.1.

Screening and Isolation of Bacteria

CellulaseproducingbacteriawereisolatedfromsoilsbythedilutionpourplateorspreadplatemethodusingCMC
agarmedia.Theplateswereincubatedat45,50,and55Cfor24hours.Tovisualizethehydrolysiszone,theplates
werefloodedwithanaqueoussolutionof0.1%Congoredfor15 minandwashedwith1 M NaCl[ 17].Toindicate
thecelluloseactivityoftheorganisms,diameteroftheclearzonearoundcoloniesonCMCagarwasmeasured.
Besides,amorequantitativeassaymethodwasusedtodeterminethecelluloseactivityoftheselectedbacterial
isolateinliquidmedium.Thecellulaseactivityofeachculturewasmeasuredbydeterminingtheamountofreducing
sugars liberated by using a DNS method [18]. A bacterial isolate with the highest activity was selected for
optimizationofcelluloseproduction.
2.2. Bacterial Identification
Thebacterialisolateswerepresumptivelyidentifiedbymeansofmorphologicalexaminationandsomebiochemical
characterizations.Theparametersinvestigatedincludedcolonialmorphology,gramreactions,endosporeformation,
catalaseproduction,VPreaction,indoleproduction,starchhydrolysis,citrateutilization,andgelatinehydrolysis.
TheresultswerecomparedwithBergeysManualofDeterminativeBacteria[19].
2.3. Enzyme Production Medium
Production medium contained (g/L) glucose 0.5gm, peptone 0.75gm, FeSO 40.01gm, KH2PO40.5gm, and
MgSO40.5gm. Ten millilitres of medium were taken ina 100mLconical flask. The flasks were sterilized in
autoclaveat121Cfor15min,andaftercooling,theflaskwasinoculatedwithovernightgrownbacterialculture.
Theinoculatedmediumwasincubatedat37Cinshakerincubatorfor24h.Attheendofthefermentationperiod,
theculturemediumwascentrifugedat5000rpmfor15mintoobtainthecrudeextract,whichservedasenzyme
source.
2.4. Enzyme Assay
CellulaseactivitywasmeasuredfollowingthemethodofMiller[18].Briefly,areactionmixturecomposedof0.2
mLofcrudeenzymesolutionplus1.8mLof0.5%carboxymethylcellulose(CMC)in50 mMsodiumphosphate
buffer(pH7)wasincubatedat37Cinashakingwaterbathfor30min.Thereactionwasterminatedbyadding3
mLofDNSreagent.Thecolourwasthendevelopedbyboilingthemixturefor5 min.ODofsampleswasmeasured
at575nmagainstablankcontainingallthereagentsminusthecrudeenzyme.
2.5. Process Optimization for Maximum Cellulase Production
2.5.1. pH
FlaskswithbrothcontainingtheoptimumconcentrationofsubstrateandcarbonsourcearetakenandthepHofthe
brothisadjustedto7.0,8.0,9.0,10.0,and11.0indifferentflasksusing1 N HCland1 N NaOHandsterilized.The
culturesareinoculatedandincubatedatparticulartemperature.Attheendofincubationperiod,thecellfreeculture
filtrateisobtainedandusedasenzymesource.
2.5.2. Temperature
ProductionmediumatpH7wasinoculatedwithovernightgrownselectedbacterialstrain.Thebrothwasincubated
atdifferenttemperaturesfrom35,40,45,50,55,and60Cfor24h.Attheendofincubationperiod,thecellfree
culturefiltrateisobtainedandusedasenzymesource.
2.5.3. Carbon Sources
Theeffectofvariouscarbonsourcessuchasstarch,glucose,maltose,lactose,andfructoseattheconcentrationof1
to5%wasexaminedintheproductionmedium.
2.5.4. Nitrogen Sources
Variousnitrogensourceslikeyeastextract,peptone,urea,andammoniumsulphatewereexaminedfortheireffect
onenzymeproductionbyreplacing0.5%peptoneintheproductionmedium.

2.5.5. Agro-Based Waste Material

Tofindoutthesuitabilityofagrobasedwasteassubstrateforenzymeproduction,different substrates,thatis,
groundnut cake, coconut cake, soy cake, and wheat bran, are taken in the growth medium under submerged
condition.Theenzymeactivityismeasuredafter24hforenzymeproduction.