enzymatic saccharification of pretreated sunflower stalks

Biomass and Bioenergy 23 (2002) 237–243

Enzymatic sacchari�cation of pretreated sun�ower stalksSanjeev K. Sharma ∗, Krishan L. Kalra, Harmeet S. Grewal

Department of Microbiology, College of basic sciences and Humanities, Punjab Agricultural University,Ludhiana 141 004, Punjab, India

Received 29 October 2001; received in revised form 27 March 2002; accepted 27 March 2002

Abstract

The sun�ower stalks were pretreated by steam explosion (at 1:05 kg=cm2 for 0:5–1:5 h) and sodium hydroxide (0:25–1:5% w=v NaOH at 1:05 kg=cm2 for 0:5–1:5 h) using solid : liquid ratio of 0:05 g=ml and subsequently sacchari�ed enzymat-ically. Steam explosion at 1:05 kg=cm2 pressure for 1:5 h was found to be the optimum pretreatment. Maximum enzymaticsacchari�cation of 57.8% was observed by treating 5% (w=v) pretreated sun�ower stalks with T. reesei Rut-C 30 cellulase(25 FPU=g) at 50

◦C, pH 5.0 for 72 h. ? 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Sun�ower stalks; Enzymatic sacchari�cation; Steam explosion; Sodium hydroxide pretreatment; Cellulase

1. Introduction

Lignocellulosic materials such as agriculturalresidues, food processing wastes, wood, municipalsolid wastes and wastes from pulp and paper industryare considered as low cost and abundant raw materialsfor bioconversion into sugars which can be fermentedto fuel ethanol.

In lignocellulosic materials cellulose, a linearpolymer of glucose is associated with hemicelluloseand surrounded by lignin seal. Lignin, a complexthree-dimensional polyaromatic matrix prevents en-zymes and acids from accessing some regions ofthe cellulose polymers. Crystallinity of the cellulosefurther impedes acid and enzymatic hydrolysis [1,2].

The pretreatment of lignocellulosics is primarilyemployed to increase the accessible surface area of

∗ Corresponding author. P�zer Limited, 178-178A, IndustrialArea, Phase-I, Chandigarh 160 002, India. Tel.: +91-172-650578;fax: +91-172-655178.

E-mail addresses: sanjeev.sharma@p�zer.com (S.K. Sharma),[email protected] (K.L. Kalra).

cellulose to enhance the conversion of cellulose to glu-cose. The commonly used methods for breakdown ofcellulose to glucose are acid and enzymatic hydrol-ysis. Each method has its advantages and disadvan-tages, but the overriding factor in the long run must below energy requirement and low pollution. Enzymatichydrolysis is not only energy sparing, because of therelatively mild reaction conditions but also avoids theuse of toxic and corrosive chemicals.

Various crop residues like wheat straw, rice straw,corn stalks and cobs, groundnut shells, etc., have beenused for ethanol production but there is no report tothe best of our knowledge on utilization of sun�owerstalks for ethanol production. This crop was cultivatedin an area of 2.2 million hectares with production of1.50 million metric tons in India in 1998 [3]. Thisresult in huge quantity of sun�ower stalks annuallywhich do not �nd any suitable end use and are gen-erally burnt in the �elds causing environmental pollu-tion. Therefore, sun�ower stalks, as lignocellulosics,aFord a renewable and low-cost raw material for theproduction of fermentable sugars.

0961-9534/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved.PII: S 0961 -9534(02)00050 -8

238 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237–243

The main objective of our work is to �nd the optimalconditions for the pretreatment and enzymatic saccha-ri�cation of sun�ower stalks and ultimately to fermentthe sugars to ethanol. In this paper, we report on theoptimization of pretreatment and enzymatic sacchari-�cation of sun�ower stalks.

2. Materials and methods

2.1. Materials

The sun�ower stalks used in the present study werecollected from the experimental farm of Department ofPlant Breeding, Punjab Agricultural University, Lud-hiana, India. The stalks were washed 2–3 times withwater to remove extraneous matter. The sun driedstalks were chopped into 2–3′′ pieces with the helpof an electric chopper and further dried in oven at70◦C to constant weight. Oven dried sun�ower stalkswere then ground (40 mesh) with electric grinder. Theground substrate was stored at room temperature tillfurther use.

2.2. Microorganisms

T. reesei Rut-C 30 NRRL 11460 used in the presentstudy for cellulase production was procured from theARS Patent Culture Collection, United States Depart-ment of Agriculture, Peoria, IL, USA. Culture wasmaintained on PDA slants at 40◦C and subculturedfortnightly.

2.3. Enzyme production

The cellulase was produced by T. reesei Rut-C30 under submerged batch conditions using Andreotii[4] basal medium supplemented with 1% cellulose.One hundred milliliter of basal medium was dispensedinto each of 250 ml Erlenmeyer �asks containing 1 gcellulose. The �asks were autoclaved at 1:05 kg=cm2

for 20 min, cooled to room temperature and inocu-lated with 10 ml of fungal culture pregrown on GYEmedium. Flasks were then placed on rotary shaker(200 rpm) at 28◦C for 8 days. After incubation culturebroth was �ltered and unpuri�ed culture �ltrate wasused as cellulase enzyme in further studies. The cul-ture �ltrate had a �lter paper activity of 1:05 IU=ml,

a CMCase activity of 4:62 IU=ml and a cellobiaseactivity of 0:42 IU=ml as measured by the methodssuggested by Mandels et al. [5].

2.4. Analytical methods

Moisture, crude fat and ash analysis were conductedaccording to AOAC procedures [6]. Protein was deter-mined by the Kjeldahl method. Cellulose content wasdetermined by the method of Crampton and Maynard[7]. Hemicellulose and lignin were determined bythe methods described by Goering and Vansoest [8]and reducing sugars were determined by the DNSmethod [9].

2.5. Pretreatment of sun5ower stalks

Powdered substrate was subjected to physical(steam explosion) and chemical pretreatments priorto enzymatic sacchari�cation. Steam explosion wasperformed in a vertical pressure-cooker-type auto-clave at 1:05 kg=cm2 for 0.5, 1.0 and 1:5 h followedby sudden depressurization by fully opening thesteam exhaust valve of autoclave. Sodium hydrox-ide (0:25–1:5% w=v) pretreatment of substrate wascarried out in an autoclave at 121◦C for 0.5, 1.0 and1:5 h [10]. Solid : liquid ratio in both steam explosionand sodium hydroxide treatment was maintained at0:05 g=ml. In both cases solids recovered by �ltrationwere repeatedly washed with distilled water untilwash water turned to pH 7.0 and oven dried at 60◦C.

2.6. Enzymatic sacchari8cation

The steam exploded pulp of sun�ower stalks ob-tained after pretreatment was sacchari�ed using crudeculture �ltrate of T. reesei Rut-C 30 in 0:1 M citratebuFer (pH 4.8) in stoppered Erlenmeyer �asks. The�asks were shaken at 150 rpm at 50◦C. The initialsolid : liquid ratio used was 4 g=100 ml. The enzymesubstrate ratios studied were 5–25 FPU=g as a littleincrease in hydrolysis eLciency has been reportedfor higher enzyme concentrations [11,12]. Sampleswere withdrawn after intervals of 12 h, centrifugedat 5000 rpm for 20 min and the supernatant wasanalyzed for reducing sugars. To determine eFective-ness of diFerent pretreatments enzyme substrateratio was maintained at 10 FPU=g and substrate was

S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237–243 239

sacchari�ed for 48 h. Other sacchari�cation condi-tions were optimized using enzyme concentration of25 FPU=g of substrate. EFect of substrate concentra-tion (4:0–8:0% w=v), temperature (40–60◦C) and pH(4:0–6:0) on enzymatic sacchari�cation of sun�owerstalks was also studied. The extent of sacchari�cationof sun�ower stalks was calculated using the factor0.9x (reducing sugar concentration obtained=potentialsugar concentration in the pretreated substratesubjected to hydrolysis) [10]. Experiments wereperformed in triplicates and results are analyzed bycomplete randomized factorial design and averagevalues were represented.

3. Results

3.1. Physical and chemical characteristics ofsun5ower stalks

Sun�ower stalks used in the present study werecharacterized for physical and chemical components.The moisture content (on wet weight basis) was9.20%. The ash (%), cellulose (%), hemicellulose(%), lignin (%), crude fat (%) and protein (%) con-tents were 4.57, 38.50, 33.50, 17.50, 1.95 and 2.00on dry weight basis, respectively. Rest 1.98% werethe hot water and organic solvent extractives.

3.2. Pretreatment of sun5ower stalks by physicaland chemical methods

Fig. 1 shows the eFect steam explosion on substratesusceptibility for enzymatic sacchari�cation. Withthe increase in autoclaving time from 0.5 to 1:5 h at1:05 kg=cm2, enzymatic hydrolysis has been continu-ously improved being maximum at 1:05 kg=cm2 for1:5 h (reducing sugars 277:60 mg=g). The autoclavingtime at a pressure of 1:05 kg=cm2 could not be in-creased beyond 1:5 h due to the technical limitationsof the autoclave used in the present studies.

Fig. 2 depicts the eFect of sodium hydroxide con-centration (0:25–1:50% w=v) alongwith autoclaving(0:5–1:5 h at 1:05 kg=cm2) on the enzymatic sac-chari�cation of sun�ower stalks. It was observedthat sodium hydroxide at 0.5% concentration at1:05 kg=cm2 for 1:5 h was more eFective pretreatmentas compared to the other concentrations and time

0.5 1 1.5

Autoclaving Time (hrs) at 1.05 kg/cm²

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265

270

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(mg/

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0.25% (w/v) NaOH

0.50% (w/v) NaOH0.75% (w/v) NaOH

1.00% (w/v) NaOH

1.25% (w/v) NaOH1.50% (w/v) NaOH

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Fig. 2. EFect of sodium hydroxide alongwith autoclavingat 1:05 kg=cm2 on the enzymatic hydrolysis of sun�ower stalks.Incubation time 48 h.

combinations for subsequent enzymatic hydrolysis ofthe substrate (reducing sugars 185:5 mg=g).

It can be concluded that steam explosion atautoclaving pressure of 1:05 kg=cm2 for 1:5 h is thebest pretreatment for sun�ower stalks among theperformed experiments.


3.3. Chemical characteristics of the pretreatedsubstrate

Optimally pretreated sun�ower stalks (with steamat 1:05 kg=cm2 pressure for 1:5 h followed by suddendepressurization) have been analyzed for chemi-cal components. Pretreated sun�ower stalks contain51.0% cellulose, 17.0% hemicellulose and 14.6%lignin. Therefore, by comparison to the chemicalcomponents in the untreated stalks it is clear thatpretreatment solubilized 12.57% cellulose, 66.31%hemicellulose and 44.94% lignin. Extraction yield(fraction of sun�ower stalks recovered after pretreat-ment) was 66.0%.

3.4. Enzymatic sacchari8cation

Enzymatic sacchari�cation of pretreated sun�owerstalks was carried out by culture �ltrate of T. reeseiRut-C 30. The various parameters, viz., enzyme con-centration, incubation period, substrate concentration,hydrogen ion concentration and temperature were op-timized to achieve maximum sacchari�cation of thepretreated sun�ower stalks.

3.4.1. E9ect of enzyme concentration andincubation period on the rate of sacchari8cation

The eFect of T. reesei Rut-C 30 enzyme (concen-trations 5–25 FPU=g of substrate) and the incubationperiod (12–72 h) on the hydrolysis of sun�ower stalkshas been studied and results are presented in Table 1.Sun�ower stalks after sacchari�cation with 5 FPU=genzyme for 12 h yielded 112:63 mg=g reducing sugarswith a corresponding sacchari�cation of 14.91%. Thelevel of reducing sugars signi�cantly (P¡ 0:05) in-creased to 375:70 mg=g with 49.73% sacchari�cationby increasing the enzyme concentration to 25 FPU=gand incubation period to 72 h. The initial increase inthe enzyme dose from 5 to 10 FPU=g led to nearly1:3–2:0-fold increase in the amount of released sugars.However, thereafter the increase observed was less.Likewise the rate of hydrolysis was fast up to 36 h andthen slowed down, resulting in comparatively lowerrate of hydrolysis between 36–48, 48–60 and 60–72 hof incubation. T

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Fig. 3. EFect of substrate concentration on the enzymatic sac-chari�cation of pretreated sun�ower stalks. Enzyme concentration25 FPU=g, incubation period 72 h, temperature 50

◦C, pH 4.8.

3.4.2. E9ect of substrate concentration on theenzymatic sacchari8cation

The results of the eFect of substrate concentration(4:0–8:0% w=v) on enzymatic sacchari�cation areshown in Fig. 3. The rate of sacchari�cation increasedup to substrate concentration of 5.0%. Further increasein the substrate concentration decelerated the rate ofhydrolysis. Maximum sacchari�cation of 56.5% wasachieved at substrate concentration of 5.0%.

3.4.3. E9ect of temperature on enzymaticsacchari8cation

The sacchari�cation of pretreated sun�ower stalkswas carried out at temperature ranging from 40◦C to60◦C. Fig. 4 indicate that the initial hydrolysis rate in-creased with increasing hydrolysis temperature. Max-imum sacchari�cation (56.4%) was observed at 50◦Cwith corresponding reducing sugars 426:2 mg=g. De-creased sacchari�cation was observed at temperatureshigher than the optimum.

3.4.4. E9ect of pH on enzymatic sacchari8cationThe sacchari�cation of pretreated sun�ower stalks

was carried out at a range of pH values (4:0–6:0) andresults are presented in Fig. 5. Maximum sacchari�-cation of 57.8% was observed at pH 5.0 with corre-sponding reducing sugars of 436:6 mg=g. Decreasedproduction of reducing sugars as well as percent

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Temperature (°C)

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250

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55

60

Rea

ding

sug

ars

(mg/

g)Fig. 4. EFect of temperature on the enzymatic sacchari�cationof pretreated sun�ower stalks. Enzyme concentration 25 FPU=g,incubation period 72 h, substrate concentration 5% (w=v), pH 4.8.

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40.5

50.5

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Fig. 5. EFect of pH on the enzymatic sacchari�cation of pretreatedsun�ower stalks. Enzyme concentration 25 FPU=g, incubation pe-riod 72 h, substrate concentration 5% (w=v), temperature 50

◦C.

sacchari�cation was obtained at pH values lesser orhigher than optimum.

In view of the results obtained it can be concludedthat optimum sacchari�cation of sun�ower stalkscould be achieved by treating 5% (w=v) pretreatedstalks with 25 FPU=g T. reesei Rut-C 30 cellulase at50◦C, pH 5.0 for 72 h.


4. Discussion

Ground sun�ower stalks (40 mesh) were pretreatedand then sacchari�ed by the T. reesei Rut-C 30 cellu-lase. Reducing sugars thus formed have the potentialfor conversion to bioethanol. Steam explosion pre-treatment by autoclaving at a pressure of 1:05 kg=cm2

for 1:5 h followed by sudden depressurization wasfound to be the best among diFerent time and pressurecombinations tried. Other workers [13–15] have alsoobserved signi�cant increase in enzymatic hydrolysisof the cellulose substrate by pretreatment with steamexplosion method. Pretreatment with 0:5% (w=v)sodium hydroxide at an autoclaving pressure of1:05 kg=cm2 for 1:5 h was more eFective among dif-ferent concentration and time combinations tried forsubsequent enzymatic hydrolysis of the substrate.Results are similar to the conclusions of diFerentauthors [10,16]. A sodium hydroxide concentrationof 1% and steam pressure of 1 kg=cm2 for 1 h hasbeen reported as optimum for deligni�cation of waterhyacinth [17]. At higher pretreatment severities it islikely to have a better access to the �ber. This shouldbe tested since the optimum obtained in this work isat the highest severity experiment.

Among two types of pretreatments studied steamexplosion was found to be the best for subsequentenzymatic hydrolysis of sun�ower stalks. This mightbe due to less lignin content in the sun�ower stalksas sodium hydroxide basically acts as deligni�ca-tion agent. Also at higher temperatures sodium hy-droxides might cause important material loss [18].Petreatment of sun�ower stalks by steam explosionunder optimized conditions solubilized 12.57%cellulose, 66.31% hemicellulose and 44.94% lignin.Extraction yield obtained was 66.0%. Accordingto Szczodrak [11] hydrothermal action favor thepentosan degradation giving a 93.5% loss of thiscomponent after the autohydrolysis reaction of wheatstraw. Dekker and Wallis [19] reported that autohy-drolysis of sun�ower seed hulls at 200◦C for 5 min,followed by explosive de�bration solubilized 78% ofthe total hemicellulose and 85% of the pectic sub-stances. The remaining exploded pulp was reportedto be highly susceptible to hydrolysis by cellulase.

Enzymatic sacchari�cation of the pretreated sun-�ower stalks was carried out by T. reesei Rut-C 30cellulase. Enzyme concentration of 25 FPU=g of the

substrate and incubation time of 72 h resulted in themaximum sacchari�cation (49.73%) of the substrate.The sacchari�cation rate at the start of incubationperiod was higher and then it slowed down. Thisbehavior might be due to the decrease in the extentof adsorbed enzyme, transformation of the structureof cellulose into less digestible form and inhibitionof the enzyme action by the accumulated hydrolysisproducts [20]. The initial (1:3–2:0-fold) increase inthe reducing sugars with increase in enzyme dosefrom 5 to 10 FPU=g and slow increase in reducingsugars afterwards, might be due to the less adsorptioneLciency for higher enzyme concentrations than fordiluted ones [10]. Saturation of the cellulose surfacewith enzyme might be the other reason behind it.Slow rate of sacchari�cation after 60 h has also beenreported earlier [12,21,22].

The substrates concentration of 5.0% (0:05 g=ml)resulted in the maximum sacchari�cation. Furtherincrease in substrate concentration decelerated therate of hydrolysis. Substrate concentrations over0:05–0:075 g=ml have been shown to cause substrateinhibition both with pure cellulose and pretreated lig-nocellulosic materials [10,20,23]. However, substrateconcentration of 6% [24] and 10% [22] has also beenfound adequate to release optimum reducing sugars.Stirring diLculties, reduction of the aqueous movablephase and end product inhibition can hinder the enzy-matic hydrolysis of pretreated lignocellulosic residuesat higher substrate concentration [11,20].

Most suitable temperature for enzymatic hydrolysisof sun�ower stalks was found to be 50◦C. At temper-atures lower or higher than this less sacchari�cationwas observed. Reduced sacchari�cation at higher tem-perature could be attributed to the thermal inactivationof endoglucanase I and cellobiohydrolase I [25,26].The temperature of 50◦C was also found optimum forenzymatic sacchari�cation of diFerent lignocellulosicmaterials [11,27–29]. However, a hydrolysis temper-ature of 40◦C was found optimum for high glucoseyield and low enzyme deactivation [21] A pH value of5.0 was found to be optimum for enzymatic saccha-ri�cation of sun�ower stalks. Decreased sacchari�ca-tion was observed at pH values lesser or higher thanthe optimum. This might be due to the requirement ofthe cellulase enzyme for speci�c hydrogen ion con-centration in the reaction mixture for eLcient perfor-mance. The results with respect to optimum pH are


in close agreement with those of the earlier workers[11,27,28,30].

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enzymatic saccharification of pretreated sunflower stalks

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