Bioresource Technology 101 (2010) 5366–5373

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Bioresource Technology

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Biocatalytic approach for the utilization of hemicellulose for ethanolproduction from agricultural residue using thermostable xylanase andthermotolerant yeast

Vishnu Menon, Gyan Prakash, Asmita Prabhune, Mala Rao *

Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 December 2009Received in revised form 25 January 2010Accepted 26 January 2010Available online 12 March 2010

Keywords:Hemicellulose, Thermostable xylanaseThermotolerant yeastBiosurfactantEthanol

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.01.150

* Corresponding author. Tel.: +91 20 25902228; faxE-mail address: mb.rao@ncl.res.in (M. Rao).

A hydrolysis of 62% and 50% for OSX (Oat spelt xylan) and WBH (Wheat bran hemicellulose) wereobtained in 36 h and 48 h using Accellerase™ 1000 at 50 �C wherein thermostable xylanase from alkalo-thermophilic Thermomonospora sp. yielded 67% (OSX) in 3 h and 58% (WBH) in 24 h at 60 �C, favouring areduction in process time and enzyme dosage. The rate of hydrolysis with thermostable xylanase wasincreased by 20% with the addition of nonionic surfactant tween 80 or biosurfactant sophorolipid. Thesimultaneous saccharification and fermentation (SSF) of OSX and WBH using thermostable xylanaseand D. hansenii in batch cultures produced 9.1 g/L and 9.5 g/L of ethanol, respectively and had a shorteroverall process time than the separate hydrolysis and fermentation (SHF). The immobilized yeast cells inCa-alginate matrix produced ethanol with a yield of 0.46 g/g from hemicellulosic hydrolysates and werereused six times with 100% fermentation efficiency.

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1. Introduction

In collation to first generation bioethanol from starch andmolasses, the development of second generation bioethanol fromlignocellulosic biomass serves many advantages from both ener-getic and environmental point of views (Zhang et al., 2009). Ligno-cellulosic biomass in general comprises of 40–50% cellulose, 25–30% hemicellulose and 15–20% lignin (Wiselogel et al., 1996). Theeffective utilization of all the three components would play a sig-nificant role in the economic viability of cellulose to ethanol pro-cess. Cellulose bioconversion to sugars/ethanol requires mainlythree steps, the first and the most crucial step being pretreatmentand the second being hydrolysis by cellulase complex and then fer-mentation. Most of the effective pretreatment processes involvingacid, alkali or solvents, remove lignin and to a large extent hemi-cellulose is not utilized (Hendriks and Zeeman, 2009). Convention-ally the acid hydrolysis pretreatment is used for the removal ofhemicellulose for further production of ethanol and value addedproducts. During the process a range of inhibitory compoundsare generated which are degradation products of hemicelluloseand lignin (Almeida et al., 2007). However enzymatic hydrolysisof hemicellulose to monomeric sugars will minimize the presenceof inhibitory compounds enabling better fermentation efficiencyand cleaner process.

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Xylan is the major constituent of hemicellulose, the secondmost abundant renewable resource and is a heteropolysaccharideswith a homopolymeric backbone of b-D-xylose (Kulkarni et al.,1999). Thermostable xylanases offer advantages in the hydrolysisof hemicellulosic substrates over their mesophilic counterparts,higher specific activity decreases the amount of enzymes, en-hanced stability allowing improved hydrolysis performance andincreased flexibility with respect to process configuration, all con-tributing towards the overall improvement of the economy of theprocess (Viikari et al., 2007). The enzymatic saccharification athigher temperature would potentially reduce the reaction timeand the enzyme loading.

Considering the significance of xylose fermentation to ethanolat higher temperature we have isolated thermotolerant yeast fromrotten grapes. The yeast is identified to be Debaromyces hansenii,grows and ferments xylose at 40 �C producing predominantly eth-anol under the experimental conditions.

When enzymatic hydrolysis is performed together with fermen-tation, it is referred to as simultaneous saccharification and fer-mentation (SSF) (Takagi et al., 1997). However, the process stepscan also be performed sequentially, i.e. separate hydrolysis and fer-mentation (SHF). The avoidance of end products inhibition andthereby increasing the saccharification rate and the ethanol yieldare one of the significant reasons for using SSF; however thereare several additional potential advantages as the presence of eth-anol in the culture medium causes the mixture to be less vulnera-ble to invasion by undesired microorganisms. In addition the

V. Menon et al. / Bioresource Technology 101 (2010) 5366–5373 5367

combination of hydrolysis and fermentation decreases the numberof vessels needed and thereby reduces the investment costs. Thedecrease in capital investment has been estimated to be larger than20% (Olofsson et al., 2008). The disadvantage being the hydrolysishas to be carried out at lower temperature to be compatible withthe yeast fermentation system.

From environmental engineering aspects, various waste and un-der-utilized lignocellulosic agricultural residues can serve as feedstocks for production of biofuel, as the economic value of theseby-products as animal feeds is decreasing (Lynd et al., 2005).Wheat bran (WB) a by-product of wheat milling industry, consti-tutes a significant under-utilized source of sugars and is similarto other high hemicellulosic biomass such as corn fiber, both ofwhich represents a potential low cost feed stock for productionof ethanol (Maes and Delcour, 2001; Palmarola-Adrados et al.,2005). Approximately 78.40 million metric tones of wheat is pro-duced in India (world production, 607 million metric tones), fromwhich almost 15.68 million metric tones of bran is generated(www.igc.org.uk).

The current investigations explore the potential of enzymatichydrolysis of hemicellulose from agricultural by-product, wheatbran using a thermostable hemicellulase from an alkalothermo-philic Thermomonospora sp. and subsequent fermentation with athermotolerant pentose fermenting yeast. In addition to WB, oatspelt xylan (OSX) is also studied as a model system under the con-sonant experimental conditions. The study also focuses for the firsttime the effect of biosurfactant, sophorolipid on the increasedhydrolysis of hemicellulosic substrates and simultaneous sacchar-ification and fermentation (SSF) in which hemicellulose is used to-gether with thermotolerant yeast.

2. Methods

2.1. Raw materials

Oat spelt xylan (OSX) was purchased from Sigma–Aldrich Co.,St. Louis, MO USA and Wheat bran (WB) was obtained locally.The proximate composition of OSX is 75% xylose, 10% arabinoseand 15% glucose (Sigma) and of WB is hemicellulose 38.50 wt.%,cellulose 13 wt.%, starch 21.95 wt.%, klason lignin 9.35 wt.% andcrude protein 17.30 wt.% (Miron et al., 2001). WB was washedand boiled in water at 60 �C for 30 min and was passed throughseveral hot water washes. Subsequently was air dried and usedin hydrolysis experiments.

2.2. Isolation of hemicellulose from WB

The washed wheat bran was processed to obtain wheat branhemicellulose (WBH). The washed bran (2%) was treated with alka-line hydrogen peroxide solution (pH 11.5) at 2% concentration and60 �C for 4 h (Almeida et al., 2007; Maes and Delcour, 2001). Thesolution was centrifuged at 4800g for 15 min and the supernatantwas treated with ethanol in a ratio of 3:1. The precipitated hemi-cellulose was separated by centrifugation at 9600g for 15 min at4 �C. The precipitate was dried under vacuum to remove traces ofethanol. The dried powder was used for hydrolysis.

2.3. Chemicals

All chemicals were of analytical grade. 3,5-dinitrosalysilic acid(DNS) was obtained from Sigma–Aldrich Co., St. Louis, MO USA.Ethanol was purchased from Les Alcools De Commerce Inc., Bramp-ton, Ontario.

2.4. Microorganisms and cultivation conditions

Alkalothermophilic Thermomonospora sp. was maintained onLuria Bertani wheat bran slants at pH 10 and 50 �C according toGeorge et al. (2001a).

2.5. Thermotolerant yeast

Thermotolerant yeast was isolated from rotten grapes byenrichment in a media containing (g/L): xylose, 20.0; malt extract,3.0; yeast extract, 3.0; peptone, 5.0; agar, 20.0; at pH 6.0 ± 0.2 at40 �C. After enrichment for 48 h, the sample was used to isolatethe yeast. The yeast was further purified by single colony plating.The yeast was identified as Debaromyces hansenii by 18s rDNA.Inoculum was prepared by growing the organism on a rotary sha-ker at 172 g for 48 h at 40 �C, in a growth medium containing (g/L):xylose, 40.0; malt extract, 3.0; yeast extract, 3.0; peptone, 5.0; agar,20.0; at pH 6.0 ± 0.2. After 48 h the inoculum was centrifuged at4800g for 20 min and the supernatant was discarded. The cellswere washed twice with sterile 0.9% NaCl solution and centrifugedagain. The supernatant was removed and the cells were weighedand used for fermentation studies.

2.6. Enzyme production

Thermomonospora was grown in a modified Reses medium at50 �C for 96 h on a rotary shaker maintained at 180g. At the end of96 h the fermentation broth was centrifuged at 9600g for 15 minat 4 �C. The supernatant was concentrated using ammoniumsulphate (0–90%) and was the source of enzyme. The preparationcontained 120 U/ml and 0.3 U/ml of xylanase and b-xylosidase,respectively.

The Commercial enzyme Accellerase™ 1000 was a kind giftfrom Dr. Raj Lad and Dr. Surendra Bade, Danisco US Inc., GenencorDivision, USA. The enzyme showed 2250 U/ml of xylanase and0.6 U/ml of b-xylosidase.

2.7. Enzyme assay

Xylanase and b-xylosidase were measured according to stan-dard procedure recommended by Commission on Biotechnology,IUPAC (Ghose and Bisaria, 1987). One unit of enzyme activity is de-fined as the amount of enzyme required to liberate one l mole ofreducing sugar per minute under the assay conditions.

2.8. Immobilization of debaryomyces in Ca-alginate beads

D. hansenii cells were harvested after 48 h of growth at 40 �C bycentrifugation at 4800g for 20 min, washed and 1 g (wet cell weight)was added to 10 ml of 2% (w/v) Na-alginate as described by Kierstanand Bucke (1977). The suspension was then extruded dropwisethrough a 5 ml syringe into a gently stirred cold solution of CaCl2

and hardened at 4 �C for 1 h in this solution. Particle integrity and ab-sence of microbial contamination were ensured by means of opticalmicroscopy. The beads (mean diameter of 2 mm) were washed withdistilled water and used for fermentation.

2.9. Enzymatic hydrolysis

The hydrolysis of oat spelt xylan (OSX), wheat bran (WB) andwheat bran hemicellulose (WBH) were carried out at different sub-strate concentrations (2.5% and 5%) with two concentrations (150and 300 U/g) of Thermomonopsora xylanase and commercial en-zyme from Accellerase™ 1000 at various temperatures (40, 50,60 and 70 �C). The hydrolysis was carried out in a stoppered flaskin 25 ml reaction volume containing appropriate concentrations

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of substrates in 50 mM phosphate buffer, pH 7.0 for Thermomonos-pora xylanase and 50 mM acetate buffer, pH 4.8 for Accellerase un-der static condition. Hydrolysis was terminated by boiling at100 �C for 5 min at the end of stipulated time intervals, filteredand the filtrate was assayed for reducing sugar by dinitrosalicylicmethod (Mandels and Weber, 1969). Appropriate substrate and en-zyme controls were performed.

The surface additives, a nonionic surfactant tween 80 and a bio-surfactant, sophorolipid (1% w/v) were used for studying their ef-fect in improving enzymatic saccharification of OSX and WBH.

2.10. Simultaneous saccharification and fermentation

SSF was carried out in stoppered flasks containing 2.5% of OSXand WBH at 40 �C with 150 U/g Thermomonospora xylanase in a to-tal reaction mixture volume of 25 ml along with 10% Debaryomy-ces cells under static condition. Nutrients were added to SSF flaskat concentration of (g/L); Urea; 5, MgSO4 7H2O; 5, K2HPO4; 2 Ali-quots were withdrawn at regular intervals and centrifuged. Thesupernatant was distilled and the distillate was used for ethanolestimation by Gas Chromatography.

2.11. Separate hydrolysis and fermentation

Ethanol production from hydrolysates obtained after enzymaticsaccharification of OSX and WBH by free and immobilized D.hansenii were investigated. The enzymatic hydrolysate was pre-pared in a similar manner as the hydrolysis experiments with2.5% substrate concentration and 150 U/g Thermomonospora xylan-ase at 50 �C for 12 h for OSX and 48 h for WBH. The hydrolysatewas filtered and supplemented with nutrients (g/L); Urea; 5,MgSO4 7H2O; 5, K2HPO4; 2 and inoculated with 10% free andimmobilized yeast and fermentation was carried out at 40 �C. Ali-quots were withdrawn, sugar and ethanol were determined. An at-tempt was also made to test the efficacy of the yeast to fermentsynthetic medium containing xylose (20 g/L) at 40 �C.

2.12. Reuse of immobilized yeast

The hydrolysates obtained in SHF were used to investigate theability of thermotolerant Debaryomyces for reprocessing. The Ca-alginate immobilized cells (10%) were added to hydrolysates sup-plemented with nutrients and fermentation was performed at40 �C for 48 h. After which the Ca-alginate beads were retrievedand reused repeatedly with fresh charge of hydrolysates and theethanol formed was assayed at the end of each cycle of operation.

2.13. Analytical methods

The hydrolysates were analyzed by High performance liquidchromatography (HPLC) (Waters) for the presence of carbohy-drates using Waters Sugar Pack Column 6.5 � 300 mm. The mobile

Table 1Enzymatic hydrolysis of OSX at 2.5% and 5% substrate concentration with Thermomonospo

40 �C 50 �C

Substrate concentration (g%) 2.5 5 2.5 5

Enzyme dosage(U/g) 150 300 150 300 150 300 15

Time (h) Percentage hydrolysis

1 38.75 40.82 30.23 33.84 50.35 53.01 472 41.05 43.96 35.77 38.45 53.17 55.52 513 48.23 50.18 40.96 42.68 58.62 61.18 574 52.42 54.36 45.41 48.25 60.66 66.12 6112 54.25 56.13 50.28 51.69 66.71 68.28 64

phase used was Milli Q water with 100 lM EDTA and 200 lMCaCl2. The flow rate was maintained at 0.4 ml/min keeping theoven temperature at 70 �C. The sugars were detected by Waters2410 refractive index detector. Ethanol was estimated by GasChromatography (GC) (Master DANI) with a BP1 (Fused silicabonded phase) column (30.0 � 0.32 mm) at oven temperature of85 �C and flame ionization detector (FID) at 200 �C. The ethanolstandards were prepared using commercial ethanol. Nitrogen witha flow rate of 0.5 ml/min was used as carrier gas.

3. Results and discussion

3.1. Enzymatic hydrolysis of hemicellulosic substrates

The thermostable xylanase from alkalothermophilic Thermomo-nospora shows optimum xylanase activity at 70 �C with 82% activ-ity at 80 �C (George et al., 2001b), whereas the commercial enzymeAccellerase from a mesophilic fungus Trichoderma reesei shows atemperature optimum of 50 �C with 58% activity at 60 �C and25% activity at 70 �C. Saccharification of Oat spelt xylan with ther-mostable xylanase and Accellerase with respect to varying enzymeand substrate concentrations at different temperatures were pur-sued. A maximum hydrolysis of 67% was obtained in 3 h at 70 �Cusing thermostable xylanase with an enzyme dosage of 150 U/gand a substrate loading of 25 g/L. At higher substrate concentration(50 g/L), a hydrolysis of 67% was obtained in 3 h at 70 �C with anenzyme dosage of 150 U/g as compared at 50 �C wherein same per-centage of hydrolysis was achieved with 300 U/g in 12 h (Table 1).The hydrolysis pattern with Accellerase concurs with the optimumtemperature of the enzyme. A maximum hydrolysis of 62% was ob-tained in 36 h at 50 �C, with double the enzyme units (300 U/g) ascompared with thermostable xylanase (150 U/g). The hydrolysisrates were significantly less at 60 and 70 �C and below the opti-mum temperature (data not shown).

The hydrolysis of wheat bran (WB) at 25 g/L with thermostablexylanase yielded a maximum saccharification of 38% at 50 �C in48 h and at 60 �C in 24 h, with 76% conversion based on the totalavailable substrate wherein the percentage hydrolysis was 32% at50 �C and 28% at 60 �C in 48 h with Accellerase. At higher substrateloading (50 g/L), the hydrolysis was 30% with thermostable xylan-ase and was 25% with Accellerase (data not shown). A maximumsaccharification of 58% and 62% was obtained for WBH in 48 hand 24 h at 50 �C and 60 �C, respectively with thermostable xylan-ase. The percentage hydrolysis was 50% and 52% with Accelleraseunder the agnate conditions (Fig. 1). The hydrolysis at 60 �C usingthermostable enzyme is more pronounced in the case of WBH thanWB. The lower hydrolysis rate of WB is probably due to the pres-ence of inhibitory compounds including lignin.

Effectively overcoming the recalcitrance structure of lignocellu-lose and releasing the locked polysaccharide is one of the mostimportant and urgent R & D priorities for the emerging cellulosicethanol and biobased chemical industries. To our knowledge there

ra xylanase at different temperatures.

60 �C 70 �C

2.5 5 2.5 5

0 300 150 300 150 300 150 300 150 300

.82 52.61 54.60 56.89 53.18 55.12 52.18 55.98 50.88 52.84

.38 54.38 58.33 60.18 60.87 62.34 60.28 63.85 60.57 61.98

.28 60.18 66.59 68.15 65.99 68.02 67.18 68.17 66.95 67.98

.81 63.47 66.78 68.59 66.07 68.99 67.29 69.08 67.25 68.06

.19 67.54 66.99 69.01 66.67 69.07 67.75 69.56 67.65 68.59

Fig. 1. Saccharification performance on WBH at 2.5% (a and b) and 5% (c and d) substrate concentration with Thermomonospora xylanase (a and c) and Accellerase (b and d) at40, 50 and 60 �C. Xylanase dosage: 150 U/g (solid lines) and 300 U/g (dashed lines).

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is only one report on the acid pretreatment and hydrolysis ofwheat bran with 80% conversion based on the theoretical total su-gar yield using a commercial cellulase (Palmarola-Adrados et al.,2005). WB is even explored as a source for the production of biohy-drogen using mixed anaerobic cultures during anaerobic degrada-tion process (Pan et al., 2008).

3.2. Effect of surfactants on hemicellulose hydrolysis

Enzyme loading is one of the important factors to the econom-ics of biofuel production based on enzymatic conversion of cellu-lose to ethanol. Effect of nonionic surfactant, tween 80 and abiosurfactant, sophorolipid each representative of chemical andbiological surfactants were studied on the hydrolysis of OSX andWBH with Thermomonospora xylanase. The rate of hydrolysis inpresence of sophorolipid and tween 80 at 50 and 60 �C for OSX

and WBH was increased by 20%. The enzyme dosage required toachieve the maximum hydrolysis of 74% for OSX (Fig. 2a) and70% for WBH (Fig. 2c) at 50 �C were reduced to half in the presenceof surface active additives. Similar results were obtained at 60 �C ina shorter period of hydrolysis (3 h) in the presence of surfactants.(Fig. 2b and d).

The positive effect of chemical surfactants has also been ob-served during the hydrolysis of various pure and lignocellulosicsubstrates (Kristensen et al., 2007). There is paucity of reports onthe effect of biosurfactant on hydrolysis. Few workers have shownthat addition of biosurfactant on the enzymatic hydrolysis of puremicrocrystalline cellulose and steam exploded wood containingonly cellulose had an increase in the rate of hydrolysis in the rangeof 80–300% (Helle et al., 1993). Unlike previous studies, which havefocused on materials with little hemicellulose content (Kristensenet al., 2007), the present investigation reveals the positive pro-

Fig. 2. Effect of surface active additives on the enzymatic hydrolysis of OSX (a and b) and WBH (c and d) at 50 and 60 �C using 150 U/g (solid lines) and 300 U/g (dashed lines)of Thermomonospora xylanase.

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nounced effect of tween 80 and sophorolipid addition on thehydrolysis (20%) of hemicellulosic substrates. However the resultson xylan hydrolysis are less pronounced which may be due to thenature of the substrate being non crystalline and fewer nonactivebinding sites. The current investigation demonstrates for the firsttime the effect of sophorolipid on the increased rate of hydrolysisand decreased enzyme dosage using only hemicellulosic sub-strates; OSX and WBH.

3.3. Simultaneous saccharification and fermentation

Simultaneous saccharification and fermentation (SSF) kineticsof oat spelt xylan (OSX) and wheat bran hemicellulose (WBH) at40 �C using xylanase from Thermomonospora and free cells ofthermotolerant D. hansenii is depicted in Fig. 3(a and b). Sacchar-ification of OSX and WBH were also carried with similar dosageof enzymes at 40 �C. In order to compare the saccharificationwith SSF the ethanol values were converted to xylose equiva-lents. With a xylanase dosage of 150 U/g and substrate loadingof 2.5% of OSX and WBH, the saccharification ceased after 54%and 38% conversions, respectively. The SSF carried out underthe agnate substrate and enzyme dosage showed 20% and 30%enhancement in hemicellulose conversion. Maximum ethanolconcentrations of 9.1 g/L and 9.5 g/L were obtained in SSF with

OSX and WBH, respectively. These concentrations were attainedin 36 h for OSX and 48 h for WBH from the onset of SSF. The in-creased ethanol yield in SSF systems is evidently due to removalof xylose formed during hydrolysis which causes end productinhibition.

Thermotolerance is clearly an important topic for SSF and ther-motolerant yeast strains, e.g. Fabospora fragilis, Saccharomyces uva-rum, Candida brassicae, C. lusitaniae, and Kluyveromyces marxianus,have been evaluated for future use in SSF, to allow fermentationat temperatures closer to the optimal temperature for the en-zymes. However, in all these cases saccharification of pure cellu-lose (e.g. Sigmacell-50) or washed fibers, in defined fermentationmedium, were applied. SSF of cellulose with mixed cultures of dif-ferent thermotolerant yeast strains have also been carried out(Olofsson et al., 2008).However, there is scarcity of literature fromSSF experiments in which hemicelluloses have been used togetherwith thermotolerant strains.

3.4. Separate hydrolysis and fermentation

Separate Hydrolysis and subsequent fermentation were carriedout using free cells of D. hansenii at 40 �C. The hemicellulosichydrolysates from the enzymatic saccharification of OSX andWBH contained 17.98 g/L and 15.67 g/L of reducing sugars andproduced 7.55 g/L and 6.42 g/L of ethanol with yield of 0.42 g/g

Fig. 3. Saccharification and SSF were carried out by incubating OSX (a) and WBH (b) with 150 U/g Thermomonospora xylanase at 2.5% substrate concentration at 40 �C. TheSSF, in addition, contained 10% Debaryomyces cells.

Table 2Ethanol production from enzymatic hydrolysates of OSX and WBH with free and immobilized Debaryomyces in SHF system.

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

Free Immobilized Free Immobilized Free Immobilized

OSX 0 17.98 – – – – – –4 14.33 2.68 2.88 0.15 0.16 0.67 0.7212 12.64 3.88 5.21 0.22 0.29 0.32 0.4324 8.72 6.89 7.24 0.38 0.40 0.29 0.3036 5.48 7.55 8.38 0.42 0.46 0.21 0.2348 1.98 7.05 7.15 0.39 0.40 0.15 0.15

WBH 0 15.67 – – – – – –4 12.89 1.98 2.38 0.13 0.15 0.50 0.6012 9.27 3.18 4.21 0.20 0.27 0.27 0.3524 6.83 5.33 6.27 0.34 0.40 0.22 0.2636 3.19 6.42 6.89 0.41 0.44 0.18 0.1948 1.06 6.08 6.53 0.39 0.42 0.13 0.14

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and 0.41 g/g and volumetric productivity of 0.21 g/L/h and 0.18g/L/h, respectively (Table 2). As a control, when D. hansenii wasgrown in a synthetic medium containing 20 g/L xylose, 9 g/L etha-nol with a yield of 0.45 g/g, productivity of 0.25 g/L/h and a theo-retical conversion efficiency of 90% was achieved.

Considering that no investigations on enzymatic hydrolysis andfermentation of hemicellulosic hydrolysates from WB are reported,the present results are compared with ethanol production fromhemicellulosic hydrolysates using lignocellulosic substrates. Theethanol concentrations and yields are summarized in Table 3. A re-cent study by Wilkins et al. (2008) reveals an ethanol productionby Kluyveromyces sp. from D-xylose at 40 �C and 45 �C with a yieldof 0.15 g/g and 0.08 g/g, respectively under anaerobic condition. H.polymorpha could ferment both glucose and xylose up to 45 �C(Ryabova et al., 2003). The ability of Debaryomyces sp. to produce

xylitol from commercial D-xylose and wood hydrolysates generat-ing high xylitol: ethanol (>4) has been studied (Girio et al., 1994).Debaryomyces sp. used both pentoses and hexoses to similar ex-tents in sugar mixtures and a preference for one carbohydratedid not inhibit the consumption of other. This could be important,since the usual substrates of hemicelluloses, which often consistsof sugar mixtures (Breuer and Harms, 2006). The present studiesshow that the newly isolated yeast D. hansenii is able to fermentxylose efficiently with an ethanol yield of 0.45 g/g in syntheticmedium and 0.42 g/g and 0.41 g/g in hemicellulosic hydrolysatesof OSX and WBH, respectively in SHF models at 40 �C. A compari-son of SHF with SSF system using Debaryomyces indicated thatthe bioprocess was completed in approximately 36 h and 48 h forOSX and WBH, respectively with SSF system as opposed to 48 hand 84 h with SHF.

Table 3Comparison of recent data with present work on ethanol production using hemicellulosic hydrolysate obtained from lignocellulosic residues.

Substrate Pretreatment Detoxification Microorganisms Ethanol(g/L)

Ethanol yield(g/g)

Reference

Prosopsis julifora Acid hydrolysis Over-liming Pichia stipitis NCIM 3498 7.13 0.39 Gupta et al. (2009)Water hyacinth Acid hydrolysis – Pichia stipitis – 0.425 Kumar et al. (2009)Olive tree Acid hydrolysis Over-liming and activated

charcoalPichia stipitis – 0.42 Diaz et al. (2009)

Bagasse Steam pretreatment – Pichia stipitis CBS 6054 19.5 0.22 Rudolf et al. (2008)Sunflower seed hull Acid hydrolysis Over-liming combined with Na2SO3 Pichia stipitis NRRLY-7124 11 0.32 Telli-Okur and Eken-

Saracoglu (2008)Bagasse Acid hydrolysis Over-liming or electrodialysis Pachysolen tannophilus DW 06 21 0.35 Cheng et al. (2008)Wheat straw Acid hydrolysis Boiling and overliming Pichia stipitis – 0.41 Nigam (2001)Oat spelt xylan Enzymatic hydrolysis – Debaromyces hansenai 7.55 0.42 Present workWheat bran Enzymatic hydrolysis – Debaromyces hansenai 6.42 0.41

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3.5. Immobilization and reuse

D. hansenii cells were immobilized in Ca-alginate matrix as de-scribed in materials and method. The fermentation of enzymatichydrolysates from OSX and WBH containing 17.98 g/L and15.67 g/L of reducing sugars using immobilized cells produced8.38 g/L and 6.89 g/L of ethanol with a yield and volumetric pro-ductivity of 0.46 g/g and 0.44 g/g and 0.23 g/L/h and 0.18 g/L/h,respectively (Table 2). The Ca-alginate immobilized yeast wasreprocessed for 10 cycles using a batch mode at 40 �C. The immo-bilized yeast was reused six times without any apparent loss incapacity to ferment. The immobilized yeast produced ethanol witha productivity of 0.23 g/L/h and 0.19 g/L/h and theoretical conver-sion efficiency of 93.63% and 87.77% for OSX and WBH hydroly-sates, respectively for 6 successive batches. Then the productivitydeclined with final conversion efficiencies of 64% in the 10th cycle.The immobilized yeast was not compatible with the SSF systemand the beads were disintegrated after the first reuse and werenot pursued further.

4. Conclusion

The present studies analyze the comparative data on hydrolysisof hemicellulosic substrates at different temperatures with ther-mostable xylanase from alkalothermophilic Thermomonospora sp.and a commercial enzyme (Accellerase™ 1000) from Trichodermareesei. At higher temperature, rapid hydrolysis of hemicellulosicsubstrates, OSX and WBH were achieved. Our results for the firsttime demonstrate the pronounced positive effect of chemical aswell as biosurfactant on the hydrolysis rates of hemicellulose.The ethanol yield for hemicellulosic substrates was higher in SSFwith an overall reduction in process time as compared to SHFand constitutes the first report of SSF system with hemicelluloseand thermotolerant yeast.

Acknowledgements

MR acknowledges the financial support from CSIR EmeritusScheme. VM acknowledges the junior research fellowship fromCSIR Emeritus Scheme, Govt. of India.

References

Almeida, J.R.M., Modig, T., Petersson, A., Hahn-Hägerdal, B., Liden, G., Gorwa-Grauslund, M.F., 2007. Increased tolerance and conversion of inhibitors inlignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol.Biotechnol. 82 (4), 340–349.

Breuer, U., Harms, H., 2006. Debaryomyces hansenii – an extremophilic yeast withbiotechnological potential. Yeast 23, 415–437.

Cheng, K.K., Cai, B., Zhang, J., Ling, H., Zhou, Y., Ge, J., Xu, J., 2008. Sugarcane bagassehemicellulose hydrolysate for ethanol production by acid recovery process.Biochem. Eng. J. 38 (1), 105–109.

Diaz, M.J., Ruiz, E., Romero, I., Cara, C., Moya, M., Castro, E., 2009. Inhibition of Pichiastipitis fermentation of hydrolysates from olive tree cuttings. World J.Microbiol. Biotechnol. 25, 891–899.

George, S.P., Ahmad, A., Rao, M.B., 2001a. Studies on carboxymethyl cellulaseproduced by alkalothermophilic actinomycetes. Bioresour. Technol. 77, 171–175.

George, S.P., Ahmad, A., Rao, M.B., 2001b. A novel thermostable xylanase fromThermomonospora sp: influence of additives on thermostability. Bioresour.Technol. 78, 221–224.

Ghose, T.K., Bisaria, V.S., 1987. Measurement of hemicellulose activities. Part 1:Xylanases (Recommendations of Commission on Biotechnology IUPAC). PureAppl. Chem. 59 (12), 1739–1752.

Girio, F.M., Roseiro, J.C., Sa-Machado, P., et al., 1994. Effect of oxygen transfer rate onlevels of key enzymes of xylose metabolism in Debaryomyces hansenii. EnzymeMicrob. Technol. 16, 1074–1078.

Gupta, R., Sharma, K.K., Kuhad, R.C., 2009. Separate hydrolysis, fermentation (SHF)of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanolby Saccharomyces cerevisiae, Pichia stipitis-NCIM 3498. Bioresour. Technol. 100,1214–1220.

Helle, S.S., Duff, S.J.B., Copper, D.G., 1993. Effect of surfactants on cellulosehydrolysis. Biotechnol. Bioeng. 42, 611–617.

Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility oflignocellulosic biomass. Bioresour. Technol. 100 (2), 10–18.

Kierstan, M., Bucke, C., 1977. Immobilization of microbial cells, subcellularorganelles and enzymes in calcium alginate gels. Biotechnol. Bioeng. 19,387.

Kristensen, J.B., Borjesson, J., Bruun, M.H., Tjerneld, F., Jorgensen, H., 2007. Use ofsurface active additives in the enzymatic hydrolysis of wheat strawlignocellulose. Enzyme Microb. Technol. 40 (4), 888–895.

Kulkarni, N., Shendye, A., Rao, M., 1999. Molecular and biotechnological aspects ofxylanases. FEMS Microbiol. Rev. 23, 411–456.

Kumar, A., Singh, L.K., Ghosh, S., 2009. Bioconversion of lignocellulosic fraction ofwater-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanolby Pichia stipitis. Bioresour. Technol. 100 (13), 3293–3297.

Lynd, L.R., Van Zyl, W.H., McBride, J.H., Laser, M., 2005. Consolidatedbioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16,577–583.

Maes, C., Delcour, J.A., 2001. Alkaline hydrogen peroxide extraction of wheat brannon-starch polysaccharides. J. Cereal Sci. 34 (1), 29–35.

Mandels, M., Weber, J., 1969. The production of cellulases. Advances in Chemistry95, 391–414.

Miron, J., Yosef, E., Ben-Ghedalia, D., 2001. Composition and in vitro digestibility ofmonosaccharide constituents of selected by-product feeds. J. Agric. Food. Chem.49, 2322–2326.

Nigam, J.N., 2001. Ethanol production from wheat straw hemicellulose hydrolysateby Pichia Stipitis. J. Biotechnol. 2001 (87), 17–27.

Olofsson, K., Bertilsson, M., Lidén, G., 2008. A short review on SSF – an interestingprocess option for ethanol production from lignocellulosic feedstocks.Biotechnol. Biofuels 1, 7.

Palmarola-Adrados, B., Choteborska, P., Galbe, M., Zacchi, G., 2005. Ethanolproduction from non-starch carbohydrates of wheat bran. Bioresour. Technol.96, 843–850.

Pan, C., Fan, Y., Hou, H., 2008. Fermentative production of hydrogen from wheatbran by mixed anaerobic cultures. Ind. Eng. Chem. Res. 47, 5812–5818.

Rudolf, A., Baudel, H., Zacchi, G., Hahn-Hagerdal, B., Liden, G., 2008. SimultaneousSaccharification and fermentation of steam-pretreated bagasse usingSaccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054. Biotechnol.Bioeng. 99 (4), 783–790.

Ryabova, O.B., Chmil, O.M., Sibirny, A.A., 2003. Xylose and cellobiose fermentationto ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha.FEMS Yeast Res. 4, 157–164.

Takagi, M., Abe, S., Suzuki, S., Emert, G.H., Yata, N., 1997. A method for production ofalcohol direct from cellulose using cellulase and yeast. In: Processbioconversion symposium, pp. 551–571.

Telli-Okur, M., Eken-Saracoglu, N., 2008. Fermentation of sunflower seed hullhydrolysate to ethanol by Pichia Stipitis. Bioresour. Technol. 99, 2162–2169.

V. Menon et al. / Bioresource Technology 101 (2010) 5366–5373 5373

Viikari, L., Alapuranen, M., Puranen, T., Vehmaanperä, J., Siika-aho, M., 2007.Thermostable enzymes in lignocellulose hydrolysis. Adv. Biochem. Eng.Biotechnol. 108, 121–145.

Wilkins, M.R., Mueller, M., Eichling, S., Banat, I.M., 2008. Fermentation of xylose bythe thermotolerant yeast strains Kluyveromyces marxianus IMB2, IMB4, andIMB5 under anaerobic conditions. Process Biochem. 43, 346–350.

Wiselogel, A., Tyson, J., Johnsson, D., 1996. In: Wyman, C.E. (Ed.), Handbook ofbioethanol: production and utilization. Taylor and Francis, Washington.

Zhang, Y-HP., Berson, E., Sarkanen, S., Dale, B.E., 2009. Sessions 3 and 8:pretreatment and biomass recalcitrance: fundamentals and progress. Appl.Biochem. Biotechnol. 153, 80–83.