270 Chiang Mai J. Sci. 2012; 39(2)

Chiang Mai J. Sci. 2012; 39(2): 270-280http://it.science.cmu.ac.th/ejournal/Contributed Paper

Optimization of Solid State Fermentation for ReducingSugar Production from Agricultural Residues of SweetSorghum by Trichoderma harzianumAnusith Thanapimmetha [a,b], Korsuk Vuttibunchon [a],Boosaree Titapiwatanakun [c] and Penjit Srinophakun [a,b,c]*[a] Department of Chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand.[b] National Center of Excellence for Petroleum, Petrochemicals and Advanced Materials, S&T Postgraduate

Education and Research Development Office (PERDO), Bangkok 10330, Thailand.[c] KU-Biodiesel Project and Center of Excellence for Jatropha, Kasetsart University, Bangkok 10900, Thailand.*Author for correspondence; e-mail: fengpjs@ku.ac.th

Received: 28 July 2011Accepted: 27 December 2011

ABSTRACTSweet sorghum is an attractive feedstock for ethanol production. The juice

extracted from the fresh stem contains sucrose, glucose, and fructose and can be readilyfermented to alcohol. The solid fraction residue, the so-called bagasse, is a lignocellulosewhich can further be processed to ethanol. In this work, optimal conditions for reducingsugar production from sweet sorghum bagasse under Solid State Fermentation (SSF) byTrichoderma harzianum was investigated. Response Surface Methodology (RSM) usingBox-Behnken Design (BBD) for optimization of culture conditions was performed; theseparameters were initial moisture content, inoculum size and incubation time. The reducingsugar production from sweet sorghum bagasse by means of SSF was achieved aftersequential of acid and alkaline pretreatments. The experimental data showed that thehighest value of reducing sugar from cellulose pulp were produced at initial moisturecontent of 77.5%, inoculum size as 10.5% and 56 hours of incubated time. Under theseconditions, reducing sugar of 10.34% (g/g dry materials) was obtained.

Keywords: Sweet sorghum bagasse; Box-Behnken Design; Trichoderma harzianum.

1. INTRODUCTIONSweet sorghum is recognized as an

alternative potential feedstock for ethanolproduction, since it is a high biomass andsugar yielding crop. It contains soluble(glucose and sucrose) and insolublecarbohydrates (cellulose and hemicellulose)[1]. The juice extracted from the fresh stem

can be easily converted to ethanol. Theremaining solid residue is called bagasse,which is a byproduct representing about30% of the plant fresh weight. Bagasse, animportant residue from sweet sorghumprocessing, can become an importantbiomass source for saccharification and

Chiang Mai J. Sci. 2012; 39(2) 271

fermentation for bioethanol production.Sweet sorghum bagasse defined as the

lignocellulosic biomass is mainly composedof cellulose, hemicellulose and lignin.Cellulose is a linear polymer that comprisesglucose subunits linked by β-1,4 glycosidicbonds. These long chains are connectedtogether by hydrogen bonds and Van derWaals forces. Cellulose is usually presentedas a crystalline form while amorphouscellulose represents as small amount ofnon-organized cellulose chains forms.Hemicellulose is a polysaccharide with alower molecular weight than cellulose. Itis composed of xylose, mannose, galactose,glucose, arabinose and glucuronic acids, andare linked together by β-1,4- and sometimesby β-1,3-glycosidic bonds. Lignin isresponsible for forming a physical seal thatis an impenetrable barrier in the plant cellwall [2].

In order to obtain sugar, it is necessaryto degrade the polymers to monomer,which can be done by either physical, orchemical, or biological methods. Chemicallypretreatment with enzymatic hydrolysisof lignocellulosic material has receivedattention due to its potential as an environ-mentally friendly process, which mayprovide an efficient and specific processtechnique [3]. For the degradation oflignocellulosic materials, a broad range ofcellulolytic enzymes are necessary.Cellulose can be hydrolysed by action ofcellulase such as β-glucosidases, cellobio-hydrolases and endoglucanases [3]. Enzymehydrolysis of hemicellulose is much morecomplex. Complete breakdown of thisheterogeneous biopolymer requires theaction of several hydrolytic enzymes in thegroup of hemicellulases such as xylanase,arabinase, etc [4].

Numerous bacterial and filamentousfungi can produce cellulolytic enzymes.

One of the most extensively studiedcellulolytic microorganisms is Trichodermaspp. which is also industrially used forenzyme production. In the natural habitat,filamentous fungi can be growing on thesolid lignocellulosic particles. Accordingly,the growth of fungi can facilitate theproduction of enzyme. The optimumapproach for enzyme production is basedon Solid State Fermentation (SSF), whichinvolves the growth of microorganisms onmoist solid substrates in the absence offree water [5]. The use of SSF has manyadvantages, such as no need for complexand sophisticated machinery, easy productrecovery, low energy demand, high volume-tric productivity and often a high yield ofproducts [6]. Moreover, the SSF can useinexpensive and widely available agriculturalresidues as substrates [7].

In this present work, sweet sorghumbagasse in different forms, namely untreatedbagasse, cellulignin, and cellulose pulp, areanalyzed for the percentages of chemicalcompositions. These three substrates areused for comparison of reducing sugarproduction by Trichoderma harzianum.In this experiment, moisture content,inoculum size and incubation time were thechosen as three factors to be optimizedfollowed the Box-Behnken Design (BBD).

2. MATERIALS AND METHODS2.1 Substrate preparation and pretreat-ment

Sweet sorghum (Sorghum bicolor (L.)Moench ) bagasse from the cultivar ofSuwan-4 was obtained from Suwan farm,Kasetsart University. This material wasthoroughly washed and dried at 65oC untilconstant weight was obtained. The air-driedbagasse was then milled and subsequentlysieved to a size particle of 2-4 mm. 1%H2SO4 solution was added to the untreated

272 Chiang Mai J. Sci. 2012; 39(2)

material at ratio of 1:20, then, autoclavedat 121oC for 20 min for an efficienthemicellulose removal from sweet sorghumbagasse. After reaction the solid residue(cellulignin) was separated, washed withwater until the pH was neutral, and driedat 65oC.

The cellulignin was, then, treated with4%(w/v) NaOH solution at a solid:liquidwith ratio of 1:20 and consequentlyautoclaved at 121oC for 20 min, conditionspreviously optimized for an efficient ligninremoval from sweet sorghum bagasse. Atthe end of the reaction, the residual solidmaterial (cellulose pulp) was separated,washed with water to remove the residualalkaline, and dried at 65oC.

2.2 MicroorganismsThe fungal strain Trichoderma harzia-

num, was obtained from Uniseeds Co.,LTD. The inoculum of T. harzianum wasin powder form and contained 108 sporesper gram.

2.3 Optimization of reducing sugar pro-duction under SSF

Reducing sugar production under SSFwas conducted in 250 ml flask covered withcotton. Each flask containing 5 g driedsweet sorghum bagasse, which used as thecarbon source. Standard Mandel mediumin 50 mM sodium citrate buffer (pH 4.8)that contained of 0.3 g/l urea, 1.4 g/l(NH4)2SO4, 2.0 g/l KH2P O4, 0.4 g/l

CaCl2⋅2H2O, 0.3 g/l MgSO4⋅4H2O, 0.75g/l peptone, 0.25 g/l yeast extract, 5 mg/lFeSO4⋅7H2O, 1.6 mg/l MnSO4⋅4H2O,1.4 mg/l ZnSO4⋅7H2O and 20 mg/l CoCl2⋅6H2O, was added to substrate for adjustinginitial moisture content before autoclavingat 121oC for 20 min. Each flask was inocu-lated with fungal spore of T. harzianumand incubated at 25oC.

Response surface methodology (RSM)was applied for optimization of the reducingsugars production. Cellulose pulp was selectedas substrate for the whole experiments.Three parameters namely size of inoculum(X1), moisture content (X2), and incubatedtime (X3) with the ranges of minimum (-1),maximum (+1) and central point (0) wereassigned as design of experiment of reducingsugar production.

The levels of parameters for experimentaldesign are shown in Table 1. The total of15 experimental runs with three variableswas designed according to a Box-BehnkenDesign (BBD) using the statistical softwareMINITAB release 14. The behavior of thesystem was explained by the followingquadratic model equation.

Y = β0+β1X1+β2X2+β3X3+β11X12+β22X2

2+β33X3

2 +β12X1X2+β13X1X3 +β23X2X3

(1)

Where Y is the predicted response, β0

intercept β1, β2 and β3 linear coefficient, β11,β22 and β33 square coefficient, and β12, β13 andβ23 interaction coefficients.

Range and levelsFactors Symbol -1 0 1

Inoculum size (%w/w) X1 5 10 15Moisture content (%w/w) X2 70 80 90Incubated time (hours) X3 36 48 60

Table 1. Experimental range and coded levels of factors for reducing sugars production.

Chiang Mai J. Sci. 2012; 39(2) 273

3. ANALYTICAL METHODSThe composition of materials (untreated

bagasse, cellulignin and cellulose pulp) werethen analyzed by the methods of Goeringand Vansoest (1970) [8] to evaluate cellulose,hemicellulose, lignin and ash content in thebiomass.

After suitable periods of time, reducingsugar was extracted from the fermentedmedium by adding 100 ml distilled waterto each flask. The flasks were then shakenat 200 rpm for 2 hr at 60oC. The suspendedmaterials and fungal biomass were separatedusing filter paper.

The total reducing sugars in the filtratewere determined by the 3,5-dinitrosalicylicacid (DNS) method described by Miller(1959) [9]. The composition of reducingsugars (glucose, xylose and arabinose) inhydrolysates was determined by HPLCwith a refractive index (RI) detector andRezex RPM Monosaccharide 00H-0135-K0(300x7.8 mm) column. The samples werefiltered through syringe filters (Regeneratedcellulose: 0.45 μm) and thus injected inchromatograph under the conditions:column temperature 75oC, distilled wateras mobile phase at a flow rate of 0.8 ml/min and injection volume of 10 μl. Theconcentration of these compounds wasobtained from the calibration curves of thestandard solution.

4. RESULT AND DISCUSSIONS4.1 Chemical Composition of Substrates

The enzymatic hydrolysis of cellulosewas reported to be affected by many factorsincluding porosity (accessible surface area)of materials, cellulose fiber crystallinity,hemicelluloses and lignin content[10]. Cellulose, hemicelluloses, lignin andashes content of sweet sorghum bagassebefore and after pretreatment are shownin Table 2.

Sweet sorghum bagasse was initiallypretreated with dilute sulfuric acid at hightemperature to solubilize the hemicellulosesfraction. While acid hydrolysis occurred,the acid also hydrolyzed the poly-saccharides, especially hemicelluloses thatwas easier to be hydrolyzed than cellulose[11]. The structural complexity of cellulosewas more than that of hemicelluloses, thencellulose hydrolysis could only be occurredwith strong acids. An acid pretreatment by1% H2SO4 effectively reduced the content(% dry weight) of cellulose and hemicellu-lose by 8.48% and 28.02%, respectively(data not shown). Therefore, the celluloseand lignin fractions remained almost thesame in solid phase [12]. Subsequently, thesolid residue (cellulignin) reacted withsodium hydroxide to solubilize the lignin..This was typical of alkaline pretreatment,which generally had a stronger effect on

Composition Untreated Cellulose(%dry weight) bagasse Cellulignin pulp

Cellulose 54.64 73.27 95.62Hemicellulose 28.15 0.21 0.13Lignin 16.07 25.35 3.67Ash 1.14 1.17 0.58

Table 2. Chemical compositions of sweet sorghum bagasse in the original form (untreatedbagasse), after the acid pretreatment (cellulignin) and after sequential acid and alkalinepretreatments (cellulose pulp).

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lignin than cellulose and hemicelluloses [13].Lignin could be well-solubilized in bases.However, upon an alkaline pretreatmentwith 4% NaOH at high temperature, thissituation still reacted with the bonds ofhemicelluloses and cellulose. Pretreatedbiomass was swollen, which led to theminimization in degree of polymerization,decrease in crystallinity, disruption of thelignin structure, and separation of structurallinkages between lignin and carbohydrates[14]. As a result shown in Table 2, thepurity of cellulose fraction was increasefrom 54.64% (from untreated bagasse) to95% (from cellulose pulb).

4.2 Effect of Pretreatment on ReducingSugar Production

In this respect, different forms of sweetsorghum bagasse (untreated bagasse,cellulignin and cellulose pulp) were testedfor the production of reducing sugar byT. harzianum under SSF. An inoculum(10% w/w) was added to a medium of 70%(w/w) moisture content and incubated at25oC for 96 hours. As shown in Figure 1,

the highest reducing sugar of 8.95% (w/w),was obtained with cellulose pulp as thesubstrate for 2 days of incubation time,followed by untreated bagasse andcellulignin of 4.79 and 4.14 % (w/w),respectively. The hemicellulose and ligninremoval were relatively resistant tomicrobial digestion. On the contrary, thetreatment with T. harzianum did causeextensive changes in structure andaccessibility of cellulose that had moredigestible surface area and more swelling[15]. Because of the highest percentages ofobtained reducing sugar, cellulose pulp wasthen selected as substrate for the optimiza-tion of reducing sugar production.

4.3 Optimization of Reducing SugarProduction under SSF

In the present study, BBD is used tofind the optimal conditions of reducingsugar production from cellulose pulpderived from sweet sorghum bagasse underSSF by T. harzianum. The performing of15 experiments of three variables namelyinoculum size, moisture content and

Figure 1. Comparison of reducing sugar production by T. harzianum under SSF usingdifferent form of sweet sorghum bagasse (untreated bagasse, cellulignin and cellulosepulp) as the substrate (10% inoculum, 70% moisture content).

10.00

8.00

6.00

4.00

2.00

0.00

Red

ucin

g su

gar

(% g

/g d

ry s

ubst

rate

)

0 12 24 36 48 60 72 84 96

Chiang Mai J. Sci. 2012; 39(2) 275

incubated time, were chosen as shown inTable 3. Details of three different rangesin each parameter were explained inTable 1. The maximum reducing sugar was9.97% (g/g dry substrate) at the mid valuesof the parameters condition, which wasshown as experimental run number 15 inTable 3.

The statistical software MINITABrelease 14 was used in design of experiments,to determine the coefficients of linear,quadratic and interaction terms, as well asto build the quadratic model and 3Dresponse surface plots.

The BBD was used to study the linear,quadratic and interaction effects of variousparameters on reducing sugar production.The experimental results were analyzed by

regression analysis consisting of the effectlinear, quadratic and interaction which gavethe following regression equation withreducing sugar production as a function ofinoculum size (X1), moisture content (X2),and incubated time (X3):

Y = -211.251+2.555X1+4.086X2+1.828X3-0.096X1

2-0.026X22-0.015X3

2 +0.004X1X2 -0.017X1X3+0.000X2X3 (2)

Reducing sugar (Y) at specific combina-tion of three variables could be predictedby substituting the corresponding valuesof each variable in Eq. (2). The predictedvalues from Eq. (2) of reducing sugar ofeach experimental runs were shown in thefinal column of Table 3.

number Inoculum Moisture Time(X1) (X2) (X3)

Experimental Predicted

1 -1 -1 0 6.05 ± 0.11 5.292 1 -1 0 6.21 ± 0.21 6.423 -1 1 0 2.87 ± 0.15 2.664 1 1 0 3.83 ± 0.20 4.595 -1 0 -1 0.10 ± 0.05 0.696 1 0 -1 4.64 ± 0.48 4.247 -1 0 1 7.92 ± 0.45 8.318 1 0 1 8.42 ± 0.56 7.839 0 -1 -1 3.13 ± 0.13 3.3110 0 1 -1 1.50 ± 0.05 1.1311 0 -1 1 8.59 ± 0.35 8.9612 0 1 1 6.86 ± 0.32 6.6813 0 0 0 9.80 ± 0.28 9.7814 0 0 0 9.56 ± 0.46 9.7815 0 0 0 9.97 ± 0.41 9.78

Level of experimental factors Reducing sugar

Run % (g/g dry substrate)

Table 3. Reducing sugar from cellulose pulp in experiments obtained by BBD.

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The probability value (p-value) was atool for evaluating the significance andcontribution of each parameter and thestatistical polynomial model equation. Thesmaller p-value was an indicator of highsignificance of corresponding coefficient[16]. The regression coefficient in theresponse surface model for the linear,quadratic and interaction effects of thevariables were shown along with p-value inTable 4. The p-value suggested that thecoefficient for the linear effect of inoculumsize (p < 0.05), moisture content (p < 0.01)and incubated time (p<0.01) were

statistically significant for reducing sugarproduction.

Analysis of variance (ANOVA) fittedfor the model (data not shown). The p-valueof the regression model (p<0.001) impliedthat the model was significant. In addition,the coefficient of variation (R2 = 0.98)indicated a high correlation between theobserved and predicted values from modelEq. (2). Therefore, this equation could beused for predicting the amount of reducingsugar production under conditions variedwith only three variables in the experimentalrange.

Term Coefficient p-valueConstant -211.251 0.001**Inoculum size (X1) 2.555 0.017*Moisture content (X2) 4.086 0.001**Time (X3) 1.828 0.004**Inoculum × Inoculum -0.096 0.001**Moisture × Moisture -0.026 0.001**Time × Time -0.015 0.003**Inoculum × Moisture 0.004 0.608Inoculum × Time -0.017 0.040*Moisture × Time 0.000 0.948

* p < 0.05, ** p < 0.01

Table 4. Estimated regression coefficient and corresponding p-value for reducing sugarproduction.

4.4 Evaluation of the Interactions ofEach Parameter

The two-dimensional (2D) contourplots and three-dimensional (3D) responsesurface of the interactions were presentedin Figure 2A-C. These observations alsoidentified the optimal conditions with themaximum response for the levels of thefactors in the design of experiments. Themaximum response was referred by the

surface confined in smallest ellipse inthe contour plot. The perfect interactionbetween the independent variables couldbe shown when elliptical contours wereobtained [16].

Moisture content had influence onreducing sugar production. The optimalinitial moisture content of medium inreducing sugar production was observedat 77.5% (w/w). The optimal moisture

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Figure 2. Contour plot and response surface.A. Effects of inoculum size and initial moisture content at a constant incubated time.B. Effects of inoculum size and incubated time at a constant initial moisture content.C. Effects of initial moisture content and incubated time at a constant inoculum size.

A

B

C

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content had been approximated to theprevious report on scaling up of cellulaseproduction by T. harzianum on a mixtureof sugar cane bagasse and wheat bran inSSF system [17]. Fungi were well-knownto favor a moist environment whichpositively affected their growth. Theappropriate moisture of substrate was oneof the critical factors influencing the SSF.It was observed that the moisture enabledbetter utilization of the substrate bymicroorganisms and the efficiency of masstransfer in the solid phase depended on thesubstrate characteristics. However, furthertoo much increase in moisture contentnegatively influenced the volume ofproduction, resulting as water. It reducedsurface area of the biomass, and made thewater film thicker, which reduced theaccessibility of the air to the biomass. Sincethe transfer of oxygen affected the growthand metabolism, the substrate shouldcontain suitable amount of water toenhance mass transfer. Therefore, thewater content of solid substrates was oneof the key factors in SSF [18].

Optimum inoculum density wasimportant consideration for SSF process.An increase in inoculum size would ensurea rapid proliferation and biomass synthesis.However, over crowding number of sporesin the inoculum led to the competition forthe nutrients resulted in the decreasedmetabolic activity of the organism. Withoptimum inoculum size for the reducingsugar production, there was a balancebetween proliferating biomass andavailability of nutrients that supportedproduction of reducing sugar [19]. Incurrent study, the optimal inoculum sizeof T. harzianum was 10.5% (w/w) ~5x107

spores/g dry substrate.Application of RSM with BBD predicted

that the maximum reducing sugar production

must occur at decoded values of conditionparameters as 10.5% (w/w) inoculum size,77.5% (w/w) moisture content, and 56hours of incubated time. The reducingsugar should reach 10.80% (g/g drysubstrate). A repeat fermentation forreducing sugar production by T. harzianumunder optimal conditions was carried outfollowing the validation of optimizedparameters. After performing the fermen-tation under optimal conditions, theobtained reducing sugar was 10.34%(g/g dry substrate). Since the differencebetween predicted and actual result wasonly about 4.35%, it should be regarded asacceptable.

4.5 Analysis of Reducing Sugar Composi-tion

The compositions of reducing sugarexisting in the enzymatic hydrolysates ofcellulose pulp under the optimal conditionsby T. harzianum, were analyzed by HPLC.Standard curves of cellobiose, glucose,xylose, arabinose and mannose concentra-tion were described first, all showing goodlinear relativity. The retention time ofcellobiose, glucose, xylose, arabinose andmannose concentration are 8.73, 10.20,10.90, 12.54 and 12.82 min, respectively.Based on these standard curves, theconcentration of reducing sugar in theenzymatic hydrolysates of cellulose pulpcould be calculated by the peak areaThe percentage of dry weight (% w/w) wassummarized as in table 5, which was 1.11%of cellobiose, 7.57% of glucose, 0.91% ofxylose and 0.29% of arabinose andmannose. Glucose and cellobiose obtainedfrom the degradation of cellulose, whichwas the major element of cellulose pulp asshown in Table 2. Cellobiose was adisaccharide, derived from the condensa-tion of two glucose molecules linked in a

Chiang Mai J. Sci. 2012; 39(2) 279

β(1→4) bond. The result showed thatglucose was the main component ofreducing sugar in the enzymatic hydro-lysates. Glucose was the most desiredreducing sugar to acquire, since it could beeasily fermented to ethanol by variouskinds of yeast.

5. CONCLUSIONSThe results obtained in this study had

shown that filamentous fungus T. harzianumwas able to produce cellulolytic enzyme forreducing sugar production from sweetsorghum bagasse as lignocellulose biomass.The optimal conditions obtained througha statistical BBD were successfully deter-mined to maximize the reducing sugarproduction. The result from the secondorder polynomial model developedindicated that the optimal conditions forreducing sugar production from cellulosepulp under SSF by T. harzianum were theinitial moisture content of 77.5%, inoculumsize of 10.5%(w/w) and 56 hours ofincubated time which give the maximumreducing sugar of 10.34% (g/g dry substrate).The reducing sugar obtained containsglucose as the major component.

ACKNOWLEDGEMENTSThis study was supported by the

Graduate School Kasetsart UniversityResearch and Development Institute

(KURDI), National Center of Excellencefor Petroleum, Petrochemicals andAdvanced Materials, S&T PostgraduateEducation and Research DevelopmentOffice (PERDO), Center for advancedStudies in Industrial Techechnology (NRU),Department of Chemical Engineering,Faculty of Engineering, Kasetsart University.

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