer. vs radial profile (A

QAIR=250 mL/min; B QAIR=400 mL/min) and the influence of SG
concentration in average liquid velocity (C) and liquid flow (D) in the
downcomer. Legend: ! - 0% (wt.WET BASIS/vol.) SG;
" - 2% (wt.WET BASIS/vol.) SG; ! - 4% (wt.WET BASIS/vol.) SG;
# - 6% (wt.WET BASIS/vol.) SG; $ - QAIR=250 mL/min; % - QAIR=400 mL/min.
The
SG
e ffe c t
in
p s e u d o-h o m o g e n e o u s
phase
is
a ffe c te d b y th e S G p ro p e rtie s (d e n s ity , w e tta b ility , h y d ro p h o b ic ity ),

w h ic h in d u c e s a s m a ll d ra g fo rc e w h e n v L d is h ig h e r th a n 1 0 c m /s a s it
o c c u rs a t 4 0 0 m L /m in . B y th e v a lu e s fro m lin e a r liq u id v e lo c ity in
d o w n c o m e r, is p o s s ib le to o b ta in e d th e lin e a r v e lo c ity in ris e r, a s w e ll
a s th e s u p e rfic ia l v e lo c ity u s in g E q . V II- 2 0 a n d V I- 2 1 (F re ita s , 2 0 0 2 ). In
210
Chapter VII
T a b le V III- 3 a re p re s e n t th e s e v a lu e s th a t w ill b e u s e d in th e m o d e llin g

s e c tio n .
Table VII-3. Values of riser linear and superficial velocities calculated from
equations 9 and 10 for all studied situations.
Qgi
(mL/min)
250
400
Qgi
(mL/min)
250
400
eSd %
vLd /
eGd %
eSd %
ULd /
A /A
(wt./vol.) (cm/s) (vol./vol.) (vol./vol.) (cm/s) d r
0.00
9.1
0.0
0.0
9.1
3.7
0.25
9.0
0.0
2.0
8.8
3.7
0.50
8.6
0.0
3.9
8.2
3.7
0.75
8.1
0.0
5.9
7.6
3.7
0.00
10.7
0.0
0.0
10.6
3.7
0.25
10.1
0.0
2.0
9.9
3.7
0.50
10.1
0.0
3.9
9.7
3.7
0.75
10.7
0.0
5.9
10.1
3.7
eSd %
(wt./vol.)
Ad/Ar
ULr /
(cm/s)
0.00
3.7
33.3
1.1
33.7
3.7
0.25
3.7
32.5
1.0
33.5
3.7
0.50
3.7
30.2
0.9
31.8
3.7
0.75
3.7
28.0
0.9
30.0
3.7
0.00
3.7
38.8
2.9
39.6
3.7
0.25
3.7
36.5
1.1
37.6
3.7
0.50
3.7
35.6
09
37.3
3.7
0.75
3.7
36.9
0.7
39.5
3.7
eGr %
vLr /
A /A
(vol./vol.) (cm/s) d r
VII.4.4 Solids-phase measurements

T o c o m p le te th e a n a ly s is o f th e th re e-p h a s e s y s te m , it w a s a ls o
im p o rta n t to s tu d y a n d a n a ly s e th e s o lid-p h a s e (S G ) b e h a v io u r. A
p re v io u s s tu d y s h o w s th a t, fo r th is ris e r le n g th , th e s o lid s d is trib u tio n
is a lm o s t c o n s ta n t e v e n a t lo w g a s flo w s (C h a p te r V ). S o it w a s
c o n s id e re d
in
th is
w o rk
th a t
s o lid s
h o ld-u p
and
d is trib u tio n
is
h o m o g e n e o u s th ro u g h o u t th e e n tire iG L R .
In th is c a s e , th e s tu d y w a s fo c u s e d o n th e d o w n c o m e r a n d u s in g th e
im a g e s fro m H S C , th e a v e ra g e s o lid v e lo c ity in th e d o w n c o m e r w a s
m a n u a lly d e te rm in e d . T h e re s u lts a re p re s e n te d in F ig u re V II- 2 1 .
211
Chapter VII
VSd / (cm/s)!
12.0!
11.0!
10.0!
9.0!
8.0!
200! 250! 300! 350! 400! 450!
QAIR / (mL/min)!
Figure VII-21. Solids Velocity (cm/s) in downcomer versus the air flow rate.
The values were obtained by HSC videos and determined manually. The SG
load was 2% (wt.WET BASIS/vol.).
A s e x p e c te d a n d a s fo r th e liq u id-p h a s e th e s o lid s v e lo c ity in c re a s e d

w ith th e g a s -flo w in c re a s e . It is in te re s tin g to v e rify th a t th is in c re a se is
s im ila r to th e in c re a s e th a t o c c u rs in liq u id p h a s e , w h ic h in d ic a te s th a t
th e a s s u m p tio n o f a p s e u d o-h o m o g e n e o u s p h a s e c a n b e u s e d in o rd e r
to s im p lify th e a p p lic a tio n o f h y d ro d y n a m ic m o d e ls in th e th re e-p h a s e
re a c to r.
H a v in g in m in d a ll th e te c h n iq u es a p p lie d in th is w o rk , it w a s
p o s s ib le to d e te rm in e th e a v e ra g e v e lo c ity o f liq u id- a n d s o lid -p h a s e in
th e iG L R s d o w n c o m e r. W ith th e s e v a lu e s it w a s p o s s ib le to d e te rm in e
b y c a lc u la tio n th e a v e ra g e v e lo c ity o f th e d iffe re n t p h a s e s in th e ris e r
m a in ly th e liq u id a n d th e p s e u d o-h o m o g e n e o u s p h a s e s . In T a b le V II- 4
a re in d ic a te d th e s e v a lu e s . T h e p s e u d o-h o m o g e n e o u s p h a s e v e lo c ity
w a s d e te rm in e d c o n s id e rin g th e e q u a tio n :
v Hd = v Ld ! (1" # Sd ) + v Sd ! # Sd
Eq. VII24
T h is e q u a tio n (E q . V II 2 4 ) m e a n s th a t b o th liq u id a n d s o lid -p h a s e

h a v e in flu e n c e in th e a v e ra g e v e lo c ity o f p s e u d o-h o m o g e n e o u s flo w .
L o o k in g a t T a b le V II- 4 , it is p o s s ib le to v e rify th a t th e p s e u d oh o m o g e n e o u s v e lo c ity is s im ila r to th e liq u id v e lo c ity a t b o th g a s flo w s .
T h is is b e c a u s e th e a m o u n t o f liq u id is a b o u t 9 8 % (v o l./v o l.). H o w e v e r it
212
Chapter VII
is a ls o in te re s tin g to n o te th a t s o lid s v e lo c ity a re 1 .3 % a n d 9 .8 % h ig h e r

th a n liq u id v e lo c ity in th e d o w n c o m e r fo r 2 5 0 m L /m in a n d 4 0 0 m L /m in
M o re o v e r,
th e
p s e u d o-h o m o g e n e o u s
v e lo c itie s
in
d o w n c o m e r a re 0 .0 2 % a n d 0 .2 % h ig h e r th a n liq u id v e lo c ity fo r th e
re fe rre d g a s flo w s . T h is re s u lt v a lid a te s th e a p p lic a tio n o f a p s e u d oh o m o g e n e o u s p h a s e to b e u s e d la te r in h y d ro d y n a m ic m o d e ls . T h e ris e r
v e lo c itie s o f liq u id a n d p s e u d o-h o m o g e n e o u s p h a s e s w e re d e te rm in e d
a c c o rd in g th e fo llo w in g e q u a tio n s , re s p e c tiv e ly (C h is ti 1 9 8 9 ; F re ita s
a n d T e ix e ira , 1 9 9 8 ):
" A % 1( )Gd ( ) Sd
v Lr = v Ld ! $ d ' !
# Ar & 1( )Gr ( ) Sr
Eq. VII25
" A % 1( ) Gd
v Hr = v Hd ! $ d ' !
# Ar & 1( ) Gr
Eq. VII26
F in a lly in T a b le V II- 4 a re p re s e n te d th e v a lu e s o f g a s v e lo c ity re la tiv e

to
th e
p s e u d o -h o m o g e n e o u s
phase.
T h is
v a lu e
was
c a lc u la te d
a c c o rd in g to th e re la tiv e v e lo c ity e q u a tio n b e tw e e n th e s e tw o p h a s e s .

A s th e y flo w b o th u p w a rd s th e e qu a tio n fo r s lip v e lo c ity in ris e r is :
Uslip,r = vGr + v Hr
Eq. VII27
T h e re s u lts p re s e n te d in T a b le V II- 4 a llo w u s to a ls o d e te rm in e ,

a c c o rd in g to E q . V 5 th e c irc u la tio n tim e o f th e g a s-lift re a c to r, w h ic h
is 9 .0 0 s a n d 8 .0 0 s fo r 2 5 0 m L /m in a n d 4 0 0 m L /m in re s p e c tiv e ly .
C o n s id e rin g th e p re v io u s ly d e te rm in e d m ix in g tim e o f 2 2 .6 a n d 1 2 .7 6 s ,
it is p o s s ib le to e s ta b lis h a re la tio n b e tw e e n T c a n d T m fo r th e s e g a s
flo w s .
A c c o rd in g
to
M o lin a
et
a l.
(1 9 9 9 )
tm
is
a ffe c te d
by
tw o
m e c h a n is m s . T h e d riv in g fo rc e is th e re la tiv e v e lo c ity b e tw e e n g a s a n d

liq u id in th e ris e r th a t p ro m o te s a x ia l m ix in g , a s th e g a s tra n s p o rts th e
liq u id u p w a rd s . A ls o th e d is ta n c e in w h ic h th e fo rm e r m e c h a n is m
o c c u rs (tw ic e th e d is p e rs io n d is ta n c e ) d ire c tly a ffe c ts th e m ix in g tim e .
T h e e q u a tio n p ro p o s e d b y M o lin a e t a l. (1 9 9 9 ) fo r t m d e te rm in a tio n is :
" U ! U Lr %
t m = 5.03$ Gr
'
# 2hD &
!0.797
Eq. VII28
213
Chapter VII
In T a b le V II- 4 it is p o s s ib le to o b s e rv e th a t E q . V II 2 8 is w e ll
a d ju s te d to th e d a ta o b ta in e d a t 2 5 0 m L /m in , b u t th e re s u lt is v e ry
d iffe re n t fo r th e o th e r g a s flo w (4 0 0 m L /m in ). T h e m a in re a s o n fo r th a t
is th e re la tiv e v e lo c ity in th e ris e r. A s th e g a s flo w is a lm o s t d o u b le ,
a lth o u g h th e p s e u d o -h o m o g e n e o u s p h a s e v e lo c ity d o e s n o t in c re a s e
to o m u c h , th e g a s v e lo c ity s h o u ld b e h ig h e r. T h u s w ith h ig h e r g a s flo w
it w a s e x p e c te d to in c re a s e th e re la tiv e v e lo c ity . A s s u c h d id n o t o c c u r,
th e m ix in g tim e d e te rm in e d b y E q . V II- 2 8 w a s o v e re s tim a te d w h e n
c o m p a re d w ith th e m ix in g tim e fro m e x p e rim e n ta l re s u lts . T h is re s u lt is
in
a g re e m e n t
w ith
th e
p re v io u s
re s u lts
w h e re
th e
gas
flo w
was
u n d e re s tim a te d a t 4 0 0 m L /m in . In fa c t it c o n firm s th a t th e lo w e r
a c q u is itio n ra te d id n o t a llo w a c o rre c t e v a lu a tio n fro m o p tic a l p ro b e o f
T U v a lu e s a t th is c o n d itio n . T h a t e v e n tu a lly le d to a n u n d e re s tim a tio n o f
v B a n d c o n s e q u e n tly U G r a n d Q G (p re v io u s ly re p o rte d ). In a d d itio n , if w e
u s e th e th e o re tic a l g a s lin e a r v e lo c ity (a ro u n d 9 4 c m /s ) b a s e d o n ly o n
th e g a s h o ld -u p a t th is c o n d itio n (2 % S G (v o l./v o l.)) a n d g a s flo w
(4 0 0 m L /m in ) th e e s tim a te d m ix in g tim e w ill b e 1 2 .1 9 s w h ic h is 4 %
lo w e r th a n th e d e te rm in e d e x p e rim e n ta lly .
Table VII-4. Velocity values of different phases in different zones of GL
reactor for 2%(vol./vol.) of solids.
Parameter
Gas Flow
250
400
mL/min
mL/min
9.0
10.1
9.1
11.1
9.0
10.2
33.4
37.6
33.4
37.7
97.5
103.9
9.00
8.0
22.3
12.8
19.9
21.1
10.7%
65.3%
vLd /(cm/s)*
vSd /(cm/s)**
vHd /(cm/s)#
vLr /(cm/s)#
vHr /(cm/s)#
Uslip_r /(cm/s)#
tC+ / (s)
tm ++/ (s)
tmTEO+/ (s)
Error
* Determined by PIV
** Determined manually using HSC videos
# Calculated value using equations VII-26, VII-27, VII-28 and VII-29.
+ Calculated using Equation VI-5
++ Determined by injection tracer technique using H2SO4
214
Chapter VII
VII.4.5 Model optimization results

T h e m o d e l u s e d it w a s a m u lti-p a ra m e te r m o d e l w e re th e o p tim iz a tio n
p a ra m e te rs (C , U b t , ! ) w e re a d ju s te d to th e e x p e rim e n ta l d a ta . In F ig u re
V II- 2 2 it is p re s e n te d th e re la tio n s b e tw e e n th e re s u lts o b ta in e d fro m
e x p e rim e n ta l d a ta a n d th e m o d e l. G e n e ra lly th e g a s h o ld-u p a n d
s u p e rfic ia l liq u id v e lo c ity in th e ris e r w ere in a g re e m e n t a n d e rro rs a re
w ith in 2 0 % fo r g a s h o ld -u p a n d 1 0 % fo r liq u id s u p e rfic ia l v e lo c ity .
T h e s e e rro rs a re c o n s id e re d to b e a c c e p ta b le a n d in a c c o rd a n c e w ith
th e e rro rs o b ta in e d b y F re ita s (2 0 0 2 ). It is a ls o im p o rta n t to c o n s id e r
th e e x p e rim e n ta l e rro rs fro m o p tic a l p ro b e n e w m e th o d p lu s P IV
m e th o d .
20%
0.02
UlrMODEL / (m/s)
eMODEL / (vol./vol.)
10%
0.60
0.03
0.01
0.00
0.40
0.20
0.00
0.00
0.01
0.02
0.03
0.00
0.20
0.40
0.60
UlrEXP/ (m/s )
eEXP/ (vol./vol.)
Figure VII-22. Modelization Values versus Experimental results. Legend: ! 250 mL/min; ! - 400 mL/min.
In T a b le V II- 5 it c a n b e fo u n d th e re s u lts o f m o d e l p a ra m e te rs a fte r

o p tim iz a tio n fo r e a c h s o lid c o n d itio n . T h e d is trib u tio n p a ra m e te r (C )
w a s n e a r u n ity a s e x p e c te d (F re ita s e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ).
H o w e v e r, th is d o e s n o t m e a n th a t p lu g flo w p re v a ils b e c a u s e th e
m a g n itu d e o f C is re la te d w ith th e g a s h o ld-u p d is trib u tio n ra th e r th a n
p h a s e v e lo c ity p ro file (F re ita s e t a l., 1 9 9 9 ; L u e t a l., 1 9 9 5 ). T h is is c a n
b e c o n firm e d b y th e o p tic a l p ro b e re s u lts a b o v e w h e re n o s ig n ific a n t
d iffe re n c e s w e re fo u n d in ra d ia l p o s itio n .
215
Chapter VII
C o n s id e rin g th e ! p a ra m e te r a n d fo r a ir-w a te r s y s te m , it is p o s s ib le
to d e te rm in e a v a lu e o f " = 1 .3 7 (E q . V II 1 6 ), w h ic h is s im ila r to th e
p ro p o s e d v a lu e fo r " (" = 2 ) (G a rc ia -C a lv o a n d L e t n ) fo r tw o -p h a s e
s y s te m . H o w e v e r, w h e n S G a re a d d e d " v a lu e s d e c re a s e . T h is d e c re a s e
c a n b e a ttrib u te d to th e h y d ro p h o b ic p ro p e rtie s o f S G th a t a c t in th e g -l
in te rfa c e .
Table VII-5. Parameters obtained after optimization.
% Solids
(vol./vol.)
Ubt
Error
0.00
0.897
0.014
0.160
14.65%
2.00
0.999
0.008
0.406
18.74%
3.90
1.010
0.006
0.714
23.66%
5.90
1.065
0.005
0.632
21.83%
G e n e ra lly U b t in c re a s e s w ith s o lid lo a d w h ic h is in a g re e m e n t, a n d in

th e s a m e ra n g e , w ith th e re s u lts o b ta in e d b y o th e r a u th o rs (F re ita s e t
a l., 1 9 9 9 ). U b t is th e b u b b le te rm in a l v e lo c ity in th e d rift-flu x m o d e l a n d
it
is
assumed
th a t
b u b b le s
do
not
in te ra c t
or
a re
a ffe c te d
by
n e ig h b o u rin g b u b b le s . M o re o v e r th e n o rm a l v a lu e s fo r U b t a re b e tw e e n
0 .2 m /s a n d 0 .4 5 m /s (L u e t a l., 1 9 9 5 ). T h e h ig h e r v a lu e s o b ta in e d c a n
b e a c lu e th a t e x p la in s c o a le s c e n c e a s b ig g e r b u b b le s ris e fa s t th e n
th e ir te rm in a l v e lo c ity . F re ita s e t a l (2 0 0 2 ) c o n sid e re d th a t th e ris e r
s m a ll
c ro s s
s e c tio n
a re a
was
th e
fa c to r
th a t
in c re a s e s
b u b b le
in te ra c tio n a n d c a u s e th e fo rm a tio n o f b u b b le s ris in g w ith h ig h e r s p e e d

in th e ris e r c e n tre (L u e t a l., 1 9 9 5 ). T h is e x p la n a tio n is c o n s is te n t w ith
th e o c c u rre n c e of c o a le s c e n c e in g -l-s s y s te m .
VII.4.6 Fermentation results

In th is c h a p te r o u r o b je c tiv e w a s a ls o to d o a n e x p e rim e n t th a t a llo w
us
to
s tu d y
how
th e
b io m a s s
(fre e
im m o b iliz e d )
can
a ffe c t
h y d ro d y n a m ic b e h a v io u r o f th e b io re a c to r a t lo c a l s c a le a fte r th e s ta rtu p s ta g e . In o rd e r to d o th a t, it w a s p e rfo rm e d a fe rm e n ta tio n th a t

s im u la te s th e in itia l s ta g e o f a c o n tin u o u s p rim a ry A F B fe rm e n ta tio n .
216
Chapter VII
P re v io u s s tu d ie s re p o rte d th a t th is s ta g e c a n ta k e fro m s e v e n to tw e lv e
d a y s a t d ilu tio n ra te o f 0 .2 h - 1 a n d 0 .1 5 h - 1 , re s p e c tiv e ly (B r n y ik , e t a l.
2 0 0 4 ). In o u r c a s e , th e d ilu tio n ra te w a s b e tw e e n 0 .0 5 h - 1 a n d 0 .0 9 h - 1
a n d th e tim e to a c h ie v e th e s ta b iliz a tio n o f iB io w a s a b o u t 1 3 d a y s . It is
p o s s ib le to o b s e rv e th a t th e te m p e ra tu re w a s h ig h e r th a n n o rm a lly
re p o rte d w h ic h , e v e n a t lo w e r liq u id flo w , le a d to a re d u c tio n in th e
tim e
fo r
b io film
s ta b iliz a tio n
in
th e
c a rrie r
(T a b le
V II- 6 ).
The
s ta b iliz a tio n o f iB io o c c u rre d a ro u n d th e 8 t h d a y .

In T a b le V II- 6 a re p re s e n te d th e re s u lts fro m th e fin a l d a y o f
fe rm e n ta tio n . A t th a t tim e , g a s-p h a s e p ro p e rtie s w e re a ls o m e a s u re d in
o rd e r to h a v e a c lu e if y e a s t c e lls (fre e + im m o b iliz e d ) in flu e n c e G L
re a c to r h y d ro d y n a m ic s . A t 1 3 t h d a y o f fe rm e n ta tio n th e to ta l a m o u n t o f
b io m a s s w a s a b o u t 1 4 .7 g /L b e in g iB io o n ly 1 0 % o f to ta l y e a s t b io m a s s
p re s e n t. T h e fin a l S G c o n c e n tra tio n le a d s to a s o lid h o ld-u p n e a r
4 % (w t. W E T
BASIS
/v o l.). T h e re s u lts a fte r fe rm e n ta tio n s h o w e d fo r a s im ila r
s o lid c o n c e n tra tio n th a t g a s h o ld -u p in c re a s e d u p to 2 2 % (fo r c o rre c te d

v a lu e ). B u b b le v e lo c ity d e c re a s e d b y 2 % in d ic a tin g th a t y e a s t c e lls
h a v e m o re in flu e n c e in G L b io re a c to r h y d ro d y n a m ic th a n S G . It is
re p o rte d
th a t
yeast
c e lls
(fre e
and
im m o b iliz e d )
become
m o re
h y d ro p h o b ic w h e n e x p o n e n tia l g ro w th s ta rts in th e c a rrie r. H o w e v e r

d u rin g th e fe rm e n ta tio n e th a n o l is p ro d u c e d a n d , a s it is a c o a le s c e n c e
in h ib ito r, th e in c re a s e o f g a s h o ld-u p o c c u rs . K e n n a rd e t J a n e k e h
(1 9 9 1 ) fo u n d th a t fro m 0 g /L to 2 g /L o f e th a n o l in s o lu tio n b u b b le
d ia m e te r
d e c re a s e s
s ig n ific a n tly
and
gas
h o ld-u p
in c re a s e s . F o r e th a n o l c o n c e n tra tio n a b o v e 2 g /L th is e ffe c t w a s n o t

n o te d (K e n n a rd a n d J a n e k e h , 1 9 9 1 ). Z a h ra d n ik e t a l. (1 9 9 7 ) fo u n d th a t
lim it e th a n o l c o n c e n tra tio n w a s a ro u n d 5 .0 6 g /L in a c c o rd a n c e w ith
O o lm a n e t B la n c h (1 9 8 6 ) th a t s u g g e s te d a v a lu e o f 6 .4 8 g /L (K e n n a rd
a n d J a n e k e h , 1 9 9 1 ). In B C re a c to r a t th e m a x im u m v a lu e o f e th a n o l
c o n c e n tra tio n d e fin ed b y Z a h ra d n ik e t a l. (1 9 9 7 ) th e g a s h o ld-u p m a y
in c re a s e b y 2 0 % . A F B c a n c o n ta in u p to 0 .5 % (v o l./v o l.) o f e th a n o l
(a ro u n d 3 .9 g /L ). In o u r c a s e e th a n o l a t 1 3 t h d a y h a d a c o n c e n tra tio n o f
6 g /L a b o v e a ll lim it c o n c e n tra tio n d e fin e d in lite ra tu re , s o th e 2 0 % g a s
h o ld -u p in c re a s e o b s e rv e d c a n b e c o n s id e re d d u e to e th a n o l p re s e n c e .
217
Chapter VII
M o re o v e r th is fa c t c o n firm s th a t th e in flu e n c e o f y e a s t c e lls in o p tic a l

p ro b e m e a su re m e n t is re a l. T h is w a s o u t o f th e s c o p e in th is th e s is ,
h o w e v e r fu rth e r s tu d ie s s h o u ld b e d o n e to a c c e s s th e re a l in flu e n c e o f
y e a s t c e lls in o p tic a l p ro b e m e a s u re m e n ts .
C o n s id e rin g
SG
as
c a rrie r
fo r
yeast
c e lls
in
c o n tin u o u s
fe rm e n ta tio n s s e e m s id e al fo r im m o b iliz a tio n , n o t o n ly d u e to th e ir h ig h

c e lls lo a d c a p a c ity , b u t a ls o b e c a u s e e v e n if th e y in flu e n c e th e iG L R
h y d ro d y n a m ic s
th is
has
little
e ffe c t
when
c o m p a re d
by
th e
m ic ro o rg a n is m s e ffe c t m a in ly if c o m p o u n d s w h ic h a r k n o w n to h a v e
h y d ro d y n a m ic in flu e n c e s u c h a s e th a n o l a re p ro d u c e d .
Table VII-6. Fermentation and Gas-phase properties obtained at last day of
fermentation.
Day
13
Free Cells /
(g/L)
13.1
iBio
(gBio/gCarrier)
0.3
WCARRIER /
g
27.7
eS
% (wt.WET BASIS/vol.)
eG /
vB / (cm/s)
chB / mm
% (vol./vol.)
1.90.1
97.226.0
5.73.4
4
(1.10.1)*
(61.59.5)*
(3.91.0)*
* values corrected considering free cells influence in Optical probe measuring
mechanism.
M o re o v e r a t th is im m o b iliz a tio n le v e ls a lm o s t a ll S G s u rfa c e is

c o lo n iz e d b y y e a s t c e lls , w h ic h h a v e d iffe re n t s u rfa c e p ro p e rtie s a n d
lo w e r h y d ro p h o b ic p ro p e rtie s . T h e n th e n e g a tiv e e ffe c t o f S G is a ls o
re d u c e d b y th e y e a s t c e lls th a t a re im m o b iliz e d o n th e c a rrie r (B r n y ik
e t a l., 2 0 0 4 ). O v e ra ll, m o re in v e s tig a tio n s e e m s to b e n e c e s s a ry to
re a lly a c c e s s b io m a s s in flu e n c e o n g a s-lift b io re a c to rs h y d ro d y n a m ic s .
VII.5 Conclusions
A t th e e n d o f th is C h a p te r it w a s p o s s ib le to s tu d y a n d b e tte r
u n d e rs ta n d th e S G e ffe c t o n g a s-lift b io re a c to r h y d ro d y n a m ic s , w ith
a n d w ith o u t a c tiv e y e a s t c e lls . C o n s id e rin g o n ly th e g -l-s s y s te m
c o m p o s e d b y A ir-W a te r-S G , it is c le a r th a t S G d u e to th e ir p a rtic u la r
s u rfa c e p ro p e rtie s d e c re a s e g a s h o ld-u p in c re a s in g b u b b le v e lo c ity a n d
218
Chapter VII
b u b b le c h o rd . H o w e v e r th o s e re s u lts a re m o re re lia b le fo r th e lo w e s t
g a s flo w te s te d (2 5 0 m L /m in ) a s a t th e h ig h e s t g a s flo w th e n e w
in je c tio n s y s te m u s e d (a p p lie d in th e o p tic a l p ro b e ) s e e m e d n o t to b e
e n o u g h fo r a c o rre c t m e a s u re m e n t o f b u b b le v e lo c ity . In a d d itio n th e
e x p e rim e n ts m a d e w ith H S C a llo w e d u s to c o n firm th e o p tic a l p ro b e
re s u lts
and
to g e th e r
w ith
P IV
m e a s u re m e n ts ,
it
was
p o s s ib le
to
c o n c lu d e th a t a n a s s u m p tio n o f a p s e u d o -h o m o g e n e o u s p h a s e c a n b e
m a d e fo r th is s y s te m , th is b e in g u s e d o n th e m o d e l p ro p o s e d . T h e
d e v e lo p e d m o d e l s h o w s a g o o d a g re e m e n t w ith th e e x p e rim e n ta l d a ta .
It s e e m s th a t S G p ro m o te s c o a le s c e n c e a n d th is w a s p ro v e d b y th e
in c re a s e o f b u b b le te rm in a l v e lo c ity u p to 4 % (v o l./v o l.) o f S G . F in a lly
it w a s p ro v e d th a t y e a s t c e lls in flu e n c e O P m e a s u rin g m e c h a n is m b y
in c re a s in g 4 2 .7 4 3 .3 1 % , 3 6 .7 4 4 .4 2 % a n d 3 0 .2 6 3 .5 7 % th e lo c a l g a s
h o ld -u p , b u b b le v e lo c ity a n d c h o rd s iz e , re s p e c tiv e ly . C o n s id e rin g th is
a n d a c o rre c tio n v a lu e a fte r fe rm e n ta tio n , it w a s n o te d th a t g a s h o ld-u p
g e n e ra lly in c re a s e s by 2 2 % in th e p re s e n c e o f c e lls . A s S G re d u c e g a s
h o ld -u p , it is c le a r th a t im m o b iliz e d y e a s t c e lls a n d e th a n o l p ro d u c tio n
p la y a n im p o rta n t ro le o n g a s-lift b io re a c to r h y d ro d y n a m ic s h a v in g a
h ig h e r
in flu e n c e
th a n
th e
s o lid-p h a s e
(S G ),
at
le a s t
fo r
th e
SG
c o n c e n tra tio n c o n s id e re d .
VII.6 References
Bla, M., Ki, M., and Marko, J. (2004). Scale influence on the hydrodynamics of an
internal loop airlift reactor. Chemical Engineering and Processing, 43(12), 15191527.
Boyer, C., Duquenne, A.-M., and Wild, G. (2002). Measuring techniques in gas-liquid
and gas-liquid-solid reactors. Chemical Engineering Science, 57, 3185-3185.
Brnyik, T., Vicente, A., Machado Cruz, J., and Teixeira, J. (2001). Spent grains a
new support for brewing yeast immobilisation. Biotechnology Letters, 23(13),
10731078.
Brnyik, Toms, Vicente, A. A., Oliveira, R., and Teixeira, J. A. (2004).
Physicochemical
surface
properties
of
brewing
yeast
influencing
their
Cartellier, Alain. (1990). Optical probes for local void fraction measurements:
Characterization of performance. Review of Scientific Instruments, 61(2), 874-886.
Cartellier, A. and Achard, J. L. (1991). Local phase detection probes in fluid/fluid
two-phase flows. Review of Scientific Instruments, 62 (2), 279-303.
Cartellier, A. (1992). Simultaneous void fraction measurement, bubble velocity, and
size estimate using a single optical probe in gas-liquid two-phase flows. Review
of Scientific Instruments, 63(11), 5442-5453.
219
Chapter VII
Cartellier, A. (1998a). Measurement of gas phase characteristics using new

monofibre optical probes and real time signal processing. Nuclear Engineering and
Desing, 184, 393-408.
Cartellier, A. and Barrau, E. (1998b). Monofiber optical probes for gas detection and
gas velocity measurements: conical probes. International Journal of Multiphase
Flow, 24(8), 12651294.
Cartellier, A., and Rivire, N. (2001). Bubble-induced agitation and microstructure in
uniform bubbly flows at small to moderate particle Reynolds numbers. Physics of
Fluids, 13, 2165-2181.
Chaumat, H., Billetduquenne, A., Augier, F., Mathieu, C. and Delmas, H. (2005).
Application of the double optic probe technique to distorted tumbling bubbles in
aqueous or organic liquid. Chemical Engineering Science, 60(22),
Dias, S. G., Frana, F. A. and Rosa, E. S. (2000). Statistical method to calculate
local interfacial variables in two-phase bubbly flows using intrusive crossing
probes. International Journal of Multiphase Flow, 26, 1797-1830.
Ferreira, A., Pereira, G., Teixeira, J. A. and Rocha, F. (2011). Statistical tool
combined with image analysis to characterize hydrodynamics and mass transfer in
a bubble column. Chemical Engineering Journal. 180 (2012) 216
228.
Freitas, C. (2002). Bioreactores Multifsicos - Caracterizao Hidrodinmica e de
Transferncia de Massa (in Portuguese). PhD thesis, University of Minho.
8094.
Hamad, F. A. and He, S. (2010). Evaluation of hot-film, dual optical and Pitot tube
probes for liquidliquid two-phase flow measurements. Flow Measurement and
Instrumentation, 21(3), 302-311.
Hong, M., Cartellier, A. and Hopfinger, E. J. (2004). Characterization of phase
detection optical probes for the measurement of the dispersed phase parameters
in sprays. International Journal of Multiphase Flow, 30(6), 615-648.
Kennard, M. and Janekeh, M. (1991). Two- and three-phase mixing in a concentric
draft tube gas-lift fermentor. Biotechnology and bioengineering, 38(11), 1261-70.
Lu, W-J., Hwang, S-J., and Chang, C-M. (1995). Liquid velocity and gas holdup in
Maceiras, R., lvarez, E. and Cancela, M. A. (2010). Experimental interfacial area
measurements in a bubble column. Chemical Engineering Journal, 163(3), 331336.
Mena, P. C., Ruzicka, M. C., Rocha, F. A., Teixeira, J. A. and Drahos, J. (2005a).
columns. Chemical Engineering Science, 60(22), 6013-6026.
Mena, P. C., Pons, M. N., Teixeira, J. A., and Rocha, F. A. (2005b). Effect of Solids
on Gas-Liquid Mass Transfer and Bubble Characteristics in Three-Phase Systems.
7th World Congress of Chemical Engineering.
Mena, P. C., Pons, M. N., Teixeira, J. A., and Rocha, F. A. (2005b). Using image
analysis in the study of multiphase gas absorption. Chemical Engineering Science,
60(18), 5144-5150.
Mena, P. C., Rocha, F. A., Teixeira, J. A., Sechet, P. and Cartellier, A. (2008).
Measurement of gas phase characteristics using a monofibre optical probe in a
three-phase flow. Chemical Engineering Science, 63(16), 4100-4115.
Molina, E., Contreras, A. and Chisti, Y. (1999). Gas holdup, liquid circulation and
mixing behaviour of viscous newtonian media in a split-cylinder airlift bioreactor.
Trans IChem E, 77(Part C), 2732.
Olivieri, G., Marzocchella, a, Vanommen, J. and Salatino, P. (2007). Local and global
hydrodynamics in a two-phase internal loop airlift. Chemical Engineering Science,
62(24), 7068-7077.
220
Chapter VII
Olivier, J.-M.M., (1999). Instrumentation temps rel pour la caractrisation

dcoulements diphasiques bulles laide de sondes optiques (in French).
Masters thesis. Institut National Polytechnique de Grenoble.
Onken, U. E. and Weiland, P. (1983). Airlift fermenters: construction behaviour and
uses. Advances in Biotechnology Procedings, 1, 67-95.
Poupot, C. and Cartellier, A. (1999). Single and double optical probes in air-water
two-phase flows!: real time signal processing and sensor performance.
International Journal of Multiphase Flow, 25, 229-256.
Shen, X., Mishima, K. and Nakamura, H. (2008). Error reduction, evaluation and
correction for the intrusive optical four-sensor probe measurement in multidimensional two-phase flow. International Journal of Heat and Mass Transfer,
51(3-4), 882-895.
Unadkat, H., Rielly, C. D., Hargrave, G. K. and Nagy, Z. K. (2009). Application of
fluorescent PIV and digital image analysis to measure turbulence properties of
solid liquid stirred suspensions. Chemical Engineering Research and Design, 87,
573-586.
Vejrazka, J., Vecer, M., Orvalho, S., Sechet, P., Ruzicka, M. C. and Cartellier, A.
(2010). Measurement accuracy of a mono-fiber optical probe in a bubbly flow.
International Journal of Multiphase Flow, 36, 533-548.
Xie, T., Ghiaasiaan, S. M., Karrila, S., and Mcdonough, T. (2003). Flow regimes and
Zon, M. V., Hamersma, P. J., Poels, E. K., and Bliek, A. (2002). Coalescence of
freely moving bubbles in water by the action of suspended hydrophobic particles.
Chemical Engineering Science, 57, 48454853.
221
Chapter VIII
VIII. Chapter VIII - Continuous Primary

Alcohol-free Beer Fermentation: Influence
of Yeast Strain, Reactor Conditions,
Carrier Type and Ageing
V III.1 O b je c tiv e s
V III.2 In tro d u c tio n
V III.3 M a te ria l a n d M e th o d s
V III.4 R e s u lts a n d D is c u s s io n
V III.5 C o n c lu s io n s
V III.6 R e fe re n c e s
222
Chapter VIII
VIII.1 Objectives
In th is c h a p te r c o n tin u o u s A F B p rim a ry fe rm e n ta io n is s tu d ie d a n d
th e in flu e n c e o f s e v e ra l p a ra m e te rs is e v a lu a te d , s u c h a s :
- th e e ffe c t o f d iffe re n t y e a s t s tra in s , re a c to r ty p e s a n d c a rrie rs in
A F B p rim a ry fe rm e n ta tio n p e rfo rm a n c e a n d fla v o u r c o m p u n d s fo rm a tio n
- th e in flu e n c e o f th e g a s-p h a s e c o m p o s itio n in c o n tin u o u s p rim a ry
A F B fe rm e n ta tio n p e rfo rm a n c e a n d in th e fla v o u r c o m p u n d s , u s in g a n
iG L R a n d S G a s a c a rrie r.
- th e in flu c e n c e o f y e a s t a g e in g in a c o n tin u o u s p rim a ry A F B u s in g a n
iG L R a n d S G a s c a rrie r.
VIII.2 Introduction
The
a p p lic a tio n
of
c o n tin u o u s
p rim a ry
fe rm e n ta tio n
fo r
AFB
p ro d u c tio n a t in d u s tria l s c a le is n o t y e t im p le m e n te d (C h a p te r II.3 .1 ).

T h e re a s o n s b e h in d th is a re m a in ly : u n b a la n c e d fla v o u r; e n g in e e rin g
p ro b le m s
(p ro c e s s
h y g ie n e );
c o n ta m in a tio n
is s u e s ;
a c c u ra c y
of
e c o n o m ic p ro s p e c ts ; th e u s e o f lim ite d y e a s t s tra in s th a t o n ly a llo w th e

c o n tin u o u s p ro d u c tio n o f a lim ite d ty p e o f b e e rs ; a n d tra d itio n in
b re w in g in d u s try (B r n y ik e t a l, 2 0 0 5 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ).
A m o n g a ll d iffe re n t re a c to r c o n fig u ra tio n a n d s y s tem s u s in g IC T
a lre a d y s tu d ie d (s e e C h a p te r II a n d III) it is p o s s ib le to d is tin g u is h tw o
m a in ty p e s th a t h a v e b e e n m o re s tu d ie d : p a c k e d-b e d (V a n Ie rs e l e t a l,
1 9 9 5 ; V irk a ja rv i a n d P o h ja la , 2 0 0 0 ) a n d g a s -lift (B e z b ra d ic a e t a l, 2 0 0 7 ;
B r n y ik , e t a l, 2 0 0 4 ) re a c to rs . B o th h a v e a d v a n ta g e s a n d d is a v a n ta g e s
in c o n tin u o u s A F B p ro d u c tio n . B e tw e e n th e s e tw o re a c to r ty p e s th e
c h o ic e
depends
m a in ly
on
th e
ty p e
of
c a rrie r
and
th e
ty p e
of
im m o b iliz a tio n m e th o d a p p lie d . T h u s it is d iffic u lt to c o m p a re a ll

s y s te m s p re s e n t in lite ra tu re w ith a c c u ra c y .
223
Chapter VII
VIII.2.1 Gas-lift reactor

T h e g a s -lift is a re a c to r w ith o u t m o v in g p a rts , w h e re th e m ix in g a n d
c irc u la tio n a re p ro m o te d b y th e g a s -p h a s e in je c te d (p e rfe c tly a g ita te d
re a c to r). T h is c o n fe rs to g a s-lifts re a c to rs g o o d m a s s tra n s fe rs a n d
m ix in g p ro p e rtie s , w h ic h a s s o c ia te d w ith th e lo w v a lu e o f te n s io n
fo rc e s
fie ld
make
th is
re a c to r
id e a l
fo r
u s in g
IC T
w ith
h ig h
im m o b iliz a tio n lo a d c a p a c ity a n d c o n s e q u e n tly h ig h e r p ro d u c tio n s c a n

b e o b ta in e d (F o n s e c a a n d T e ix e ira , 2 0 0 7 ). S e v e ra l c o n fig u ra tio n s o f
g a s -lift re a c to r c a n b e fo u n d in lite ra tu re (s e e C h a p te r V ) fo r d iffe re n t
p u rp o s e s . In a th re e-p h a s e iG L R , th e g a s p h a s e is in je c te d in to th e
ris e r tu b e th ro u g h a d is trib u to r to p ro v id e th e d riv in g fo rc e fo r liq u id
c irc u la tio n a n d e n e rg y fo r th e s o lid-p h a s e d is trib u tio n (B r n y ik e t a l.,
2 0 0 4 a ).
F o r c o n tin u o u s A F B p ro d u c tio n th e iG L R is u s u a lly u s e d in m a in
fe rm e n ta tio n (B e z b ra d ic a e t a l., 2 0 0 7 ; B r n y ik e t a l., 2 0 0 4 ; D o m n y e t
a l., 1 9 9 9 ; L e h n e rt e t a l., 2 0 0 8 ). T h e im m o b iliz a tio n o f y e a s t c e lls in th e
c a rrie r m a y o c c u r b y a d s o rp tio n o n to th e s o lid p h a s e (c a rrie r), d u rin g
th e firs t d a y s o f fe rm e n ta tio n (B r n y ik e t a l, 2 0 0 4 b ) o r b y p re v io u s
e n tra p m e n t p rio r to fe rm e n ta tio n (B e z b ra d ic a e t a l., 2 0 0 7 ; D e c a m p s e t
a l , 2 0 0 4 ). N e v e rth e le s s , d e ta c h m e n t o f b io m a s s u n d e r c o n d itio n s o f
m e c h a n ic a l s tre s s (B e z b ra d ic a e t a l., 2 0 0 7 ; B r n y ik e t a l., 2 0 0 4a ),
g ra d u a l d is in te g ra tio n a n d w a s h o u t o f c a rrie r (B r n y ik e t a l, 2 0 0 4 a ,
2 0 0 4 b ) m a y in flu e n c e s ta b ility o f im m o b iliz e d b io m a s s in a lo n g -te rm
fe rm e n ta tio n . T h is p ro b le m is m o re s ig n ific a n t if th e im m o b iliz a tio n
o c c u rs b y a d s o rp tio n a n d it is n e c e s s a ry to o b ta in a b a la n c e b e tw e e n
m ix in g tim e , s e d im e n ta tio n , a n d th e m a x im u m lo a d o f im m o b iliz e d
b io m a s s (B r n y ik e t a l, 2 0 0 4b ). M o re o v e r in lo n g-te rm c o n tin u o u s
fe rm e n ta tio n s , re p la c in g fre s h c a rrie r m ig h t b e n e e d e d to m a in ta in c e ll
c o n c e n tra tio n a n d a v o id /re d u c e c e ll a g e in g p ro b le m s . It w a s o b s e rv e d
th a t, in a ro u n d o n e w e e k a fte r a d d in g n e w c a rrie r, th e im m o b iliz e d
b io m a s s a c h ie v e d th e v a lu e s b e fo re a d d itio n (B r n y ik e t a l, 2 0 0 4 b ). T h e
m a in d is a d v a n ta g e s o f u s in g a iG L R to p re fo rm m a in fe rm e n ta tio n a re
224
Chapter VIII
fo a m in g fo rm a tio n a n d th e c o m p le x ity o f s c a le-u p (P ilk in g to n e t a l. ,

1 9 9 8 ).
C o n tin u o u s p rim a ry fe rm e n ta tio n o f A F B in a n iG L R is n o rm a lly
d iv id e d
in
tw o
s ta g e s .
The
in itia l
one,
a ls o
c a lle d
s ta rt-u p ,
is
c h a ra c te riz e d b y : a ir a s g a s-p h a s e ; h ig h e r te m p e ra tu re (a ro u n d 1 2 C );
lo w e r d ilu tio n ra te (0 .0 5 h - 1 to 0 .1 5 h - 1 ); m e d iu m
a s liq u id p h a s e
(in s te a d o f b e e r w o rt). T h is c o n d itio n s a re id e a l to a ra p id y e a s t c e lls

g ro w th a n d im m o b iliz a tio n o f th e s e o n to S G . N o rm a lly d ilu tio n ra te
used
is
b e lo w
th e
m a x im u m
yeast
g ro w th
ra te
( M A X )
at
g iv e n
te m p e ra tu re to a llo w th e in c re a s e o f y e a s t c e lls in s id e th e re a c to r. T h e n
th e s e c o n d s ta g e , w h ic h c o rre s p o n ds to th e p rim a ry fe rm e n ta tio n
b e g in s .
The
m a in
a re :
CO2
te m p e ra tu re (8 C ); h ig h e r d ilu tio n ra te (0 .2 h
-1
as
g a s -p h a s e ;
lo w e r
-1
to 0 .4 h ) a n d w o rt a s
m e d iu m (B r n y ik e t a l, 2 0 0 2 , 2 0 0 4b ; L e h n e rt e t a l., 2 0 0 8 , 2 0 0 9 ).
N o rm a lly th e g a s p h a s e p re s e n t in G L R re a c to rs v a rie s fro m th e firs t
to th e s e c o n d s ta g e . In th e firs t o n e th e m a in a im is to in c re a s e y e a s t
c e ll g ro w th a s w e ll a s th e fo rm a tio n o f a s o lid y e a s t b io film a ro u n d
c a rrie r p a rtic le s . B e in g s o th e g a s-p h a s e is a ir to p ro m o te y e a s t g o w th .
W h e n th e b io film
g ra d u a lly
changed
is fo rm e d a ro u n d th e c a rrie r th e g a s -p h a s e is
to
C O 2 in
o rd e r to
m a in ta in e d
th e
re a c to r a t
a n a e ro b ic c o n d itio n s (B r n y ik e t a l., 2 0 0 4 a ; L e h n e rt e t a l., 2 0 0 8 , 2 0 0 9 ).

T h e g a s -p h a s e le a v in g th e re a c to r c a n b e re c o v e r a n d th e C O 2 is th e n
p u rifie d a n d re-u s e d in th e iG L R (K o u rk o u ta s e t a l., 2 0 0 4 ).
In
o rd e r
to
m a in ta in
a n a e ro b ic
c o n d itio n s
it
is
im p o rta n t
to
u n d e rs ta n d a ll o x y g e n flu x p re s e n t in an iG L R o p e ra tin g in c o n tin u o u s

m o d e . O x y g e n is im p o rta n t to s y n th e tiz e e s te ro ls fo r m e m b ra n e lip id s
p ro d u c tio n , th a t w ill b e u s e d d u rin g y e a s t g ro w th . L e h n e rt e t a l (2 0 0 8 )
o b ta in e d b e tte r re s u lts a t m in im u m o x y g e n tra n s fe r ra te (O T R ) u s e d
(1 m g /(L . h )). If th e w o rt b a rre l is n o t m a in ta in e d a t a n a e ro b ic c o n d itio n s ,
s o m e o x y g e n c a n b e d is s o lv e d in th e in flo w m e d iu m . M o re o v e r w h ile
th e w o rt is a d d e d to th e re a c to r, th e b a rre l h e a d s p a c e in c re a s e s w h ic h
w ill in c re a s e s lig h ty th e d is s o lv e d o x y g e n in th e w o rt. B e in g s o th e O T R
a ls o d e p e n d s o n th e in flo w th a t e n te rs in th e G L R w h e n o p e ra tin g in
225
Chapter VII
c o n tin u o u s m o d e , (O T R D O ). W h e n th e in flo w is m a in ta in e d a t 8 C th e
a v e ra g e o x y g e n c o n c e n tra tio n (C O D
AVG
) is 3 m g /L (M a c ie ira , 2 0 0 8 ) a n d
th e a v e ra g e O T R is c a lc u la te d b y : O T R T O T A L = O T R O S + O T R D O
If
th e
g a s -p h a s e
O T R D O = D *C O D
AVG
is
CO2
th e
O T R O S = 0 m g /(L . h )
and
th e
m g /(L h ), w h e re D is th e d ilu tio n ra te . C o n s id e rin g th a t
d ilu tio n ra te n o rm a lly a re n o t a b o v e 0 .3 h - 1 (L e h n e rt, 2 0 0 9 ) w h e n A F B s

a re p ro d u c e d in c o n tin u o u s th e fin a l O T R w ill a lw a y s b e in fe rio r to th e
o n e s p re s e n t in th e lite ra tu re (L e h n e rt e t a l., 2 0 0 8 , 2 0 0 9 ; M a c ie ira ,
2 0 0 8 ). T h e s e m in im u m o x y g e n c o n d itio n s a re c o n s id e re d to b e th e id e a l
to e n s u re a g o o d fin a l p ro d u c t q u a lity in te rm s o f fla v o u r c o m p o u n d s
a n d a t th e s a m e tim e to m a in ta in y e a s t c e ll g ro w th a t a m in im u m .
VIII.2.2 Packed-bed reactor

T h e p a c k e d -b e d re a c to r (P B R ) o r p lu g flo w re a c to r, is u s u a lly u s ed
fo r m a tu ra tio n a n d in th a t c a s e lo w te m p e ra tu re s (0 C to 5 C ) a re
a p p lie d (D m n y e t a l., 1 9 9 8 ; V a n Ie rs e l e t a l., 1 9 9 5 ; V irk a ja rv i a n d
P o h ja la , 2 0 0 0 ; P e rp te a n d C o llin , 1 9 9 9b ). T h e s e c o n d itio n s le a d to : (1 )
lo w y e a s t g ro w th a n d c o n s e q u e n tly a lo w ris k o f c lo g g in g ; (2 ) h ig h
v ia b ility o v e r lo n g e r p e rio d s ; (3 ) e n o u g h re d u c tio n o f a ld e h y d e s a n d
o th e r c a rb o n y ls ; (4 ) lo w e th a n o l fo rm a tio n ; (5 ) s im p lic ity o f o p e ra tio n . In
th e o ry ,
th e
p lu g
flo w
p re s e n t
in
th is
re a c to r
o p e ra tin g
at
id e a l
c o n d itio n s a llo w to m im ic k e d w e ll th e d iffe re n t s ta g e s p re s e n t in b a tc h

fe rm e n ta tio n s . H o w e v e r in p ra c tic e , it is v e ry d iffic u lt to a c h ie v e th e
id e a l c o n d itio n s . In a d d itio n , it is re p o rte d th e o c c u rre n c e o f c h a n n e lin g
(d e v ia tio n s to id e a l p lu g re g im e ), m a s s tra n s fe r lim ita tio n s , d iffic u ltie s
in C O 2 e v a c u a tio n (g a s p o c k e ts ), c o m p re s s io n o f c a rrie r m a te ria ls a n d
fo u lin g (B r n y ik e t a l., 2 0 0 5 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ; P ilk in g to n e t
a l. , 1 9 9 8 ).
226
Chapter VIII
VIII.3 Material and Methods

VIII.3.1 Microorganisms and medium
T h e m ic ro o rg a n is m s u s e d in th e e x p e rim e n ts w e re fo r in itia l s tu d y : a
b o tto m fe rm e n tin g b re w in g y e a s t (S a c c h a ro m y c e s p a s to ria n u s ) s tra in
W 9 6 (c o lle c tio n o f R e s e a rc h In s titu te o f B re w in g a n d M a ltin g P ra g u e ,
C z e c h R e p u b lic ); a n d S a c c h a ro m y c e s c e re v is ia e B Y 4 7 4 3 w ith d is ru p tio n
in th e K G D 2 (! -k e to g lu ta ra te d e h y d ro g e n a s e ) g e n e . F o r s tu d in g th e
a g e in g a n d g a s c o m p o s ito n e fe c t a n in d u s tria l P o rtu g u e s e b o tto m y e a s t
s tra in fro m U N IC E R , S A w a s u s e d .
Table VIII-1. Wort used in this chapter (VIII) for alcohol free-beer production
Name
Type of
Wort/Medium
Sugar ContentAVG
(g/L)
WA
powder wort
concentrate
32.6
WB
SMM
concentrated wort
Synthetic mineral
medium
Manufacturer
39.3
Research Institute of
Brewing and Malting, Plc.,
Czech Republic
UNICER SA, Portugal
20
T h e p o w d e r w o rt c o n c e n tra te (W A ) w a s p re p a re d b y d is s o lu tio n o f
1 k g in 2 0 L o f p re v io u s ly s te riliz e d w a te r (1 2 1 C , 6 0 m in ) in o rd e r to
a c h ie v e d 5 P w o rt. T h e n s o lu tio n w a s h e a te d to 1 0 0 C d u rin g 3 0 m in .
T h e p H o f w o rt w a s a d ju s te d to 4 .5 w ith th e a d d itio n o f la c tic a c id
(8 0 % w t./v o l., IF C F o o d , L td ., C z e c h R e p u b lic ). F o r e a c h e x p e rim e n t
th e in o c u lu m w a s g ro w n in 5 0 0 m L o f w o rt a t 8 C fo r 4 8 h in a n o rb ita l
shaker
(1 5 0 rp m ).
If
n e c e s s a ry ,
th e
c e lls
w e re
c o lle c te d
by
c e n trifu g a tio n (5 0 0 0 rp m , 5 C , 1 0 m in ).
T h e c o n c e n tra te d w o rt fro m U n ic e r (W B ) w a s d ilu te d in o rd e r to
a c h ie v e a n a v e ra g e c o n c e n tra tio n o f 5 .3 P . T h e p H w a s a d ju s te d w ith
la c tic a c id to 4 .5 a n d th e w o rt w a s s te riliz e d a t 1 0 0 C d u rin g 6 0 m in .
T h e S M M c o m p o s itio n u s e d to s tu d y th e e ffe c ts o f a g e in g in a
c o n tin u o u s p rim a ry fe rm e n ta tio n o f A F B is p re s e n te d in T a b le V III- 2 .
227
Chapter VIII
Table VIII-2. Composition of the SMM and respective concentrations.

Compound
Concentration
KH2PO4
5 g/L
MgSO4 . 7 H2O
0.4 g/L
(NH4)2SO4
Yeast Extract
Glucose
Antifoam
3-methylbutanal
2-methylpropanal
Hexanal
Furfural
2 g/L
2 g/L
20 g/L
10 mL/10 L
200 mg/L
100 mg/L
100 mg/L
100 mg/L
B a rre ls w ith 2 0 L o f S M M w e re s te riliz e d b y a u to c la v in g a t 1 2 0 C ,

1 0 0 k P a fo r 4 0 m in .
VIII.3.2 Carrier preparation

F o r th is w o rk tw o ty p e s o f c a rrie r w e re te s te d: c o rn c o b s a n d s p e n t
g ra in s . E a c h o n e w a s p re p a re d a c c o rd in g to th e m e th o d s d e s c ib re d
b e lo w .
VIII.3.2.1 Corncobs
C o rn c o b s w ith a d ia m e te r b e tw e e n 2 a n d 2 .5 c m a n d to ta l le n g th o f
c a . 1 0 c m w e re c u t in to c y lin d e rs w ith a m a x im u m h e ig h t o f 0 .6 c m .
T h e n e a c h p ie c e w a s cu t in tw o p ie c e s o f th e s a m e s iz e a lo n g th e
w id th . T h e s e h a lf c y lin d e r p a rtic le s w e re u s e d a s c a rrie r in P B R . F o r
G L R th e c o rn c o b s w e re d is in te g ra te d in a k n ife m ill (C u ttin g M ill S M
2 0 0 0 , R e ts c h G m b H , G e rm a n y ) in to p a rtic le s w ith a m a x im u m d ia m e te r
o f 0 .1 ! c m . T h e n 1 0 0 g (d ry b a s e ) o f p re v io u s ly m ille d c o rn c o b s w e re
fu rth e r s e p a ra te d in to tw o fra c tio n s (e q u iv a le n t d ia m e te r: 1 m m a n d 0 .5
mm)
u s in g
p o rta b le
s ie v e
shaker
(M o d e l
A n a ly s e tte ,
F rits c h ,
G e rm a n y ). S m a lle r p a rtic le s w ith a n e q u iv a le n t d ia m e te r low e r th a n 0 .5

mm
w e re
d is c a rd e d
due
to
th e ir
lo w
s e d im e n ta tio n
c o n s e q u e n tly e a s y w a s h o u t fro m iG L R re a c to r.
228
v e lo c ity
and
Chapter VIII
P rio r to u s e , a ll c o rn c o b p a rtic le s w e re a u to c la v e d (1 2 1 C , 6 0 m in )
a s a s u s p e n s io n (1 0 % (w t./v o l.)) in d is tille d w a te r in o rd e r to re m o v e th e
s o lu b le c o m p o u n d s th a t m ig h t in flu e n c e th e b e e r fla v o u r (B r n y ik e t a l.,
2 0 0 6 ).
VIII.3.2.2 Spent
grains
S p e n t g ra in s w e re p re p a re d a c c o rd in g to m e th o d p re s e n t in s e c tio n
III.2 .4 .1 .
VIII.3.3 Packed-bed reactor

T h e P B R u s e d w a s m a d e o f g la s s a n d h a d a to ta l w o rk in g v o lu m e o f
2 .1 2 L . T h e d im e n s io n s o f th e re a c to r a re : to ta l h e ig h t 5 1 c m ;
d ia m e te r 8 c m a n d p a c k e d b e d h e ig h t 3 8 .5 c m . In flo w w a s a t
b o tto m o f th e re a c to r a n d th re e s a m p lin g s p o in ts w e re lo c a te d a t 3 ,
1 7 .6 a n d 3 1 .4 c m fro m th e b o tto m o f th e re a c to r (p o in t I,II a n d III in
F ig u re V III- 1 ). T h e o u t flo w is lo c a te d 4 1 c m fro m b o tto m . T h e re a c to r
w a s m a in ta in e d a t 8 C b y p la c in g it in to a c o n tro lle d te m p e ra tu re c o ld
ro o m .
T h e P B R fille d w ith c o rn co b p a rtic le s (2 1 0 g d ry w e ig h t) h a d a s o lid
v o id a g e o f 0 .3 6 . T h e c h a rg e d P B R w a s a u to c la v e d a t 1 2 1 C fo r 2 0 m in .
A fte r th is th e P B R w a s w a s h e d w ith 1 0 L o f s te rile d is tille d w a te r a n d
fin a lly th e w a te r w a s a llo w e d to d ra in . T h e n 5 0 0 m L o f p re v io u s ly g ro w n
in o c u lu m w a s a d d e d a n d th e re m a in in g re a c to r v o lu m e w a s fille d w ith
fre s h w o rt. A fte r 2 4 h o f b a tc h g ro w th , th e re a c to r w a s c o n tin u o u s ly fe d
w ith w o rt, u s in g a p e ris ta ltic p u m p (F ig u re V III- 1 ). T h e c o n tin u o u s
s y s te m w a s c o n s id e red to re a c h s te a d y s ta te a fte r fiv e re s id e n c e tim e s .
229
Chapter VIII
Figure VIII-1. Experimental layout of the continuous packed-bed reactor: 1 =

cold room, 2 = wort barrel, 3 = peristaltic pump, 4 = PBR, 5 = cooling system;
I, II and III = sampling points, IV = beer outflow, V = gas outlet.
VIII.3.4 Gas-lift reactor characteristics and startup

T h e g a s -lift re a c to r la y o u t (F ig u r e V III- 2 ) a n d s ta rtu p w a s id e n tic a l
fo r b o th u s e d c a rrie rs (s p e n t g ra in s , c o rn co b s ) a n d it w a s d e s c rib e d in
L e h n e rt e t a l. (2 0 0 8 , 2 0 0 9 ). T h e to ta l a m o u n t o f s p e n t g ra in s a n d m ille d
c o rn c o b p a rtic le s in g a s-lift re a c to r w a s 4 0 a n d 5 5 g d ry w e ig h t,
re s p e c tiv e ly . A ir flo w (0 .2 5 L /m in ) w a s s u p p lie d in th e firs t 1 2 d a y s o f
fe rm e n ta tio n to p ro m o te y e a s t g ro w th a n d w h e n a fu lly d e v e lo p e d y e a s t
b io film w a s fo rm e d a ro u n d th e c a rrie r p a rtic le s th e in te n s ity o f a e ra tio n
w a s d e c re a s e d b y s w itc h in g to a ir + C O 2 m ix tu re , w h ile th e to ta l g a s
flo w ra te w a s k e p t c o n s ta n t (0 .2 5 L /m in ). T h e s w itc h in g to a fu lly C O 2
(0 .2 5 L /m in ) g a s flo w w a s g ra d u a l to a v o id c e ll s tre s s . A s in P B R , th e
c o n tin u o u s s y s te m w a s c o n s id e re d to re a c h s te a d y s ta te a fte r fiv e
re s id e n c e tim e s . T h e iG L R u s e d in a ll e x p e rim e n ts h a s a w o rk in g
v o lu m e o f 3 L (L e h n e rt e t a l., 2 0 0 8 , 2 0 0 9) w ith th e e x c e p tio n o f th e
re a c to r u s e d in th e s e c tio n V III.4 .5 , w h ic h h a s 6 L (s a m e u s e d in s e c tio n
V II.4 .6 ).
230
Chapter VIII
Figure VIII-2. Laboratory scale installation for primary beer fermentation: 1air supply; 2-mass flow controller; 3-gas sterilization filter; 4-CO2 bottles; 5rotameters; 6-peristaltic pump; 7-thermostated cold room; 8-wort barrel; 9gas outlet; 10-gas-lift reactor; I. beer sampling; II. carrier sampling.
VIII.3.5 Batch fermentations

T h e b a tc h fe rm e n ta tio n s w e re s ta rte d b y c o o lin g th e s te rile w o rt (2 L )
to 8 C a n d a e ra tin g (7 0 0 m L /m in ) fo r 1 5 m in . T h e fin a l o x y g e n
c o n c e n tra tio n in a e ra te d w o rt w a s a p p ro x im a te ly 8 m g /L . T h e p itc h in g
ra te w ith p re v io u s ly g ro w n a n d c e n trifu g e d c e lls w a s 3 ! 1 0 6 c e ll/m L .
In itia lly th e fe rm e n ta tio n w a s p e rfo rm e d a t 8 C fo r 2 4 h r a n d th e n th e
p a rtia lly fe rm e n te d w o rt w a s c o o le d to 2 C fo r 4 8 h r.
VIII.3.6 Yeast cells analysis

VIII.3.6.1 Methylene
blue staining
B re w in g y e a s t v ia b ility w a s d e te rm in e d a ls o b y m e th y le n e b lu e
m e th o d w h ic h is a p p lic a b le to a ll s a m p le s c o n ta in in g b re w in g y e a s t.
231
V ia b le
c e lls
compound.
m e th y le n e
Chapter VIII
re d u c e
The
b lu e
m e th y le n e
yeast
b lu e
s u s p e n s io n
s o lu tio n
in
e n z y m a tic a lly
was
te s t
tu b e
d ilu te d
u n til
to
w ith
a
c o lo u rle s s
1 % (w t./v o l.)
s u s p e n s io n
w ith
a p p ro x im a te ly 5 -1 0 c e lls in a m ic ro s c o p ic fie ld w a s o b ta in e d . T h e
c o u n tin g w a s m a d e u s in g a m a g n ific a tio n o f 4 0 0 tim e s d u rin g a
m a x im u m o f 5 m in u te s . C e lls s ta in e d d a rk b lu e w e re c o n s id e re d to b e
d e a d a lo n g w ith b ro k e n , s h riv e lle d a n d p la s m o ly z e d c e lls . C e lls s ta in e d
lig h t b lu e s h o u ld b e c o n s id e re d a liv e a n d th e y e a s t c e lls th a t w e re
b u d d e d w e re c o u n te d a s o n e c e ll if th e b u d is le s s th a n o n e h a lf th e
s iz e o f th e m o th e r c e ll. V ia b ility w a s c a lc u la te d fro m th e ra tio b e tw e e n
to ta l a n d v ia b le c e lls (A S B C , 1 9 9 6 ).
VIII.3.6.2 Free
biomass concentration
A s a m p le o f 1 0 m L w a s re m o v e d fro m th e re a c to r o u tflo w a n d it w a s
c e n trifu g e d fo r 3 m in a t 5 0 0 0 rp m . T h e s u p e rn a ta n t w a s re m o v e d a n d, if
n e c e s s a ry , th e s e d im e n t w a s d ilu te d (1 :5 ra tio ) w ith d is tille d w a te r. T h e
o p tic a l d e n s ity o f th e s o lu tio n w a s m e a s u re d w ith s p e c tro p h o to m e te r a t
a w a v e le n g th o f 6 0 0 n m . T h e re s u lts o f b io m a s s c o n c e n tra tio n w e re
c a lc u la te d u s in g a c a lib ra tio n c u rv e , b io m a s s v s . a b s o rb a n c e .
VIII.3.6.3 Immobilized
biomass determination
D u e to d iffic u ltie s w ith c o lle c tin g b io c a ta ly s t (c a rrie r + im m o b iliz e d

c e lls ) s a m p le s fro m th e ru n n in g P B R (B r n y ik e t a l., 2 0 0 6 ) th e v a lu e s o f
im m o b iliz e d b io m a s s o n c o rn c o b p a rtic le s w e re o n ly d e te rm in e d a fte r
fin is h in g th e fe rm e n ta tio n . S a m p le s o f b io c a ta ly s ts (3 to 4 g ) w e re
c o lle c te d a n d p la c e d in 20 0 m L o f 1 5 g /L a q u e o u s s o lu tio n o f N a C l
(p H = 3 ). T h e s u s p e n s io n w a s th e n a g ita te d a t 7 0 0 rp m fo r 3 0 m in . T h e n
th e c e ll s u s p e n s io n a b s o rb a n c e (6 0 0 n m ) w a s re a d a n d th e c e ll b io m a s s
w a s c a lc u la te d b y u s in g a c a lib ra tio n c u rv e .
T h e a m o u n t o f b io m a s s a d h e re d to m ille d c o rn c o b s p a rtic le s (C c 3
s e e c h a p te r III.3 .2 ) w a s d e te rm in e d in 5 0 m L s a m p le s ta k e n fro m iG L R
to a 1 0 0 m L E rle n m e y e r fla s k . T h is s a m p le c o n ta in e d a p p ro x im a te ly
0 .5 g o f d ry b io c a ta ly s t. T h e n th e b u lk o f th e liq u id p h a s e w a s re m o v e d
232
Chapter VIII
w ith a s y rin g e a n d th e c a rrie r w a s w a s h e d w ith 3 x 7 5 m L o f d is tille d

w a te r in o rd e r to re m o v e th e fre e c e lls c a p tu re d b e tw e e n th e c a rrie r
p a rtic le s . T h e s e o p e ra tio n s w e re c a rrie d o u t v e ry c a re fu lly a v o id in g
s h o c k s th a t w o u ld d e ta c h th e im m o b iliz e d c e lls fro m th e s u rfa c e o f th e
c a rrie r. A fte r d ry in g (4 0 C fo r 4 8 h ), th e b io c a ta ly s t w a s w e ig h te d a n d
th e n a d d e d in to 5 0 m L o f N a C l (1 5 g /L a t p H = 3 ). T h e a tta c h e d c e lls
w e re re m o v e d fro m th e c a rrie r b y s tro n g m ix in g (6 0 0 rp m ) fo r 3 0 m in .
T h e a m o u n t o f re le a s e d b io m a s s w a s d e term in e d a fte r s e ttlin g th e
c a rrie r b y m e a s u rin g th e a b s o rb a n c e (A 6 0 0 ) o f th e b u lk liq u id a t 6 0 0 n m
a n d w a s c o rre la te d to c e ll d ry w e ig h t b y a c a lib ra tio n c u rv e . T h e w h o le
p ro c e d u re o f m e c h a n ic a l c e ll re m o v a l w a s re p e a te d w h ile th e b u lk
liq u id c o n ta in e d a s ig n ific a n t a m o u n t o f re le a s e d c e lls (A 6 0 0 > 0 .1 )
(B r n y ik e t a l., 2 0 0 1 ). T h is p ro c e d u re w a s re p e a te d in d u p lic a te fo r
e a c h s a m p le .
T h e iB io in S G w a s d e te rm in e d a c c o rd in g to th e m e th o d d e s c rib e d b y
B r n y ik e t a l. (2 0 0 4 a ). B rie fly a s a m p le c o n ta in in g a p p ro x im a te ly 1 .0 g
o f d ry b io c a ta ly s t w a s ta k e n fro m th e re a c to r in a fo rm o f s lu rry th ro u g h
a s a m p lin g p o rt (p o in t II in F ig u re V III- 2 ) to a p re v io u s ly w e ig h t
E rle n m e y e r fla s k . T h e b u lk liq u id w a s rem o v e d w ith a s y rin g e , a n d th e
c a rrie r w a s w a s h e d w ith 2 0 0 m L (2 x 1 0 0 m L ) o f d is tille d w a te r. T h e n
th e fla s k + b io c a ta ly s t w e re d rie d a t 1 0 5 C fo r 1 2 h a n d w e ig h t. A fte r
1 0 0 m L o f 3 % (w t./v o l.) N a O H s o lu tio n w a s a d d e d a n d s h a k e n a t
1 2 0 rp m fo r 2 4 h . D u rin g th is tim e th e a tta c h e d b io m a s s w a s c o m p le te ly
re m o v e d fro m th e c a rrie r a n d th is w a s v e rifie d w ith m ic ro s c o p y . T h e
b io m a s s fre e s u s p e n s io n w a s re m o v e d fro m th e S G p a rtic le s a n d th e
E rle n m e y e r c o n ta in in g o n ly S G w a s d rie d a t 1 0 5 C fo r 5 h . T h e a m o u n t
o f y e a s t b io film w a s d e te rm in e d fro m th e w e ig h t d iffe re n c e b e fo re a n d
a fte r th e tre a tm e n t w ith c a u s tic . C o rre c tio n s o f th e b io m a s s w e ig h t fo r
th e lo s s e s o f S G its e lf (a p p ro x im a te ly 6 .3 % o f in itia l w e ig h t) d u rin g th e
w a s h in g p ro c e d u re s w e re c a rrie d o u t w ith b la nk e x p e rim e n ts w ith c le a n
c a rrie r.
233
Chapter VIII
VIII.3.6.4 Microbial
adhesion to solvents (MATS) analysis
T h e c e lls w e re h a rv e s te d fro m y e a s t s u s p e n s io n b y c e n trifu g a tio n a t

6 0 0 0 rp m , 8 C fo r 5 m in , d is c a rd in g th e s u p e rn a ta n t a n d re-s u s p e n d in g
th e c e lls in 1 5 0 m M N a C l s o lu tio n a t a c o n c e n tra tio n o f a b o u t 4 x 1 0 7
c e lls /m L (o p tic a l d e n s ity O .D . o f 0 .8 a t 4 0 0 n m ). A h ig h io n ic s tre n g th
e le c tro ly te w a s u s e d to a v o id c h a rg e in te rfe re n c e b y a m a s k in g c e lls
c h a rg e . A t th e e n d , th e o p tic a l d e n s ity o f th e s u s p e n s io n w a s m e a s u re d
(O .D . 0 ) a t a w a v e le n g th o f 4 0 0 n m .
T h e n 3 m L o f w a s h e d y e a s t s u s p e n s io n w e re v o rte x e d fo r 6 0 s e c o n d s
w ith 0 .5 m L o f th e o rg a n ic s o lv e n t (c h lo ro fo rm , h e x a d e c a n e , e th y l
a c e ta te , a n d d e c a n e ). T o e n s u re th e c o m p le te s e p a ra tio n o f th e tw o
p h a s e s th e m ix tu re w a s k e p t s till fo r 1 0 m in . T h e n 2 m L s a m p le fro m
th e a q u e o u s p h a s e w e re re m o v e d a n d th e O . D . w a s m e a s u re d a t
4 0 0 n m . T h e c e lls a ffin ity fo r e a c h s o lv e n t w a s c a lc u la te d b y E q . V III
1 . E a c h a s s a y w a s p e rfo rm e d in trip lic a te .
#
O.D. &
%affinity = 100 ! %1"
(
$ O.D.0 '
VIII.3.6.5 Glycogen
E q . V III 1
and neutral lipids (NL) content
Im m o b iliz e d c e lls w e re h a rv e s te d a n d k e p t a s d e s c rib e d b e lo w a n d

g ly c o g e n ,
n e u tra l
lip id s ,
and
bud
s c a rs
w e re
a n a ly s e d
by
flo w
c y to m e try .
T h e flo w c y to m e tric m e a s u re m e n ts w e re ta k e n u s in g a P a rte c P a s III
(P a rte c G m b H , M n s te r, G e rm a n y ) a n aly z e r e q u ip p e d w ith a n a rg o n io n
la s e r (1 5 m W la s e r p o w e r w ith e x c ita tio n w a v e le n g th 4 8 8 n m ).
T h e re la tiv e c o n te n t o f g ly c o g e n in c e lls w a s d e te rm in e d u s in g
A c rifla v in e , w h ic h is a flu o re s c e n t d y e a b le to c o v a le n tly b in d to
g ly c o g e n
a fte r
p e rm e a b ilis a tio n
of
th e
c e ll
m e m b ra n e
by
e th a n o l
(fix a tio n ). A n a liq u o t o f 0 .5 m L (O D = 0 .8 a t 6 0 0 n m ) o f th e s a m p le w a s

re m o v e d a n d 1 0 L o f A c rifla v in e (S ig m a A ld ric h , G e rm a n y ) s o lu tio n
(1 m g /m L in P B S , s to re d a t 4 C ) w e re a d d e d . In c u b a tio n o c c u rre d a t
ro o m te m p e ra tu re in d a rk n e s s fo r 3 0 m in . T h e s a m p le w a s th e n
234
Chapter VIII
a n a ly z e d b y flo w c y to m e try in F L 1 c h a n n e l (5 3 0 n m ). T h e re la tiv e

c o n te n t o f n e u tra l lip id s w a s d e te c te d a fte r re a c tio n w ith 2 0 m l o f n ile
re d s o lu tio n (0 .1 m g /m l a c e to n e ). R e a c tio n w a s c a rrie d o u t in d a rk a t
ro o m te m p e ra tu re fo r 1 0 m in . S a m p le w a s s u b s e q u e n tly w a s h e d th re e
tim e s
w ith
PBS
(8 g /L N a C l;
1 .1 4 g /L N a 2 P O 4 ;
0 .2 g /L K C l;
0 .8 g /L K H 2 P O 4 in d is tille d w a te r) a n d a n a ly s e d b y flo w c y to m e try in

F L 3 c h a n n e l (6 3 0 n m ).
VIII.3.6.6 Bud
scars staining
A n a liq u o t o f 0 .5 m L (O D = 0 .8 a t 6 0 0 n m ) o f th e s a m p le w a s w a s h e d
tw ic e in P B S . A fte r it w a s re-s u s p e n d e d in 0 .5 m L P B S a n d 2 0 m L o f
A le x a F lu o r 4 8 8 -la b e lle d w h e a t-g e rm a g g lu tin in (le c tin fro m T ritic u m
v u lg a ris ; S ig m a -A ld ric h , U K ), a t a c o n c e n tra tio n o f 1 m g /m L . C e lls w e re
g e n tly a g ita te d a t ro o m te m p e ra tu re fo r 1 5 m in in d a rk , h a rv e s te d b y
c e n trifu g a tio n (5 0 0 0 r.p .m . fo r 3 m in ) a n d w a s h e d th re e tim e s in P B S .
T h e s ta in e d c e ll c u ltu re w a s re-s u s p e n d e d in 0 .5 m L P B S a n d e x a m in e d
u s in g flo w c y to m e try w ith a 4 8 8 n m a rg o n io n la s e r fo r flu o re s c e n c e
e x c ita tio n . F lu o re s c e n c e w a s d e te c te d in F L 1 c h a n n e l (5 3 0 n m ).
VIII.3.7 Out-flow analytical methods

VIII.3.7.1 Ethanol
determination
E th a n o l w a s a n a ly z e d b y H P L C (P u m p L C P 4 0 0 0 , C o lu m n o v e n L C O
1 0 1 , E C O M L td ., P ra g u e , C z e c h re p u b lic ) u s in g a P o ly m e r IE X C a fo rm
c o lu m n (2 5 0 x 8 m m , W a tre x In te rn a tio n a l In c ., S a n F ra n c is c o , U S A ), a n d
a R ID K 1 0 2 re fra c tio n in d e x d e te c to r (L a b o ra to rn p!s tro je P ra h a ,
P ra g u e , C z e c h re p u b lic ). E lu tio n w a s p e rfo rm e d w ith d e g a s s e d a n d
d e m in e ra liz e d w a te r a t 8 5 C a n d th e flo w ra te w a s 0 .7 m L /m in .
VIII.3.7.2
Higher alcohols, esters and organic acids method A
T h e fla v o u r a n d a ro m a c o m p o u n d s (H A a n d E S ) w e re m e a s u re d
a c c o rd in g to th e c u rre n t E u ro p e a n B re w e ry C o n v e n tio n re c o m m e n d e d
235
Chapter VIII
m e th o d s (E B C , 1 9 8 7 ). T h e c o n c e n tra tio n o f O A w a s d e te rm in e d b y a
c a p illa ry z o n e e le c tro p h o re tic a n a ly z e r (E A 1 0 1 , L a b e c o-V illa , L td .,
S lo v a k R e p u b lic ), w ith s e p a ra tio n c a p illa rie s (P T F E , le n g th 9 0 m m ,
d ia m e te r 0 .8 m m , 1 5 0 A ) c o n n e c te d to a n a ly tic a l c a p illa ry (le n g th
9 0 m m ; d ia m e te r 0 .3 m m ; 2 0 A ) a n d a d e te c to r (c o n d u c tiv e a n d U V
d e te c tio n a t 2 5 4 n m ). T h e a n a ly s is w a s c a rrie d o u t b y a le a d in g
e le c tro ly te
(5 m M
HCl
10 mM
g ly c y lg ly c in
0 .0 5 %
h y d ro x y e th y lc e llu lo s e ) a n d a fin is h in g e le c tro ly te (1 0 m M c a p ro ic a c id ).

The
of
re d u c in g
s u g a rs
was
d e te rm in e d
s p e c tro p h o to m e tric a lly a fte r re a c tio n w ith 3 ,5-d in itro s a lic y lic a c id a t
530 nm.
VIII.3.7.3
Higher alcohols, esters and aldehydes method B
T h e s e m e th o d s w e re u s e d to d e te rm in e th e v o ltile c o m p o u n d s fro m
th e s a m p le s o b ta in e d in s e c tio n V II.4 .5 . T h e q u a n tific a tio n o f th e m a jo r
v o la tile (a c e ta ld e h y d e , e th y l a c e ta te , 1-p ro p a n o l, 2 -m e th y l-1 -p ro p a n o l,
2 -m e th y l-1 -b u ta n o l, 3 -m e th y l-1 -b u ta n o l, 2 -p h e n y le th a n o l) a n d m in o r
(ty ro s o l, is o a m y l a c e ta te , e th y l c a p ry la te , 2-p h e n y le th y l a c e ta te , e th y l
h e x a n o a te ,
e th y l
(h d ro x y m e th y l)fu rfu ra l
d e c a n o a te ,
(H M F ))
2-p h e n y le th y l
compounds
w e re
a c e ta te ,
p e rfo rm e d
w ith
5th e
fo llo w in g m e th o d s .
M a jo r v o la tile c o m p o u n d s
T o 5 m L o f s a m p le , filte re d p re v io u s ly th ro u g h a 0 .2 m m e m b ra n e
filte r (W h a tm a n M E 2 4 ), 1 0 0 L o f in te rn a l s ta n d a rd (4 -n o n a n o l, M e rc k
re f. 8 1 8 7 7 3 , in a lc o h o lic s o lu tio n a t 4 .1 g /L ) w e re a d d e d .
M a jo r v o la tile c o n s titu e n ts w e re a n a ly z e d d ire c tly , b y in je c tin g 1 L
of
s a m p le
in
th e
s p lit
mode
on
C h ro m p a c k
C P -9 0 0 0
gas
c h ro m a to g ra p h e q u ip p e d w ith a S p lit/S p litle s s in je c to r a n d a fla m e

io n iz a tio n d e te c to r. A c a p illa ry c o lu m n , c o a te d w ith C P-W a x 5 7 C B
(5 0 m ! 0 .2 5 m m i.d ., 0 .2 m film th ic k n e s s ; C h ro m p a c k ), w a s u s e d .
T h e te m p e ra tu re s o f th e in je c to r a n d d e te c to r w e re b o th s e t to 2 5 0 C .
T h e o v e n te m p e ra tu re w a s h e ld a t 6 0 C , fo r 5 m in , th e n p ro g ra m m e d to
ris e fro m 6 0 C to 2 2 0 C , a t 3 C /m in , a n d fin a lly h e ld a t 1 0 m in a t
236
Chapter VIII
2 2 0 C . H e liu m G H E 5 ! (P ra x a ir) a t 1 2 5 k P a w a s u s e d a s c a rrie r g a s ,

a n d th e s p lit v e n t w a s s e t to 1 5 m L /m in . Id e n tific a tio n o f v o la tile
c o m p o u n d s w a s c a rrie d o u t b y c o m p a rin g re te n tio n in d ic e s w ith th o s e
of
p u re
s ta n d a rd
compounds.
Q u a n tific a tio n
was
p e rfo rm e d
w ith
s o ftw a re S ta r C h ro m a to g ra p h y W o rk s ta tio n , v e rs io n 6 .4 1 (V a ria n ),

a fte r th e d e te rm in a tio n o f d e te c to rs re s p o n s e fa c to r fo r e a c h v o la tile
compound.
M in o r v o la tile c o m p o u n d s
E x tra c tio n o f v o la tile c o m p o u n d s w e re d e te rm in e d a c c o rd in g th e
m e th o d p ro p o s e d b y O liv e ira e t a l. (2 0 0 6 ). In a 1 0 m L c u ltu re tu b e
(P y re x , re f. 1 6 3 6 /2 6 M P ), 8 m L o f b e e r, 3 .5 8 2 g o f in te rn a l s ta n d a rd (4 n o n a n o l) a n d
E x tra c tio n
was
m a g n e tic
done
by
s tir b a r (2 2 .2 m m ! 4 .8 m m ) w e re
s tirrin g
th e
s a m p le
w ith
added.
400 L
of
d ic h lo ro m e th a n e (M e rc k , re f. 1 .0 6 0 5 0 ) d u rin g 1 5 m in , o v e r a m a g n e tic
s tirre r. A fte r c o o lin g a t 0 C d u rin g 1 0 m in , th e m a g n e tic s tir b a r w a s
re m o v e d
and
th e
o rg a n ic
phase
was
d e ta c h e d
by
c e n trifu g a tio n
(R C F = 5 1 1 8 , 5 m in , 4 C ) b e in g th e e x tra c t re c o v e re d in to a v ia l, u s in g a
P a s te u r p ip e tte . T h e n , th e a ro m a tic e x tra c t w a s d rie d w ith a n h y d ro u s
s o d iu m s u lp h a te (M e rc k , re f. 1 .0 6 6 4 9 ) a n d p ic k e d u p a g a in in to a n e w
v ia l. E a c h s a m p le w a s e x tra c te d in trip lic a te . T h e e x tre a c ts w e re
a n a liz e d b y g a s c h ro m a to g ra p h ic . T h e a n a ly s is o f v o la tile c o m p o u n d s
w a s p e rfo rm e d u s in g a G C M S s y s te m c o n s titu te d b y a V a ria n 3 8 0 0
C h ro m a to g ra p h ,
w ith
1079
in je c to r,
and
an
io n-tra p
mass
s p e c tro m e te r V a ria n S a tu rn 2 0 0 0 . A 1 L in je c tio n (s p litle s s fo r 3 0 s ;

s p lit ra tio o f 3 0 m L /m in ) w a s m a d e in to a c a p illa ry c o lu m n , c o a te d w ith
V F -W a x m s (3 0 m ! 0 .1 5 m m i.d ., 0 .1 5 m fi lm th ic k n e s s , V a ria n ). In je c to r
a n d tra n s fe r lin e te m p e ra tu re s w e re b o th s e t to 2 5 0 C . T h e o v e n
te m p e ra tu re w a s h e ld a t 6 0 C , fo r 2 m in , th e n p ro g ra m m e d to ris e fro m
6 0 C to 2 3 4 C , a t 3 C /m in , a n d fro m 2 3 4 C to 2 5 0 C , a t 1 0 C /m in ,
th e n h e ld 1 0 m in a t 2 5 0 C . T h e c a rrie r g a s w a s h e liu m G H E 5! (P ra x a ir)
a t a c o n s ta n t flo w ra te o f 1 .3 m L /m in . T h e d e te c to r w a s s e t to
e le c tro n ic im p a c t m o d e (7 0 e V ), w ith a n a c q u is itio n ra n g e fro m 3 5 m /z
to 2 6 0 m /z , a n d a n a c q u is itio n ra te o f 6 1 0 m s . Id e n tific a tio n o f vo la tile s
237
Chapter VIII
w a s p e rfo rm e d w ith V a ria n M S W o rk s ta tio n s o ftw a re , v e rs io n 6 .9 .3 , b y

c o m p a rin g re te n tio n in d ic e s w ith th o s e o f p u re s ta n d a rd c o m p o u n d s .
V o la tile c o m p o u n d s w e re q u a n tifie d a s 4-n o n a n o l e q u iv a le n ts (O liv e ira
e t a l., 2 0 0 6 ).
VIII.3.7.4 Determination
of vicinal diketones
T o ta l V D K s (2 ,3 -b u ta n o d io n e (d ia c e ty l) a n d 2 ,3-p e n ta n o d io n e ) w e re
m e a s u re d
a c c o rd in g
to
th e
c u rre n t
E u ro p e a n
B re w e ry
C o n v e n tio n
re c o m m e n d e d m e th o d s (E B C , 1 9 9 9 ).
VIII.3.8 Determination of residence time distribution

C a rrie r p re p a re d w a s a d d e d to b o th P B R a n d iG L R a n d th e w o rk in g
v o lu m e o f liq u id p h a s e in th e re a c to rs w a s c a lc u la te d . T h e n a p u ls e o f
2 m L o f m e th y le n e b lu e (3 .5 g /L ) w a s in je c te d a t th e in flo w o f b o th
re a c to rs a n d s a m p le s (1 .5 m L ) w e re c o lle c te d a t re g u la r in te rv a ls (3
m in ) in th e o u tflo w . T h e a m o u n t o f m e th y le n e b lu e in th e o u tflo w w a s
d e te rm in e d b y s p e c tro p h o to m e tric m e a s ure m e n t (6 0 0 n m ) a n d th e s e
d a ta w e re u s e d to p lo t re a l (e x p e rim e n ta l) re s id e n c e tim e d is trib u tio n
(R T D ) c u rv e s .
& %(1% " ) 2 $ Pe )

! (" ) = 1/2 Pe /# $ exp(
+ where Pe = (uL/D);" = t /,
4"
'
*
% -(1- " D ) 2 Pe (
! (" D ) = 1/2 Pe /# $ exp'
* where :
4" D
&
)
E q . V III 2
E q . V III 3
" D = t /+ D; + D = (VR , VD ) /QL

N(N # " ) n $1
! (" ) =
exp($N" )
(N $ 1)!
The
d a ta
fro m
PBR
E q . V III 4
w e re
o b ta in e d
at
h y d ra u lic
re s id e n c e tim e o f 1 6 .7 m in . T h e a x ia l d is p e rs io n in P B R w a s e v a lu a te d
b y a d ju s tin g th e d is p e rs io n p a ra m e te r (D /u L ) o f th e m o d e l (E q . V III 2 )
s u g g e s te d b y L e v e n s p ie l (1 9 9 9 ). In o rd e r to o b ta in m o re in fo rm a tio n
a b o u t th e h y d ro d y n a m ic b e h a v io r in th is re a c to r, th e e x p e rim e n ta l d a ta
fro m P B R w e re a ls o s im u la te d w ith a m u ltip a ra m e te r m o d e l (E q . V III 3 )
238
Chapter VIII
a n d a C S T R in s e rie s m o d e l (E q . V III 4 ) s u g g e s te d b y s a m e a u th o r
(L e v e n s p ie ld , 1 9 9 9 ).
T h e R T D d e te rm in a tio n in th e iG L R w a s d o n e a t g a s flo w ra te o f 0 ,2 5
L /m in , w h ic h c o rre s p o n d s to th e n o rm a l g a s flo w u s e d in th is re a c to r
fo r c o n tin u o u s fe rm e n ta tio n o f a lc o h o l-fre e b e e r (L e h n e rt e t a l, 2 0 0 8 ),
a n d a t a h y d ra u lic re s id e n c e tim e o f 3 9 .2 m in . T h e h y d ro d y n a m ic
b e h a v io r in s id e th e iG L R w a s d e te rm in e d b y a d ju s tin g th e R T D c u rv e to
th e C S T R in s e rie s m o d e l E q . V III 4 s u g g e s te d b y L e v e n s p ie l (1 9 9 9 ).
T h e e x p e rim e n ta l v a lu e s o n b o th re a c to rs w e re tre a te d b y n on -lin e a r
a d ju s tm e n t u s in g S o lv e r fro m E x c e l (M ic ro s o ft C o rp .). S o lv e r w a s
a p p lie d o n th e m in im iz a tio n o f th e s q u a re s u m o f th e e rro rs b e tw e e n th e
th e o re tic a l (m o d e l) a n d e x p e rim e n ta l v a lu e s .
VIII.3.9 Fermentations
In th e C h a p te r V II s e v e ra l c o n tin u o u s fe rm e n ta tio ns w e re p e rfo rm e d
e ith e r in iG L R o r in P B R . T o s tu d y th e in flu e n c e o f y e a s t s tra in , c a rrie r
ty p e a n d re a c to r d e s ig n th e fe rm e n ta tio n s d o n e a re p re s e n te d in T a b le
V III- 3 .
Table VIII-3. Conditions used for optimizing continuous primary AFBs
fermentation.
R e a c to r
PBR GLR
GLR
PBR
B a tc h
Y e a s t s tra in
W96 W96
W96 !KGD2 !KGD2
B a tc h
B a tc h a
W96
In d u s tria l
C a rrie r
Cc
Cc
SG
Cc
T e m p e ra tu re (C )
8 /2
NAa
NA Information not available
The
s tu d y
of
gas
c o m p o s itio n
in
p rim a ry
c o n tin u o u s
AFB
fe rm e n ta ito n w a s p e rfo rm e d in a iG L R u s in g S G a s c a rrie r (IV .3 .5 ). T h e

s a m e re a c to r s y s te m a n d im m o b iliz a tio n m e th o d w a s u s e d in th e s tu d y
o f th e e ffe c t o f a g e in g in c o n tin u o u s p rim a ry A F B fe rm e n ta tio n (IV .3 .6 ).
239
Chapter VIII
VIII.4 Results and Discussion

VIII.4.1 General considerations
It h a s b e e n s ta te d th a t c o n tin u o u s b e e r fe rm e n ta tio n c o u ld , u n d e r
fa v o u ra b le c irc u m s ta n c e s , p ro d u c e a c o n s id e ra b le e c o n o m ic a l b e n e fit
th ro u g h tim e s a v in g s . In s p ite o f th is e x p e c ta tio n , m o s t o f th e in d u s tria l
im p le m e n ta tio n a tte m p ts s tu m b le d o n fla v o u rs d e fe c ts a n d e n g in e e rin g
c o m p le x n e s s o f c o n tin u o u s ly fe rm e n te d b e e rs (B r n y ik e t a l., 2 0 0 8 ;
M e n s o u r e t a l., 1 9 9 2 ; W illa e rt e t a l., 2 0 0 6 ). C o n v e rs e ly to re g u la r b e e r,
th e fla v o u r d is s im ila rity o f A F B is u s u a lly b e tte r a c c e p te d b y th e
c o n s u m e rs .
T h e re fo re ,
s u ita b ly
m a tc h e d
yeast
s tra in
w ith
im m o b iliz a tio n m e th o d (c a rrie r) a n d p ro ce s s c o n fig u ra tio n c o u ld b e a n

a ttra c tiv e a lte rn a tiv e fo r th e in d u s try to s a tis fy th e A F B s w o rld w id e
g ro w in g m a rk e t.
VIII.4.1.1 Measurement
and modeling of mixing regimes in two
different bioreactors
F ro m th e p o in t o f v ie w o f re a c to r d e s ig n th e re a re tw o fu n d a m e n ta l
a n d m a n y c o m b in e d a p p ro a c h e s to d e s ig n a m u lti-re a c to r c o n tin u o u s
b e e r fe rm e n ta tio n s y s te m . T h e fu n d a m e n ta l s y s te m s a re b a s e d o n e ith e r
a s e rie s o f c o n tin u o u s s tirre d-ta n k re a c to rs (C S T R ) o r p lu g flo w -lik e
re a c to rs (P F R ), w h ile th e c o m b in e d s y s te m s u s e re a cto r v e s s e ls w ith
b o th ty p e s o f m ix in g re g im e . T h e g o a l o f th e s e m u lti-re a c to r s y s te m s is
to m im ic th e s e q u e n tia l s u b s tra te u p ta k e a n d b y-p ro d u c t fo rm a tio n
ty p ic a l fo r fe rm e n ta tio n in b a tc h (B r n y ik e t a l., 2 0 0 8 ; W illa e rt e t a l.,
2 0 0 6 ).
U n lik e
fo r
re g u la r
be e rs ,
th e
c o n tin u o u s
AFBs
p ro d u c tio n
re q u ire s o n ly a s in g le re a c to r s y s te m . N e v e rth e le s s , th e k n o w le d g e o f
th e h y d ro d y n a m ic m ix in g re g im e in s id e th e re a c to r fo r A F B p ro d u c tio n
c a n h e lp a v o id in g th e p o te n tia l e n g in e e rin g p ro b le m s , c h o o s in g th e
m o s t c o n v e n ie n t c a rrie r m a te ria l a n d u n d e rs ta n d in g th e b e h a v io u r o f
im m o b iliz e d y e a s t.
240
C o m p a rin g
th e
Chapter VIII
and
th e o re tic a l
re s id e n c e
tim e
d is trib u tio n (R T D ) c u rv e s fo r b o th re a c to rs , a n e a rly p e rfe c tly m ix e d

re g im e a n d a p lu g flo w w ith s ig n ific a n t a x ia l d is p e rs io n c a n b e s e e n fo r
th e G L R a n d P B R , re s p e c tiv e ly (F ig u re V III- 3 ).
In th e c a s e o f iG L R , th e b e s t fit fo r e x p e rim e n ta l tra c e r re s p o n s e w a s
a c h ie v e d w ith a n o n -lin e a r a d ju s tm e n t o f C S T R -in -s e rie s m o d e l (T a b le
V III- 4 ), w h ic h re p re s e n ts a n a lm o s t id e a lly m ix e d re a c to r a s e x p e c te d .
T h e C S T R m o d e l a d ju s ts th e n u m b e r o f c o n tin u o u s a g ita te d ta n k s in
s e rie s to th e e x p e rim e n ta l d a ta . In th is c a s e th e a d ju s te d p a ra m e te r
w a s n e a rly 1 , in d ic a tin g th e p e rfe c tly m ix e d c ha ra c te r o f th e s tu d ie d
G L R . A s d is p e rs io n m o d e l is n o rm a lly a p p lie d fo r P F R it w a s u s e le s s to
a p p ly it fo r th is ty p e o f re a c to r. O n th e o th e r h a n d it is c o m m o n ly
k n o w n th a t m o re th a n 1 0 C S T R in s e rie s m im ic w e ll th e p lu g-flo w
re g im e , w h ic h m e a n s th a t th is m o d e l c a n b e a p p lie d to P B R , w h e re a
p lu g -flo w re g im e is e x p e c te d .
A ll te s te d m o d e ls (C S T R in s e rie s , d is p e rs io n w ith a n d w ith o u t d e a d
z o n e s ) c a n b e a d ju s te d to p lu g -flo w re g im e . T h e in itia l o b s e rv a tio n s o f
th e e x p e rim e n ta l d a ta in d ic a te d th e p re s e n c e o f plu g flo w re g im e in o u r
P B R , th e re fo re in th is c a s e a ll m o d e ls w e re a p p lie d (F ig u r e V III- 3 a n d
T a b le V III- 4 ).
F o r th e a x ia l d is p e rs io n m o d e l, th e fittin g p a ra m e te r D /u L = 0
c o rre s p o n d s to id e a l p lu g flo w , a n d D /u L = ! to id e a l m ix e d flo w . T h e
o b ta in e d e x p e rim e n ta l d is p e rs io n c o e ffic ie n t fo r P B R (D /u L = 0 .2 3 )
s u g g e s ts th e p re s e n c e o f a s ig n ific a n t a x ia l d is p e rs io n (T a b le V III- 4 ).
T h is h ig h d is p e rs io n w a s n o t o b ta in e d n e ith e r b y th e C S T R in s e rie s o r
b y th e d is p e rs io n m o d e ls . B o th fa ile d in s im u la tin g th e e x p e rim e n ta l
d a ta (F ig u r e V III- 3 ). T h e P B R in itia lly d id n o t fit in to th e c o n c e p t o f
id e a lly m ix e d o r p lu g flo w -lik e re a c to rs . T h is d iffe re n c e in d ic a te d th e
p re s e n c e o f d e a d z o n e s in s id e th e P B R , w h ic h c a u s e d th e C S T R a n d
d is p e rs io n m o d e ls to b e b e y o n d th e lim its o f th e ir v a lid ity (L e v e n s p ie l,
1 9 9 9 ).
241
Chapter VIII
Table VIII-4. Hydrodynamic properties of packed-bed (PBR) and gas-lift

(GLR) reactors.
Experiment/
Model
Parameter
GLR
Errora
PBR
Errora
Experiment
D/uLd
0.23
CSTRb in series
Nc
1.05
0.14
5.07
4.54
Dispersion
D/uLd
0.16
10.47
VDZe
437.97
D/uLd
0.03
Dispersion with
dead zones
0.79
Sum of the errors between the experimental curves and theoretical

models as obtained from Solver application.
b
Continuous stirred tank reactor
c
Number of CSTR in series
d
Dimensionless dispersion parameter (u linear velocity, L height of the
carrier bed, D axial dispersion coefficient)
e
Volume of dead zones (mL)
In o rd e r to a c h ie v e a b e tte r fit to e x p e rim e n ta l d a ta , a m u ltip a ra m e te r m o d e l w a s a p p lie d . T h e c h o s e n m o d e l w a s a m o d ific a tio n o f

th e d is p e rs io n m o d e l, ta k in g in to a c c o u n t th e p re s e n c e o f d e a d z on e s ,
a n d d e s c rib e d s a tis fa c to rily th e e x p e rim e n ta l d a ta (F ig u r e V III- 3 A ). T h e
m o d ifie d d is p e rs io n m o d e l h a s tw o m a in p a ra m e te rs : th e d is p e rs io n
c o e ffic ie n t (D /u L ) a n d th e v o lu m e o f d e a d z o n e s V D Z (T a b le V III- 4 ).
T h e s ig n ific a n t v o lu m e o f d e a d z o n e s in s id e th e P B R (c a 3 3 % ) b o th
d e c re a s e s th e re a l w o rk in g v o lu m e o f P B R a n d h in d e rs th e c irc u la tio n
o f th e liq u id p h a s e in s id e th e re a c to r. T h e o c c u rre n c e o f d e a d z o n e s
p ro m o te s th e fo rm a tio n o f n o n -p ro d u c tiv e a re a s w ith m a s s tra n s p o rt
lim ita tio n s . In a d d itio n th e s e lim ita tio n s c a n in flu e n c e n e g a tiv e ly th e
fin a l p ro d u c t q u a lity b e c a u s e th e m a s s tra n s fe r lim ita tio n s m ig h t le a d to
a u to ly s is o f s ta rv in g c e lls (V e rb e le n e t a l., 2 0 0 6 ; V irk a ja rv i a n d L in k o ,
1 9 9 9 ).
C o n s id e rin g th e P B R c o n fig u ra tio n th e m a in d e a d z o n e s m a y b e a t
th e b o tto m n e a r th e in flo w , b e c a u s e th e liq u id w a s n o t w e ll d is p e rs e d .
In o rd e r to d e c re a s e th e v o lu m e o f d e a d z o n e s in P B R a p o ro u s
d is trib u tio n la y e r (g la s s s p h e re s , s a n d , e tc .) c a n b e in s e rte d o n th e
b o tto m o f th e re a c to r, p ro m o tin g a b e tte r liq u id flo w d is trib u tio n
242
Chapter VIII
th ro u g h o u t th e c ro s s s e c tio n a re a o f th e s y s te m (R ic h a rd s o n e t a l.,
2 0 0 2 ).
Figure VIII-3. Experimental residence time distribution (RTD) curves (!) for
PBR (A) and GLR (B) vs. mathematical simulations (models) of RTD curves: CSTR in series model; - - - dispersion model; ! ! dispersion model with dead
zones.
T h e s iz e o f c a rrie r p a rtic le s a p p lie d in P B R a ls o in flu e n c e s th e liq u id

flo w d is trib u tio n a n d c o n s e q u e n tly th e u tiliz a tio n o f b ig g e r p a rtic le s
m ig h t re d u c e d is p e rs io n ; a s a re s u lt, a ty p ic a l p lu g flo w re g im e m ig h t
be
a c h ie v e d
if
d e s ire d
(R ic h a rd s o n
et
a l.,
2 0 0 2 ).
G e n e ra lly ,
th e
g ra n u la rity o f th e b e d s h o u ld re fle c t a c o m p ro m is e b e tw e e n th e c a rrie r

a n d re a c to r s iz e , b io m a s s g ro w th ra te a n d th e d e s ire d flo w p a tte rn
in s id e th e c o lu m n .
VIII.4.2 Fermentations performance and flavour compounds

A F B s a re o fte n c h a ra c te riz e d b y th e la c k o f fla v o u r c o m p o u n d s fo u n d
in re g u la r b e e rs (P e rp te a n d C o llin , 1 9 9 9a , 1 9 9 9 b ). T h e re fo re th e tw o
243
Chapter VIII
s tra in s s tu d ie d in th is th e s is w e re e v a lu a te d b a s e d o n to ta l H A , E S a n d
O A c o n te n t, a s w e ll a s H A /E S ra tio . T h e c o m p a ris o n w a s m a d e e ith e r
b e tw e e n re a l o r h y p o th e tic a lly d ilu te d o r c o n c e n tra te d A F B s . W h ile
d ilu tio n o f m o re c o n c e n tra te d b e e rs is n o w a d a y s a c o m mo n p ra c tic e ,
th e h y p o th e tic a l c o n c e n tra tio n o f A F B s is b a s e d o n a s im p lify in g
a s s u m p tio n th a t fu rth e r fe rm e n ta tio n w o u ld in c re a s e th e c o n te n t o f
e a c h fla v o u r a c tiv e c o m p o u n d lin e a rly b y th e s a m e fa c to r a s th a t o f
e th a n o l.
VIII.4.2.1 Ethanol
and higher alcohols
T h e fin a l e th a n o l c o n c e n tra tio n a t d iffe re n t fe rm e n ta tio n s (s e e ) w a s

s tro n g ly in flu e n c e d b y th e p ro c e s s c o n d itio n s a n d th e p ro d u c tio n
s tra in .
The
re s u lts
o b ta in e d
under
c o m p a ra b le
c o n d itio n s
(e g .
b a tc h
fe rm e n ta tio n ) im p ly th a t th e g e n e tic a lly m a n ip u la te d ! K G D 2 s tra in

p ro d u c e s le s s e th a n o l a n d a t a c o n s id e ra b ly lo w e r y ie ld th a n th e s tra in
W 9 6 (T a b le V III- 5 ). T h e c o m p a ris o n o f e th a n o l fo rm a tio n in c o n tin u o u s
s y s te m s is le s s s tra ig h tfo rw a rd , s in c e th e tria ls d iffe r in b io m a s s
c o n te n t a n d d ilu tio n ra te s . H o w e ve r, th e y ie ld o f e th a n o l fo rm a tio n b y
th e ! K G D 2 s tra in in c o n tin u o u s s y s te m s is a g a in s ig n ific a n tly lo w e r
th a n fo r th e s tra in W 9 6 (T a b le V III- 5 ).
In o rd e r to m a k e e a s ie r th e c o m p a ris o n o f fla v o u r a c tiv e b y -p ro d u c ts
(H A , E S a n d O A ) fo rm e d in d iffe re n t s y s tem s tw o d iffe re n t a n a ly s is w e re
m a d e : (1 ) b a s e d o n A F B s w ith d iffe re n t d e g re e o f a tte n u a tio n a n d
e th a n o l c o n te n t a n d /o r (2 ) o n h y p o th e tic a l p ro d u c ts u n ifie d b y th e
le g a lly
a d m itte d
m a x im u m
e th a n o l
c o n te n t
(0 .5 %
by
v o lu m e
or
3 .9 4 5 g /L in th e E U c o u n trie s ). T h e s e re s u lts a re p re s e n te d in T a b le
V III- 6 b e lo w .
T h e re s u lts in d ic a te th a t th e ! K G D 2 s tra in s h o w e d a n in c re a s e d H A
fo rm a tio n , p a rtic u la rly w h e n c o m p a rin g th e h y p o th e tic a l p o te n tia l fo r
H A fo rm a tio n a t u n ifie d 0 .5% (v o l./v o l.) e th a n o l c o n te n t (T a b le V III- 6 ).
244
Chapter VIII
Table VIII-5. Production conditions and basic parameters of real alcohol-free

beers produced in continuous PBR and iGLR with SG and Cc as carriers, and
in batch fermentations.
Batch
Batcha
W96
Industrial
8/2
8/2
NAd
0.28
0.15
0.19
1.20
0.07
4.8
3.8
17.5
8.1
1.08
1.26
NAd
0.50
0.39
1.14
0.25
0.22
0.36
!0.50
23.90
25.78
14.13
24.34
25.14
25.78
NAd
Yielde (gE/gURS) 0.453
0.451
0.487
0.239
0.232
0.416
NAd
Reactor
PBR
GLR
GLR
Yeast strain
W96
W96
W96
Carrier
Cc
Cc
SG
Cc
0.30
0.07
XIBb (gDB/gDC)
0.05
XTOTc (g/L)
Temperature
(C)
Dilution rate
(hr-1)
% Ethanol
(vol/vol)
Reducing
sugars (g/L)
PBR
Batch
!KGD2 !KGD2
Commercial AFBs produced in the Czech republic by limited fermentation

using bottom fermenting yeast strains.;
b
Immobilized biomass concentration (gDry Biomass/gDry Carrier).;
c
Total biomass concentration.;
d
Information not available.;
e
Yield of ethanol (gEthanol/gUtilized Reducing Sugars)
T h is
a p p lie s
b o th
fo r
lim ite d
b a tc h
and
c o n tin u o u s
PBR
w ith
c o rn c o b s (C c ). S im u lta n e o u s ly it c a n b e s e e n th a t th e re a l H A c o n te n t
in
most
of
th e
AFBs
p ro d u c e d
under
la b o ra to ry
c o n d itio n s
was
s ig n ific a n tly lo w e r th a n in th e c o m m e rc ia l p ro d u c ts . T h e o n ly e x c e p tio n

is th e s tra in W 9 6 im m o b iliz e d in s p e n t g ra in s (S G ) in G L R , w h ic h c a n b e
e x p la in e d b y th e h ig h e s t to ta l b io m a s s p re s e n t (T a b le V III- 5 ) th a t, in
tu rn , c a n b e re la te d w ith H A p ro d u c tio n (W illa e rt a n d N e d o v ic , 2 0 0 6 ).
T h e s tra in ! K G D 2 w a s a b le to p ro d u c e a m o u n ts o f H A c o m p a ra b le to
in d u s tria l A F B s o n ly w h e n th e h y p o th e tic a l H A c o nte n t c o rre s p o n d in g to
m a x im u m a llo w e d e th a n o l c o n c e n tra tio n w a s c o n s id e re d (T a b le V III- 5 ).
In te rm s o f E S fo rm a tio n u n d e r c o m p a ra b le c o n d itio n s (b a tc h a n d
P B R w ith C c ) th e ! K G D 2 s tra in s e e m s to b e a g a in s u p e rio r to s tra in
W 9 6 . T h e h ig h e r e s te r fo rm a tio n b y ! K G D 2 s tra in is e v e n m o re o b v io u s
fo r h y p o th e tic a l A F B s w ith 0 .5 % (v o l/v o l) e th a n o l c o n te n t (T a b le V III- 6 ).
U n d e r la b o ra to ry c o n d itio n s th e E S c o n te n t o f c o m m e rc ia l A F B s w a s in
245
Chapter VIII
fa c t a c h ie v e d o n ly in th e iG L R w ith s tra in W 9 6 a n d S G a n d th e o re tic a lly

it c o u ld b e re a c h e d o r s u rp a s s e d b y s tra in ! K G D 2 b o th in b a tc h a n d
c o n tin u o u s P B R w ith C c (T a b le V III- 6 ).
T h e H A /E S ra tio in re g u la r b e e rs is c o n s id e re d fa v o ra b le in th e ra n g e
3 to 4 (K u n z e , 2 0 0 4 ) b u t in th e th re e a n a ly z e d c o m m e rc ia l A F B s
p ro d u c e d in th e C z e c h R e p u b lic th is ra tio w a s in th e ra n g e 4 to 1 0 .
T h e re fo re a ll th e re a l A F B s p ro d u c e d d u rin g th is w o rk a re c o m p a ra b le
to th e c o m m e rc ia l A F B s . A m o n g th e m th re e c o n tin u o u s ly fe rm e n te d
p ro d u c ts w e re re la tiv e ly c lo s e (5 .0-5 .7 ) a n d o n e p ro d u c t (b a tc h , ! K G D 2 )
w a s in th e ra n g e fo r re g u la r b e e rs (T a b le V III- 6 ). T h e c o n c e n tra tio n o f
e a c h in d iv id u a l H A a n d E S w a s b e lo w th e ta s te th re s h o ld s fo u n d in
re g u la r b e e rs (s e e T a b le II- 4 ). O n th e o th e r h a n d w h e n c o m p a re d w ith
th e th re s h o ld s in w a te r, a g e n e ra l o v e rp ro d u c tio n o f is o a m y l a lc o h o l
w a s o b s e rv e d b o th in c o n tin u o u s a n d b a tc h s y s te m s . T h is fa c t w a s a ls o
v e rifie d fo r c o m m e rc ia l C z e c h b e e rs . T h is e ffe c t w a s n o t p e rc e iv e d in
te rm s o f th e c h a ra c te ris tic fla v o u r o n th e te s te d s a m p le s . S o m e a u th o rs
m e n tio n e d
th a t
th e
th re s h o ld
of
compounds
in
AFBs
s h o u ld
be
c o m p a ra b le to th e th re s h o ld o f th e c o m p o u n d in w a te r in s te a d o f
c o m p a rin g w ith th e tre s h o ld in re g u la r b e e rs , a s it is d o n e b y th e
m a jo rity o f th e s tu d ie s (P e rp te a n d C o llin , 1 9 9 9a ). In th is c a s e , fo r a ll
s a m p le s , th e is o a m y l a lc o h o l h a s b ig g e r c o n c e n tra tio n th a n th e n o rm a l
tre s h o ld in w a te r a n d a s tro n g b a n a n a fla v o u r s h o u ld b e n o te d .
H o w e v e r it w a s n o t d e te c te d e v e n in th e c o m m e rc ia l C z e c h A F B s ..
T h e re fo re , a n d c o n s id e rin g th a t re g u la r b e e r th re s h o ld s a re u s e d b y
a lm o s t e v e ry s tu d ie s (B r n y ik e t a l., 2 0 0 5 ; W illa e rt a n d N e d o v ic , 2 0 0 6 )
in th e p re s e n t th e s is th e s a m e c rite ria w e re b e u s e d . In fa c t it is v e ry
im p o rta n t to e v a lu a te th e c o rre c t fla v o u r c o m p o n d s tre s h o ld in A F B s .
T h e m a in re a s o n is to h a v e a c o rre c t e v a lu a tio n o f th e in flu e n c e th a t
e a c h c o m p o u n d h a s in th e fin a l A F B fla v o u r. F ro m T a b le II- 4 th e
tre s h o ld o f e th a n o l in w a te r is 9 9 0 m g /L w h ile in re g u la r b e e rs it is
1 4 0 0 0 m g /L . In A F B th e m a x im u m e th a n o l c o n te n t a llo w e d in E U
c o u n trie s is 3 9 0 0 m g /L (T a b le II- 4 ).
246
Chapter VIII
Table VIII-6. Higher alcohol (HA), Esters (ES) and Organic Acids (OA) content
(mg/L) of real AFBs produced in continuous PBR and iGLR with SG and Cc as
carriers, and in batch fermentations.
Reactor
PBR
GLR
GLR
PBR
Batch
Batch
Batcha
Strain
Carrier
W96
Cc
W96
SG
0.31
!KGD2
Cc
0.47
0.94b
!KGD2
0.69
1.57b
W96
0.26
0.36b
Industrial
-
Isobutanol
1-Butanol
0.03
0.010.01
Amyl Alcohol
0.93
W96
Cc
0.20
0.26b
0.08
0.1 b
0.38
0.49 b
2.69
3.44 b
0.28
0.36 b
3.62
4.65 b
0.30
0.38 b
Isoamyl Alcohol 2.92

2-Phenylethanol 0.24
1.15c
2.06
3.87
1.20
8.32
1.36
0.66
1.32
2.91
5.82
0.24
0.48
4.27
8.55
0.56
1.12
0.08
0.16
0.11
0.22
Total HA
4.43
Ethyl acetate
0.79
Amyl acetate
0.04
0.02
Isoamyl acetate 0.06
0.12
Ethyl caprylate
0.10
0.13
0.04
2-Phenylethyl
acetate
0.06
Total ES
0.89
1.60
HA/ES
5.0
0.40
0.51
9.2
5.2
0.75
1.50
5.7
0.21
0.48
2.39
5.43
0.43
0.98
3.71
8.44
0.74
1.68
0.06
0.13
0.11
0.25
0.20
0.45
1.11
2.52
3.35
1.640.35
5.780.75
1.730.82
10.32.14
1.560.55
0.010.02
0.180.09
0.010.01
0.020.03
0.37
0.52 b
2.16
3.0 b
0.15
0.21b
2.94
4.09b
0.31
0.43b
0.820.18
0.31
0.43
9.5
1.790.68
7.13.2
22.5
21.8
29.8
38.4
34.56.4
13 c
28.8 b
43.6 b
67.7 b
53.3b
77.7
81
145
129
Citrate
85.3
83.6 c
111.322.2
99.6 b
162 b
329.5 b
179.2 b
65
24
19.5
87.8
87.225.6
Malate
40.5
32.2 c
83.3 b
48 b
44.3 b
122 b
46.8
214.6
276
57.8
Lactate
66.6
44.3 c
15648.4d
60 b
429.2 b 627 b
80.3 b
12.2
71.8
124
45
12.46.7
Succinate
8.6
9.8 c
15.6 b
143.6 b 282 b
62.4 b
7.8
7.3
10.7
14
Propionate
9.0
2.8 c
7.41.6
10 b
14.6 b
24.3 b
19.4 b
232
420.4
605
372
408.953
Total OA
233.4
185.7c
297.3b
841b
1375b
516.7b
a
Three different commercial AFBs produced in the Czech republic were
analyzed to obtain the average content of their flavour active compounds.; b
Values were obtained after multiplication of the experimental higher alcohol
concentration by a factor corresponding to the ratio between the maximum
allowed (0.5% (vol/vol)) and experimentally determined ethanol content for
the given AFB.; c Values were obtained after dilution of the fermented AFB to
ethanol content of 0.5% (vol/vol.); d Acidification with lactic acid was
indicated on two out of three commercial AFBs used in this work
Pyruvate
23.3
247
Chapter VIII
T h e re fo re it is e s s e n c ia l to d e te rm in e n e w tre s h o ld s fo r th e e th a n o l
ra n g e n o rm a lly fo u n d in A F B s ; th is w a s n o t fo u n d in th e lite ra tu re , a n d
it w a s n o t th e m a in s c o p e o f th is th e s is .
In o rd e r to m in im iz e th e ris k o f m ic ro b io lo g ic a l c o n ta m in a tio n , th e pH
o f w o rt in b a rre ls w a s a d ju s te d to 4 .5 w ith la c tic a c id p rio r to
fe rm e n ta tio n (s e e s e c tio n IV .2 .1 ). A s la c ta te is n o t a fe rm e n ta tio n b yp ro d u c t, it w a s e x c lu d e d fro m th e d a ta s h o w n in T a b le V III- 6 . In te rm s
o f O A fo rm a tio n th e y e a s t s tra in w ith d is ru p tio n in th e K G D 2 g e n e
behaved,
b o th
in
lim ite d
b a tc h
and
c o n tin u o u s
(P B R
w ith
Cc)
fe rm e n ta tio n s , a s e x p e c te d (S e le c k y e t a l, 2 0 0 3 ). It s h o w e d s ig n ific a n tly

h ig h e r O A fo rm a tio n a s c o m p a re d to s tra in W 9 6 a n d c o m m e rc ia l A F B s
(T a b le V III- 6 ). T h e g e n e tic a lly m a n ip u la te d s tra in p ro d u c e d a m o u n ts o f
O A (e x c e p t m a la te ) in c o n c e n tra tio n s c o m p a ra b le w ith th o s e fo u n d in
re g u la r a lc o h o lic b e e rs (s e e C h a p te r.II.4 .3 ); th is h a p p e n e d b o th in b a tc h
a n d c o n tin u o u s o p e ra tio n (B rig g s e t a l., 2 0 0 4 ).
B e s id e s , th e fo rm a tio n o f la c ta te b y ! K G D 2 s tra in w a s h ig h e r th a n
th e o v e ra ll c o n te n t (la c ta te a s b re w in g b y-p ro d u c t p lu s la c ta te a d d e d
fo r a c id ific a tio n ) o f th is O A in c o m m e rc ia l A F B s (T a b le V III- 6 ). T h e to ta l
O A c o n te n t a tta in e d th e h ig h e s t v a lu e s in lim ite d b a tc h fe rm e n ta tio n ,
fo r b o th y e a s t s tra in s (T a b le V III- 6 ). T h e W 9 6 s tra in s h o w e d a s lig h tly
h ig h e r fo rm a tio n o f c itra te a n d m a la te , b o th in b a tc h a n d c o n tin u o u s
s y s te m s , w h ile th e a v e ra g e O A c o n c e n tra tio n in c o m m e rc ia l A F B s
re v e a le d a re la tiv e ly lo w s u c c in a te c o n te n t (T a b le V III- 6 ).
VIII.4.3 Effect of yeast strain

T h e A F B s p ro d u c e d b y th e tw o s tu d ie d s tra in s (W 9 6 a n d ! K G D 2 )
w e re ra th e r d iffe re n t in a ll a n a ly z e d p a ra m e te rs . T h e ir d iffe re n t b e h a v io r
can
be
shown
on
th e
e a s ily
c o m p a ra b le
e x a m p le s
of
lim ite d
fe rm e n ta tio n in b a tc h m o d e a n d c o n tin u o u s fe rm e n ta tio n in P B R w ith

c o rn c o b s (C c ) a s c a rrie r. T h e ! K G D 2 s tra in s h o w e d (in b o th s y s te m s )
h ig h e r H A , E S a n d O A (T a b le V III- 6 ) fo rm a tio n , p a rtic u la rly w h e n
c o m p a rin g th e h y p o th e tic a l p o te n tia l fo r fo rm a tio n o f v o la tile s a t a n
248
u n ifie d
Chapter VIII
0 .5 % (v o l/v o l)
e th a n o l
c o n te n t.
U n d e rs ta n d a b ly ,
th e
to ta l
o rg a n ic a c id (O A ) fo rm a tio n b y th e ! K G D 2 s tra in s u rp a s s e s th e la g e r
y e a s t s tra in s d u e to th e O A o v e rp ro d u c tio n c a u s e d b y in c o m p le te T C A
c y c le o p e ra tio n (Z a w is la k e t a l., 1 9 9 9 ; S e le c k y e t a l., 2 0 0 3 ). K e e p in g in
m in d th a t v o la tile s (H A , E S ) a n d O A s a re d e s ira b le fo r th e fla v o u r o f
A F B s th e re s u lts s h o w th a t th e p e rfo rm a n c e o f th e ! K G D 2 s tra in w a s
s ig n ific a n tly b e tte r th a n th a t o f W 9 6 in lim ite d b a tc h fe rm e n ta tio n , w h ile
th is a d v a n ta g e s lig h tly d im in is h e d in c o n tin u o u s P B R w ith C c . T h is
s lig h t re d u c tio n in c o n tin u o u s P B R p ro v e d th e im p o rta n c e of te s tin g in
c o n tin u o u s s y s te m s y e a s t s tra in s th a t h a v e g o o d p e rfo rm a n c e in b a tc h
(S e le c k y e t a l., 2 0 0 3 ). T h e im p o rta n c e o f a c a re fu l s tra in s e le c tio n fo r
A F B p ro d u c tio n c a n b e d e m o n s tra te d a ls o b y c o m p a rin g th e p ro d u c ts
o f o u r tw o s tu d ie d s tra in s w ith th e a v e ra g e c o m p o s itio n o f th re e
c o m m e rc ia l A F B s p ro d u c e d b y lim ite d b a tc h fe rm e n ta tio n . W h ile th e
s tra in W 9 6 s e e m s to b e s u ita b le fo r A F B p ro d u c tio n o n ly in c o n tin u o u s
G L R w ith S G , th e ! K G D 2 s tra in h a s a p o te n tia l fo r s u ffic ie n t O A a n d
v o la tile fo rm a tio n d u rin g b o th c o n tin u o u s (P B R w ith C c ) a n d b a tc h A F B
fe rm e n ta tio n . T h e fa c t th a t d iffe re n t y e a s t s tra in s p ro d u c e v o la tile s a n d
O A s in d iffe re n t p ro p o rtio n s s u p p o rts th e p ro m is in g p o te n tia l o f s tra in
s e le c tio n fo r im p ro v in g th e q u a lity o f in d u s tria l A F B s .
B e s id e s th e fa v o ra b le fe rm e n ta tio n p e rfo rm a n c e , th e ! K G D 2 s tra in
tu rn e d o u t to b e u n a b le to firm ly a d h e re to S G . T h e re a s o n fo r lo w
b io film fo rm a tio n b y ! K G D 2 s tra in o n S G lie s p ro b a b ly in d iffe re n t
s u rfa c e p ro p e rtie s o f th e s e c e lls to g e th e r w ith iG L R ty p ic a l tu rb u le n t
re g im e (e s p e c ia lly in th e ris e r). T h e p ra c tic a l c o n s e q u e n c e o f th is fa c t
w a s th a t th e u s e o f ! K G D 2 s tra in in c o n tin u o u s G L R w ith S G fa ile d .
H o w e v e r, th e c h o ic e o f a p ro p e r re ac to r a n d c a rrie r ty p e (P B R w ith C c )
s h o w e d th a t a w e ll p e rfo rm in g , b u t lo o s e ly a d h e rin g s tra in c a n b e u s e d
in
c o n tin u o u s
fe rm e n ta tio n
s y s te m .
In
PBR
th e
yeast
c e lls
im m o b iliz a tio n m e th o d is s lig h t d iffe re n t th a n in iG L R . In iG L R if th e

a d s o rp tio n is w e a k, th e lo o s e ly a tta c h e d c e lls m a y b e m o re e a s ily
re le a s e d to th e fe rm e n ta tio n b u lk d u e to th e tu rb u le n c e ty p ic a l o f th is
ty p e o f re a c to rs . In th e P B R th e liq u id v e lo c ity is q u ite s m a lle r,
c o n s e q u e n tly b o th b u lk tu rb u le n c e a n d v e lo c ity a re lo w e r in th is ty p e o f
249
Chapter VIII
re a c to r. T h e n , b io film fo rm a tio n in th e c a rrie r is fa v o u re d (h ig h tic k n e s s )

and
m o re y e a s t c e lls a re re ta in e d in s id e th e P B R . In a d d itio n ,
s e d im e n ta tio n o f c e lls m a y b e p re s e n t th u s a llo w in g e v e n h ig h e r y e a s t

c e ll c o n c e n tra tio n s to b e re a c h e d . T h is is c le a r if th e y e a s t c e ll p ro file
tro u g h th e re a c to rs le n g th is a n a ly s e d (F ig u re V III- 4 ).
A!
Reactor Lenght / cm!
% Ethanol (vol./vol.)!
0!
0.2!
0.4!
41!
31!
18!
3!
Free Cells / (g/L)!

0.0!
Reactor Lenght / cm!
0.6!
0.2!
0.4!
0.6!
B!
0.8!
41!
31!
18!
3!
Figure VIII-4. Ethanol (A) and free cells (B) concentration troughout the PBR
length (from 0 cm to 41 cm) for S. pastorianus strain W96 ( ) and S.
cerevisiae BY4743 ( )
F ig u r e V III- 4 s h o w s th a t th e b io m a s s c o n c e n tra tio n is h ig h e r a t th e

P B R b o tto m fo r b o th s tra in s . T h e m a in re a s o n s fo r th is p h e n o m e n o n
a re : (1 ) h ig h s u g a r c o n c e n tra tio n in th a t a re a ; (2 ) n e a r fre s h w o rt in flo w
w ith h ig h d is s o lv e d o x y g e n ; (3 ) s e d im e n ta tio n o f y e a s t c e lls . T h e firs t
tw o fa c to rs in c re a s e y e a s t g ro w th in te n s ity a n d th e th ird o n e in c re as e s
y e a s t c e ll in th e b o tto m b y s e d im e n ta tio n o f c e lls th a t a re d e ta c h e d
fro m h ig h e r p o s itio n s o f th e P B R . E v e n c o n s id e rin g th e s e re a s o n s , it is
v e ry d iffic u lt to a s s e s s th e re a l im p a c t o f e a c h fa c to r in th e fin a l
b io m a s s c o n c e n tra tio n a t th e re a c to r b o ttom .
250
Chapter VIII
VIII.4.4 Effect of reactor design and carrier type

T h e c o m b in e d e ffe c t o f re a c to r d e s ig n a n d c a rrie r m a te ria l o n
fo rm a tio n o f fla v o u r a c tiv e c o m p o u n d s (H A , E S , O A ) c a n b e s h o w n b y
th e m o s t s u ita b le s tra in : th e b o tto m fe rm e n tin g la g e r u s e d (s tra in W 9 6 ),
w h ic h w a s te s te d in tw o d iffe re n t c o n tin u o u s re a c to rs (P B R , iG L R ) w ith
tw o c a rrie rs (C c , S G ), p lu s in lim ite d b a tc h fe rm e n ta tio n .
A s p re v io u s ly s ta te d , th e s tra in W 9 6 te n d s to p ro d u c e lo w e r a m o u n ts
o f H A a n d E S (T a b le V III- 6 ) b o th in c o n tin u o u s a n d b a tc h a rra n g e m e n t
c o m p a rin g to ! K G D 2 a n d in d u s tria l s tra in s . H o w e v e r, in th e iG L R w ith
S G , th e s tra in W 9 6 p ro d u c e d a n A F B w ith H A (8 .3 2 m g /L ) a n d E S
(1 .6 m g /L ), a fte r d ilu tio n o f b e e r to 0 .5 % (v o l./v o l.) e th a n o l, c o m p a ra b le
w ith A F B s p ro d u c e d b y in d u s tria l b a tc h p ro c e s s . A s e x p e c te d , the
p ro d u c tiv ity in c o n tin u o u s s y s te m (3 .6 h re s id e n c e tim e ) is s ig n ific a n tly
h ig h e r th a n th a t in b a tc h o p e ra tio n (7 2 h re s id e n c e tim e ).
T h e d iffe re n t c o m p o s itio n o f A F B fro m c o n tin u o u s iG L R w ith S G ,
c o m p a rin g to o th e r tria ls w ith s tra in W 9 6 , c a n b e m o s t p rob a b ly
a s c rib e d to th e s ig n ific a n tly h ig h e r to ta l b io m a s s c o n c e n tra tio n w ith a
la rg e
p ro p o rtio n
a d h e s io n
b e lo n g s
of
to
im m o b iliz e d
th e
most
b io m a s s
g e n tle
(T a b le
and
V III- 5 ).
n a tu ra l
A lth o u g h
m e th o d s (S m o g ro v ic o v a n d D o m n y , 1 9 9 9 ), th e im m o b iliz e d b io m a s s
p re s e n t o n S G in a fo rm o f th ic k y e a s t b io film is c e rta in ly e x p o s e d to
s e v e re m a s s tra n s fe r lim ita tio n s a n d s p e c ific m ic ro e n v iro n m e n t w ith
c o n c e n tra tio n g ra d ie n ts o f s u b s tra te s a n d p ro d u c ts s tu d ie s (B r n y ik e t
a l., 2 0 0 5 ). T h e d ire c t e v a lu a tio n o f im m o b iliz a tio n e ffe c ts o n c e ll
p h y s io lo g y is d iffic u lt, b u t a n in c re a s e d p ro d u c tio n o f H A a n d E S h a s
a lre a d y b e e n d e s c rib e d in th e c a s e o f c e lls im m o b iliz e d b y a tta c h me n t
(S h e n e t a l., 2 0 0 3 ). T h e p e rfo rm a n c e o f th e s tra in W 9 6 in c o n tin u o u s
iG L R w ith S G a s a c a rrie r w a s a n e x c e p tio n , p ro v in g th a t e v e n a s tra in
th a t s e e m s to b e le s s s u ita b le fo r A F B p ro d u c tio n c a n u n d e r fa v o ra b le
c o n d itio n s p ro d u c e a n a c c e p ta b le fin a l p ro d u c t (T a b le V III- 6 ).
T h e h y p o th e s is th a t th e b io film th ic k n e s s m a y p la y a n im p o rta n t ro le
in b y -p ro d u c t fo rm a tio n b y s tra in W 9 6 c a n b e a ls o s u p p o rte d b y th e
251
Chapter VIII
e x p e rim e n t w ith th e s a m e s tra in in th e iG L R w ith C c . In th is c a s e , th e

b io m a s s c o n c e n tra tio n w a s lo w e r th a n in th e c o n tin u o u s G L R w ith S G
(T a b le V III- 5 ) a n d it le d to a p ro d u c t w ith a s im ila r c o m p o s itio n a s
a c h ie v e d b y th e s tra in W 9 6 in b a tc h a n d P B R w ith C c (T a b le V III- 6 ).
N e v e rth e le s s in th e iG L R w ith C c th e e ffe c t o f th e lo w d ilu tio n ra te
(0 .0 7 h r - 1 , T a b le V III- 5 ) h a s to b e c o n s id e re d a s w e ll, b e c a u s e a t
p ro lo n g e d re s id e n c e tim e o f w o rt in th e c o n tin u o u s re a c to r a m o re
s ig n ific a n t s trip p in g o f v o la tile c o m p o u n d s b y d riv in g g a s c a n b e
e x p e c te d (L e h n e rt e t a l., 2 0 0 9 ; V irk a ja rv i a n d L in k o , 1 9 9 9 ).
A n o th e r
p ro c e s s
p a ra m e te r
th a t
sig n ific a n tly
d iffe rs
b e tw e e n
c o n tin u o u s im m o b iliz e d c e ll s y s te m s a n d b a tc h fe rm e n ta tio n s a n d

s im u lta n e o u s ly s tro n g ly in flu e n c e s th e p ro d u c tio n o f H A , E S a n d O A is
th e o x y g e n s u p p ly (L e h n e rt e t a l., 2 0 0 8 ). T h e to ta l o x y g e n lo a d o f w o rt
w a s a p p ro x im a te ly 8 a n d 3 m g /L fo r b a tc h a n d c o n tin u o u s s y s te m s ,
re s p e c tiv e ly . H o w e v e r, th e s u p p ly o f o x y g e n n o t o n ly d iffe rs o n th e to ta l
a m o u n t b u t a ls o in its su p p ly d u rin g th e fe rm e n ta tio n tim e . B a tc h
fe rm e n ta tio n is c h a ra c te riz e d b y h ig h in itia l o x y g e n c o n c e n tra tio n (c a . 8
m g /L ) a n d s u b s e q u e n t d e c lin e w h ile in th e c o n tin u o u s s y s te m s o x y g e n
is c o n s ta n tly s u p p lie d (0 .2-0 .9 m g /(L h )) in to th e fe rm e n ta tio n s y s te m
th ro u g h w o rt in flo w .
T h e c o n te n t o f O A w a s th e h ig h e s t fo r b o th y e a s t s tra in s in lim ite d
b a tc h fe rm e n ta tio n (T a b le V III- 6 ). T h e re fo re th is d iffe re n c e c a n b e m o s t
p ro b a b ly a s c rib e d to th e c o m b in e d e ffe c t o f im m o b iliz a tio n (c h a n g e s in
m e ta b o lic fu n c tio n s in d u c e d b y c o n ta c t o f c e lls w ith s o lid c a rrie r) a n d
re a c to r p ro p e rtie s (s u p p ly o f C O 2 a n d O 2 , re a c to r m ix in g ) (S h e n e t a l.,
2 0 0 4 ). T h e d iffe re n c e s in o x y g e n s u p p ly to c o n tin u o u s a n d b a tc h
fe rm e n ta tio n s c a n b e im p o rta n t a s re g a rd s O A fo rm a tio n s in c e th e
d e g re e o f re s p iro-fe rm e n ta tiv e m e ta b o lis m in flu e n c e s th e a c tiv ity o f
e n z y m e s in v o lv e d in th e T C A c y c le (Y a m a u c h i e t a l., 1 9 9 5 ).
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Chapter VIII
VIII.4.5 Effect of gas-phase in the final product

B o th p u rific a tio n p ro c e s s e s o f C O 2 o r in d u s tria l C O 2 a re e x p e n s iv e
a n d its u tiliz a tio n a t in d u s tria l s c a le m u s t re d u c e a t m in im u m th e
in d u s tria l n e e d s . T h a t is w h y it is e x tre m e ly im p o rta n t to a c h ie v e a
c o m p ro m is e b e tw e e n th e m in im u m g a s-flo w a n d a g o o d im m o b iliz a tio n
lo a d c a p a c ity (B r n y ik e t a l., 2 0 0 4 b ). In o rd e r to m a in ta in a n a e ro b ic
c o n d itio n s
d u rin g
p rim a ry
fe rm e n ta tio n
CO2
is
u s u a lly
e m p lo y e d .
G e n e ra lly , in s y s te m s u s in g iG L R fo r p rim a ry b e e r o r A F B fe rm e n ta tio n ,

a ir
is
in itia lly
in je c te d
to
p ro m o te
g ro w th ,
w h ic h
w ill
in c re a s e
im m o b iliz a tio n , th e n th e g a s -p h a s e is g ra d u a lly c h a n g e d to p u re C O 2

(B r n y ik e t a l., 2 0 0 6, 2 0 0 4 a ; L e h n e rt e t a l., 2 0 0 8 , 2 0 0 9 ).
T h is s e c tio n d is c u s s e s th e e ffe c t o f g ra d u a lly c h a n g in g th e g a sp h a s e fro m a ir to C O 2 a n d th e u s e o f o th e r in e rt g a s e s , w h ic h c a n
m a in ta in th e a n a e ro b ic c o n d itio n s (s u c h a s n itro g e n , N 2 ). D is s o lv e d C O 2
c a n a ffe c t y e a s t c e lls m e ta b o lis m a n d p e rfo rm a n c e b y: (1 ) a lte rin g
m e m b ra n e s tru c tu re in flu e n c in g th e c o m p o u n d s d iffu s io n ; (2 ) a lte rin g
th e s tru c tu re o f a m in o a c id s , p e p tid e s a n d p ro te in s ; a n d (3 ) in flu e n c in g
e n z y m e s re s p o n s ib le fo r c a rb o x ila tio n /d e s c a rb o x ila tio n re a c tio n s . T h e
C O 2 d is s o lu tio n d e p e n d s o n re a c to r d e s in g , p re s e n c e /a b s e n c e o f s o lid
p a rtic le s , p H , te m p e ra tu re , a n d m e d iu m p ro p e rtie s . H ig h c o n c e n tra tio n s
o f d is s o lv e d C O 2 d e c re a s e th e p H in th e b u lk a n d e n te r in th e y e a s t c e ll
c y to p la s m . In o rd e r to m a in ta in c y to p la s m p ro p e rtie s th e c e lls s p e n t
e n e rg y to fo rc e C O 2 to le a v e a n d a d e c re a s e in H A a n d E S fo rm a tio n is
re p o rte d (S h e n e t a l., 2 0 0 4 ). O n th e o th e r h a n d N 2 is a n in e rt g a s a t
g e n e ra l te m p e ra tu re s a n d p re s s u re . A t 1 0 C a n d a tm o s p h e ric p re s s u re
N 2 s o lu b ility in w a te r is a ro u n d 2 4 m g N 2 /k g H 2 O , w h ile fo r C O 2 a t s a m e
c o n d itio n s is a ro u n d 2 5 0 0 0 m g C O 2 /k g H 2 O . In a d d itio n , N 2 s e e m s to b e
e c o n o m ic a lly b e tte r th a n C O 2 . C o n s id e rin g th e a v a ila b ility o f N 2 in
a tm o s p h e re it w ill b e c h e a p e r fo r b re w e ry in d u s try to c o lle c t a nd p u rify
N 2 in s te a d o f C O 2 . C o n s id e rin g th is a n d th e a d v e rs e e ffe c ts o f C O 2 o n
c e ll w a ll m e m b ra n e it w a s s u g g e s te d to s tu d y th e e ffe c t o f d iffe re n t
g a s -p h a s e s in p rim a ry A F B fe rm e n ta tio n in a n iG L R .
253
It
is
a ls o
v e ry
Chapter VIII
im p o rta n t
to
u n d e rs ta n d
th e
oxygen
s u p p ly
in
c o n tin u o u s fe rm e n ta tio n s o f b e e r b e c a u s e it in flu e n c e s y e a s t c e lls

m e ta b o lis m a n d c o n s e q u e n tly th e fo rm a tio n o f a c tiv e v o la tile a n d
fla v o u r c o m p o u n d s (H A , E S ). T h e in flu e n c e o f th e g a s-p h a s e in b e e r
fla v o u r w a s te s te d in o rd e r to u n d e rs ta n d h o w c o u ld th e g a s -p h a s e
in flu e n c e th e v o la tile c o m p o u n d s . F o r th a t s e v e ra l m ix tu re s o f a ir, C O 2
and
N 2 w e re
te s te d
(2 0 % C O 2 v s
8 0 % a ir; 5 0 % C O 2 v s
5 0 % a ir; ;
8 0 % C O 2 v s 2 0 % a ir; 1 0 0 % C O 2 ; 5 0 % C O 2 v s 5 0 % N 2 ; 1 0 0 % N 2 ). D u rin g
th e s e fe rm e n ta tio n s s a m p le s fo r H A a n d E S a na ly s is w e re ta k e n w h e n
s te a d y s ta te w a s a c h ie v e d (s e c tio n V III.3 .4 ). P a ra m e te rs a s re a c to r a n d
w o rt b a rre l te m p e ra tu re (8 C ) a n d re s id e n c e tim e (1 4 .3 h ) w e re k e p t
c o n s ta n t. C o n s id e rin g th e d iffe re n c e s in s o lu b ility o f N 2 a n d C O 2 in th e
w o rt, a n d k n o w in g th a t C O 2 d e c re a s e s th e p H in th e b u lk , a c h a n g e in
p H w a s e x p e c te d , th u s p H w a s m o n ito re d d a ily . F ig u r e V III- 5 A s h o w s
th a t w h e n C O 2 w a s u s e d th e b u lk p H w a s s lig h ty b e lo w 4 .5 , e v e n
k e e p in g th e in flo w p H c o n s ta n t a t 4 .5 ; th e o p p o s ite w a s o b s e rv e d w h e n
p u re N 2 w a s in je c te d . In te rm s o f fre e b io m a s s it w a s d e te c te d a n
in c re a s e o f 5 0 % o n fre e b io m a s s c o n c e n tra tio n w h e n p u re N 2 w a s u s e d .
O n th e o th e r h a n d im m o b iliz e d b io m a s s d id n o t s h o w re a l d iffe re n c e s
e ith e r in p u re C O 2 o r N 2 . T h e s e d iffe re n c e s c a n b e e x p la in e d b y th e C O 2
d is s o lu tio n in th e b u lk m e d iu m . T h e e ffe c t o f C O 2 o n c e lls g ro w th h a s
b e e n v e rifie d to re d u c e fre e a n d im m o b iliz e d c e ll g ro w th b y 3 0 % a n d
1 0 % re s p e c tiv e ly (S h e n e t a l., 2 0 0 4 ).
In o u r c a s e th e in e x is te n c e o f
c h a n g e s in iB io c o n c e n tra tio n c a n b e e x p la in d e d b y d iffe re n c e s in th e

im m o b iliz a tio n m e th o d a n d b y th e m e th o d u s e to d e te rm in e im m o b iliz e d
c e lls m a s s , w h ic h h a s a n e rro r o f 6 .3 % (s e c tio n V II-3 .6 .3 ).
S u g a r u p ta k e a n d e th a n o l fo rm a tio n p a tte rn s s h o w th a t w h e n th e
ra tio o f C O 2 in C O 2 /A IR m ix tu re w a s in c re a s e d (fro m th e 1 5 t h d a y to th e
2 5 t h d a y ) s u g a r c o n s u m p tio n d e c re a s e s m a in ly th e m a lto s e u p ta k e ,
w h ic h le d to a d e c re a s e o f e th a n o l p ro d u c tio n (F ig u r e V III- 6 A /B ). B y
th e a n a ly s is o f F ig u r e V III- 5 B a n d F ig u re V III- 6 it is p o s s ib le to re la te
m a lto s e c o n s u m p tio n w ith fre e -c e lls c o n c e n tra tio n , u n d e r a n a e ro b ic
c o n d itio n s . B e tw e e n th e 3 1 s t d a y a n d th e 3 5 t h d a y , w h e n p u re N 2 w a s
in je c te d , th e in c re a se o n fre e -c e lls c o n c e n tra tio n w a s a c c o m p a n ie d b y
254
Chapter VIII
a s im u lta n e o u s d e c re a s e o f m a lto s e c o n c e n tra tio n .

A!
120%!
100%!
80%!
pH!
4.50!
60%!
4.00!
40%!
20%!
3.50!
CO2/Air or CO2/N2 !
5.00!
0%!
14!
24!
34!
44!
Time / days!
()'#
($!"#
(),#
(!!"#
()$#
'!"#
!)+#
&!"#
!)&#
%!"#
!)*#
$!"#
!)!#
!"#
(!#
$!#
*!#
%!#
-.$/012#32#-.$/4$##
-5677#-7889#/#:;/8<#
32#
##1=>.#:;=13?@AA#/;-@221B2<#
!"
,!#
C1?B#/#D@EA#
Figure VIII-5.A pH evolution vs gas composition during primary AFB

fermentation; B Free and Immobilized biomass evolution vs gas-phase
composition; ! - pH; " - iBIO (gBIOMASS/gDRY CARRIER); ! - Concenration of free
cells (g/L); -- : CO2/Air mixture; -- : CO2/N2 mixture; _ : Wort barrel pH.
M a lto trio s e c o n c e n tra tio n w a s c o n s ta n t a n d a ro u n d 5 g /L u n d e r

a n a e ro b ic c o n d itio n s . A s fo r g lu c o s e c o n c e n tra tio n , a s lig h t re la tio n
w ith im m o b iliz e d b io m a s s c a n b e o b s e rv e d in d ic a tin g th a t im m o b iliz e d
b io m a s s c o n s u m e s p re fe ra b ly g lu c o s e . Im m o b iliz e d c e lls a re re p o rte d
to h a v e c h a n g e s in c e ll w a ll c o m p o s itio n b u t th e s e c ha n g e s d o n o t
depend
on
th e
g a s-p h a s e
(S h e n
et
a l.,
2 0 0 4 ).
The
g a s -p h a s e
c o m p o s itio n c a n a ffe c t th e m e m b ra n e s o lu b ility a n d s e le c tiv ity to s o m e
255
Chapter VIII
c o m p o u n d s , le a d in g im m o b iliz e d c e lls to c o n s u m e g lu c o s e in s te a d o f
m a lto s e . H o w e v e r, th is re la tio n is n o t s tra igh tfo rw a rd a s it o c c u rs fo r
m a lto s e a n d fre e c e lls a n d fu rth e r s tu d ie s m u s t b e u n d e rta k e n .
120%!
100%!
C Xavg / (g/L)!
15.00!
80%!
10.00!
60%!
40%!
5.00!
20%!
0.00!
CO2/Air or CO2/N2 !
A!
20.00!
0%!
10!
20!
30!
40!
50!
Time / Days!
120%!
100%!
80%!
60%!
40%!
20%!
0%!
1.50!
1.00!
0.50!
0.00!
10!
20!
30!
40!
50!
CO2/Air or CO2/N2 !
% Ethanol (vol./vol.)!
B!
2.00!
Time / Days!
Figure VIII-6. A Sugars evolution vs gas composition during primary AFB

fermentation; B Ethanol evolution vs gas-phase composition; ! - Glucose;
" - Maltose; ! - Maltotriose;! - Ethanol ; -- : CO2/Air mixture; -- : CO2/N2
mixture.
In
a d d itio n ,
th e
e v a lu a tio n
of
some
v o la tile
compounds
was
p e rfo rm e d a n d is p re s e n te d in T a b le V III- 7 . A s it w a s e x p la in e d in
s e c tio n V II.3 .7 .3 th e m e th o d u s e d in th is s e c tio n is d iffe re n t fro m th e
m e th o d p ro p o s e d b y th e E u ro p e a n B re w e ry C o n v e n tio n (E B C , 1 9 8 7 ). It
is im p o rta n t to n o tic e th a t th e m e th o d s u s e d a re : (1 ) v a lid a n d re p o rte d
b y o th e r a u th o rs ; (2 ) a ll s a m p le s w e re a n a ly s e d b y th e s a m e p ro c e d u re
a n d th e re fo re a re p e rfe c tly c o m p a ra b le . M o re o v e r th e ra n g e o f th e
v a lu e s p re s e n t in T a b le V III- 7 fo r s e v e ra l c o m p o u n d s (e .g . 1-p ro p a n o l
a n d e th y l a c e ta te ) is s im ila r to th e v a lu e s p re s e n t in litera tu re a s
d is p la y e d in T a b le II - 4 (L e h n e rt e t a l., 2 0 0 8 ).
A firs t lo o k a t T a b le V III- 7 s h o w s th a t e th a n o l p ro d u c tio n e x c e e d e d
256
Chapter VIII
th e m a x im u m a llo w e d in E U le g is la tio n fo r A F B s (C C E , 1 9 9 2 ). In fa c t in
b re w in g in d u s try b e e rs m a d e in b a tc h a re n o t e x ac tly e q u a l in te rm s o f
e th a n o l c o n te n t, a n d in o rd e r to o b ta in a s ta n d a rd fin a l p ro d u c t s o m e
p ro c e s s e s a re a p p lie d . W h e n , a t th e e n d o f a b a tc h fe rm e n ta tio n , th e
fin a l e th a n o l c o n te n t o f a tra d itio n a l b e e r is h ig h e r th a n u s u a l th is b e e r
c a n b e d ilu te d in ste riliz e d w a te r o r m ix e d w ith o th e r b e e r fro m o th e r
b a tc h fe rm e n te n ta tio n in o rd e r to o b ta in th e re q u ire d e th a n o l c o n te n t.
In o u r c a s e , to a c h ie v e th e le g a l e th a n o l c o n te n t a d ilu tio n is re q u ire d .
The
th e o te ric a l
re s u lts
fro m
th is
d ilu tio n
(c o n s id e rin g
fo r
each
c o m p o u n d th e s a m e fa c to r a s fo r e th a n o l) a re p re s e n te d in b ra c k e ts in
T a b le V III- 7 . T h e re fo re , th is c o rre c te d v a lu e s a re th e o n e s u s e d to b e
c o m p a re w ith th e lite ra tu re a n d w ith th e re s u lts o b ta in e d fo r th e
c o m m e rc ia l P o rtu g u e s e A F B s a n a ly s e d (C o m m 1 a n d C o m m 2 ).
An
a n a lis y s
on
T a b le
V III- 7
shows
s tro n g
in flu e n c e
of
th e
c o n d itio n s u s e d (g a s-p h a s e c h a n g e ) o n th e fin a l c o n c e n tra tio n o f H A .

T h e to ta l p ro d u c tio n o f H A is in th e s a m e ra n g e o f c o m m e rc ia l be e rs
b u t b e lo w th e tre s h o ld v a lu e s (T a b le II 4 ). G e n e ra lly, th e to ta l c o n te n t
o f H A in c o n tin u o u s A F B p ro d u c e d is h ig h e r th a t C o m m 1 a n d lo w e r th a n
C o m m 2 . In C o m m 1 th e to ta l H A c o n te n t is m a in ly s u p p o rte d b y th e
c o n te n t o f 2 -p h e n y le th a n o l (fla v o u r tre s h o ld : 1 2 5 m g /L in M e ilg a a rd
(1 9 7 5 )), w h ic h is q u ite h ig h e v e n w h e n it is c o m p a re d w ith th e c o n te n t
o f th is a lc o h o l in c o m m e rc ia l C z e c h A F B s (T a b le V III- 6 ).
A m o n g th e c o n tin u o u s A F B s a m p le s , a s e x p e c te d , th e H A p ro d u c tio n
is h ig h e r w h e n a ir is p re s e n t. T h is is s im ila r to th e e x p e rim e n ts
p e rfo rm e d b y L e h n e rt e t a l. (2 0 0 8 ), w h o d e m o n s tra te a n in c re a s e o f H A
w ith a n in c re a s e o f O T R . A s e x p la in e d , th e O T R d e p e n d s b o th fro m th e
in itia l o x y g e n o n th e in flo w w o rt a n d th e tra n s fe r fro m th e g a s-p h a s e to
th e fe rm e n ta tio n b u lk . In o u r c a s e th e liq u id flo w in th is s itu a tio n w a s
c o n s ta n t th e re fo re o n ly d iffe re n c e s in th e g a s-p h a s e a re c o n s id e re d .
C o m p a rin g th e tw o a n a e ro b ic g a s-p h a s e s (p u re N 2 w ith p u re C O 2 ) it
is p o s s ib le to o b s e rv e th a t w ith p u re N 2 th e a m o u n t o f to ta l H A is h ig h e r
th a n w ith p u re C O 2 . C o n s id e rin g th e h ig h s o lu b ility o f C O 2 a n d its
a d v e rs e e ffe c ts it is b e lie v e d th a t y e a s t c e lls in th e p re s e n c e o f h ig h
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Chapter VIII
a m o u n ts o f d is s o lv e d C O 2 a lte r th e ir m e ta b o lis m to c o u n te r-b a la n c e

s u c h C O 2 a d v e rse e ffe c ts , le a d in g to a n u n d e rp ro d u c tio n o f H A .
M o re o v e r th e H A is re la te d to y e a s t m e ta b o lis m in te n s ity , w h ic h w a s
h ig h e r in p u re N 2 th a n in p u re C O 2 (F ig u r e V III- 5 ). T h e v a lu e s o f to ta l
H A o b ta in e d w h e n p u re C O 2 (9 .7 m g /L ) w a s a p p lie d is s im ila r to th e
v a lu e s o b ta in e d p re v io u s ly (8 .3 2 m g /L in T a b le V III- 6 ) w h e n S G w e re
u s e d a s c a rrie r in a n iG L R .
C o n c e rn in g th e E S c o n te n t in T a b le V III- 7 it c a n b e o b s e rv e d th a t
th e y a re in th e s a m e ra n g e o f c o m m e rc ia l A F B s a n d b e lo w th e ir
th re s h o ld (T a b le II 4 ). A s re p o rte d in c h a p te r II (s e c tio n II.4 .2 ) E S
p ro d u tio n is in flu e n c e d b y o x y g e n c o n te n t a n d th e m e tb o lis m in te n s ity
(L e h n e rt e t a l., 2 0 0 8 ; S h e n e t a l., 2 0 0 3 ; V a n Ie rs e l e t a l., 1 9 9 9 ; W illa e rt
a n d N e d o v ic , 2 0 0 6 ). In o u r c a s e lo w E S p ro d u c tio n o c c u rs in th e
p re s e n c e o f a ir, b e in g h ig h e r w h e n in p re s e n ce o f N 2 o r C O 2 . W h e n in
th e p re s e n c e o f a ir a d u a l e ffe c t o c c u rs , w h ic h in flu e n c e s E S s y n th e s is .
In o n e h a n d y e a s ts m e ta b o lis m is h ig h e r (h ig h H A p ro d u c ito n ), b u t in
th e o th e r h a n d th e p re s e n c e o f o x y g e n re p re s s e s th e A T T a s e a c tiv ity . In
th e p re s e n c e o f o x y g e n y e a s t c e lls m e ta b o lis m is d riv e n to re p ira to ry
m e ta b o lis m a n d a w a y fro m fe rm e n ta tiv e m e ta b o lis m . T h e re fo re th e id e a l
s itu a tio n is to a c h ie v e a c o n d itio n w h e re y e a s t m e ta b o lis m is in c re a s e d
in a n a e ro b ic (o r c lo s e to a n a e ro b ic ) c o n d itio n s . A s s h o w e d b efo re
(F ig u re V III- 5 a n d F ig u r e V III- 6 ) in th e p re s e n c e o f N 2 th e re w a s a n
in c re a s e o f y e a s t c e lls in o u r iG L R (m a in ly fre e c e lls ). T h is is a n
in d ic a to r th a t w h e n N 2 is u s e d a s g a s-p h a s e in iG L R le s s s tre ss is
in d u c e d a n d y e a s t m e ta b o lis m in c re a s e s . In a d d itio n , a s th e re is fe w
o x y g e n a v a ila b le (o n ly th e d is s o lv e d o x y g e n fro m th e in flo w ) th e A T T a s e
a c tiv ity is n o t n e g a tiv e ly in flu e n c e d . T h e re fo re it is n o t s u rp ris in g th a t
th e b e s t c o n d itio n a s fa r a s E S p rod u c tio n is c o n c e rn e d w a s a c h iv e d
w h e n p u re N 2 w a s in je c te d in th e iG L R . A s fo u n d b y L e h n e rt a n d c ow o rk e rs (2 0 0 8 ) it is im p o rta n t th a t s o m e o x y g e n is p re s e n t (fo r H A
p ro d u c tio n
and
s te ro ls
s y n th e s is )
in
c o n tin u o u s
AFBs
p ro d u c tio n .
H o w e v e r it is a ls o im p o rta n t to re d u c e th e a d v e rs e e ffe c t o f C O 2 in th e
fe rm e n ta tio n p e rfo rm a n c e a n d th a t w a s a c h ie v e d w ith N 2 . N e v e rth e le s s
C O 2 is a n im p o rta n t c o m p o u n d in b e e r fla v o u r b e in g c o n s id e re d o n e o f
258
Chapter VIII
th e p rim a ry fla v o u r c o n s titu e n ts (B rig g s , e t a l. 2 0 0 4 ). T h e re fo re a n A F B

p ro d u c e d o n ly in th e p re s e n c e o f N 2 w ill la c k th e C O 2 in flu e n c e in th e
fin a l A F B fla v o u r. In fa c t th is w a s c o n firm e d b y th e d a ily d e g u s ta tio n
te s t p e rfo rm e d . H o w e v e r th is w a s ju s t a m e re in d ic a tio n a n d o f c o u rs e
a t in d u s tria l s c a le C O 2 c a n a lw a y s b e a d d e d . O f c o u rs e C O 2 is
e x p e n s iv e b u t a d d in g o n ly C O 2 a t th e e n d o f th e p ro c e s s w ill re d u c e th e
c o s ts to m in im u m v a lu e s a s its in je c tio n c a n b e fu lly c o n tro lle d .
O b s e rv in g th e c o m p o s itio n o f th e d iffe re n t a n a ly s e d E S (T a b le V III- 7 ) it
c a n b e v e rifie d , a s e x p e c te d th a t e th y l a c e ta te is in h ig h e s t a m o u n t
b e in g fo llo w e d b y e th y l c a p ry la te . If e th y l a c e ta te is in th e s a m e ra n g e
in c o n tin u o u s a n d c o m m e rc ia l A F B s th e o p p o s ite d o e s n o t o c c u r w ith
e th y l c a p ry la te . T h e s y n th e s is o f th is s p e c ific E S is n o t y e t fu lly
u n d e rs to o d , a s w e ll a s th e ir in flu e n c e in A F B fin a l fla v o u r (B r n y ik e t
a l., 2 0 0 8 ; V e rs tre p e n e t a l., 2 0 0 3 ; W illa e rt a n d N e d o v ic , 2 0 0 6 ). H o w e v e r
it s e e m s th a t im m o b iliz a tio n in d u c e s its p ro d u c tio n . In th e re a c to r u s e d
(iG L R ) th e re a re p h e n o m e n a , s u c h a s g a s s trip p in g , th a t s h o u ld b e
ta k e n in to a c c o u n t b e c a u s e th e y m a y re d u c e th e a m o u n ts o f v o la tile
c o m p o u n d s in th e fin a l p ro d u c t. M a c ie ira (2 0 0 8 ) fo u n d th a t lo s s e s o f
v o la tile c o m p o u n d s b y g a s s trip p in g m a in ly o c c u r fo r a ld e h y d e s a n d
d ia c e ty l (h ig h e r th a n 6 0 % fo r D ! 0 .2 h - 1 ) w h ile s u c h lo s s e s a re re d u c e d
fo r E S (a ro u n d 3 0 % in s im ila r c o n d itio n s ). T h e e x c e p tio n in E S w a s
e th y l c a p ry la te , w ith lo s s e s b y s trip p in g o f u p to 5 0 % (D ! 0 .2 h - 1 ).
C o n s id e rin g th a t th e g a s c o m p o s itio n is n o t c h a n g in g s ig n ific a n tly th e
g a s s trip p in g , it c a n b e c o n c lu d e d th a t e th y l c a p ry la te fo rm a tio n is
s o m e h o w in d u c e d in im m o b iliz e d y e a s t in a c o n tin u o u s A F B p ro d u c tio n
p ro c e s s u s in g S G a s c a rrie r a n d a n iG L R . H o w e v e r, a n d a s E S s y n th e s is
is a ls o s tra in s p e c ific , th is re s u lt c a n a ls o b e a c h a ra c teris tic o f th e
s tra in u s e d in th is s e c tio n (s e e s e c tio n V II.3 .1 ).
259
Chapter VIII
Table VIII-7. Mean concentration and associated error (p=0.05) of volatile compounds during different stages of
continuous fermentations.
Compound
100% Air
20% Air
and
80% CO2
50% CO2
and
50%N2
100% N2
100% CO2
Comm.1
Comm2.
Ethanol (g/L)
1-propanol (mg/L)
2-methyl-1-propanol (mg/L)
Amyl-alcohol (mg/L)
Isoamyl-alcohol (mg/L)
2-phenylethanol (mg/L)
tyrosol* (g/L)
9.2 (3.9)
4.80.4 (2.0)
3.10.2 (1.3)
2.10.7 (0.9)
14.12.3 (6.0)
3.90.6 (1.7)
3.70.5 (1.6)
5.4 (3.9)
2.80.6 (2.0)
1.20.1 (0.9)
1.50.2 (1.1)
11.11.8 (8.0)
2.80.9 (2.0)
2.10.9 (1.5)
6.4 (3.9)
2.20.4 (1.3)
1.50.1 (0.9)
1.60.4 (1.0)
8.41.3 (5.1)
2.20.4 (1.3)
2.71.3 (1.6)
5.2 (3.9)
2.70.5 (2.0)
3.70.7 (2.8)
3.20.3 (2.4)
11.41.6 (8.6)
2.90.9 (2.2)
6.02.3 (4.5)
4.6 (3.9)
1.80.2 (1.5)
1.10.3 (0.9)
1.40.0 (1.2)
5.70.4 (4.8)
1.40.8 (1.2)
tr
< 3.9
1.00.1
0.70.3
0.60.1
2.70.3
0.90.4
tr
< 3.9
0.90.1
1.20.2
1.50.3
71.2
25.42.5
24.75.2
Total HA
28 (11.9)
19.4 (14.0)
15.9 (9.7)
23.9 (17.9)
11.4 (9.7)
5.9
36
ethyl acetate (mg/L)

Isoamyl acetate* (g/L)
Ethyl caprylate* (g/L)
2-Phenylethyl acetate* (g/L)
ethyl hexanoate* (g/L)
ethyl decanoate* (g/L)
1.60.7 (0.7)
22.11.5 (9.4)
225.28.8 (95.5)
130.52.6 (55.3)
67.15.2 (28.4)
41.94.5 (17.8)
1.20.3 (0.9)
9.60.7 (6.9)
109.612.0 (79.2)
22.31.9 (16.1)
32.91.5 (23.8)
11.82.6 (8.5)
2.10.6 (1.3)
44.75.2 (27.2)
378.472.2 (230.6)
209.214.8 (127.5)
66.46.8 (40.5)
134.227.2 (81.8)
5.20.5 (3.9)
199.7105.8 (149.8)
639.7338.4 (479.8)
163.574.6 (122.6)
134.571.5 (100.9)
252.8153.2 (189.6)
0.90.3 (0.8)
22.51.6 (19.1)
600.840.9 (509.4)
44.43.6 (37.6)
78.05.0 (66.1)
270.816.2 (229.6)
1.50.2
14.20.4
3.91.1
12.00.1
3.40.2
4.71.4
4.01.1
297.4148.1
67.325.6
88.736.8
52.328.2
8.03.8
Total ES
HA/ES
acetaldehyde (mg/L)
2.1 (0.9)
12.6
8.72.6 (3.7)
1.4 (1.0)
13.8
5.92.0 (4.3)
2.9 (1.8)
5.1
3.00.2 (1.8)
6.6 (4.9)
3.5
3.70.8 (2.8)
1.9 (1.6)
5.8
2.00.3 (1.7)
1.6
3.8
3.71.8
4.6
7.8
2.00.7
Total AL
8.7 (3.7)
5.9 (4.3)
3.0 (1.8)
3.7 (2.8)
2.0 (1.7)
3.7
2.0
Legend: In brackets are present the theoretically values obtained if a dilution of the fermented AFB was done to obtain
an ethanol content of 0.5% (vol/vol) or 3.9 g/L; Comm1 and Comm2: Commercial Protugues AFBs.
260
F in a lly
T a b le
Chapter VIII
V III- 7
a ls o
shows
th e
amount
of
a ld e h y d e s .
As
e x p e c te d in th e p re s e n c e o f a ir (h ig h e r m e ta b o lis m ) th e a c e ta ld e h y d e
c o n c e n tra tio n is h ig h e r. H o w e v e r, in th e a b s e n c e o f a ir th e v a lu e s o f
to ta l a ld e h y d e s a re c o m p a ra b le w ith th e c o m m m e rc ia l b e e rs a n d b e lo w
th e th re s h o ld . A s in p u re N 2 th e y e a s t g ro w th in c re a s e d , a n in c re a s e o f
a c e ta ld e h y d e w a s a ls o e x p e c te d , w h ic h a c c o rd in g ly to th e d a ta in
T a b le V III- 7 w a s n o t o b s e rv e d . A ld e h y d e s a re re s p o n s ib le fo r th e w o rty
o ff-fla v o u rs a n d a s th e ir th re s h o ld is lo w (1 0 m g /L , s e e T a b le II- 4 ), th e y
c o n trib u te la rg e ly fo r A F B s o ff-fla v o u rs . S tu d ie s p e rfo rm e d in iG L R b y
L e h n e rt a n d c o -w o rk e rs (2 0 0 8 ) s h o w e d th a t a ld h e y d e s lo s s e s b y g a s
s trip p in g a re v e ry h ig h fo r th is re a c to r d e s ig n (a b o v e 8 0 % ), w h ic h is
o n e m o re a d v a n ta g e o f u s in g iG L R fo r c o n tin u o u s A F B p ro d u c tio n
(L e h n e rt e t a l., 2 00 8 ; M a c ie ira , 2 0 0 8 ).
VIII.4.6 Effect of ageing: preliminary studies

T h e c o n tin u o u s fe rm e n ta tio n fo r te s tin g a g e in g e ffe c t o n fe rm e n ta tio n
p e rfo rm a n c e a n d in y e a s t p h y s io lo g y w a s d iv id e d in tw o p e rio d s (F ig u re
V III- 7 ):
A In th e firs t p e rio d c a rrie r lo s s e s w e re re p la c e d , m a in ta in in g th e
a m o u n t o f c a rrie r in s id e th e re a c to r a p p ro x im a te ly c o n s ta n t;
B In th e s e c o n d s ta g e n o c a rrie r w a s a d d e d ; th is w a s c o n s id e re d a s
a n a g e in g p e rio d .
T h is a llo w e d c o m p a rin g th e tw o s itu a tio n s , b e in g th e firs t 3 3 d a y s
(P e rio d A ) d e s c rib e d a s a c lo s e d s te a d y-s ta te , w h ile th e s u b s e q u e n t 2 3
d a y s (P e rio d B ) c h a ra c te ris e d b y a g ra d u a l a g e in g o f th e im m o b iliz e d
b io c a ta ly s t. A fte r th e a g e in g p e rio d o n e litre o f re a c to r c o n te n ts (c a .
1 /3
of
th e
to ta l
re a c to rs
v o lu m e),
in c lu d in g
spent
g ra in s
(c a .
8 % (v o l./v o l.) a n d m e d iu m w a s re p la c e d (a t th e 5 6 t h d a y ); th is a llo w e d

to u n d e rs ta n d b e tte r th e e ffe c t o f c a rrie r re p la c e m e n t th a t o c c u rre d
d u rin g p e rio d A .
261
Chapter VIII
F ig u r e V III- 7 s h o w s th e e v o lu tio n o f c a rrie r in s id e th e re a c to r a n d

im m o b iliz e d
fin is h e d
b io m a s s
a fte r
at
8 C.
Im m o b iliz e d
a p p ro x im a te ly
14 days,
b io m a s s
a tta in in g
c o n c e n tra tio n o f a p p ro x im a te ly 1 .7 g b i o m a s s /g d r y
g ra d u a l d e c re a s e to a ro u n d 1 .2 g b i o m a s s /g d r y
a c c u m u la tio n
carrier
carrier
m a x im u m
, fo llo w e d b y a
, v a lu e a t w h ic h a n
e q u ilib riu m w a s e s ta b lis h e d (F ig u re V III- 7 ). T h e 1 /3 o f th e re a c to r

v o lu m e
re p la c e d
s y n th e tic
model
by
s u s p e n s io n
m e d iu m
le d
to
of
c le a n
sudden
c a rrie r
suspended
d e c re a s e
of
th e
in
to ta l
im m o b iliz e d b io m a s s . H o w e v e r, th is w a s s u b s e q u e n tly fo llo w e d b y a

g ra d u a l a d h e s io n o f c e lls o n to th e c le a n s p e n t g ra in p a rtic le s re s u ltin g
in a n in c re a s in g a m o u n t o f im m o b iliz e d b io m a s s . T h e c a rrie r lo s s e s th a t
o c c u r d u e to o u tflo w lo s s e s (a n a v e ra g e o f 0 .2 5 g D R Y
CARRIER
/d a y w a s
c o n s id e re d ) a n d im m o b iliz e d b io m a s s s a m p le s w e re m o n ito re d a n d
c o n tro lle d .
Figure VIII-7. Variation of immobilized biomass concentration (!) and carrier

amount inside the reactor (!) during the experiment. Black arrows identify
carrier addition at fermentation days (left to right): 18th (6.9 g); 20th (2.4 g);
22nd (3.8 g); 26th (3.8 g); 33rd (2.9 g) and 56th (12.9 g).
D u rin g th e w h o le c o n tin u o u s fe rm e n ta tio n e x p e rim e n t th e fre e c e ll

v ia b ility re m a in e d re la tiv e ly c o n s ta n t. A s lig h t d e c re a s e w a s o b s e rv e d
fro m a p p ro x im a te ly 9 2 .5 % to 8 8 % v ia b le fre e c e lls u n til th e 1 7 t h d a y o f
262
Chapter VIII
fe rm e n ta tio n , re m a in in g re la tiv e ly c o n s ta n t a ro u n d th e la s t v a lu e fo r th e
re s t o f th e tim e . T h is e q u ilib riu m re s u lts fro m th e b a la n c e b e tw e e n
y e a s t c e ll g ro w th a n d y e a s c e lls w a s h-o u t fro m th e re a c to r. R e g a rd in g
th e v ia b ility o f im m o b iliz e d c e lls , it w a s o b s e rv e d th a t u n d e r th e
c o n d itio n s o f P e rio d A th e v ia b ility ( 8 5 % ) w a s re la tiv e ly c o n s ta n t,
h o w e v e r w h e n th e c a rrie r re p la c e m e n t s to p p e d d u rin g th e s e c o n d
p e rio d (P e rio d B ), a d e c re a s e in im m o b iliz e d c e ll v ia b ility w a s d e te c te d
up
to
77%
of
c e ll
v ia b ility .
A fte r
th e
c a rrie r
re p la c e m e n t,
th e
im m o b iliz e d b io m a s s in c re a s e d a ga in to v a lu e s s im ila r to th o s e e x is tin g

a t th e e n d o f P e rio d B . T h is c a n b e e x p la in e d b y th e m e c h a n is m o f
a d h e s io n o f th e n e w c e lls o n to s p e n t g ra in p a rtic le s (B r n y ik e t a l.,
2 0 0 4 a ). W h e n th e fre e b io m a s s a d h e s io n to s pe n t g ra in s re s to re d th e
m a x im u m im m o b iliz e d b io m a s s c o n c e n tra tio n (6 9 t h d a y o f fe rm e n ta tio n )
th e v ia b ility o f im m o b iliz e d c e lls w a s s im ila r to th e o b ta in e d b e fo re
(P e rio d A ).
T h e o re tic a lly th e im m o b iliz e d c e lls in fu lly d e v e lo p e d b io film re m a in
in s id e th e re a c to r fo r lo n g p e rio d s o f tim e a n d n a tu ra lly e x c e e d th e ir
c a p a b ility to g ro w re a c h in g its H a y flic k lim it, w h ic h is fin a lly le a d in g
th e m to d ie . T h e b io c a ta ly s t (c a rrie r + c e lls ) re p la c e m e n t o c c u rre d
e v e ry tim e c le a n c a rrie r w a s a d d e d , b e c a u s e n o n-c o lo n iz e d c a rrie r
s u rfa c e w a s in tro d u c e d in to re a c to r a llo w in g th e a d h e s io n o f fre e c e ll
p o p u la tio n . F re e c e lls h a v e a h ig h e r v ia b ility th a t h e lp e d to m a in ta in th e
h ig h v ia b ility v a lu e s o f th e c e lls im m o b iliz e d o n to th e c a rrie r. T h is
k e e p s th e im m o b iliz e d c e ll v ia b ility s im ila r to th a t o f th e fre e c e lls a n d
as
th e
to ta l
amount
of
im m o b iliz e d
c e lls
is
h ig h e r,
th e
e th a n o l
p ro d u c tio n w a s h ig h w ith lo w g lu c o s e p re s e n t T a b le V III- 8 .

Table VIII-8. Ageing Fermentation performance parameters at different
stages.
Stage
Period A
Period B
End of
Fermentation
Concentration* (g/L)
Glucose
Ethanol
3.51
9.29
5.69
5.18
2.33
7.23
Y etanol /
0.50
0.49
Total VDKs
/ (mg/L)
0 .0 6 0 .0 0 7
0 .0 4 0 .0 0 1
0.46
0 .0 6 0 .0 0 9
glucose
* Values corrected considering maximum carrier load (8% (vol./vol.))
263
Chapter VIII
T h e a d d itio n o f a la rg e r a m o u n t o f c le a n c a rrie r (5 6 t h d a y ) s u p p o rts

th e p re v io u s id e a . A fte r 5 6 t h d a y o f fe rm e n ta tio n a n in c re a s e o f g lu c o s e
c o n s u m p tio n a n d e th a n o l fo rm a tio n w a s n o te d . T h e fin a l a m o u n t o f
g lu c o s e a n d e th a n o l in th e o u tflo w a t th e e n d o f th e fe rm e n ta tio n is
s im ila r to v a lu e s o b ta in e d in th e p e rio d o f c a rrie r a d d itio n (P e rio d A
s e e F ig u re V III- 7 ). T h e re is n o s ig n ific a n t d e c re a se o r a c le a r te n d e n c y
o f th e e th a n o l/g lu c o s e y ie ld . T h is re s u lt in d ic a te s th a t p o s s ib ly n o
m e ta b o lic c h a n g e s w e re p re s e n t in im m o b iliz e d c e lls ; h o w e v e r, is
e x p e c te d th a t d iffe re n t re s u lts w ill o c c u r if c e ll v ia b ility d e c re a s e s
fu rth e r. T a b le V III- 8 s h o w s th e v a lu e s o f to ta l V D K s p ro d u c tio n ; o v e r
e v e ry p e rio d s s tu d ie d th e to ta l V D K s c o n c e n tra tio n is s ig n ific a n tly
b e lo w th e ta s te th re s h o ld c o n s id e re d fo r la g e r b e e rs (S e e T a b le II- 4 ). A
re la tio n s h ip b e tw e e n th e in c re a s e o f im m o b ilize d b io m a s s w ith th e
in c re a s e o f V D K s s e e m s to b e p re s e n t b o th in P e rio d A a n d a fte r
re p la c in g 1 /3 o f re a c to r v o lu m e .
VIII.4.6.1 Yeast
physiology and cell properties
T h e in tra c e llu la r c o m p o u n d s o f th e im m o b iliz e d b io m a s s s ta in e d b y

flu o re s c e n t d y e s d u rin g th e c o n tinu o u s e x p e rim e n t w e re c h itin rin g s ,
g ly c o g e n a n d n e u tra l lip id s . D u rin g P e rio d A th e v a lu e s o f th e s e
c o m p o u n d s s e e m to b e n e a r to s te a d y s ta te c o n d itio n . O n th e c o n tra ry ,
in P e rio d B a n in c re a s e o f th e in te n s ity o f flu o re s c e n c e o f c h itin rin g s
(b u d s c a rs ) a n d n e u tra l lip id s a c c o m p a n ie d th e d e c re a s e o f v ia b ility .
T h e n u m b e r o f b u d s c a rs p re s e n t o n th e c e ll s u rfa c e is d ire c tly
re la te d to th e n u m b e r o f tim e s a c e ll h a s d iv id e d , a n d c a n re p re s e n t a
b io m a rk e r
fo r
re p lic a tiv e
c e ll
age
e s tim a tio n .
The
fa c t
th a t
th e
flu o re s c e n c e b u d s c a r s ig n a l in c re a s e d b e fo re th e in c re a s e o f d e a d c e ll
n u m b e r m e a n s th a t it c a n b e a p p lie d a s a n in d ic a to r o f th e im m o b iliz e d
b io m a s s a g e in g w ith p ra c tic a l im p lic a tio n s . T h is in d ic a to r (flu o re s c e n t
b u d s c a r s ig n a l) c o u ld b e u s e d fo r s c h e du lin g th e re p la c e m e n t o f a g e d
b io c a ta ly s t d u rin g lo g te rm fe rm e n ta tio n s .
264
Chapter VIII
Intensity of
flourescence!
35!
% Dead cells!
95%!
30!
90%!
25!
20!
85%!
15!
10!
80%!
5!
0!
75%!
10!
20!
30!
40!
50!
60!
70!
Time / days!
Figure VIII-8. Relationships between immobilized dead/non viable cells (!)

and intracellular glycogen ("), neutral lipids (#), and bud scars (!) during
the course of the fermentation experiment.
B e s id e s th e b u d s c a rs a n a c c u m u la tio n o f n e u tra l lip id s (N L ) w a s a ls o

o b s e rv e d in re p lic a tiv e ly a g e d c e lls , w h ic h c a n a ls o b e u s e d a s a n
in d ic a to r to p re d ic t a g e in g . H o w e v e r, th e p h y s io lo g ic a l re a s o n fo r lip id
a c c u m u la tio n m a y n o t b e e n tire ly re la te d w ith a g e in g a n d fu rth e r
re s e a rc h e ffo rts w ill b e re q u ire d . G ly c o g e n c o n c e n tra tio n d id n o t s h o w
a s ig n ific a n t v a ria tio n o v e r th e s tu d ie d p e rio d s .
M ic ro b ia l a d h e s io n te s ts to s o lv e n ts (M A T S ) F ig u re V III- 9 w e re
c a rrie d
out
to
e v a lu a te
th e
changes
of
re la tiv e
c e ll
s u rfa c e
h y d ro p h o b ic ity fo r fre e a n d im m o b iliz e d c e lls d u rin g th e d iffe re n t

p e rio d s s tu d ie d . B o th fre e a n d im m o b iliz e d y e a s t s h o w e d a s ig n ific a n tly
h ig h e r a d h e s io n to c h lo ro fo rm , a n e le c tro n a c c e p to r s o lv e n t, s u g g e s tin g
th u s a s tro n g e le c tro n d o n o r n a tu re o f th e u s e d y e a s t b a s e d o n L e w is
a c id -b a s e (e le c tro n d o n o r/a c c e p to r) in te ra c tio n s (F ig u r e V III- 9 ).
R e g a rd in g fre e c e lls , th e m o s t s ig n ific a n t c h a n g e o c c u rs d u rin g th e
im m o b iliz a tio n p ro c e s s a t th e b e g in n in g o f th e e x p e rim e n t. A s s u m in g
th a t
th e
yeast
b io film
is
in
d y n a m ic
e q u ilib riu m
(a d s o rp tio n /d e s o rp tio n ) w ith th e s u rro u n d in g fre e c e lls , th e in c re a s e d

a d h e s io n to th e h y d ro p h o b ic s o lv e n ts c o u ld b e e x p la in e d w ith th e
s e le c tio n p re s s u re in s id e th e c o n tin u o u s re a c to r fa v o u rin g th e s e le c tio n
o f h y d ro p h o b ic c e lls a b le to e ith e r a d h e re to c a rrie r a n d /o r flo c c u la te .
265
Chapter VIII
Cloroform !
&!
Hexadecane!
100%!
Ethyl Acetate &

Decane!
100%!
80%!
80%!
60%!
60%!
40%!
40%!
20%!
20%!
0%!
0%!
0!
10!
20!
30!
40!
50!
60!
70!
Time / Days!
Cloroform !
&!
Hexadecane!
100%!
Ethyl Acetate &

Decane!
100%!
80%!
80%!
60%!
60%!
40%!
40%!
20%!
20%!
0%!
0%!
0!
20!
40!
60!
80!
Time / Days!
Figure VIII-9. MATS analysis results for free cells (Top) and immobilized cells
(Bottom). Results are expressed as a percentage of cells adhered to a given
solvent. Legend: Period A: up to 30th day; Period B: from 30th to 56th day; ! Cloroform; " - Hexane; # - Decane; ! - Ethyl Acetate.
M o re o v e r, th e a d h e s io n p ro p e rtie s o f im m o b iliz e d c e lls s h o w , fo r th e

s a m e fe rm e n ta tio n tim e , th e s a m e v a lu e s a s fre e c e lls , s u g g e s tin g th u s
th e e x is te n c e o f a d y n a m ic e q u ilib riu m b e tw e e n fre e a n d im m o b iliz e d
m ic ro b ia l
p o p u la tio n s .
s ig n ific a n t
d e cre a s e
in
a d h e s io n
of
im m o b iliz e d y e a s t to n o n -p o la r s o lv e n ts (d e c a n e a n d h e x a d e c a n e ) w a s
o b s e rv e d
d u rin g
th e
a g e in g
p e rio d
of
th e
c o n tin u o u s
e x p e rim e n t
(F ig u re V III- 9 ). T h e n a tu re o f th is p h e n o m e n o n is n o t c le a r, b u t it c a n
b e m o s t p ro b a b ly lin k e d w ith th e a lte ra tio n o f c e ll s u rfa c e d u rin g y e a s t
a g e in g . F u rth e r e x p e rim e n ts w ill h a v e to b e c a rrie d o u t in o rd e r to
e lu c id a te th is o b s e rv a tio n .
266
Chapter VIII
VIII.5 Conclusions
T h e p ro d u c tio n o f A F B b y lim ite d fe rm e n ta tio n , a s c o m p a re d to
re g u la r b e e r, h a s its o w n p a rtic u la ritie s th a t re q u ire e ith e r a lte ra tio n in
p ro d u c tio n p ro c e d u re a n d /o r th e u s e o f a s p e c ia l y e a s t s tra in . T h e
s a m e s p e c ia l a p p ro a c h is re q u ire d fo r c o n tin u o u s fe rm e n ta tio n w ith
im m o b iliz e d y e a s t in o rd e r to e x p lo it its p o te n tia l p ro d u c tiv ity c o m b in e d
w ith s u ffic ie n t p ro d u c t q u a lity .
It w a s s h o w n th a t th e la b o ra to ry y e a s t s tra in w ith d is ru p tio n in th e
K G D 2 (! -k e to g lu ta ra te d e h y d ro g e n a s e ) g e n e h a d s u ffic ie n t p o te n tia l fo r
v o la tile s fo rm a tio n (H A , E S ), w h ile it p ro d u c e d a n a m o u n t o f o rg a n ic
a c id s c o m p a ra b le to re g u la r b e e r fe rm e n ta tio n . In te rm s o f fla v o u r
fo rm a tio n
th is
g e n e tic a lly
m a n ip u la te d
la b o ra to ry
s tra in
p e rfo rm e d
e q u a lly w e ll in b a tc h a n d c o n tin u o u s P B R . H o w e v e r, it w a s u n a b le to
fo rm b io film a ro u n d s p e n t g ra in p a rtic le s a n d th e re fo re its u s e w a s n o t
p o s s ib le in th e iG L R .
C o n v e rs e ly th e b o tto m fe rm e n tin g s tra in W 9 6 a d h e re d w e ll to b o th o f
th e s o lid s u p p o rts , b u t th e fo rm a tio n o f fla v o u r a c tiv e c o m p o u n d s w a s
in s u ffic ie n t w ith th e e x c e p tio n o f im m o b iliz a tio n o n to S G in iG L R . T h is
s y s te m a rra n g e m e n t p ro v e d th a t e v e n a s tra in th a t s e e m s to b e le s s
s u ita b le fo r A F B p ro d u c tio n c a n , u n d e r a p p ro p ria te c o n d itio ns , p ro d u c e
a n a c c e p ta b le fin a l p ro d u c t.
T h e in flu e n c e o f th e ty p e o f g a s -p h a s e in th e iG L R s h o w e d to b e
m o re in te n s e fo r fre e c e lls th a n fo r im m o b iliz e d c e lls . U n d e r a n a e ro b ic
c o n d itio n s w ith N 2 a n in c re a s e in fre e c e lls c o n c e n tra tio n a n d in
m a lto s e c o n s u m p tio n w a s v e rifie d . M o re o v e r th e re s u lts in d ic a te th a t
th e re is a re la tio n b e tw e e n m a lto s e u p ta k e a n d fre e c e lls c o n c e n tra tio n .
T h e re s u lts o b ta in e d w ith C O 2 a n d N 2 s u g g e s te d th a t N 2 is a g o o d
s u b titu te a s g a s -p h a s e in iG L R u s e d fo r c o n tin u o u s A F B p rim a ry
fe rm e n ta tio n . T h e e v a lu a tio n o f fla v o u r a c tiv e c o m p o u n d s (H A ,E S a n d
A L ) s h o w e d th a t in th e p re s e n c e o f N 2 th e fo rm a tio n o f im p o rta n t
fa lv o u r
a c tiv e
compounds
as
ES
267
was
enhanced.
P a rtic u la ry
th e
Chapter VIII
fo rm a tio n o f e th y l c ra p y la te w a s h ig h e r in th e c o n tin u o u s s y s te m te s te d
(iG L R w ith S G ).
T h e a g e in g e ffe c ts e x p e rim e n ts s u g g e s t th a t d u rin g c a rrie r a d d itio n
p e rio d c o n d itio n s c lo s e to s te a d y-s ta te w e re a c h ie v e d w ith a lm o s t
c o n s ta n t (1 ) g lu c o s e a n d e th a n o l c o n te n t in th e o u tflo w ; (2 ) s p e c ific
m e ta b o lic ra te s ; (3 ) V D K fo rm a tio n ; a n d (4 ) fre e a n d im m o b iliz e d c e lls
v ia b ility . T h e c o n s ta n t c e ll v ia b ility m a y b e re la te d to e q u ilib riu m
b e tw e e n c e ll a d h e s io n , g ro w th a n d w a s h o u t in flu e n c e d b y re g u la r
a d d itio n o f c le a n , n o n-c o lo n iz e d c a rrie r s u rfa c e . It a p p e a rs th a t re g u la r
re p la c e m e n t o f s m a ll a m o u n ts o f b io c a ta ly s t w ith c le a n c a rrie r c o u ld b e
a s u ita b le s tra te g y to m a in ta in a lo n g-te rm v ia b ility a n d c o n s ta n t
fe rm e n ta tio n c o n d itio n s in s id e o f a b io re a c to r. O n th e o th e r h a n d d u rin g
a g e in g
p e rio d
g ra d u a l
d e c re a s e
of
im m o b iliz e d
c e lls
v ia b ility
o c c u rre d . A re la tio n s h ip b e tw e e n in c re a s e o f b u d s c a rs a n d n e u tra l

lip id s a n d d e c re a s e o f im m o b iliz e d c e lls v ia b ility w a s o b s e rv e d . T h e s e
p h e n o m e n a , e s p e c ia lly fo r b u d s c a rs , c o u ld s e rv e a s a n in d ic a to r fo r
s c h e d u lin g th e b io c a ta ly s t re p lac e m e n t o f lo n g -te rm fe rm e n ta tio n s . T h e
h id d e n e ffe c t o f s m a ll b io c a ta ly s t a d d itio n s w a s d e m o n s tra te d b y th e
re p la c e m e n t o f 1 /3 o f re a c to r v o lu m e b y a s u s p e n s io n o f c le a n c a rrie r
p a rtic le s .
It
was
b io m a s s ,
w h ic h
fo llo w e d
was
by
sudden
s u b s e q u e n tly
d e c re a s e
g ra d u a lly
of
im m o b iliz e d
re p la c e d
by
c e ll
c o lo n iz a tio n o f th e a v a ila b le c a rrie r s u rfa c e . A s re g a rd s d e a d c e ll

n u m b e r, th e re s u lt w a s a n im p ro v e m e n t in im m o b iliz e d c e ll v ia b ility .
VIII.6 References
ASBC, T. C. and the E. C. of. (1996). Microbiology, Yeast - 3. Methods of Analysis of
the American Society of Brewing Chemist (p. 3).
Bezbradica, D., Obradovic, B., Leskosek-Cukalovic, I., Bugarski, B. and Nedovic, V.
Briggs, D. E., Boulton, C. A., Brookes, P. A. and Stevens, R. (2004). Brewing
Science and practice.
Brnyik, T., Vicente, A., Machado Cruz, J., and Teixeira, J. (2001). Spent grainsa
new support for brewing yeast immobilisation. Biotechnology Letters, 23(13),
10731078.
Brnyik, T., Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2002).
grains. Journal Of The Institute Of Brewing, 108(4), 410-415.
268
Chapter VIII
Brnyik, T., Vicente, A. A., Kuncov, G., Podrazk!, O., Dostlek, P., and Teixeira, J.
A. (2004a). Growth model and metabolic activity of brewing yeast biofilm on the
surface of spent grains: a biocatalyst for continuous beer fermentation.
Biotechnology progress, 20(6), 1733-40.
Brnyik, T., Vicente, A. A., Machado Cruz, J. M., and Teixeira, J. A. (2004b).
Continuous Primary Fermentation of Beer with Yeast Immobilized on Spent
GrainsThe Effect of Operational Conditions. Journal of American Society of
Brewing Chemist, 62(1), 29-34.
Brnyik, Toms, Vicente, A. A., Oliveira, R., and Teixeira, J. A. (2004c).
Physicochemical
surface
properties
of
brewing
yeast
influencing
their
Brnyik, T., Vicente, A. A., Dostlek, P., and Teixeira, J. A. (2005). Continuous beer
fermentation using immobilized yeast cell bioreactor systems. Biotechnology
progress, 21(3), 653-63.
Brnyik, Toms, Silva, D. P., Vicente, A. A., Lehnert, R., Silva, J. B. A., Dostlek, P.,
and Teixeira, J. A. (2006). Continuous immobilized yeast reactor system for
complete beer fermentation using spent grains and corncobs as carrier materials.
Journal of Industrial Microbiology and Biotechnology, 33(12), 1010-8.
Decamps, C., Norton, S., Poncelet, D., and Neufeld, R. J. (2004). Continuous pilot
plant-scale immobilization of yeast in!!-carrageenan gel beads. AIChE Journal,
50(7), 1599-1605.
Dmny, Z., Smogrovicov, D., Gemeiner, P., Sturdk, E., Ptkov, J., and
EBC, A. (1987). Dimethyl Sulphide and Other Lower Boiling Point Volatile
Compounds in Beer by Gas Chromatography. Analysis.
EBC, A. (1999). Vicinal Diketones in Beer: Gas Chromatographic Method.
Evellin, F., Perpte, P., and Collin, S. (1999). Yeast ADHI Disruption: A Way to
Promote Carbonyl Compounds Reduction in Alcohol-Free Beer Production. Journal
of American Society of Brewing Chemist, 57(3), 109-113.
Fonseca, M. M., and Teixeira, J. A. (2007). Reactores Biologicos (in Portuguese).
Lidel-Edies Tcnicas, p. 482.
Kourkoutas, Y., Bekatorou, A., Banat, I. M., Marchant, R., and Koutinas, A. A.
(2004). Immobilization technologies and support materials suitable in alcohol
beverages production: a review. Food Microbiology, 21(4), 377397.
Kunze, W. (2004). Techology brewing and malting. (3rd ed., p. 939). Berlin: VBL
Berlin.
Lehnert, R., Kurec, M., Brnyik, T., and Teixeira, J. A. (2008). Effect of Oxygen
Lehnert, R., Novk, P., Macieira, F., Kurec, M., Teixeira, J. A., and Brnyik, T.
(2009). Optimisation of Lab-Scale Continuous Alcohol-Free Beer Production.
Czech Journal of Food Science, 27(4), 267-275.
Levenspield, O. (1999). Chemical Reaction Engineering. (W. Anderson and K. Santor,
Eds.)Distribution (3rd ed., p. 684). New York: John Wiley and Sons.
Macieira, F. F. (2008). Continuous fermentation of real alcohol- free beer.
IChem E, 77(Part C), 2732.
Levenspield, O. (1999). Chemical Reaction Engineering. (W. Anderson and K. Santor,
Eds.)Distribution (3rd ed., p. 684). New York: John Wiley and Sons.
Macieira, F. F. (2008). Continuous fermentation of real alcohol-free beer. MSc Thesis
Oliveira, M., Faria, M., Filomena, S., Barros, F., and Arajo, I. M. (2006). C6-alcohols
as varietal markers for assessment of wine origin. Analytica Chimica Acta, 563,
300-309.
Perpte, P, and Collin, S. (1999a). Contribution of 3-methylthiopropionaldehyde to
the worty flavour of alcohol-free beers. Journal of agricultural and food chemistry,
47(6), 2374-8.
Perpte, Philippe, and Collin, S. (1999b). Fate of the worty favours in a cold contact
fermentation. Food Chemistry, 66, 359-363.
269
Chapter VIII
Pilkington, P., Margaritis, A., Mensour, N., and Russell, I. (1998). Fundamentals of
immobilized yeast cells for continuous beer fermentation: a review. Journal of the
Institute of Brewing, 104(1), 1931. London: Harrison and Sons, 1904-.
Richardson, J. F., Harker, J. H., and Backhurst, J. R. (2002). Flow of Fluids through
granular beds and packed bed columns. Vol. 2. Chemical Engineering. Particle
Technology and Separation Processes (5th ed., pp. 191-236). Oxford:
Butterworth-Heinemann.
Selecky, R., Smogrovicova, D., and Sulo, P. (2008). Beer with Reduced Ethanol
Content Produced Using Saccharomyces cerevisiae Yeasts Deficient in Various
Tricarboxylic Acid Cycle Enzymes. Journal Of The Institute Of Brewing, 114(2), 97101.
Shen, H., Moonjai, N., Verstrepen, K., and Delvaux, F. (2003). Impact of attachment
immobilization on yeast physiology and fermentation performance. Journal of the
American Society of Brewing Chemists, 61(2), 7987.
Shen, H.-Y., De Schrijver, S., Moonjai, N., Verstrepen, K. J., Delvaux, F., and
Delvaux, F. R. (2004). Effects of CO2 on the formation of flavour volatiles during
fermentation with immobilized brewers yeast. Applied microbiology and
biotechnology, 64(5), 636-43.
Smogrovicov, D., and Domny, Z. (1999). Beer volatile by-product formation at
different
fermentation
temperature
using
immobilized
yeasts.
Process
Biochemistry, 34(8), 785-794.
Van Iersel, M. F. M., Meersman, E., Swinkels, W., Abee, T., and Rombouts, F. M.
(1995). Continuous production of non-alcohol beer by immobilized yeast at low
temperature. Journal of Industrial Microbiology and Biotechnology, 14(6), 495
501.
Virkajarvi, I., and Pohjala, N. (2000). Primary fermentation with immobilized yeast:
some effects of carrier materials on the flavour of the beer. Journal of the
Institute of Brewing, 106(5), 31118.
immobilized yeast a review on flavour formation and control strategies. Jounal
Yamauchi, Y., Okamoto, T., Murayama, H., Kajino, K., Nagara, A., and Noguchi, K.
(1995). Rapid maturation of beer using an immobilized yeast bioreactor. 2.
Balance of total diacetyl reduction and regeneration. Journal of Biotechnology,
38, 109-116.
270
Chapter IX
IX. Chapter IX Conclusions and Future Work
T h e m a in a im o f th is th e s is w a s th e d e v e lo p m e n t o f a c o n tin u o u s
A F B s y s te m th a t w o u ld o v e rc o m e th e in d u s tria l b a tc h s y s te m s a n d th e
re p o rte d c o n tin u o u s m e th o d s . T o re a c h th e s e o b je c tiv e s , th is th e s is
w a s d iv id e d in tw o m a in p a rts : A F B c o n tin u o u s fe rm e n ta tio n a n d
b io re a c to r h y d ro d y n a m ic s . O v e ra ll, th e o b ta in e d re s u lts im p ro v e d th e
e x is tin g k n o w le d g e o n A F B p ro d u c tio n a n d p o in te d o u t fo r th e w o rk
th a t n e e d s to b e d o n e in o rd e r to d e liv e r to th e b re w e ry in d u s try a
re lia b le a n d e c o n o m ic a lly a d v a n ta g e o u s s o lu tio n fo r c o n tin u o u s A F B
p ro d u c tio n .
In th e firs t p a rt o f th e w o rk , b re w e rs y e a s t im m o b iliz a tio n s tu d ie s o n
c o rn
cobs
(C c )
and
spent
g ra in s
(S G )
w e re
fo llo w e d
by
th e
c h a ra c te riz a tio n o f th e in flu e n c e o f s e v e ra l p a ra m e te rs in c o n tin u o u s

p rim a ry A F B fe rm e n ta tio n . T h e m a in c o n c lu s io n s w e re :
- N o n -tre a te d C c a n d S G (s e e T a b le III- 4 ) a re e ffic ie n t c a rrie rs fo r
y e a s t im m o b iliz a tio n ;
- C h e m ic a l m o d ific a tio n w ith D E C o f th e s e c a rrie rs d o e s n o t im p ro v e its
y e a s t im m o b iliz a tio n c a p a c ity ;
Y e a s t s tra in , re a c to r ty p e a n d c a rrie r a re k e y p a ra m e te rs to b e
c o n s id e re d in th e s e le c tio n o f th e b e s t c o n tin u o u s A F B p ro c e s s . B o tto m
y e a s t s tra in s p e rfo rm e d b e tte r in iG L R u sin g S G a s c a rrie r, w h ile
m u ta n t y e a s t s tra in s a llo w e d fo r b e tte r re s u lts in P B R w ith C c a s
c a rrie r. T h is , to g e th e r w ith th e u s e o f b o tto m fe rm e n tin g y e a s t s tra in s
b y th e P o rtu g u e s e b re w e rie s a n d th e E U la w s c o n c e rn in g th e u s e o f
g e n e tic a lly m o d ifie d y e a s ts, a llo w e d u s to s e le c t th e iG L R w ith S G a s
c a rrie r a s th e m o s t a d e q u a te s y s te m fo r c o n tin o u s A F B p ro d u c tio n .
In th is s y s te m , th e e ffe c t o f in tro d u c in g C O 2 a n d N 2 in th e g a s -p h a s e
w a s s tu d ie d , b e in g s h o w n th a t N 2 h a s a p o s itiv e in flu e n c e in th e
fo rm a tio n o f fla v o u r a c tiv e c o m p o u n d s .
271
Chapter IX
Y e a s t a g e in g w a s a ls o s tu d ie d a n d in a lo n g-te rm fe rm e n ta tio n th e
c o n s ta n t a d d in g o f fre s h c a rrie r p ro v e d to b e a s u ita b le s tra te g y fo r
m a in ta in in g a s te a d y p e rfo rm a n c e o f th e c o n tin u o u s p rim a ry A F B
M o re o v e r
bud
sc a rs
w e re
d e te rm in e d
as
s u ita b le
in d ic a to r o f th e lo s s o f iB io v ia b ility in lo n g te rm fe rm e n ta tio n s .
In th e s e c o n d p a rt o f th e th e s is , th e h y d ro d y n a m ic a s p e c ts re la te d
w ith th e o p e ra tio n o f im m o b iliz e d c e ll s y s te m s w e re s tu d ie d , s ta rtin g
fro m th e in flu e n c e o f S G in B C re g im e flo w s ta b ility fo llo w e d b y th e
g lo b a l a n d lo c a l s tu d y o f h y d ro d y n a m ic p a ra m e te rs s u c h a s : m ix in g
tim e ,
gas
h o ld -u p ,
liq u id
v e lo c ity ,
s o lid s
d is trib u tio n .
The
m a in
c o n c lu s io n s w e re :
- S G , a c e llu lo s e-b a s e d c a rrie r, re d u c e s H o R re g im e s ta b ility a n d a
re la tio n b e tw e e n th e p s e u d o-v is c o s ity in d u c e d b y th e p re s e n c e o f th e s e
p a rtic le s a n d c ritic a l flo w re g im e p a ra m e te rs w a s e s ta b lis h e d ;
- F o r th e s e v e ra l iG L R c o n fig u ra tio n s , th e b e s t m ix in g p a tte rn w a s
o b ta in e d fo r th e h ig h e s t A d /A r ra tio (3 .6 7 ) a n d ris e r le n g th (1 .4 m );
- A n im p ro v e d m e th o d u s in g a s a n O P w a s d e v e lo p e d a n d a p p lie d to
m e a s u re g a s-p h a s e p ro p e rtie s in g-l-s s y s te m w ith S G a s s o lid-p h a s e
- T h e e ffe c t o f S G in iG L R h y d ro d y n a m ic s a t u s e d c o n c e n tra tio n s
w a s n e g lig ib le a n d th e e v a lu a tio n o f its im p o rta n c e w a s o n ly p o s s ib le
u s in g a d v a n c e d te c h n iq u e s s u c h a s O P a n d H S C ;
-
model
fo r
th re e-p h a s e
iG L R
u s in g
SG
was
d e v e lo p e d a n d g o o d a g re e m e n t w ith e x p e rim e n ta l d a ta w a s o b ta in e d ;
- T h e e th a n o l p ro d u c e d b y th e y e a s t w a s fo u nd to h a v e a b ig g e r
e ffe c t
th a n
6 % (w t. W E T
SG
BASIS
/v o l.)
on
is
g a s-p h a s e
th e
p ro p e rtie s ;
a d e q u a te
amount
SG
to
lo a d in g
be
use
up
fo r
to
c e lls
im m o b iliz a tio n in a lc o h o lic fe rm e n ta tio n a s it w ill n o t in d u c e a n y

re le v a n t h y d ro d y n a m ic m o d ific a tio n s
- S G a re a s u ita b le c a rrie r to p e rfo rm a lc o h o lic fe rm e n ta tio n a s it
d o e s n o t in flu e n c e iG L R h y d ro d y n a m ic s u n d e r fe rm e n ta tio n c o n d itio n s .
272
Chapter IX
- A s a fin a l a p p o in tm e n t a n d c o n s id e rin g th a t a lo t h a s s till to b e

d o n e , s o m e s u g g e s tio n s fo r fu tu re w o rk a re p re s e n te d :
A s y s te m a tic s tu d y to im p ro v e y e a s t a tta c h e m e n t in S G s h o u ld
be
d o n e b y m o d ify in g th e c o n v e n tio n a l p re-tre a tm e n t b y a s im p le r a n d le s s

c o s tly o n e ;
T o p e rfo rm a s y s te m a tic s tu d y in o rd e r to u n d e rs ta n d th e u s e o f N 2
a s th e g a s -p h a s e in b ig in d u s tria l iG L R s a n d its e ffe c t o n A F B
p ro p e rtie s . A n e c o n o m ic a l e v a lu a tio n c o m p a rin g N 2 u s e a s g a s p h a s e
a n d C O 2 re u tiliz a tio n s h o u ld b e p e rfo rm e d ;
L o n g te rm fe rm e n ta tio n s tra te g ie s m u s t b e d e v e lo p e d to ta c k le w ith
y e a s t a g e in g e ffe c ts a n d d e a l w ith th e lo s s o f iB io v ia b ility w ith o u t th e
n e e d o f s to p a n d s ta rt p ro c e d u re s ;
F o r th e O P m e a s u re m e n ts , to d e v e lo p th e in je c tio n s y s te m s o th a t
d a ta a c q u is itio n m a y b e p e rfo rm e d a h ig h g a s flo w ra te s a n d h ig h
a c q u is itio n ra te .
T o s tu d y th e in flu e n c e th a t y e a s t c e lls h a v e in O P m e a s u re m e n t
m e c h a n is m a s w e ll a s to d e v e lo p a s y s te m th a t a llo w s O P to b e u s e d
o n lin e
in
o rd e r
to
o b ta in
g a s-p h a s e
273
p ro p e rtie s
e v o lu tio n
d u rin g
Chapter X
X. Apendix I
M a tla b p ro g ra m u s e d to d e te rm in e d th e g a s-p h a s e p ro p e rtie s in g -l-s
(S G ) s y s te m u s in g a n O P .
clear all; close all
Solids='I:\Dados
IBM\Resume\Data\0.25%\';
Gas_Flow='A
250mLmin-1\';
Position='Central\';
Path=[Solids
Gas_Flow
Position];
fileList=dir([Path,'ASSAY*.*']);
nposition = numel(fileList); %number of files
for n=1:numel(fileList); Filename=[Path fileList(n).name];
% Aquisition board configuration
MaxVoltage_carte = 10; MinVoltage_carte=-MaxVoltage_carte; Precision_carte = 12;
Freq_ech = 0.01; % in MHz
%Reading the size and number of points in the file
info_fic = dir(Filename); nb_pt_fic = info_fic.bytes/4; nb_pt_bloc=Freq_ech*1E6*10*20;
nb_bloc = ceil(nb_pt_fic/nb_pt_bloc); duree_bloc = nb_pt_bloc/(Freq_ech*1E6);
% Range of aquisition board
du=(MaxVoltage_carte-MinVoltage_carte)/(2^Precision_carte); fid=fopen(Filename,'r');
for i = 1 : nb_bloc; %1
Data_Binaire
=
fread(fid,nb_pt_bloc,'int','b');
i_c
=
1:2:nb_pt_bloc;
Data_Binaire_1=Data_Binaire(i_c);
Data_Binaire_2
=
Data_Binaire(i_c+1);
bloc(i).Data_Volt_1=Data_Binaire_1.*du; bloc(i).Data_Volt_2=Data_Binaire_2.*du;
end %1
% Assemble all blocks in two signals: optical probe and valve injections
Probe_array=[]; Valve_array=[];
for i = 1 : nb_bloc; %2
Probe_array=[Probe_array
bloc(i).Data_Volt_1'];
Valve_array=[Valve_array bloc(i).Data_Volt_2'];
end %2
Final=[Probe_array' Valve_array' Time_array];
% Parameters
Spl_rate
=
Freq_ech*1E6;
Inj_time
=
0.5;
dt=1/Spl_rate;
Prb_volt=Final(:,1);
Inj_volt_0=Final(:,2);
Zero_pos=find(Inj_volt_0<0);
Inj_volt=[];
Inj_volt=Inj_volt_0;
Inj_volt(Zero_pos,1)=[0];
Zero_pos_1=find(Inj_volt>3);
add0=Zero_pos_1-Inj_time*Spl_rate*0.375;
add1=
union(Zero_pos_1,add0); % este artificio permite-me utilizar as frequencias de corte que
quiser.
add2=
Zero_pos_1+Inj_time*Spl_rate*0.75;
Zero_pos_2=
union(add1,add2);
neg_elements=find(Zero_pos_2<=0);
Zero_pos_2(neg_elements)=[1];
Prb_volt_2=[];
Prb_volt_2=Prb_volt;
Prb_volt_2(Zero_pos_2,1)=[0];
X=find(Prb_volt_2);
Prb_volt_3=Prb_volt_2(X); Time_array_2=Time_array(X); % time correction
clear Data_Binaire_1; clear Data_Binaire_2; clear Final; clear Prob_volt_2; clear Inj_volt_0;
clear Inj_volt;
%Gas level General and Plate level definition
noise=0.4;
Overshoot_in_Prob_volt=find(Prb_volt_3>5);
First_max=find(Prb_volt_3>3,1,'first');
Prb_volt_3(Overshoot_in_Prob_volt,1)=[Prb_volt_3(First_max)];
InGasGeral=find(Prb_volt_3>(max(Prb_volt_3)-0.05*(max(Prb_volt_3)-min(Prb_volt_3))));
GasLevelGeral = mean(Prb_volt_3(InGasGeral)); NivPlat=GasLevelGeral-2*noise;
%Filter acquisition problems
Y=diff(X);
end_block=find(Y>1)-1;
begin_block=find(Y>1)+1;
begin_block=[X(1,1);begin_block]; number_block=numel(begin_block);
if number_block>numel(end_block); end_block=[end_block;numel(Prb_volt_3)]; end
% Erros in blocks due to acquisition issues
Blocks_diff=end_block-begin_block;
Blocks_diff_pos=find(Blocks_diff>0);
end_block=end_block(Blocks_diff_pos);
begin_block=begin_block(Blocks_diff_pos);
number_block_corr=numel(begin_block);
for i=1:number_block_corr; %3
bloc(i).Prb_volt_4=Prb_volt_3(begin_block(i):end_block(i));
bloc(i).Time_array_3=Time_array_2(begin_block(i):end_block(i));
noise=0.4;
bloc(i).InLiquid=find(bloc(i).Prb_volt_4<(min(bloc(i).Prb_volt_4)+0.025*(max(bloc(i).Prb_volt_4
)-min(bloc(i).Prb_volt_4))));
% Solving problems when block are too small
if numel(bloc(i).Prb_volt_4)<3; bloc(i).InLiquid =1; end
bloc(i).LiquidLevel
=
mean(bloc(i).Prb_volt_4(bloc(i).InLiquid));
bloc(i).treshold=bloc(i).LiquidLevel+noise; % treshold in Volts
% To determine when a block does not have bubbles
274
Chapter X
if max(bloc(i).Prb_volt_4)<2; bloc(i).treshold=[2]; end

Taq=[]; indexStart =[]; indexStop =[];
% Bubble Detection
try
Prb_Boolean = (bloc(i).Prb_volt_4>bloc(i).treshold); detectChange=xor(Prb_Boolean(1:end-1),
Prb_Boolean(2:end));
detectRise=detectChange
&
Prb_Boolean(2:end);
detectFall=detectChange&Prb_Boolean(1:end-1);
indexStart=find(detectRise);
indexStop=find(detectFall);end
% calculate time of signal start/stop and bubble start/stop
bloc(i).Trise = bloc(i).Time_array_3(indexStart); bloc(i).Tfall=bloc(i).Time_array_3(indexStop);
if numel(bloc(i).Trise)<numel(bloc(i).Tfall); bloc(i).Tfall=bloc(i).Tfall(2:numel(bloc(i).Tfall),1);
end
if
numel(bloc(i).Tfall)<numel(bloc(i).Trise);
bloc(i).Trise=bloc(i).Tfall(1:numel(bloc(i).Trise)1,1); end
% Tm determination
nr_Bubbles_in_block=numel (bloc(i).Tfall-bloc(i).Trise);
for j=1:nr_Bubbles_in_block; %4
bloc(j).Prb_volt_5=bloc(i).Prb_volt_4(indexStart(j):indexStop(j)+1);
bloc(j).InGas=find(bloc(j).Prb_volt_5>(min(bloc(j).Prb_volt_5)+0.5*(max(bloc(j).Prb_volt_5)min(bloc(j).Prb_volt_5)))); bloc(j).GasLevel=mean(bloc(j).Prb_volt_5(bloc(j).InGas));
%Find Last Mounting point (2nd point for Tm determination)
bloc(j).Index_First_Gas_Point=find(bloc(j).Prb_volt_5>bloc(j).GasLevel*0.9,1,'first');
bloc(j).First_Gas_Point=bloc(j).Prb_volt_5(bloc(j).Index_First_Gas_Point);
%Tm for each bubble
bloc(j).Tm=numel(bloc(j).Prb_volt_5(1:
bloc(j).Index_First_Gas_Point))*dt;
bloc(j).CC1=(bloc(i).Tfall(j)(bloc(i).Trise(j)+bloc(j).Index_First_Gas_Point*dt))/(bloc(i).Trise(j)+bloc(j).Index_First_Gas_Poi
nt*dt-bloc(i).Trise(j));
if nr_Bubbles_in_block<1; bloc(j).CC1=[2]; end
if bloc(j).Prb_volt_5(bloc(j).Index_First_Gas_Point)<NivPlat; bloc(j).Tm=[0]; end
if bloc(j).CC1<0.66; bloc(j).Tm=[0]; end
%Tg for each bubble
bloc(j).Tg=bloc(i).Tfall(j)-bloc(i).Trise(j); bloc(j).Ta=bloc(i).Trise(j); end %(j)4
% Voidage Determination
bloc(i).sumTg=sum(bloc(i).Tfall-bloc(i).Trise);
bloc(i).nrBubbles=numel(bloc(i).Tfall);
bloc(i).Taq=end_block(i)*dt-begin_block (i)*dt; bloc(i).voidage=bloc(i).sumTg/bloc(i).Taq*100;
% Assemble all Tm and Tg in an array of the main block (i)
bloc(i).Tg1=[]; bloc(i).Tm1=[]; bloc(i).Ta1=[];
for j=1:nr_Bubbles_in_block;
bloc(i).Tg1=[bloc(i).Tg1
bloc(j).Tg];
bloc(i).Tm1=[bloc(i).Tm1
bloc(j).Tm];
bloc(i).Ta1=[bloc(i).Ta1 bloc(j).Ta];
end; end %(i)3
bloc(n).Tg_assay=[]; bloc(n).Tg_assay=[bloc(n).Tg_assay bloc(1:number_block_corr).Tg1];
bloc(n).Tm_assay=[]; bloc(n).Tm_assay=[bloc(n).Tm_assay bloc(1:number_block_corr).Tm1];
bloc(n).Ta_assay=[]; bloc(n).Ta_assay=[bloc(n).Ta_assay bloc(1:number_block_corr).Ta1];
bloc(n).voidage_assay=[];
bloc(n).voidage_assay=[bloc(n).voidage_assay bloc(1:number_block_corr).voidage];
bloc(n).nrBubbles_assay=[];
bloc(n).nrBubbles_assay=[bloc(n).nrBubbles_assay bloc(1:number_block_corr).nrBubbles];
bloc(n).Taq_assay=[]; bloc(n).Taq_assay=[bloc(n).Taq_assay bloc(1:number_block_corr).Taq];
bloc(n).Sec_intital_time=[];
bloc(n).Sec_intital_time=[bloc(n).Sec_intital_time begin_block(1:number_block_corr).*(dt/0.57
5)];
bloc(n).Sec_final_time=[];
bloc(n).Sec_final_time=[bloc(n).Sec_final_time end_block(1:number_block_corr).*(dt/0.575)];
end %(n) 2
Sect_Results_C_A_250_mLmin_025_1=[bloc(1).voidage_assay'
bloc(1).nrBubbles_assay'
bloc(1).Taq_assay' bloc(1).Sec_intital_time bloc(1).Sec_final_time];
% Assemble all Tg,Tm and Ta
Center_A_250_mLmin_025_1=[bloc(1).Tg_assay' bloc(1).Tm_assay' bloc(1).Ta_assay'];
save('\Center_A_250_mLmin_025%','Center_A_250_mLmin_025_1','Center_A_250_mLmin_0
25_2','Center_A_250_mLmin_025_3','Sect_Results_C_A_250_mLmin_025_1','Sect_Results_C_
A_250_mLmin_025_2','Sect_Results_C_A_250_mLmin_025_3');fclose(fid);
275

Continuous Fermentation of Alcohol-Free Beer - Bioreactor Hydrodynamics and Yeast Physiology

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UMinho|2012

Continuous fermentation of alcohol-free

Andr Manuel de Oliveira Mota

Continuous fermentation of alcohol-free

Andr Manuel de Oliveira Mota

Continuous fermentation of alcohol-free

Doctoral Dissertation for PhD degree in Biological and

Supervisor of the thesis:

AUTORIZADA A REPRODUO PARCIAL DESTA TESE APENAS PARA EFEITOS

Universidade do Minho, ___/___/______

If I have seen further it is by standing on ye sholders of Giants

T h e a c c o m p lis h m e n t o f th is th e s is w a s fin a n c ia lly s u p p o rte d b y a

p ro p e rtie s fo r th e c o n tin u o u s fe rm e n ta tio n o f a lc o h o l-fre e b e e r w a s

a ffe c te d b y th e fe rm e n ta tio n m e ta b o lite s (e .g . e th a n o l) th a n th e s o lid s .

K e y w o rd s : a lc o h o l-fre e b e e r, c o n tin u o u s fe rm e n ta tio n , b io re a c to rs ,

a p lic a d o s a d o is tip o s d e b iore a c to re s - " g s -lift" (p a rtc u la s m e n o re s )

s is te m a trif s ic o . P a ra ta l re c orre u -s e a t c n ic a s m a is re fin a d a s (fib ra s

e s tu d a d o s o p ro d u to fin a l q u e a p re s e n ta v a m e lh o r q u a lid a d e fo i o b tid o

fis io lo g ia d a s le v e d u ra s im o b iliz a d a s . O s re s u lta d o s in d ic a ra m q u e o

e n c o n tra m -s e : a a d i o re g u la r d e s u p o rte fre s c o ( s p e n t g ra in s ) e o

M a d a le n a V ie ira , J e a n-M a rc ) in a ll th re e in s titu tio n s I h a v e w o rk e d .

Alcohol-free beer: definition, history and market .............................. 2!

I.2! Research Aims ...........................................................................5!

Techniques to remove alcohol from regular beer ............................. 9!

Continuous beer fermentation using cell immobilization systems ..... 15!

Industrial applications ................................................................ 25!

Higher alcohols .........................................................................

II.2! Continuous Beer Production ...................................................... 14!

II.3! Continuous Alcohol-free Beer Production ................................... 24!

II.5! References .............................................................................. 40!

Immobilization of biocatalysts: history and application..................

Determination of corncobs chemical composition .........................

General considerations ..............................................................

III.3! Material and Methods ............................................................... 53!

III.4! Results and Discussion ............................................................. 58!

III.5! Conclusions ............................................................................. 67!

Bubble columns: flow regime transition ....................................... 72!

Apparatus and measurements .....................................................

IV.4.1! Spent grains characterization .....................................................

IV.3! Material and Methods ............................................................... 77!

IV.4! Results and Discussion ............................................................. 83!

IV.5! Conclusions ............................................................................ 96!

V.Chapter V Global Hydrodynamic Properties of Three-phase iGLR ..... 99!

Gas-lift reactor design and configurations ................................... 101!

Gas-lift reactor: dimensions and characteristics ........................... 110!

Solid particles distribution ......................................................... 114!

V.3! Material and Methods ..............................................................110!

V.4! Results and Discussion ............................................................114!

V.5! Conclusions ...........................................................................134!

CFD simulations in iGLR: advantages and limitations ................... 142!

Experimental setup .................................................................. 144!

Auxiliary simulation results ....................................................... 153!

VI.3! Material and Methods ..............................................................144!

VII.! Chapter VII Local Hydrodynamic Properties of Three-phase iGLR

Measurement of local gas-phase properties ............................... 170!

Experimental apparatus for gas hold-up measurements ............... 186!

Calibration of new system ........................................................ 196!

VIII.3! Material and Methods ........................................................... 227!

Microorganisms and medium ................................................... 227!

General considerations ........................................................... 240!

VIII.4! Results and Discussion ......................................................... 240!

VIII.5! Conclusions ......................................................................... 267!

Chapter IX Conclusions and Future Work ................................. 271!

Universidade do Minho, _/_/______