CITRIC ACID PRODUCTION

Marin Berovi
1
and Matic Legia
2
1
Department of Chemical, Biochemical and Ecology Engineering
Faculty of Chemistry and Chemical Technology, University of Lu!lana
2
"ational #nstitute of Chemistry
$adrihova 1%, &1''' Lu!lana, (lovenia
CONTENT
1. INTRODUCTION
2. UE AND OCCURRENCE
!.TRAIN "OR CITRIC ACID PRODUCTION
#. BIOC$EMITR%
#.1 In&'(ence o& t)e trace *eta'+
,. UBTRATE
,.1 Mo'a++e+
,.2 (cro+e
,.! -r(.+
,.# tarc)
,., $-dro'
,./ A'0ane+
,.1 Oi'+ and &at+
/.PRODUCTION PROCEE
/.1 (r&ace .roce++ on 'i2(id +(3+trate
/.2 o'id +tate &er*entation
/.! (3*erged &er*entation
/.!.1. (3*erged &er*entation (+ing Aspergillus niger
/.!.2 (3*erged &er*entation (+ing -ea+t+
/.!.! (3*erged &er*entation (+ing i**o3i'i4ation o& *icro5organi+*+
/.# o'id +tate &er*entation
1. PRODUCT RECO6ER%
1.1 (r&ace .roce++
1.2 (3*erged &er*entation
1.! Preci.itation
1.# o'vent e7traction
1., Ion e7c)ange
1./ Li2(id *e*3rane+
1.1 Micro.oro(+ )o''o8 &i3re+
1.9 E'ectrodia'-+i+
1.: U'tra&i'tration
9. ECONOMIC APECT
:. CITRIC ACID ;ORLD PRODUCTION
1<. LITERATURE
1. INTRODUCTION
Citric acid is the main organic acid produced today !y fermentation) The history of citric
acid actually started in 1*+, -ith .) (cheele /10 -ho first isolated it from the lemon
uice as calcium citrate, -hich treated -ith sulphuric acid gave citric acid in the li1uid
phase)
#n 1+2+ Li!ieg considered that citric acid is actually three car!o3ylic acid and in 1++'
4rimeu3 and 5dam /20 synthesised citric acid from glycerole6derived 1,2
dichloroacetone for the first time chemically /10 )
.ehmer in 1+%2 -as the first -ho o!served the presence of citric acid as !y6product of
calcium o3alate produced !y a culture Penicillium glaucum fermenting sugar /1,20) The
result of this fermentation had encouraged him to patent the process for citric acid
production /20) 7n this !ase in 1+%, the first industrial fermentation, using on the
open tray system -as !uilt) Ten years later the factory -as closed, as the fermentation
-as considered to long and fre1uent contamination occured /,0)
5fter .ehmer several other researchers follo-ed /8,&0 , !ut reasona!le advance in
citric acid production appeared -ith 9ahors:y in 1%12, -ho first patented a ne- strain 6
Aspergillus niger /*0) Follo-ing the fundamental investigations !y Thom an Currie
1%1& /+0) Currie 1%1* /%0 oppended the -ay for industrial citric acid fermentation using
a ne- micro6organism) $is most important finding -as that Aspergillus niger could gro-
-ell at lo- p$ values around 2)8 to 2)8) This lo- p$ prevented contamination
-hich -as common in .ehmer;s process)
#n 1%2+ !eet molasses, as a cheap sugar source -as for first used in C<echoslova:ia)
Difficulties, ho-ever, -ere encountered -ith this source due to its trace metals content)
Using a patent of =e<<adroli 1%2+ /1'0, this pro!lem -as effectively solved !y
using potassium he3acianoferrate as a chelating agent for the trace metals in !eet
molasses su!strate)
7riginally first industrial citric acid fermentations -ere carried out as surface cultures)
The introduction of the su!merged fermentation -as a significant improvement)
5mong the studied preceding the commercial implementation of su!merged
fermentation the -or: of >er1uin 1%2+ /110 it should !e mentioned as the first one for its
shill and precision compara!le to Currie;s -or: on the surface process /%0) 7n ?apan in
the si3ties a ne- process emerged !y using n6al:anes as a car!on source) 5 yeast of the
genus Candida, -as used, -hich produced apprecia!le amounts of citric and isocitric acid
/12,120 )
The .orld production of this 26hidro3y6propan61,2,26three car!o3y acid, !y
fermentation, is rapidly increasing) 5lthough in (outh 5merica, =e3ico and 4reece
there are still e3isting some factories -here citric acid is isolated from unripe citrus
fruits, today over %% @ of the .orld;s output of citric acid is produced micro!ial !y
various fermentation processes, su!strates and microorganisms)
The traditional method of preparing citric acid !y e3traction from the uice of lemons,
limes and pineapple -astes is still in practice in the developing .orld, !ut its production is
not significant, as it comprises less .orld production)
Aarious chemical syntheses of citric acid have appeared in the chemical and patent
literature since the first one !ased on the reaction of glycerol6derived 1)2 dichloroacetone
-ith cyanide !y 4rimau3 and 5dam in 1++' /20 ) $o-ever none of these has reached a
commercial status competitive -ith fermentation processes)
2. UE AND OCCURRENCE
Uses &' @ of citric acid product is mainly used in the food and !everage industry, !ecause
of its general recognition as safe having pleasant taste, high -ater solu!ility and chelating
and !uffering properties) Citric acid is used e3tensively in car!onated !everages to provide
taste and complement fruit and !erry flavours) #t also increases the effectiveness of
antimicro!ial preservatives) The amount of acid used depends on the flavour of the
product) #t usually may very from 1)8 to 8 @ /1 6 1,0)
7n am and ellies it is used for taste and for p$ adustment in the final product) For
optimum gelation p$ has to !e adusted in very narro- limits /180) Citric acid is usually
added as a 8' @ solution to assure good distri!ution through the !atch) #n confectionery
industry ')8 6 2)' @ it is used as flo-ing agent /1&0) The chelating and p$ adusting
properties of citric acid ena!le it to optimi<e the sta!ility of fro<en food products !y
enhancing the action of antio3idants and inactivating en<ymes) #t also helps to prolong the
shellfire of fro<en fish and shellfish /1*0)
Citric acid also inhi!its colour and flavour deterioration in fro<en fruit /1+0) 5mounts in
concentration of ')''8 6 ')'2 @ citric acid are used as an antio3idant synergism in fats, oils
and fat containing foods /1&0) 5s a flavour adunct citric acid is used in sher!ets and ice
creams /1&0)
Temperament of total citric acid production are used in pharmaceutical industry as oral
pharmaceutical li1uids, eli3irs and suspensions to !uffer and maintain sta!ility of active
ingredients and to enhance the activity of preservatives) 5ddition of ')'2 @ citric acid to
li1uid dosage forms comple3es trace iron and copper ions and retards degradation of active
ingredients /1%0)
Citric acid is a standard ingredient in cosmetic formulations for p$ adustment, and in
antio3idant systems as a metallic6ion chelator /2'0) The detergent6!uilding properties of
citrate una!le it to !e used as a rapidly !iodegrada!le environmentally accepta!le
phosphate su!stitute in nonphosphate detergent po-ders /210)Citric acid6!ased metal
cleaning formulations efficiently remove metal o3idation products from the surface of
ferrous and non6ferrous metals /220 )Citrates have !een reported to assist in platting of
copper /220 , nic:el /2,0 , chromium, lead /280 and various heavy metals /1&0)
Aarious other uses of citric acid and its salts and esters -ere reported also in photography as
a component of printing plate emulsions in various !leaches, fi3ers and sta!ilisers /2&0
in oil -ell treatment and cements /1&0 in te3tile industry /2*0 in paper industry /2+0
ta!acco industry /2%0) Citric acid is also an preferred nucleating or !lo-ing agent in
polymeric foams for food and !everage use and its esters are used as plastifi<ers in the
preparation of polymer compositions /1&0)
5s the !y6products of citric acid fermentation various en<ymes Bamilolytic, pectolytic )))C
-ere referred /2%0 )
!.TRAIN "OR CITRIC ACID PRODUCTION
=any strains e3crete traces of citric acid as a meta!olite of primary meta!olism) #t is a result
of some severe irregularity of meta!olism caused !y genetic deficiency or !y meta!olic
im!alances)
#n the history of citric acid fermentation, in the last hundred years, various strains of genera
fungi, yeast and !acteria -ere reported such as: Penicillium luterum, Penicillium
purpurogenum, Penicillium restrictum, Penicillium janthinellum, Penicillium citrinum,
Paecilomyces divaricatum, Mucor piriformis, Trichoderma viride, Sacharomycopsis
lipolitica, Arthrobacter paraffineus, Corynebacterium sp. et al) /2',210)
$o-ever, only mutants of Aspergillus and yeasts genus Candida have almost e3clusively
!een utili<ed)
5part from Aspergilus niger the follo-ing species of Aspergillus have !een reportedD
Aspergillus niger, Aspergillus entii, Aspergillus aamori, Aspergillus foetidus, Aspergillus
fenicis, Aspergillus fonsecalus, Aspergillus fumaricus, Aspergillus luchensis, Aspergillus
saitoi, and Aspergillus usumii.
From the genus Candida the follo-ing have to !e mentionedD Candida lipolytica, Candida
tropicalis, Candida guilliermondii, Candida intermedia, Candida parapsilosis, Candida
!eylanoides, Candida fibriae, Candida subtropicali", Candida oleophila. =utants of
Aspergillus niger, Aspergillus entii and on paraffine su!strats Candida lypolitica are used
in industrial production /220)
For industrial citric acid production filamentous fungus Aspergillus niger is far the most
used micro6organism) #n the second half of the 2'th century, progress in life sciences and
accumulating :no-ledge a!out meta!olic events stimulated several research groups to study
the !iochemical !asis of citric acid accumulation !y Aspergillus niger) They investigated
-hy, and under -hich circumstances, citric acid is accumulated so that productive strains
might !e improved further and yields increased) 5lthough a num!er of !iochemical events
-ere found to !e responsi!le for citric acid overflo-, differences e3isted amongst individual
high6 producing strains) Eesearch during last t-o decades, has resulted in generally accepted
theory that descri!es the meta!olic path-ays used and the regulation events that are
significant during citric acid accumulation /220)
#. BIOC$EMITR%
There have !een many theories proposed to e3plain the phenomena of citric acid
accumulation !y Aspergillus niger /2,62+ 0 , !ut so far no complete e3planation is
availa!le) #t can !e said that citric acid accumulates !y an induced a!normality in the meta6
!olism of the mould during the operation of the Tricar!o3ylic 5cid Cycle /TC50 postulated
!y Fre!s in 1%2*, under its original name Gcitric acid cycleG /,*0 The TC5 Cycle is a
cyclic se1uence of reactions of almost universal occurrence in mitochondria in aero!ic
organisms) #t is catalysed !y multien<yme system, that accepts the acetyl group of acetyl6
Co en<yme 5 as fuel and dismem!ers it to yield car!on dio3ide and hydrogen atoms /,', ,20
)
7n each turnaround -ithin the TC5 cycle, one molecule of acetic acid Bt-o car!on
atomsC enters as acetyl6Co en<yme, condenses -ith a molecule of the four6car!on
compound o3aloacetic acid to form citric acid, the si36car!on compound) Citric acid is them
degraded through a reaction se1uence, that yields t-o molecules of C7
2
and regenerates
the four6car!on o3aloacetic acid) 5nother turn of the cycle may no- start !y the reaction
of the o3aloacetic acid -ith another molecule of acetyl6Co en<yme 5) Thus in each turn of
the cycle one molecule of acetic acid enters, t-o molecules of 5T> and C7
2
are formed
and a molecule of o3aloacetate is utilised to form citrate, !ut is regenerated at the end of
the cycle /,,0)
The degree of involvement of TC5 cycle during the accumulation is indeed controversial)
(hu et al)1%8, found that ,' @ of the citric acid -as formed from recycled dicar!o3ylic
acid /,80) #n contrast to that other researchers -ere a!le to demonstrate some degree of
recycling /,&,,*0) #t -as also considered /,+0 that the citric acid accumulated came from
the disappearance of acoinitase and isocitric dehydrogenase, since prior) to the
accumulation of citric acid all en<ymes of TC5 cycle -ere present)
Citric acid is e3creted from the cells in response to unfavoura!le intracellular condition
caused !y increased levels of tricar!o3ylic acids BTC5C) 5 crucial prere1uisite for overflo-
of citric acid from A. niger cells is therefore increased level of Fre!s cycle intermediates
caused !y anaplerotic reactions) E3tensive studies have revealed that there are three main
meta!olic events that replenish tricar!o3ylic acid cycle intermediates and predispose the cell
to product overflo-)
Fast upta:e of glucose !ased on simple diffusion
Unrestricted meta!olic flo- through glycolysis, ma:ing precursors for synthesis of
the TC5 cycle intermediates readily availa!le
Uncoupled "5D$ re6o3ydation resulting in lo-er levels of 5T> and therefore
decreased ana!olic reaction
7nly the activities of certain en<ymes of individual A. niger cells can lead to such
intracellular conditions)
4lucose upta:e rate has !een identified as an important factor in the rate of citric acid
production /,%,8'0) By using mathematical modelling it -as sho-n that glycolytic
reactions of A. niger are limited !y the supply of the initial su!strate and the removal of final
product) T-o glucose carriers have !een identified, the first, a high6affinity carrier that is
e3pressed at all times, and the second, a lo-6affinity carrier that is e3pressed only in the
presence of high concentrations of glucose /810) $o-ever =ischa: et al) B1%+,C /820 and
Torres et al) B1%%&C /810 reported that !oth glucose carriers are inhi!ited !y citric acid
under production conditions) #f the entry of glucose is primarily via the glucose carriers, the
effect of citric acid concentration on the o!served glucose upta:e should !e pronounced) By
contrast, a simple diffusion model fits all of the o!served data under citric acid e3cretion
conditions, e3plaining the o!served relationship !et-een specific upta:e rate and glucose
concentration, -hich -ould not e3ist under carrier6saturated conditions /820) Finally,
!ecause simple diffusion is an inevita!le physical process, it is not capa!le of !eing regulated
directly !y the organismsH this may, in itself, account for dramatic overproduction of citric
acid under the conditions used in this process) The simple nature of this mechanism also
e3plains the similarity of the upta:e relationship from the different sources, despite the use
of different strains and gro-ing conditions)
7n the other hand de6regulated meta!olic flu3 through glycolysis is a prere1uisite for rapid
synthesis of citric acid) #n glycolysis, the reactions catalysed !y he3o:inase,
phosphofructo:inase and pyruvate :inase are virtually irreversi!le) The activities of these
en<ymes are regulated !y reversi!le !inding of allosteric effectors or !y covalent
modification) "ormally in eu:aryotic organisms, the phosphofructo:inase is the most
important control element) $o-ever, in Aspergillus niger, during the gro-th on high sugar
concentrations that are needed for rapid citric acid formation, the control of glycolysis is
shifted from &6phosphofructo616:inase level to glyceraldehyde step /8,0) #n the literature
t-o attempts can !e found to influence the efficiency of this path-ay !y genetic
modification of the en<ymes involved) Firstly, !y disrupting trehalose6&6phosphate synthase
gene Bggs5C, the synthesis of trehalose6&6phosphate, a potent inhi!itor of glycolysis -as
prevented, yet citric acid accumulation improved only slightly /880) (econdly, moderate
overe3opression of the :ey regulatory en<ymes, &6phosphofructo616:inase and pyruvate
:inase did not enhance acid production /8&0)
De6regulated glycolysis leading to strong anaplerosis is characteristic for a productive phase
of A. niger cells, ho-ever significant physiological changes are ta:ing place in the cells
during the early stages of gro-th in high initial sucrose or glucose medium that have crucial
impact on overall productivity and yield) #n fact there could !e no citric acid detected in the
su!strate during the first 2, hours of gro-th /8*0, -hile a relatively slo- accumulation rate
initiates only during the second day of fermentation, follo-ed !y a sudden increase in
specific productivity after-ards /8+,8%,&'0)
By flu3 distri!ution e3periments pentose phosphate B>>C path-ay -as found to !e
predominant during the germination of spores, follo-ed !y a s-itch to glycolysis /&1,&20)
#nitial phases of gro-th are characterised also !y polyol formation and glucosamine
accumulation) >olyols especially glycerol, -hose intracellular concentration can reach up to
1*8 m=, may play an important role as an osmoregulator in A. niger cells during the
gro-th in high sucrose medium /&20)
Because the enhanced glycolytic flu3 is a prere1uisite for increased anaplerosis,
understanding the mechanism of the s-itch in car!ohydrate meta!olism from the >>
path-ay to glycolysis is of crucial importance) 5lthough the initial inhi!ition at the level of
TC5 cycle en<ymes has !een e3tensively studied in the past, some authors in their recent
revie-s on citric acid accumulation !y A. niger e3clude the hypothesis of an inhi!ition of
TC5 cycle in the phase -here acid accumulation starts /&20)
$o-ever !y measuring intracellular citrate concentration in the cells, lo- levels of citrate
-ere recorded in germinating spores follo-ed !y a constant rise up to 1' m= !efore 2,
hours of fermentation /&20)
5n inhi!ition of "5D>6dependant isocitrate dehydrogenase B#CD$C !y glycerol -as
initially proposed to trigger an increase of intracellular citrate /&,0 on the !asis of :inetic
measurements performed on un6purified en<yme in the homogenate /&,,&80, ho-ever later
tests on partially purified en<yme sho-ed no inhi!ition !y glycerol /&80) "5D>6specific
isocitrate dehydrogenase -as found to !e predominantly located in mitochondrial
compartment -hen glucose -as used as sole car!on source /&&0, -hile only minor
activities of "5D6dependent en<yme -ere detected in A. niger cells /&*0)
$o-ever, "5D>6isocitrate dehydrogenase -as found to !e inhi!ited !y citrate /&*0) and
reduced meta!olic flo- at the early stages of fermentation through the TC5 cycle at the
stage of #CD$ could !e predicted from studies !ased on the distri!ution of different mar:ed
C
12
atoms in glucose !y /8,0) 5t the early stages of citrate formation their model indicated a
su!strate cycle of o3aloacetate to pyruvate -as su!stantial in comparison to the flu3 of
o3aloacetate to citrate) (imultaneously, a significant pyruvateIphosphoenolpyruvate
su!strate cycle -as predicted) Later in the fermentation there -as decreased operation of
the pyruvateIphosphoenolpyruvate su!strate cycle and a net flu3 to citrate) The 1uestion of
-hat triggers the initial increase in citrate concentration remains une3plained) #t might !e
that another su!stance structurally related to citrate and meta!olically formed from glycerol
could cause initial deactivation of mitochondrial TC5 cycle en<yme or the mass action effect
of intermediates from glucose might cause the increase)
The initial increase in intracellular citrate concentration could cause a decrease in glucose
degradation through the >> path-ay) #t -as sho-n that &6phosphogluconate
dehydrogenase, one of the regulatory en<ymes of the o3idative step of >> path-ay is
inhi!ited !y citrate, -ith apparent F
i
value of ')+ m= /&+0)
The direct conversion of he3oses to pyruvate via glycolysis !ecomes predominant during
the productive phase of citric acid accumulation that starts after a!out 2, hours and
accelerates after ,' to 8' hours of gro-th in a !atch system) Eegulation of the central part
of he3ose meta!olism ta:es place at several levelsD at the transcriptional level, !y regulating
the activity of allosteric en<ymes !y specific effectors and as revealed recently /&%,*'0,
even !y post translational modification)
#n the glycolytic flu3, &6phosphofructo616:inase BEC 2)*)1)11C is the most important control
element) #t catalyses essentially irreversi!le reaction of glycolysis, the phosphorylation of
fructose6&6phosphate using =g65T> to form fructose61,&6!isphosphate and releasing =g6
5D>) (i3 organic allosteric ligands either increase or decrease su!strate !inding affinity and
concomitantly determine overall en<yme activity /*10) The en<yme attracted the interest of
investigators, due to its a!ility to maintain a high glycolytic flu3 in spite of elevated
intracellular concentrations of citrate, a -ell :no-n inhi!itor of >FF1, -hich -as reported
to reach concentrations !et-een , m= /8+0 and 1' m= /&20)
Eecently, another en<yme e3hi!iting >FF1 activity -as isolated from A. niger mycelium
-ith molecular mass of ,% :Da) 5 fragment of identical si<e could !e also o!tained in vitro
!y the proteolytic cleavage of the purified native >FF1 -ith proteinase F, -hich regained
its activity after the phosphorylation of the protein molecule !y catalytic su!unit of c5=>6
dependent protein :inase) The native en<yme as a sole >FF1 en<yme could !e isolated only
from the early stages of gro-th on a minimal medium, -hile a ,% :Da fragment seemed to
appear later and -as activated concurrently -ith a sudden change in the gro-th rate) There
is a strong evidence that the native >FF1 en<yme undergoes spontaneous posttranslational
modification at the early stages of the fungal development)
By measuring :inetic parameters of !oth >FF1 forms found in A. niger cells, 5T> proved to
!e a strong inhi!itor of the short >FF1 fragment, !ut the negative effect of 5T> seemed to
!e suppressed !y physiological concentrations of fructose62,&6!isphosphate) The same
effector significantly increased the A
ma3
and the affinity of the fragmented protein to-ard the
su!strate, -hile it does not affect the ma3imal velocity of the native protein /&%0) #n A.
niger a!out & J= of fructose62,&6!isphosphate -ere detected under citric acid e3creting
conditions /8&0) The studies on fructose62,&6!isphosphate formation sho-ed that its
synthesis is stimulated after the transfer of A. niger mycelium from lo- B1@C to high B1,@C
initial sucrose medium simultaneously -ith a rapid increase in c5=> level /*20) 7ther >FF1
stimulators, 5=> and ammonium ions, increased the activity of the shorter fragment more
intensely than the activity of the native protein, -hile citrate, a -ell :no-n allosteric
inhi!itor of eu:aryotic >FF1 en<ymes, sho-ed moderate inhi!ition of the native en<yme,
-hile no inhi!ition of the fragment could !e o!served !y concentrations up to 1'm= /*'0)
Finetic data so far o!tained support the hypothesis that the posttranslational modification is
needed for the formation of a highly active >FF1 en<yme in6sensitive to normal feed !ac:
control !y citrate)
5nother phenomenon ta:ing place at the early stages of A. niger gro-th in a high citric acid
yielding medium seems to !e relevant for the development of high citric acid yielding
mycelium) "amely, the shorter fragment is inactive immediately after proteolytic cleavage
and must !e phosphorylated in order to regain activity /&%,*20) >F5 -as found to !e
capa!le of the appropriate phosphorylation, -hich led to the re6activation of ,% :Da
fragment /&%0) Finases are normally under the tight control of specific regulatory su!units
and cyclic 5=> is :no-n to induce >F5 en<yme) #n A. niger 5&' a spontaneous increase in
the concentration of cyclic 5=> -as recorded after 2, hours of gro-th in a citrate yielding
medium) Further analyses have sho-n that the amount of c5=> formed depends on the
initial concentration of sucrose in the medium) Under higher sucrose conditions the c5=>
pea: appeared earlier and -as higher, -hile in lo-er sucrose media a flattened pea: -as
o!served later in fermentation /*,0) 5 spontaneous increase in cyclic 5=> concentration
could !e caused !y intracellular acidification since a drop of intracellular p$ is :no-n to
stimulate the E5(6adenylate cyclase signalling path-ay in a num!er of fungal species,
including Saccharomyces cerevisiae /*80)
#n the citrate accumulating strain B5&'C gro-n in a high sucrose medium, intracellular
acidification -as indeed recorded at the early stages of gro-th /&2,*&0, -hile in another A.
niger strain B".121C no change in cytoplasmic and vacuolar p$ could !e detected !y >
21
"=E techni1ue during the gro-th of immo!ilised cells /**0) (ignificant differences in
mem!rane $
K
65T>ase activities of !oth strains -ere descri!ed) #n the 5&' B"EEL 22*'H
5TCC 11 ,1,C strain the activity of proton pumps -as a 1uarter of that in 518+ BCB(
12',,%H ",''C, a strain -hich is related to the ".121 strain /*&0) =oreover, under
identical gro-th conditions strain 518+ e3truded protons more rapidly into the medium
-hen ammonium ions -ere used as a sole nitrogen source than the 5&' strain, indicating
that proton pumps of the latter strain perhaps cannot e3trude all the protons that are
released into the cytosol after initial increase in intracellular citric acid concentration and
ammonium assimilation /*&0) Citric acid, -hich can reach concentration up to 1' m= in
the cells /&20, dissociates at neutral p$ values BpF
2
L8),C releasing t-o protons) 7n the
other hand under citric acid accumulating conditions the ammonium salts are the preferred
source of nitrogen) A. niger consumes ammonium very rapidly and it is normally depleted
from the medium !et-een ,' to 8' hours of fermentation, -hich is -ell !efore the fungus
stops gro-ing /8%0) The amount of protons e3creted from the !iomass appeared to !e
directly related to the initial ammonium concentration /8%0) #t is -orth noting that
ammonium ions are ta:en up !y an uniport mechanism, ho-ever after the incorporation of
"$
,
K
ion as an amino group, t-o protons are released -hich must !e pumped !ac: to the
medium !y $
K
65T>ases in order to maintain electroneutrality in the cells) (toichiometric
modelling of the early stages of the fermentation revealed that ammonium ions com!ine
-ith a car!on6containing meta!olite inside the cells in a ratio 1D1, to form an organic
nitrogen compound, -hich is immediately e3creted !y the mycelium) The compound -as
proven to !e glucosamine /8%0) Characteristically, the ma3imal rate of acid overflo- -as
recorded only after the depletion of ammonium from the medium, although increase in dry
!iomass -as o!served at later phases of gro-th as -ell) The en<yme responsi!le for
glucosamine formation must !e glucosamine6&6phosphate deaminase -hich catalyses
amination of fructose6&6phosphate to produce glucosamine6&6phosphate) (ince the en<yme
competes for the same su!strate Bfructose6&6phosphateC as >FF1, rapid accumulation of
glucosamine must significantly decrease the meta!olic flu3 through glycolysis at the early
stages of gro-th, ho-ever !etter understanding of the phenomenon must a-ait more
detailed characterisation of deaminase :inetics)
Aspergillus niger is -ell :no-n for its strong e3tra and intracellular proteolytic activity
/*+0) 5lthough proteases are normally strictly compartmentalised in the cells and are
activated from their pre6pro forms only in the vacuoles, some lea:age through the tonoplast
into the cytosol must occur, since cleavage of the native >FF1 en<yme ta:es place) #n A.
niger cells increased protein degradation -as reported under the manganese deficient
conditions, -hich -as also reflected !y increased intracellular proteinase activities
/*%,+'0)
For efficient citric acid fermentation, lac: of trace metal ions, particularly =n
2K
ions from
the medium is of maor importance) =uch has !een speculated a!out the principal
physiological role of manganese ions in citric acid overflo- in past, -hich seem to affect
meta!olism on various levels /&20) .hether manganese ions are someho- involved in
increased cytosolic protease activities, and concomitantly, in the posttranslational
modification of >FF1, -ill have to a-ait further investigation)
During the idiophase, the phase of ma3imal product formation /+10, no significant
inhi!ition of the TC5 cycle could !e o!served and an increased level of all tricar!o3ylic
acids, -ith an e3ception of 26o3o6acids, can !e detected in the mycelium /+20) The only
plausi!le e3planation of the phenomenon is accelerated glucose meta!olism, -hich -as
confirmed also !y testing mutants -ith increased citric acid productivity in respect to the
parental strain /+20) $o-ever, another t-o en<ymatic reactions that appear in A. niger cells
play a note-orthy role in citric acid overflo-) Cytosolic pyruvate car!o3ylase /+,0 and
malate dehydrogenase isoen<yme /+80 are catalysing the conversion of pyruvate first to
o3aloacetate and finally to malate) #n the late phase of fermentation car!on dio3ide fi3ation
!y pyruvate car!o3ylase !ecame an important anaplerotic reaction /+&0, -hile increased
concentration of malate in the cytosol finally serves as a counter ion for citrate e3port from
the mitochondrial compartment !y a tricar!o3ylic acid carrier /+*0)
The formation of citric acid is dependent on strong aerationH dissolved o3ygen tensions
higher than those re1uired for the vegetative gro-th of A. niger stimulate citric acid
fermentation /++,+%0) The !iochemical !asis of this o!servation is related to the presence of
an alternative, cyanide6resistant respiratory path-ay, -hich is re1uired for the re6o3ydation
of glycolytically produced "5D$, -hen high o3ygen tension is maintained) The en<yme
responsi!le for the additional respiratory path-ay is an alternative o3idase, -hich catalyses
reduction of o3ygen to -ater -ithout the translocation of protons across the inner
mitochondrial mem!rane, and thus functions as a non6energy6conserving mem!er of the
respiratory electron chain)
The alternative respiration seems to !e constitutively present in citric acid producing strains
/%',%10) The alternative o3idase is synthesised in the cytosol and translocated into the
mitochondria /%20) 5lthough ao"#1 gene encoding alternative o3idase from A. niger cells
has !een isolated, cloned and characterised /%20, no transformants carrying multiple gene
copies or strains -ith disrupted gene -ere prepared and tested for intracellular 5T>
concentrations andIor citric acid overflo-) $o-ever, it is generally accepted that the
presence of uncoupled "5D$ re6o3ydation results in lo-er levels of 5T> and therefore
decreases ana!olic reactions)
5fter citrate first accumulates in the cytosol it must pass the plasma mem!rane to !e
e3creted into the su!strate) #t -as assumed that citrate, a charged meta!olite cannot cross
the lipid !ilayer -ithout support of the transport protein and an active, p$ driven, $
K
6
symport dependent system -as proposed that -as functional only under the manganese
deficient gro-th conditions /%,0) $o-ever, recent thermodynamic calculations presented
for citrate overflo- from A. niger cells at the p$ value 2 of the su!strate suggest that a
passive transport step suffices for citrate e3cretion /%80)
Future perspectives
#n last several decades enough :no-ledge on !iochemical mechanisms leading to citric acid
overflo- have accumulated to generally understand the phenomenon, ho-ever many details
still remain une3plained) Eecently, several genomes of 5spergillus species have !een fully
se1uenced and the information pu!lishedD A. fumugatus /%&0, A. nidulans /%*0, A. ory!ae
/%+0, -hile the information of several other genomes including A. niger -ill !e released in
near future) 7n the !asis of data from se1uence analyses, and physiological information
pu!lished from A. niger and related filamentous fungi, in silico model of the central car!on
meta!olism of A. niger has !een constructed /%%0 and is regularly updated) 5pplication of
the stoichiometric model together -ith recent discoveries on the posttranslational
modification of the :ey regulatory en<yme of glycolysis -ill present a po-erful tool for
further improvement of the primary meta!olism in A. niger that -ill result in stronger
anaplerosis and increased productivity)
#.1 In&'(ence o& t)e trace *eta'+
#n citric acid technology a!sence of iron and manganese in the fermentation su!strate plays
the most crucial role /1'',1'10) Trace element nutrition is specially highlighted !y the fact
that an optimal nutrient medium for citric acid fermentation -ill not allo- high production
unless the trace elements content is carefully controlled /1'20) 7n the other hand, if the
trace element nutrition is correct, other factors Bsugar concentration, phosphate and the
othersC have only less pronounced effects /1'20)
#ron ions in higher concentration than 1)8 mgIl strongly affect cellular morphology, !y
promoting unproductive filamentous mycelial gro-th form /1',,1'80) #n further insights
into the importance of metal ions, the presence of manganese ions to citric acid
fermentation -as reported !y Clar: et al) 1%&& /1'&0) 5s little as 1 Jgl of manganese could
completely ruined the production yield of and caused organismMs morphology to s-itch
from micro!ial pellets, :no-n as citric acid productive form, to unproductive filamentous
gro-th)
#n oposite of this in the most recend research !y Berovic et al)2''& /1'*0
it -as found that -hen fungal !iomass reaches its stationary phase even in a case -hen fed
media contains unusually high ammounts of manganese ions up to 2'' JgIl the presence of
heavy metal ions do not effect on mycelial gro-th neither on citric acid !iosynthesis)
=anganese deficient lo-er than 1'
6*
= rised chitin and reduced N6glucan production)
=anganese levels also affect lipid synthesis -hich in turn affects cell mem!rane composition
/1'+0) #t also e3hi!its effects on D"5 synthesis of A.niger. Under manganese limitation,
D"5 formation -as not inhi!ited !ut E"5 synthesis -as impaired /1'%0) 7n the other side
manganese deficiency in A.niger cultivation also results in significantly lo-er lipid levels
due primarily to reduction of triglycerides and -ith little effect on free acids and sterols
/11'0) #n any-ay the influence of manganese ions on 5)niger is very comple3 and it
o!vious represents the most critical metal ion in citric acid fermentation /1'2,1'80)
#n recent articles the attention to A.niger metal ion tolerance -as related to action of
elevated manganese ion concentration and effects of copper and <inc antagonism to iron
and manganese /111,1120 and to various genetic manipulation for metal resistance strain
improvement /112,11,,1180)
,. UBTRATE
=ost processes are !ased on molasses, although the use of cleaner sources is gaining
ground) .hatever the source, its cost and preparation in order to permit optimal
fermentation conditions are t-o important aspects of the technology in citric acid
production) The !asic su!strate for citric acid fermentation in plants using the surface
method of fermentation is !eet or cane molasses) >lants using su!merged fermentation
can use not only !eet or cane molasses, !ut a su!strate of higher purity such as
hydrolysed starch, technical and pure glucose, refined or ra- sugar, purified and
condensed !eet or cane uice) This is !ecause use of a pure su!strate may result in
increases in yield, or reduction in fermentation time /11&0)
,.1 Mo'a++e+
=olasses is a -idely used su!strate, coming in a variety of 1ualities) $igh 1uality
molasses is usually demanded for citric acid production) Cane and !eet molasses are not
identical in compositionH often one type -ill !e preferred to the other) They are
sometimes mi3ed to ta:e advantage of the additional nutrients arising from the
differences in composition)
Besides su!strate type Bsugar !eet, sugar caneC, the chemical composition of molasses
depends on many factors such as soil and climate conditions, fertili<ation type, crop
method, time and conditions of storage, production technology, technical e1uipment of
plant, etc /11&0)
Beet molasses
Beet molasses consists of a!out &86+' per cent dry su!stance and 2'628 per cent -ater)
The main ingredient of molasses is sucrose, ,,68, per cent !y -eight) 7ther sugars
Bcar!ohydratesC -hich can !e found in higher amounts are inverted sugar '),61)8 per
cent, raffinose ')862)' per cent and :estose and neo:estose ')&61)& per cent) Eaffinose
is a natural part of sugar !eet, -hile :estose is the result of micro!ial action during
sugar !eet treatment) 7ther sugars in molasses are ara!inose, 3ylose and mannose in
amounts of ')861)8 per cent) 5ll sugars Be3cept sucroseC are included in the non6
nitrogen organic su!stances of molasses) >roducts of chemical and thermal sugar
decomposition Bmela6noidines, caramelC and organic acids also !elong to this group)
Caramel consists of sugar anhydride and colouring mattersH melanoidines are made in
hot solution as the result of a reaction !et-een reducing sugars and amino acids) #n
addition to the non6volatile dar: coloured compounds, there are a!out ,' volatile
compounds as aliphatic aldehyde, methylglyo3al, diacetyl, acetoin, acetone,
o3ymethylfurfurol and others /11&0)
The non6volatile organic acids present in molasses are glutaric, malonic, succinic,
aconitic, malic and lactic acidH the remainder are o3alic, citric and tartaric acid) These
can all react -ith calcium to form insolu!le salts that can influence the precipitation and
recovery of the citric acid crystals) =olasses contain such volatile acids as formic,
acetic, propionic, !utyric and valeric acid) 5lmost all organic acids, volatile and non6
volatile, are potassium or calcium salts) =olasses containing higher amounts Bover 1
per centC of volatile acids are normally too dar: to !e used as feedstoc: for the citric
acid fermentation)
"itrogen compounds contained in molasses are mostly !etaine Ba!out &'6*' per cent
of total nitrogenC, amino acids B2'62' per cent of nitrogenC, protein B26, per cent of
nitrogenC and traces of nitrogen in ammonium nitrate and amide) The amino acids
content in molasses depends on the soil and climate conditions and !eet cultivation))
Betaine comes from !eet and is not used !y micro6organisms as a nitrogen source) The
content of mineral su!stances in !eet molasses amounts to +)861,)' per cent /11&0)
Besides this factors one of the most relevant parameters for high yielding ciric acid
fermentation is also the amount of particular microelements in different molasses)
The p$ of molasses depends on the sugar e3traction technology) #t -as considered that a
neutral, or slightly al:aline molasses gave the !est citric acid yields) Citric acid production
needs molasses -ith lo- !uffer a!ility, to ma:e possi!le the re1uired rapid fall of medium
p$ during fermentation /11&0)
Cane molasses
Cane molasses differs from !eet molasses in its chemical composition) #t contains less
sucrose and more inverted sugar, has lo-er content of nitrogen and raffinose, more
intensive colour and lo-er !uffer capacity)
Beet and cane molasses can also contain other su!stances -hich appear in small amounts,
!ut are often crucial in deciding -hether the molasses are suita!le for use in citric acid
!iosynthesis) These are pesticides, fungicides and her!icides used in !eet and cane
cultivation and also su!stances used for defoaming in sugar production process) 5ll have
mostly to3ic properties and negatively affect molasses usa!ility) #n general !eet molasses is
more suita!le for citric acid fermentation than cane molasses) #t is especially relevant in
su!merged fermentation -here the 1uality of the su!strate is more important for
productivity and fermentation yield)
The microflora of molasses can !e an agent of negative influence on yield and productivity
of fermentation) =olasses -ill al-ays contain a certain num!er and type of microorganisms,
sometimes the count can !e higher than 1' ''' per g of molasses) The most common
microorganisms in molasses are species of $acillus, sometimes yeasts of Candida species,
and very rarely, moulds of Penicillium, Aspergillus and other species /11&0)
The !asic operation in molasses preparation is a treatment for heavy metal ions
removal) >otassium ferrocyanide or other comple3 compounds are commonly used)
5nother compound comple3ing -ith heavy metals is the sodium salt of ethylene6
diamineacetic acid BEDT5C) 7ther heavy metal comple3ing compounds can also !e
used, e)g) sodium polyphosphates, potassium rhodanate, 2,,6dinitrophenols and +6
o3y1uinoline) =olasses media are sometimes purified !y ionites, especially on cation
e3changer) "ot all microelements should !e removed during this process, as some of
them are necessary for gro-th of the Aspergillus niger mycelium /11&0)
,.2 (cro+e
Eefined sugar of !eet or cane is almost pure sucrose -hich Aspergillus niger strains
ferment very -ell /11*0) >reparation of a refined sugar solution as a fermentation medium
is !ased on its diluting -ith -ater to a concentration of 18622 per cent, adding necessary
nutrients B"$
,
"'
2
, F$
2
>'
,
, =g('
,
C and acidifying -ith sulphuric acid to p$ 2)&62)' /11+0)
Batch medium is sterili<ed in the fermentation vessel) 5ll the ingredients of the fermentation
medium are added straight into the !ioreactor or are prepared separately !y diluting in hot
-ater B+86%8OCC and then pumped into the !ioreactor) #n this case, sugar is diluted to 8'6&'
per cent concentration and pumped into the fermenter that has had an e3act amount of
sterile -ater added, resulting in a total sugar concentration of 18622 per cent)
,.! -r(.+
(yrups of !eet or cane sugar can also !e used as !asic su!strate for the su!merged citric
acid fermentation) The great advantage -ith this su!strate is its purityH ho-ever, the 1uality
of the syrups deteriorates rapidly during storage) Because of this they can only !e used
during the sugar campaign season and only if the citric acid plant is not too far from the
sugar factory !ecause of the large transport costs)
>reparation of the syrups for fermentation entails dilution -ith -ater to a sugar con6
centration of 1862' per cent, addition of necessary nutrients B"$
,
"'
2
, F$
2
>'
,
, =g('
,
,
B"$
,
C
2
C
2
'
,
C, acidification -ith hydrochloric or sulphuric acid to p$ ,68 and sterili<ation at
121OC for ')861 hours /11%0)
,.# tarc)
The production of citric acid from sources of starch such as corn, -heat, tapioca and
potato is -idely used) The suita!ility of these su!strates for citric acid fermentation
depends on their purity and method of hydrolysis) 5cid hydrolysis, en<ymatic
hydrolysis, or a com!ination of the t-o, are used) >reparation of starch su!strates for
fermentation is !ased on their en<ymatic li1uefaction and saccharification to a defined
hydrolysis level) 5dditional nutrients are added, depending on -hich starch is used) The
p$ is adusted to 26, using hydrochloric or sulphuric acid and the medium is sterili<ed
at 121 OC for ')861 hour)
4ood citric acid yields have !een also o!tained using de3trose syrup, o!tained !y
en<ymatic hydrolysis of starch) This method is no- employed also inindustrial scale) #n
this case it is especially important to restrict the amount of heavy metals !elo- critical
levelsH heavy metals should therefore !e removed !y ion e3change)
.hen using an Aspergillus niger strain resistant to higher concentrations of heavy
metals, practically the same yield may !e o!tained on decationi<ed and non6
decationi<ed de3trose syrup /12'0)
,., $-dro'
This is a paramolasses o!tained as a !y6product during crystalline glucose production
from starch) Because of the high glucose content B,'6,8 per centC and high purity
coefficient it is a very good su!strate for citric acid production) >reparation of hydrol
for fermentation involves dilution to a sugar concentration of 1861+ per cent, addition
of necessary nutrients and adustment of p$ -ith hydrochloric or sulphuric acid to 2)'6
,)') The solution is sterili<ed at 121 OC for ')8 hour and cooled to 22628OC /1210)
,./ A'0ane+
The lo- price of al:anes, coupled -ith the a!ility of many organisms to utili<e them,
produced maor changes in the fermentation industry during the 1%&'s and 1%*'s)
Citric acid production, using Candida lipolytica, is a typical e3ample and has !een the
su!ect of many patents /122,1220) There are fe- industrial citric acid processes that
are !ased on al:anes) #n these processes isocitric acid -ould also !e produced at
concentrations that -ould cause product recovery pro!lems, as -ell as reduced citric
acid yields /12,0) 5 fourfold increase in price since 1%*2 no longer ma:es al:anes a
cheap su!strate)
,.1 Oi'+ and &at+
For citric acid production, oils are no- !eing used as principal car!on source in a manner
analogous to the previous use of al:anes) .ith palm oil as car!on source, a yield of citric
acid of 1,8 per cent using a mutant of Candida lipolytica has !een reported /1280 B#:eno
et ah, 1%*8C) There are e3amples of oil !eing added in small concentrations to Aspergillus
niger fermentation /12&0 and even !eing used as a sole car!on source for Aspergillus niger
fermentation) #t -as found that citric acid could !e produced on these su!strates -ith good
yield /12*0) These oils and fats may replace al:anes in several fermentations, !ut it is
unli:ely that they -ill remain at their current lo- prices)
/. PRODUCTION PROCEE
5lthough in citric acid industrial scale production in past surface or emerged production in
earlier years of t-enty centuries dominated over traditional method of preparing citric acid
!y e3traction from various uices at the present time a much greater emphasis is placed on
the use of su!merged culture production) Batch techni1ues in stirred tan: or air lift
!ioreactors are in general use) Aery promising results -ere o!tained in fed6!atch process
/2+,2%,,'0 and !y continuous fermentation /12+61210 -here various :inds of !ioreactors as
stirred tan: reactors /1'',1'2,1'&0, airlift reactors /122,1220, e3ternal loop reactors
/12,,1280, magnetic drum contactors /12&0, reciprocated et reactors, !iodisc reactor
/12*0, deep et reactors /12+,12%0, in hollo- fi!er /1,'0 or !y use of fi3 !ed reactor /1,10)

(everal report of citric acid fermentation using immo!ili<ed A. niger cells on various :inds
of carriers as glass /1,20, polyurethane foams /1,20, entrapment in calcium alginate !eds
/1,,,1,8,1,&0 polyacrylamide gels /1,*,1,+0 agar /1,%0 agarose /18'0 cellulose carriers
/181,1820 metal screens and polyester felts /182,18,0)
The traditional method of preparing citric acid !y e3traction from the uice of lemons,
limes and pineapple -astes is still in practice in the developing -orld, !ut its production is
not significant, as it comprises less .orld production /188,18&0)
Aarious chemical syntheses of citric acid have appeared in the chemical and patent
literature since the first one !ased on the reaction of glycerol6derived 1)2 dichloroacetone
-ith cyanide !y 4rimau3 and 5dam in 1++' /20 ) $o-ever none of these has reached a
commercial status competitive -ith fermentation processes)
/.1 (r&ace .roce++ on 'i2(id +(3+trate
The surface fermentation process, using li1uid su!strate is the oldest production method
and accounts for 861' @ of The .orld supply of citric acid)
This process is still in use !ecause of lo- investment, and energy cost for the cooling
and heating system, and due to simple technology, despite to the higher la!our costs as
compared to su!merged fermentation) The system consists of fermentation rooms in -hich
a large num!er of trays are mounted one over the other in sta!le rac:s) The trays are
generally made of high purity aluminium or special stainless steel) Their si<e varies from 2 3
2)8 3 ')18 m to 2)8 3 , 3 ')18 m, -ith usage li1uid depths of ')'+ to ')12 m) >rovision is
made for continuous filling and draining !y appropriate overflo- devices) 5eration is
provided !y climati<ed sterile air circulation, -hich serves the purpose of temperature
regulation and only to a lesser e3tent that of supplying o3ygen and controlling humidity)
5ir is introduced in to the fermentation cham!er in an almost laminar flo- manner /2*0 )
=olasses su!strates is generally employed as a su!strate containing 18 6 2' @ of
sucrose, added nutrients, /various natural polymers /,2,,20, acidified -ith, e)g), phosphoric
acid to a p$ &)' 6 &)8 and heat heated at temperature 11' PC for 18 to ,8 min)
(u!se1uently, potassium he3acyanoferrate is added to the hot su!strate, to precipitate or
comple3 trace metals /Fe, =n, 9n0 and to act in e3cess as a meta!olic inhi!itor restricting
gro-th and promoting acid production /,20) For come molasses com!ined treatment -ith
tricalcium phosphate, hydrochloric acid and (ephade3 -as used /,&0 )
#noculation is performed in t-o -ays, as a suspension of conidia added to the cooled
medium, or as a dry conidia mi3ed -ith sterile air and spread as an aerosol over the trays
/2*0)The temperature is :ept constant at 2' PC during the fermentation !y means of air
current) Aentilation is also important for gas e3change !ecause the rate of citric acid
production drops if car!on dio3ide in the atmosphere increases over 1' @) .ithin 2,
hours after inoculation, the germinating spores start forming a 262 cm cover !lan:et of
mycelium floating on the surface of the su!strate) 5s a result of the upta:e of ammonium
ions the p$ of the su!strate falls to 2)') 5fter 2' hours the idiophase !egins) #f too much
iron ions are present, o3alic acid is produced and a yello-ish pigment is formed, -hich later
complicates the recovery process) The fully developed mycelium floats as a thic: -hite
layer on the nutrient solution) Through evaporation, the temperature can !e maintained
constant, !ut the culture loses 2' 6 ,' @ of its original volume) The fermentation process
stops after + 6 1, days)
For recovery, the mycelium and nutrient solution are removed from the cham!ers) Due to
its volume, the mycelium must !e carefully -ashed in sections) 7n some cases,
mechanical presses are also used to o!tain more citric acid from the cells)
/.2 o'id +tate &er*entation
(urface process employing solid su!strate may use fi!rous residues from apple /,,0 ,
grape pommace /,80, -heat !ran or rice starch containing residual pulps from starch
manufacture /,&0 potato /,&0 and s-eet potato /,*0 )
#n this process, !ased on the traditional :oi process :no-6ho-, the Aspergillus niger
strains are not sensitive to trace elements as in surface fermentation -ith li1uid su!strate or
in su!merged process /,+0 )
7n the solid state fermentation process the solid su!strate is soa:ed -ith -ater up to &8 6
*' @ of -ater content) 5fter the removal of e3cess -ater, the mass undergoes a steaming
process) 5fter sterilisation, sterile starch paste is inoculated !y spreading Aspergillus
niger conidia in the form of aerosol or as a li1uid conidia suspension on the su!strate
surface /2*0)
The p$ of the su!strate is a!out 8 to 8)8, and incu!ation temperature 2+ to 2' PC) 4ro-th
can !e accelerated !y adding Q6amylase, although the fungus can hydroly<e starch -ith
its o-n Q6 amylase) During the citric acid production p$ dropped to values !elo- 2 /,'0 )
The solid state surface process ta:es 8 to + days at the end of -hich the entire is e3tracted
-ith hot -ater) 7n other cases, mechanical passes are also used to o!tain more citric acid
from the cells)
Using cane !agasse as the su!strate !y solid state fermentation citric acid -as o!tained in
& days /18*0) Total .orld production of citric acid !y solid state fermentation -as in 1%%'
a!out 28')''' tons /188,18&0 )
/.! (3*erged &er*entation
5n effective alternative to surface fermentation processes is the su!merged process)
5lthough ta:ing a longer fermentation time it has several advantagesD lo-er investment !y
a factor of 2)8, 28 @ lo-er total investment and la!our costs, more effective process
control and sterility) The disadvantages are the higher energy costs and more sophisticated
control -hich re1uires more highly trained personal)
Three main factors especially important for highly yielding citric acid production in
su!merged processes are /,'0 D
6 1uality of the stainless steel for the construction of the !ioreactor
6 mycelium structure
6 o3ygen transfer
Batch techni1ues in stirred tan: or air lift !ioreactors are in general use) Aery promising
results -ere o!tained in fed6!atch process /2+,2%,,'0 and !y continuous fermentation /12+6
1210 -here various :inds of !ioreactors as stirred tan: reactors /1'',1'2,1'&0, airlift
reactors /122,1220, e3ternal loop reactors /12,,1280, magnetic drum contactors /12&0,
reciprocated et reactors, !iodisc reactor /12*0, deep et /12+,12%0 hollo- fi!er /1,'0 or !y
use of fi3 !ed reactor /1,10)
(everal report of citric acid fermentation using immo!ili<ed A. niger cells on various :inds
of carriers as glass /1,20, polyurethane foams /1,20, entrapment in calcium alginate !eds
/1,,,1,8,1,&0 polyacrylamide gels /1,*,1,+0 agar /1,%0 agarose /18'0 cellulose carriers
/181,1820 metal screens and polyester felts /182,18,0) Bioreactors for citric acid
production must !e either protected from acids or constructed of special stainless steel)
5t p$ value 2 the heavy metals leached from ordinary steel fermenter -alls can inhi!it the
formation of citric acid /,'0 )
Aarious su!strates as !eet /,2,18+61&10 and cane molasses /1&2,1&2,1&,0, media /1&86
1&*0, starch hydroli<ates/1&+61*20, C%6C22 paraffines /2+,2%0 and consume oil /1*261*80
have !een reported) The concentration of car!on source in fermentation su!strate is of
great importance) =a3imum citric acid production is usually achieved at car!on
concentrations as high as 1, 6 2* @)
/.!.1. (3*erged &er*entation (+ing Aspergillus niger
7n the case of !eet molasses su!strate the reducing sugar content is usually 12 6 18 @)
The ro- molasses is previously clarified !y sulphuric acid and neutralised) >otassium
he3acyanofernate is added to the preparatory su!strate for the purpose of suppressing,
!y means of comple3 formation, any detrimental effect of metal ions, particularly iron and
to prevent a too rapid gro-th of the mycelium)
"utritive salts, such as ammonium nitrate or potassium dihydrogen phosphate may !e
added) For su!strate preparation common tap -ater can !e used) 7-ing to its content of
salts it is generally more suita!le than deionised -ater)p$ of the su!strate should !e adusted
to 8)8 to 8)%, -hich is the most suita!le for the germinated conidia aggregation) (u!strate is
sterilised !y heat, mostly !y continuous sterili<ation /1&+0)
#n the case of the relatively pure sucrose containing su!strates, fermentation is generally run
at the medium sucrose concentration of 18 6 2* @) 5fter ion e3change of the cations, the
filtered solution is sterilised su!se1uently !y heating and after cooling to 8' PC fed to
!ioreactor) The !ioreactors filled up to its -or:ing volume and nutrients are added) The
p$ is adusted to an initial value of 2)8 to 2)' /2*0 )
The process can usually run in one or t-o stages, using hydrophilic spores suspensions /,'0
or germinated conidia from the propagator stage /1&+0 ) The use of germinated conidia
may shorten the fermentation cycle from 12 to 2, hours /2*0) 5mounts of spores are 8 to 28
3 1'
&
per litre of su!strate /2*0 and conidia 1'
1'
per litre /1&+0 ) #t has !een proved
useful to incu!ate the spore suspension for & to + hours in saline solution -ith added
surface active agents prior to inoculation, thus shortening the fermentation cycle for 12
hours /1&80 )
7n the t-o stage fermentation process, germinated conida are produced in the first stage at
p$ 8)+, -ith a!sence of phosphate, at lo- dissolved o3ygen level and at a sugar
concentration of * to % @ at temperature of 22 PC /1&%0 )
For citric acid production, the spherical mycelia pellet gro-th form is -idely used
/1*2,1*,0) 5n effective pellet formation is prefera!ly performed !y a higher shear stress
effected !y aeration and agitation of the su!strate) The development of the hyphae and the
aggregation generally re1uires a period from % to 28 hours at temperature of 22 PC) The first
t-o or three days of fermentation, i)e) the period of initial mycelial gro-th and pellet
formation, are decisive for the success of the fermentation) $eavy metals in the medium
BFe, =nC, e3ceeding concentration of iron ions higher than 1)8 mgIl and manganese 1 Rg Il
strongly affect cellular morphology, !y promoting unproductive filamentous mycelial gro-th
form /2*0 )
2, hours after inoculation the production of citric acid start) =ycelial aggregates and
spherical pellets, the productive form can !e detected at the first and the second ma3imum
of the redo3 potential curve /1*8,1*&0 )
The start of citric acid production is follo-ed !y an e3cessive foaming, therefore an
effective foam control system is essentially needed /1**0 )
The additions of silicone antifoam agents can reduce the dissolved o3ygen
concentration /1*+,1*%0 or influence increasingly the pseudoplastic rheological
!ehaviour of the fermentation !roth /1*,0 )7n the production phase the aeration is set
from ')2 v)v)m), in germination phase to 1 v)v)m) The change of p$ in this phase is from 8)8
to 2)8, for !eet molasses su!strate, and to 2)2 for the sucrose su!strate) The p$ of ,)8 6 2 is
also characteristic for the fed !atch fermentation production phase, -here !ioreactor is
only filled to ,' @ of its -or:ing volume -ith the propagation su!strate containing * @ of
sucrose content, and fulfilled -ith citric acid production su!strate of 1* @ sucrose content
/2*0 )
The temperature in the production phase is from 2+ to PC) Temperature change from 22 to
2+ PC is also the !ase of some industrial processes and patents /1+'0 )
(u!merged citric acid fermentation using starch hydrolisates as a car!on sources is also an
effective alternative to standard processes on !eet molasses or sucrose su!strate) 5n
advance step in this technology is L#F7 process !ased on various starch hydrolisates /1&%0)
7n the first step starch is treated !y termosta!ile Q6amilase and temperature of 1'2 PC)
7ften additional en<ymatic treatment !y com!ination of pullulanose and fungal Q6 amylase
is need) "utrient salts -ere added to the starch hydrolisate and su!strate is after
continuous sterili<ation used in propagation and production stage)
For the separation of the product and -aste !iomass tangential flo- filtration is used)
Compared to classic Ca citrate precipitations -ith &,' @ !ases, only 2+ @ of the citric
acid -as lost) The !enefit of this process are simpler isolation more pure product and no
calcium sulphate)
(u!merged citric acid fermentation on -heat flavour hydrolisates designed is ine3pensive
technology -ith more pure product has a lot of perspectives in the future production
Comparing to using !eet molasses and sucrose su!strates, this is also a cheaper process)
7ne of pro!a!ly the most perspective su!merged citric acid technology on sucrose
su!strate !y LeSnia: et al) B++ 6 %' @ yields on industrial 18' m
2
reactors0 /2+0) 5s an
alternative to !atch and fed6!atch process is Aspergillus niger continuous citric acid
fermentation developed !y B) Fristiansen and co6-or:ers /12+,12%0 )
/.!.2 (3*erged &er*entation (+ing -ea+t+
Candida strains are also used novel process that permits production of citric acid from C%
to C2' normal paraffins) Citric acid yields up to %8 @ -ere claimed) 7n 1%*, >fi<er
patented a continuous process for fermentation !y Candida lypolitica using a single
bioreactor to -hich paraffin -as continuously added and fermented !roth continuously
-ithdra-n /1*10 )
7n citric acid fermentation stirred tan: bioreactors, -ith usual of capacities from 8' to 18'
m
2
and air lift bioreactors up to 22' m
2
, are used) The fermentation is a gro-th associated
process -hich lasts from & to + days)
/.!.! (3*erged &er*entation (+ing i**o3i'i4ation o& *icro5organi+*+
#t is -orth noting that some of the pro!lems arising in the do-nstream processing of citric
acid produced !y su!merged cultivation, especially in a continuous process, might !e
minimi<ed !y immo!ili<ation of micro6organisms in the !ioreactor) The successful
application of immo!ili<ed micro6organisms as living !iocatalysts, involving more careful
handling and often having higher production rates than free micro6organisms, has prompted
a rapid development of this techni1ue) Citric acid production !y immo!ili<ed 5) niger has
!een performed on a la!oratory scale -ith the use of calcium alginate gel /1+1,1+20,
polyacrylamide gel /1+2,1+,0, polyurethane foam /1+8,1+&,1+*0 and cryopolymeri<ed
acrylamide /1++0 ) The profita!le effect of the immo!ili<ation of 5) niger mycelium in vie-
of the citric acid recovery from the fermentation !roth depends on the type of the support
material and process conditions)
/., o'id +tate &er*entation
The solid culture process is completed -ithin %& hours under optimal conditions B%C) The
most common organism used in solid6state fermentation is A. niger. $o-ever, there have
also !een reports -ith yeasts) The strains -ith large re1uirements of nitrogen and
phosphorus are not ideal microorganisms for solid culture due to lo-er diffusion rate of
nutrients and meta!olites occurring at lo-er -ater activity in solid6 state process) The
presence of trace elements may not affect citric acid production so harmfully as it does in
su!merged fermentation, thus, su!strate pretreatment is not re1uired) This is one of the
important advantages of the solid culture /1+%,1%%'0)
1. PRODUCT RECO6ER%
7n pletion of the citric acid fermentation the o!tained solution contains, !esides the
desira!le product, mycelium and varying amounts of other impurities, e)g) mineral salts,
other organic acids, proteins, etc) The method of citric acid recovery from the fermentation
!roth may vary depending on the technology and ra- materials used for the production
/1%10) (eparation of !iomass from fermentation !roth ta:es place in first step of the
recovery process) separated mycelia retain a!out 18 @ of the citric acid formed during
fermentation) The mycelia are then -ashed and pressed in filter presses dried and often
used as a protein rich feed for cattle) #f o3alic acid is formed as a side product due to
su!optimal fermentation control, it can !e precipitated as calcium o3alate at p$ !elo- 2)'
/1%20)
1.1 (r&ace .roce++
#n the surface process the fermentation fluid is drained off the trays and hot -ater is
introduced to -ash out the remaining amount of citric acid from the mycelial mats)
5lthough it is a relatively simple procedure in the case of surface fermentation, -here
!iomass is in the form of 262 cm cover !lan:et on the su!strate surface) Thorough
-ashing at this stage is necessary, !ecause the mycelium retains a!out 18 per cent of the
product formed in the fermentation) #n this vessel the mycelium is heated to a!out 1''OC !y
steam) The solution containing 26, per cent of citric acid is added to the fermentation fluid,
-hereas the filtration ca:e, containing not more than ')2 per cent of citric acid, is dried to
yield a protein6rich feed6stuff /1%20)
1.2 (3*erged &er*entation
#n the su!merged fermentation the mycelium is far more difficult to separate from the
fermentation !roth) 5fter the fermentation process is completed the mycelium containing
!roth is heated to a temperature of *'OC for a!out 18 minutes, to o!tain partial coagulation
of proteins, and then filtered) Eotating Aacuum drum or !elt discharge filters or in
centrifuges are used in this case /,'0 )#f the mycelium is to !e used as a feedstuff, the filter
aid must also !e digesti!le, e)g) from cellulosic materials) #f during the fermentation process
o3alic acid is formed it has to !e removed from the !roth) This is usually achieved !y
increasing the p$ of the fermentation fluid -ith the calcium hydro3ide to p$ L 2)*62)% at a
temperature of *'6*8OC) Calcium o3alate thus precipitated may !e removed from the
solution !y nitration or centrifugation, and the citric acid remains in solution as the mono6
calcium citrate)
Eecovery of citric acid from pretreated fermentation !roth may !e accomplished !y several
proceduresD classical method of precipitation, solvent e3traction, ion6e3change and some
more sophisticated methods such as electrodialysis, ultra6 and nanofiltration or application
of li1uid mem!ranes B4lus< and Leda:o-ic<C /1%20)
1.! Preci.itation
The standard method of citric acid recovery has involved precipitating the insolu!le tri6
calcium citrate !y the addition of an e1uivalent amount of lime to the citric acid solution)
(uccessful operation of the precipitation depends on citric acid concentration, temperature,
p$ and rate of lime addition) To o!tain large crystals of high purity, mil: of lime containing
calcium o3ide B1+'628' :gIm
2
C is added gradually at a temperature of %'OC or a!ove and
p$ !elo-, !ut close to, *) The concentration of citric acid in the solution should !e a!ove
18 per cent) The process of neutrali<ation usually lasts a!out 12'618' minutes) The
minimum loss of citric acid due to solu!ility of calcium citrate is ,68 per cent) Calcium
citrate is then filtered off and su!se1uently treated -ith concentrated sulphuric acid B&'6*'
per centC to o!tain citric acid and the precipitate of calcium sulphate BgypsumC) 5fter
filtering off the gypsum a solution of 2862' per cent of citric acid is o!tained) The filtrate is
treated -ith activated car!on to remove residual impurities or may !e purified in ion6
e3change columns) The purified solution is then concentrated in vacuum evaporators at
temperature !elo- ,'OC Bto avoid carameli<ationC, crystalli<ed) 5 conventional
crystallisation scheme consists of a !atch vacuum6pan evaporator or a forced circulating
evaporator coupled -ith au3iliary tan:age and appropriate centrifuge e1uipment) .ithin
these systems the crystals formed are separated !y centrifugation and the mother li1uor is
fed !ac: to the activated car!on stage) Both !atch and continuous units have !een
employed in this cooperation depending of process adapta!ility and economics /,'0 )
The drying of citric acid monohydrate is usually performed in conventional rotary
drying e1uipment or in fluidised !ed dryers) 5s anhydrous citric acid is hygroscopic, care
must !e ta:en to achieve the final moisture specification during drying and to avoid
storage in areas of high temperature and humidity /1%20 )
The disadvantage of this technology is the large amount of lime re1uired for citric acid
neutrali<ation and of sulphuric acid for calcium citrate decomposition) =oreover, it results
in the formation of large amounts of li1uid and solid -astes Bsolution after calcium citrate
filtration and gypsumC) For one tonne of citric acid, 8*% :g of calcium hydro3ide, *&8 :g of
sulphuric acid and 1+m
2
of -ater are consumed and appro3imately one tonne of -aste
gypsum is produced /1%20)
.ith the aim of decreasing the amount of lime and sulphuric acid !y a!out one third,
/1%20) has proposed recovery of citric acid !y precipitation of di6calcium acid citrate) 5n
additional advantage of this method is that di6calcium acid citrate has a definite crystalline
structure and -ashes cleaner than the amorphous tri6calcium citrate)
1.# o'vent e7traction
5n alternative method of citrate6free recovery of citric acid from a fermentation !roth
is e3traction !y means of a selective solvent -hich is insolu!le or only sparingly solu!le
in the a1ueous medium /1%,,1%8,1%&0) The solvent should !e chosen so as to e3tract
the ma3imum amount of citric acid and the minimum amount of impurities) The citric
acid can then !e recovered from the e3tract either !y distilling off the solvent or !y
-ashing the e3tract -ith the -ater) From the a1ueous solution purified citric acid is
su!se1uently crystalli<ed !y concentration)
Aarious organic solvents -hich are partly or -holly immisci!le -ith -ater, such as
certain aliphatic alcohols, :etones, ethers or esters /1%*0, organophosphorus
compounds, such as tri6n6!utylphosphate BTB>C /1%+0 and al:ylsulpho3ides, /1%%0
and -ater6insolu!le amines or a mi3ture of t-o or more of such amines are used /2''6
2'20)
1., Ion e7c)ange
The efficiency of the ion6e3change separation process may !e greatly enhanced !y
applying a simulated moving !ed counter6current flo- system) #t consists of at least
t-o static !eds, connected -ith appropriate valving so that the feed mi3ture is passed
through one adsor!ent !ed -hile the desor!ent material can !e passed through the
other) >rogressive changes in the function of each ion6e3change !ed simulate the
counter6current movement of the adsor!ent in relation to li1uid flo-) #n such a system,
the adsorption and desorption operations are continuously ta:ing place, -hich allo-s
!oth continuous production of an e3tract and a raffinate stream and the continual use of
feed and desor!ent streams /2'20)
The disadvantage of the ion6e3change method may !e seen in the fact that elution of
citric acid from the adsorption !ed may re1uire a large amount of desor!ent, due to the
tailing effect :no-n in chromatography, causing considera!le dilution of the resulting
citric acid solution) The periodical regeneration of the ion6e3change resins !y inorganic
!ases may also !e a source of un-anted effluent -astes)
1./ Li2(id *e*3rane+
Li1uid mem!ranes containing mo!ile carriers consist of an inert, micro6porous support
impregnated -ith a -ater6immisci!le, mo!ile ion6e3change agent) The mo!ile carrier,
-hich is held in the pores of the support mem!rane !y capillarity, acts as a shuttle,
pic:ing up ions from an a1ueous solution on one side of the mem!rane, carrying them
across the mem!rane and releasing them to the solution on the opposite side of the
mem!rane /2',0) For citric acid separation !y li1uid mem!ranes, the tertiary amines
-hich give the !est results also in solvent e3traction can also !e used)
Eecently more sophisticated methods of citric acid separation -ith the application of
li1uid mem!ranes are !eing developed /2'862'*0)
1.1 Micro.oro(+ )o''o8 &i3re+
=icroporous hollo- fi!res have !een employed !y Basu and (ir:ar B1%%1C /2'+0) #n this
case the permeator consists of t-o sets of identical hydropho!ic microporous hollo- fi!res)
7ne set carries the feed solution of citric acid and the other the strip solution flo-ing in the
lumen) The organic li1uid mem!rane is contained in the shell side !et-een these t-o sets of
hollo- fi!res) This techni1ue has !een sho-n to !e promising for citric acid
separation even in the large scale, as the e3tent of citric acid recovery of up to %% per
cent -as linear -ith the mem!rane area, suggesting easy scale6up /1%20)
1.9 E'ectrodia'-+i+
This process ena!les separation of salts from a solution and their simultaneous conversion
into the corresponding acids and !ases using electrical potential and mono6 or !ipolar
mem!ranes) Bipolar mem!ranes are special ion e3change mem!ranes -hich, in an electrical
field, ena!le the splitting of -ater into $
K
and 7$G ions /2'%0) By integrating !ipolar
mem!ranes -ith anionic and cati6onic e3change mem!ranes a three or four compartment
cell may !e arranged, in -hich electrodialytic separation of salt ions and their conversion
into !ase and acid ta:es place/21'0)
Before the fermentation solution comes to the electrodialysis some pretreatment steps are
normally necessaryD filtration of the !roth, removal of ionogenic su!stances Bespecially Ca
KK
and =g
KK
ionsC and neutrali<ation !y means of sodium hydro3ide) #n the su!se1uent
electrodialytic step the sodium citrate solution is converted into !ase and citric acid, -hich
is simultaneously concentrated and for the most part purified) The produced "a7$ may !e
reused for the neutrali<ation /2110)
The energy consumption Be3cluding pumpingC for the separation of 1 :g of citric acid using
!ipolar mem!ranes is in the range of &)1 3 1'
2
to *)2 3 1'
2
:.s /2120) Due to lo- mass
transfer at lo- p$ values it is advantageous to adust the p$ of the feed acid stream to *)8
/212,21,0)
1.: U'tra&i'tration
Continuous separation and concentration of citric acid may !e also achieved !y ultra andI or
nanofiltration) verified in a la!oratory scale a t-o6stage mem!rane process for citric acid
recovery from the !roth o!tained in 5) niger cultivation on sucrose) >olysulphone
mem!rane -ith cut6off 1' ''' used in the first stage allo-ed the product to pass through to
the permeate stream, -hile the retentate stream contained most of peptides and proteins
from the !roth) The reection coefficient for the product in this step -as 2 per cent, for the
reducing sugars 1, per cent and for the proteins 1'' per cent) Tighter nanofiltration
mem!rane -ith cut6off 2'' in the second stage reected appro3imately %' per cent of citric
acid and &' per cent of reducing sugars Bmono6saccharidesC 5 similar t-o6stage mem!rane
techni1ue -as adapted !y Bohd<ie-ic< and Bod<e: B1%%,C /2180 for simultaneous
separation and concentration of pectinolytic en<ymes and citric acid from a fermentation
!roth)
Eecovery of citric acid via calcium salt precipitation is a comple3 process) #n this
process calcium citrate is formed in further !y adding a lime slurry at a neutral p$) 5fter
sufficient reaction time, the slurry is filtered and the precipitate -ashed free of solu!le
impurities) The resulting calcium citrate is then acidified -ith sulfuric acid) This reaction
converts calcium citrate to calcium sulfate and citric acid in the presence of free sulphuric
acid) Calcium sulphate is then filtered and -ashed free of citric acid solution) Both the
calcium citrate and calcium sulphate reactions are generally performed in agitated reactors
and filtrated commercially availa!le filtration e1uipment)
The a1ueous citric acid solution is demineralised at this step !y strong cation e3change
resin in the $
K
form BDo-e3 8'C and an anion e3change resin of medium strength) The
purified citric acid solution is su!se1uently evaporated in a multi stage evaporator at
temperature of ,' PC to avoid carameli<ation /2*0 )
The clear citric acid solution undergoes a series of crystallisation steps to achieve the
physical separation of citric acid from the remaining trace impurities) 5 conventional
crystallisation scheme consists of a !atch vacuum6pan evaporator or a forced circulating
evaporator coupled -ith au3iliary tan:age and appropriate centrifuge e1uipment) .ithin
these systems the crystals formed are separated !y centrifugation and the mother li1uor is
fed !ac: to the activated car!on stage) Both !atch and continuous units have !een
employed in this cooperation depending of process adapta!ility and economics /,'0 )The
drying of citric acid monohydrate is usually performed in conventional rotary drying
e1uipment or in fluidised !ed dryers) 5s anhydrous citric acid is hygroscopic, care must
!e ta:en to achieve the final moisture specification during drying and to avoid storage in
areas of high temperature and humidity /2*0)
(olvent e3traction is an alternative recovery process, -hich involves the e3traction of
citric acid from fermentation !rutish using hydrocar!ons such asD n6octanol, C1' or C11
isoparaffin, !en<ene, :eroseneH ethersD n or iso !utyleterH estersD n6!utylacetateH :etonesD
methyl iso!utyl:etone /1'80 or various aminesD trilaurylamine /1'&0 ) The recovery
process !y solvent e3traction consists of selectively transferring citric acid via a solvent
from an a1ueous solution containing various !y6products to another a1ueous solution in
-hich the citric acid is more concentrated and contains su!stantially less !y products) The
final processing steps !egin -ith a different -ash of the a1ueous solution !y the
hydrocar!on solvent, follo-ed !y the passage of the acid solution through a conventional
se1uence of evaporator6crystalli<er steps to complete the manufacturing process)
5nhydrous citric acid and its monohydrate can !e stored in dry form -ithout
difficultiesH ho-ever, high humidity and elevated temperatures should !e avoided to prevent
ca:ing) There fore the use of pac:ing materials -ith a desiccant is suggested /1'*0 )
The citric acid recovery process leads to considera!le accumulation of -aste products)
=ove than &' @ of it !elongs to gypsum /calcium sulphate0 ) .hich still contains
potassium he3acyanoferrote, charcoal and organic compounds from molasses ma:ing it so
unsuita!le as a !uilding material) The -aste mycellium from su!merged and surface
fermentation can !e dried and used as an animal protein rich feed or alternatively as
fertiliser)
9. ECONOMIC APECT
5lthough the surface production process is from the vie-point of energy re1uirements, a
less e3pensive, there are a lot of disadvantages in it) This involves larger space
re1uirements for production and isolation, higher steam re1uirement and higher sterility
re1uests) 7ne of the greatest pro!lems of this production process sterility) =ain
advantages of the su!merged fermentation process areD shorter fermentation time B&6*
daysC , higher level of process sterility and control of process parameters, simpler
process operations, lo-er space re1uirements, process reproduci!ility and higher yields)
(chierholt / 21& 0 compared the economy of surface and su!merged fermentation process
for the citric acid production D
Capacities of 2'' m
2
and 18' m
2
in % days of fermentation time at productivity *2 tons and
12 tons per day, -ere compared) 7n his -or: he concludes that the !uilding investment
costs connected -ith the surface fermentation process are 2)8 times higher than those
connected -ith the su!merged fermentation) Contrary to this, the e3penses on e1uipment
are considera!ly higher at su!merged fermentation, and more than &' @ of those e3penses
consist of complicated component as are !ioreactors and more sophisticated instrumental
control, -hich are su!ect to relatively high -ear)
The total investment costs for the su!merged process are a!out 28 @ lo-er for higher
capacities and 18 @ lover for smaller capacities than for surface fermentation) The more
favoura!le total investment costs for the su!merged process are in contrast to considera!ly
higher production costs for any capacity) Especially evident is the high consumption of
electric energy -hich is a!out 2' @ higher as much as that re1uired at surface fermentation)
The la!our costs in highly developed countries are for surface fermentation considera!ly
higher)
7n countries -here cooling -ater temperature e3ceed 2' PC additional e3penses for
cooling the !ioreactors are incurred !y installation of coolling aggregates for su!merged
process) The su!merged fermentation is sensitive to short interruptions or !rea:do-ns
in aeration, -hich results not only in losses of yield, !ut also in total !rea:do-n of the
respective !atch) 5t surface fermentation, the resulting citric acid solution or fermentation
!roth is much more concentrated than at su!merged fermentation, effected !y higher
evaporation rates during fermentation)
>roduction of citric acid !y surface solid state or !y isolation from citrus uices does not
represent a significant percentage on The .orld scale) 5lthough !oth processes are from all
aspects very cheap processes, they are in use mostly in the countries -ith old tradition
B#taly, 4reece, 5siaC)
:. CITRIC ACID ;ORLD PRODUCTION
Development of citric acid fermentation industry during the nearly passed century has
aroused a great deal of interest) Formerly, the ra- material, calcium citrate, -as produced
almost entirely from citrus products, #taly !eing !y far the largest producer) The !ul: of
the #talian production of calcium citrate -as shipped to England, France and the United
(tates) Because of the development of the fermentation process and the increased output of
citrus materials, import in the United (tates has practically ceased since 1%2*) The
fermentation process has to a large e3tent developed also in Europe) Large 1uantities of
fermentation !ased citric acid have !een produced in England, Belgium and
C<echoslova:ia and pro!a!ly Eussia) The former dominant position occupied !y the #talian
producers of this commodity has thus !een last through ne- methods introduced !y
scientific research)
The first successful commercial development of the citric acid fermentation process
-as achieved in the United (tates) =iles and later Charles >fi<er Company gradually
developed in to .orld leading companies) The United (tates citric acid production in
1%2% -as ,%'' tons per year) .hile in 1%*+ the production !y =iles -as 2%''' tons and
!y >fi<er /U)()0 ,28'' tons, and raised in 1%%' to &&)''' tons !y =iles and 1'8)''' tons
!y >fi<er /U)()0 )>fi<er overall production, including the U)() and other countries B#rish
Eepu!lic, "igeria, Tai-an, 5rgentinaC , ta:es a!out 2' @ of The .orld citric acid
production) From 1%*+ until 1%%' >fi<er increased its production !y 2,)8 @
5lthough The .orld greatest producers are in the United (tates, The .orld;s greatest
production continentally !elongs to Europe -ith 28'''' tons per year, produced in
si3teen countries) The yearly production in "orth 5merica -as in 1%%' a!out 218)''' tons
follo-ed !y 5sia -ith &&)''' tons, 5frica 1,)''' tons, 5ustralia a!out +)''' tons and
(outh 5merica -ith *)''' tons) #n 5sia citric acid production is characterised also !y the
use of traditional solid state production on the foad industry -astes and !y su!merged
technogies !ased on) various yeast strains)
Ta3'e I. T)e ind(+tria' ;or'd .rod(ction o& citric acid in 1::<
T)e greate+t ;or'd .rod(cer+ /1880
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
U(5 >fi<er 1'8)''' tons
=iles &&)'''
Belgium Citri1ue BelgeU 88)'''
5ustria ?ung!un<lauer ,')'''
#reland >fi<er 2&)'''
4erm)Fed)Eep) Biochemic Laden!urg 2')'''
#taly Biacor 28)'''
=e3ico Vuimica =e3ama 1%)'''
(oviet Union (tate 5uthorityU 1+)'''
4reat Britain ?ohn W E) (turge 1,)'''
#srael Cadot >etroch +)'''
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
.orld >roduction 1%%' 8%+)'''
U(urface production
Citric acid is a commodity chemical produced and consumed throughout The .orld) #t is
used mainly in the food and !everage industry, primarily as an acidulant) #t is estimated that
over &8@ of the citric acid produced is consumed for food and !everages) 4lo!al
production of citric acid in 2'', -as a!out 1), million tonnes estimated !y Business
Communications Co) !ut in 2''8 -as a!out 1,&'' thousand metric tons) The maority of
production capacity and consumption -as in China, .estern Europe and the United (tates)
China is estimated to account for at least half of glo!al production capacity, -hile .estern
Europe and the United (tates com!ined account for a!out a third) .estern Europe, the
United (tates and China com!ined are estimated to account for &8X*'@ of glo!al citric acid
consumption) The citric acid industry continues to !e influenced !y increased supply from
China and a!undant glo!al capacity) #n recent years, plant closures have occurred as a result
of competition, and prices have continued to decline)
1930 1940 1950 1960 1970 1980 1990 2000 2010
0
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1600000
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n
e
s
)
Fig)1 Citric acid .orld production
From the first industrial fermentations, The .orld production has increased e3ponential
from 8''' tons in 1%2% until the present production in 2''& of a!out 1&''''' tons per
year Fig 1) /188,18&0)
#n 2''8, the top si3 producing companies accounted for a!out 82@ of the .orldMs total
capacity for citric acid) ChinaMs capacity -as +'' thousand metric tons B8'@ of -orld
capacityC, most of -hich is unrefined citric acid) The glo!al production capacity of the
.orldMs si3 largest citric acidXproducing companies, plus China B-hich produces mostly
unrefined citric acidC, is sho-n !elo-)
Fig)2 .orld capacity for citric acid producer in 2''& /18&0
The follo-ing pie chart sho-s .orld consumption of citric acid !y region in 2''8D
Fig)2) .orld consumption of citric acid !y region in 2''8 /18&0
7ver half of glo!al consumption of citric acid is used for the !everage industry) The food
industry consumes a!out 18X2'@, follo-ed !y detergent and soaps B18X1*@C,
pharmaceuticals and cosmetics B*X%@C, and industrial uses B&X+@C)
#n the United (tates, the citric acid mar:et -ill continue to gro- mainly as a result of
gro-th in the !everage mar:et) "e- product introductions and continued use in diet colas,
fruit6flavored -aters, iced teas and sports drin:s -ill lead to higher gro-th) Li1uid
detergent gro-th -ill also contri!ute to gro-ing citric acid demand) "e- gro-th -ill also
!e seen in industrial applications, as rene-a!le resources continue to gro-) #n Canada, citric
acid use may increase significantly as a result of use in oil recovery)
#n Europe, the mar:et is impacted !y price, -hich is driven do-n !y a com!ination of
strong competition from Chinese product and an a!undance of glo!al capacity) European
producers are contending -ith imports from Chinese producers) .hile the average prices
-ere declining, Chinese imports of citric acid to .estern Europe gre- from roughly ,&
thousand metric tons in 1%%% to 1'% thousand metric tons in 2'',) Chinese competition is
mainly in citric acid monohydrate Bsolid formC and among citrates in sodium citrate, the
most6used6form of citric acid salts) Chinese suppliers have started to adopt .estern pricing
practices, -hich might lead to a more sta!ili<ed price) #n the future, European
manufacturers of citric acid and citrates might concentrate on the production of citric acid
solutions Busing solid form produced in6house or importedC andIor higher6value citrates)
The citric acid mar:et continues to face pressure from 5sian imports and increased glo!al
supply causing selling prices to decline) $o-ever, tight supplies from Europe caused !y
closures, and high energy and freight costs are some of the factors leading to higher citric
acid prices) The overall glo!al mar:et for citric acid is e3pected to gro- at an average
annual rate of 2)8X,)8@ in the ne3t fe- years)
LITERATURE
/ 1 0 .ehmer C), Chem)) 9entr), 2, ,8* B1+%2C
/ 2 0 4rimou3 E), 5dam >), C)E) 5cad) (ci) >aris %', 1282 B1++'C
/ 2 0 .ehmer C), Compt) Eend) 11*, 222 B1%+2C
/ , 0 .ehmer C), U)() >atent 818'22 B1%+,C
/ 8 0 =a<Y >), >errier 5), 5nn) #nst) >asteur 1+, 822, B1%',C
/ & 0 Buchner E), .Zstenfeld $) Biochem) 9tg) 1*, 2%8, B1%'%C
/ * 0 9ahors:y B) U)() >atent 1 '&828+ B1%12C
/ + 0 Thom C), Currie ?)"), ?) 5gric) Ees) *, 1, B1%1&C
/ % 0 Currie ?)"), ?) Biol) Chem) 22, 18, B1%1*C
/1'0 =e<<adroli 4), Fr) >atent +22 &21 B1%2+C
/110 >er1uin L)$)C), Dissertation, Tech) University Delft B1%2+C
/120 E[hr =), Fu!ice: C)>), Fomine: ?), \Citric 5cid] #n Biotechnology, Ed $)?)
Eehm, 4) Eeed Aol) 2, ,2', Aerlag Chem),
/120 Tana:a F), Fimura F), ^amaguchi F), ?ap >at) 12&** B1%&+C
/1,0 Fu:ui (), Tana:a 5), 5dv) Biochem Eng 1*, 1, B1%+'C
/180 Crueger .), Crueger 5), #n \Biotechnology], (cience Tech) #nc) B1%+,C
(underland, =)5), Usa
/1&0 Buchard E)F), =errit E)4) B1%*%C \Citric 5cid] #n \Fir:67thmers
Encyclopedia 7f Chemical Technology] 2
rd
Ed) Aol) &, >) 18', .iley, "e- ^or:
/1*0 Chem) 5!str) (ervice, \Ca File], Colum!us, 7hio, Usa B1%%'C
/1+0 Chem6#ntell, Chem) #nt) (ervice, London, 4reat Britain B1%%'C
/1%0 =oledina F)$), ?) Food) (ci) ,2, *8% B1%**C
/2'0 .ells C)E), =artin D)C), Tichenor D)5), ?) 5m) Dietetic 5ssoc) &1, &&8 B1%*2C
/210 Bhatacharya (), ?) #ndian Chem) (oc) 21, 221 B1%8,C
/220 =ac Donald L) 5m) >erfum) *& B*C, 22 B1%&1C
/220 Frummel $)F), 4ault T).), U)() >at 2, %+8 &&%, B1%*&C
/2,0 $orner D)C), Electroplat) =et) Finish, *8, B1%&+C
/280 (mith C).), =unton C)B), Electroplat) =et) Finish) 2%, ,18, B1%,1C
/2&0 Fr) >at) +12, 8,+, B1%2*C
/2*0 U)() >at 2, ,*,'%2, B1%,%C
/2+0 Fa-asa:i =), $arada F), "ippon (has:in 4a::aishi 2+ B1C 2*, B1%*8C
/2%0 $ushedec: $)E), U)() >at 2, 212 %2+ B1%&8C
/2'0 (inha ?), Bae ?)T), >ar: ?)>), Changes in morphology of Paecilomyces japonica and
their effect on !roth rheology during production of e3o6!iopolymers) 5ppl
=icro!iol Biot 8&D B162C ++6%2, 2''1
/210 .olsche: =)F), Fu!ice: C)>), Biochemistry of citric acid accumulation !y 5spergillus,
#nDCitric acid !iotechnology Eds) B)Fristiansen, =)=attey, ?)Linden, 2268,, Taylor
W Francis >ress,London, 1%%%
/220 =attey =), Biochemistry of citric acid production !y yeasts, #nDCitric acid
!iotechnology Eds) B)Fristiansen, =)=attey, ?)Linden, 11622, Taylor W Francis
>ress,London, 1%%%
/220 .hitta:er 5), Long >)5), >rocess Biochem) +, 2*, B1%*2C
/2,0 =artin ()=), #nd) Eng) Chem) ,%, 1221, B18*C
/280 Clar: D)(), Canad) ?) =icro!iol) +, 122, B1%&2C
/2&0 Chmiel 5), >r<emysl Fermentacyny # Eolny, 1*, 11612, 2% B1%*2C
/2*0 Cimerman 5), ?ohanides A), _:afar (), #n \5!stracts 7f Fifth #nternational
Fermentation (ymposium] Ed) $) Dell-eg, ,'8 B1%*&C Berlin
/2+0 Futerman:ie-ic< =), LeSnia: .), >remysl Ferm) 7-oco-o6.ar<y-ny
11, 2&, B1%+,C
/2%0 =artin ?)F), Demain 5)L), #n \The Filantous Fungi] Aol) 2, Ed) (mith ?)E), Berry
D)E), Eduard 5rnold, B1%**C London
/,'0 "oyes E), Chem) >roc) Ee-) \Citric 5cid >roduction >roces] Aol) 2*, ,2, B1%&%C
/,10 Berovi` =), >h)D) Thesis, University 7f Lu!lana B1%+&C
/,20 Berovi` =), =)(c), Thesis, University 7f Lu!lana B1%*%C
/,20 .a:sman ()5), Faro- E)7), U)() >atent 2,2%,'21, B1%,&C
/,,0 >rescott ()C), Dunn C)4), \#ndustrial =icro!iology] =c 4ra-6$ill, "e- ^or:,
B1%8%C
/,80 >erlman D), (ich C)?), >rogress #nd) =icro!iol), 2, 1&%, B1%&'C
/,&0 =ayrath ?), >roc) Biochem), 2, 28, B1%&*C
/,*0 Fre!s $)5), ?ohnson .)5) En<ymologia ,, 1,+ B1%2*C
/,+0 Lehninger 5)L), \Biochemistry] 2
nd
Ed), "orth >u!) #nc) "e- ^or: B1%*8C
/,%0 Torres "A B1%%,aC =odelling approach to control of car!ohydrate meta!olism during
citric acid accumulation !y Aspergillus nigerD #) =odel definition and sta!ility of
steady state) Biotechnol Bioeng ,,D1',6111
/8'0 Torres "A B1%%,!C =odelling approach to control of car!ohydrate meta!olism) during
citric acid accumulation !y Aspergillus nigerD ##) (ensitivity 5nalysis) Biotechnol
Bioeng ,,D112611+
/810 Torres "A, Eiol6Cimas ?=, .olsche: = et al B1%%&!C 4lucose transport !y
Aspergillus nigerD the lo- affinity carrier is only formed during gro-th on high
glucose concentrations) 5ppl =icro!iol Biotechnol ,,D*%'6*%,
/820 =ischa: =, Fu!ice: C>, E[hr = B1%+,C Citrate inhi!ition of glucose upta:e in
Aspergillus niger) Biotechnol) Lett) /, ,286,2')
/820 .ayman F =, =attey = B2'''C (imple diffusion is the primary mechanism for glucose
upta:e during the production phase of the Aspergillus niger citric acid process)
Biotechnol Bioeng &*D,816,8&
/8,0 >e:sel 5, Torres "A, Liu ? et al B2''2C 12C6"=E analysis of glucose meta!olism
during citric acid production !y Aspergillus niger) 5ppl =icro!iol Biotechnol
8+D18*61&2
/880 5risan65tac #, .olsche: =F, Fu!ice: C> B1%%&!C Trehalose6&6phosphate synthase 5
affects citrate accumulation !y Aspergillus niger under conditions of high
glycolytic flu3) FE=( =icro!iol Lett 1,'D**6+2
/8&0 Euiter 4?4, >anneman $, Aisser ? B1%%*C 7vere3pression of phosphofructo:inase
and pyruvate :inase in citric acid6producing Aspergillus niger) Biochim Biophys
5cta 122,D21*622&
/8+0 E[hr =, 9ehentgru!er 7, Fu!ice: C> B1%+1C Finetics of !iomass formation and
citric acid production !y Aspergillus niger on pilot plant scale) Biotechnol) Bioeng)
22, 2,2262,,8)
/8%0 >apagianni =), .ayman F, =attey = B2''8C) Fate and role of ammonium ions during
fermentation of citric acid !y Aspergillus niger) 5ppl Environ =icro!iol *1D*1*+6
*1+&
[60] Legia M, Mattey M (1986b) Glycerol synthesis by Aspergillus
niger under citric acid accuulating conditions! "n#ye Microb
$echnol 8%60&'609
/&10 E[hr =, Fu!ice: C>, 9ehentgru!er 7 et al B1%+*C 5ccumulation and partial re6
consumption of polyols during citric acid fermentation !y Aspergillus niger) 5ppl
=icro!iol Biotechnol 2*D228622%
/&20 LegiSa =, Fidri` ? B1%+%C #nitiation of citric acid accumulation in the early stages of
Aspergillus niger gro-th) 5ppl =icro!iol Biotechnol 21D,826,8*
/&20 Faraffa L, Fu!ice: C> B2''2C Aspergillus niger citric acid accumulationD do -e
understand this -ell -or:ing !lac: !o3a 5ppl =icro!iol Biotechnol &1D1+%61%&
/&,0 LegiSa =, =attey = B1%+&aC 4lycerol as an initiator of citric acid accumulation in
Aspergillus niger. En<yme =icro! Technol +D28+628%
/&80 5risan65tac #, Fu!ice: C> B1%%&aC 4lycerol is not an inhi!itor of mitochondrial
citrate o3idation !y Aspergillus niger) Microbiology 1#2D 2%2*62%,2)
/&&0 Corde-ener ?, Busin: E and Aisser ? B1%+%C 5 permea!ili<ed cell assay system for
studying en<yme regulation and localisation in Aspergillus niger. &. Microbiol.
Methods 1'D 22162,')
/&*0 =attey = B1%**C Citrate regulation of citric acid production in Aspergillus niger)
'(MS Microbiol. )ett. 2, *16*,)
/&+0 LegiSa =, =attey = B1%++C Citrate regulation of the change in car!ohydrate
degradation during the initial phase of the citric acid production !y Aspergillus
niger) (n!yme Microbiol. Technol. +D 2262&)
/&%0 =esoedni: (, LegiSa = B2''8C) >osttranslational modification of &6phosphofructo616
:inase in Aspergillus niger) 5ppl Environ =icro!iol *1D1,2861,22
/*'0 =la:ar T, LegiSa = B2''&C Citrate inhi!ition resistant from of &6phosphofructo616
:inase from Aspergillus niger) 5ppl Environ =icro!iol *2D,8186,821
/*10 >oorman E5, Eandolph 5, Femp E4 et al B1%+,C Evolution of
phosphofructo:inase66gene duplication and creation of ne- effector sites) "ature
2'%D,&*6,&%
/*20 Fu!ice:6>ran< E=, =o<elt =, E[hr = et al B1%%'C Changes in the concentration of
fructose62,&6!isphosphate in Aspergillus niger during stimulation of acidogenesis
!y elevated sucrose concentration) Biochim Biophys 5cta 1'22D28'6288
/*20 LegiSa =, Ben`ina = B1%%,C Evidence for the activation of &6phosphofructo616:inase
!y c5=>6dependent protein :inase in Aspergillus niger) FE=( =icro!iol Lett
11+D22*6222
/*,0 4radiSni:64rapulin =, LegiSa = B1%%*C 5 spontaneous change in the intracellular
cyclic 5=> level in Aspergillus niger is influenced !y the sucrose concentration in
the medium and !y light) 5ppl Environ =icro!iol &2D2+,,62+,%
/*80 Thevelein ?=, de .inde ?$ B1%%%C "ovel sensing mechanisms and targets for the
c5=>6protein :inase) 5 path-ay in the yeast Saccharomyces cerevisiae) =ol
=icro!iol 22D%',6%1+
/*&0 ?ernec F, LegiSa = B2'',C 5 drop of intracellular p$ stimulates citric acid
accumulation !y some strains of Aspergillus niger) ? Biotechnol 112D2+%62%*
/**0 $esse (?, Euiter 4?4, Di:ema C et al B2''2C #ntracellular p$ homeostasis in the
filamentous fungus Aspergillus niger) Eur ? Biochem 2&%D2,+862,%,
/*+0 van den $om!ergh, ?>, van de Aondervoort >?, Fraissinet6Tachet L et al B1%%*C
Aspergillus as a host for heterologous protein productionD the pro!lem of
proteases) Trends Biotechnol 18D28&62&2
/*%0 =a $, Fu!ice: C>, E[hr = B1%+8C =eta!olic effects of manganese deficiency in
Aspergillus nigerD evidence for increased protein degradation) 5rch =icro!iol
1,1D2&&62&+
/+'0 (chreferl 4, Fu!ice: C>, E[hr = B1%+&C #nhi!ition of citric acid accumulation !y
manganese ions in Aspergillus niger mutants -ith reduced citrate control of
phosphofructo:inase) ? Bacteriol 1&8D1'1%61'22
/+10 E[hr =, Fu!ice: C> B1%+1C Eegulatory aspects of citric acid fermentation !y
Aspergillus niger) >rocess Biochem 1&D2,62*
/+20 Fu!ice: C>, E[hr = B1%*+C The role of the tricar!o3ylic acid cycle in citric acid
accumulation !y Aspergillus niger) Eur ? 5ppl =icro!iol Biotechnol 8D2&262*1
/+20 (chreferl6Funar 4, 4rot< =, E[hr =, Fu!ice: C> B1%+%C #ncreased citric acid
production !y mutants of Aspergillus niger -ith increased glycolytic capacity)
FE=( =icro!iol) Letts) 8%, 2%*62'')
/+,0 ?a:litsch .=, Fu!ice: C>, (crutton =C B1%%1C #ntracellular organisation of ctrate
production in Aspergillus niger) Can) ?) =icro!iol) 2*, +226+2*)
/+80 =a $, Fu!ice: C>, E[hr = B1%+1C =alate dehydrogenase isoen<yme in Aspergillus
niger) FE=( =icro!iol) Letts) 12, 1,*6181)
/+&0 Fu!ice: C>, 9ehentgru!er 7, E[hr = B1%*%C 5n indirect method for studying fine
control of citric acid accumulation !y Aspergillus niger) Biotechnol) Letts 1, ,*6
82)
/+*0 Fu!ice: C> B1%++C The role of the citric acid cycle in fungal organic acid
fermentation) Biochem) (oc) (ymposia 8,, 112612&)
/++0 Fu!ice: C>, 9ehentgru!er 7, El6Fala: $, E[hr = B1%+'C Eegulation of citrate
synthase from the citric acid producing fungus Aspergillus niger) Biochim)
Biophys) 5cta, &18, ,,%6,8*)
/+%0 Da-son =., =addo3 #(, Boag #F, Broo:s ?D B1%++C 5pplication of fed6!atch
culture to citric acid production !y 5spergillus nigerD the effects of dilution rate and
dissolved o3ygen tension) Biotechnol) Bioeng) 22, 22'622&)
/%'0 Firimura F, $iro-atari ^, Usami ( B1%+&C 5lterations of respiratory systems in
Aspergillus niger under the conditions of citric acid fermentation) 5gric) Biol)
Chem) ,1, 12%%612'2)
/%10 Firimura F, ^oda =, (himi<u $, (ugano (, =i<uno m, :ino F, Usami ( B2'''C
Contri!ution of cyanide6insensitive respiratory path-ay, cataly<ed !y the
alternative o3idase, to citric acid production in Aspergillus niger) Biosci)
Biotechnol) Biochem) &,, 1' 2'2,62'2%)
/%20 Firimura F, =atsui T, (ugano (, Usami ( B1%%&C Enhancement and repression of
cyanide6insensitive respiration in Aspergillus niger) FE=( =icro!iol) Letts) 1#1,
281628,)
/%20 Firimura F, ^oda =, Usami ( B1%%%C Cloning and e3pression of the cD"5 encoding
an alternative o3ydase gene from Aspergillus niger .U62222L) Curr) 4enet) 2,,
,*26,**)
/%,0 "eti: 5, Torres "A, Eiol ?=, Fu!ice: C> B1%%*C Upta:e and e3port of citric acid !y
Aspergillus niger is reciprocally regulated !y manganese ions) Biochim) Biophys)
5cta, 122&, 2, 2+*6%,)
/%80 Burgstaller . B2''&C Thermodynamic !oundary conditions suggest that a passive
transport step suffices for citrate e3cretion in 5spergillus and >enicillium)
=icro!iology 182, 2, ++*6+%2)
/%&0 "ierman .C, >ain 5, 5nderson =? et al B2''8C 4enomic se1uence of the pathogenic
and allergenic filamentous fungus Aspergillus fumigatus. "ature ,2+D11816118&
/%*0 4alagan ?E, Calvo (E, Cuomo C et al B2''8C (e1uencing of Aspergillus nidulans and
comparative analysis -ith A. fumigatus and A. ory!ae. "ature ,2+D11'861118
/%+0 =achida =, 5sai F, (ano = et al B2''8C 4enome se1uencing and analysis of
Aspergillus ory!ae. "ature ,2+D118*611&1$)
/%%0 David $, 5:esson =, "ielsen ? B2''2C) Eeconstruction of the central car!on
meta!olism of Aspergillus niger) Eur ? Biochem 2*'D,2,26,282
/1''0 Eoehr =) B1%%+C, 5 century of citric acid fermentation and research, 'ood
Technol.$iotechnol. Aol) 2&, 2D 1&* X 1*2)
/1'10 Tomlinson "), Cam!ell ?)?)E), Trussel >)C) B1%8'C The influence of <inc, copper, iron and
manganese on production of citric acid !y A.niger. &.$acteriol. 8%D 21*622,)
/1'20 Fu!ice: C)>) Eoehr =) B1%+&C Citric acid fermentation) CEC Critical Eevie-s in
Biotechnology, Aol)2, ,D 22162*2)
/1'20 (hu >) ?ohnson =)?) B1%,+C The interdependence of medium constituents in citric acid
fermentation !y su!merged fermentation) &.$acteriol) 8&D 8**68+&)
/1',0 Euiter 4)?)4), Fu!ice: C)>), Aisser ?),B2''2C >roduction of organic acids !y fungi,
#n D 7sie-ac< $)D) ed) The Mycota * +ndustrial Applications, Berlin,
$eidel!erg D (pringer6Aerlag, 212622*)
/1'80 Euiter 4)?)4), Fu!ice: C)>), Aisser ?),B1%%+C =eta!olic engineering of the glycolytic
path-ay in A.niger. 'ood Technol.$iotechnol. Aol) 2&, 2, 1+8 X 1+%)
/1'&0 Clar: D)(), #to F), $oritsu $) B1%&&C Effects of manganese and other heavy metals on
su!merged citric acid fermentation of molasses) $iotechnol. $ioeng. +D ,&86,*1)
/1'*0 Berovic =), Aodopivec =), =ilicic (), Chemical Biochemical Engineering Vuaterly,
Bin pressC 2''&
/1'+0 9id-ic: =)?)B1%%2 C 7rganic 5cids) #nD Fin:elstein D)B) and Ball C) eds)
$iotechnology of 'ilamentous 'ungi. Boston D Butter-orth6$einemann, pp)2'26
22,)
/1'%0 =a $), Fu!ice: C)>) Eoehr =) B1%+8C =eta!olic effects of manganese deficiency in
A.niger D evidence for increased protein degradation) Arch.Microiol. 1,1D 2&&62&+.
/11'0 7rthofer E), Fu!ice: C)>)H Eoehr =), B1%*%C Lipid levels and manganese deficiency in citric
acid producing strains of A.niger, '(MS Microbiol.)ett. 8D ,'26,'&)
/1110 =irminachi F), 9ang 5), Eoehr =) B2''2C Citric acid fermentations and heavy metal ions)#)
Effects of iron, manganese and copper, Acta $iotechnol. Aol)22, 26,D 2&262*2)
/1120 9ang 5), Eoehr =)B2''2C Citric acid fermentations and heavy metal ions)##) The
action of elevated manganese ion concentration) Acta $iotechnol. Aol)22, 26,D
2*862+2)
/1120 $a1 #6U), 5li (), Vuadeer =)5), #1!al ?) B2''1C Effect of copper ions on mould morphology
and citric acid productivity !y 5spergillus niger using molasses !ased media, Process
$iochemistr.
/11,0 4upta (),(harma C)B)B2''2C Biochemical studies of citric acid production and accumulation
!y A.niger mutants, ,orld &.Microbiol.$iotechnol) 1+ D 8, 2*%62+2)
/1180 LeSnia: .) >iet:ie-ic< ?), >odgors:i .) B2''2C Citric acid fermentation from starch and
de3trose syrups !y trace metal resistant mutant of A.niger) $iotechnol.)ett. Aol) 2, , 12D
1'&861'&*)
/11&0 LeSnia: .), Citric acid !iotechnology D Fermentation su!strates BEds) Fristiansen B),
=attey =), Linden ?) Beds)C London, Taylor Francis Ltd) 1%%%, pp)1,%618% )
/11*0 Lesnia: .),1%+%) Polish Technical -evie, 8, 1+8)
/11+0 Lesnia: .), 1%*2) (tudies on (u!merged Citric 5cid Fermentation, >hD Thesis,
University of .rocla-, >oland)
/11%0 Futerman:ie-ic< =), Lesnia: .), Bolach E), 1%+') Pr!em. 'erm. i .oc.#,ar!y,&,
2*
/12'0 >iet:ie-ic< ?),>odgors:i .), Lesnia: .), 1%%&) Proceedings of the +nternational
Conference on Advances in Citric Acid Technology Bratislava, (lova: Eepu!lic, p)
%)
/1210 Lesnia: .), >odgors:i .), and >iet:ie-ic< ?) 1%+&) Pr!em. 'erm. i .oc.#,ar!y, &,
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/1220 =aldonaldo >, Charpentier =, 1%*8) /erman Patent 0112345.
/1220 Fimura F), "a:anishi T, 1%+8) /erman Patent 0 641 064.
/12,0 .otato-ic< =), (o!ies<c<ans:i ?), 1%+16) Acta Microbiologica Polonica, 2', &%)
/1280 #:eno ^), =asuda =), Tanno F), 7omori #), Ta:ahashi "), 1%*8) &ournal of 'er#
mentation Technology, 82, *82)
/12&0 4utcho ()?), 1%*2) Chemicals by 'ermentation B"oyes Data Corporation, >ar:
Eidge, "^,U(5C)
/12*0 Elimer E), 1%%,) (tudies on Use of >lant Fats for Citric 5cid >roduction !y
Aspergillus niger,>hD thesis, University of .rocla-, >oland)
/12+0 Fristiansen B), (inclair C)4) B1%*%C >roduction of citric acid in continuous culture,
$iotech.$ioeng. 21D 2%*62'2)
/12%0 Fristiansen B), Charley E) B1%+1C Continuous process for citric acid production) #n D
=oo6^oung =) ed) Advanced $iotechnology Aol)1) "e- ^or: D >ergamon
>ress, &%6*&)
/12'0 (aha =)L), Ta:ahashi F), Continuous citric acid fermentation !y magnetic rotating
!iological contactors using), Aspergillus niger 5? 11*1*2, ? Ferment) Bioeng
+,D B2C 2,,62,+ 1%%*)
/1210 (ommariva C), 9illi =), Converti 5), Continuous citric acid fermentation in a
supported !iomass reactor, Chem) Eng) Technol) 2'D B8C 2,+6282,1%%*)
/1220 4odo (, Flein ?, Bales A, =i3ing time in airlift reactors during citric acid
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