Theinterestintheconversionofplantbiomasstorenewablefuelssuchasbioethanolhasledtoanincreasedinvestigationintotheprocessesregulatingbiomasssaccharification.ThefilamentousfungusAspergillusnigerisanimportantmicroorganismcapableofproducingawidevarietyofplantbiomassdegrADIngenzymes.InA.nigerthetranscriptionalactivatorXlnRanditsclosehomolog,AraR,controlsthemain(hemi-)cellulolyticsystemresponsIBLeforplantpolysaccharidedegradation.Sugarcaneisusedworldwideasafeedstockforsugarandethanolproduction,whilethelignocellulosicresidualbagassecanbeusedindifferentindustrialapplications,includingethanolproduction.Theuseofpentosesugarsfromhemicellulosesrepresentsanopportunitytofurtherincreaseproductionefficiencies.Inthepresentstudy,wedescribeaglobalgeneexpressionanalysisofA.nigerXlnR-andAraR-deficientmutantstrains,grownonaD-xylose/L-arabinosemonosaccharidemixtureandsteam-explodedsugarcanebagasse.DifferentgenesetsofCAZyenzymesandsugartransporterswereshowntobeindividuallyorduallyregulatedbyXlnRandAraR,withXlnRappearingtobethemajorregulatoroncomplexpolysaccharides.Ourstudycontributestounderstandingofthecomplexregulatorymechanismsresponsibleforplantpolysaccharide-degradinggeneexpression,andopensnewpossibilitiesfortheengineeringoffungiabletoproducemoreefficientenzymaticcocktailstobeusedinbiofuelproduction.

Thedevelopmentofaviablelignocellulosicethanolindustryrequiresmultipleimprovementsintheprocessofconvertingbiomasstoethanol.Akeystepistheimprovementoftheplantsthataretobeusedasbiomassfeedstocks.Tofacilitatetheidentificationandevaluationoffeedstockplants,itwouldbeusefultohaveamethodtoscreenlargenumbersofindividualplantsforenhanceddigestibilityinresponsetocombinationsofspecificpretreatmentsandenzymes.Thispaperdescribesahigh-throughputdigestibilityplatform(HTDP)forscreeningcollectionsofgermplasmforimproveddigestibility,whichwasdevelopedundertheaUSPicesoftheDepartmentofEnergy-GreatLakesBioenergyResearchCenter(DOE-GLBRC).Akeycomponentofthisplatformisacustom-designedworkstationthatcangrindanddispense1–5mgquantitiesofmorethan250differentplanttissuesamplesin16h.Theotherstepsintheprocessing(pretreatment,enzymedigestion,andsugaranalysis)havealsobeenlargelyautomatedandrequire36h.Theprocessisadaptabletodiverseacidicandbasic,low-temperaturepretreatments.TotalthroughputoftheHTDPis972independentbiomasssamplesperweek.Validationoftheplatformwasperformedonbrownmidribmutantsofmaize,whichareknowntohaveenhanceddigestibility.Additionalvalidationwasperformedbyscreeningapproximately1,200ArabidopsismutantlineswithT-DNAinsertionsingenesknownorsuspectedtobeinvolvedincellwallbiosynthesis.Severallinesshowedhighlysignificant(p < 0.01)increases=""in=""glucose=""and=""xylose=""release=""(20–40%=""above=""the=""mean).=""the=""platform=""should=""be=""useful=""for=""screening=""populations=""of=""plants=""to=""identify=""superior=""germplasm=""for=""lignocellulosic=""ethanol=""applications=""and=""also=""for=""screening=""populations=""of=""mutant=""model=""plants=""to=""identify=""specific=""genes=""affecting=""digestibility.="">

Thepretreatmentofcelluloseusingionicliquids(ILs)hasbeenshowntobeaneffectivemethodforimprovingtheenzymatichydrolysisofcellulose;thistechniqueaffordsafastandcompletesaccharificationofcelluloseintoreducingsugars(Dadietal.,BiotechnolBioeng.2006;95:904–910;LiuandChen,ChineseSciBull.2006;51:2432–2436;Zhaoetal.,JBiotechnol.2009;139:47–54).Motivatedbytheseadvances,thisstudyexaminestheeffectofIL-pretreatmentontheenzymatichydrolysisofpurifiedxylan(asamodelsystemofhemicellulose)andswitchgrass(asareallignocellulose).TheIL-pretreatmentresultedinnoimprovementinthehydrolysisofxylan.Thelikelyreasonisthatpurexylanhasalowdegreeofpolymerization(DP),andisreadilybiodegradedevenwithoutanypretreatment.However,inrealcellulosicmaterials(suchasswitchgrass),xylanisentrappedwithinthecellulosicmatrix,andcannotbeconvenientlyaccessedbyenzymes.OurdatademonstratethattheIL-pretreatmentofswitchgrasssignificantlyimprovedtheenzymaticsaccharificationofbothcellulose(96%D-glucoseyieldin24h)andxylan(63%D-xyloseyieldin24h).ThecompositionalanalysisofswitchgrasssuggestsalowerlignincontentafterIL-pretreatment.Inaddition,theinfraredspectrumofregeneratedswitchgrassindicatesalowersubstratecrystallinity,whereastheenzymeadsorptionisothermfurtherimpliesthattheregeneratedsubstrateismoreaccessibletoenzymes.ThisstudyhasfurtherconfirmedthatIL-pretreatmentisaneffectivetoolinenhancingtheenzymatichydrolysisofcellulosicbiomass,andallowingamorecompletesaccharification.

Solventogenicclostridiaarewell-knownsincealmostacenturyduetotheiruniquecapabilitytobiosynthesizethesolventsacetoneandbutanol.Basedonrecentlydevelopedgeneticengineeringtools,atargeted3-hydroxybutyryl-CoAdehydrogenase(HBD)-negativemutantofClostridiumacetobutylicumwasgenerated.Interestingly,theentirebutyrate/butanol(C₄)metabolicpathwayofC.acetobutylicumcouldbeinactivatedwithoutaseveregrowthlimitationandindicatedthegeneralfeasibilitytomanipulatethecentralfermentativemetabolismforproductpatternalteration.CellextractsofthemutantC.acetobutylicumhbd::int(69)revealedclearlyreducedthiolase,HbdandcrotonasebutincreasedNADH-dependentalcoholdehydrogenaseenzymeactivitiesascomparedtothewildtypestrain.NeitherbutyratenorbutanolweredetectedinculturesofC.acetobutylicumhbd::int(69),andtheformationofmolecularhydrogenwassignificantlyreduced.Insteadupto16and20g/lethanolwereproducedinglucoseandxylosebatchcultures,respectively.Furthersugaradditioninglucosefed-batchfermentationsincreasedtheethanolproductiontoafinaltiterof33g/l,resultinginanethanoltoglucoseyieldof0.38g/g.

L-MalicacidandfumaricacidareC₄dicarboxylicorganicacidsandconsideredaspromisingchemicalbuildingblocks.Theycanbeappliedasfoodpreservativesandacidulantsinrustremovalandaspolymerizationstarterunits.MoldsofthegenusAspergillusareabletoproducemalicacidinlargequantitiesfromglucoseandothercarbonsources.InordertoenhancetheproductionpotentialofAspergillusoryzaeDSM1863,productionandconsumptionratesinanestablishedbioreactorbatch-processbasedonglucoseweredetermined.At35°C,upto42g/Lmalicacidwasproducedina168-hbatchprocesswithfumaricacidasaby-product.Inprolongedshakingflaskexperiments(353h),thesuitabilityofthealternativecarbonsourcesxyloseandglycerolatacarbon-to-nitrogen(C/N)ratioof200:1andtheinfluenceofdifferentC/Nratiosinglucosecultivationsweretested.Whenusingglucose,58.2g/Lmalicacidand4.2g/Lfumaricacidwereproduced.Whenapplyingxyloseorglycerol,bothorganicacidsareproducedbuttheformationofmalicaciddecreasedto45.4and39.4g/L,respectively.Whereasthefumaricacidconcentrationwasnotsignificantlyalteredwhencultivatingwithxylose(4.5g/L),itisclearlyenhancedbyusingglycerol(9.3g/L).Whenusingglucoseasacarbonsource,anincreaseordecreaseoftheC/Nratiodidnotinfluencemalicacidproductionbuthadanenormousinfluenceonfumaricacidproduction.ThehighestfumaricacidconcentrationsweredeterminedatthehighestC/Nratio(300:1,8.44g/L)andlowestatthelowestC/Nratio(100:1,0.7g/L).

TheyeaststrainsCryptococcuspodzolicus,TrichosporonporosumandPichiasegobiensiswereisolatedfromsoilsamplesandidentifiedasoleaginousyeaststrainsbeneficialfortheestablishmentofmicrobialproductionprocessesforsustainablelipidproductionsuitableforseveralindustrialapplications.WhenculturedinbioreactorswithglucoseasthesolecarbonsourceC.podzolicusyielded31.8%lipidperdrybiomassat20°C,whileT.porosumyielded34.1%at25°CandP.segobiensis24.6%at25°C.Theseamountscorrespondtolipidconcentrationsof17.97g/L,17.02g/Land12.7g/Landvolumetricproductivitiesof0.09g/Lh,0.1g/Lhand0.07g/Lh,respectively.DuringthecultureofC.podzolicus30g/lgluconicacidwasdetectedasby-productintheculturebrothand12g/LgluconicacidinT.porosumculture.Theproductionofgluconicacidwaseliminatedforbothstrainswhenglucosewassubstitutedbyxyloseasthecarbonsource.Usingxyloselipidyieldswere11.1g/Land13.9g/L,correspondingto26.8%and33.4%lipidperdrybiomassandavolumetricproductivityof0.07g/Lhand0.09g/Lh,forC.podzolicusandT.porosumrespectively.Thefattyacidprofileanalysisshowedthatoleicacidwasthemaincomponent(39.6to59.4%)inallthreestrainsandcouldbeapplicableforbiodieselproduction.Palmiticacid(18.4to21.1%)andlinolenicacid(7.5to18.7%)arevaluableforcosmeticapplications.P.segobiensishadaconsiderableamountofpalmitoleicacid(16%content)andmaybesuitableformedicalapplications.

ThetypicalSerbiandietischaracterisedbyhighintakeofcerealproductsandalsolegumesareoftenused.Thecontentoftotalfibreaswellascertainfibrefractionswasdeterminedincereals,cerealproducts,andcookedlegumes.Thecontentoftotalfibreincookedcerealsandcerealproductsrangedfrom2.5to20.8g/100g,andincookedlegumesfrom14.0to24.5g/100g(ondrymatterbasis).Distributionofanalysedfibrefractionsandtheirquantitiesdifferedsignificantlydependingonfoodgroups.Fructansandarabinoxylanswerethemostsignificantfibrefractionsinryeflakes,andβ-glucaninoatflakes,celluloseandresistantstarchwerepresentinsignificantamountsinpeasandkidneybeans.Whenthesizeofregularfoodportionswastakenintoconsideration,thebestsourcesoftotaldietaryfibrewerepeasandkidneybeans(morethan11g/serving).Thesamefoodswerethebestsourcesofcellulose(4.98and3.56g/serving)andresistantstarch(3.90and2.83g/serving).Highintakeofarabinoxylansandfructanscouldbeaccomplishedwithcookedwheat(3.20gand1.60g/serving,respectively).Oat(1.39g/serving)andbarleyflakes(1.30g/serving)canberecommendedasthebestsourcesofβ-glucan.

InapreviousstudycrystalsofPseudomonasfluorescenssubspeciescellulosaxylanaseA(XYLA)containingxylopentaoserevealedthattheterminalnonreducingendglycosidicbondoftheoligosaccharidewasadjacenttothecatalyticresiduesoftheenzyme,suggestingthatthexylanasemayhaveanexo-modeofaction.However,aclusterofconservedresiduesinthesubstratebindingcleftindicatedthepresenceofanadditionalsubsite,designatedsubsiteF.AnalysisofthebiochemicalpropertiesofXYLArevealedthattheenzymewasatypicalendo-β1,4-xylanase,providingsupportfortheexistenceofsubsiteF.Thethree-dimensionalstructureoffourfamily10xylanases,includingXYLA,revealedseveralhighlyconservedresiduesthatareonthesurfaceoftheactivesitecleft.Toinvestigatetheroleofsomeoftheseresidues,appropriatemutationsofXYLAwereconstructed,andthebiochemicalpropertiesofthemutatedenzymeswereevaluated.N182Ahydrolyzedxylotetraosetoapproximatelyequalmolarquantitiesofxylotriose,xylobiose,andxylose,whilenativeXYLAcleavedthesubstratetoprimarilyxylobiose.ThesedatasuggestthatN182islocatedattheCsiteoftheenzyme.N126AandK47Awerelessactiveagainstxylanandaryl-β-glycosidesthannativeXYLA.ThepotentialrolesofAsn-126andLys-47inthefunctionofthecatalyticresiduesarediscussed.E43AandN44A,whicharelocatedintheFsubsiteofXYLA,retainedfullactivityagainstxylanbutweresignificantlylessactivethanthenativeenzymeagainstoligosaccharidessmallerthanxyloseptaose.ThesedatasuggestthattheprimaryroleoftheFsubsiteofXYLAistopreventsmalloligosaccharidesfromformingnonproductiveenzyme-substratecomplexes.

Lignocellulosicwasteisanaturallyabundantbiomassandisthereforeanattractivematerialtouseinsecondgenerationbiorefineries.Microbialgrowthonthemonosaccharidespresentinhydrolyzedlignocelluloseishoweverassociatedwithseveralobstacleswhereofoneisthelackofsimultaneousuptakeofthesugars.WehavestudiedtheaerobicgrowthofEscherichiacolionD-glucose,D-xylose,andL-arabinoseandforsimultaneousuptaketooccur,boththecarboncataboliterepressionmechanism(CCR)andtheAraCrepressionofxyloseuptakeandmetabolismhadtoberemoved.ThestrainAF1000isaMC4100derivativethatisonlyabletoassimilatearabinoseafteraconsiderablelagphase,whichisunsuitableforcommercialproduction.ThisstrainwassuccessfullyadaptedtogrowthonL-arabinoseandthisledtosimultaneousuptakeofarabinoseandxyloseinadiauxicgrowthmodefollowingglucoseconsumption.Inthisstrain,adeletioninthephosphoenolpyruvate:phosphotransferasesystem(PTS)forglucoseuptake,theptsGmutation,wasintroduced.Theresultingstrain,PPA652arasimultaneouslyconsumedallthreemonosaccharidesatamaximumspecificgrowthrateof0.59 h^-1,55%higherthanfortheptsGmutantalone.Also,noresidualsugarwaspresentinthecultivationmedium.ThepotentialofPPA652araisfurtheracknowledgedbytheperformanceofAF1000duringfed-batchprocessingonamixtureofD-glucose,D-xylose,andL-arabinose.Theconclusionisthatwithouttheremovalofbothlayersofcarbonuptakecontrol,thisprocessresultsinaccumulationofpentosesandleadstoareductionofthespecificgrowthrateby30%.

β-Xylosidasesparticipateinxylanbiodegradation,liberatingxylosefromthenon-reducingendofxylooligosaccharides.ThefungusPenicilliumpurpurogenumsecretestwoenzymeswithβ-D-xylosidaseactivitybelongingtofamily43oftheglycosylhydrolases.Oneoftheseenzymes,arabinofuranosidase3(ABF3),isabifunctionalα-L-arabinofuranosidase/xylobiohydrolaseactiveonp-nitrophenyl-α-L-arabinofuranoside(pNPAra)andp-nitrophenyl-β-D-xylopyranoside(pNPXyl)withaK_Mof0.65and12mM,respectively.Theother,β-D-xylosidase1(XYL1),isonlyactiveonpNPXylwithaK_Mof0.55mM.Thexyl1genewasexpressedinPichiapastoris,purifiedandcharacterized.Thepropertiesofbothenzymeswerecomparedinordertoexplaintheirdifferenceinsubstratespecificity.Structuralmodelsforeachproteinwerebuiltusinghomologymodelingtools.Moleculardockingsimulationswereusedtoanalyzetheinteractionsdefiningtheaffinityoftheproteinstobothligands.Thestructuralanalysisshowsthatactivecomplexes(ABF3–pNPXyl,ABF3–pNPAraandXYL1–pNPXyl)possessspecificinteractionsbetweensubstratesandcatalyticresidues,whichareabsentintheinactivecomplex(XYL1–pNPAra),whileotherinteractionswithnon-catalyticresiduesarefoundinallcomplexes.pNPAraisacompetitiveinhibitorforXYL1(K_i=2.5mM),confirmingthatpNPAradoesbindtotheactivesitebutnottothecatalyticresidues.

Inthiswork,anovellaboratory-scaledirectsteam-injectionapparatus(DSIA)wasdevelopedtoovercomethemaindrawbackoftheconventionalbatch-drivenlabrigs,namelythelongtimeneededtoheatfiberslurryfromroomtoreactiontemperaturesgreaterthan150°C.Thenovelapparatusmainlyconsistedofthreeunits:(i)amechanically-stirredbioreactorwheresaturatedsteamat5–30barcanbeinjected;(ii)anautomaticon–offvalvetoflashsuddenlythereactionmediumafteraprefixedreactiontime;(iii)acycloneseparatortorecoverthereactedslurry.Thissystemwastestedusing0.75dm³ofanaqueoussolutionofH₂SO₄(0.5%,v/v)enrichedwith50kgm^-3ofeithercommercialparticlesofAvicel®andLarchxylanor0.5mmsievedparticlesofTamarixjordanis.Eachslurrywasheatedtoabout200°Cbyinjectingsteamat28barfor90s.Theprocessefficiencywasassessedbycomparingthedissolutiondegreeofsuspendedsolid(Y_S),aswellasxylose(Y_X),glucose(Y_G),andfurfural(Y_F)yields,withthoseobtainedinaconventionalsteamautoclaveat130°Cfor30or60min.TreatmentofT.jordanisparticlesinDSIAresultedinY_SandY_Gvaluesquitesimilartothoseobtainedinthesteamautoclaveat130°Cfor60min,butinalessefficienthemicellulosesolubilization.Alimitedoccurrenceofpentosedegradationproductswasobservedinbothequipments,suggestingthathydrolysispredominatedoverdegradationreactions.ThesusceptibilityoftheresidualsolidfractionsfromDSIAtreatmenttoaconventional120hlongcellulolytictreatmentusinganenzymeloadingof5.4FPUg^-1wasmarkedlyhigherthanthatofsampleshydrolysedinthesteamautoclave,theircorrespondingglucoseyieldsbeingequalto0.94and0.22gpergramofinitialcellulose,respectively.Thus,T.jordanisresultedtobeavaluablesourceofsugarsforbioethanolproductionasprovedbypreliminarytestsinthenovellabrigdevelopedhere.

Plant-degradingenzymescanbeproducedbyfungionabundantlyavailablelow-costplantbiomass.However,enzymessetsaftergrowthoncomplexsubstratesneedtobebetterunderstood,especiallywithemphasisondifferencesbetweenfungalspeciesandtheinfluenceofinhibitorycompoundsinplantsubstrates,suchasmonosaccharides.Inthisstudy,AspergillusnigerandTrichodermareeseiwereevaluatedfortheproductionofenzymesetsaftergrowthontwo“secondgeneration”substrates:wheatstraw(WS)andsugarcanebagasse(SCB).A.nigerandT.reeseiproduceddifferentsetsof(hemi-)cellulolyticenzymesaftergrowthonWSandSCB.ThiswasreflectedinanoverallstrongsynergisticeffectinreleasingsugarsduringsaccharificationusingA.nigerandT.reeseienzymesets.T.reeseiproducedlesshydrolyticenzymesaftergrowthonnon-washedSCB.Thesensitivitytonon-washedplantsubstrateswasnotreducedbyusingCreA/Cre1mutantsofT.reeseiandA.nigerwithadefectivecarboncataboliterepression.TheimportanceofremovingmonosaccharidesforproducingenzymeswasfurtherunderlinedbythedecreaseinhydrolyticactivitieswithincreasedglucoseconcentrationsinWSmedia.ThisstudyshowedtheimportanceofremovingmonosaccharidesfromtheenzymeproductionmediaandcombiningT.reeseiandA.nigerenzymesetstoimproveplantbiomasssaccharification.

Theprocessoflignocellulosicbiomassroutinelyproducesastreamthatcontainssugarsplusvariousamountsofaceticacid.Asacetateisknowntoinhibitthecultureofmicroorganismsincludingoleaginousyeasts,littleattentionhasbeenpaidtoexplorelipidproductiononmixturesofacetateandsugars.HerewedemonstratedthattheyeastCryptococcuscurvatuscaneffectivelyco-fermentacetateandsugarsforlipidproduction.Whenmixturesofacetateandglucosewereapplied,C.curvatusconsumedbothsubstratessimultaneously.Similarphenomenawerealsoobservedforacetateandxylosemixtures,aswellasacetate-richcornstoverhydrolysates.Moreinterestingly,thereplacementofsugarwithequalamountofacetateascarbonsourceaffordedhigherlipidtitreandlipidcontent.Thelipidproductshadfattyacidcompositionalprofilessimilartothoseofcocoabutter,suggestingtheirpotentialforhighvalue-addedfatsandbiodieselproduction.Thisco-fermentationstrategyshouldfacilitatelipidproductiontechnologyfromlignocelluloses.

Thepresentinvestigationwasaimedtoevaluatethereleaseofbothantioxidantsandcellulosicfibrefromdifferentagro-foodwastes.Cost-effectiveandeasilyavailableagro-foodresidues(brewers’spentgrains,hazelnutshells,orangepeelsandwheatstraw)wereselectedandsubmittedtoadouble-stepacid/alkalinefractionationprocess.Theobtainedacidandalkalineliquorswereanalysedfortotalphenolscontentandantioxidantcapacity.Thefinalfibreresiduewasanalysedforthecellulose,ligninandhemicellulosecontent.Thetotalphenolscontentandantioxidantcapacityoftheacidliquorswerehigherthanthealkalinehydrolysates.Orangepeelsandwheatstrawgave,respectively,thehighest(19.70±0.68mg/g_dm)andthelowest(4.70±0.29mg/g_dm)totalphenolsrelease.Correlationbetweenantioxidantcapacityoftheliquorsandtheirorigindependedontheanalyticalassayusedtoevaluateit.Alltheacidliquorswerealsorichinsugardegradationproducts(mainlyfurfural).HPLCanalysisrevealedthatthemostabundantphenoliccompoundintheacidliquorswasvanillinforbrewers’spentgrains,hazelnutshellsandwheatstraw,andp-hydroxybenzoicacidfororangepeels.Wheatstrawservedasthebestrawmaterialforcelluloseisolation,providingafinalresiduewithahighcellulosecontent(84%)whichcorrespondedto45%oftheoriginalcellulose.Theappliedprocessremovedmorethan90%ofthehemicellulosefractioninallthesamples,whiledelignificationdegreerangedfrom67%(inhazelnutshells),to93%(inbrewers’spentgrains).Itwasnotpossibletoselectauniquerawmaterialforthereleaseofhighestlevelsofbothtotalphenolsandcellulose.