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Cellulase Production from Soil Bacteria

1. Bacteria that produce cellulase enzymes were isolated from soil samples using agar plates containing carboxymethyl cellulose (CMC) to identify colonies with cellulolytic activity. The isolate with the highest cellulase activity was selected for further study. 2. The selected bacterial isolate was identified using morphological and biochemical tests according to Bergey's Manual of Determinative Bacteria. 3. Various process parameters for the production of cellulase enzymes by the selected bacterial isolate were optimized, including pH, temperature, carbon sources, nitrogen sources, and agricultural waste substrates.

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67 views2 pages

Cellulase Production from Soil Bacteria

1. Bacteria that produce cellulase enzymes were isolated from soil samples using agar plates containing carboxymethyl cellulose (CMC) to identify colonies with cellulolytic activity. The isolate with the highest cellulase activity was selected for further study. 2. The selected bacterial isolate was identified using morphological and biochemical tests according to Bergey's Manual of Determinative Bacteria. 3. Various process parameters for the production of cellulase enzymes by the selected bacterial isolate were optimized, including pH, temperature, carbon sources, nitrogen sources, and agricultural waste substrates.

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aj_6
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.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.

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