49â51. © 1997 Chapman & Hall ... Biotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine,. Co.
Biotechnology Letters, Vol 19, No 1, January 1997, pp. 49–51
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Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3
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M Boyle, N Barron and AP McHale* Biotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine, Co. Derry, BT52 1SA, Northern Ireland
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The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.
Introduction Agriculture residues including grain crop straw represent a significant source of fermentation feed-stock for the production of fuel ethanol (Bridgewater and Double, 1994). The main polysaccharides in straws derived from a variety of sources are cellulose (37–40%), hemicellulosic fractions (30–35%) and small amounts of water soluble, pectic, 80% ethanol soluble, and sodium chlorite soluble materials (Lawther et al., 1995; Moniruzzaman, 1995; Mamma et al., 1995; Stewart et al., 1995). In most biological processes concerned with the conversion of the cellulosic reserves in straw to ethanol, systems require either pretreatment of the straw with enzymes and subsequent fermentation to ethanol or simultaneous saccharification and fermentation of those materials to ethanol in a single stage process. The latter system has obvious advantages in that it would improve both the economics and kinetics of substrate conversion to ethanol (Barron et al., 1995). In order to render the cellulose in lignocellulosic materials accessible to degradative enzymatic attack it is usually necessary to pretreat the substrate and methods such as treatment of phosphoric acid, sodium hydroxide and hypochlorites have been employed (Nilsson et al., 1995, Wyman et al., 1993). Since the optimal operating temperature of most cellulolytic systems ranges between 40 and 60ºC it would be advantageous to choose a fermenting microorganism capable of growth and ethanol production within that range. Recently we have described the isolation of a © 1997 Chapman & Hall
thermotolerant yeast strain which is capable of growth at 45–52ºC and ethanol production at 45–50ºC (Banat et al., 1992). The yeast strain also produced ethanol from cellulosic substrates in a simultaneous saccharification and fermentation configuration (Barron et al., 1995; Barron et al., 1996). In the study presented here we describe ethanol production by Kluyveromyes marxianus IMB3 in a simultaneous saccharification system designed to convert straw to ethanol at 45ºC. Materials and methods Microorganism and growth conditions Kluyveromyces marxianus IMB3 was grown on malt extract agar plates at 45ºC. In the simultaneous saccharification and fermentation system the organism was grown in shake flasks containing 100ml of growth medium (Barron et al., 1995) supplemented with straw at the indicated concentrations. Media were also supplemented with a commercial cellulase preparation (2% [v/v]) derived from Trichoderma reesei (Clampzyme from Finn Feeds International, UK). In our laboratory a 3% (v/v) solution of the enzyme liberated 0.8mmoles of glucose reducing equivalents per min. from 4% (w/v) milled filter paper at 50ºC (within the first 5 min. of the reaction). Reducing sugar concentration was determined using the dinitrosalicylate method (Miller, 1959). Straw pre-treatment Barley straw was obtained locally. The straw was pulverized using a bench-top mill with a 1mm screen. For each series of fermentations 2, 4 and 6g of untreated Biotechnology Letters · Vol 19 · No 1 · 1997
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straw were placed in 200ml conical flasks and incubated with 5M NaOH (final concentration in each flask was 10% [w/v]) for 3 hours at room temperature. The solid pretreated material was recovered by filtration and rinsed to neutrality with distilled water. The rinsed residual solid material was placed in 200ml shake flasks together with 100ml aliquots of medium supplemented with 2% (w/v) cellulase.
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Determination of ethanol concentration Samples harvested from fermentations were clarified by centrifugation at 5,000 3g for 10 min. Ethanol concentrations were determined using a gas chromatograph (GLC 8410, Perkin Elmer) as described previously (Banat et al. 1992).
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Results and discussion Recently we described the use of the thermotolerant, ethanol-producing yeast strain, K. marxianus IMB3 in converting cellulose to ethanol using an SSF system (Nilsson et al., 1995). In those studies it was found that the untreated substrate (milled filter paper) was converted to ethanol and the concentrations were in the region of 25% of the maximum theoretical yield. It was subsequently felt however that in practical terms, substrates other than the more defined celluloses used in those previous studies would need to be accommodated by the SSF system. To this end barley straw was pulverized, incorporated into the SSF system at 45ºC and ethanol production was monitored over a 70h period. The straw was added to the system at concen-
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Figure 1 Ethanol production by Kluyveromyces marxianus IMB3 during growth on 2 (j), 4 (m) and 6 (d) % (w/v) straw at 45ºC in media supplemented with 2% (v/v) cellulase activity. Samples were harvested at the indicated times and the ethanol concentration was determined as described.
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Figure 2 Ethanol production by Kluyveromyces marxianus IMB3 during growth at 45°C on media containing 2 (j), 4 (m) and 6 (d) % (w/v) straw pretreated with NaOH (based on original untreated straw content) and supplemented with 2% (v/v) cellulase. Samples were harvested at the indicated times and the ethanol concentration was determined as described.
trations of 2, 4 and 6% (w/v) and it was found concentrations of ethanol produced were quite low even at the highest concentration of substrate (Fig. 1). The maximum ethanol concentrations represented 48, 36 and 12.3% of the maximum theoretical yield, respectively. In comparison with previously obtained yields using this microorganism and more purified forms of cellulose (Barron et al., 1995) these yields, particularly at the higher substrate concentrations were low. In addition, the overall concentrations of ethanol were also quite low. It should be mentioned however that in systems containing the 6% (w/v) straw, adequate mixing of cultures represented a significant problem. Pretreatment of lignocellulosic materials with agents such as phosphoric acid and sodium hydroxide increases enzymatic degradation of cellulose fractions (Nilsson et al., 1995; Wyman et al., 1993). Since yields of ethanol were relatively low in the above described experiments it was decided to study the effects of NaOH pretreatment on ethanol production by the SSF system. In these experiments the pretreated straw content is based on the original untreated material added to each series of fermentations. Maximum ethanol concentrations obtained from fermentations containing 2, 4 and 6% (w/v) pretreated straw were 3.9, 8 and 12g/L, respectively (Fig. 2). Since the original straw added to each system was assumed to containing 40% cellulose at most and since the straw content of each system was based on the original quantity of untreated straw added to that system, these ethanol concentrations represented
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95, 98 and 98% of the maximum theoretical yield, respectively. On the basis of previous studies with this organism in SSF systems concerned with cellulose conversion to ethanol, these yields are extremely high (Nilsson et al., 1995; Barron et al., 1995; Barron et al., 1996). It should however be noted that although the organism is not capable of converting xylose (unpublished preliminary results) to ethanol, it would be capable of converting C6 sugars (galactose and mannose) derived from hemicellulose fractions to ethanol. This may account for the apparently high yields in our studies. In a recent study Zayed and Myer (1996) obtained 17g of ethanol from 100g of wheat straw in a single-batch process involving the use of cellulase from Trichoderma viride. Our results, reporting yields of 20g ethanol/100g straw compare very favourably with that study and confirm our earlier suggestion that this SSF system may play an important role in commercial processes concerned with conversion of lignocellulosic materials to ethanol.
References Banat, I.M., Nigam, P. & Marchant, R. (1992) World J. Microbiol. Biotechnol. 8, 259–263. Barron, N., Marchant, R., McHale, L. & McHale, A.P. (1995) Appl. Microbiol. Biotechnol. 43, 518–520. Barron, N., Marchant, R., McHale, L. & McHale, A.P. (1996) World J. Microbiol. Biotechnol. 12, 103–104. Bridgewater, A.V. & Double, J.M. (1994) Intl. J. Energy Res. 18, 79–95. Lawther, J.M., Sun, R.C. & Banks, W.B. (1995) J. Ag. Food Chem. 43, 667–675. Mamma, D., Christakopoulos, P., Koullas, D., Kekos, D., Macris, B. & Koukios, E. (1995) Biomass Bioeng. 8, 99–103. Miller, G.L. (1959) Anal. Chem. 31, 426–428. Moniruzzaman, M. (1995) World J. Microbiol. Biotechnol. 11, 646–648. Nilsson, U., Barron, N., McHale, L. & McHale, A.P. (1995) Biotechnol. Letts. 17, 985–988. Stewart, D., Wilson, H.M., Hendra, P.J. & Morrison, I.M. (1995) J. Ag. Food Chem. 43, 2219–2225. Wyman, M., Chen, S. & Doan, K. (1993) Trans. IChemE. 71, 141–143. Zayed, G. & Myer, O. (1996) Appl. Microbiol. Biotechnol. 45, 551–555.
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