Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanol

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1 Industrial Crops and Products 20 (2004) Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanol Zs. Kádár, Zs. Szengyel, K. Réczey Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, Szt. Gellért tér 4, Budapest H-1521, Hungary Received 1 April 2002; accepted 22 December 2003 Abstract During the past decades considerably large efforts have been made to optimize the production of lignocellulose derived fuel ethanol production in order to develop a process configuration which is economically feasible and competitive with gasoline. One of the process alternatives uses cellulase enzymes for the conversion of cellulose content of lignocellulosic biomass to fermentable glucose. Due to the relatively similar process conditions in the enzymatic hydrolysis and ethanol fermentation, the option of carrying out these two-steps together in one vessel exists. The application of simultaneous saccharification and fermentation (SSF) for the conversion of lignocellulosics to alcohol would result in a more cost-effective process. In the present study various lignocellulosic substrates, i.e. Solka Floc, OCC waste cardboard, and paper sludge, were examined in SSF experiments for the production of ethanol. Two yeast strains were compared, a commercially available baker s yeast and a thermotolerant Kluyveromyces marxianus, in two types of SSF experiments, i.e. isothermal SSF and SSF with temperature profiling. The results showed that OCC waste and paper sludge could be used as substrates for ethanol production in SSF. There was no significant difference observed between Saccharomyces cerevisiae and K. marxianus when the results of SSF were compared. The ethanol yields were in the range of g/g for both strains used. SSF resulted in higher ethanol yields compared to non-isothermal SSF (NSSF; SSF with temperature profiling) Elsevier B.V. All rights reserved. Keywords: Simultaneous saccharification and fermentation; Paper sludge; Ethanol 1. Introduction Since the technical revolution the carbon dioxide concentration in the atmosphere has been increasing gradually. However, during the past century the net carbon dioxide production has increased exponentially Corresponding author. Tel.: ; fax: address: kati reczey@mkt.bme.hu (K. Réczey). because of the tremendous expansion in the transportation sector resulting in notably changes in the earth s ecosystem. In order to prevent irreversible changes and reduce the impact of greenhouse gases on the earth s climate international collaboration is needed. Several countries have decided that in their energy production, renewable sources are going to play an important role. The first rather effective step in this process, since the majority of CO 2 is produced by the transportation /$ see front matter 2004 Elsevier B.V. All rights reserved. doi: /j.indcrop

2 104 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) sector, would be using biomass derived so called alternative fuels. One of the candidates, which could substitute fossil fuels, is ethanol. Today ethanol is produced in USA from cornstarch based process (Claassen et al., 1999). However, the economic competitiveness with gasoline still remains an issue. One of the main cost contributive parameters of biomass originated ethanol is the cost of the raw material. For the reduction of overall production, cost cheap raw materials, such as industrial wastes, have to be used. Further cost reduction can be obtained if the conversion efficiency of the raw material is increased to the maximum. In Hungary approximately 50,000 t of paper sludge is produced annually. Paper sludge is the solid waste stream of the papermaking industry containing the short cellulose fibers, which leave the process. Usually this stream is deposed off, which has a significant cost-increasing factor on the paper production. Another option for utilizing the organic content of this waste stream is heat and electricity generation by direct combustion. However, the high water and inorganic matter content, which can be as high as 30 wt.% dry matter, would result in substantial energy loss. Producing a value added product, such as fuel ethanol, from the cellulose present in the paper sludge, could provide an economically more attractive option. Due to the high and rather accessible cellulose content (50 60%) of paper sludge, it could be a potential feedstock for fuel ethanol production (Lynd et al., 2001). However, the high ash content of paper sludge could be a problem for several reasons. First of all the handling of the raw material can be difficult. Secondly, the economy of the process highly depends on the utilization of the solid residue i.e. the lignin, in the case of for instance wood based fuel ethanol production, which constitutes approximately 25 wt.% of the raw material (Claassen et al., 1999). Lignin can be burnt to provide energy required for the processing. When paper sludge is used, the solid residue will contain mostly inorganic ash. Obviously, energy cannot be obtained from this waste. Alternatively, this by-product could be sold as an additive for the construction industry. There are several technologies available for the conversion of lignocellulosics to fuel ethanol. The main difference between these technologies is the catalyst used for the brake-down of polysaccharides in the raw material. In the one-step concentrated acid, or in the two-step dilute acid technologies, acids are used to convert the hemicellulose and cellulose fraction to monomeric sugars. In the enzymatic process, the hemicellulose fraction is hydrolyzed by means of acids or bases, while cellulase enzymes are used for the conversion of cellulose. Although the current market price of cellulases makes the process less favorable compared to technologies using acid catalysts, working with enzymes, due to the significantly milder processing conditions applied, makes it possible to combine the cellulose hydrolysis with the ethanol fermentation. Simultaneous saccharification and fermentation (SSF) technique provides the possibility to overcome the main disadvantage of the enzymatic hydrolysis i.e. decreasing the enzyme loading and therefore the production cost. In spite of the economical advantage of SSF over separate hydrolysis and fermentation (SHF), the critical problem with SSF is the difference in temperature optima of the cellulases and the fermenting microorganism. Saccharomyces strains are well known as good ethanol producing microorganisms, however they require an operating temperature of 35 C. Fungal cellulases, which are most frequently applied in the cellulose hydrolysis have an optimum temperature of 50 C. At lower temperatures, the substantially lower hydrolysis rates would be unfavorable in terms of increased processing time. A possible solution to solve this problem is using thermotolerant yeast strains instead of Saccharomyces strains, which would allow higher processing temperatures, thus increased rates of the hydrolysis. Szczodrak and Targoňski (1988) screened 58 yeast strains from 12 different genera for their ability to grow and ferment at temperatures above 40 C. It was reported that some of the Fabospora and Kluyveromyces strains were able to grow at as high temperature as 46 C. Fabospora fragilis CCY cultivated at 43 C produced 56 g/l ethanol from 140 g/l glucose, which corresponds to 0.4 g/g (g ethanol/g glucose) ethanol yield. However, when the cultivation temperature was increased to 46 C the performance of the strain significantly decreased, and 0.25 g/g ethanol yield was obtained. In another study Saccharomyces, Candida and Kluyveromyces strains were examined by Ballesteros et al. (1991) for their ability to ferment glucose at temperatures above 40 C. Similarly to the results

3 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) obtained by Szczodrak and Targoňski (1988) Candida and Saccharomyces strains proved to be less thermotolerant than Kluyveromyces strains. When Kluyveromyces marxianus L.G. was cultivated at 42 C on glucose containing medium 37.6 g/l ethanol concentration was obtained with an ethanol yield of 0.4 g/g. Bollók and Réczey (2000) evaluated five different Kluyveromyces strains based on the examination of their growth on agar slants and in shake flask cultures at different temperatures. On glucose medium in aerobic cultivation, K. marxianus Y01070 proved to be the best thermotolerant strain of all examined strains. In SSF experiments Ballesteros et al. (1991) achieved best conversion of 10% Solka Floc cellulose substrate to ethanol using both K. marxianus L.G. and K. fragilis L.G. at temperatures up to 42 C, where 0.5 and 0.46 ethanol yields were achieved, respectively. The ethanol yields were reduced at 45 C because of cell death. Barron et al. (1995) found that the K. marxianus IMB3 is capable of producing ethanol at as high temperature as 45 C using milled paper as substrate. The obtained ethanol yield was 0.11 g/g (21% of the theoretical). Nilsson et al. (1995) reported that the ethanol yield could be further increased using the same substrate. Pretreatment of the milled paper with phosphoric acid increased the accessibility of the substrate resulting in considerably higher ethanol yield of 0.21 g/g. SSF experiments on paper sludge were carried out by Lynd et al. (2001). The results showed wide variability with respect to the sludge processing. It was concluded that the prediction of the results of SSF experiments was a difficult task, however some general conclusions could be made. Most commercially available cellulase enzyme preparations have low -glucosidase activity. This enzyme is essential, because it converts the cellobiose to glucose, which then can be fermented by the yeast. Supplementing the cellulases with -glucosidase from external source increased the theoretical ethanol yield from 63 to 84%. It was also shown that neither the composition of growth medium (rich or lean) nor the cellulase loading had any significant effect on the results and similar results were obtained at 10 and 20 FPU/g substrate enzyme loading. Using rich medium the ethanol yield was 83% of the theoretical, while performing the fermentation in lean medium 86% ethanol yield was obtained. Increasing the enzyme loading from 10 FPU/g cellulose to 20 FPU/g cellulose resulted in slightly lower, 81% of theoretical, ethanol yield when compared to the results, 87% of theoretical ethanol yield, obtained at lower cellulase loading. Huang and Chen (1988) examined the SSF technique with temperature profiling using Solka Floc as the substrate. The fermenting microorganism was Zymomonas mobilis, and culture medium was supplemented with Trichoderma reesei cellulases. Two different strategies were applied: temperature cycling and profiling, to enhance the SSF fermentation. In a cycling study the temperature was changed periodically square-pulse-function wise between the optimal fermenting temperature, 37 C, and the highest tolerable temperature, 40 C, of Z. mobilis. In the profiling experiment during the initial phase, the temperature was controlled between 30 and 37 C to allow optimal condition for the propagation of the cells. After the cells entered into their active ethanol production phase, therefore the hydrolysis reaction became the rate limiting step of SSF process, the temperature was increased to 40 C. The results showed that with temperature cycling the ethanol productivity (the final ethanol concentration divided by the reaction time) could be increased from 0.49 g/(l h) to 0.62 g/(l h). However, similar ethanol yield, 0.23 g/g, was obtained as with traditional SSF. In contrary, with temperature profiling the ethanol yield obtained was significantly higher, 0.32 g/g, than that obtained with isothermal SSF. Unfortunately, the productivity, 0.32 g/(l h), was reduced due to the increased processing time required for the prehydrolysis. Since in SSF the rate limiting step is the hydrolysis, it is possible that a pre-hydrolysis at optimum temperature for enzymes could improve the process. In the present study, SSF and non-isothermal SSF (NSSF) experiments with temperature profiling were carried out. The ethanol production of thermotolerant yeast (K. marxianus) and ordinary baker s yeast was also investigated on different cellulosic waste materials, i.e. old corrugated cardboard and paper sludge, produced in high amount in Hungary. Solka Floc, a purified spruce cellulose powder, was used in control experiments.

4 106 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) Materials and methods 2.1. Microorganisms K. marxianus Y01070 was obtained from the National Collection of Agricultural and Industrial Microorganisms, Szent István University (Budapest, Hungary) and maintained on agar slants containing 5 g/l glucose, 1 g/l peptone, 20 g/l malt extract and 20 g/l agar. After 3 days incubation at 30 C the agar slants were stored at 4 C. For each experiments, fresh commercial compressed baker s yeast (Budafok Yeast and Spirit Factory Ltd., Budapest, Hungary) was used Inoculum preparation For the inoculum preparation of K. marxianus Y01070 a loopful of cells was added to each 750 ml E-flask containing 150 ml of sterile culture medium in which the concentration of nutrients in g/l were 50 glucose, 2.5 yeast extract, 5 peptone, 1 KH 2 PO 4, 0.3 MgSO 4 and2nh 4 Cl. The E-flasks were incubated in a rotary shaker at 30 C and 300 rpm for 24 h Enzymes Commercially available enzyme solutions, Celluclast 1.5 l and Novozym 188, were kind gifts from Novo Industri A/S (Bagsvaerd, Denmark). Iogen Cellulase was obtained from Iogen Corporation (Ottawa, Canada). Celluclast 1.5 l and Iogen Cellulase were analyzed for cellulase and -glucosidase activity, whereas only -glucosidase activity was determined in Novozym 188. Celluclast 1.5 l had a cellulase activity (in terms of filter-paper units (FPU) per milliliter of enzyme solution) of 75.8 FPU/ml and a -glucosidase activity (in international unit of 1 ml enzyme) of 38.5 IU/ml. The cellulase and -glucosidase activities in Iogen Cellulase were measured to be 99.8 FPU/ml and IU/ml, respectively. The Novozym 188 had a -glucosidase activity of IU/ml Substrates Two industrial wastes, old corrugated cardboard (OCC) and paper sludge, obtained from Dunapack Paper and Packaging Ltd. (Budapest, Hungary), were used in this study for the production of ethanol in SSF experiments. Solka Floc 200 pure cellulose powder (FS&D, Urbana, IL, USA) was used in reference fermentation tests. All three substrates were analyzed for cellulose content using the Hägglund s (1951) method with the modification that the acid hydrolysate obtained during the assay was analyzed for the concentration of glucose using HPLC, which was then used to calculate the cellulose content. The estimated cellulose contents of raw materials for OCC, paper sludge and Solka Floc 200 were 75, 45, and 95 wt.%. 3. Simultaneous saccharification and fermentation and non-isothermal SSF The SSF and NSSF experiments were performed in 750 ml E-flasks. Each flask contained 500 ml of culture medium in which the concentrations of nutrients were the same as used in the inoculum preparation except for the carbon source. Instead of glucose, 6 wt.% DM Solka Floc 200, OCC or paper sludge were used. In all cases, the culture medium was supplemented with 15 FPU of cellulase enzymes (Celluclast 1.5 or Iogen Cellulase) and 15 IU of Novozym 188 per g dry substrate. The fermentation was started with addition of yeast. The living cell content in the medium was cells/ml after inoculation, according to a relationship between optical density of inoculum and the living cell content (not shown here). The flasks were incubated in a rotary shaker at 40 C for 96 h. Samples were withdrawn regularly, centrifuged in a laboratory desktop centrifuge at 1400 g, and the supernatants were analyzed for glucose and ethanol concentrations. The ph of the fermentation broth was measured at each sampling and if necessary adjusted between 4.4 and 5.3 by addition of either 10 wt.% NaOH or H 2 SO 4. In case of NSSF experiments, the same procedure was followed except a 24 h prehydrolysis was performed at 50 C, with the same enzyme loading written above, prior to inoculation with yeast cell. After the prehydrolysis each flask was inoculated with yeast cells and incubated at 30 C Analytical methods Samples for analysis of glucose and ethanol contents were first filtered through a ME m membrane filters (Schleicher & Schuell, Dassel,

5 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) Germany) and then analyzed on an HPLC unit. Glucose and ethanol were separated on an Aminex HPX-87H (Bio-Rad, Hercules, CA, USA) at 65 C using 5 mm H 2 SO 4 solution as the mobile phase at a flow rate of 0.5 ml/min, and then detected with a refractive index detector. For analysis Gilson UniPoint software was used. The enzyme activity of industrial enzymes was determined as filter paper activity (FPA) using Mandels et al. (1976) procedure and -glucosidase activity using Berghem and Petterson s (1974) method Calculations Data obtained from the HPLC analysis was used to calculate ethanol yields, initial ethanol production rates, ethanol productivities, and cellulose conversions. For the calculation of ethanol yield (Y EtOH ), ethanol concentration in g/l was taken after 72 h of fermentation and then divided with initial cellulose concentration in g/l. It has to be noted that caution must be taken when these ethanol yields are compared with the theoretical 0.51 g ethanol/g glucose yield, since the residual cellulose content was not determined in this case, thus the amount of cellulose consumed could not be calculated. Conversion of cellulose was calculated from the ethanol concentration measured after 72 h of fermentation. Initial ethanol production rates and ethanol productivities (r 5,r1 72 ) were calculated from ethanol concentrations measured after 5 and 72 h fermentation. In case of NSSF an additional productivity (r2 72 ) was calculated after 72 h of residence time in which the prehydrolysis was taken into account, i.e. the ethanol concentration was taken after 48 h of fermentation. 4. Results and discussions In the present study the ethanol production of K. marxianus, a thermotolerant yeast strain and commercially available baker s yeast, Saccharomyces cerevisiae, was investigated in SSF experiments using OCC waste and paper sludge. Solka Floc 200, a purified cellulose powder, was used in control fermentation tests. As an alternative of SSF, NSSF with temperature program was also studied in order to increase ethanol yields. During the SSF experiment with both K. marxianus and S. cerevisiae, the rate of hydrolysis was higher than the rate of glucose consumption by the yeast cell, which resulted in glucose accumulation in the fermentation broth. Furthermore, there was no ethanol production observed until the 5th hour of fermentation. Although, it was expected that after an initial adaptation phase, the glucose concentration would be reduced to zero, it stayed at around 3 5 g/l, which is considered to be rather high (Fig. 1). It could Concentration [g/l] Time [hours] Fig. 1. SSF of Solka Floc 200 cellulose powder using S. cerevisiae ( ) and K. marxianus ( ). Continuous lines: glucose concentration in g/l. Dotted lines: ethanol concentration in g/l.

6 108 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) Table 1 SSF and NSSF of various substrates using K. marxianus and S. cerevisiae K. marxianus S. cerevisiae c EtOH Y EtOH Conversion c EtOH Y EtOH Conversion Solka Floc SSF NSSF OCC SSF NSSF Paper sludge SSF NSSF Ethanol concentrations (c EtOH ) in g/l, ethanol yields (Y EtOH ) in g EtOH/g cellulose, and cellulose conversions in percent. Data are taken after 72 h of fermentation. The experiments were carried out in duplicate, in the table means are shown. be that the cells suffered from this temperature. It was especially unexpected with the thermotolerant K. marxianus, which was thought to be performing much better at 40 C than S. cerevisiae. It can be also seen (Fig. 1) that using Solka Floc 200 as a substrate, similar ethanol concentrations, 17 g/l, were obtained with both strains. In Table 1 the ethanol concentrations, ethanol yields, and cellulose conversions obtained after 72 h of fermentation are summarized for the different substrates using both microorganisms. When the performance of the two yeast strains was compared on the same substrate, there was no significant difference observed and similar ethanol yields were obtained (Table 1). It can be stated that the generally used S. cerevisiae for the ethanol fermentation can be applied at 40 C in the SSF process. When Solka Floc substrate in the SSF fermentation was substituted with OCC or paper sludge the ethanol concentration obtained after 72 h of fermentation significantly decreased from about g/l and 9 g/l, because of the substantially lower cellulose concentration in OCC and paper sludge. However, the cellulose conversions rates calculated from the ethanol produced were in the same range, 55 60%, for both yeast strains applied (Table 1), which indicates that both OCC and paper sludge were suitable for substrates for ethanol production. The cellulose conversions and ethanol yields, g ethanol/g cellulose were considered rather low, since about 40% of the cellulose was unutilized, even on Solka Floc substrate. It is assumed that the high initial dry matter content, 6 wt.% substrate, which could cause mass transfer problems, might be responsible for the low ethanol yields obtained. The initial ethanol production rates (r 5 ) varied over a wide range, g/(l h), in the SSF experiments. When Solka Floc 200 supplemented medium was used for ethanol production the values of r 5 was 44% higher with compressed baker s yeast than with K. marxianus (Table 2). The same value for OCC medium was 148%. However, in the initial phase of fermentation the production rate of ethanol on paper sludge was 30% higher when K. marxianus was used instead of S. cerevisiae. Comparison of the ethanol productivities calculated after 72 h fermentation time did not show large variation and approximately same values were obtained with both strains on the same substrate as it is shown in Table 2. However, when the pure cellulosic substrate, Solka Floc, was replaced with paper sludge the ethanol productivity decreased to the half of obtained on Solka Floc, which is consequence of the lower cellulose content of the raw material used. In contrary to the results of Huang and Chen (1988), in the NSSF experiments lower ethanol yields, and therefore cellulose conversions were reached when compared to the data obtained with SSF using the same substrate. In the case of S. cerevisiae, 11% higher cellulose conversion was obtained with SSF than with NSSF Solka Floc 200 as the substrate. Considerably higher difference was measured on paper

7 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) Table 2 Initial ethanol production rates (r 5 ) [g EtOH/(l h)] and productivities (r1 72,r2 72 ) [g EtOH/(l h)] of SSF and NSSF experiments on various substrates K. marxianus S. cerevisiae r 5 r1 72 r2 72 r 5 r1 72 r2 72 Solka Floc SSF NSSF OCC SSF NSSF Paper sludge SSF NSSF Concentration [g/l] Time [hours] Fig. 2. Comparison of SSF ( ) and NSSF ( ) using S. cerevisiae on Solka Floc 200 cellulose powder. Continuous lines: glucose concentration in g/l. Dotted lines: ethanol concentration in g/l. sludge. With SSF operation, the cellulose conversion was 40% higher than applying the NSSF technique. Similarly to the results obtained with the S. cerevisiae on all substrates, the SSF resulted in higher ethanol yields. As it can be seen on Fig. 2, NSSF operation did not increase the ethanol yield at all. Furthermore, when the productivities calculated after 72 h of residence time (r2 72 ) are compared to the productivities calculated for SSF after 72 h of fermentation (r1 72 ), it can be seen that considerably lower values were obtained for NSSF operation (Table 2), which indicates that from an industrial point of view there is no advantage to apply the NSSF operation mode. 5. Conclusions The main objectives of the present study were to compare the performance of a non-thermotolerant yeast, S. cerevisiae, to a thermotolerant K. marxianus yeast strain, to evaluate two different potential substrates in SSF ethanol production, and to investigate the possibility of NSSF in order to increase ethanol yield. The results showed that S. cerevisiae was as good as K. marxianus in simultaneous saccharification and fermentation at 40 C using both industrial wastes, i.e. OCC and paper sludge. The results showed that both OCC and paper sludge could be used for

8 110 Zs. Kádár et al. / Industrial Crops and Products 20 (2004) ethanol production in SSF. The cellulose conversions were in the range of 55 60% in the SSF experiments for all substrates. Although, the ethanol yields were considered rather low, g ethanol/g cellulose added, they were comparable with data obtained in the literature varied between 0.11 and 0.4 g/g. The NSSF operation mode did not increase the ethanol yield at all, and slightly lower values were obtained, which is in contrary to the results published by Huang and Chen (1988). Acknowledgements The authors would like to gratefully acknowledge the National Research Fund of Hungary (OTKA, Hungary, T ), the Research and Development Division of the Ministry of Education (OM, Hungary, NKFP-OM-00231/2001), and the Research Found of the Ministry of Education (OM, Hungary, FKFP ) for their financial support. We also acknowledge the contribution by Á. Sárdi, M. Csizmadia and N. Soós. References Ballesteros, I., Ballesteros, M., Cabaòas, A., Carrasco, J., Martín, C., Negro, M.J., Saez, F., Saez, R., Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol. Appl. Biochem. Biotech. 28/29, Barron, N., Marchant, R., McHale, L., McHale, A.P., Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose. Appl. Microbiol. Biotechnol. 43, Berghem, L.E.R., Petterson, L.G., The mechanism of enzymatic cellulose degradation. Eur. J. Biochem. 46, Bollók, M., Réczey, K., Screening of different Kluyveromyces strains for simultaneous saccharification and fermentation. Acta Aliment. Hung. 29, Claassen, P.A.M., van Lier, J.B., Lopez Contreras, A.M., van Niel, E.W.J., Sijtsma, L., Stams, A.J.M., de Vries, S.S., Weusthuis, R.A., Utilisation of biomass for supply of energy carriers. Appl. Microbiol. Biotechnol. 52, Huang, S.Y., Chen, J.C.J., Ethanol production in simultaneous saccharification and fermentation of cellulose with temperature profiling. Ferment. Technol. 66, Hägglund, E., Chemistry of Wood. Academic Press, New York. Lynd, L.R., Lyford, K., South, C.R., Levenson, K., Evaluation of paper sludge for amenability to enzymatic hydrolysis and conversion to ethanol. TAPPI J. 84, 50. Mandels, M., Andreotti, R., Roche, C., Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp. 6, Nilsson, U., Barron, N., McHale, L., McHale, A.P., The effects of phosphoric acid pretreatment on conversion of cellulose to ethanol at 45 C using the thermotolerant yeast Kluyveromyces marxianus IMB3. Biotech. Lett. 17, Szczodrak, J., Targoňski, Z., Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose. Biotechnol. Bioeng. 31,