- Email: [email protected]
Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast
Bioresource Technology 77 (2001) 193±196
Short communication
Simultaneous sacchari®cation and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast S. Hari Krishna *, T. Janardhan Reddy, G.V. Chowdary Biotechnology Division, Department of Chemical Engineering, Andhra University, Visakhapatnam 530 003, India Received 4 July 2000; received in revised form 3 September 2000; accepted 21 September 2000
Abstract Simultaneous sacchari®cation and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5±3.5% w/v) compared to S. cerevisiae (2.0±2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with b-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka ¯oc > A. leptopus > sugar cane. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Simultaneous sacchari®cation and fermentation (SSF); Antigonum leptopus leaves; Sugar cane leaves; Alkaline H2 O2 pretreatment; Ethanol; Trichoderma reesei cellulase; Lignocellulose
1. Introduction Ethanol production from lignocellulosics has received major research attention due to their abundance and immense potential for conversion into sugars and fuels. So far signi®cant progress has been made using Solka ¯oc, a commercial microcrystalline cellulose (Savarese and Young, 1978) bagasse (Dwivedi and Ghose, 1979) and wheat straw (Saddler et al., 1982a). However, screening several lignocellulosics to select the ideal substrate holds the key for economic production of ethanol. The simultaneous sacchari®cation and fermentation (SSF) process increases the yields of ethanol by minimizing product inhibition as well as eliminates the need for separate reactors for sacchari®cation and fermentation (Deshpande et al., 1983). The SSF process was also shown to be superior to sacchari®cation and subsequent fermentation (Hari Krishna et al., 1999) due to the rapid assimilation of sugars by yeast during SSF. Recent economic analyses (Nguyen and Saddler, 1991; Hinman et al., 1992; VanSivers and Zacchi, 1995) still identi®es the SSF operation as the major contribu*
Present address: Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore 570 013, India. Tel.: +91-821-515-792; fax: +91-821-517-233. E-mail address: [email protected] (S. Hari Krishna).
tor (>20%) to the cost of ethanol production from biomass. The main disadvantage of SSF lies in dierent temperature optima for sacchari®cation (50°C) and fermentation (35°C). Besides, ethanol itself exerts some inhibition. In the previous studies (Hari Krishna et al., 1997, 1998, 1999, 2000), we have identi®ed sugar cane (Saccharum ocinarum) and Antigonum leptopus leaves as potential cellulosic substrates. The leaves of A. leptopus, an abundantly found weedy creeper, can be transformed to sugars in a short time due to the microcrystalline nature of its cellulose (Hari Krishna et al., 1997). Sugar cane leaves are agro-residues burnt after harvesting the crop. Utilizing these leaves would aid pollution abatement. The present study aimed at overcoming SSF problems by employing a thermotolerant yeast, Kluyveromyces fragilis, in SSF, and comparing the eectiveness of K. fragilis over the conventional yeast, Saccharomyces cerevisiae, in the SSF process.
2. Methods 2.1. Cellulase source Cellulase from T. reesei QM-9414 (Celluclastâ ) and b-glucosidase (Novozym 188) from Aspergillus niger
0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 5 1 - 6
194
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
Table 1 Simultaneous sacchari®cation and fermentation (SSF) resultsa Enzyme
Units/g substrate
Cellulase
40 FPU
Cellulase + b-glucosidase
40 FPU + 50 U
Time (h)
Ethanol yield (% w/v) A. leptopus
Sugar cane
Solka ¯oc
A
B
A
B
A
B
12 24 48 72 96 12 24
1.4 1.7 1.8 1.9 1.9 1.8 1.9
1.6 2.0 2.3 2.3 ndb 2.0 2.3
0.2 0.4 1.6 1.8 2.0 0.8 1.5
0.8 1.5 2.2 2.5 2.5 1.5 2.0
1.9 2.2 2.3 2.3 nd 2.2 2.5
1.8 2.5 2.8 3.0 3.0 2.0 2.5
48 72 96
2.1 2.1 nd
2.7 2.7 nd
2.0 2.2 2.2
2.5 2.8 2.8
2.5 2.5 nd
3.3 3.5 3.5
a The values given were the average of duplicate experiments. Conditions: SSF Basal medium (see Methods); 10% (w/v) substrate; 10% (v/v) yeast inoculum (A ± Saccharomyces cerevisiae; B ± Kluyveromyces fragilis) simultaneously added; 40°C for A, 43°C for B; initial pH 5.1. b nd: Not determined.
were generous gifts from Novo Nordisk (Bagsvaerd, Denmark). 2.2. Substrates and pretreatment A. leptopus leaves were collected from the University campus, Andhra University (Visakhapatnam, India). Sugar cane (S. ocinarum) leaves were procured from sugar cane harvesters from East Godavari District (Andhra Pradesh, India). Substrate leaves were pretreated with alkaline hydrogen peroxide NaOH H2 O2 (Gould, 1984). Solka ¯oc SW-40 (microcrystalline cellulose) was employed as the substrate of reference. 2.3. Simultaneous sacchari®cation and fermentation SSF reaction mixtures contained alkaline peroxidepretreated substrate (previously autoclaved for 15 min at 121°C), cellulase, 10% (v/v) yeast inoculum and SSF basal medium (Takagi et al., 1977) to make up the volume to 100 ml. pH was adjusted to 5.1 with 0.05 M citrate buer. Reactions were carried out in 250 ml ¯asks with 100 ml working volume on an orbital shaker at 150 rpm. 2.4. Preparation of yeast inoculum S. cerevisiae (NRRL-Y-132) and K. fragilis (NCIM 3358) were obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratories (Pune, India). The S. cerevisiae inoculum culture was prepared as described elsewhere (Hari Krishna et al., 1999). The inoculum basal medium for K. fragilis was as follows (g/l): yeast extract, 10.0; NaCl, 1.0; CaCl2 2H2 O, 0.2; KH2 PO4 , 2.0; FeCl3 6H2 O,
0.01; MgSO4 7H2 O, 1.7; NH4 Cl, 2.0 and distilled water to make up the volume to 1 l. The inoculum cultures were taken from malt agar slants and incubated with shaking for 24 h at 28°C in the inoculum media with 4% (w/v) glucose until the sugar had been exhausted. 2.5. Analytical techniques Cellulase activity was assayed as Filter Paper Units (FPU) (Mandels et al., 1976) and reducing sugars were estimated by the DNS method (Miller, 1959). Ethanol was estimated by gas chromatography (Hari Krishna et al., 2000) Ethanol was also estimated by using a chemical oxidation method (Caputi and Wright, 1969). Quanti®cations of ethanol by both GC analysis and the chemical method were found to be in good agreement.
3. Results and discussion Alkaline H2 O2 pretreatment, pH 4.5, 50°C, 40 FPU cellulase/g substrate, 2.5% substrate were found to be optimum for the sacchari®cation (Hari Krishna et al., 1997). An ideal pretreatment would reduce the lignin content and crystallinity of cellulose and increase surface area (Thompson et al., 1992). Alkaline H2 O2 treatment was identi®ed as suitable to result in quantitative yields of reducing sugars. Increase in substrate concentration (5±25%) resulted in low sacchari®cation yields due to diculty in stirring and product inhibition. To overcome inhibition, the SSF process was applied and thus, the substrate content was increased to 10% in SSF. Though 50°C was found to be optimum, the range of 40±50°C did not aect the yield signi®cantly and hence, SSF temperature was chosen as 40°C and 43°C for S. cerevisiae and K. fragilis, respectively. Increase in
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
enzyme content enhanced the yield, but only a minimal increase was found in the range of 40±120 FPU and thus, 40 FPU/g substrate was selected. The present study employed a thermotolerant yeast, K. fragilis, which can be cultured at a temperature near to that optimum for cellulase (i.e., 50°C). Table 1 summarizes the results obtained with all the substrates (A. leptopus, sugar cane and Solka ¯oc) using both S. cerevisiae and K. fragilis. It should be stressed that K. fragilis proved to be superior to S. cerevisiae as the yields obtained with K. fragilis were distinctly higher. This result was attributed to the thermotolerance of K. fragilis. But, at 50°C, the ethanol yield was found to be reduced drastically with both the yeasts (where the yields were only 0.15% and 0.35% w/v for S. cerevisiae and K. fragilis, respectively) and a signi®cant amount of sugars was found to remain unmetabolized. This was due to the thermolability of both the yeasts at the higher temperature. However, K. fragilis generally tolerated a higher ethanol content and temperature than S. cerevisiae. b-glucosidase addition was found to favor faster reactions with both the yeasts. This might have been due to the low activities of b-glucosidase in the T. reesei cellulase enzyme complex, and compensating it from external source gave better yields. This result is in good agreement with that reported by Breuil et al. (1992). Most of the researchers (Takagi et al., 1977; Saddler et al., 1982b; Deshpande et al., 1983; Spangler and Emert, 1986) reported the conversions (up to 2% w/v ethanol) in 4±6 days. We have achieved ethanol yields of 2.2% (w/v) in 72 h and 2.1% (w/v) in 48 h using celluloses from sugar cane (Hari Krishna et al., 1998) and A. leptopus (Hari Krishna et al., 1999) leaves. The conversions with A. leptopus cellulose were completed in a shorter time due to the ®ne microcrystalline nature of its cellulose. Thermotolerant yeasts capable of growth and ethanol production at temperatures above 40°C, have been investigated in the past for suitability in SSF (Szezodrak and Targonski, 1988; Spindler et al., 1989). Among 58 strains tested (Szezodrak and Targonski, 1988), Fabospora fragilis was the most suitable for ethanol production (56 g ethanol/l from 140 g glucose/l) at 43°C. Spindler et al. (1989) suggested that the use of a mixed culture of yeast is advantageous in achieving a higher product yield at 41°C. Although the research on K. marxianus strain improvement was extensive (Ballesteros et al., 1993), studies on an other thermotolerant yeast, K. fragilis, have been scanty. The present investigation shows that ethanol yields can further be improved using thermotolerant yeast for a shorter culture time. The substrates were of the following order in terms of rate of conversion: Solka ¯oc > A. leptopus > sugar cane. Though Solka ¯oc was a better substrate, low-cost raw materials should obviously be the materials of choice. The yields were of the
195
order of 2.5±3.5% (w/v) with all the substrates and the conversions were completed in about 48±72 h. In this regard, it should be noted that an overall economic process (Bothast and Saha, 1997) must include achieving a high ethanol yield (>3.5%) at high substrate loading (>10% w/v) over short residence times (<4 days), some of which were attained in this study.
Acknowledgements The authors are thankful to the University Grants Commission, New Delhi and Andhra University, Visakhapatnam for the ®nancial support.
References Ballesteros, I., Oliva, J.M., Ballesteros, M., Carrasco, J., 1993. Optimization of the simultaneous sacchari®cation and fermentation process using thermotolerant yeasts. Appl. Biochem. Biotechnol. 39, 307±315. Bothast, R.J., Saha, B.C., 1997. Ethanol production from agricultural biomass substrates. Adv. Appl. Microbiol. 44, 261±286. Breuil, C., Chan, M., Gilbert, M., Saddler, J.N., 1992. In¯uence of bglucosidase on the ®lter paper activity and hydrolysis of lignocellulosic substrates. Bioresource. Technol. 39, 139±142. Caputi, A., Wright, D., 1969. Collaborative study of the determination of ethanol in wine by chemical oxidation. J. AOAC 52, 85±88. Deshpande, V., Raman, H.S., Rao, M., 1983. Simultaneous sacchari®cation and fermentation of cellulose to ethanol using Penicillium funiculosum cellulase and free or immobilized Saccharomycesuvarum cells. Biotechnol. Bioeng. 25, 1679±1684. Dwivedi, C.P., Ghose, T.K., 1979. A model of hydrolysis of bagasse cellulose by enzyme from Trichoderma reesei QM 9414. J. Ferment. Technol. 57, 15±24. Gould, J.M., 1984. Alkaline peroxide deligni®cation of agricultural residues to enhance enzymatic sacchari®cation. Biotechnol. Bioeng. 26, 46±52. Hari Krishna, S., Prabhakar, Y., Rao, R.J., 1997. Sacchari®cation studies of lignocellulosic biomass from Antigonum leptopus Linn. Indian J. Pharma. Sci. 59, 39±42. Hari Krishna, S., Prasanthi, K., Chowdary, G.V., Ayyanna, C., 1998. Simultaneous sacchari®cation fermentation of pretreated sugar cane leaves to ethanol. Process. Biochem. 33, 825±830. Hari Krishna, S., Chowdary, G.V., Reddy, D.S., Ayyanna, C., 1999. Simultaneous sacchari®cation and fermentation of pretreated Antigonum leptopus (Linn) leaves to ethanol. J. Chem. Technol. Biotechnol. 74, 1055±1060. Hari Krishna, S., Chowdary, G.V., 2000. Optimization of simultaneous sacchari®cation and fermentation for the production of ethanol from biomass. J. Agric. Food Chem. 48, 1971±1976. Hinman, N.D., Schell, D.J., Riley, C.J., Bergeron, P., Walter, P.J., 1992. Preliminary estimate of the cost of ethanol production for simultaneous sacchari®cation and fermentation technology. Appl. Biochem. Biotechnol. 34/35, 639±649. Mandels, M., Andreotti, R.E., Roche, C., 1976. Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp. 6, 21±23. Miller, G.L., 1959. Use of dinitro salicylic acid reagent for determination of reducing sugar. Analyt. Chem. 31, 426±428. Nguyen, Q.A., Saddler, J.N., 1991. An integrated model for the technical and economic evaluation of an enzymatic biomass conversion process. Bioresource Technol. 35, 275±282.
196
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
Saddler, J.N., Brownell, H.H., Clermont, L.P., Levitin, N., 1982a. Enzymatic hydrolysis of cellulose and various pretreated wood fractions. Biotechnol. Bioeng. 24, 1389±1402. Saddler, J.N., Hogan, C., Chan, M.K.H., Louiz-Seize, G., 1982b. Ethanol fermentation of enzymatically hydrolyzed pretreated wood fractions using Trichoderma cellulases, Zymomonas mobilis and Saccharomyces cerevisiae. Canadian J. Microbiol. 28, 1311±1319. Savarese, J.J., Young, S.D., 1978. Combined enzyme hydrolysis of cellulose and yeast fermentation. Biotechnol. Bioeng. 20, 1291±1293. Spangler, D.J., Emert, G.H., 1986. Simultaneous sacchari®cation and fermentation with Zymomonas mobilis. Biotechnol. Bioeng. 28, 115±118. Spindler, D.D., Wyman, C.E., Grohmann, K., 1989. Evaluation of thermotolerant yeasts in controlled simultaneous sacchari®cation and fermentation of cellulose to ethanol. Biotechnol. Bioeng. 34, 189±195.
Szezodrak, J., Targonski, Z., 1988. Selection of thermotolerant yeast strains for simultaneous sacchari®cation and fermentation of cellulose. Biotechnol. Bioeng. 31, 300±303. Takagi, M., Abe, S., Suzuki, S., Emert, G.H., Yata, N., 1977. A method for production of alcohol directly from cellulose using cellulase and yeast. In: Ghose, T.K. (Ed.), Proceedings of Bioconversion of cellulosic substances into energy, chemicals and microbial protein. I.I.T., New Delhi, pp. 551±571. Thompson, D.N., Chen, H.C., Grethlein, H.C., 1992. Comparison of pretreatment methods on the basis of available surface area. Bioresource Technol. 39, 155±163. VanSivers, M., Zacchi, G., 1995. A techno-economical comparison of three processes for the production of ethanol from pine. Bioresource Technol. 51, 43±52.
Short communication
Simultaneous sacchari®cation and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast S. Hari Krishna *, T. Janardhan Reddy, G.V. Chowdary Biotechnology Division, Department of Chemical Engineering, Andhra University, Visakhapatnam 530 003, India Received 4 July 2000; received in revised form 3 September 2000; accepted 21 September 2000
Abstract Simultaneous sacchari®cation and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5±3.5% w/v) compared to S. cerevisiae (2.0±2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with b-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka ¯oc > A. leptopus > sugar cane. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Simultaneous sacchari®cation and fermentation (SSF); Antigonum leptopus leaves; Sugar cane leaves; Alkaline H2 O2 pretreatment; Ethanol; Trichoderma reesei cellulase; Lignocellulose
1. Introduction Ethanol production from lignocellulosics has received major research attention due to their abundance and immense potential for conversion into sugars and fuels. So far signi®cant progress has been made using Solka ¯oc, a commercial microcrystalline cellulose (Savarese and Young, 1978) bagasse (Dwivedi and Ghose, 1979) and wheat straw (Saddler et al., 1982a). However, screening several lignocellulosics to select the ideal substrate holds the key for economic production of ethanol. The simultaneous sacchari®cation and fermentation (SSF) process increases the yields of ethanol by minimizing product inhibition as well as eliminates the need for separate reactors for sacchari®cation and fermentation (Deshpande et al., 1983). The SSF process was also shown to be superior to sacchari®cation and subsequent fermentation (Hari Krishna et al., 1999) due to the rapid assimilation of sugars by yeast during SSF. Recent economic analyses (Nguyen and Saddler, 1991; Hinman et al., 1992; VanSivers and Zacchi, 1995) still identi®es the SSF operation as the major contribu*
Present address: Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore 570 013, India. Tel.: +91-821-515-792; fax: +91-821-517-233. E-mail address: [email protected] (S. Hari Krishna).
tor (>20%) to the cost of ethanol production from biomass. The main disadvantage of SSF lies in dierent temperature optima for sacchari®cation (50°C) and fermentation (35°C). Besides, ethanol itself exerts some inhibition. In the previous studies (Hari Krishna et al., 1997, 1998, 1999, 2000), we have identi®ed sugar cane (Saccharum ocinarum) and Antigonum leptopus leaves as potential cellulosic substrates. The leaves of A. leptopus, an abundantly found weedy creeper, can be transformed to sugars in a short time due to the microcrystalline nature of its cellulose (Hari Krishna et al., 1997). Sugar cane leaves are agro-residues burnt after harvesting the crop. Utilizing these leaves would aid pollution abatement. The present study aimed at overcoming SSF problems by employing a thermotolerant yeast, Kluyveromyces fragilis, in SSF, and comparing the eectiveness of K. fragilis over the conventional yeast, Saccharomyces cerevisiae, in the SSF process.
2. Methods 2.1. Cellulase source Cellulase from T. reesei QM-9414 (Celluclastâ ) and b-glucosidase (Novozym 188) from Aspergillus niger
0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 5 1 - 6
194
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
Table 1 Simultaneous sacchari®cation and fermentation (SSF) resultsa Enzyme
Units/g substrate
Cellulase
40 FPU
Cellulase + b-glucosidase
40 FPU + 50 U
Time (h)
Ethanol yield (% w/v) A. leptopus
Sugar cane
Solka ¯oc
A
B
A
B
A
B
12 24 48 72 96 12 24
1.4 1.7 1.8 1.9 1.9 1.8 1.9
1.6 2.0 2.3 2.3 ndb 2.0 2.3
0.2 0.4 1.6 1.8 2.0 0.8 1.5
0.8 1.5 2.2 2.5 2.5 1.5 2.0
1.9 2.2 2.3 2.3 nd 2.2 2.5
1.8 2.5 2.8 3.0 3.0 2.0 2.5
48 72 96
2.1 2.1 nd
2.7 2.7 nd
2.0 2.2 2.2
2.5 2.8 2.8
2.5 2.5 nd
3.3 3.5 3.5
a The values given were the average of duplicate experiments. Conditions: SSF Basal medium (see Methods); 10% (w/v) substrate; 10% (v/v) yeast inoculum (A ± Saccharomyces cerevisiae; B ± Kluyveromyces fragilis) simultaneously added; 40°C for A, 43°C for B; initial pH 5.1. b nd: Not determined.
were generous gifts from Novo Nordisk (Bagsvaerd, Denmark). 2.2. Substrates and pretreatment A. leptopus leaves were collected from the University campus, Andhra University (Visakhapatnam, India). Sugar cane (S. ocinarum) leaves were procured from sugar cane harvesters from East Godavari District (Andhra Pradesh, India). Substrate leaves were pretreated with alkaline hydrogen peroxide NaOH H2 O2 (Gould, 1984). Solka ¯oc SW-40 (microcrystalline cellulose) was employed as the substrate of reference. 2.3. Simultaneous sacchari®cation and fermentation SSF reaction mixtures contained alkaline peroxidepretreated substrate (previously autoclaved for 15 min at 121°C), cellulase, 10% (v/v) yeast inoculum and SSF basal medium (Takagi et al., 1977) to make up the volume to 100 ml. pH was adjusted to 5.1 with 0.05 M citrate buer. Reactions were carried out in 250 ml ¯asks with 100 ml working volume on an orbital shaker at 150 rpm. 2.4. Preparation of yeast inoculum S. cerevisiae (NRRL-Y-132) and K. fragilis (NCIM 3358) were obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratories (Pune, India). The S. cerevisiae inoculum culture was prepared as described elsewhere (Hari Krishna et al., 1999). The inoculum basal medium for K. fragilis was as follows (g/l): yeast extract, 10.0; NaCl, 1.0; CaCl2 2H2 O, 0.2; KH2 PO4 , 2.0; FeCl3 6H2 O,
0.01; MgSO4 7H2 O, 1.7; NH4 Cl, 2.0 and distilled water to make up the volume to 1 l. The inoculum cultures were taken from malt agar slants and incubated with shaking for 24 h at 28°C in the inoculum media with 4% (w/v) glucose until the sugar had been exhausted. 2.5. Analytical techniques Cellulase activity was assayed as Filter Paper Units (FPU) (Mandels et al., 1976) and reducing sugars were estimated by the DNS method (Miller, 1959). Ethanol was estimated by gas chromatography (Hari Krishna et al., 2000) Ethanol was also estimated by using a chemical oxidation method (Caputi and Wright, 1969). Quanti®cations of ethanol by both GC analysis and the chemical method were found to be in good agreement.
3. Results and discussion Alkaline H2 O2 pretreatment, pH 4.5, 50°C, 40 FPU cellulase/g substrate, 2.5% substrate were found to be optimum for the sacchari®cation (Hari Krishna et al., 1997). An ideal pretreatment would reduce the lignin content and crystallinity of cellulose and increase surface area (Thompson et al., 1992). Alkaline H2 O2 treatment was identi®ed as suitable to result in quantitative yields of reducing sugars. Increase in substrate concentration (5±25%) resulted in low sacchari®cation yields due to diculty in stirring and product inhibition. To overcome inhibition, the SSF process was applied and thus, the substrate content was increased to 10% in SSF. Though 50°C was found to be optimum, the range of 40±50°C did not aect the yield signi®cantly and hence, SSF temperature was chosen as 40°C and 43°C for S. cerevisiae and K. fragilis, respectively. Increase in
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
enzyme content enhanced the yield, but only a minimal increase was found in the range of 40±120 FPU and thus, 40 FPU/g substrate was selected. The present study employed a thermotolerant yeast, K. fragilis, which can be cultured at a temperature near to that optimum for cellulase (i.e., 50°C). Table 1 summarizes the results obtained with all the substrates (A. leptopus, sugar cane and Solka ¯oc) using both S. cerevisiae and K. fragilis. It should be stressed that K. fragilis proved to be superior to S. cerevisiae as the yields obtained with K. fragilis were distinctly higher. This result was attributed to the thermotolerance of K. fragilis. But, at 50°C, the ethanol yield was found to be reduced drastically with both the yeasts (where the yields were only 0.15% and 0.35% w/v for S. cerevisiae and K. fragilis, respectively) and a signi®cant amount of sugars was found to remain unmetabolized. This was due to the thermolability of both the yeasts at the higher temperature. However, K. fragilis generally tolerated a higher ethanol content and temperature than S. cerevisiae. b-glucosidase addition was found to favor faster reactions with both the yeasts. This might have been due to the low activities of b-glucosidase in the T. reesei cellulase enzyme complex, and compensating it from external source gave better yields. This result is in good agreement with that reported by Breuil et al. (1992). Most of the researchers (Takagi et al., 1977; Saddler et al., 1982b; Deshpande et al., 1983; Spangler and Emert, 1986) reported the conversions (up to 2% w/v ethanol) in 4±6 days. We have achieved ethanol yields of 2.2% (w/v) in 72 h and 2.1% (w/v) in 48 h using celluloses from sugar cane (Hari Krishna et al., 1998) and A. leptopus (Hari Krishna et al., 1999) leaves. The conversions with A. leptopus cellulose were completed in a shorter time due to the ®ne microcrystalline nature of its cellulose. Thermotolerant yeasts capable of growth and ethanol production at temperatures above 40°C, have been investigated in the past for suitability in SSF (Szezodrak and Targonski, 1988; Spindler et al., 1989). Among 58 strains tested (Szezodrak and Targonski, 1988), Fabospora fragilis was the most suitable for ethanol production (56 g ethanol/l from 140 g glucose/l) at 43°C. Spindler et al. (1989) suggested that the use of a mixed culture of yeast is advantageous in achieving a higher product yield at 41°C. Although the research on K. marxianus strain improvement was extensive (Ballesteros et al., 1993), studies on an other thermotolerant yeast, K. fragilis, have been scanty. The present investigation shows that ethanol yields can further be improved using thermotolerant yeast for a shorter culture time. The substrates were of the following order in terms of rate of conversion: Solka ¯oc > A. leptopus > sugar cane. Though Solka ¯oc was a better substrate, low-cost raw materials should obviously be the materials of choice. The yields were of the
195
order of 2.5±3.5% (w/v) with all the substrates and the conversions were completed in about 48±72 h. In this regard, it should be noted that an overall economic process (Bothast and Saha, 1997) must include achieving a high ethanol yield (>3.5%) at high substrate loading (>10% w/v) over short residence times (<4 days), some of which were attained in this study.
Acknowledgements The authors are thankful to the University Grants Commission, New Delhi and Andhra University, Visakhapatnam for the ®nancial support.
References Ballesteros, I., Oliva, J.M., Ballesteros, M., Carrasco, J., 1993. Optimization of the simultaneous sacchari®cation and fermentation process using thermotolerant yeasts. Appl. Biochem. Biotechnol. 39, 307±315. Bothast, R.J., Saha, B.C., 1997. Ethanol production from agricultural biomass substrates. Adv. Appl. Microbiol. 44, 261±286. Breuil, C., Chan, M., Gilbert, M., Saddler, J.N., 1992. In¯uence of bglucosidase on the ®lter paper activity and hydrolysis of lignocellulosic substrates. Bioresource. Technol. 39, 139±142. Caputi, A., Wright, D., 1969. Collaborative study of the determination of ethanol in wine by chemical oxidation. J. AOAC 52, 85±88. Deshpande, V., Raman, H.S., Rao, M., 1983. Simultaneous sacchari®cation and fermentation of cellulose to ethanol using Penicillium funiculosum cellulase and free or immobilized Saccharomycesuvarum cells. Biotechnol. Bioeng. 25, 1679±1684. Dwivedi, C.P., Ghose, T.K., 1979. A model of hydrolysis of bagasse cellulose by enzyme from Trichoderma reesei QM 9414. J. Ferment. Technol. 57, 15±24. Gould, J.M., 1984. Alkaline peroxide deligni®cation of agricultural residues to enhance enzymatic sacchari®cation. Biotechnol. Bioeng. 26, 46±52. Hari Krishna, S., Prabhakar, Y., Rao, R.J., 1997. Sacchari®cation studies of lignocellulosic biomass from Antigonum leptopus Linn. Indian J. Pharma. Sci. 59, 39±42. Hari Krishna, S., Prasanthi, K., Chowdary, G.V., Ayyanna, C., 1998. Simultaneous sacchari®cation fermentation of pretreated sugar cane leaves to ethanol. Process. Biochem. 33, 825±830. Hari Krishna, S., Chowdary, G.V., Reddy, D.S., Ayyanna, C., 1999. Simultaneous sacchari®cation and fermentation of pretreated Antigonum leptopus (Linn) leaves to ethanol. J. Chem. Technol. Biotechnol. 74, 1055±1060. Hari Krishna, S., Chowdary, G.V., 2000. Optimization of simultaneous sacchari®cation and fermentation for the production of ethanol from biomass. J. Agric. Food Chem. 48, 1971±1976. Hinman, N.D., Schell, D.J., Riley, C.J., Bergeron, P., Walter, P.J., 1992. Preliminary estimate of the cost of ethanol production for simultaneous sacchari®cation and fermentation technology. Appl. Biochem. Biotechnol. 34/35, 639±649. Mandels, M., Andreotti, R.E., Roche, C., 1976. Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp. 6, 21±23. Miller, G.L., 1959. Use of dinitro salicylic acid reagent for determination of reducing sugar. Analyt. Chem. 31, 426±428. Nguyen, Q.A., Saddler, J.N., 1991. An integrated model for the technical and economic evaluation of an enzymatic biomass conversion process. Bioresource Technol. 35, 275±282.
196
S. Hari Krishna et al. / Bioresource Technology 77 (2001) 193±196
Saddler, J.N., Brownell, H.H., Clermont, L.P., Levitin, N., 1982a. Enzymatic hydrolysis of cellulose and various pretreated wood fractions. Biotechnol. Bioeng. 24, 1389±1402. Saddler, J.N., Hogan, C., Chan, M.K.H., Louiz-Seize, G., 1982b. Ethanol fermentation of enzymatically hydrolyzed pretreated wood fractions using Trichoderma cellulases, Zymomonas mobilis and Saccharomyces cerevisiae. Canadian J. Microbiol. 28, 1311±1319. Savarese, J.J., Young, S.D., 1978. Combined enzyme hydrolysis of cellulose and yeast fermentation. Biotechnol. Bioeng. 20, 1291±1293. Spangler, D.J., Emert, G.H., 1986. Simultaneous sacchari®cation and fermentation with Zymomonas mobilis. Biotechnol. Bioeng. 28, 115±118. Spindler, D.D., Wyman, C.E., Grohmann, K., 1989. Evaluation of thermotolerant yeasts in controlled simultaneous sacchari®cation and fermentation of cellulose to ethanol. Biotechnol. Bioeng. 34, 189±195.
Szezodrak, J., Targonski, Z., 1988. Selection of thermotolerant yeast strains for simultaneous sacchari®cation and fermentation of cellulose. Biotechnol. Bioeng. 31, 300±303. Takagi, M., Abe, S., Suzuki, S., Emert, G.H., Yata, N., 1977. A method for production of alcohol directly from cellulose using cellulase and yeast. In: Ghose, T.K. (Ed.), Proceedings of Bioconversion of cellulosic substances into energy, chemicals and microbial protein. I.I.T., New Delhi, pp. 551±571. Thompson, D.N., Chen, H.C., Grethlein, H.C., 1992. Comparison of pretreatment methods on the basis of available surface area. Bioresource Technol. 39, 155±163. VanSivers, M., Zacchi, G., 1995. A techno-economical comparison of three processes for the production of ethanol from pine. Bioresource Technol. 51, 43±52.