Isolation of fungi from dung of wild herbivores for application in bioethanol production

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Isolation

of

fungi

from

dung

of

wild

herbivores

for

application

in

bioethanol

production

Rhulani

Makhuvele

_,

_Ignatious

_Ncube

_,

Elbert

Lukas

Jansen

van

Rensburg

_,

_Daniël

_Coenrad

_La

_Grange

b_{UnitofEnvironmentalSciencesandManagement,North-WestUniversity,PotchefstroomCampus,Potchefstroom,SouthAfrica}

a

r

t

i

c

l

e

i

n

f

o

Received14April2016 Accepted4November2016 Availableonline3June2017 AssociateEditor:EleniGomes

Keywords: Lignocellulase Biofuel Fungi Xylose Fermentation

a

b

s

t

r

a

c

t

Producingbiofuelssuchasethanolfromnon-foodplantmaterialhasthepotentialtomeet transportationfuelrequirementsinmanyAfricancountrieswithoutimpactingdirectlyon foodsecurity.Thecurrentshortcomingsinbiomassprocessingareinefficientfermentation ofplantsugars,suchasxylose,especiallyathightemperatures,lackoffermentingmicrobes thatareabletoresistinhibitorsassociatedwithpre-treatedplantmaterialandlackof effec-tivelignocellulolyticenzymesforcompletehydrolysisofplantpolysaccharides.Duetothe presenceofresidualpartiallydegradedlignocelluloseinthegut,thedungofherbivorescan beconsideredasanaturalsourceofpre-treatedlignocellulose.Atotalof101fungiwere isolated(36yeastand65mouldisolates).Sixyeastisolatesproducedethanolduringgrowth onxylosewhilethreewereabletogrowat42◦C.Thisisadesirablegrowthtemperature asitisclosertothatwhichisusedduringthecellulosehydrolysisprocess.Fromtheyeast isolates,sixisolateswereabletotolerate2g/Laceticacidandonetolerated2g/Lfurfuralin thegrowthmedia.Theseinhibitorsarenormallygeneratedduringthepre-treatmentstep. Whengrownonpre-treatedthatchgrass,Aspergillusspeciesweredominantinsecretionof endo-glucanase,xylanaseandmannanase.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Plant biomass represents the largest source of renewable energyinnature.Thesearchforrenewablesourcesofenergy requiresaglobaleffortinordertoreducetheharmful con-sequences of global warming and to meet future energy

∗ _{Correspondingauthor.}

E-mail:dclagrange@gmail.com(D.C.LaGrange).

demands.1_{SecondGenerationbiofuelsareemergingasanew}

source ofenergythat is produced from biomass. The pro-duction ofbiofuels through advanced process technologies couldaidinreducinggreenhousegasemissions.These tech-nologieswouldalsoallowtheproductionofrenewablefuels without negatively impacting directly or indirectlyon food production.2

http://dx.doi.org/10.1016/j.bjm.2016.11.013

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Plant biomass is composed of lignocellulose, which generally consists of up to 45% cellulose, 30% hemicel-luloses and 25% lignin.3 _{Cellulose and hemicelluloses are}

polysaccharides, while lignin is an aromatic heteropoly-mer binding the two polysaccharides together. Lignocel-lulosic biomass includes agricultural residues such as corn stover, straw, sugarcane bagasse, herbaceous energy crops, wood residues (sawmill and paper mill discards), and municipal waste.4 _{These materials could serve as}

a cheap, abundant and renewable energy feedstock that is essential to the functioning of industrial communities and critical to the development of a sustainable global economy.5

The products of cellulose and hemicellulose hydrolysis are substratesfor fermentationintheproductionof biofu-els/bioethanol.Hydrolysisoflignocellulosicbiomassisaslow process due to the resistant crystalline structure of cellu-loseandthephysicalbarrierofligninsurroundingcellulose, thus limiting the sites for enzymatic attack.6 _Novel

orga-nismsareneededtoprovideimprovedenzymesandreduce thecostofconvertinglignocellulosicmaterialtofermentable sugars.Alsorequiredareorganismsthatareabletoferment pentosesugarseffectively.7 _{Theseorganismsshouldalsobe}

abletoresistinhibitorsreleasedduringthepretreatmentof lignocellulose.8

Itisestimatedthatupto70%cellulosepresent in natu-ralfeed isexcreted byherbivoresmaking theirdungarich sourceof“pre-treated”lignocellulolyticmaterialforthe iso-lationoflignocellulosicorganisms.9 _{Coprophilousfungiare}

dung-lovingandencodemanyenzymesneededforthe hydrol-ysisofcellulose.10 _{Anumberofstudies reporttheisolation}

ofcellulaseproducingfungifromthedungofdomestic ani-mals,however,fewstudieshavebeendone onthedungof wildherbivores.Onestudyreporttheisolationofsixdifferent xylosefermentingyeasts speciesfromthe dungofa num-berofherbivoresinThailand.11_{Otherstudiesfocusedonthe}

breakdownofelephantdung,12 _{theproductionofhydrogen}

usingthermophilicanaerobesinelephantdung,13_the

isola-tion ofcellulolyticfungifrom the dungofelephants,14 _the

antimicrobialcompoundsproducedbyfungifromthedung ofelephant,tigerandrhinoceros15_{andlastlytheisolationof}

a␤-glucosidasefromafungusassociatedwiththerumenof buffalo.16

Yeastscurrentlyconsideredforthefermentationof pen-toses, especially xylose, are mainly Scheffersomyces stipitis, Kluyveromyces marxianus, Candida shehatae and Pachysolen tannophilus.Ethanolproductionbytheseyeastsutilizingxylose ascarbonsourceisgenerallyfivetimeslowerwhencompared toSaccharomycescerevisiaefermentingglucose.17_Otherfactors

toconsiderinsearchingforanidealxylosefermenterare resis-tancetoinhibitors, suchasfurfural andacetic acid,ability tocarryoutfermentationatlowpHandhightemperatures conditions.18

The aim of this study was to isolate xylose uti-lizing yeasts and cellulolytic moulds from decomposed dung of various herbivore species found in the Kruger National Park, South Africa. Yeast isolates were evalu-atedfortheirxylosefermentationcapabilities,whilemould isolates were screened for cellulolytic enzyme produc-tion.

Material

and

methods

Samplecollection

Fiftydecomposeddungsamples,fromwildherbivores,were collectedfromtheKrugerNationalPark,SouthAfrica.Forty dungsampleswerecollectednearthePhalaborwarestcamp and 10 samples were collected from the proximity of the Skukuzarestcamp.Anexperiencedgamerangeraidedwith theidentificationofthesourcesofthedungsamples.All sam-ples were collected into plastic bags and processedwithin 48h.

Isolationoffungi

Approximately1gofthedungsamplesweresprinkleddirectly on agar plates containing 10g/L xylose, 10g/L beechwood xylan,10g/Lavicelcelluloseor10g/Llocustbeangum (man-nan),asasolecarbonsource,6.7g/LYNB(yeastnitrogenbase, Difco),15g/Lbacteriologicalagar and0.2g/L chlorampheni-coltoinhibitbacterialgrowth.Thefungalisolates(yeastsand moulds) were purifiedthroughrepeated streakingon fresh YM(10g/Lglucose,3g/Lmaltextract,3g/Lyeastextract,5g/L peptoneand15g/Lbacteriologicalagar)platesandpure cul-tureswerestoredonYMagarslants.

Fermentationofxylosebyyeastisolates

Fermentationmedia(20g/Lxylose,10g/Lyeastextract,2g/L KH2PO4,10g/LNH4SO4,2g/LMgSO4·7H2Oand0.2g/L chloram-phenicol)ina250mlErlenmeyerflaskseachcontaining25ml ofmediawasinoculatedwithayeastisolateandincubatedat 30◦Cand150rpmfor24–120h.Theabovementionedculture wasusedtoinoculate3×100mlofthesamemediain500ml ErlenmeyerflaskstoanOD600nmof0.2andincubatedat30◦C and150rpmfor96h.Samplesof2mlweretakenevery24h. Allthesampleswerecentrifugedfor5minat2000xgand4◦C afterwhichthesupernatantswerefilteredthrougha0.22␮m syringefilterandstoredat−20◦Cuntilanalysis.

Tolerancetoinhibitorsandelevatedtemperatures

Xylosefermentingyeastisolateswerefurthertestedfortheir abilitytogrowinthepresenceof1,2,3,5,7,and10g/Lacetic acidand1,2,3and4g/LfurfuralinYMagarplates.Allplates wereincubatedat30◦Cfor48h.Themaximumgrowth tem-peraturesforalltheyeastisolatesweredeterminedusingYM slants.Theslantswereincubatedat35,37,40,42,and45◦C. Themaximumtemperatureforgrowthisconsideredthe high-esttemperaturewheregrowthoccurred.

Productionofenzymebymouldisolatesonthatchgrass basedmedium

Mould isolateswere screenedfor endoglucanase, xylanase and mannanase activity in liquid media containing 20g/L pre-treatedthatchgrass(Hyparrheniasp),4g/LKH2PO4,10g/L (NH4)HPO4, 10g/L peptone, 3g/L yeast extract and 0.1g/L chloroamphenicolin100mldeionizedwater.Pre-treatmentof

thatchgrasswasperformedbygrindingairdriedgrasstoafine powder.Diluteacidpre-treatmentusing1.2%sulphuricacid wasperformedat120◦Cfor60minatabiomass concentra-tionof10%(w/v).Afterpre-treatment,5Msodiumhydroxide (NaOH)wasusedtoadjustthepHofthethatchgrass suspen-sionto6.Erlenmeyerflasks(250ml)containing50mlofthe thatchgrassbasedmediawereinoculatedwithten4mmplugs ofagarfromafreshlyculturedplate.Allisolateswere inocu-latedinduplicateandincubatedinarotaryshakerfor5days at30◦Cand150rpm.Samplesweretakenat24hintervals.

Highperformanceliquidchromatography(HPLC)analysis

Xylose and xylitol levels were determined by HPLC using aShimadzu Prominence 20 HPLCsystem. Samplesof20␮l were injected into a Rezex RHM-monosaccharide H+ col-umn (300×7.8mm) and eluted using waterat a flow rate of0.6ml/min. The column temperature was kept at85◦C. TheseparatedcomponentsweredetectedusingaShimadzu RID10Arefractiveindexdetector.Data wasprocessedusing LC Solutions software. Sugars were identified and quan-tified by comparing with known standards of xylose and xylitol.

Gaschromatography(GC)analysis

Ethanolwasanalyzedbycapillarygaschromatographyusing a Shimadzu GC2010Plus gas chromatograph on a ZB-WAX pluscolumn(30m×0.25mmID×0.25␮m).Nitrogenwasused asthe carriergasata flowrate of17.6ml/min. Theinitial columntemperaturewas 40◦C.Afterinjectionof1␮l sam-ple,the oventemperature wasraisedto140◦C ata rateof 20◦C/min after which it was further raised to 200◦C at a rate of 50◦C/min and held at this temperature for 2min. Ethanolwasdetectedusingaflameionizationdetector(FID)at 255◦C.ThepeakdatawasprocessedusingGCSolutions soft-wareandethanolconcentrationwascalculatedusingethanol standards.

Enzymeactivityassays

Xylanaseactivitywasdeterminedbymixing45␮lof1%(w/v) birchwoodxylan in50mM citratebuffer pH5 with 5␮l of theculturesupernatant(enzyme).Theenzyme-substrate mix-turewasincubatedat50◦Cfor5min.Thereleasedreducing sugarsweredeterminedusingamodified3,5-dinitrosalicylic acid(DNS)methodwithxylosebeing usedasastandard.19

The amount of xylanase activity was then expressed in nkat/ml.20

Mannanaseactivity wasassayed using0.5%(w/v)locust beangum in50mMcitratebufferpH5.Thissubstratewas prepared by homogenizing the gum suspension at 80◦C and then heating until the mixture boiled. The mixture was cooled to room temperature and left overnight with continuous stirring. The insoluble material was removed by centrifugation at 4000×g for 5min.21 _{The assay}

mix-ture contained 45␮l of substrate solution and 5␮l of enzyme solution. The enzyme–substrate mixture was incubated at 50◦C for 10min. Released reducing sugars

were determined by the DNS method using mannose as standards.

Endoglucanaseactivitywasdeterminedbymixing25␮lof 1%carboxymethylcellulose(CMC)in50mMcitratebufferpH5 with25␮loftheenzymesolution.Theenzyme–substrate mix-turewasincubatedat50◦Cfor30min.Thereleasedreducing sugarsweredeterminedbytheDNSmethodusingglucoseas standards.

Allenzymeactivitieswereexpressedinkatalsper millil-itre(nkat/ml),where1katalistheamountofenzymeneeded to produce1molofreducing sugar from the substrate per second.

ITSandD1/D2sequencing

Allfungalisolatesweresub-culturedonYMagarat30◦C.The cultureplatesweresenttoInqabaBiotechnicalIndustries(Pty) Ltd,SouthAfricaforITSandD1/D2DNAsequencing.DNAwas extractedusingtheZRFungal/BacterialDNAMiniPrepTMKit (ZymoResearch)accordingtothemanufacturer’sinstructions. TheITS1-5.8S-ITS2regionwasamplifiedusingPCRprimers ITS-1(5-TCCGTAGGTGAACCTGCGG-3)andITS-4(5-TCC TCCGCTTATTGATATGC-3).22_{Amplificationwascarriedout}

in25␮lreactionsusingtheEconoTaqPlusGreenMasterMix (Lucingen).ThefollowingPCRconditionswereused:35cycles including an initialdenaturationat 95◦C for2min. Subse-quentdenaturationwasat95◦Cfor30s, annealingat50◦C for30sandextensionat72◦Cfor1min.Afinalextensionat 72◦Cfor10minwasfollowedbyholdingat4◦C.Additionally, theD1/D2domainofthe26SrDNAregionwasamplifiedusing primersNL1(5‘-GCATATCAATAAGCGGAGGAAAAG-3)and NL4(5-GGTCCGTGTTTCAAGACGG-3)asdescribedabove. DNAsequencingwasdoneusingABIV3.1Bigdyeaccording to manufacturer’s instructions on the ABI 3500XL Instru-ment.

Species were identified by searching databases using BLAST(http://www.ncbi.nlm.nih.gov/BLAST/).

Results

In thisstudy, 101fungal strains, withpotentialapplication in thebioethanol industry,were isolatedfrom the dungof various wild herbivores (buffalo, dassie, elephant, impala, klipspringer, kudu, rhino, wildebeest and zebra) from the KrugerNationalPark,SouthAfrica.Cellulolyticmouldswere selectedbasedontheirabilitytoutilizeplantpolysaccharides (cellulose,xylan,ormannan),whileyeastswereselectedfor theirabilitytoutilizexyloseasthesolecarbonsource.This stringentselectionusedinthestudylimitedthenumberof fungiisolated.

Yeastsfromherbivoredung

Thirty six yeasts were isolated from 50 dung samples

(Table1).Fastgrowingyeastswereselectedbypickingcolonies

appearingafter24hofincubationat30◦C.Cryptococcus lau-rentii(19strains)wasthemostdominantyeastinthedung samples followed by Candida tropicalis (7 strains), Candida xylanilytica (2 strains), Trichosporon scarabaeorum (2 strains),

Table1–Yeastisolatesobtainedfromthedungofvariouswildherbivores.Isolateswereevaluatedfortheirabilityto

growatelevatedtemperatures,inthepresenceofaceticacidandfurfuralandfortheirabilitytofermentxylose.Ethanol

andxylitoldataarereportedattheendoffermentationwhenxylosewasdepletedornotconsumedfurther.Dataare

presentedasthemean±standarddeviation(SD)of3repeats.

Dung source

Yeastname Temperature

(◦C) Aceticacid (g/L) Furfural (g/L) Ethanol (g/L) Xylitol (g/L) Fermentation time(h) Buffalo

CandidaxylanilyticaKP6my 40 1 1 – 1.6±0.06 48

CandidaxylanilyticaKP6.2ey 42 1 0 1.5±0.15 – 48

CryptococcuslaurentiiKP23ey 40 1 1 – – 48

TrichosporonjiroveciiKP6.1ey 40 1 0 – – 48

Dassie PichiakudriavzeviiKP34ey 35 3 1 2.3±0.03 nd 48

Elephant

CandidatropicalisKP21ey 40 2 1 – 11.6±0.69 48

CryptococcuslaurentiiKP2.2ay 40 1 1 – 8.4±0.36 48

CandidatropicalisKP2.1ay 40 1 1 – 1.0±0.12 96

CryptococcuslaurentiiKP40ey 37 0 0 – 12.8±0.3 96

CryptococcuslaurentiiKP39ey 40 1 1 – 1.2±0.11 72

CryptococcuslaurentiiKP36ey 40 1 1 – 0.9±0.17 96

CryptococcuslaurentiiKp28ey 40 0 0 – 0 120

CryptococcuslaurentiiKP25ey 40 1 0 – 0 96

CryptococcuslaurentiiKP2.1ey 35 1 0 – 6.5±1.2 48

CryptococcuslaurentiiKp16ey 40 1 1 – 0 96

OgataeamethanolicaKP2ey 35 1 1 1.4±0.09 2.0±0.23 72

Impala

Aureobasidiumsp.KP22ey 40 0 0 – 1.7±0.02 96

CryptococcuslaurentiiKp4ey 40 0 1 – 0.9±0.05 96

CryptococcuslaurentiiKP45ey 40 1 1 – 0 48

CryptococcuslaurentiiKP3ey 40 1 1 – 1.7±0.05 96

CryptococcuslaurentiiKP31ey 30 0 0 – 0 72

CryptococcuslaurentiiKP29ey 42 1 0 – 1.2±0.04 72

CryptococcuslaurentiiKP26ey 40 2 2 – 0 96

Klipspringer CryptococcuslaurentiiKP35ey 40 0 0 – 0 96

Kudu CryptococcuslaurentiiKP20ey 40 1 1 – 0 72

Rhino

CandidatropicalisKP47ey 40 2 0 – 13.7±0.5 96

CandidatropicalisKP43ey 42 2 1 1.4±0.03 4.3±0.63 72

CandidatropicalisKP42ey 40 2 1 1.2±0.08 12.8±0.3 72

TrichosporonscarabaeorumKP42.2ny 37 0 0 – 0.9±0.04 48

TrichosporonscarabaeorumKP42.1ny 37 0 0 – 8.8±0.14 96

Wildbees CandidatropicalisKP46ey 30 0 0 1.9±0.19 9.1±0.29 48

Zebra

CandidaalbicansKP48ey 40 0 0 – 1.0±0.12 96

CryptococcuslaurentiiKP9ey 40 1 1 – 2.0±0.23 96

CryptococcuslaurentiiKP48ny 37 0 0 – 7.9±0.06 48

CryptococcuslaurentiiKp32ey 40 0 1 – – 120

CryptococcuslaurentiiKP18ey 37 1 1 – 0.9±0.08 120

Nd–notdetermined.

Aureobasidiumsp.(1strain),Pichiakudriavzevii(1strain),Ogataea methanolica.(1strain)andTrichosporonjirovecii(1strain).Itis knownthatC.laurentiiproducemycocinsinsoilwhichcould explaintheabundanceoftheseorganismscomparedtoother species.23

Sixteenpercentofyeastisolatesinthisstudy,C.tropicalis KP42ey,KP43eyandKP46ey,C.xylanilyticaKP6.2ey,P. kudravze-viiKP34ey andO.methanolitica KP2ey, wereable toferment xylosetoethanol.Ethanolconcentrationsrangedbetween1.2

and2.3g/L(Table1).P.KudriavzeviiKP34eyproducedthe

high-estethanolconcentration(2.3±0.03g/L).

C.xylanilyticaKP6.2eyandC.tropicalisKP43eywereableto growat40and42◦C,respectively.P.kudriavzeviiKP34eywas abletogrowat35◦Candtoleratedinhibitorssuchasacetic acid(3g/L)andfurfural(1g/L).

Twentytwoyeastsproducedsignificantamountsofxylitol duringgrowth onxylose (Table1).Thehighestxylitol con-centrationobservedwas13.7±0.5g/L,producedbyC.tropicalis KP47ey.

Mouldsfromherbivoredung

A total of 65 mould isolates belonging to 16 genera, were purifiedandidentifiedfrommediacontainingplant polysac-charides (cellulose, xylan and mannan) (Fig. 1). Species belongingtothegenusAspergillusweredominantamongthe isolates (58%).The mostabundantAspergillus specieswere Aspergillus flavus (11strains), followed by Aspergillus terreus (8 strains), Aspergillusniger (4 strains), Aspergillusnomius (3 strains),Aspergillustubigensis(3strains),Aspergillusbrasiliensis (2strains),Aspergillusoryzae(2strains)andAspergilluslentulus (1strain).FourAspergillusisolatescould notbeidentifiedto specieslevel.

Trichoderma wasless prevalent,withonly6%(4isolates) ofthefungiisolatedfrompolysaccharidecontainingmedia belonging to the genus Trichoderma. Of the 4 isolates two were identified as Trichoderma konilangbra and Trichoderma harizianum,respectively.TheremainingtwoTrichoderma iso-lateswereonlyidentifiedtogenuslevel.

Fungus name

Source CMCase (h) Mannanase (h) Xylanase (h)

Buffalo Aspergillus flavus KP23.1nm 72 72 48

Buffalo Candida tropicalis KP23nm 120 168 120

Buffalo Neosartorya sp. KP5mm 48 120 120

Buffalo Sordaria sp. KP11mm 72 72 120

Buffalo Trichoderma konilangbra KP12am 120 72 120

Dassie Candida tropicalis KP34nm 120 72 120

Elephant Actinomucor elegans KP2nm 72 48 48

Elephant Aspergillus lentulus KP16am 72 72 48

Elephant Aspergillus brasiliensis KP16nm 48 72 120

Elephant Aspergillus brasiliensis KP39nm 72 72 72

Elephant Aspergillus flavus KP21mm 72 120 120

Elephant Aspergillus flavus KP33.2am 120 120 120

Elephant Aspergillus flavus KP33nm 72 24 48

Elephant Aspergillus flavus KP37.1am 72 72 48

Elephant Aspergillus niger KP2am 48 72 72

Elephant Aspergillus sp. KP36nm 72 48 168

Elephant Aspergillus terreus KP30.2nm 120 120 120

Elephant Aspergillus terreus KP30am 72 72 72

Elephant Candida sp. KP25am 120 120 72

Elephant Fusarium sp. KP28am 72 48 120

Elephant Mucor sp. KP21am 72 72 72

Elephant Penicillium sp. KP30.1nm 72 72 168

Elephant Phoma sp. KP37nm 120 120 72

Impala Aspergillus flavus KP28.1nm 120 120 120

Impala Aspergillus flavus KP29.1am 120 72 72

Impala Aspergillus flavus KP4nm 72 48 48

Impala Aspergillus niger KP27.1am 120 120 120

Impala Aspergillus niger KP27.2am 168 120 48

Impala Aspergillus niger KP31am 72 72 120

Impala Aspergillus nomius KP4mm 72 72 120

Impala Aspergillus oryzae KP3nm 72 48 48

Impala Aspergillus sp. KP29mm 48 72 48

Impala Aspergillus terreus KP1.1nm 72 120 120

Impala Aspergillus terreus KP17.1nm 72 48 168

Impala Aspergillus terreus KP17.2nm 72 48 168

Impala Aspergillus terreus KP17.2nm 168 168 168

Impala Aspergillus terreus KP31am 72 72 120

Impala Cryptococcus laurentii KP26mm 120 48 120

Impala Mucor sp. KP1.2nm 72 120 120

Impala Trichoderma sp. KP38.2nm 120 48 168

Impala Aspergillus flavus KP4nm 72 48 48

Klipspringer Aspergillus sp. KP35nm 72 48 72

Klipspringer Candida tropicalis KP35.2nm 72 72 120

Klipspringer Neosartorya fischeri KP35.1am 72 120 168

Kudu Aspergillus terreus KP20nm 72 72 120

Rhino Apiosordaria nigeriensis KP47mm 120 48 48

Rhino Aspergillus flavus KP42mm 72 72 72

Rhino Aspergillus flavus KP43.2nm 72 72 72

Rhino Aspergillus oryzae KP43.1nm 72 48 120

Rhino Aspergillus tubingensis KP47nm 72 120 48

Steenbok Phialemonium sp. KP14am 72 72 48

Termite hill Aspergillus nomius KP38.2mm 72 72 72

Termite hill Aspergillus nomius KP38mm 48 48 48

Termite hill Candida tropicalis KP38.2nm 48 48 120

Termite hill Chaetomium sp. KP38nm 72 168 168

Termite hill Mucor sp. KP38am 168 72 72

Wildebees Aspergillus tubingensis KP46nm 120 48 72

Zebra Aspergillus sp. KP32nm 72 120 72

Zebra Aspergillus tubingensis KP9.1am 72 72 120

Zebra Chaetomium brasiliense KP10nm 120 72 72

Zebra Meyerozyma sp. KP32am 72 120 120

Zebra Rhizopus microsporus KP7nm 72 48 168

Zebra Sordaria sp. KP13mm 48 48 120

Zebra Trichoderma harzianum KP10am 120 48 120

Zebra Trichoderma aasp. KP13am 168 120 168

nkat/ml nkat/ml nkat/ml 8 6 4 2 0 0 5 10 15 0 100 200 300

Fig.1–Enzymeproductionbymouldisolatesduringgrowthon2%pre-treatedthatchgrass.Hoursindicatedrepresentthe

incubationtimeatwhichmaximumenzymeactivitywasreached.Errorbarsindicatestandarddeviationfortwo

Thatchgrasscontainscellulose,hemicelluloseandlignin. Drygrassharvested atthe endofthegrowing season con-tainsapproximatelytwiceasmuchligninasthelivegrass.24

Thismakesthethatchgrassusedinthisstudyavery resis-tantcarbonsourcethatdoesnotsupportgrowthtohighcell densities.Therefore,enzymeactivitiesobtainedherearelow comparedtopublisheddata(Fig.1).Thehighestglucanase activitieswere produced byAspergillus (KP30am, KP29mm), Trichoderma(KP13am)andNeosartorya(KP5mm)spp,whilethe bestmannanaseproducerswereAspergillusspecies(KP39nm, KP33.2am, KP42mm) (Fig. 1). The best xylanase producers belong to the genera Neosartorya (KP5mm, KP35.1am) and Aspergillus(KP35nm,KP47mm).

Discussion

Ethanolisanimportantrenewablefuelthatcanreplace fos-sil transportation fuel. The use of plant biomass for the productionofbioethanolrequires theconversion ofsugars in lignocellulose, mainly glucose and xylose into ethanol. Unfortunately, most hexose fermenting organisms, includ-ingSaccharomycescerevisiae,cannotfermentxylose.Depending ontheorigin,xylosecan constituteasmuchas30% ofthe fermentablesugars,inbiomass.Itisthereforeimportantto fermentbothglucoseand xyloseforefficientconversionof biomasstoethanol.

From the 36 yeasts isolated in the study, 6 (16%) were abletoproduceethanolduringgrowthonxylose.Similarly, Moraisetal.25 _{isolated69 yeastsfrom rottenwoodwith14}

(20%)abletofermentxylose.Raoetal.26_{isolated374yeasts,}

fromrotten fruitandthe barkoftreesand only27(7%) of theseyeastswereabletofermentxylose.Lorliametal.11

iso-lated 39 yeasts from the dung ofvarious herbivoresusing xylose ascarbon source. Thirty seven(95%) ofthese yeast isolateswereabletofermentxylosetoethanol.Nineteenof theseyeastswereidentifiedasC.tropicalis.Inanotherstudy byLorliametal.2728xyloseutilizingyeastswereisolatedfrom thedungofbuffalo.Elevenoftheseisolateswereidentified asC.tropicalis.

P. kudriavzevii KP34ey, isolated from the dung of dassie produced the highest ethanol concentration on xylose (2.3±0.5g/L).ThisresultcompareswellwiththatofLorliam et al.11 _{where most yeasts (C. tropicalis) produced}

approxi-mately0.6g/LethanolwithZygoascusmeyeraeproducingthe highestethanolconcentration(3.6g/L).

Enzymatichydrolysis(saccharification) ofplant polysac-charidestypicallyoccursataround50◦C,thereforetheability ofayeasttofermentatelevatedtemperaturescould poten-tiallyleadtoreducedenergyinputcost.Thisisduetothefact thatmosthydrolyticenzymesfunctionoptimallyat approxi-mately60◦C,whilefermentationontheotherhandistypically done at30–35◦C. Hence,this requires coolingofthe sugar mixbeforeinoculation withthe fermentingorganism. Fur-ther, after fermentation the broth needs to be heated for recoveryoftheethanolthroughdistillation.Thus,ifthe fer-mentationtemperaturecould beincreasedthroughtheuse of a thermophilic organism, a significant saving in terms of energy as well as enzyme cost would be achieved. A number of yeast isolates in this study were able to grow

attemperaturesabove40◦C.P. kudriavzevii isknown forits ability to ferment at elevated temperatures.28 _{Similarly, in}

this study P. kudriavzevii KP34ey was able to grow well at 35◦C.

Furfuralandaceticacidarecommonlyproducedduringthe pre-treatmentofhemicellulosesand haveanegativeeffect on thesubsequent fermentation.29 _{Itispossibletoremove}

theseandotherinhibitorsbeforeinoculatingthefermenting organisms,howeverthisaddstothecostofethanol produc-tion.Ideally,thefermentingorganismsshouldbeabletoresist inhibitorsatlevelscommonlyfoundinpre-treatedmaterial.P. kudriavzeviiKP34eyisolatedfromthedungofdassiewasable togrowinthepresenceof3g/Laceticacidand1g/Lfurfural. This meansthat P. kudriavzevii KP34eyis agood candidate forfurtherstudy,sinceitisagoodethanolproducer,itcan tolerateelevatedtemperaturesand growinthepresenceof inhibitors.

Anumber ofyeastsisolated inthisstudy produced sig-nificant amount of xylitol during growth on 20g/L xylose. Similarly,Lorliametal.27_{reportedthatC.tropicalisproduced}

22.8g/Lofxylitolduringgrowthon6%xylose.Thepresence ofhigh concentrationsofxylitolcould beexplainedbythe redoximbalancethatoccursduringtheconversionofxyloseto xylulose,especiallyunderoxygenlimitingconditions.Xylose is reduced to xylitol by xylose reductase using NADPH as cofactor.Xylitolshouldthenbeoxidizedtoxylulosebyxylitol dehydrogenaseusingNAD+_{asco-factor.Oxidative} phosphor-ylationisnecessarytoconvertNADHtoNAD+_{.Therefore,an} accumulationofNADHleadstotheaccumulationofxylitol underoxygenlimitingconditions.30,31

Therearemanymicroorganismsincludingbacteria,fungi and actinomycetes able to degrade cellulose. Fungi are generally considered to be the main group of cellulolytic organisms.32,33_{Themajority(58%)ofmouldsisolatedinthis}

studybelongstothegenusAspergillus.Thisisnotsurprising consideringthenumberofreportsonAspergillusspecies pro-ducinglignocellulolyticenzymes.34–37_{Aspergillusisagenusof}

filamentousfungithatcontainsalargenumberofspecies.It iswellknownforitsabilitytoproduceplantpolysaccharide degradingenzymes.37–39_{Aspergilllihavebeenreportedto}

pro-ducethefourclassesofenzymesinvolvedinthehydrolysisof cellulose,namelyendoglucanases(EC3.2.1.4),exoglucanases, includingcellodextrinases(EC3.2.1.74)andcellobiohydrolases (EC 3.2.1.91)and ␤-glucosidases (EC 3.2.1.21).33,40 _The

envi-ronmental samples screened by Ja’afaru35 _{yielded 43 and}

41%TrichodermaandAspergillusisolates,respectively.In con-trast, the fungi identified from the tropical soil samples screenedbyReanprayoonandPathomsiriwong34_yieldedonly

10%Aspergillusand3%Trichoderma.

The cellulolytic systems of the mesophilic fungus, Tri-choderma reesei is one of the most thoroughly studied.33 Trichoderma species are common soil inhabiting fungiwith highlyefficientcellulolyticsystems.Numerousstrainsof Tri-chodermareeseihasbeenmutatedtoincreasetheproduction ofextracellularcellulases.41

Lignified thatchgrass isa difficultsource ofcarbon for microorganismstouse,howeveritenabledtheidentification ofpromisinglignocellulolase producing fungiinthisstudy. Thebest endo-glucanase,mannanase andxylanase strains wereAspergillussp.KP29mm,AspergillusbrasiliensisKP39nm

andAspergillussp.KP35mm.Theseorganismswillbe investi-gatedfurtheraspotentialenzymeproducersforthehydrolysis ofthatchgrass.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest

Acknowledgements

ThisresearchwasfinanciallysupportedbytheUniversityof LimpopoResearchOffice,theofficeoftheExecutiveDeanof thefacultyofScienceandAgriculture,theNationalResearch Foundation of South Africa for Grant No. 91503 and the FlemishInteruniversityCouncil(VLIR-UOS).WethankSouth African National Parks for providing access to the Kruger NationalParkforthecollectionofdungsamples.

Anyopinion,findingandconclusionorrecommendation expressedinthismaterialisthatoftheauthor(s)andtheNRF doesnotacceptanyliabilityinthisregard.

r

e

f

e

r

e

n

c

e

s

1. LyndLR,VanZylWH,McBrideJE,LaserM.Consolidated

bioprocessingofcellulosicbiomass:Anupdate.CurrOpin

Biotechnol.2005;16:577–583.

2. BansalN,TewariR,GuptaJK,SoniR,SoniSK.Anovelstrain

ofAspergillusnigerproducingacocktailofhydrolytic

depolymerisingenzymesfortheproductionofsecond

generationbiofuels.BioResources.2011;6(1):552–569.

3. ZaldivarJ,NielsenJ,OlssonL.Fuelethanolproductionfrom

lignocellulose:achallengeformetabolicengineeringand

processintegration.ApplMicrobiolBiotechnol.2001;56:17–34.

4. TaherzadehMJ,KarimiK.Pretreatmentoflignocellulosic

wastestoimproveethanolandbiogasproduction:areview.

IntJMolSci.2008;9:1621–1651.

5. PetrovaP,IvanovaV.Perspectivesfortheproductionof

bioethanolfromlignocellulosicmaterials.Biotechnol

BiotechnolEquip.2010;24:529–546.

6. PrasadMP,SethiR.Optimizationofcellulaseproduction

fromanovelbacterialisolateMesorhizobiumsp.frommarine

source.JEnzymRes.2013;4(1):39–45.

7. GirioFM,FonsecaC,CarvalheiroF,DaurteLC,MarquesS,

Bogel-LukasikR.Hemicellulosesforfuelethanol:areview.

BioresourTechnol.2010;101:4775–4800.

8. OlssonL,Hahn-HägerdalB.Fermentationoflignocellulosic

hydrolysatesforethanolproduction.EnzymeMicrobTechnol.

1996;18(5):312–331.

9. WahyudiA,CahyantoMN,SoejonoM,BachruddinZ.Potency

oflignocellulosedegradingbacteriaisolatedfrombuffalo

andhorsegastrointestinaltractandelephantdungforfeed

fiberdegradation.JIndonesianTropAnimAgric.

2010;35(1):34–41.

10.JahangeerS,KhanN,JahangeerS,etal.Screeningand

characterizationoffungalcellulasesisolatedfromthenative

environmentalsource.PakJBot.2005;37(3):739–748.

11.LorliamW,AkaracharanyaA,JindamorakotS,

SuwannarangseeS,TanasupawatS.Characterizationof

xylose-utilizingyeastsisolatedfromherbivorefaecesin

Thailand.ScienceAsia.2013;39:26–35.

12.MasungaGS,AndresenO,TaylorJE,DhillionSS.Elephant

dungdecompositionandcoprophilousfungiintwohabitats

ofsemi-aridBotswana.MycolRes.2006;110(10):1214–1226.

13.FangkumA,ReungsangA.Biohydrogenproductionfrom

mixedxylose/arabinoseatthermophilictemperatureby

anaerobicmixedculturesinelephantdung.IntJHydrogen

Energy.2011;36(21):13928–13938.

14.FarouqAA,AbdullahDK,Hooi-LingF,AbdullahN.Isolation

andcharacterizationofCoprophilouscellulolyticfungifrom

Asianelephant(Elephasmaximus)dung.JBiolAgrHealthc.

2012;2(7):44–51.

15.GaneshKumarC,MongollaP,JosephJ,NageswarYVD,Kamal

A.Antimicrobialactivityfromtheextractsoffungalisolates

ofsoilanddungsamplesfromKazirangaNationalPark,

Assam,India.JMycolMed.2010;20(4):283–289.

16.ChenHL,ChenYC,LuMY,etal.Ahighlyefficient

beta-glucosidasefromabuffalorumenfungusNeocallimastix

patriciarumW5.BiotechnolBiofuels.2012;5(24):1–24.

17.SarkarN,GhoshSK,BannerjeeS,AikatK.Bioethanol

productionfromagriculturalwastes:Anoverview.Renew

Energ.2012;37(1):19–27.

18.KuhadRC,GuptaR,PalKhasaYP,SinghA,ZhangY-HP.

Bioethanolproductionfrompentosesugars:Currentstatus

andfutureprospects.RenewSustainEnergyRev.

2011;15(9):4950–4962.

19.IlmenM,DenHaanR,BrevnovaE,etal.Highlevelsecretion

ofcellobiohydrolasesbySaccharomycescerevisiae.Biotechnol

Biofuels.2011;4:30.

20.BaileyMJ,BielyP,PoutanenK.Interlaboratorytestingof

methodsforassayofxylanaseactivity.JBiotechnol.

1992;23:257–270.

21.MudauMM,SetatiME.Partialpurificationand

characterizationofendo-␤-1,4-mannanasesfrom

Scopulariopsiscandidastrainsisolatedfromsolarsalterns.Afr JBiotechnol.2015;7(13):2279–2285.

22.ChanprasartsukO,PrakitchaiwattanaC,SanguandeekulR.

Comparisonofmethodsforidentificationofyeastsisolated

duringspontaneousfermentationoffreshlycrushed

pineapplejuices.JAgrSciTech.2013;15:

1479–1490.

23.BothaA.Theimportanceandecologyofyeastsinsoil.Soil

BiolBiochem.2011;43(1):1–8.

24.MartinSB,DaleJL.Biodegradtionofturfthatchwith

wood-decayfungi.Phytopathology.1979;70:297–301.

25.MoraisCG,CadeteRM,UetanabaroAP,RosaLH,Lachance

MA,RosaCA.D-xylose-fermentingandxylanase-producing

yeastspeciesfromrottingwoodoftwoAtlanticRainforest

habitatsinBrazil.FungalGenetBiol.2013;60(0):19–28.

26.RaoRS,BhadraB,ShivajiS.Isolationandcharacterizationof

ethanol-producingyeastsfromfruitsandtreebarks.Lett

ApplMicrobiol.2008;47(1):19–24.

27.LorliamW,AkaracharanyaA,SuzukiM,OhkumaM,

TanasupawatS.Diversityandfermentationproductsof

xylose-utilizingyeastsisolatedfrombuffalofecesin

Thailand.MicrobesEnviron.2013;28(3):

354–360.

28.HasunumaT,KondoA.Consolidatedbioprocessingand

simultaneoussaccharificationandfermentationof

lignocellulosetoethanolwiththermotolerantyeaststrains.

ProcessBiochem.2012;47(9):1287–1294.

29.JonssonLJ,AlrikssonB,NilvebrantNO.Bioconversionof

lignocellulose:inhibitorsanddetoxification.Biotechnol

Biofuels.2013;6(1):16.

30.WinkelhausenE,KuzmanovaS.Microbialconversionof

d-xylosetoxylitol.JFermentBioeng.1998;86(1):

1–14.

31.deAlbuquerqueTL,daSilvaIJ,deMacedoGR,ochaMVP.

wastes:Areview.ProcessBiochem.2014;49(11): 1779–1789.

32.MandelsM,SternbergD.Recentadvancesincellulase

technology.FermentTechnol.1976;54:267–286.

33.SolimanSA,El-ZawahryYA,El-MougithAE.Fungal BiodegradationofAgro-IndustrialWaste.InTech;2015,26pp. Availablefrom:http://dx.doi.org/10.5772/56464.

34.ReanprayoonP,PathomsiriwongW.Tropicalsoilfungi

producingcellulaseandrelatedenzymesinbiodegradation.J

ApplSci.2012;12(18):1909–1916.

35.Ja’afaruMI.ScreeningofFungiIsolatedfromEnvironmental SamplesforXylanaseandCellulaseProduction.ISRNMicrobiol; 2013,7pp.Availablefrom:

http://dx.doi.org/10.1155/2013/283423.

36.ReddyPLN,BabuBS,RadhalahA,SreeramuluA.Screening,

identificationandisolationofcellulolyticfungifromsoilsof

Chittoordistrict,India.IntJCurrMcirobiolAppSci.

2014;3(7):761–771.

37.DeviMC,KumarMS.Isolationandscreeningof

lignocellulosehydrolyticsaprophyticfungifromdairy

manuresoil.AnnBiolRes.2012;3(2):1145–1152.

38.deVriesRP,VisserJ.Aspergillusenzymesinvolvedin

degradationofplantcellwallpolysaccharides.MicrobiolMol

BiolRev.2001;65(4):497–522.

39.deVriesRP,KesterHCM,PoulsenCH,BenenJAE,VisserJ.

SynergybetweenenzymesfromAspergillusinvolvedinthe

degradationofplantcellwallpolysaccharides.CarbohydrRes.

2000;327(4):401–410.

40.LaGrangeDC,DenHaanR,VanZylWH.Engineering

cellulolyticabilityintobioprocessingorganisms.Appl

MicrobiolBiotechnol.2010;87:1195–1208.

41.ShahbaziS,IsparehK,KarimiM,AskariH,EbrahimiM.

GammaandUVradiationinducedmutagenesisin

Trichodermareeseitoenhancecellulasesenzymeactivity.IntlJ FarmAlliedSci.2014;3(5):543–554.