Technologies for valorization of

Bio-wastes
Sami SAYADI
Laboratory of Bioprocesses
Regional Excellency Pole AUF
Centre of Biotechnology at Sfax, Tunisia
e-mail: sami.sayadi@cbs.rnrt.tn
Feedstocks
Feedstocks
/
/
solid
solid
agro
agro
-
-
wastes
wastes
A feedstockis the general name given to materials that can be
transformed. Feedstocks can vary tremendously, but are normally
derived fromliving organisms and are often termed as organic
materials.
The most common and abundant formof feedstock is lignocellulosic
wastes, (more often described as green wasteor yard waste). Other
feedstocks include household kitchen waste, commercial waste from
supermarkets, restaurants, kitchens and food processors,
There are also industrial feedstocks such as paper pulp, some
biodegradable plastics and sewage sludge.
For est er
For est er
domai n
domai n
Sahar i en
Sahar i en
domai n
domai n
Cul t ur es
Cul t ur es
domai n
domai n
Bi o Bi o- -f uel f uel ? ?
Bi o Bi o- -f er t i l i zer f er t i l i zer ? ?
Bi o Bi o- -based based c hemi c al s c hemi c al s? ?
- -Smal l l and ar ea Smal l l and ar ea w i t h w i t h Ener gy Ener gy, ,
f er t i l i zer f er t i l i zer def i c i enc i es def i c i enc i es
- -Bi o Bi o- -w ast es w ast es non non val or i zed val or i zed
Available bio
Available bio
-
-
waste materials as possible
waste materials as possible
renewable resources and bio
renewable resources and bio
-
-
based
based
coumpounds
coumpounds
Agro-industrial wastessuchas
Agricultural and foresterie solid wastes (pruning )
Fishconditonningwastes
Slaughterhouseswastes
Dairywastesandwhey
Olive mill wastes (Olive wastewaters, leaves, husks)
Dates wastes and Grapes husks
ssame wastescommonlyusedin makingsweets
Sugar Beets
Grual waste(wheat) as a source of glucose syrup
Sewagesludge(more than90 WWTP in Tunisia)
Solidwastes(9 landfillsare under construction plus that of J . Chakir whichis
operatingsince1999)
Muni c i pal Muni c i pal Sol i d Sol i d Wast es Wast es
Mar k et s Mar k et s w ast es w ast es
Agr i c ul t ur al Agr i c ul t ur al Wast es Wast es (Gr een (Gr een w ast es w ast es) )
Tr i mmi ng Tr i mmi ng, , pr uni ng pr uni ng of of pal m pal m and and ol i ve ol i ve
t r ees t r ees , , f or est f or est .. ..
Agr o Agr o f ood f ood w ast es w ast es (ol i ve (ol i ve w ast es w ast es
gr ape gr ape mar c mar c
WWTP WWTP sl udge sl udge
Far mer Far mer w ast es w ast es ( (manur e manur e,..) ,..)
Ref Ref. PR . PR Krauss Krauss Tunis, 6 Tunis, 6- -7 7 december december 2006 2006

Bi o
Bi o
-
-
Wast es
Wast es
,
,
Feedst oc k s
Feedst oc k s

i n
i n
Tuni si a
Tuni si a
2 000 000 t /an 2 000 000 t /an
100 000 t /an 100 000 t /an
5 000 000 t /an 5 000 000 t /an
300 000 t /an 300 000 t /an
5 400 000 t /an
1 250 000 t /an 1 250 000 t /an
Technologies for
Technologies for
bio
bio
-
-
waste
waste
treatment
treatment
and
and
Valorisation (
Valorisation (
most
most
adapted
adapted
to
to
the
the
Tunisian
Tunisian
context
context
)
)
A broad variety of technologies for conversion of biomass
and/or waste to valuable compounds, The treatment and
utilisation of these residues and waste for energy, recycling
and added value compounds can contribute significantly to
greenhouse gas emission reduction.
Anaerobic digestion: biomethanisation
Composting
Biogaz from landfills
Solid state fermentation
New molecular processes and technologies for conversion of biomass and bio-
waste into added value chemicals should emerge
UNDER EXPLORATION
Fermentation of lignocellulosic material and production of ethanol (Norh part)
Fermentation of dates residues in ethnol (South part)
Olea
Olea
europaea
europaea
L.: olive oil tree
L.: olive oil tree
Economic, cultural, historical Economic, cultural, historical .importance in Tunisia .importance in Tunisia
Olea europaea L., which
belongs to the Oleaceae is
considered a drought-resistant
species because it thrives in
areas where water stress is
frequent such as arid and semi-
arid regions.
It has been postulated that the
mean water requirement for
olive is 2000 m
3
/ha year (Ryan
and Robards, 1998).
In desert climates, olives are
frequently used as yard trees.
Olea
Olea
europaea
europaea
L.
L.
Schematic Diagram of Olive Oil Production Processes
Selection & Weighing
Selection & Weighing
Air Suction Unit
Air Suction Unit
Leaves & Dust
Leaves & Dust
Water
Water
Washing
Washing
Washing Wastewater
Washing Wastewater
Crushing
Crushing Water
Water
Temperature adjustment
Temperature adjustment
Horizontal Centrifugation
Horizontal Centrifugation
Vertical Centrifugation
Vertical Centrifugation
Solid residue
Solid residue
Wastewater
Wastewater
Oil
Oil
Storage Tank
Storage Tank
Micro-Filtration
Micro-Filtration
Bottling
Bottling
I
I
-
-
Co
Co
-
-
c ompost i ng of t he ol i ve pr oc essi ng
c ompost i ng of t he ol i ve pr oc essi ng
sol i d w ast e (Husk s): agr onomi c t est s
sol i d w ast e (Husk s): agr onomi c t est s
(
(
pot at oe
pot at oe
)
)
Industrial test of co-compostingolive husks(C) withchickenmanure(N) Agronomictests using
potatoswererealizedContact: ridha.hachicha@enis.rnu.tn.
Abi d N. and Sayadi S (2006). Det r i ment al Ef f ec t s of Ol i ve Mi l l Wast ew at er on t he Compost i ng Pr oc ess
-of Agr i c ul t ur al Wast es. Wast e Management , 26: 1099-1107.
Olive mill effluents and leaves should be considered as
Olive mill effluents and leaves should be considered as
wastes to be valorized rather than pollutants
wastes to be valorized rather than pollutants
Minutes
0 5 10 15 20 25 30 35
V
o
l
t
s
0,00
0,02
0,04
0,06
0,08
V
o
l
t
s
0,00
0,02
0,04
0,06
0,08
F3
F2
F1
SEC-HPLC of polyphenols in OMWW
OH
OH
HO O
OH
HO
O
O
OH
OH
O
O
O
OH
O
O OH
O O
O
Cl ean Tec hnol ogy Pr oc esses w er e used t o pr oduc e hi gh added val ue
c hemi c al s suc h as hydr ox yt yr osol , ol eur opei n, pol yhydr ox ybut yr at e,
and phenol i c pol ymer s (di mmer s, t r i mer s, ol i gomer s) of phenol i c
3 t o 5 Kg of ant i ox i dant s c oul d be r ec over ed /t on of OMW mak i ng val ue
addi t i on t o OMW an at t r ac t i ve ent er pr i se (Food addi t i ves)
II
II
-
-
Production of High added value
Production of High added value
compounds from olive mill by
compounds from olive mill by
-
-
products
products
Development of recovery processes: liquid-liquid extrcation,
membrane filtration, hydrolysis,
0
100
200
300
400
500
600
700
0 40 80 120
Temps (min)
I
n
d
i
c
e

p
e
r
o
x
i
d
e

(
m

q
/

k
g
)
Temoi n 200 ppm 500 ppm
Use of recovered compounds for fats
stabilization
Olive leaves and branches are the byproducts of
farming of the olive grove and can be found in
high amounts in the olive oil industries (10% of
the total weight of the olives) and during pruning
of olive trees
Extract and
pur compounds
IC
50
[g/ml]
BHT 0 .87
Hydroxytyrosol 0.58
Enzymatic
hydrolysis extract
0.65
Oleuropein 1.19
Ethyl acetate
extract
1.25
CH
3
OH/H
2
O
extract
1.57
III
III
-
-
Highly added value products from olive leaves
Highly added value products from olive leaves
and branches
and branches
Bouaziz M, Fki I, J emai H. Ayadi M. Sayadi S. Food Chem. (2007), in press
Bouaziz M., Bouallagui Z., Sayadi S. J. Arid Land Studies, 2006, 54, p 435-438
3,4-dihydroxyphenyl ethanol production through
enzymatic hydrolysis of olive leaf extract
Destabilisation of the molecule by the cleavage of the osidic bound
(Feuilles doli vi er)
OMW by-product
4-hydroxyphnyl thanol,
3,4-dihydroxyphnyl thanol,
Acide para-coumarique
Acide syringique,
Acide frulique,
4-Acide phnyl actique,
Acide vanillique,
oleuropeine,
etc
Bacteria, fungi, enzymes
High added
value product
Antioxydants
Aroma
Preservation C
Examples
3,4DHPE
Enzyme
ORAC
4-hydroxyphnyl thanol
oleuropeine,
IV
IV
-
-
Clean technologies for the bioconversion of
Clean technologies for the bioconversion of
available aromatics in highly added value products
available aromatics in highly added value products
ORAC
Heterogenous catalysis
Heterogenous catalysis
Ar. Hydroxylase
CH
2
CH
2
OH
OH
CH
2
CH
2
OH
OH
OH
3,4-di-hydroxyphnyl thanol
Yield 85 %
ORAC
ORAC
4-hydroxyphnyl thanol
Ex 1
Ex 1
-
-
hydroxylation of
hydroxylation of
tyrosol
tyrosol
by resting cells
by resting cells
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18
Time (h)
Y
i
e
l
d

(
%
)
0,8
0,9
1
1,1
1,2
1,3
1,4
1,5
O
D

(
6
0
0

n
m
)
Bouallagui, Z. and Sayadi, S. J . Agric. Food Chem. (2006)
L L
OH
N. Allouche, M. Damak, R. Ellouz, and S. Sayadi : App. Environ. Microbiol., 2004, p. 21052109
natural labelled products
avoid purification steps of intracellular enzymes
reuse of the cellular catalyst;
stabilized enzyme activities and protection against toxicity
Reaction implicating a cascade of enzyme
Cofactors (NADH, NADPH,); later on regenerated
during the metabolism
OH
OH OH
OH
OH
OH
OH
OH
p - h y d r o x y p h en y l ac e t i c
ac i d
Ty r o s o l Hy d r o x y t y r o s o l 3,4- d i h y d r o x y p h e n y l -
ac et i c ac i d
OH
Ri n g c l eav ag e
O
O
OH
EX.2 Hydroxylation of OMW phenolic precursors by immobilized cells
NADH,H
+
+ O
2
NAD
+
+H
2
O
m-t yr osol (3-hydr ox yphenyl et hanol )
and o-t yr osol (2-hydr ox yphenyl et hanol )
w er e c ompl et el y t r ansf or med i nt o 3-
hydr ox yphenyl ac et i c ac i d and 2-
hydr ox yphenyl ac et i c ac i d r espec t i vel y
vi a t he ox i dat i on of t he si de c hai n
c ar bon of t he t r eat ed subst r at es
4-hydr ox yphenyet hanol (p-t yr osol ) and
4-hydr ox yphenyl ac et i c ac i d w er e
r espec t i vel y bi o-c onver t ed i nt o 3,4
di hydr ox yphnyl t hanol and 3,4-
di hydr ox yphnyl ac et i c ac i d,
r espec t i vel y .
Example Example 3: BIOCONVERSION OF 2 PRECURSORS P 3: BIOCONVERSION OF 2 PRECURSORS P- -TYROSOL TYROSOL
AND P AND P- -HYDROXYPHENYL ACETIC ACID HYDROXYPHENYL ACETIC ACID
- - STEREOSPECIFICITY OF ACTION STEREOSPECIFICITY OF ACTION
Minutes
0 10 20 30 40 50
V
o
lt
s
0,0
0,5
1,0
1,5
V
o
lt
s
0,0
0,5
1,0
1,5
Channel A
EX T0
EX T0
Minutes
0 10 20 30 40 50
V
o
lt
s
0,0
0,2
0,4
0,6
V
o
lt
s
0,0
0,2
0,4
0,6
Channel A
EX Tf
EX Tf
Aromatic Enrichment conditions Isolated strains
Tyrosol
50 g/l NaCl
37 C
150 r.p.m
5 mM tyrosol
12 strains
YAS1,2,3
FE1,2,3
IM1,2,3
SL1,2,3
p-coumaric acid
100 g/l NaCl
37 C
150 r.p.m
5 mM p-coumaric
4 strains
IMPA, IMPB
IMPC, IMPD
Ferulic acid
100 g/l NaCl
37 C
150 r.p.m
5 mM ferulic
2 strains
MAR
MBR
Ex ampl e 4: Bi oc onver si on of ar omat i c st r uc t ur es i n ex t r eme
c ondi t i onsBy-pr oduc t s of ol i ve f er ment at i on/br i ne
Abdel k af i S, Sayadi S, Ben Al i Gam Z, Casal ot L, Labat M (2006) FEMS Mi c r obi ol .
Let t .,
Abdel k af i S, M. Labat , M. Chamk ha, Casal ot L, Sayadi S (2005) FEMS Mi c r obi ol .
Let t .
Degradation pathway of
Degradation pathway of
ferulic
ferulic
acid by
acid by
Halomonas
Halomonas
elongata
elongata
strain MAR
strain MAR
Metabolis of ferulic acid
Oxidation C
3
-aliphatic chain
0
20
40
60
80
100
1 2 3 4 5
Repeated time of biotransformation
Y
i
e
l
d

o
f

v
a
n
i
l
l
i
c

a
c
i
d

(
%
)
Accumulation of vanillic acid by resting cells
Ex 5: Degradation of tyrosol by the use of catalyst (Al-
Fe)PILC in the presence of UV and Hydrogen
peroxide
0
20
40
60
80
100
0 5 10 15 20 25
Temps (h)
C
o
n
v
e
r
s
i
o
n

d
u

t
y
r
o
s
o
l

(
%
UV/H
2
O
2
/(Al-Fe)PILC
UV/H
2
O
2
H
2
O
2
/(Al-Fe)PILC
Conditions [tyrosol] = 500 ppm
[H
2
O
2
] = 2.10
-2
M
[Catalyst] = 500 ppm
= 254 nm
Najjar W., AzabouS., Sayadi S. and Ghorbel A. Appl. Cat: B Env. (2007), 74:11-18
AzabouS., Najjar W., Gargoubi A., Ghorbel A. and Sayadi S. App. Cat. B Env(2007), 77:166-174
CH
2
OH
HO
Tyrosol
CH
2
OH
HO
HO
Hydroxytyrosol
0
0,1
0,2
0,3
0,4
0,5
0,6
0 5 10 15 20 25 30
Ti me(h)
tyrosol
Hydroxytyrosol
3,4-DHPA
t =0 h
t =4 h
Ex 5
Ex 5
-
-
hydroxylation of
hydroxylation of
tyrosol
tyrosol
by (Al
by (Al
-
-
Fe)PILC
Fe)PILC
Azabou S., Najjar W., Ghorbel A. and Sayadi S. J .Agric. Food. Chem(2007), 55:4877-4882.
Ex: 6 Enzymatic synthesis of lipophilic
compound from p-hydroxyphenyl ethanol
The hydrophilic character of some polyphenols limits their application
Synthesis of lipophilic derived products
Lipase;
acylant;40C
Aissa I., Bouaziz M., Ghamgui H., Kamoun A.,Miled N.,Sayadi S.,Gargouri Y.
J. Agric. Food Chem. 2007, 55,p 1029810305
monoacetylated compound (+95%)
Peroxide values of refined husk oils stored at 50C and supplemented
with: (x) BHT at 200 ppm, () OMW extract at 200 ppm, () OMW
extract at 500 ppm, () hydroxytyrosol at 200 ppm, () control.
I. FKI, N. ALLOUCHE AND S. SAYADI, Food Chem., 2005, 93:197-204.
Some activities of the obtained antioxidants
Refined husk oil stabilization
Peroxide values of refined
olive oils stored at 50C and
supplemented with: (x) BHT
at 200 ppm, () OMW extract
at 200 ppm, () OMW
extract at 500 ppm, ()
hydroxytyrosol at 200 ppm,
() control.
The oxidation of refined husk and olive oils stored at 50 after the addition of
OMWW extract was measured by peroxide values (PV) and conjugated diene
formation CD (data not shown).
Both parameters CD and PV measure the primary product of lipid oxidation.
Oils treated with purified hydroxytyrosol and BHT exhibited the lowest
peroxide values as compared with the control sample.
0
1,5
3
1 2 3 4 5 6
Treatment
A
I
Effect of HCD and OMW phenolic extracts on
serum lipids levels in rats
HCD diet increase of AI compared with the rats fed normal
diet.
HCD diet + MeOH extracts, purified compounds and OMW
decrease of AI.
I. FKI, M. BOUAZIZ, Z. SAHNOUN AND S. SAYADI, 2005, Bioorganic and Medicinal Chem., 13: 5362 5370.
ABTS
Poly R-478
RBBR
Negative strain Positive strain
Precoce (24h)
Amongst 400 fungal strains isolated
on solide medium, 67 strains are
able to oxidyze ABTS and
decolorize the chromogenes
Screening fromlocal biotopes of fungal strains producing
laccases and peroxidases on solid medium + chromogene
N CBS LiP MnP Lac
Collection Strain (U/l) (U/l) (U/l)
CTM 10125 Phlebia Sp - 182 (8) 19 (7)
CTM 10133 Oxyporus latemarginatus 4 (7) 108 (8) -
CTM 10136 Oxyporus latemarginatus 6,5 (2) 202 (11) -
CTM 10154 Trametes trogii 25 (5) 67,2 (6) 8990 (10)
CTM 10155 Polyporus Sp 35 (4) 96,7 (2) 7393 (10)
CTM 10156 Trametes trogii 5,5 (1) 56,5 (7) 9956 (7)
CTM 10313 Stereum annosum - - 97 (9)
CTM 10476 Trichoderma atroviride - - 9005 (6)
Phylogenic characterization of novel strains able to produce Lac, MnP
and/or LiP
0
10
20
30
40
50
60
0 1 2 3 4 5 6 7 8 9 10
0
5000
10000
15000
20000
25000
30000
Residual BOD5
Residual COD
Biomass
Laccase activity
Time (days)
B
i
o
m
a
s
s
;

R
e
s
i
d
u
a
l

B
O
D
5
;

R
e
s
i
d
u
a
l

C
O
D


(
g

l
-
1
)

L
a
c
c
a
s
e

a
c
t
i
v
i
t
y

(
U

l
-
1
)
Ti me c our se of gr ow t h and l ac c ases pr oduc t i on by Tr amet es t r ogi i DSM 17786
c ul t ur ed i n 7-l i t r es bi or eac t or on OMW:w at er (80:20)-based medi a suppl ement ed w i t h ur ea
(2 g L
-1
) and Resi dual BOD
5
and COD c onc ent r at i ons.
ChakrounH., Sayadi S., Machichi T and DhouibH. J ournal of chemical technology
and biotechnology (2009), in press
.
Laccases production on OMW in 7litres fermenter by Trametes trogii
for further polymerization/oxidation reactions
-
-
Laccase
Laccase
mediated
mediated
polymers
polymers
synthesis
synthesis
laccases catalyze the oxidative coupling of phenolic
compounds.
Formation of polymerized products ranging from dimers to
pentamers (NMR), during the oxidation of several olive
phenolics by T. togii laccases.
These proposed procedures are alternative approaches to
obtain valuable chemicals such polymers of catechol and
tyrosol in an environmentally friendly way which could
encourage their use in medical and food industries .
Minutes
0 5 10 15 20 25 30 35 40 45 50
V
o
lts
0,0
0,5
1,0
Detector A (280nm)
10 0,5h
10 0,5h
Minutes
0 5 10 15 20 25 30 35 40 45 50
V
o
lts
0,0
0,2
0,4
0,6
Detector A (280nm)
15 18h
15 18h
0
20
40
60
80
100
0 2 4 6 8
I
n
h
i
b
i
t
i
o
n

o
f

b
i
o
l
u
m
i
n
e
s
c
e
n
c
e

o
f

V
i
b
r
i
o

f
i
s
h
e
r
i

I
B

(
%
)
Time
(h)
Fraction S+ Laccase Fraction S+ Laccase of of Trametes Trametes
versdicolor versdicolor
After After 30 min 30 min
2,5 g/l of phenolic monomers polymerized
Formation of insoluble matter
Minutes
0 10 20 30 40 50
V
o
l
t
s
0.0
0.2
0.4
0.6
0.8
V
o
l
t
s
0.0
0.2
0.4
0.6
0.8
Channel A
Laccase 28h119
Laccase 20028h123
HPLC after enzymaticreaction
Polymres
Laccases for the synthesis of polymers from
olive phenolic wastes
Minutes
0 10 20 30 40 50
V
o
lt
s
0.0
0.5
1.0
1.5
V
o
lt
s
0.0
0.5
1.0
1.5
Channel A
Laccase To' 119
Laccase To' 119
1
7 9
2 3 4 5
6
8
10