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Optimizationofthemediumfortheproductionofcellulasebythe
Trichodermaviride usingsubmergedfermentation
Gautam S.P.1,Bundela P.S.2,Pandey A.K.3, Jamaluddin4,Awasthi M.K.2,SarsaiyaS.2
1CentralPollutionControlBoard,NewDelhi,India.
2Regionaloffice,M.P.PollutionControlBoard,VijayNagar,Jabalpur(M.P.),India
3MycologicalResearchLaboratory,DepartmentofBiologicalSciences,RaniDurgavati
University,Jabalpur(M.P.),India.
4 YeastandMycorrhizaBiotechnologyLaboratory,DepartmentofBiologicalSciences,
RaniDurgavatiUniversity,Jabalpur(M.P.),India.
surendra_sarsaiya@yahoo.co.in

ABSTRACT
The main purpose of this study to reduced the production cost of cellulase by using
alternative carbon source such as municipal solid waste residue andoptimizedfermentation
medium for high yielding. In the present investigation, to isolate the novel cellulase
producingfungi,Trichodermaviridefrommunicipalsolidwasteandtooptimizethephysico
chemical and nutritional parameters for cellulase production.Municipal solid waste residue
andyeastextractwerefoundtobethebestcombinationofcarbonandnitrogensourceforthe
productionof cellulase byTrichodermasp. Optimal concentration of municipal solid waste
residue and yeast extract for the production of cellulase were 45% (w/v) and 1.0% (w/v),
respectively. Optimum temperature and pH of the medium for the cellulase production by
Trichodermaviridewas45Cand6.5.CellulaseproductionfromTrichodermaviridecanbe
anadvantageastheenzymeproductionrateisnormallyhigherascomparedtootherfungi.
Keywords:Fermentation,optimization,biodegradation,cellulase,municipalsolidwaste
1.Introduction
Globallytheestimatedquantityofthewastesgenerationwas12billiontonesinayear2002
ofwhich 11billion tones were industrialwastes and 1.6billion tones were municipal solid
waste.About90billiontonessolidwastesareexpectedtobegeneratedannuallybytheyear
2025. Annually, Asia alone generates 4.4 billion tones of solid wastes and municipal solid
waste comprises 790 million tones of which about 48 million tones are generated in India.
Unscientific disposal of waste causes an adverse impact on all components of the
environment and human health (Lee et al., 2007). Agricultural waste is also caused
environmentalpollution.Theirconversionintousefulproductsmayamelioratetheproblems
they cause. Municipal solid waste (MSW) is composed of 4050% cellulose, 912%
hemicellulose and 1015% lignin on a dry weight basis. Complete cellulose hydrolysis to
glucosedemandstheactionofexoglucanases(alsocellobiohydrolyses),endoglucanasesand
glucosidases. Exoglucanases (1,4Dglucan cellobiohydrolase, EC3.2.1.91) areusually
active on crystalline cellulose and are lacking from incomplete cellulase systems.
Endogluconases (1,4 Dglucan4glucanohydrolase, EC 3.2.1.4) are more active against
theamorphousregionsofcelluloseandtheycanalsohydrolysesubstitutedcelluloses,suchas
carboxymethylcellulose(CMC)andhydrxyethylcellulose(HEC).Cellobiohydrolasescleave
disaccharide (cellobiose) units either from nonreducing or reducing ends, whereas

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endoglucanases hydrolyse the cellulose chain internally. glucosidase (EC 3.2.1.21) are
neededtocleavecellobioseandothersolubleoligosaccharidestoglucose(Beguin,1990).
Intherecentyears,oneofthemostimportantbiotechnologicalapplicationsistheconversion
of agricultural wastes and all lignocellulosics into products of commercial interest such as
ethanol, glucose and single cell products (Ojumu et al., 2003). The key element in
bioconversionprocessoflignocellulosicstotheseusefulproductsisthehydrolyticenzymes
mainly cellulases (Ojumu et al., 2003 Fan et al., 1987 Immanuel et al., 2007). The
bioconversions of cellulosic materials are now a subject of intensive research as a
contribution to the development of a largescale conversion process beneficial to mankind.
Such process would help alleviate shortages of food and animal feeds, solve modern waste
disposalproblemanddiminishmansdependenceonfossil fuelsbyprovidingaconvenient
and renewable source of energy in the form of glucose. A diverse spectrum of cellulolytic
microorganismmainlyfungi(Falconetal.,1995)andBacteria(McCarthy,1987)havebeen
isolatedandidentifiedovertheyearsandthisstillcontinuetogrowrapidly.Oneofthemost
extensively studied fungi is Trichoderma reesei, which converts native as well as derived
cellulosetoglucose.Mostcommonlystudiedcellulolyticorganismincludingfungalspecies
Trichodermasp.,Humicolasp.,Penicilliumsp.and Aspergillussp.
In this investigation, a cellulase producing strain of Trichoderma viride, isolated from
municipalsolidwaste,weresubjectedtooptimizationofmediaandcultivationparametersfor
cellulaseproduction.
2.MaterialsandMethods
2.1.Materials
Municipal solidwastewascollectedfromdifferentdumping sitesofJabalpur(M.P.),India.
FungalcultureofTrichodermaviridewasisolatedfromcompostedmunicipalsolidwastein
June 2009. It was grown at 300C, maintained on potato dextrose agar containing potato
2.5%(w/v),dextrose2.0%andagar1.5%at420Candthenidentifiedbyapreviousstudy
(BarnettandHunter1972).
2.2.Enzymeproduction
Theculturewasgrownin150mlErlenmeyerflaskthatcontained50mlofbasalsaltmedium.
The pH of the medium was adjusted to6.5prior to sterilization. The flask were inoculated
with 2 agar discs (2 mm in diameter) of 7 days old culture from PDA plates and were
incubatedunderstationaryconditionat4020Cupto7days.Thecrudeenzymewerefiltered
andcentrifugedat11000xgfor10min.
2.3.Enzymeassay
Filterpaperactivity(FPase)fortotalcellulaseactivity intheculturefiltratewasdetermined
accordingtothestandardmethod(HankinandAnagnostakis1975).Aliquotsofappropriately
dilutedculturedfiltrateasenzymesourcewasaddedtowhatmanno.1filterpaperstrip(1x6
cm 50 mg) immersed in one milliliter of 0.05 M Sodium citrate buffer of pH 5.0. After
incubationat5020Cfor1hrs,thereducingsugarreleasedwasestimatedbydinitrosalicylic
acid(DNS)method(Miller1959).Oneunitoffilterpaper(FPU)activitywasdefinedasthe

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amount of enzyme releasing 1 mole of reducing sugar from filter paper per ml per min.
Endoglucanaseactivity(CMCase)wasmeasuredusingareactionmixturecontaining1mlof
1%carboxymethylcellulose(CMC)in0.5Mcitrateacetatebuffer(pH5.0)andaliquotsof
suitably diluted filtrate. The reaction mixture was incubated at 5020C for 1 h and the
reducingsugarproducedwasdeterminedbyDNSmethod.glucosidaseactivitywasassayed
bythemethodofPointing(1999).Oneunit(IU)ofendoglucanaseactivitywasdefinedasthe
amountofenzymereleasing1moleofreducingsugarpermin.
2.4.Optimizationofcultureconditionsforenzymeproduction
2.4.1.Effectofincubationperiodinenzymeproduction
Fermentationperiodisimportantparameterforenzymeproductionby A.niger.Inthisstudy,
experimentsfermentationwascarriedoutupto7daysandproductionratemeasuredat24h
intervals.
2.4.2.EffectofpHandtemperatureonenzymeproduction
ThemostsuitablepHofthefermentationmediumwasdeterminedbyadjustingthepHofthe
culturemediumatdifferentlevelsintherangeofpH3to9usingdifferentbuffers.Inorderto
determinetheeffectivetemperatureforcellulaseproductionbytheA.niger,fermentationwas
carriedoutat100Cintervalsintherangeof20to8020C.
2.4.3.Effectofcarbonsourcesonenzymeproduction
Effectofvariouscarboncompoundsviz.,cellulase,CMC,glucose,sucroseandmaltosewere
used for studying. The broth was distributed into different flasks and 0.5 to 3.0 % of each
carbonsourceswerethenaddedbeforeinoculationofthestrainandaftercultureinoculation,
theflaskswereincubatedfor7daysat4520C.
2.4.4.Effectofnitrogensourcesonenzymeproduction
Inthepresentstudy,todetecttheappropriatenitrogensourceforcellulaseproductionbythe
Trichoderma viride. The influence of peptone, beef extract, ammonium nitrate, sodium
nitrate and yeast extract were studied. The fermentation medium was supplemented with
organic and inorganic compounds at 0.5 to 3.0% level, replacing the prescribed nitrogen
sourceofthefermentationmedium.
2.4.5.Effectofcationsonenzymeproduction
Effect of cations oncellulase activity were determined by using different cations (Na+, K+,
Ca++,Mg++ &EDTA).Thebrothwasdistributedintodifferentflaskand10mMto80mMof
each cations were then added and after culture inoculation, the flask were incubated at
4520Cfor7days.

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2.4.6.Effectofmunicipalsolidwasteresidueinenzymeproduction
In the present study, to detect the appropriate municipal solid waste residue for cellulase
production by Trichoderma viride. The fermentation medium was supplemented with
municipalsolidwasteresidueat1.0to6.0%level,replacingtheprescribedcarbonsourceof
thefermentationmedium.
2.5.Statisticalanalysis
Data presented on the average of three replicates (SE) obtained from there independent
experiments.
3.ResultsandDiscussion
3.1.Effectofincubationperiodonenzymeproduction
Theincubationperiodisdirectlyrelatedwiththeproductionofenzymeandothermetabolic
upto a certain extent. Trichoderma sp. showed the most active cellulolytic species along
different incubation period. The incubation period to achieve peak cellulase activity by the
isolateTrichodermasp.was6th days(Figure3)whichsuitableforcommercialpointofview
(Kangetal.,2004).Itmightbeduetothedepletionofnutrientsinthemediumwhichstressed
the fungal physiology resulting in the inactivation of secretary machinery of the enzymes
(Nochureetal.,1993).
3.2.EffectofpHonenzymeproduction
Cellulase yield by Trichoderma viride appeartodependonpH value. Results illustratedby
Figure 1 clearly show that cellulase production, expressed as enzyme activity, gradually
increased as thepH value increased from56 andreached its maximum atpH of5.5being
exoglucanase (2.16 U/ml), endoglucanase (1.94 U/ml) and glucosidase (1.71 U/ml). The
enzymeactivitygraduallyincreasedwhenincreasingthepHuptotheoptimumfollowedbya
gradualfullinactivity.ItwasalsonotedthattheenzymeactivitywasstableatpHrangeof
5.08.0.EffectofpHoncellulaseproductionbythesefungisupportsthefindingsofLeeetal.
(2002)whoreportedthatCMCase,AvicelaseandFPaseactivitiesexhibitapHoptimumof
approximately4,whilethepHoptimumofglucosidasewasbetweenpH5and6.
3.3.Effectoftemperatureonenzymeproduction
LikepH,temperatureisalsoanimportantfactorthatinfluencesthecellulaseyield.Maximum
enzyme production byTrichoderma viride was found to be exoglucanase (1.95 U/ml) and
endoglucanaseactivity(1.88U/ml)andglucosidase(1.88U/ml)activitiesbetween40500C
(Figure 2). Many workers have reported different temperatures for maximum cellulase
production either in flask or in fermentor studiesusingTrichoderma sp. suggesting that the
optimal temperature for cellulase production also depends on the strain variation of the
microorganism(Muraoetal.,1988,Luetal.,2003).
3.4.Effectofcarbonsourcesonenzymeproduction

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Data presented in table 1 shown that cellulase production by Trichoderma viride was
significantly influencedbythetypeofcarbonsourceinthebasalsaltmedium.Sucrosewas
themosteffectiveasasolecarbonsourceforcellulaseenzymeproduction,resultsincreased
in enzyme activity, being exoglucanase (2.68 U/ml), endoglucanase (2.17 U/ml) and
glucosidase(2.06U/ml)wereobtainedinculturemediumcontaining1.0%Sucrosefollowed
by glucose, cellulose maltose and CMC. Cellulase production increased with increases in
initial sugar concentration from 1.0 to 1.5% while further increases in sugar concentration
slightlyreducedthe yield.MendalsandReese(1957)alsoreportedthatmaximum yieldsof
cellulasewereobtainedon1%differentcarbonsubstrateusing T.viride.Cellulaseproduction
commended on reaching nitrogenlimiting conditions and the yield of cellulase decreased
whenexcesspeptonewaspresented,variousinorganicnitrogensourceshavebeenoptimized
bydifferentworkersforcellulaseproduction(Sherief etal. 2010Solomon etal. 1997Leeet
al. 2010).
Table1: Effectofcarbonsources(%)oncellulasesproduction(U/ml)by Trichoderma
viride
Differentcarbonsources
Conc.
Glucose
Maltose
(%)
FPase
CMCase glucosidase
FPase
CMCase glucosidase
0.5
0.680.04 0.490.03
0.410.02
0.440.05 0.350.04 0.470.05
1.0
2.310.08 1.930.07
1.910.09
1.890.08 1.910.07 1.900.08
1.5
1.710.06 1.430.05
1.670.07
1.610.08 1.460.05 1.630.06
2.0
0.970.05 0.980.04
0.910.05
1.260.06 0.880.05 1.100.05
2.5
0.620.04 0.740.04
0.550.04
0.810.05 0.530.04 0.720.04
3.0
0.260.02 0.340.03
0.370.03
0.630.04 0.320.03 0.390.03
Differentcarbonsources
Conc.
Cellulose
Sucrose
(%)
FPase
CMCase glucosidase
FPase
CMCase glucosidase
0.5
0.410.05 0.700.06
0.350.04
0.670.05 0.500.06 0.420.04
1.0
1.990.08 1.890.07
2.080.07
2.680.07 2.170.07 2.060.08
1.5
1.710.07 1.330.07
1.810.07
2.210.08 1.510.06 1.510.08
2.0
1.110.07 0.940.06
0.920.06
1.550.05 0.860.05 0.900.06
2.5
0.830.06 0.680.05
0.710.05
0.840.04 0.640.04 0.740.05
3.0
0.560.04 0.550.04
0.590.04
0.440.03 0.310.03 0.310.04
Conc.
Differentcarbonsources
(%)
Carboxymethylcellulose
FPase
CMCase
glucosidase
0.5
0.410.03
0.370.04
0.410.05
1.0
1.940.06
1.720.05
1.880.07
1.5
1.710.05
1.630.06
1.730.08
2.0
0.630.05
0.850.05
1.410.06
2.5
0.420.04
0.640.04
0.860.05
3.0
0.320.03
0.380.04
0.430.04

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Table 2: Effectofnitrogensources(%)oncellulasesproduction(U/ml)by Trichoderma


viride
Differentnitrogensources
Conc.
Peptone
Yeastextract
(%)
FPase
CMCase glucosidase
FPase
CMCase glucosidase
0.5
0.710.04 0.320.05 0.370.03
0.310.04 0.320.05
0.370.03
1.0
2.210.08 1.930.07 1.940.06
2.210.08 1.930.07
1.940.06
1.5
1.890.07 1.620.06 1.710.07
1.890.07 1.620.06
1.710.07
2.0
1.520.07 0.840.06 0.910.06
1.520.07 0.840.06
0.910.06
2.5
0.910.06 0.630.05 0.460.05
0.910.06 0.630.05
0.460.05
3.0
0.530.04 0.480.04 0.390.05
0.530.04 0.480.04
0.390.05
Differentnitrogensources
Conc.
Ammoniumnitrate
Sodiumnitrate
(%)
FPase
CMCase glucosidase
FPase
CMCase glucosidase
0.5
0.240.04 0.190.03
0.150.03
0.310.03 0.220.03
0.110.03
1.0
1.730.06 1.640.06
1.690.06
1.760.07 1.560.05
1.740.05
1.5
1.470.06 1.230.06
1.440.06
1.530.06 1.440.05
1.110.05
2.0
0.890.05 0.820.05
0.880.04
0.990.06 0.840.04
0.680.05
2.5
0.640.04 0.670.05
0.630.04
0.650.04 0.670.04
0.540.04
3.0
0.300.04 0.310.04
0.280.03
0.410.04 0.510.03
0.310.03

Table3.Effectofcations(mM)oncellulasesproduction(U/ml)by Trichodermaviride
Conc.
(%)
0.5
1.0
1.5
2.0
2.5
3.0
Conc.
(mM)
10
20
30
40
50
60
70
80

FPase
0.310.04
2.210.08
1.890.07
1.520.07
0.910.06
0.530.04

FPase
1.870.07
1.640.09
1.070.05
0.790.05
0.540.04
0.380.04
0.370.03
0.320.03

Differentnitrogensources
Beefextract
CMCase
0.320.05
1.930.07
1.620.06
0.840.06
0.630.05
0.480.04
Differentcationsources

Na+
CMCase glucosidase
FPase
1.730.09
1.990.08
1.890.11
1.320.07
1.940.09
1.670.09
1.080.06
1.660.07
1.390.06
0.840.06
0.960.06
0.820.05
0.430.07
0.510.06
0.650.04
0.380.05
0.430.05
0.410.05
0.360.05
0.410.04
0.370.04
0.350.04
0.290.03
0.280.04

glucosidase
0.370.03
1.940.06
1.710.07
0.910.06
0.460.05
0.390.05
K+
CMCase glucosidase
1.920.08
1.970.12
1.840.08
1.900.09
1.610.06
1.730.08
0.570.06
0.870.06
0.410.05
0.640.06
0.360.05
0.530.05
0.310.04
0.430.04
0.220.03
0.320.03

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Table3.Continued.

Conc.
(mM)

Differentcation sources
++

FPase
10
1.760.09
20
1.710.08
30
1.540.07
40
0.970.05
50
0.780.06
60
0.540.05
70
0.410.04
80
0.290.03
Conc.(mM)

10
20
30
40
50
60
70
80

Ca
CMCase
1.890.12
1.670.09
1.520.07
0.910.08
0.750.06
0.560.06
0.460.05
0.320.03

EDTA
glucosidase
FPase
CMCase glucosidase
2.020.14
1.890.09 1.610.08 1.670.08
1.890.10
1.760.09 1.510.08 1.540.09
1.720.08
1.640.08 1.380.07 1.320.07
1.430.08
1.370.07 1.250.06 0.970.06
0.800.07
0.890.06 0.910.05 0.730.05
0.640.06
0.630.06 0.660.05 0.440.05
0.510.05
0.470.05 0.380.05 0.340.04
0.390.03
0.310.04 0.300.04 0.210.03
Differentcationsources
Mg++
FPase
CMCase
glucosidase
1.730.08
1.680.09
1.790.13
1.680.08
1.510.08
1.530.09
1.420.07
1.420.07
1.440.07
0.270.06
0.970.07
1.130.06
0.830.06
0.650.06
0.720.06
0.550.05
0.430.05
0.510.05
0.350.04
0.380.04
0.430.05
0.310.03
0.320.03
0.390.03

3.5.Effectofnitrogensourcesonenzymeproduction
Toevaluatetheeffectofnitrogensourceoncellulaseformation,thenitrogensourcesinthe
basal medium was replaced by different nitrogen sources. Data revealed that the
supplementationoforganicandinorganic nitrogensources(rangedfrom0.5to3.0%(w/v)
peptone, beef extract, yeast extract, ammonium nitrate and sodium nitrate) stimulated the
cellulaseyieldandactivity.Themaximumenzymeactivitieswereobtainedwithyeastextract
(1.0%)whichbroughtaboutanimprovementinallthethreecellulasecomponents,including
exoglucanase(2.40U/ml),endoglucanase(2.28U/ml)andglucosidase(1.99U/ml).where
peptone also produce second most cellulase producing nitrogen sourcebyTrichoderma sp.
(table2)Itwasreportedthatgoodcellulaseyieldcanbeobtainedwithammoniumcompound
asthenitrogensource.Thoughtheadditionoforganicnitrogensourcessuchasbeefextract
andpeptoneresultedinincreasedgrowthandenzymeproduction,wasreportedbefore,they
werenotaneffectivereplacementforinorganicnitrogensourcesbecauseoftheirhighercost
(Sunetal.,1999).
3.6.Effectofcationsonenzymeproduction
Inthe presented study, it was found that allof these cations leaded toreduction in enzyme
activity with increase the concentration (10 mM to80mM). Most ions such as Na+ and K+

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E n zym aticactivity(U /m l)....

E nzymaticactivity(U /ml)...

did not significantly reduce the expression of maximum activity. It was found that the
additionof10mMofCa++ intheproductionmediumwasrequirementfortheexpressionof
full enzymatic activity (table 3). EDTA has been cause reduction of enzyme activity with
increase the concentration in the production medium. Mg++ was also strongly affecting the
cellulaseproductionby Trichodermaviridewhentheconcentrationwasincreased.
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0

2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0

4
FPase

6
pH
CMCase

glucosidase

30

40

FPase

Figure 1 Effect of pH on cellulase


production(U/ml)by Trichodermaviride

50
60
70
Temperature
CMCase
glucosidase

80

Figure2Effectoftemperatureoncellulase
production(U/ml)by Trichodermaviride

2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0

2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0

E nzym aticactivity(U /m l)
.
...

E nzym aticactivity(U /ml)....

20

2
FPase

3
4
5
Incubationperiod
CMCase

glucosidase

2
FPase

3
4
MSWresidue(%)
CMCase

glucosidase

Figure 3 Effect of incubation period on


Figure 4 Effect of MSW residue on
cellulase
production
(U/ml)
by
cellulaseproduction(U/ml)byTrichoderma
Trichodermaviride
viride
3.7.Effectofmunicipalsolidwasteresidueinenzymeproduction
The resultsrevealed (Figure4)that MSW residue (5.0%) was the best carbon substrate for
FPase, CMCase and glucosidase by Trichoderma sp. 4.0% MSW residue was best for
cellulase [exoglucanase (1.77 U/ml), endoglucanase (1.95 U/ml) and glucosidase (1.66
U/ml)] enzyme production. these results were confirmed by the results of AboState et al.
(2010).Thisvariationmaybeattributedtothechemicalnatureandnutrientavailabilityofthe
usedsubstrates.
4. ACKNOWLEDGEMENTS
The authors are thankful to Madhya Pradesh Pollution Control Board Bhopal and Head,
DepartmentofBiologicalSciences,R.D.University,Jabalpur,forlaboratory facilities.Also
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thanks to Municipal Corporation of Jabalpur for his support. Ministry of Environment and
Forest,NewDelhiisalsothankfullyacknowledgedforfinancialsupport.
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