BASE Biotechnol. Agron. Soc. Environ.201014(S2),541-548 Agrofuels & Energies

FermentativehydrogenproductionbyClostridium butyricumCWBI1009andCitrobacter freundiiCWBI952inpureandmixedculturesLaurentBeckers,SergeHiligsmann,ChristopherHamilton,JulienMasset,PhilippeThonartUniv.Liege(ULg).WalloonCentreofIndustrialBiology(WCIB).BoulevardduRectorat,29/40-P.70.B-4000Liege(Belgium).E-mail:lbeckers@ulg.ac.be

This paper investigates the biohydrogen production by twomesophilic strains, a strict anaerobe (Clostridium butyricumCWBI1009)andafacultativeanaerobe(Citrobacter freundiiCWBI952).Theywereculturedinpureandmixedculturesinserumbottleswithfivedifferentcarbonsources.ThehydrogenyieldsofpureC. freundiiculturesrangedfrom0.09molH2.molhexose

-1 (with sucrose) to 0.24 molH2.molhexose-1 (with glucose). Higher yields were obtained by the pure cultures of

Cl. butyricum ranging from 0.44 molH2.molhexose-1 (with sucrose) to 0.69 molH2.molhexose

-1 (with lactose). This strain alsofermented starch whereasC. freundii did not. However, it consumed the other substrates faster and produced hydrogenearlierthanCl. butyricum.ThisabilityhasbeenusedtopromotethegrowthconditionsofCl. butyricuminco-culturewithC. freundii,sinceCl. butyricumisextremelysensitivetothepresenceofoxygenwhichstronglyinhibitsH2production.ThisapproachcouldavoidtheadditionofanyexpensivereducingagentsintheculturemediasuchasL-cysteinesinceC. freundiiconsumestheresidualoxygen.Thereafter,co-cultureswithglucoseandstarchwereinvestigated:hydrogenyieldsdecreasedfrom0.53molH2.molhexose

-1forpureCl. butyricumculturesto0.38molH2.molhexose-1formixedculturewithglucosebutslightly

increasedwithstarch(respectively0.69and0.73molH2.molhexose-1).After48hoffermentation,metabolitesanalysisconfirmed

withmicrobialobservation,revealedthatthecellconcentrationofC. freundiidramaticallydecreasedorwasstronglyinhibitedbythedevelopmentofCl. butyricum.Keywords.Biohydrogen,Clostridium butyricum,Citrobacter freundii,mixedculture,biochemicalhydrogenpotential,batch,substrate.

Production d’hydrogène par Clostridium butyricum CWBI1009 et Citrobacter freundii CWBI952 en cultures pures et mixtes.Cetarticleétudie laproductiondebiohydrogènepardeuxsouchesmésophiles,uneanaérobiestricte(Clostridium butyricumCWBI1009)etuneanaérobiefacultative(Citrobacter freundiiCWBI952).Cessouchesontététestéesenculturesbatchpuresetmixtessurcinqsubstratsdifférents.LesrendementsdeconversiondesculturespuresdeC. freundiis’étalentde0,09molH2.molhexose

-1(sursaccharose)à0,24molH2.molhexose-1(surglucose).Desrendementsplusélevéssontatteintspar

Cl. butyricum,de0,44molH2.molhexose-1(sursaccharose)à0,69molH2.molhexose

-1(surlactose).Cettedernièresouchedégradel’amidon,tandisqueC. freundiineleconsommepas.Cependant,cettedernièreassimilelesautressubstratsplusrapidementetproduitdel’hydrogèneplustôt.CetavantageaétéutilisépourpromouvoirlesconditionsdecroissancedeCl. butyricumenco-cultureavecC. freundii,étantdonnéqueCl. butyricumestextrêmementsensibleauxtracesd’oxygènedanslemilieu.Cecipermettraitd’éviterl’ajoutd’agentsréducteurscouteuxdanslemilieudeculture(telsquelaL-cystéine)danslamesureoùC. freundiiconsommel’oxygènerésiduel.Cetypedeco-cultureaététestésurglucoseetsuramidon.Ellesonteupourconséquence de diminuer les rendements de conversion sur glucose (de 0,53molH2.molhexose

-1 pour les cultures pures deCl. butyricumà0,38molH2.molhexose

-1pourlesculturesmixtes),tandisqu’ilsontlégèrementaugmentésuramidon(passantrespectivementde0,69and0,73molH2.molhexose

-1).Après48hdefermentation,lesanalysesmétaboliques,confirméespardesobservationsmicrobiologiques,ontmontréquelenombredecellulesdeC. freundiiontdrastiquementdiminuéouquecettedernièresoucheaététrèsfortementinhibéeparledéveloppementdeCl. butyricum.Mots-clés.Biohydrogène,Clostridium butyricum,Citrobacter freundii, culturemixte,potentielbiochimiqued’hydrogène,batch,substrat.

542 Biotechnol. Agron. Soc. Environ. 201014(S2),541-548 BeckersL.,HiligsmannS.,HamiltonCh.etal.

1. IntroduCtIon

Hydrogen (H2),whetherburnedoruseddirectly in afuelcell, isaverypromisingcleanenergyvector forthe decrease of our environmental impact since itsutilizationgeneratesonlywatervapor.Nevertheless,H2isstillmainlyproducedbysteamreformingofmethane,aprocessreleasinglargeamountoffossilCO2 in theatmosphere. In the last few years, there has been anincreasinginteresttofindnewH2productionprocesseswithalmostnocarbonemission(Balat,2009;Holladayet al., 2009;Moriarty et al., 2009).One of themostpromisingandinvestigatedprospectsisthebiologicalproductionofhydrogen through thedegradationofalargespectrumofcarbonsourcesbyanaerobicbacteriain a process called “dark fermentation” (Das et al.,2001;Levinetal.,2004;Nathetal.,2004a;Das,2009;Hallenbeck,2009).

The best describedmesophilic strains are, on theone hand, strict anaerobic bacteria from the genusClostridium that have the potential to reach highexperimental hydrogen yields (about two molesof hydrogen per mole of hexose consumed). Andon the other hand, facultative anaerobes such asEnterobacteriaceae that present lower experimentalyields (~ 1 molH2.molhexose) but can achieve higherproductionrates(Hallenbecketal.,2002;Hawkesetal.,2002;Kotayetal.,2008).Themainpurposetoenhancefermentative hydrogen production is to improvehydrogenyieldsforanefficientenergyrecoveryfromthesubstrate.Thetwospeciesinvestigatedinthiswork,Clostridium butyricumCWBI1009(Massetetal.,2010)andCitrobacter freundii CWBI952 (Hamilton et al.,2010), have a maximum theoretical hydrogen yieldof4and2molH2.molhexose

-1 respectivelydependingonthemetabolicpathway followed for the fermentationof thecarbonsource (Nandietal.,1998;Nathetal.,2004b;Kraemeretal.,2007;Ohetal.,2008a).

Clostridia are however extremely sensitive to thepresenceofoxygenwhichstronglyinhibitsH2evolvingenzymes(Heinekey,2009).Thiscanbeavoidedwiththe addition of an expensive reducing agent such asL-cysteine.However, theuseofsuchanagent isnotsuitable for a large-scale cost effective biohydrogenproductionprocess(Dasetal.,2008;Yuanetal.,2008).

Hydrogen may evolve through the fermentationprocesses of simple carbohydrates such as glucose,sucrose,lactoseandmaltoseormorecomplexonessuchasstarchorevencellulose(Uenoetal.,1995;Davila-Vazquezetal.,2008;Magnussonetal.,2008).Onlyafewstudieshaveinvestigatedthehydrogenproductionwith these different substrates on pure cultures incomparisonwithco-cultures(Yokoietal.,1995;Nathetal.,2006;Chenetal.,2008;Panetal.,2008).Thisis why this work compares the hydrogen andmajormetabolitesproduction(i.e.acetate,butyrate,formate,

lactate,ethanolandsuccinate)inpureC. freundiiandCl. butyricum cultures with five different substrates.These experimentswere carried out in serumbottlesbatch cultures based on the biochemical hydrogenpotential(BHP)testproceduredescribedbyLin(Linetal.,2007).

Furthermore, a co-culture of C. freundii andCl. butyricum in thesameBHPculturemayenhancehydrogenproduction.Suchaculturewouldnotrequiretheadditionofany reducingagents sinceC. freundiiconsumes oxygen and provides the anaerobicconditionsrequiredforCl. butyricumgrowth.ThishasalreadybeenshowninamixedcultureofEnterobactersp.andCl. butyricumgrowingonstarch(Yokoietal.,1998; 2002). However, little is known about thisconsortiumonothersubstrates.Inthiswork,co-cultureswere monitored on glucose and also on starch forcomparison. They are discussed in comparison withtheresultsfoundintheliterature.

2. MAtErIAls And MEthods

2.1. Microorganisms conservation and preculture media

Thetwospeciesculturedinthiswork,i.e.Citrobacter freundii CWBI952 and Clostridium butyricumCWBI1009werepreviouslyisolatedandcharacterizedin our laboratory (Hamilton et al., 2010; Massetetal.,2010).Fortheconservationofthestrains1mlof a previous 25 ml culture, hermetically stopperedand incubated at 30°C, was transferred weekly to afresh tube filled with sterile MD medium. The MDmediumcontainedperliterofdeionizedwater:glucosemonohydrate(5g),caseinpeptone(5g),yeastextract(0.5 g), KH2PO4 (2 g), MgSO4.7H2O (0.5 g) andL-cysteine hydrochloride (0.5 g). All the chemicalsusedwereofanalyticalorextrapurequalityandweresuppliedbyMerck,UCBandSigma.CaseinpeptoneandyeastextractweresuppliedbyOrganotechnie(LaCourneuve,France).

2.2. Experimental procedures and culture media

The BHP (Biochemical Hydrogen Potential) testswerecarriedoutin270mlsterileglassserumbottlesfilled with 200 ml of MD medium and differentcarbon sources in order to determine the hydrogenproductionpotentialof thestrains.Thesterilecarbonsource (i.e. glucose monohydrate, maltose, sucrose,lactoseorstarchinsolutionindeionizedwater)werepreparedandaddedseparatelyatafinalconcentrationof 4.3 gCOD.l

-1 to preventMaillard reactions betweencarbohydrates and amino acids. The same approachwasfollowedwithL-cysteinehydrochloridetoafinal

BiohydrogenproductionbyCl. butyricumandC. freundii 543

concentrationof0.5g.l-1.Theeffectivefinalsubstrateconcentration was measured by HPLC. Differencesbetweentheinitialconcentrationsofthecarbonsourcesrangedfrom3.9to4.8gCOD.l

-1duetohydrationofthecompounds.AfterthepreparationoftheMDmediumand adjustment of the pH to 7.3 the bottles weresterilized.ThecarbonsourceandL-cysteinesolutionwerethenaddedbeforeinoculationofthemediumwith5mlofafreshpreculturetube(incubatedfor48h)forthepurestrainsculturesandwith3mlofeachstrainforthemixedcultures.ThebottleswerethencappedwithabutylstopperasdescribedbyLin(Linetal.,2007)andflushedwithnitrogengastoremovetheremainingoxygeninthebottlegasphase.Thebottleswerethenincubatedat 30°C±0.5°Cwith anorbital stirring at150rpm.EachBHPtestwasconductedintriplicateforthestandarddeviationestimations.

2.3. Analytical methods

ThecellconcentrationofC. freundiiwasdeterminedbyconsecutivedilutions inpeptonedwater.A100µlsampleofthethreefinaldilutionswasspreadonPCAPetridishesbeforeincubationat30°Cfor18to24h.ThismethodwasalsousedtoconfirmtheabsenceofothermicroorganismsthanC. freundii.ThePCA(PlateCountAgar)mediumcontainedperliterofdeionizedwater:glucosemonohydrate(1g),caseinpeptone(5g),yeastextract(2.5g),agar(15g).Thecellconcentrationof Cl. butyricum or of the mixed culture weredeterminedbymicroscopicobservationsonaBürkercounting chamber (meanof 10 counts) after dilutionand cell settlement in a 0.4% (final concentration)fresh formaldehyde solution.The absence of aerobiccontaminantswasverifiedbyspreading100µlofthecultureonPCAmediumplatesandincubatingfor48hat30°C.Theabsenceofbacterialgrowthconfirmedtheabsenceofaerobiccontaminants.

The volume of biogas produced was measuredwith a sterile syringe and needle introduced throughthe previously sterilized septum. The proportion ofhydrogengaswasdeterminedusingagaschromatograph(GC) (Hewlett-Packard 5890 Series II) fitted witha thermalconductivitydetector (TCD)anda30mx0.32mmGASPROGSCcapillarycolumn(Altech)inserieswitha20mx0.25mmCarboPLOTP7column(Chrompak).The temperaturesof the injection,TCDchambersandtheovenweremaintainedat90°,110°and55°Crespectively.Nitrogenwasusedasthecarriergasinthecolumnataflowrateof20ml.min-1.

Theliquidsamplesharvestedduringorattheendofthefermentations(ca.from48to96h)werecentrifugedat 13,000 g for 10 min. The obtained supernatantswere thenfiltered througha0.2µmcelluloseacetatemembrane (Midisart Sartorius) and analyzed byHPLC.TheHPLCanalysiswas carriedout using an

Agilent1100series(HPChemstationsoftware)withaSupelcogelC-610HcolumnprecededbyaSupelguardHpre-column(oven temperature40°C),0.1%H3PO4(in milliQ water) as the isocratic mobile phase at aflow rateof0.5ml.min-1 andadifferential refractionindex detector (RID, heated at 35°C). The methodlastedfor35minatamaximumpressureof60bars.Theconcentrationsof thecompounds in the samplesare determined in comparisonwith standard samplesanalyzed for the establishment of calibration curves.Thedatafortheglucose(oranyothercarbonsource)andmetabolite concentrationswereused to calculatethemassbalance(MB)oftheglucoseconversionusingtheequation:

MB=ΣNi.ΔCi

NG.ΔCG

(1)

whereNiisthenumberofcarbonatomsinamoleculeofmetabolitei;∆Ciistheconcentrationofmetaboliteieffectivelyproduced(i.e.thedifferencebetweenthefinaland initial concentrations);NG is thenumberofcarbonatomsinthesubstratemolecule(i.e.6)and∆CGistheconcentrationoftheglucoseconsumed(Eq.(1)).

3. rEsults And dIsCussIon

3.1. hydrogen production by Citrobacter freundii CWBI952 and Clostridium butyricum CWBI1009 using five different substrates

The investigation of hydrogen and metabolitesproduction with five different substrates (glucose,maltose,sucrose, lactoseandstarch)wasfirstcarriedoutwithpureculturesofCitrobacter freundiiCWBI952andClostridium butyricumCWBI1009in270mlbatchserumbottles.Cellconcentrationsintheserumbottlesatthebeginningofthefermentationwereof4.3.10+07and 1.2.10+07 CFU.ml-1 respectively. The hydrogenproduction performanceswere compared in terms ofhydrogenyieldsallowingafirmbasisforacomparisonofthetwostrains.Thecumulativevolumesofhydrogenproduced and the hydrogen yields are reported intable 1alongwiththesubstrateconversionratiosandthefinalcellconcentrations(48hafterinoculation).

Theseresultsclearlyshowthatbetterperformanceswere obtained with Cl. butyricum compared toC. freundiiwhichproducedhydrogen lessefficiently.With starchC. freundii produced no hydrogen. Thishadalreadybeenreportedintheliteratureandisduetoitslackoffunctionalamylases(Kotayetal.,2009).Although no hydrogenwas produced byC. freundii,biomassincreasedto3.3.10+08CFU.ml-1probablydueto the consumption of other less accessible carbonsources in the media, i.e. casein peptone and yeast

544 Biotechnol. Agron. Soc. Environ. 201014(S2),541-548 BeckersL.,HiligsmannS.,HamiltonCh.etal.

extract.BycontrastCl. butyricumisabletoproducehydrogenbydegradingstarch.

Foreverysubstrateinvestigated,higherhydrogenyieldswereobtainedwithCl. butyricum.Inaddition,hydrogenwasmoreabundantinthebiogasproducedbyCl. butyricumcomparedtoC. freundii(onaverage70%vs62%respectively,datanotreportedhere).

The differences in the performances of the twostrains are due to the differentmetabolic pathwaysfollowed (Ying et al., 2004; Oh et al., 2008b).Enterobacteriaceae produce hydrogen via thedecomposition of formate up to a maximum yieldof2molH2.molhexose

-1.WhereasClostridia follow themixed acid fermentation and provide a maximumyield that is twice as highwhen producing acetateand butyrate. However, the yields obtained in thisinvestigation, ranging from0.1 to 0.24 and0.49 to0.69molH2.molhexose

-1forC. freundiiandCl. butyricumrespectively, are lower than those published inseveralstudies[(0.88molH2.molhexose

-1forC. freundii(Hamiltonetal.,2010)and1.69molH2.molhexose

-1forCl. butyricum(Massetetal.,2010)].Thisdifferencecan be explained by the absence of pH regulationin the serum bottle experiments. During thefermentationofthecarbonsources,acidmetaboliteswere produced leading to a rapid decrease in pH(set at 7.3 at the beginning). Since no buffer suchasHPO4

2-/H2PO4-wasused in themedia thepHof

theculturesrapidlydroppedtolevelslowerthantheoptimalpHforhydrogenproduction(5.9and5.2forC. freundii andCl. butyricum respectively) therebyreducingthefinalyieldreached.InadditionthefinalpH levels were much lower (ranging from 4.13 to4.67) inducing a strong inhibitory effect, not onlyon the hydrogen production but also on substrateconsumption.

3.2. Metabolites analysis for C. freundii and Cl. butyricum with different substrates

The main soluble metabolites (ethanol, lactate,acetate, succinate and formate for C. freundii andbutyrate, lactate, acetate, formate and ethanol forCl. butyricum)wereanalyzedbyHPLCattheendoftheculture(48h).Thecarbonmassbalanceforthesemetabolitesisindicatedinthefigure 1intermsofthepercentage of substrate’s carbon content converted.Due to the difference in the metabolic pathwaysinvolved,butyrateisonlyproducedbyCl. butyricumandsuccinateonlybyC. freundii. Inadditionmoreof the carbon source is converted to ethanol byC. freundii(tentimesmorethanwithCl. butyricum).

WiththeC. freundiiculturesonlysmallamountsofformateweredetectedindicatingthatonlyalittlemorehydrogen,enhancingtheyieldsbyabout20%,couldhavebeenproducedthroughtheactivityoftheFormateHydrogenLyase(FHL)enzymaticcomplexdecomposing formate in hydrogen (Kim et al.,2008).Othermetabolites,mainlyethanolandlactateaccountingforabout20%oftheglucoseconverted,are of less interest in this context since they donot lead to the production of hydrogen.When theculturesweremaintainedinoptimalconditions,thesemetaboliteswere stillproduced in similar amounts.This indicates that very little or no adaptation inC. freundii’smetabolic pathwayoccurs in responsetotheseconditions(Hamiltonetal.,2010).

WithClostridia’smetabolicpathway,onlyacetateand butyrate lead to the coproduction of hydrogen,accountingfor15.9%and24.5%respectivelyoftheglucose converted. In a previous work, regulatedbatch cultures at the optimal pH for hydrogenproduction were carried out with Cl. butyricum

table 1. Substrateconversionratios,finalhydrogenproduction,hydrogenyieldandcellconcentrationin270mlbatchculturewithfivedifferentsubstratesduring48h—Taux de conversion du substrat, production d’hydrogène finale, rendement de conversion en hydrogène et concentration cellulaire en culture batch de 270 ml avec cinq substrats différents pendant 48 h.

substrate conversion hydrogen production hydrogen yield Final cell ratio (ml) (molH2.molhexose

-1) concentration(CFU.ml-1)

Citrobacter Glucose 98.2% 46.1±5.7 0.24±0.03 1.0.10+09 freundii Maltose N.D. N.D. N.D. N.D.CWBI952 Sucrose 92.5% 19.0±2.8 0.10±0.02 1.3.10+09 Lactose 99.4% 35.3±7.2 0.18±0.04 5.4.10+08 Starch 0 0 N.D.(0) 3.3.10+08

Clostridium Glucose 89.3% 95.9±2.0 0.58±0.01 2.6.10+08 butyricum Maltose 97.2% 100.8±2.0 0.51±0.01 2.6.10+08CWBI1009 Sucrose 99.1% 98.3±0.5 0.52±0.00 2.2.10+08 Lactose 93.3% 123.9±2.0 0.69±0.00 1.4.10+08 Starch 85.6% 79.1±2.1 0.49±0.02 2.1.10+08

N.D.:Notdetermined—Indéterminé.

BiohydrogenproductionbyCl. butyricumandC. freundii 545

and these two metabolites accounted for 12.7%and 40.3% respectivelywith a yield of 1.69molH2.molhexose

-1(Massetetal.,2010).Thelowerconversionrateofcarbohydrateintoacetateandbutyrateshownhereexplainsthelowhydrogenyieldsobtainedintheserumbottlescultures.Therefore,itcanbeassumedthatCl. butyricumadaptsitsmetabolismtoproducemore favorable metabolites and hydrogen whengrowingunderoptimalpHconditions.Thesynthesisoflactateisnotaccompaniedbyhydrogenproduction.Inthisstudy,nolactatewasproducedfromlactosebyCl. butyricum.Thisobservationmaybelinkedtothehigher hydrogen yields reached compared to othersubstrates(table 1)andtothehigherconversionrateofthecarbonsourceintobutyrate(35.3%withlactoseand24.5%withglucose).Bycontrast,aconsiderableamount of lactate was produced from the othercarbohydrates (e.g. 37.3% of the initial glucose).Cl. butyricum also synthesizes formate accountingfor2to3%ofthesubstrate.Thismetabolitecouldbeusedinaco-cultureofthetwospeciestopotentiallyproducemorehydrogenviaC. freundii’smetabolism.Yokoi et al. (1998) have already investigated thistopic, reporting that suchamixedcultureproducedhydrogenefficientlyandwithouttheneedtoaddanexpensivereducingagent.

3.3. Batch fermentation with co-culture of C. freundii and Cl. butyricum on glucose and starch

MixedculturesofCl. butyricumandC. freundiiwereculturedin270mlserumbottleswithglucoseorstarch.Purecultureswerecarriedoutinthesameconditionsforcomparison.L-cysteinewasaddedineachcultureaccording to the media description in materials andmethods.Theresultsoftheseexperimentsarereportedintable 2indicatingthehydrogenproductionmeasuredafter24hofcultureandattheendofthefermentation(96 h on glucose and 48 h on starch) and hydrogenyields.

Theresultsoftheseexperimentswithstarchareinaccordancewith the results obtained byYokoi et al.(2002): a sustainable hydrogen production, in termsof hydrogen production and yield, is possible withstarch. Inour culturesC. freundii seemed to surviveand, as reported byYokoi,was subsequently able tore-establishviablegrowthconditionforCl. butyricumincaseofoxygencontaminationintheculturemedia.The results presented in the table 2 suggest thatC. freundiiestablishesanaerobicconditionsandallowsproductionofhydrogentobeginearlierthaninapureCl. butyricumculture(54mlwerealreadyproduced24hafterinoculation).

Figure 1.CarbonmassbalanceforClostridium butyricumandCitrobacter freundiiin270mlbatchfermentationwithdifferentcarbonsources,expressedinpercentageofcarbonconvertedfromtheconsumedcarbonsource(succinate,lactate,formate,acetate, ethanol,butyrate, carbondioxide)—Bilans carbone de fermentations avec Clostridiumbutyricum et Citrobacterfreundii en culture batch de 270 ml avec différentes sources de hydrocarbonées, exprimés en pourcentage de carbone converti provenant du substrat consommé (succinate, lactate, formiate, acétate, éthanol, butyrate, dioxyde de carbone).

Car

bon

con

vert

ed fr

om g

luco

se (%

)

40

35

30

25

20

15

10

5

0

C. freundii g

lucose

C. freundii s

ucrose

C. freundii la

ctose

C. freundii s

tarch

Cl. butyri

cum glucose

Cl. butyri

cum glucose

Cl. butyri

cum sucrose

Cl. butyri

cum lactose

Cl. butyri

cum starch

succinate

lactate

formate

acetate

ethanol

butyrate

carbon dioxide

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546 Biotechnol. Agron. Soc. Environ. 201014(S2),541-548 BeckersL.,HiligsmannS.,HamiltonCh.etal.

Toourknowledgesuchasustainableconsortiumwithasimplesugarsuchasglucosehasnotyetbeenstudied.Duringthefirst24hoffermentationnohydrogenwasproducedbyCl. butyricumwhileC. freundiihadalreadyprovidedmorethan95%ofitstotalhydrogenproduction.The sustainable influence ofC. freundii in the mixedculturewithglucoseallowedtheproductionofhydrogento begin earlier than with the pure Cl. butyricumculture.However, the use of amixed culture in batchfermentation decreased the final hydrogen yield from0.53to0.39molH2.molhexose

-1bycomparisonbetweenthemixedcultureandthepureCl. butyricumculture.TestsonPCAmedia,carriedout24hafterinoculation,didnotshowanyaerobicbacterialdevelopmentindicatingthatC. freundiididnotsurviveorthatthecellconcentrationhaddramaticallydecreased.Thissuggeststhatthestrainenhanced the initiation of hydrogen production, butwas then rapidly overgrown or strongly inhibited byCl. butyricum.

Thisisconfirmedbytheresultspresentedinfigures 2and3depicting theanalysisofmetabolitesandcarbonmass balance.They show that lactate andbutyrate arethemainmetabolites from the degradation of glucoseandstarch.Thesetwometabolitesarecommonlyfoundin Cl. butyricum culture supernatants. However, thepresenceofsuccinateindicatesthatC. freundiigrewatthebeginningofthefermentation.

The data obtained on starch degradation confirmthat the metabolites (high butyrate and low ethanolconcentrations)seemedtocorrespondtothefermentationperformed byCl. butyricum and thatC. freundii wasstronglyinhibitedundertheseconditions.

Theproductionofsuccinateandthehighamountofethanoldetectedinthesupernatantsoftheglucosemediaindicates thatC. freundii was active at the beginningof thefermentationandgrewfaster thanCl. butyricumconsumingpartof theglucose.Thiswouldexplain thedecrease in thefinalhydrogenyields (see table 2).AsC. freundii was not found in themedia at the end of

the fermentation, it had by then been overgrown byCl. butyricum. InhibitionofC. freundiigrowthmaybealimitingfactorifasuddeninfiltrationofoxygenoccursin the absence of a reducing agent, since the strictlyanaerobicstrainCl. butyricummaythenstarttosporulate.

4. ConClusIon

Our work highlights the fact that mixed cultures ofCitrobacter freundiiCWBI952andClostridiumbutyricumCWBI1009 can efficiently maintain the production ofhydrogenatacceptableyieldscomparedtopureculturesofCl. butyricum.Moreover,itcouldconsumeefficientlymanydifferentcarbonsourcessinceCl. butyricumwasable to degrade simple carbohydrates or even starch.HoweverthesurvivalofC. freundiiincompetitionwithCl. butyricumwascompromisedsinceitwasovergrownespeciallywiththeglucosesubstrate.FurtherworkwillbedonetofindotherfacultativeaerobicstrainswhichareabletoenhanceanaerobicconditionsintheculturemediawithoutbeingovergrownbyCl. butyricumandwithoutdecreasingthehydrogenyield.

Acknowledgements

L. Beckers and S. Hiligsmann both equally contributed tothe paper. L. Beckers is grateful to the FRS-FNRS (Fondsde laCommunautéfrançaisedeBelgiquepour laRechercheScientifique) for supporting his work and researches.C. Hamilton is to be thanked for his guidance through theredaction of this paper. J. Masset is a recipient of a FRIAfellowship (Fonds pour la Formation à la Recherche dansl’Industrie et l’Agriculture). This work was also supportedbyanARCproject(ActiondeRecherchesConcertées,ARC-07/12-04) and theWalloon Region. The publication of thisarticle has been allowed by the organization of the secondthematic day organized by the GEPROC doctoral thematicschool.

table 2.HydrogenproductionandyieldsfromglucoseandstarchfermentationinpureormixedculturewithClostridiumbutyricumandCitrobacter freundii—Production d’hydrogène et rendements de conversion en hydrogène de cultures pures ou mixtes avec Clostridiumbutyricum et Citrobacterfreundii sur glucose et amidon.

Cumulative hydrogen production (ml) hydrogen yield 24 h End of fermentation (molH2.molhexose

-1)

Glucose PureC. freundii 38.4±1.2 40.3±5.4 0.25±0.03 Pure Cl. butyricum 0 99.6±8.8 0.53±0.04 MixedC. freundii and Cl. butyricum 54.4±2 62.6±2.7 0.38±0.02Starch PureC. freundii N.D. N.D. N.D. PureCl. butyricum N.D. 92.0±5.7 0.69±0.04 MixedC. freundiiandCl. butyricum 44±1.4 96.5±0.7 0.73±0.01

N.D.:Notdetermined—Indéterminé;Citrobacterfreundiiisnottestedinpurecultureonstarchbecauseitdoesnotdegradestarch—Citrobacterfreundiin’a pas été testée avec l’amidon dans la mesure où cette souche ne dégrade pas ce substrat;Theyieldsarecalculatedattheendofthefermentation—Les rendements de conversions sont calculés en fin de fermentation.

BiohydrogenproductionbyCl. butyricumandC. freundii 547

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Figure 3.Carbonmassbalance in270mlbatchpure andmixed culture of Citrobacter freundii and Clostridiumbutyricumwithglucoseandstarch,expressedinpercentageof carbon converted from the consumed carbon source(succinate,lactate,formate,acetate,ethanol,butyrate,carbondioxide) — Bilans carbone de fermentations en cultures pures et mixtes de 270 ml avec Clostridium butyricum et Citrobacter freundii sur glucose et amidon, exprimés en pourcentage de carbone converti provenant du substrat consommé (succinate, lactate, formiate, acétate, éthanol, butyrate, dioxyde de carbone).

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Figure 2. Metabolites analysis in 270ml batch pure andmixed culture of Citrobacter freundii and Clostridium butyricum with glucose and starch substrates, expressedin mmol.l-1 (residual hexose, succinate, lactate, formate,acetate, ethanol, butyrate)—Analyses des métabolites en solution pour les cultures pures et mixtes avec Clostridium butyricum et Citrobacter freundii sur glucose et amidon, exprimées en mmol.l-1 (hexose résiduel, succinate, lactate, formiate, acétate, éthanol, butyrate).

A:Analysis24hafterinoculation(pureCl. butyricumculturesarenotanalyzedsincehydrogenwasnotyetproduced)—Analyses 24 h après inoculation (les cultures pures de Cl.butyricum ne sont pas analysées dans la mesure où aucune production d’hydrogène n’a alors commencé);B:Analysisattheendofthefermentation—Analyses en fin de fermentation.

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