Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis

Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis

Bioresource Technology 102 (2011) 6473–6478 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/loca...

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Bioresource Technology 102 (2011) 6473–6478

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis K. Srilekha Yadav, Shaik Naseeruddin, G. Sai Prashanthi, Lanka Sateesh, L. Venkateswar Rao ⇑ Department of Microbiology, Osmania University, Hyderabad 500 007, AP, India

a r t i c l e

i n f o

Article history: Received 16 January 2011 Received in revised form 3 March 2011 Accepted 8 March 2011 Available online 16 March 2011 Keywords: Rice straw Concentrated hydrolysate Yeast Co-culture Fermentation

a b s t r a c t Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses). Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L 1. However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L 1 by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L 1, with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L 1 h 1, 0.4 g g 1 and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Energy security and climate change imperatives require large scale substitution of petroleum based fuels. The depletion of petroleum-based fuels and environmental problems has stimulated the development of inexpensive production of biofuels (Demain, 2009). Bioethanol, not only reduces the reliance on oil imports and alleviates uncertainties caused by the fluctuations of oil price, but also secures reductions in environmental pollution problems due to its high oxygen content (Huang et al., 2008). Lignocelluloses from agricultural, industrial and forest residuals account for the majority of the total biomass present in the world and regarded as the largest known renewable carbohydrate. This has placed attention on the utilization of fermentable sugars from lignocellulose source for biofuel production (Jørgensen et al., 2007). Rice straw is one of the abundant lignocellulosic waste materials in the world. It is the most important staple crop for more than 70% of Indians. India is the second largest producer of paddy in the world after China and has 30,000 varieties of paddy crops (Maiorella, 1985). On the other hand, every kilogram of rice grain harvested is accompanied by the production of 1–1.5 kg of the straw. Rice straw predominantly contains cellulose (32–47%), hemicellulose (19– 27%) and lignin (5–24%). The cellulose and hemicellulose content

⇑ Corresponding author. Tel./fax: +91 40 27090661; mobile: +91 9391011277. E-mail address: [email protected] (L. Venkateswar Rao). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.019

of rice straw can be hydrolyzed either chemically or enzymatically (Maiorella, 1985). Prior to ethanol fermentation by microorganism, the feedstock needs to be processed by saccharification technology in order to release fermentable sugars. To date, dilute sulphuric acid hydrolysis is one of the most promising pretreatment method and is extensively employed in industries. Dilute-sulfuric acid hydrolysis, a chemical hydrolysis is carried out either before the enzyme hydrolysis or it is used for direct conversion of lignocellulose to corresponding sugars (Taherzadeh et al., 1997). In dilute acid hydrolysis, the hemicellulose fraction is depolymerized at lower temperature than the cellulose fraction. If higher temperature or longer retention times are applied, the monosaccharides formed will be further hydrolyzed to other compounds. It is therefore suggested that the hydrolysis process to be carried out in at least two stages, the first stage at relatively milder conditions during which the hemicellulose fraction is hydrolyzed and a second stage can be carried out by enzymatic hydrolysis or dilute-acid hydrolysis at higher temperatures during which the cellulose is hydrolyzed (Palmqvist and Hahn-Hägerdal, 2000). An increase at the initial hydrolysate sugar concentration provides an increased ethanol concentration which has a major effect on the energy demand; especially at concentrations below 4 wt% of ethanol (Dehkhoda et al., 2008). The increase in sugar concentration in the water-soluble fraction can be achieved either by evaporation of water or less addition of water to the hydrolysis process or concentrating at 70 °C under vacuum to obtain a initial sugar content of approximately 100 g L 1 (Deng, 2006).

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A number of by-products are however formed during the hydrolysis, and the compounds (e.g. furfural, hydroxymethylfurfural, etc.) which are the prominent ones inhibit yeast metabolism (Taherzadeh et al., 1997). The inhibition of these inhibitors can be avoided either by different detoxification methods (e.g., by over liming) prior to fermentation or by in situ detoxification by yeast. Traditional microorganisms (e.g., Saccharomyces cerevisiae and Zymomonas mobilis) used for ethanol fermentation do not metabolize pentoses. Further the discovery of xylose-fermenting yeasts has attracted widespread interest, as the economy of production of liquid fuels from lignocellulosic materials is much improved by the efficient fermentation of both hexose and pentose sugars (Hinman et al., 1989). The conversion of xylose to ethanol allows potentially high ethanol yields from lignocellulose as nearly all the sugars are converted to ethanol. Some yeasts such as Pichia stipitis can ferment xylose and other important hexoses with relatively high yields and rate of fermentation but they have low ethanol tolerance, and ethanol concentrations above 30 to 35 g L 1 inhibit their reactions (Laplace et al., 1991). Therefore for efficient conversion of all sugars to ethanol, co-fermentation of hexoses and pentoses to ethanol is a prime research target In this aspect, we have carried out co-culture experiments with the rice straw acid hydrolysate so that both hexoses and pentoses can be totally utilized for the production of ethanol. Therefore in the present study, an attempt has been made to carry out co-culturing using S. cerevisiae and P. stipitis where both the hexose and pentose sugars can simultaneously get fermented to ethanol.

2. Methods 2.1. Raw material The rice straw which was used in all the experiments was procured from the local market, Hyderabad. It was dried at 60 °C for 1 day before the experiments were carried out. Then the dried rice straw was cut into 5 cm in size and further processed in a laboratory disintegrator to attain 4 mm size by milling.

2.1.1. Analysis of chemical composition of rice straw The cellulose, lignin and hemicellulose fractions of rice straw were analyzed using standard NREL (National Renewable Energy Laboratory) methods.

2.1.2. Delignification Hundred grams of Rice straw was delignified with 0.2 M KOH at 10% level (1:10 ratio w/v) for 4 h at room temperature i.e. 30 ± 20 °C. The contents were filtered with muslin cloth and the biomass was repeatedly washed with tap water until pH becomes neutral. The residue was dried to constant weight and later subjected to biphasic acid hydrolysis. The filtrate was used to estimate sugar loss and percentage of lignin removal.

2.2. Pretreatment of the delignified rice straw 2.2.1. Acid hydrolysis Fifty grams of the above delignified rice straw was taken in 1 L flask for biphasic sulphuric acid hydrolysis at solid to liquid ratio (1:10). Initially it was carried out with 1% acid at 121 °C (15 PSI) for 45 min and in second phase with 2% acid at 130 °C (23 PSI) for 60 min. Then the hydrolysates obtained from both the phases were mixed after cooling and sugars, phenolics and furans were estimated (see Section 2.7).

2.2.2. Concentration of hydrolysate Concentration of rice straw hydrolysate to increase sugar concentration was carried out by vacuum distillation unit (Spectrochem Pvt. Ltd, Hyderabad). During the vacuum distillation, the boiling temperature of the liquid was maintained at 80 °C using vacuum. Concentration and fermentability of the hydrolysates was assessed in accordance to the fermentation experiments by Dehkhoda et al. (2008). The volume of the dilute acid hydrolysate used for concentration was 330 ml. Sugars, phenolics and furans were checked before and after the concentration process. 2.2.3. Detoxification After vacuum distillation, the hydrolysate was added with calcium oxide with stirring, until the pH of the hydrolysate reaches 10.0. Then it was incubated for half an hour followed by centrifugation (3000g, 20 min) and filtration. Later the pH of the hydrolysate was brought back to pH 6 using concentrated H2SO4 (Chandel et al., 2007). After overliming, 3.5% of activated charcoal was added to the hydrolysates and stirred for 1 h. The mixture was again centrifuged (3000g, 20 min) and vacuum filtered (Martinez et al., 2000). Sugars, phenolics and furans were estimated before and after detoxification process. The treated hydrolysate was then used for the fermentation studies. 2.3. Microorganism and maintenance 2.3.1. Hexose yeast S. cerevisiae OVB 11 was an isolate of our laboratory from the toddy in hot summer. It is thermo tolerant, osmotolerant and inhibitor tolerant yeast which was confirmed by our previous studies. It was maintained on YEPD (yeast extract, peptone, and dextrose) medium consisted of yeast extract. 10 g L 1; peptone, 20 g L 1; glucose, 20 g L 1; and agar, 25 g L 1, pH: 5.0. 2.3.2. Pentose yeast Two pentose yeasts P. stipitis (NCIM 3498) and Candida shehatae (NCIM 3501) were procured from NCIM culture collection centre. It was maintained on MGYP (malt extract, glucose, yeast extract, peptone, and xylose) medium consisted of malt extract, 5 g L 1; yeast extract. 5 g L 1; peptone, 20 g L 1; glucose, 5 g L 1; xylose 30 g L 1 and agar, 25 g L 1, pH: 5.0. 2.4. Selection of pentose yeast For ethanol production, two different pentose utilizing yeasts (P. stipitis NCIM 3498 & C. shehatae NCIM 3501) were examined to select the best one based on their ethanol production and their ability to utilize maximum sugar. Each 50 ml of modified MGYP inoculum media containing malt extract-0.5%, glucose + xylose-1.0%, yeast extract-0.5%, peptone0.5%, pH-5.5 (Pasha et al., 2007) is inoculated with P. stipitis and C. shehatae separately and incubated at 30 °C and 150 rpm for 24 h. This 24 h old inoculum is transferred to fermentation medium. Fermentation medium (20 ml) was taken in each flask with varying concentration of xylose (1–6%) in two sets each inoculated with inoculum of C. shehatae and P. stipitis, one in each set and incubated at 30 °C, 150 rpm for 48 h. Samples were collected at 24 and 48 h and estimated for ethanol by gas chromatography (Shimadzu (GC-2011), Japan) and residual sugars by DNS method (Miller, 1959). 2.5. Inoculum development for co-culture fermentation For the seed culture, glucose and xylose were used as sole carbon source for S. cerevesiae (OVB 11) and P. stipitis (NCIM 3498),

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respectively, at a concentration of 2 g L 1 in medium. lnocula were grown aerobically in erlenmeyer flasks at 30 °C on a rotary shaker at 150 rpm for 24 h. 2.6. Ethanol fermentation by co-culture The detoxified hydrolysates (150 ml) were taken along with supplementation of 0.1% (w/v) yeast extract, peptone, NH4Cl, KH2PO4 and 0.05% of MgSO4.7H2O, MnSO4.5H2O, CaCl2.2H2O, FeCl3.2H2O and ZnSO4.7H2O in two 150 ml conical flasks (75 ml each) adjusting the pH to 5.5 and autoclaved at 110 °C for 20 min (Pasha et al., 2007). After cooling the media to 40 °C, shake flask fermentation was carried out. In one flask both P. stipitis (NCIM 3498) and thermotolerant yeast S. cerevisiae OVB11 inoculum were added at a time which is assumed as CO1. In other flask only P. stipitis was added initially and S. cerevisiae OVB11 was added at 24 h which is considered as CO2. Both the inocula containing OD600 3.0 (10% level) were transferred to the respective supplemented hydrolysates for fermentation at 30 °C, 150 rpm for first 18 h and then in static mode till the end of fermentation i.e. 72 h. Samples were collected at various intervals and centrifuged at 600 g for 10 min at 4 °C and analyzed for residual sugars, ethanol and growth. Fermentation efficiency was calculated as: Practical yield/Theoretical yield  100. Practical yield is the ethanol produced and the theoretical yield is 0.511 per gram of sugar consumed. 2.7. Analytical methods 2.7.1. Total reducing sugars The total reducing sugars, released after acid hydrolysis were estimated by DNS method (Miller, 1959). 2.7.2. Fermentation inhibitors The fermentation inhibitors (i.e. furans and phenolics) were analyzed by spectroscopic analysis. Phenolics estimation was carried out by Folin ciocalteus method (Singleton and Rossi, 1965), and furans by Martinez et al. (2000). 2.7.3. Cell density Cell density was measured turbidometrically at 600 nm. Fermentation broth was diluted 10 times using sterile water and then turbidity of solution was detected with a UV–VIS spectrophotometer 117 (Systronics India Pvt. limited). 2.7.4. Dry cell mass determination For dry cell mass determination, 10 ml of culture samples were filtered, washed and dried to a constant mass at 104 °C. 2.7.5. Ethanol Ethanol produced was analyzed by gas chromatography (GC) (Shimadzu (GC-2011), Japan) using ZB-Wax column (30 mm  025 mm) with a flame ionization detector (FID). The analysis was performed according to NREL (National Renewable Energy Laboratory) procedure LAP #001 (David, 1994). The column temperature was 150 °C (isothermal), program run time: 5.5 min, ethanol retention time: about 2.3 min and the carrier gas was nitrogen (16 kPa), injector temperature: 175 °C, detector temperature: 250 °C, flow rate: 40 ml/min, spilt ratio: 1/50, velocity of H2 flow: 60 ml/min, sample quantity: 1 ll. One part of the supernatant was filtered by 0.22 lm cellulose acetate filters for GC analysis.

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3. Results and discussion 3.1. Chemical composition of rice straw The pulverized rice straw was found to contain 35% cellulose, 22% hemicellulose, 12% lignin, 17% ash, 5% moisture, 95% total solids and 13.4% Silica. The presence of cellulose and hemicellulose together make the total holocellulose content of the substrate (57%), which is also the potential sugar concentration in the pretreated substrate. It can be fairly compared with the extensively explored lignocelluloses (sugarcane bagasse, 67.15%; corn stover, 58.29%; wheat straw, 54% and sorghum straw, 61%) for ethanol production (Chandel et al., 2007). 3.2. Delignification and acid hydrolysis The presence of lignin in cellulosic substrates hinders the saccharification of them into monomeric sugars. Alkaline pretreatment technique is one of the delignification processes with significant solubilization of hemicellulose as well. The mechanism of alkaline hydrolysis is saponification of intermolecular ester bonds cross linking hemicellulose and lignin. Therefore to overcome the lignin barrier, lignocelluloses are usually pretreated initially with alkali to dissolve the lignin caused by the breakdown of ether linkages. (Lee, 1997). Efficient delignifier should remove a maximum of lignin and minimum of sugars (not more than 5%) (Taherzadeh and Karimi, 2007). Therefore we have used 0.2 M potassium hydroxide (KOH) for delignification for 4 h at room temperature i.e. 30 ± 2 °C. We could achieve nearly 80% lignin loss (0.08/g) and 2% sugar loss (0.02 g/g). A slight improvement in hemicellulose yield using potassium hydroxide rather than sodium hydroxide was reported according to Curling (2005). Our delignification data was also well supported by Zuluaga et al. (2009), who stated that KOH removed most of the lignin and filtrate samples exhibited minor quantities of xylose, thus indicating that removal of xylose increased with a higher concentration of KOH. Acid hydrolysis of pretreated rice straw was carried out for the depolymerization of cell wall carbohydrate fraction into fermentable sugars. In our study, biphasic sulphuric acid saccharification was done with 1% at 121 °C and 2% at 130 °C which generated 16 g L 1 sugars (Fig. 2) with a holocellulose hydrolysis efficiency of 52.1%. In this work, we have used intermediate conditions, so as to prevent the formation of degradation products at concentrations that can inhibit the subsequent fermentation process. Dilute sulphuric acid pretreatment usually involves at concentrations of 0.3–2% (w/w) to hydrolyze hemicellulose. Although lignin is also solubilized during acid hydrolysis, it recondenses forming an altered lignin polymer (Torget et al., 1991). Lee and Dong-Hun (2009) reported that maximum attainable hemicellulose (xylose + galactose + arabinose) yield was about 80% using rice straw which was pretreated using dilute sulphuric acid at reaction conditions covering two levels of reaction temperature (140, 150 °C) and five levels of acid concentrations (1–3%). The maximum glucose yields reported were between 16% and 18%. Roberto et al. (2003) reported that xylose maximum recovery was 20.5 g L 1 and glucose recovery was 6.3 g L 1 respectively, with the use of 1.6% H2SO4 during 30 min. 3.3. Selection of pentose yeast C. shehatae and P. stipitis are the most promising yeasts for xylose to ethanol conversion (Ligthelm et al., 1988). Ethanol fermentation with various concentrations of xylose (1–6%) was performed with Pichia stiptis (NCIM 3498) and C. shehatae (NCIM 3501).

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P. stipitis (NCIM 3498) was selected for pentose fermentation than C. shehatae (NCIM 3501) for co-culture in combination with hexose fermenting Saccharomyces cerevesiae OVB 11 for ethanol production. The ethanol produced was 24 g L 1 with a yield of (0.4 g/ g ± 0.01) at D-xylose concentration of 6% (Fig. 1). In agreement to our studies, Ligthelm et al. (1988) also reported ethanol yield at 0.47 g/g using D-xylose by P. stipitis under limitedoxygen condition. Recent studies of ethanol production in our laboratory by same P. stipitis (NCIM 3498) using S. spontaneum enzymatic hydrolysates in batches yielded (g/g) 0.36 ± 0.011, 0.384 ± 0.022, 0.391 ± 0.02, and 0.40 ± 0.01 from detoxified acid hydrolysate and NaOH- and aqueous ammonia(AA) pretreated substrate hydrolysates, respectively (Chandel et al., 2010). The hemicellulosic hydrolysate of Prosopis juliflora containing 18.24 g L 1 sugars, when fermented with the same strain of P. stipitis (NCIM 3498) produced 7.13 g L 1 ethanol with a yield of 0.39 g/g and productivity of 0.30 g L 1 h 1 after 24 h (Gupta et al., 2009).

Fig. 3. Furans present before and after concentration and detoxification process.

3.4. Concentration of hydrolysate for ethanol fermentation Concentration of rice straw hydrolysate was carried out to increase the sugar concentration for ethanol fermentation by vacuum distillation. The fermentable sugars (glucose and xylose) in the rice straw hydrolysate reached to 30 g L 1 compared to the initial hydrolysate (16 g L 1) (Fig. 2). The concentration factor represented a 2-fold increase of fermentable sugars. The hydrolysate was evaporated with the main purpose of increasing the sugar content. Furfural concentration reached 0.21 g L 1 after vacuum distil-

Fig. 4. Phenolics present before and after concentration and detoxification process.

lation from initial concentration of 0.15 g L 1. (Fig. 3) and phenolics concentration reached to 1.58 g L 1 from initial concentration of 0.95 g L 1 (Fig. 4). The doubling of concentration of fermentable sugars in the hydrolysate indicates that vacuum distillation had not decomposed the carbohydrates (Dehkhoda et al., 2008). Fig. 1. Ethanol production using Pichia stipitis and Candida shehatae with various xylose concentrations.

Fig. 2. Sugars present before and after concentration and detoxification process.

3.5. Detoxification To reduce the effect of microbial inhibitors caused by the acid hydrolysis, partial neutralization, over liming and activated charcoal treatments were used which improved the bioconversion of the sugars into ethanol. The acid hydrolysate when treated with calcium oxide and activated charcoal brought about maximum reduction in furans from 0.2 mg/L to 0.025 mg/L (88.4% removal) and total phenolics from 0.95 g L 1 to 0.14 g L 1 (84.6% removal) (Figs. 2–4) however, sugar concentration reduced to 30 g L 1 from the initial concentration of 31 g L 1 (3% loss) during concentration process. Based on ethanol yield and fermentative xylose conversion, over liming at pH 10-conditioned hydrolysate produced the best results, 75% xylose utilization and 76% ethanol yield (Mohagheghi et al., 2006). The fermentation using the non-detoxified hydrolysate led to 4.9 g L 1 ethanol at 120 h of incubation, with a yield of 0.20 g/g and a productivity of 0.04 g L 1 h 1. The detoxification by pH alteration and active charcoal adsorption led to 6.1 g L 1 ethanol in 48 h, with a yield of 0.30 g/g and a productivity of

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0.13 g L 1 h 1 (Larissa et al., 2008). Adaptation of yeast cells to the wood hydrolysate and detoxification methods, such as using charcoal and over lime, had some beneficial effects on ethanol production using the concentrated wood hydrolysate (Lee et al., 1999). Some reports showed the similar trends of decrease in inhibitors present in acid hydrolysates by overliming while activated charcoal being hydrophobic in nature removes the hydrophobic inhibitory compound i.e., furan and phenolics more effectively. Raising the pH of the hydrolysate to 10.0 with Ca (OH)2 and readjustment to pH 6.5 with 6 N H2SO4 revealed efficient detoxification of lignocellulosic hydrolysates (Chandel et al., 2010). Optimal over liming resulted in a 51 ± 9% reduction of total furans, a 41 ± 6% reduction in phenolic compounds, and a 8.7 ± 4.5% decline in sugar (Martinez et al., 2001). Treatment of hydrolysate with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively (Chandel et al., 2007). 3.6. Monoculture ethanol fermentation using S. cerevesiae (OVB 11) and co-culture ethanol fermentation using S. cerevesiae (OVB 11) and P. stipitis Batch fermentation of ethanol using S. cerevesiae (OVB 11) utilizing 30 ± 0.2 g L 1 of sugars produced maximum ethanol of 7.5 g L 1 at 36 h of incubation with a yield 0.3 g/g 1 and productivity of 0.20 g l 1 h 1. However, the ethanol production was slightly declined after 36 h of incubation (Fig. 5). The ethanol efficiency was low i.e. 55%. The reason may be due to most of the xylose was left unfermented by S. cerevesiae (OVB 11), as the hydrolysate contains both xylose and glucose. On the other hand batch fermentation of ethanol using co-culture of S. cerevesiae (OVB 11) and P. stipitis (NCIM 3498) produced maximum ethanol at 36 h of incubation (12 g L 1) with an efficiency of 95% and declined slowly after that. (Figs. 5 and 6). The volumetric ethanol productivity was 0.33 g L h with a yield of 0.4 g/g 1. The increased ethanol yield and ethanol efficiency is due to the total conversion of both sugars i.e glucose and xylose. A batch culture with pretreated sugarcane bagasse hemicellulose hydrolysate produced ethanol of 19 g L 1 with a yield of 0.34 g g 1 and productivity of 0.57 g L 1 h 1 (Cheng et al., 2008). The maximum ethanol concentration reported by Ferrari et al. (1992) was 12.6 g L 1, with a yield of 0.35 g/g in fermentation time

Fig. 6. Fermentation efficiency (%) of monoculture (Saccharomyce cerevisia OVB 11) and co-culture (Saccharomyces cerevisia OVB 11 and Pichia stipitis).

of 75 h. The complete conversion of glucose and xylose mixture (50 g L 1) was obtained using a respiratory deficient mutant of Saccharomyces diastaticus co-cultivated with P. stipitis in continuous culture. Using the co-culture process, the maximum ethanol concentration was 215 g L 1 (Yp/s = 045 g/g) and the maximum volumetric ethanol productivity was 43 g/ (litre  h) (Laplace et al., 1993. Mingyu et al. (2006) also reported that fermentation of detoxified hydrolysate by adapted co-culture (S. cerevisiae + Pachysolen tannophilis) generated an exceptionally high ethanol yield on total sugar of 0.49 g/g, corresponding to 96.1% of the maximal theoretical value after 48 h of incubation. Our studies were supported by Sornvoraweat et al.(2009) who also reported that co-culture of S. cerevisiae and Candida tropicalis produced maximum ethanol in comparison to monoculture of S. cerevisiae using acid hydrolysate of cassava peels. 4. Conclusion Concentrating the sulphuric acid hydrolysate of rice straw which is considered as cheap lignocellulosic substrate for bioethanol production can fulfill the demand of high sugar concentration in industrial scale and also it is possible to ferment this hydrolysate with a fair yield of ethanol because sugar concentration in the hydrolysate will be increased by 2-fold. Hence, the concentrated and detoxified hydrolysate was fermented with co-culture of S. cerevisiae (OVB 11) and P. stipitis (NCIM 3498) leading to conversion of both hexoses and pentoses in the hydrolysate with high ethanol yields than the ethanol produced with monoculture of S. cerevisiae (OVB 11). Acknowledgement We are grateful to Department of Biotechnology (DBT ISLARE), Ministry of Science and Technology (Government of India) for the financial assistance. We thank Dr. Smitha Panda for assisting in the manuscript preparation. References

Fig. 5. Ethanol production using monoculture (Saccharomyces cerevisiae OVB 11) and co-culture (Saccharomyces cerevisiae OVB 11 and Pichia stipitis).

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