Brazilian Archives of Biology and Technology - Microbial Production of Citric Acid

27/12/2014
BrazilianArchivesofBiologyandTechnologyMicrobialproductionofcitricacid
BrazilianArchivesofBiologyandTechnology
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Braz.arch.biol.technol.vol.42no.3Curitiba1999
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http://dx.doi.org/10.1590/S151689131999000300001
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Microbialproductionofcitricacid
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LucianaP.S.VandenbergheI,IICarlosR.SoccolI*AshokPandeyI
JeanMichelLebeaultII
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I LaboratriodeProcessosBiotecnolgicos,DepartamentodeEngenharia
CitedbySciELO
Qumica,UniversidadeFederaldoParan,CEP81531970CuritibaPR,
Brazil
II LaboratoiredeProcdsBiotechnologiques,GnieChimique,Universit
deTechnologiedeCompigne,60205Compigne,Cedex,France
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ABSTRACT
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Citricacidisthemostimportantorganicacidproducedintonnageandis
extensivelyusedinfoodandpharmaceuticalindustries.Itisproducedmainlybysubmergedfermentationusing
AspergillusnigerorCandidasp.fromdifferentsourcesofcarbohydrates,suchasmolassesandstarchbased
media.However,otherfermentationtechniques,e.g.solidstatefermentationandsurfacefermentation,and
alternativesourcesofcarbonsuchasagroindustrialresidueshavebeenintensivelystudiedshowinggreat
perspectivetoitsproduction.Thispaperreviewsrecentdevelopmentsoncitricacidproductionbypresentinga
briefsummaryofthesubject,describingmicroorganisms,productiontechniques,andsubstrates,etc.
Keywords:Citricacid,Aspergillusniger,submergedfermentation,solidstatefermentation,substrates
RESUMO
Ocidoctricoocidomaisproduzidoemtermosdetonagemeextensivamenteutilizadopelasindstrias
alimentciaefarmacutica.produzidoprincipalmenteporfermentaosubmersautilizandoofungoAspergillus
nigerelevedurasdogneroCandidasp.partirdediferentesfontesdecarbono,comoaglicoseemeios
basedeamido.Noentanto,outrastcnicasdefermentao,e.g.fermentaonoestadoslidoeemsuperfcie,
efontesalternativasdecarbonotemsidointensamenteestudadasmostrandograndeperspectivasparao
processo.Opresentetrabalhoapresentaumresumodosltimosavanossobreaproduodocidoctrico,
descrevendodemaneirasucintaostrabalhosmaisrecentes,descrevendomicrorganismos,tcnicasdeproduo
esubstratosempregados,etc.
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S151689131999000300001
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INTRODUCTION
Citricacid(C6H8O7,2hydroxy1,2,3propanetricarboxylicacid),anaturalconstituentandcommon
metaboliteofplantsandanimals,isthemostversatileandwidelyusedorganicacidinthefieldoffood(60%)
andpharmaceuticals(10%).Ithasgotseveralotherapplicationsinvariousotherfields.Currently,theglobal
productionofcitricacidisestimatedtobearound736000tones/year(QumicaeDerivados,1997),andthe
entireproductioniscarriedoutbyfermentation.InBrazil,almosttheentiredemandofcitricacidismetthrough
imports.Thereisconstantincrease(3.54%)eachyearinitsconsumption,showingtheneedoffindingnew
alternativesforitsmanufacture.
Historicaldevelopments
CitricacidwasfirstisolatedbyKarlsScheelsin1874,inEngland,fromthelemonjuiceimportedfromItaly.
Italianmanufacturershadmonopolyforitsproductionforalmost100years,anditwassoldathighcost.This
ledextensiveattemptsallovertheworldtofindalternativeswayforitsproduction,whichincludedchemical
andmicrobialtechniques.In1923,Wehmerobservedthepresenceofcitricacidasabyproductofcalcium
oxalateproducedbyacultureofPenicilliumglaucum.Otherinvestigationsshowedtheisolationoftwovarieties
offungibelongingtogenusCitromyces(namelyPenicillium).However,industrialtrialsdidnotsucceeddueto
contaminationproblemsandlongdurationoffermentation(Rohretal.,1983).Theindustrialprocesswasfirst
openbyCurrie,in1917,whofoundthatAspergillusnigerhadthecapacitytoaccumulatesignificantamountsof
citricacidinsugarbasedmedium.Healsoshowedthathighconcentrationsofsugarfavoureditsproduction,
whichoccurredunderlimitationofgrowth.Inthethirties,someunitswereimplantedinEngland,inSoviet
Union,andinGermanyforthecommercialproduction.However,thebiochemicalbasiswasonlyclearedinthe
fiftieswiththediscoveryoftheglycolyticpathwayandthetricarboxylicacidcycle(TCA).Consequently,an
improvedprocessemployingsubmergedfermentationwasdevelopedinUnitedStates(AboudZeidandAshy,
1984).
Althoughmethodswerewelldevelopedtosynthesiscitricacidusingchemicalmeansalso,bettersuccesses
wereachievedusingmicrobialfermentations,andovertheperiodoftime,thistechniquehasbecomethe
methodofultimatechoiceforitscommercialproduction,mainlyduetoeconomicadvantageofbiological
productionoverchemicalsynthesis(Mattey,1992).Muchattentionhasbeenpaidonresearchtoimprovethe
microbialstrains,andtomaintaintheirproductioncapacity.
Applicationsofcitricacid
Citricacidismainlyusedinfoodindustrybecauseofitspleasantacidtasteanitshighsolubilityinwater.Itis
worldwideacceptedas"GRAS"(generallyrecognizedassafe),approvedbytheJointFAO/WHOExpert
CommitteeonFoodAdditives.Thepharmaceuticalandcosmeticindustriesretain10%ofitsutilizationandthe
remainderisusedforvariousotherpurposes.Table1presentsmainapplicationsofcitricacid.
MICROORGANISMSUSEDFORCITRICACICPRODUCTION
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Alargenumberofmicroorganismsincludingbacteria,fungiandyeastshavebeenemployedtoproducecitric
acid.Mostofthem,however,arenotabletoproducecommerciallyacceptableyields.Thisfactcouldbe
explainedbythefactthatcitricacidisametaboliteofenergymetabolismanditsaccumulationrisesin
appreciableamountsonlyunderconditionsofdrasticimbalances.KubicekandRohr(1986)reviewedthestrains
reportedtoproducecitricacid.Table2showsthemicroorganismsusedtoproducecitricacid.Amongthese,
onlyA.nigerandcertainyeastssuchasSaccharomycopsissp.areemployedforcommercialproduction.
However,thefungusA.nigerhasremainedtheorganismofchoiceforcommercialproduction.Themain
advantagesofusingthismicroorganismare:(a)itseaseofhandling,(b)itsabilitytofermentavarietyof
cheaprawmaterials,and(c)highyields.
Strainsselectionandimprovement
Thetwoprincipalmethodsofselectingpopulations,namely,"thesinglesporetechnique"andthe"passage
method"havebeenusedforselectingcitricacidproducingmicroorganismsThesinglesporetechniquehasthe
disadvantagethatmineralacidororganicacids(gluconicacid,oxalicacid)simulatethepresenceofcitricacid.
Rohretal.(1979)improvedthismethodbyincorporatingaspecificstainforcitricacid(paradimethylamino
benzaldehyde),insteadofusingtheindicator.
Themostemployedtechniquetoimprovecitricacidproducingstrainshasbeenbyinducingmutationsin
parentalstrainsusingmutagens.Amongphysicalmutagens,gradiation(BonatelliandAzevedo,1983Gunde
Cimerman,1986Islametal.,1986)andUVradiation(Pelechovaetal.,1990)haveoftenused.Toobtain
hyperproducerstrains,frequentlyUVtreatmentcouldbecombinedwithsomechemicalmutagens,e.g.
aziridine,NnitrosoNmethylureaorethylmethanesulfonate(Musilkovaetal.,1983).Byusingasuitable
selectiontechniqueonmodelmediumwithnonspecificcarbonsources,astrainyieldinghighamountsofcitric
acidfromunusualsubstratescanbeobtainedfromthemutantsproduced.
Anotherapproachforstrainimprovementhasbeentheparasexualcycle,asfirstdescribedbyPontecorvoetal.
(1953).AccordingtoDasandRoy(1978),diploidsdisplayedhighercitricacidyieldscomparedtotheirparent
haploids,buttendedtobelessstable(BonatelliandAzevedo,1983).Protoplastfusionappearedtobea
promisingtooltoextendtherangeofgeneticmanipulationofA.nigerwithrespecttocitricacidproduction.
Kirimuraetal.(1988a)studiedprotoplastfusionofproductionstrains.Theywereabletoobtainfusantswith
acidproductioncapacitiesexceedingthoseoftheparentstrainsinsolidstatefermentation,butnotin
submergedfermentation.Someotheraspectsofstrainimprovementcouldbetheresistancetodetrimental
constituentsoffermentationrawmaterials,capabilityofutilizingrawmaterials(starch,cellulose,pectin
containingandotherwastematerials).However,thereisnosingleeffectivetechniquetoachievehyper
producingmutantsandmuchremainstobedoneinthisarea.
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PRODUCTIONTECHNIQUESANDRAWMATERIALS
Althoughcitricacidismostlyproducedfromstarchorsucrosebasedmediausingliquidfermentation,avariety
ofrawmaterialssuchasmolasses,severalstarchymaterialsandhydrocarbonshavealsobeenemployed.Rohr
etal.(1983)classifiedrawmaterialsusedforcitricacidproductionintotwogroups:(i)withalowashcontent
fromwhichthecationscouldberemovedbystandardprocedures(e.g.caneorbeetsugar,dextrosesyrupsand
crystallizeddextrose)(ii)rawmaterialswithahighashcontentandhighamountsofothernonsugar
substances(e.g.caneandbeetmolasses,crudeunfilteredstarchhydrolysates).
Severalattemptshavebeenmadetoproducecitricacidusingmolasses,whichispreferreddueitslowcostand
highsugarcontent(4055%).Thecompositionofmolassesdependsonvariousfactors,e.g.thekindofbeetand
cane,methodsofcultivationofcropsandfertilizersandpesticidesappliedduringcultivation,conditionsof
storageandhandling(e.g.transport,temperaturevariations),productionprocedures,etc.Both,caneandbeet
molassesaresuitableforcitricacidproduction.However,beetmolassesispreferredduetoitslowercontentof
tracemetals.Generally,canemolassescontainscalcium,magnesium,manganese,ironandzinc,whichhavea
retardingeffectonthesynthesisofcitricacid.Consequently,somepretreatmentisrequiredforthe
removal/reductionoftracemetals.Despitethat,canemolassespossesdifficultiesinachievinggood
fermentationyields.
Variousotheragroindustrialresiduessuchasapplepomace,cassavabagasse,coffeehusk,wheatstraw,
pineapplewaste,sugarbeetcosset,kiwifruitpeel,etc.havebeeninvestigatedwithsolidstatefermentation
techniquesfortheirpotentialtobeusedassubstratesforcitricacidproduction(PandeyandSoccol,1998,
Pandeyetal.1999,Vandenbergheetal.,1999a,b,c).Infact,theseresiduesareverywelladaptedtosolid
stateculturesduetotheircellulosicandstarchynature.However,despitethefactthatthesesolidresidues
providerichnutrientstothemicroorganisms,andaregoodsubstratesforgrowthandactivityofmicro
organisms,muchremainstobedonefordevelopingcommerciallyfeasibleprocessutilizingtheseresidues
(Pandey1992,1994,PandeyandSoccol1998).
Liquidfermentation
Submergedfermentation:Thesubmergedfermentation(SmF)processisthecommonlyemployedtechnique
forcitricacidproduction.Itisestimatedthatabout80%ofworldproductionisobtainedbySmF.Several
advantagessuchashigheryieldsandproductivityandlowerlabourcostsarethemainreasonsforthis.Two
typesoffermenters,conventionalstirredfermentersandtowerfermentersareemployed,althoughthelatteris
preferredduetotheadvantagesitoffersonprice,sizeandoperation(Rohretal.,1983).Preferentially,
fermentersaremadeofhighgradesteelandrequireprovisionofaerationsystem,whichcanmaintainahigh
dissolvedoxygenlevel.Fermentersforcitricacidproductiondonothavetobebuiltaspressurevesselssince
sterilizationisperformedbysimplysteamingwithoutapplyingpressure.Coolingcanbedonebyanexternal
waterfilmovertheentireoutsidewallofthefermenter.
InSmF,differentkindsofmediaareemployedsuchassugarandstarchbasedmedia(Table3).Molassesand
otherrawmaterialsdemandpretreatment,additionofnutrientsandsterilization.Inoculationisperformed
eitherbyaddingasuspensionofspores,orofprecultivatedmycelia.Whensporesareused,asurfactantis
addedinordertodispersetheminthemedium.Forprecultivatedmycelia,aninoculumsizeof10%offresh
mediumisgenerallyrequired.Normally,submergedfermentationisconcludedin5to10daysdependingonthe
processconditions.Itcanbecarriedoutinbatch,continuousorfedbatchsystems,althoughthebatchmode
morefrequentlyused.
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Surfacefermentation:Thefirstindividualprocessforcitricacidproductionwastheliquidsurfaceculture
(LSC),whichwasintroducedin1919bySocitdesProduitsOrganiquesinBelgium,andin1923byChasPfizer
&Co.inUS.Afterthat,othermethodsoffermentation,suchassubmergedfermentationweredeveloped.
Althoughthistechniqueismoresophisticated,surfacemethodrequiredlesseffortinoperationandinstallation
andenergycost(GrewalandKalra,1995).
Intheclassicalprocessforcitricacidmanufacture,theculturesolutionisheldinshallowtrays(capacityof50
100L)andthefungusdevelopsasamycelialmatonthesurfaceofthemedium.Thetraysaremadeofhigh
purityaluminiumorspecialgradesteelandaremountedoneoveranotherinstableracks.Thefermentation
chambersareprovidedwithaneffectiveaircirculationinordertocontroltemperatureandhumidity.
Fermentationchambersarealwaysinasepticconditions,whichmightbeconservedprincipallyduringthefirst
twodayswhensporesgerminate.FrequentcontaminationaremainlycausedbyPenicilia,otherAspergilli,yeast
andlacticbacteria(Rohretal,1983Morgant,1988).Refinedorcrudesucrose,canesyruporbeetmolasses
aregenerallyusedassourcesofcarbon.Whenapplied,molassesisdilutedto1520%andistreatedwith
hexacyanoferrate(HFC).
Solidstatefermentation
Solidstatefermentation(SSF)hasbeentermedasanalternativemethodtoproducecitricacidfromagro
industrialresidues(Pandey1991,1992,1994,Soccol1994,PandeyandSoccol1998).Citricacidproductionby
SSF(theKojiprocess)wasfirstdevelopedinJapanandisasthesimplestmethodforitsproduction.SSFcanbe
carriedoutusingseveralrawmaterials(Table4).Generally,thesubstrateismoistenedtoabout70%moisture
dependingonthesubstrateabsorptioncapacity.TheinitialpHisnormallyadjustedto4.56.0andthe
temperatureofincubationcanvaryfrom28to30C.ThemostcommonlyorganismisA.niger.Howeverthere
alsohavebeenreportswithyeasts(MaddoxandKingston,1983Tisnadjajaetal.,1996).Oneoftheimportant
advantagesofSSFprocessisthatthepresenceoftraceelementsmaynotaffectcitricacidproductionso
harmfullyasitdoesinSmF.Consequently,substratepretreatmentisnotrequired.
Differenttypesoffermenterssuchasconicalflasks,glassincubatorsandtrays,etc.havebeenusedforcitric
acidfermentationinSSF.Vandenbergheetal.(1999a,b)usedErlenmeyerflasksandglasscolumnsforthe
productionofcitricacidfromgelatinizedcassavabagasse.Higheryieldswereobtainedinflaskswithoutany
aeration,andverylittlesporulationwasobserved.Thesameyieldswerefoundincolumnreactorsonlywith
variableaeration.ThisshowedgreatperspectivetouseSSFprocessforcitricacidproductioninsimpletray
typefermenters.
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FACTORSAFFECTINGCITRICACIDPRODUCTION
Mediumanditscomponents
Carbonsource:Citricacidaccumulationisstronglyaffectedbythenatureofthecarbonsource.Thepresence
ofeasilymetabolizedcarbohydrateshasbeenfoundessentialforgoodproductionofcitricacid.Hossainetal.
(1984)showedthatsucrosewasthemostfavourablecarbonsourcefollowedbyglucose,fructoseandgalactose.
Galactosecontributedtoaverylowgrowthoffungianddidnotfavourcitricacidaccumulation.Othersourcesof
carbonsuchassorbose,ethanol,cellulose,manitol,lactic,malicandaacetoglutaricacid,allowalimited
growthandlowproduction.Starch,pentoses(xylosesandarabinoses),sorbitolandpyruvicacidslowdown
growth,thoughtheproductionisminimal(Yokoya,1992).
AccordingtoKovats(1960),initialsugarconcentrationwascriticalforcitricacidproductionandotherorganic
acidsproducedbyA.niger.Xuetal.(1989)reportedthatA.nigerstrainsneededaninitialsugarconcentration
of1014%asoptimalnocitricacidwasproducedatsugarconcentrationoflessthan2.5%.Honeckeretal.
(1989)showedthatimmobilizedcellsofA.nigerneededlowerconcentrationsofsucrosethanfreecellsculture,
inordertoobtainhighyields(200gofcitricacid/Lforfreecellsculture,and120g/Lforimmobilizedcells).
Maddoxetal.(1985)reportedtheinfluenceofdifferentsourcesofcarbononcitricacidproductionbyA.niger
andSaccharomycopsislipolytica.Glucose,maltose,galactose,xyloseandarabinoseweretested.Fermentation
wascarriedoutin8and4days,respectively,at30Cand180rpm.BetterresultswerefoundforA.nigerwith
0.45gofcitricacid/gofglucosecorrespondingto27g/L.S.lipolyticaproduced0.41g/gofglucoseor9g/L
whichwasnotsobad.
Aspresentedpreviously,severalrawmaterialscanbeemployedsuccessfullyforcitricacidproduction.There
aresomecriticalfactors(costs,needofpretreatment),whichshouldbeconsideredforsubstratedetermination.
Oneanotheraspectisthepresenceoftraceelements,whichcanactasinhibitorsorstimulants.Consequently,
sometimesitisnecessarytoconduceapretreatment,e.g.precipitationoftracemetalsofmolassesby
potassiumferrocyanide.
Nitrogensource:Citricacidproductionisdirectlyinfluencedbythenitrogensource.Physiologically,ammonium
saltsarepreferred,e.g.urea,ammoniumsulfate,ammoniumchlorure,peptone,maltextract,etc.Nitrogen
consumptionleadstopHdecrease,whichisveryimportantpointincitricacidfermentation(Rohretal.,1983,
KubicekandRohr,1986).However,itisnecessarytomaintainpHvaluesinthefirstdayoffermentationprior
toacertainquantitybiomassproduction.Ureahasatamponeffect,whichassurespHcontrol(Raimbault,
1980).Theconcentrationofnitrogensourcerequiredforcitricacidfermentationis0.1to0.4N/liter.Ahigh
nitrogenconcentrationincreasesfungalgrowthandtheconsumptionofsugars,butdecreasestheamountof
citricacidproduced(Hangetal.,1977).
Phosphoroussource:Presenceofphosphateinthemediumhasagreateffectontheyieldofcitricacid.
Potassiumdihydrogenphosphatehasbeenreportedtobethemostsuitablephosphoroussource.Shuand
Johnson(1948)reportedthatphosphorousatconcentrationof0.5to5.0g/Lwasrequiredbythefungusina
chemicallydefinedmediumformaximumproductionofcitricacid.Phosphateisknowntobeessentialforthe
growthandmetabolismofA.niger(ShankaranandandLonsane,1994).Lowlevelsofphosphatefavourcitric
acidproduction,however,thepresenceofexcessofphosphatewasshowntoleadtotheformationofcertain
sugaracids,adecreaseinthefixationofCO2,andthestimulationofgrowth.Phosphatesactsatthelevelof
enzymeactivityandnotatthelevelofgeneexpression(Kubiceketal.,1979).Itisinterestingtonotethat
differentstrainsrequiredistinctnitrogenandphosphorousconcentrationsinthemedium.Infact,nitrogenand
phosphorouslimitationisacrucialfactorincitricacidproductionasthereisaninteractionbetweenthem.
Consequently,thestudyoftheircombinedeffectisnecessary(Pintadoetal.,1993Chen,1994).Pintadoetal.
(1998)reportedhowtheculturingmodalityconditionsthebehaviorofthemicroorganismsreferringtothe
tendenciesofproductionasafunctionofthelevelsofNandP.Theauthorusedasfirstorderanempirical
modelbasedonrotatabledesigntostudytheeffectofbothnutrients.Asexpected,forthetwostudiedstrains,a
similarbehaviorwasnoticed,showinganimprovementtowardslowlevelsofNandPinsubmergedculture,and
towardhighlevelsinsolidstateculture,andwithsuperiorproductionsforthelastone.Shankaranandand
Lonsane(1994)affirmedthatthespecificityofsolidstatecultureislargelyduetoalowerdiffusionrateof
nutrientsandmetabolites,whichoccursinlowwateractivityconditions.Consequently,strainswithlarge
requirementsofNandPseemstobedisfavored,duetotherestrictionofaccessibilitytothenutrientsinthe
medium.
Traceelements:Traceelementnutritionisprobablythemainfactorinfluencingtheyieldofcitricacid.A
numberofdivalentmetalssuchaszinc,manganese,iron,copperandmagnesiumhavebeenfoundtoaffect
citricacidproductionbyA.niger.However,itiscrucialtotakeintoaccounttheinterdependenceofmedium
constituentsinSmFand,probably,inSSF.ZincfavouredtheproductionofcitricacidifaddedwithKH2PO4.On
theotherhand,thepresenceofmanganeseionsandironandzinc(inhighconcentrations)couldcausethe
reductionofcitricacidyieldsonlyinphosphatefreemedium.ShankaranandandLonsane(1994)noticedthat
therewerefewdifferencesintheresponseofA.nigertometalionsandmineralsinSSFandinSmFsystems.
SSFsystemswereabletoovercometheadverseeffectsofthehighconcentrationsofthesecomponentsinthe
medium.Asaconsequenceofthis,theadditionofchelatingagentssuchaspotassiumferrocyanidetothe
mediumprovedtobeofnouse.
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Copperwasfoundtocomplementtheabilityofironatoptimumlevel,toenhancethebiosynthesisofcitricacid.
ManganesedeficiencyresultedintherepressionoftheanaerobicandTCAcycleenzymeswiththeexceptionof
citratesynthetase.Thisledtooverflowofcitricacidasanendproductofglycolysis(KubicekandRohr,1978).A
lowlevelofmanganese(ppm)wascapabletoreducetheyieldofcitricacidby10%.Citricacidaccumulation
decreasedbytheadditionofiron,whichalsohadsomeeffectonmycelialgrowth.BenuzziandSegovia(1996)
reportedthatthepresenceofdifferentcopperconcentrationsinthepelletformationmediumwasvery
importantinordertoenhanceasuitablestructure,relatedtocellularphysiology,forcitricacidproduction.The
optimalinitialCuSO4.5H2Oconcentrationwas78mg/L.
Magnesiumisrequiredbothforgrowthaswellasforcitricacidproduction.Optimalconcentrationofmagnesium
sulfatewasfoundintherangeof0.020.025%(Kapooretal.,1983).
Loweralcohols:Additionofloweralcoholsenhancescitricacidproductionfromcommercialglucoseandother
crudecarbohydrate.Appropriatealcoholsaremethanol,ethanol,isopropanolormethylacetate.Theoptimal
amountofmethanol/ethanoldependsuponthestrainandthecompositionofthemedium,generallyoptimum
rangebeing13%.Theeffectofmethanolorethanolhavebeenextensivelystudiedbymanyauthors(Hamissa,
1978MannomaniandSreekantiah,1988Georgievaetal.,1992Dasguptaetal.,1994).
MannomaniandSreekantiah(1987)reportedthatadditionofethanolresultedintwofoldincreaseincitrate
synthetaseactivityand75%decreaseinaconitaseactivity.WhereastheactivitiesofotherTCAcycleenzymes
increasedslightly.Theyalsofoundthatcoconutoilinfluencedcitricacidproductioninasucrosemediumwhen
addedat3%(v/w).Alcoholshavebeenshowntoprincipallyactonmembranepermeabilityinmicroorganisms
byaffectingphospholipidcompositiononthecytoplasmaticmembrane(Orthoferetal.,1979).HoweverMeixner
etal.(1985)arguedagainstaroleofmembranepermeabilityincitricacidaccumulation.IngramandButtke
(1984)foundthatalcoholsstimulatecitricacidproductionbyaffectinggrowthandsporulationthroughtheaction
notonlyonthecellpermeabilitybutalsothespatialorganizationofthemembrane,orchangesinlipid
compositionofthecellwall.
Miscellaneous:Somecompoundswhichareinhibitorsofmetabolismsuchascalciumfluoride,sodiumfluoride
andpotassiumfluoridehavebeenfoundtoacceleratethecitricacidproduction,while,potassiumferrocyanide
hasbeenfoundtodecreasetheyield.Therearemanycompounds,whichactinmanywaystofavourcitricacid
accumulation.Someofthemarecapabletoimpairtheactionofmetalionsandothertoxiccompoundsinfluence
growthduringtheinitialphase.Someoftheseare:4Methylumbelliferone,3hydroxi2naphtoic,benzoicacid,
2naphtoicacid,ironcyanide,quaternaryammoniumcompounds,amineoximes,starch,EDTA,vermiculite,etc.
Processparameters
pH:ThepHofaculturemaychangeinresponsetomicrobialmetabolicactivities.Themostobviousreasonis
thesecretionoforganicacidssuchascitric,aceticorlacticacids,whichwillcausethepHtodecrease.Changes
inpHkineticsdependhighlyalsoonthemicroorganism.WithAspergillussp.,Penicilliumsp.andRhizopussp.,
pHcandropveryquicklyuntillessthan3.0.ForothergroupsoffungisuchasTrichoderma,Sporotrichum,
Pleurotussp.,pHismorestable(between4and5).Besides,thenatureofthesubstratealsoinfluencespH
kinetics(Raimbaultetal.,1997).
Generally,apHbelow2.0isrequiredforoptimumproductionofcitricacid.AlowinitialpHhastheadvantage
ofcheckingcontaminationandinhibitingoxalicacidformation.ApHof2.2wasreportedtobeoptimumforthe
growthofthemouldaswellasfortheproductionofcitricacid(SrivastavaandDe,1980)whereas,ahigherpH
i.e.5.4and6.06.5hasbeenfoundoptimumforcitricacidproductioninmolassesmedium(Roukosuand
Anenih,1980).
Aeration:Aerationhasbeenshowntohaveadeterminanteffectoncitricacidfermentation(Rohretal.,1983
Dawsonetal.,1986).Increasedaerationratesledtoenhancedyieldsandreducedfermentationtime(Grewal
andKalra,1995).
Theinfluenceofdissolvedoxygenconcentrationoncitricacidformationhasbeenexamined.Itisimportantto
maintaintheoxygenconcentrationabove25%saturationandinterruptionsinoxygensupplymaybequite
harmful(Kubiceketal.,1980).Thehighdemandofoxygenisfulfilledbyconstructingappropriateaeration
devices,whichisalsodependentontheviscosityofthefermentationbroth.Thisisanadditionalreasonwhy
smallcompactpelletsarethepreferredmycelialformsofA.nigerduringfermentation(KubicekandRohr,
1986).Whentheorganismturnsintofilamentousdevelopments,e.g.duetometalcontamination,thedissolved
oxygentensionrapidlyfallstolessthan50%ofitspreviousvalue,evenifthedryweighthasnotincreasedby
morethan5%.Aerationisperformedduringthewholefermentationwiththesameintensitythroughthe
mediumatarateof0.5to1.5vvm.However,becauseofeconomicreasons,it'susuallypreferredtostartwith
alowaerationrate(0.1to0.4vvm).Highaerationratesleadtohighamountsoffoam,especiallyduringthe
growthphase.Therefore,theadditionofantifoamingagentsandtheconstructionofmechanical"defoamers"are
requiredtotacklethisproblem.
PRODUCTRECOVERY
Therecoveryofcitricacidfromliquidfermentationisgenerallyaccomplishedbythreebasicprocedures,
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precipitation,extraction,andadsorptionandabsorption(mainlyusingionexchangeresins).Citricacid
extractionhasbeendescribedbytheFoodandDrugAdministration(1975)oftheUnitedStatesandbyColin
(1960,1962).Citricacidextractedbythismethodhasbeenrecommendedsuitableforuseinfoodanddrugs.
Precipitationistheclassicalmethodanditisperformedbytheadditionofcalciumoxidehydrate(milkoflime)
toformtheslightlysolubletricalciumcitratetetrahydrate.Theprecipitatedtricalciumcitrateisremovedby
filtrationandwashedseveraltimeswithwater.Itisthentreatedwithsulphuricacidformingcalciumsulphate,
whichisfilteredoff.Motherliquorcontainingcitricacidistreatedwithactivecarbonandpassedthroughcation
andanionexchangers.Severalanionexchangeresinsarecommerciallyavailable.Finally,theliquoris
concentratedinvacuumcrystallizersat2025C,formingcitricacidmonohydrate.Crystalizationat
temperatureshighertothisisusedtoprepareanhydrouscitricacid.
CONCLUSIONSANDPERSPECTIVES
Sincethebeginningofthiscentury,citricacidproductionhasbeenintensivelystudiedandgreatalternativesto
thisprocesshavebeenfoundtofollowitsgreatdemand.Theuseofalternativerawmaterialstoproducecitric
acidbySmF,LSC,andSSFseemstobeasuitablepossibility.However,itisnecessarytoadapttherighttypeof
rawmaterialtotherighttechniquee.g.cassavabagasseemployedassubstrateinSSF,orcellulosehydrolysate
usedinSmF.Theneedofsomepretreatmentofrawmaterialsmayenhancethefermentationefficiency.One
area,whichneedsattentionisthedevelopmentofcontinuousculturetechniqueswhichhavebeenattemptedbut
onlyatthelaboratoryscale.Anotherareaisthestrainimprovementwithimprovedsubstrateutilization
efficiency.
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Received:May16,1999
Revised:July20,1999
Accepted:August16,1999.
*Authorforcorrespondence
Tecpar
RuaProf.AlgacyrMunhozMader,3775CIC
81350010CuritibaPRBrazil
Tel.:+554133163052/33163012
Fax:+554133462872
babt@tecpar.br
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