Bioresource Technology 100 (2009) 1214–1220

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Bioresource Technology

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Separate hydrolysis and fermentation (SHF) of Prosopis juliflora,a woody substrate, for the production of cellulosic ethanolby Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Rishi Gupta, Krishna Kant Sharma, Ramesh Chander Kuhad *

Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110 021, India

a r t i c l e i n f o

Article history:Received 19 April 2008Received in revised form 20 August 2008Accepted 20 August 2008Available online 2 October 2008

Keywords:Lignocellulosic biomassProsopis julifloraAcid hydrolysisEnzymatic hydrolysisFermentation

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.08.033

* Corresponding author. Tel.: +91 124 24112972; faE-mail address: kuhad85@gmail.com (R.C. Kuhad)

a b s t r a c t

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol.In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pre-treatment to produce more sugar, to be fermented to ethanol. Dilute H2SO4 (3.0%, v/v) treatment resultedin hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byprod-ucts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated sub-strate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%, w/v) andsodium chlorite (3.0%, w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M cit-rate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of b-glucosidase permL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%)was achieved after 28 h incubation at 50 �C. The fermentation of both acid and enzymatic hydrolysates,containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.

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1. Introduction

Worldwide high demand for energy, uncertainty of petroleumresources and concern about global climatic changes has led tothe resurgence in the development of alternative liquid fuels. Eth-anol has always been considered a better choice as it reduces thedependence on reserves of crude oil and promises cleaner combus-tion leading to a healthier environment. Developing ethanol as fuelbeyond its current role of fuel oxygenate, would require lignocell-ulosics as a feedstock because of its renewable nature, abundanceand low cost (Saha et al., 2005).

Lignocelluloses are mainly comprised of cellulose, a polymer ofsix-carbon sugar, glucose; hemicellulose, a branched polymer com-prised of xylose and other five-carbon sugars and lignin consistingof phenyl propane units. The presence of lignin limits the fullestusage of cellulose and hemicellulose. To convert these energy richmolecules into simpler forms, it is necessary to remove the ligninfrom lignocellulosic materials. A number of pretreatments suchas concentrated acid hydrolysis (Liao et al., 2006), dilute acidhydrolysis (Cara et al., 2008), alkali treatment (Carrillo et al.,2005), sodium sulphite treatment (Kuhad et al., 1999; Kapooret al., 2008), sodium chlorite treatment (Sun et al., 2004), steam

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explosion (Ohgren et al., 2005), ammonia fiber explosion (Teymo-uri et al., 2005) lime treatment (Kim and Holtzapple, 2005), and or-ganic solvent treatment (Xu et al., 2006) have been used frequentlyto remove lignin and improve the saccharification of the cell wallcarbohydrates.

Of these methods, dilute acid treatment and enzymatic hydroly-sis have been the most popular ones. Dilute acid hydrolysis is a fastand convenient method to perform but it leads to the accumulationof fermentation inhibitory compounds such as furfurals, hydroxymethyl furfurals (HMF) and phenolics. These compounds, depend-ing on their concentration in the fermentation media, can inhibitmicrobial cell and affect the specific growth rate and cell-massyield. Several treatments e.g., ion exchange (Canilha et al., 2004;Chandel et al., 2007), overliming (Martinez et al., 2001; Chandelet al., 2007), activated charcoal adsorption (Mussatto et al., 2004;Canilha et al., 2004; Chandel et al., 2007), and laccase oxidationtreatment (Chandel et al., 2007) have been reported for thedetoxification of hydrolysate to improve the fermentability of acidhydrolysates into ethanol. However, the combination of pH adjust-ment by overliming followed by activated charcoal adsorption hasbeen shown to improve the detoxification of hemicellulosic hydro-lysate (Converti et al., 1999).

The acid hydrolysis pretreatment removes the hemicellulosicportion and some fraction of lignin but rest of the lignin remainsintact to the cellulosic substrate. Kaya et al. (2000) had reportedthat during enzymatic hydrolysis of lignocellulosic biomass,

R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220 1215

cellulase components, b-glucosidase and endoglucanase have morebinding affinity towards lignin than to the carbohydrates, resultingin lower efficiency of saccharification. Hence, to achieve maximumhydrolysis of cellulosics, which is a prerequisite for ethanol fer-mentation, an appropriate delignification treatment of biomass isrequired. In the present work, the combination of sodium sulphiteand sodium chlorite for the delignification of cellulosic biomasshas been attempted.

The cellulosic and hemicellulosic sugars obtained through acidand enzymatic hydrolysis can efficiently be used for ethanol fer-mentation either by separate fermentation of individual hydroly-sate or fermentation of mixed hydrolysate using co-culture.However, in co-culture cultivation, optimum growth conditionsof the yeasts would be different and might result in lowerefficiency and lower product yield. Hence, for better efficiency ofethanol production, the approach of separate hydrolysis andfermentation (SHF) was preferred (Olsson and Hahn-Hagerdal,1993).

In the present study, Prosopis juliflora (Mesquite), a perennialdeciduous thorny shrub, the common vegetation of semi-aridregion of Indian subcontinent, was used as a raw material for theproduction of cellulosic ethanol. The mesquite has recently beensuggested to be used as raw material for long-term sustainableproduction of cellulosic ethanol (Hopkins, 2007). Its nature totolerate drought, grazing, heavy soil, sand as well as saline dry flatsand no competence with animal feed demand made it a potentiallow value substrate for ethanol production. Here, an attempt wasmade to saccharify P. juliflora into reducing sugars and eventuallyto ethanol fermentation.

2. Methods

2.1. Raw material and chemicals

Prosopis juliflora wood, collected from University of Delhi SouthCampus, New Delhi, India, was comminuted by a combination ofchipping and milling to attain a particle size of 1–2 mm using alaboratory knife mill (Metrex Scientific Instrumentation, Delhi, In-dia). The processed substrate was washed thoroughly and driedovernight at 60 �C.

Commercial cellulase from Trichoderma reesei (ATCC 26921)(6.5 FPU/mg), b-glucosidase (Novozyme 188) (250 U/g) from Asper-gillus niger and 3,5-dinitrosalicylic acid (DNS) were purchased fromSigma, St. Louis, Missouri, USA. Ethanol was purchased from Merck(Darmstadt, Germany). Rest of the chemicals and media compo-nents of highest purity grade were purchased locally.

2.2. Micro-organisms and culture conditions

Pichia stipitis NCIM 3498 was procured from National ChemicalLaboratory (NCL), Pune, India and was maintained on agar slantscontaining (g/L): xylose, 20.0; yeast extract, 4.0; peptone, 5.0;KH2PO4, 1.5; MgSO4 � 7H2O, 0.5; agar, 20.0 at pH 5.0 ± 0.2 and tem-perature 30 �C. Saccharomyces cerevisiae, was procured from theculture collection of University of Delhi South Campus, New Delhi,India, and maintained on agar slants containing (g/L): glucose,30.0; yeast extract, 3.0; peptone, 5.0; agar, 20.0 at pH 6.0 ± 0.2and temperature 30 �C.

Inoculum of P. stipitis was prepared as described by Nigam(2001) using (g/L): xylose, 50.0; yeast extract, 3.0; malt extract,3.0; peptone, 5.0 at pH 5.0 ± 0.2 and temperature 30 �C. Saccharo-myces. cerevisiae inoculum was grown for 24 h at 30 �C in a culturemedium containing (g/L): glucose, 30.0; yeast extract, 3.0; peptone,5.0; (NH4)2HPO4, 0.25 at pH 6.0 ± 0.2 (Chen et al., 2007). Cells werecultured to an optical density of 0.6–0.8 at 620 nm.

2.3. Proximate chemical composition analysis of the substrate

The chemical composition of P. juliflora wood was analysed forholocellulose, Klason lignin, pentosans, ash and moisture content.The plant material was extracted with alcohol–benzene (1:2 v/v)to remove wax, resin etc. The extractive-free wood dust was pro-cessed for chemical analysis following the TAPPI (1992) protocols,(a-Cellulose–TAPPI Method T203 om–83; Klason lignin–TAPPIMethod T222 om–83; Pentosans–TAPPI Method T223 hm–84;Moisture–TAPPI Method T208 om–84 and Ash–TAPPI method211om–93).

2.4. Dilute acid pretreatment

The dilute sulphuric acid pretreatment of wood dust wasoptimized at varied temperatures (100–140 �C), treatment time(15–60 min) and acid concentrations (1.0–5.0%, v/v) at 10.0%(w/v) consistency, in a 20.0 L plastic vessel (Carboy, Tarson Pvt.Ltd., Kolkata, India), using an autoclave (Russian make). The acidhydrolysate after treatment was recovered by filtering the contentsthrough double-layered muslin cloth. The remaining wood dustwas washed with tap water till neutral pH. The hydrolysate wasanalysed for sugars, phenolics, acetic acid and furans and theleftover plant biomass was dried overnight till constant weightand used for further experiments.

2.5. Detoxification of acid hydrolysate

The acid hydrolysate was overlimed at room temperature byadding dried lime [Ca(OH)2] till the pH reached 10.0, with constantstirring for 30 min by an overhead stirrer (Remi Motors Ltd,Mumbai, India). After overliming, the hydrolysate was neutralizedwith concentrated H2SO4 and centrifuged at 10,000g for 15 min toremove the precipitate formed during neutralization. Theoverlimed hydrolysate was further detoxified by treating withactivated charcoal (1.5%, w/v) with constant stirring at room tem-perature for 30 min and the sugar syrup was recovered throughvacuum filtration.

2.6. Chemical delignification of acid pretreated woody biomass

Acid pretreated residue of P. juliflora was delignified by treatingwith sodium sulphite (5.0–20.0%, w/v) alone and combination ofsodium sulphite (5.0%, w/v) and sodium chlorite (3.0%, w/v) andautoclaved at different temperatures (100–140 �C) for differenttime intervals (15–60 min). The delignified material was thenfiltered through double-layered muslin cloth and the leftovercellulosic residue was washed repeatedly with water till neutralpH. The cellulosic residue was dried overnight at 60 �C till constantweight and hydrolyzed enzymatically.

2.7. Enzymatic hydrolysis of delignified cellulosic substrate

Enzymatic hydrolysis of cellulose (delignified acid treated plantmaterial) was carried out at a 5.0% (w/v) consistency in 0.05 Mcitrate phosphate buffer (pH 5.0) containing 0.005% sodium azide.Before enzyme loading, slurry was acclimatized by incubating at50 �C on a rotatory shaker (Innova-40, New Brunswick Scientific,Germany) at 150 rpm for 2 h. Thereafter, a mixture of 3.0 U of filterpaper cellulase (FPase) and 9.0 U of b-glucosidase per mL of citratephosphate buffer was added to preincubated cellulose slurry andreaction continued for 36 h. Samples were withdrawn at regularinterval of 4 h, centrifuged at 10,000g for 15 min and the superna-tant was analysed for total reducing sugars released. The extent ofhydrolysis was calculated as follows:

1216 R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220

Saccharificationð%Þ

¼ Reducing sugar concentration obtainedPotential sugar concentration in the pretreated substrate

� 100

Different surfactants (1.0%, v/v or w/v) including nonionicsurfactants (Tween 20, Tween 40, Tween 60, Tween 80 andTriton X-100), polyethylene glycols (PEG 4,000, PEG 6,000 andPEG 10,000) and ionic surfactants (sodium dodecyl sulphite(SDS) and cetyl trimethyl ammonium bromide (CTAB)) wereused for studying their effect in improving enzymatic sacchari-fication.

2.8. Ethanol fermentation

The acid and enzymatic hydrolysates were fermented in anin situ sterilizable fermenter (B-Lite Sartorius India Ltd., Banga-lore, India) having geometric volume of 13.5 L and working vol-ume of 10.0 L. The acid hydrolysate (9.0 L) containing 18.24 g/Lsugars was supplemented with nutrients (g/L) NH4Cl, 0.5;KH2PO4, 2.0; MgSO4 � 7H2O, 0.5; Yeast extract, 1.5; CaCl2 � 2H2O,0.1; FeCl3 � 2H2O, 0.1 and ZnSO4 � 7H2O, 0.001 (pH 5.5 ± 0.2) andinoculated with 10.0% (v/v) culture of P. stipitis (OD 0.6). Whilecellulosic hydrolysate having 37.47 g/L sugars supplementedwith yeast extract, 3.0 g/L and (NH4)2HPO4, 0.25 g/L was fer-mented with S. cerevisiae (10.0%, v/v; OD 0.6). The agitation of150 revolutions per min (rpm), aeration 0.4 liter per minute(lpm), temperature 30 �C and pH 5.5 ± 0.2 were maintainedthroughout the process. The pH was adjusted with 2 N HCl and2 N NaOH, as required. A 10.0% (w/v) solution of siliconeantifoaming agent was used for controlling foam as and whenrequired. The dissolved oxygen was monitored continuously.Samples were withdrawn at regular intervals of 4 h and centri-

Table 1Release of sugars and phenolics during the dilute sulphuric acid (H2SO4) hydrolysis of P. j

Time (min) 15 30

Temperature Acid concentration Sugar(mg/g)

Phenolics(mg/g)

Sugar(mg/g)

100 �C 1% 94.36 6.38 114.61(14.25)a (2.19) (17.31

2% 125.75 6.80 150.00(18.99) (2.34) (22.66

3% 144.29 7.56 159.05(21.8) (2.6) (24.03

4% 142.42 8.21 149.13(21.51) (2.82) (22.53

5% 139.17 8.88 142.70(21.02) (3.05) (21.56

120 �C 1% 108.10 6.71 122.07(16.33) (2.30) (18.44

2% 144.19 7.07 163.10(21.78) (2.43) (24.64

3% 165.24 7.83 179.37(24.96) (2.69) (27.10

4% 151.27 9.00 164.61(22.85) (3.09) (24.86

5% 146.99 9.21 157.46(22.20) (3.16) (23.79

140 �C 1% 117.57 7.68 130.75(17.76) (2.64) (19.75

2% 150.94 8.00 167.67(22.80) (2.75) (25.33

3% 172.65 8.47 176.77(26.08) (2.91) (26.7)

4% 163.87 9.61 154.05(24.75) (3.30) (23.27

5% 155.24 10.435 148.87(23.45) (3.59) (22.49

a Values in parenthesis are sugar and phenolic yield with respect to total carbohydra

fuged at 10,000g for 15 min at 4 �C. The cell free supernatantswere used for the determination of ethanol produced and sugarconsumed. An attempt was also made to test the efficacy of theyeast to increase ethanol production by growing it in mediumhaving initial sugar load of 100 g/L.

2.9. Analytical methods

The hydrolysates were analysed using high performance liquidchromatography (HPLC) (Shimadzu Kyoto, Japan) for the presenceof carbohydrates. Aminex (Bio-Rad, Hercules CA USA) column(300.0 � 7.8 mm) was used with 0.04 M H2SO4 as an eluent withflow rate of 0.5 mL/min keeping oven temperature at 60 �C withRID detector. Furans, furfurals and lignin derivatives were esti-mated with Luna 5 U C18 column (250.0 � 4.6 mm). The analysiswas done by using acetonitrile: 1.0% phosphoric acid (22:78) asmobile phase with flow rate 0.5 mL/min and oven temperature35 �C with UV–detector. Ethanol was estimated by Gas chroma-tography (GC) (Perkin Elmer, Clarus 500) with an elite-wax (crossbond-polyethylene glycol) column (30.0 m � 0.25 mm) at oventemperature of 85 �C and flame ionization detector (FID) at200 �C. The ethanol standards were prepared using commercialgrade ethanol (Merck, Darmstadt, Germany). Nitrogen with a flowrate of 0.5 mL/min was used as carrier gas. Total reducing sugarswere estimated by the DNS method and the total phenolicsreleased were determined by the Folin–Ciocalteu reagent method(Singleton et al., 1999) using vanillin as standard. The optical den-sity (A600 nm) of culture filtrate was measured using a doublebeam spectrophotometer (Specord 200). Dry biomass of yeastcells was measured after drying the yeast pellets at 70 �C till con-stant weight.

uliflora wood at different temperatures

45 60

Phenolics(mg/g)

Sugar(mg/g)

Phenolics(mg/g)

Sugar(mg/g)

Phenolics(mg/g)

7.59 138.71 9.04 142.45 9.60) (2.61) (20.95) (3.11) (21.52) (3.30)

8.13 161.00 9.24 161.83 10.51) (2.79) (24.32) (3.18) (24.44) (3.61)

8.30 176.67 4.55 177.35 11.565) (2.85) (26.69) (1.56) (26.79) (3.97)

9.48 164.45 10.61 166.27 11.71) (3.26) (24.84) (3.64) (25.12) (4.02)

9.71 156.75 10.62 159.29 12.24) (3.34) (23.68) (3.65) (24.06) (4.20)

7.91 148.56 9.37 154.69 9.37) (2.72) (22.44) (3.22) (23.37) (3.22)

8.40 185.80 9.51 189.05 9.51) (2.88) (28.07) (3.27) (28.56) (3.27)

8.80 200.50 10.05 204.84 10.05) (3.02) (30.29) (3.45) (30.94) (3.45)

9.81 190.88 10.93 198.62 10.93) (3.37) (29.34) (3.76) (29.94) (3.76)

10.02 190.34 10.56 188.34 11.06) (3.44) (28.75) (3.63) (28.45) (3.80)

8.36 140.23 9.67 156.86 9.835) (2.87) (21.18) (3.32) (23.69) (3.38)

8.74 182.56 10.61 181.95 10.95) (3.00) (27.58) (3.65) (27.48) (3.76)

8.36 169.14 11.47 160.79 11.86(2.87) (25.55) (3.94) (24.29) (4.07)10.14 149.07 11.75 135.41 12.03

) (3.48) (22.52) (4.04) (20.45) (4.13)8.36 126.86 12.23 114.69 12.87

) (2.87) (19.16) (4.20) (17.32) (4.42)

te and total lignin content of the substrate.

R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220 1217

3. Results

3.1. Proximate chemical composition of P. juliflora

Prosopis juliflora wood contained 66.20% holocellulose (47.50%a-cellulose and 18.70% pentosans), 29.10% Klason lignin, 2.68%moisture and 2.02% ash content.

3.2. Dilute acid hydrolysis of P. juliflora

The woody material when hydrolyzed with different concentra-tion of dilute H2SO4, at 100 �C and 120 �C, the release in sugarincreased with increase in acid concentration upto 3.0% (v/v)H2SO4 and it declined thereafter. While at 140 �C, the acid concen-tration beyond 2.0% (v/v) resulted in continuous decrease inrelease of sugar. The maximum sugars (204.84 mg/g) werereleased, when the woody material was treated with 3.0% H2SO4

at 120 �C for 60 min. However, no significant difference in sugarreleased was observed when hydrolysis was carried either for 45or 60 min. (Table 1). The acid hydrolysis also resulted in releaseof phenolics ranging from 2.0–4.5% (w/w of phenolics present insubstrate). Under optimized hydrolysis conditions (3.0% H2SO4,120 �C, 60 min), the hydrolysate was found to contain furfural(0.34 mg/mL), HMF (0.58 mg/mL) and caffeic acid (0.13 mg/mL).

3.3. Detoxification of acid hydrolysate

The sequential detoxification of acid hydrolysate using overlim-ing and activated charcoal resulted in a drastic decrease in the con-centrations of inhibitors. Overliming resulted in reduction of HMF

Table 2Release of phenolics during the delignification of acid treated biomass of P. juliflora

Time (min) 15 30 45 60

Temperature Chemicalsb

(%, w/v)Phenolics(mg/g)

Phenolics(mg/g)

Phenolics(mg/g)

Phenolics(mg/g)

100 �C S 5 92.21 105.54 149.98 149.98(31.69)a (36.27) (51.54) (51.54)

S 10 125.54 137.76 167.76 170.87(43.14) (47.34) (57.65) (58.72)

S 15 152.21 158.87 174.43 181.1(52.3) (54.6) (59.94) (62.23)

S 20 163.32 169.98 187.76 191.1(56.12) (58.41) (64.52) (65.67)

S + C 5 + 3 187.42 194.11 212.76 216.89(64.41) (66.7) (73.11) (74.53)

120 �C S 5 111.54 126.87 156.65 163.32(38.33) (43.6) (53.83) (56.12)

S 10 154.43 165.1 191.1 201.54(53.07) (56.73) (65.67) (69.26)

S 15 180.21 193.32 206.65 214.65(61.93) (66.43) (71.01) (73.76)

S 20 187.1 200.43 211.1 214.43(64.29) (68.88) (72.54) (73.69)

S + C 5 + 3 255.26 271.1 270.47 270.28(87.72) (93.16) (92.95) (92.8)

140 �C S 5 127.76 143.32 189.98 187.76(43.9) (49.25) (65.29) (64.52)

S 10 189.98 198.87 231.1 234.43(65.29) (68.34) (79.41) (80.56)

S 15 207.76 238.87 241.1 241.1(71.4) (82.09) (82.85) (82.85)

S 20 215.54 244.43 244.43 246.65(74.07) (84) (84) (84.76)

S + C 5 + 3 260.15 270.44 270.37 270.25(89.04) (92.81) (92.6) (92.51)

a Values in parenthesis are yield of phenolics with respect to total lignin contentpresent in substrate.

b Delignifying chemicals used; S, sodium sulphite and C, sodium chlorite.

(51.18%), furfural (59.0%), acetic acid (12.4%) and caffeic acid(20.08%) followed by further detoxification with activated charcoaladsorption with additional removal of HMF (38.24%), furfural(29.31%), acetic acid (45.26%) and caffeic acid (74.92%).

3.4. Delignification of pretreated wood

A regular increase in delignification of woody material was ob-served with the increase in concentration of sodium sulphite from5.0% to 20.0% (w/v) (Table 2). The treatment of woody biomasswith 20.0% (w/v) sodium sulphite at 140 �C and 60 min resultedin release of 246.65 mg/g of phenolics. However, when sodiumchlorite (3.0%, w/v) was used along with sodium sulphite (5.0%,w/v), it reduced the concentration of sodium sulphite from 20.0%(w/v) to 5.0% (w/v), and released maximum phenolics at 120 �Cfor 30 min. Moreover, at optimized conditions, the release ofphenolics (271.10 mg/g) was found to be 8.4% (w/w) higher thanthe phenolics released by the sodium sulphite alone (20.0%, w/v,140 �C and 60 min).

3.5. Enzymatic saccharification of delignified cellulosic material

During the time course of enzymatic saccharification of deligni-fied cellulosic substrate, a regular increase in release of sugars wasobserved till 28 h of incubation, which remained almost constantthereafter, however, after attaining the maximum rate of sacchar-ification (49.58 mg/g/h, 4 h), the saccharification rate decreasedregularly showing the reciprocal relationship with saccharificationyield (Fig. 1). Moreover, the maximum yield of saccharification,586.16 mg/g, was achieved after 28 h incubation, with saccharifi-cation rate of 20.60 mg/g/h (Fig. 1).

Different ionic and nonionic surfactants were evaluated fortheir ability to improve the enzymatic hydrolysis of delignified P.juliflora. The amount of sugar released in the presence of Tween80 increased maximally by 47.51% compared to control and fol-lowed by PEG 4,000 (43.81%) and Tween 40 (39.17%). However,the quantity of sugar released in presence of ionic surfactantse.g., CTAB and SDS, decreased by 60.12% and 48.24%, respectively(Fig. 2).

3.6. Fermentations of hemicellulosic and cellulosic hydrolysates

The hemicellulosic hydrolysate containing 18.24 g/L sugarswhen fermented with P. stipitis produced 7.13 g/L ethanol with ayield of 0.39 g/g and productivity of 0.30 g/L/h after 24 h. After24 h of fermentation, P. stipitis produced biomass (5.96 g/L) withyield and productivity, 0.33 g/g and 0.25 g/L/h, respectively (Table

0

100

200

300

400

500

600

700

12 16 20 24 28 32 360

10

20

30

40

50

60Sugar yield

Sacc

hari

fica

tion

rat

e (m

g/g/

h)

Saccharification rate

Time(h)4 8

Suga

r y

ield

(m

g/g)

Fig. 1. Enzymatic saccharification profile of delignified P. juliflora.

0

100

200

300

400

500

600

700

800

900

Suga

r yi

eld

Surfactants

T-20 T-40 T-60 T-80 T X-100 CTAB SDS PEG4000

PEG10000

PEG20000

Control

Fig. 2. Effect of different surfactants on the enzymatic hydrolysis of delignified P. juliflora after 36 h. (T, Tween (1%, v/v); TX, Triton (1%, v/v); CTAB, cetyl trimethyl ammoniumbromide (1%, w/v); SDS, sodium dodecyl sulphite (1%, w/v); PEG, polyethylene glycol (1%, w/v)).

Table 3Fermentation profile of detoxified acid hydrolysate using P. stipitis

Time (h) Ethanol (g/L) Sugar (g/L) Ethanol yield (g/g) Ethanol productivity (g/L/h) Biomass (g/L) Biomass yield (g/g) Biomass productivity (g/L/h)

0 0.17 18.24 0.01 0.00 0.15 0.01 0.004 0.19 17.43 0.01 0.05 1.47 0.08 0.378 1.08 15.09 0.06 0.14 2.91 0.16 0.3612 3.33 11.17 0.18 0.28 3.59 0.20 0.3016 4.41 9.02 0.27 0.3 4.55 0.25 0.2820 6.24 5.39 0.34 0.31 5.29 0.29 0.2624 7.13 3.51 0.39 0.3 5.96 0.33 0.2528 6.95 2.87 0.38 0.25 6.12 0.34 0.2232 6.71 2.05 0.37 0.21 6.23 0.34 0.19

Table 4Fermentation profile of enzymatic hydrolysate using S. cerevisiae

Time (h) Ethanol (g/L) Sugar (g/L) Ethanol yield (g/g) Ethanol productivity (g/L/h) Biomass (g/L) Biomass yield (g/g) Biomass productivity (g/L/h)

0 0.49 37.47 0.03 0.00 0.17 0.00 0.004 11.61 15.29 0.28 2.44 1.91 0.05 0.488 16.13 5.51 0.43 2.02 3.90 0.10 0.4912 17.47 3.84 0.47 1.46 6.05 0.16 0.5016 18.52 1.86 0.49 1.16 8.02 0.21 0.5020 16.81 1.67 0.45 0.84 7.99 0.21 0.4024 14.90 1.52 0.40 0.62 7.95 0.21 0.33

0

20

40

60

80

100

120

0 12 16 20 24 280.0

1.0

2.0

3.0

4.0

5.0

6.0

Suga

r an

d E

than

ol c

once

ntra

tion

(g/

L)

Time (h)

Eth

anol

Yie

ld (

g/g)

and

Pro

duct

ivit

y (g

/L/h

)

Ethanol production (g/L)Sugar consumption (g/L)Ethanol Yield (g/g)Ethanol Productivity (g/L/h)

4 8

Fig. 3. Time course for the production of ethanol from glucose (100 g/L).

1218 R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220

3). Fermentation of cellulosic hydrolysate (37.47 g/L) using S.cerevisiae, gave maximum ethanol (18.52 g/L) with yield (0.49 g/g) and productivity (1.16 g/L/h) after 16 h, whereas, the biomasswas found to be 8.02 g/L having yield (0.21 g/g) and productivity(0.50 g/L/h) (Table 4). However, S. cerevisiae, when grown inmedium containing 100 g/L glucose produced 42.5 g/L ethanolwith a yield of 0.43 g/g and productivity 2.66 g/L/h (Fig. 3).

4. Discussion

Dilute acid pretreatment method is commonly used to sacchar-ification of any lignocellulosic biomass. It has the dual advantage ofsolubilizing hemicellulose and further converting it to fermentablesugars. In the present work, dilute sulphuric acid pretreatment ofmilled wood of P. juliflora was optimized to achieve maximumsugar yield at minimum severity conditions. The optimum pre-treatment conditions i.e., 3.0% (v/v) acid at 120 �C for 45 min, gave

R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220 1219

a saccharification yield of 200.50 mg/g. While any further increasein pretreatment stringency caused the increase in release of toxiccompounds without much effect on sugar yield. Increase in toxiccompounds with corresponding increase in acid concentration,suggested to use the minimal acid concentration with reducedinhibitory compound and achieving maximum sugar hydrolysis.

The mechanistic rationale for the inhibitory action of furfuraland HMF on yeast cultures could be the result of decrease in theactivities of aldehyde dehydrogenase (AlDH), pyruvate dehydroge-nase (PDH) and alcohol dehydrogenase (ADH) (Modig et al., 2002).Detoxification of acid hydrolysate using Ca(OH)2 effectivelyreduced the level of inhibitors. This may be the result of eitherpolymerization or chemical transformations of these inhibitors athigher pH (Martinez et al., 2001). Previous overliming reportsshowed the similar trends of decrease in inhibitors present in acidhydrolysates (Martinez et al., 2001; Chandel et al., 2007). Whileactivated charcoal being hydrophobic in nature removes thehydrophobic inhibitory compound i.e., furan and phenolics moreeffectively (Saha, 2004; Chandel et al., 2007). Our results showedthat detoxification of acid hydrolysate using Ca(OH)2 followed byactivated charcoal adsorption had resulted in almost complete re-moval of majority of fermentation inhibitory compounds exceptacetic acid, which was removed partially (58%). However, the leftover concentration of acetic acid was lower than the minimuminhibitory concentration i.e., 5.0 g/L as reported by Taherzadehet al. (1997). Moreover, the yeast can grow on a media containingacetic acid up to 20 g/L at pH (5.5) (Taherzadeh et al., 2000). Con-verti et al. (1999) had also shown the detoxification of hydrolysatethrough sequential steps of overliming and activated charcoaladsorption. However, removal of phenolics using laccase had alsobeen reported (Chandel et al., 2007).

The chemical delignification of lignocellulosic material has pre-viously been reported to achieve better enzymatic saccharificationas compared to untreated sample (Saha, 2004; Kapoor et al., 2008).Earlier, Kuhad and coworkers had reported sodium sulphite (13.7%,w/v) for efficient delignification of biomass (Kuhad et al., 1999;Kapoor et al., 2008). In the present study, the maximum delignifi-cation was observed in P. juliflora when pretreated with combina-tion of sodium sulphite (5.0%, w/v) and sodium chlorite (3.0%, w/v).It is interesting to note that the overall concentration of sodiumsulphite (20.0%, w/v) was drastically reduced to 5.0% (w/v) withaddition of merely 3.0% (w/v) sodium chlorite as delignificationagent. Sodium chlorite is an elemental chlorine free compound,which at high temperature produces chlorous (HClO2) and hypo-chlorous acids (HOCl). The subsequent oxidation of chlorous byhypochlorous acid regenerates chlorine dioxide (ClO2) that furtherreact with lignin (Hamzeh et al., 2006). Chlorine dioxide has hadtremendous success as a replacement for chlorine because it is eas-ily substituted in a conventional chlorination stage without anyspecial modification, produces fewer potentially toxic byproductsand causes less damage to the wood fibers (Svenson et al., 2005).

During the enzymatic saccharification of delignified substrate, aregular decrease in the rate of hydrolysis was observed after 4 h ofsaccharification, which may be due to the end product inhibition ofthe enzymes (Kuhad et al., 1999). However, to weaken the feed-back inhibition caused by the cellobiose accumulation, b-glucosi-dase at a 3-fold concentration to FPase was used. In the presentexperiment, 5.0% (w/v) substrate consistency was found to obtainmaximum hydrolysis, however, further increase in substrate con-sistency led to the reduced extent of hydrolysis (data not shown).This decrease in hydrolysis efficiency may be because of mixingand heat transfer problem due to the rheological properties of adense fibrous suspension, which ultimately cause insufficientadsorption of the cellulase to the cellulose (Chen et al., 2007). Asthe enzymatic saccharification of cellulose involve transport of en-zyme molecules and soluble sugars between the solid substrate

and bulk reaction solution, a modification of the surface and inter-facial properties of the reaction system may improve saccharifica-tion (Hemmatinejad et al., 2002). For further improvement in thesaccharification efficiency of delignified P. juliflora, various ionicas well as nonionic surfactants were investigated. Among differentsurfactants tested, Tween 80, a nonionic surfactant has supportedthe enzymatic saccharification of delignified P. juliflora maximally(Fig. 2). Kaar and Holtzapple (1998) had also proposed that Tweenprotects the enzymes from thermal deactivation during the enzy-matic hydrolysis. This may be the result of reduced contact ofenzyme with the air-liquid interface due to surface activity of thesurfactant. The reduction in surface tension of the solution inhibitsthe non-productive attachment of the exoglucanase to the ligninsurface and allows the saccharifying exoglucanase greater accessto cellulose, which results in increase of sugar release (Hemma-tinejad et al., 2002).

The acid and enzymatic hydrolysates of P. juliflora werefermented by P. stipitis and S. cerevisiae, respectively. Several evi-dences suggested that separate fermentation by substrate specificorganisms work better instead of using mixed hydrolysate witheither single culture or co-culture method (Delgenes et al., 1996).Co-culture fermentation associates both hexose and pentose fer-menting yeasts that trade-off for oxygen requirement betweenthe two microorganisms. Whereas, ethanol production from mixedhydrolysates using single culture, circumvent the fact that utiliza-tion of xylose becomes a subject of glucose catabolite repression(Olsson and Hahn-Hagerdal, 1993). The acid hydrolysate of ligno-cellulosics comprises mainly pentose sugars and very few microor-ganisms e.g., Candida shehatae, P. stipitis and Pachysolentannophilus, which can utilize pentose sugars efficiently have beenidentified so far (Abbi et al., 1996). Among different pentose utiliz-ing yeasts, P. stipitis has shown great potential by having broadsubstrate specificity and no absolute vitamin requirement forpentose utilization (du Preez et al., 1986). In our experiment, theethanol yield (0.39 g/g) from hemicellulosic hydrolysate using P.stipitis was very much in agreement with previous reports havingyield, 0.37 g/g from aspen wood (Delgenes et al., 1996), 0.39 g/gfrom D-xylose (du Preez et al., 1986) and 0.36 g/g from P. juliflora(Kapoor et al., 2008). However, the ethanol yield obtained from cel-lulosic hydrolysate using S. cerevisiae (0.49 g/g) was even higherthan the previous results of ethanol yield 0.40 g/g from sweet sor-ghum (Mamma et al., 1995) and 0.48 g/g from corncob (Chen et al.,2007). A low aeration rate of 0.4 lpm was maintained throughoutthe process, as low aeration conditions are attributed to obligatehypoxic induction of essential fermentative enzymes (Mc Millanand Boynton, 1994) and also in slightly aerobic conditions the pro-duction of ethanol enhanced by lowering down the production ofglycerol as byproduct (Alfenore et al., 2004). However, S. cerevisiaewhen grown in medium having 100 g/L sugar produced 42.5 g/Lethanol, suggesting thereby to increase the initial sugar loadingeither by concentration of the hydrolysates or by supplementationwith sugar to improve cellulosic ethanol production.

5. Conclusion

Mesquite (P. juliflora) wood could serve as novel material for theproduction of ethanol. The separate hydrolysis and fermentationcould prove better in improving the ethanol production fromcellulosic hydrolysates by minimizing the problem of cataboliterepression. The ethanol production may be enhanced by increasingthe initial sugar load in cellulosic hydrolysates. There is still a needto develop; (i) a more efficient and economic pretreatment process(ii) a hyper-cellulase producing strain for improved saccharifica-tion and, (iii) an improved recombinant yeast strain capable ofutilizing both pentose and hexose sugars, which in turn wouldincrease ethanol production.

1220 R. Gupta et al. / Bioresource Technology 100 (2009) 1214–1220

Acknowledgements

Authors are grateful to Department of Biotechnology (DBT),Ministry of Science and Technology (Government of India) for thefinancial support. Authors are thankful to Dr. Ajay Singh, AdjunctProfessor, Department of Biology, University of Waterloo, Ontario,Canada, for his suggestions during the preparation of the manu-script. The assistance from Mr. Adesh Kumar and Mr. ManwarSingh Shah during the course of this work is highly acknowledged.

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