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ARTICLE Simultaneous Saccharification and Fermentation of Kanlow Switchgrass Pretreated by Hydrothermolysis Using Kluyveromyces marxianus IMB4 Lilis Suryawati,1 Mark R. Wilkins,1 Danielle D. Bellmer,1 Raymond L. Huhnke,1 Niels O. Maness,2 Ibrahim M. Banat3 1

Department of Biosystems and Agricultural Engineering, Oklahoma State University, 111 Agricultural Hall, Stillwater, Oklahoma 74078; telephone: 405-744-8416; fax: 405-744-6059; e-mail: [email protected] 2 Department of Horticulture and Landscape Architecture, Oklahoma State University, Stillwater, Oklahoma 3 School of Biomedical Sciences, University of Ulster, Coleraine, United Kingdom Received 8 January 2008; revision received 22 April 2008; accepted 28 April 2008 Published online 6 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21965

Introduction ABSTRACT: A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 2008C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 458C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D5A at 378C and pH 4.8. Fermentation by IMB4 at 45 and 418C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 378C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 458C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 458C, pH 5.5 was greater than the control after 48, 72, and 96 h ( P < 0.05). Biotechnol. Bioeng. 2008;101: 894–902. ß 2008 Wiley Periodicals, Inc. KEYWORDS: thermotolerant; ethanol; biofuels; renewable energy

Correspondence to: M.R. Wilkins

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Biotechnology and Bioengineering, Vol. 101, No. 5, December 1, 2008

Industrial ethanol production in the US has grown vastly over the past decade. The Renewable Fuels Association (Anonymous, 2007) reported US ethanol production increased from 660 million to over 18 billion liters from 1980 to 2006. Cellulosic biomass, represents a potential alternative feedstock to meet increasing demand for fuel ethanol. One unit operation in biological conversion of cellulosic biomass that has been intensely researched is simultaneous saccharification and fermentation (SSF). It has been documented that SSF reduces contamination risk due to the presence of ethanol and eliminates the need for separate reactors, thus reducing capital costs (Nigam and Singh, 1995; Philippidis et al., 1993). Additionally, SSF increases the hydrolysis rate by reducing product inhibition as the sugars are rapidly consumed by yeast during SSF (Takagi et al., 1977). SSF is constrained by the different optimum temperatures for saccharification and fermentation (Chung et al., 2005; Grohmann, 1993; Krishna et al., 1999). Saccharification by cellulase is optimum at temperatures between 40 and 508C; whereas, fermentation temperature using the most commonly used ethanolgenic yeast S. cerevisiae cannot exceed 388C (Bollok et al., 2000). Above 408C, the viability of yeast culture diminishes, which consequently affects ethanol yield. The use of lower temperature reduces enzyme activity and increases SSF time and/or enzyme usage. One approach to improve SSF is by using thermotolerant, ethanol producing microorganisms to carry out SSF at higher temperature. SSF at temperatures within the ideal range for cellulase activity may enhance hydrolysis rate and ß 2008 Wiley Periodicals, Inc.

reduce SSF time and/or enzyme usage. Thermotolerant species of Saccharomyces, Kluyveromyces, and Fabospora genera have been observed growing at temperatures above 408C and fermenting six carbon sugars at 40, 43, and 468C, respectively (Szczodrak and Targonski, 1988). Several researchers have experimented with thermotolerant strains of S. cerevisiae, K. marxianus, and K. fragilis for SSF of cellulosic substrates (Ballesteros et al., 1991, 2004; Barron et al., 1997; Bollok et al., 2000; Boyle et al., 1997; Lark et al., 1997). Among these, thermotolerant K. marxianus IMB strains isolated by Banat et al. (1992) from a distillery in India are of particular interest. IMB yeasts were reported capable of growing and producing ethanol at temperatures of up to 508C, which are more thermotolerant than most other yeasts discussed in published reports. Moreover, in their investigation the authors indicated that the ethanol tolerance of several strains of IMB can reach 9.5% (w/v). Two previous studies used the K. marxianus IMB3 strain to convert barley straw to ethanol in an SSF process at 458C, which is a temperature within the ideal range for cellulose hydrolysis by commercially available cellulases (Barron et al., 1997; Boyle et al., 1997). The present study used switchgrass as a feedstock for ethanol production. Switchgrass, a warm-season perennial herbaceous crop, was chosen because it is native to North America, thus non-invasive (McLaughlin and Walsh, 1998), and was selected as a model energy crop by the US Department of Energy (McLaughlin and Kszos, 2005). Switchgrass can potentially be used in water limited regions and under poor and low nutrient soil conditions that are not suitable for food crops (McLaughlin, 1993). The last criterion is particularly important to the Midwestern and South Central regions of the US where water resources are limited. The present study investigated the effect of fermentation temperature on SSF of hydrothermolysis-pretreated switchgrass using IMB4 yeast and compared the results with SSF of the same substrate using the ethanolgenic yeast S. cerevisiae D5A. Ethanol yield from SSF using IMB4 at temperature 37, 41, and 458C were investigated. The objectives of this study were to determine (1) the effect of temperature on ethanol yield by IMB4 and (2) whether IMB4 has any advantages over S. cerevisiae in terms of ethanol yield and productivity.

Materials and Methods

2 mm particle size using a Thomas-Wiley mill (Model 4, Arthur H. Thomas Co., Philadelphia, PA). Compositional analysis of switchgrass before pretreatment and fermentation was performed using the National Renewable Energy Laboratory (NREL) procedures LAP-001, -002, and -005 (Sluiter et al., 2004a,b,c). Absorbance reading of acid soluble lignin (ASL) was taken at 205 nm using a UV–Visible spectrophotometer (Cary 50 Bio, Varian, Inc., Palo Alto, CA) with high purity quartz cuvettes of pathlength 1 cm (Hellma Cells, Inc., Plainview, NY). The suggested 205 nm wavelength (l) and absorptivity (e) of switchgrass (110 L/g cm) were chosen based on previous work by Thammasouk et al. (1997). A mixture of 60 g dry switchgrass and 540 g water was placed in a 1-L bench top stirred reactor and pressure vessel (Parr Series 4520, Parr Instrument Company, Moline, IL), mixed at 150 rpm and heated to 2008C. It took between 35 and 40 min to heat the mixture to 2008C. The sample was then held at 2008C for 10 min. Pretreatment conditions were selected in an earlier study (Suryawati et al., 2007). After heating, the reactor was placed in an ice bath to reduce the temperature to less than 1008C within 5 min. Five batch pretreatments were conducted to prepare sufficient substrate for SSF experiments, and then the material was combined. Determination and quantification of structural carbohydrates, sugars, byproducts, and degradation products in insoluble solids and liquid prehydrolyzate from pretreatment were conducted according to NREL procedures using HPLC with an Aminex HPX-87H (organic acids, furfurals, and sugars) column (Bio-Rad, Hercules, CA) with refractive index detection (1100 Series, Agilent, Santa Clara, CA) (Sluiter et al., 2004b,c). Microorganism and Inoculum Preparation K. marxianus IMB4 was isolated from samples in an Indian distillery (Banat et al., 1992) and S. cerevisiae D5A was obtained from the American Type Culture Collection (ATCC 200062, Manassas, VA). Both yeasts were grown on liquid yeast peptone dextrose (YPD) medium containing (g/L): yeast extract 5.0, peptone 10.0, and glucose (dextrose) 50.0. All nutrients were obtained from Fisher Scientific (Pittsburgh, PA). One loopful of IMB4 or D5A cells grown on a YPD agar slant was added to 250 mL baffled culture flasks containing 100 mL of YPD medium covered with an aerobic stopper. IMB4 was incubated at 458C and D5A was incubated at 378C for 18 h while being rotated 130 rpm on a shaker (Banat et al., 1992; Dowe and McMillan, 2001).

Substrate Preparation and Pretreatment Kanlow switchgrass (P. virgatum var. Kanlow) was harvested at the Oklahoma State University Plant Sciences Research Farm and ground in a hammer mill (Model E9506, Bliss Industries, Ponca City, OK) through a 13 mm screen. The biomass was stored in a sealed plastic bag and kept refrigerated (48C) for use in all experimentation. Prior to compositional analysis, switchgrass was ground through a

SSF and Analyses Yeast fermentation medium (YFM) was prepared using deionized water consisting of (g/L): yeast extract, 5.0; KH2PO4, 20.0, (NH4)2SO4, 20.0; MgSO4 7H2O, 10.0; and MnSO4 H2O, 1.0 (Banat et al., 1992). Commercial cellulase (Fibrilase, Iogen, Ottawa, Canada) with activity of 62 FPU/mL, as determined by the procedure of Ghose

Suryawati et al.: SSF of Switchgrass Using K. marxianus IMB4 Biotechnology and Bioengineering

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(1987), was used for hydrolysis of solid substrate. SSF was conducted in 250 mL baffled flasks sealed with a rubber stopper fitted with a 1-way air valve to maintain an anaerobic environment. SSF was conducted according to NREL procedure LAP-008 (Dowe and McMillan, 2001), which was modified as described here. Each fermentation flask contained 10 mL YFM, 5 mL 1 M sodium citrate buffer at pH 4.8, washed, pretreated switchgrass to provide 41 g/L glucan, 15 FPU/g glucan of cellulase, and 10 mL yeast inoculum with optical density of 5.0. Deionized water was added to bring the total volume to 100 mL. Initial cell density was 0.14 and 0.2 g/L for IMB4 and D5A, respectively. All flasks were incubated at the specified temperature while being rotated 130 rpm on a shaker. Additional SSFs at 458C using IMB4 were performed to investigate the effect of fermentation nutrients and pH on ethanol yield. In the nutrient experiment, SSF was conducted as before except the nutrient concentration was tripled. In the pH experiment, SSF was also performed as before except 50 mM sodium citrate buffer at pH 5.5 was used. Aliquots of 4.0 mL were

% sugars degraded ¼ 100

concentration at the beginning of fermentation (g/L), 0.511 conversion factor for glucose to ethanol and 1.11 is the conversion factor for glucan to glucose. Conversion of monomer sugars, 5-hydroxymethyl furfural (HMF), and furfural to polymer sugars (e.g., glucose to glucan) was calculated by: ð% Monomer þ % DimerÞ X where X is the conversion factor for converting monomers and dimers to anhydromonomers. X ¼ 1.11 for 6 carbon monomers, 1.14 for 5 carbon monomers, and 1.05 for cellobiose (a dimer of beta-glucose), 0.78 for HMF, and 0.73 for furfural. The preservation of sugars as oligomers in prehydrolyzate was calculated by subtracting the concentration of sugars in untreated prehydrolyzate from the concentration of sugars in acid-hydrolyzed prehydrolyzate (Sluiter et al., 2004c). The % of sugars degraded during hydrothermolysis was calculated as follows: % polymer ¼

½ð% db sugar in PS ðIS=100Þ ðHS=100Þ þ g=L sugar in PH % sugar in NS

taken at 0, 6, 12, 24, 48, 72, 96, 120, 144, and 168 h and frozen immediately. At the end of fermentation, the pH values of all fermentation slurries were recorded. To account for ethanol produced from sugars present in the commercial cellulase mixture, duplicate fermentations using IMB4 and D5A were done without switchgrass, thus only the enzyme and nutrients provided substrate for the yeast. The ethanol concentration produced in the enzyme control was subtracted from the final ethanol concentration. For analyses, samples were thawed and centrifuged at 13,000g for 12 min twice. Supernatant was collected, filtered through 0.2 mm 13 mm syringe filter from Fisher Scientific, and analyzed for ethanol, organic acids, and sugar residues (cellobiose, glucose, and xylose) by HPLC on an HPX-87H column (Bio-Rad, Sunnyvale, CA) with 0.01 N H2SO4 as solvent, 0.6 mL/min flow rate at 608C using refractive index detection (1100 Series, Agilent).

where PS ¼ pretreated solids, IS ¼ % insoluble solids after hydrothermolysis, HS ¼ concentration of solids in hydrothermolysis (g/L), PH ¼ prehydrolyzate, and NS ¼ native switchgrass. IS ¼ 56.1 and HS ¼ 100 in this study.

Experimental Design and Statistical Analysis All treatment conditions were repeated in triplicate, except the IMB4 pH 5.5 experiment, which was done in duplicate. Comparisons of mean ethanol yields at 24, 48, 72, 96 and 120 h and 72 h specific ethanol productivities from IMB4 to the control (S. cerevisiae D5A at 378C and pH 4.8 buffer) were made using Dunnett’s test at a 95% confidence level ( P < 0.05; Dunnett, 1955). Analyses were performed using SAS Release 9.1 (SAS, Cary, NC).

Calculations

Results and Discussion

The percent cellulose conversion or theoretical yield of ethanol production was calculated as follows (Dowe and McMillan, 2001):

Hydrothermolysis

% Theoretical Yield ¼

½EtOHt ½EtOH0 100% 0:511 ðf ½Biomass 1:11Þ

where [EtOHt] is the concentration of ethanol at time t, [EtOH0] is the initial ethanol concentration, f is glucan fraction of dry biomass (g/g), [Biomass] is dry biomass

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The moisture composition of native switchgrass was 8.5% (wet basis). The dry matter composition of the native switchgrass is presented in Table I. Approximately 43.9% of the dry solids in native switchgrass were solubilized during hydrothermolysis. The average glucan and xylan content of washed insoluble solids after pretreatment was 56.6% and 2.4%, respectively (Table I). Hydrothermolysis resulted in greater solubilization of hemicellulose than cellulose, with 13.2% of glucan and 91.4% of xylan in the native switchgrass

Table I.

Composition of native and hydrothermolyzed Kanlow switchgrassa.

Component

Native switchgrass (% dry solids)a

Hydrothermolyzed switchgrass (% dry solids)b

36.6 0.2 21.0 0.3 1.0 0.0 2.8 0.1 0.8 0.1 16.3 0.1 2.2 0.0 5.0 0.2 12.5 2.1

56.6 1.1 2.4 0.2 n/d n/d n/d n/m n/m n/m n/m

Glucan Xylan Galactan Arabinan Mannan Klason lignin Acid soluble lignin Ash Extractives

n/d, not detected; n/m, not measured. a All values are means of four replicates one standard deviation except ash and extractives, which are means of two replicates one standard deviation. b Values are means of two replicates one standard deviation.

being solubilized during pretreatment. During pretreatment 8.6% of glucan and 65.5% of xylan was degraded. Only 4.6% of glucan and 28.0% of xylan in the native switchgrass were recovered in the prehydrolyzate, and 79.6% of glucan and 35.1% of xylan in the prehydrolyzate were preserved as oligomers. Previous studies have shown that preservation of carbohydrates as oligomers in the prehydrolyzate reduces formation of HMF and furfural during pretreatment (Weil et al., 1998). High temperature pretreatment conditions can degrade xylose and glucose causing formation of furfural and 5-hydroxymethyl furfural (HMF), respectively (Palmqvist and Hahn-Hagerdal, 2000). Prehydrolyzate composition is presented in Table II. HMF, furfural, acetic acid, and glycerol were the sugar degradation compounds and byproducts detected and measured in the prehydrolyzate. HMF and furfural are sugar degradation products, as previously described, and have been shown to inhibit growth and ethanol production by K. marxianus CECT 10875; however, inhibition of ethanol production occurred at levels greater than those observed in the prehydrolyzate from this study (Oliva et al., 2003). Only 1.2 g/L of xylan and 0.3 g/L of glucan degraded during hydrothermolysis can be accounted for by HMF and furfural present in the prehydrolyzate (Palmqvist and Hahn-Hagerdal, 2000). The rest of the degraded xylan and glucan was present as formic acid and levulinic acid, which

Table II.

Composition of prehydrolyzatea.

Component Total glucose Glucose monomers Total xylose Xylose oligomers HMF Furfural Acetic acid Glycerol a

Mean of five replicates one standard deviation.

g/L 1.9 0.1 0.4 0.0 6.7 3.3 4.4 1.1 0.2 0.1 0.9 0.1 3.7 0.2 0.6 0.0

are formed by degradation of HMF and furfural and were not measured in this study, lost as volatilized HMF and furfural, or were retained in the insoluble solids in a form that could not be hydrolyzed during the acid hydrolysis procedure used for compositional analysis (Allen et al., 2001; Laser et al., 2002; Palmqvist and Hahn-Hagerdal, 2000). Allen et al. (2001) found that insoluble solids obtained after hydrothermolysis contained more acid insoluble material than was present in corn fiber prior to pretreatment. They concluded that this ‘‘new’’ acid insoluble material was hemicellulose retained in a form that could not be hydrolyzed to monomer sugars during compositional analysis (Allen et al., 2001). Acetic acid was generated during hydrothermolysis as a result of cleavage of hemiacetal linkages in hemicellulose (Antal, 1996; Palmqvist and Hahn-Hagerdal, 2000). Acetic acid inhibition of ethanol production and cell growth by K. marxianus is highly dependant on fermentation pH (Oliva et al., 2003). Glycerol is a sugar alcohol that is not known to be inhibitory to ethanol production.

Simultaneous Saccharification and Fermentation Washed pretreated solids from five hydrothermolysis batches at 2008C for 10 min were combined and mixed thoroughly and used as substrate in SSF using thermotolerant K. marxianus IMB4 and S. cerevisiae D5A (control) at 15 FPU/g glucan. The maximum ethanol concentration that could be produced from 41 g/L glucan was 23.2 g/L. The maximum ethanol concentrations produced from fermentation of enzyme controls were 1.4 g/L using IMB4 and 1.5 g/L using D5A. Ethanol concentrations from enzyme controls were subtracted from SSF ethanol concentrations. From 0 to 72 h, ethanol concentrations increased continuously and residual glucose after 72 h remained below 2 g/L (Figs. 1 and 2). Over the course of fermentation, cellobiose concentrations remained relatively constant as it was hydrolyzed to glucose continuously, indicating sufficient beta-glucosidase activity in the enzyme preparation


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Figure 1.

Ethanol production during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 37, 41, and 458C and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading from hydrothermolyzed switchgrass, 15 FPU/g glucan cellulase loading and pH 4.8 citrate buffer. All values are means of three runs.

Figure 3. Mean cellobiose concentration during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 37, 41, and 458C, 458C pH 5.5 and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading hydrothermolyzed switchgrass, 15 FPU/g glucan cellulase loading and pH 4.8 citrate buffer. Value for IMB4 at 458C and pH 5.5 was mean of two runs; others are means of three runs.

(Fig. 3). Glucose concentrations increased and ethanol concentration remained constant after 72 and 96 h for IMB4, 458C and IMB4, 418C, respectively (Figs. 1 and 2). Fermentation continued until 168 h in the IMB4, 378C and control SSFs as evidenced by relatively low glucose concentrations and increases in ethanol concentration (Figs. 1 and 2). Ethanol yields from IMB4 SSFs using pH 4.8 buffer were greater than the control at 24, 48, and 72 h ( P < 0.05). At 24 h, only the 418C ethanol yield was greater than the control ( P < 0.05). At 48 h, 41 and 458C ethanol yields were greater than the control ( P < 0.05). At 72 h, only the 458C ethanol yield was greater than the control

( P < 0.05). After 96 h, IMB4 ethanol yields were not different than or less than the control ( P > 0.05). During SSF, acetic acid production was observed in all SSFs (Fig. 4). At the end of SSF, final pH of all SSFs ranged from 4.36 to 4.51. The greatest final acetic acid concentration (2.8 g/L) was observed for IMB4, 418C, followed by IMB4, 378C (1.7 g/L), IMB4, 458C (1.2 g/L), and the control (0.61 g/L). The final pH for IMB4, 418C was 4.36, followed by IMB4, 378C (4.40), the control (4.49) and IMB4, 458C (4.50). Acetic acid concentrations increased from 0 to 96 h in all SSFs. After 96 h, acetic acid concentrations decreased at 378C for both yeasts, but not at other temperatures.

Figure 2.

Figure 4. Mean acetic acid concentration during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 458C at pH 4.8, and ethanolgenic S. cerevisiae D5A, at 4.1% glucan loading from hydrothermolyzed switchgrass, 15 FPU/g glucan cellulase loading and pH 4.8 citrate buffer. Value for IMB4 at 458C and pH 5.5 was mean two runs; others are means of three runs.

Mean glucose concentration during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at pH 4.8 and at 37, 41, and 458C and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading hydrothermolyzed switchgrass, 15 FPU/g glucan cellulase loading and pH 4.8 citrate buffer. All values are means of three runs.

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Acetic acid produced by IMB4 may have caused inhibition of IMB4 fermentation and ethanol production. In all SSFs, the initial pH was 4.8, which was close to the pKa of acetic acid of pH 4.76, and the final pH was below pH 4.76, indicating much of the acetic acid in the medium was undissociated. Undissociated acetic acid has been shown to cause greater inhibition of cell growth than the dissociated form (Berg et al., 2007). The combination of greater temperature, ethanol concentration, and decreased pH may have caused the cessation of fermentation by IMB4 after 72 h at 458C and 96 h at 418C. A similar result was observed by a previous study, which utilized a thermotolerant strain called K. marxianus CECT 10875 at 428C for SSF of various cellulosic biomass sources (Ballesteros et al., 2004). They observed cessation of ethanol production between 72 and 82 h, which was attributed to metabolic stress caused by low glucose concentration and the presence of ethanol. They did not report acetic acid concentrations. Another study reported the effect of lignocellulosic degradation compounds on fermentation by K. marxianus CECT 10875 (Oliva et al., 2003). Cell growth was 40% less than the control of no acetic acid when 5 g/L acetic acid was added, and 47% less than the control when 10 g/L of acetic acid was added. However, they observed ethanol production was not affected by acetic acid concentrations up to 10 g/L. In the same study, the authors tested whether fermentation pH affected acid toxicity (Oliva et al., 2003). At pH 5.5, addition of 5 g/L acetic acid had little effect on ethanol yield; however, when pH was reduced to 4.0, the same concentration of acetic acid significantly decreased ethanol production by 80%. Maiorella et al. (1983) reported that acetic acid concentrations in the range of 0.5–9 g/L inhibited growth of S. cerevisiae. They attributed the inhibition to interference with cell maintenance function leading to membrane disruption. At 7.5 g/L acetic acid concentration, cell mass was reduced by 80%. To determine if increasing initial pH would result in increased ethanol production by K. marxianus IMB4, SSFs using IMB4, 458C and 50 mM citrate buffer at pH 5.5, instead of at pH 4.8 as suggested by the NREL protocol (Dowe and McMillan, 2001), were performed. Previous studies using IMB4 K. marxianus CECT 10875 reported adjusting initial pH to 5.5 when growing IMB4 on glucose (Ballesteros et al., 2004; Banat and Marchant, 1995). This pH may have provided better conditions for growth of IMB4 and therefore could result in improvement of ethanol yield. During SSF using IMB4 at 458C in pH 5.5 buffer, glucose concentration increased and ethanol concentration was constant after 96 h, indicating that glucose fermentation ceased after 96 h (Figs. 5 and 6). Fermentation continued for 24 more hours when pH 5.5 buffer was used than when pH 4.8 buffer was used. Ethanol yield using IMB4 at 458C and pH 5.5 was greater than the control at 48, 72, and 96 h ( P < 0.05), as opposed to IMB4 at 458C and pH 4.8, which was greater than the control only at 48 and 72 h. After 96 h, ethanol yield using IMB4 at 458C and pH 5.5 was not different than the control ( P > 0.05). The maximum acetic

Figure 5. Ethanol production during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 458C and pH 4.8 and 5.5, and tripled nutrient concentration, and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading from hydrothermolysis-pretreated switchgrass and 15 FPU/g glucan cellulase loading. Value for IMB4 at 458C pH 5.5 was mean of two runs; others are means of three runs. Values for IMB4-45-pH 4.8 and D5A are from Figure 1.

acid concentration produced during SSF at 458C and pH 5.5 was 0.71 g/L, which was 40% lower than that of SSF at 458C and pH 4.8 as shown in Figure 7. The final pH at 458C and pH 5.5 was 4.79, which was close to the pKa of acetic acid. Using pH 5.5 buffer as opposed to pH 4.8 buffer resulted in at least 24 more h of fermentation and a 96 h ethanol yield that was greater than the control. Also, it was thought that by having more nutrients available, IMB4 would continue growing and producing

Figure 6. Mean glucose concentration during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 37, 41, and 458C and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading from hydrothermolysis-pretreated switchgrass and 15 FPU/g glucan cellulase loading. Value for IMB4 at 458C and pH 5.5 was mean of two runs; others are means of three runs. Values for IMB4-45-pH 4.8 and D5A are from Figure 2.


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Figure 7.

Mean acetic acid concentration during 168 h simultaneous saccharification and fermentation using thermotolerant K. marxianus IMB4 at 458C pH 4.8 and 5.5 and ethanolgenic S. cerevisiae D5A at 4.1% glucan loading from hydrothermolysispretreated switchgrass and 15 FPU/g glucan cellulase loading. Value for IMB4 at 458C and pH 5.5 was mean of two runs; others are means of three runs. Values for IMB4-45pH 4.8 and D5A are from Figure 4.

ethanol past 72 h at 458C. Therefore, additional SSF experiments at 458C were performed to investigate the effect of nutrient concentration (mineral salts and yeast extract) on ethanol yield from IMB4 fermentations. In order to examine this, triplicate SSFs using IMB4 at 458C and pH 4.8 were performed as before, except the nutrient concentration was tripled. Ethanol yield from these SSFs was 56.9% theoretical after 72 h, which was less than the ethanol yield with the standard nutrient concentration (Fig. 5). Ethanol concentration remained constant and glucose concentration increased after 72 h, indicating cessation of fermentation (Figs. 5 and 6). Table III shows 72 h specific ethanol productivities of IMB4 and the control. The time of 72 h was chosen since this is when IMB4 at 458C, pH 4.8 ceased fermentation of glucose. The specific ethanol productivity of IMB4 at all conditions was greater than the control ( P < 0.05). It should be noted that all SSF flasks contained the same optical density; however, control flasks contained more cells initially due to differences in absorbance between

K. marxianus IMB4 and S. cerevisiae D5A. Cellulose hydrolysis rate increased as temperature increased; however, fermentation of glucose by IMB4 at 458C did not occur as rapidly as the rate of hydrolysis as shown by residual glucose measured during SSF (Figs. 2 and 6). Residual glucose concentrations in SSFs at 37 and 418C were close to zero, indicating that hydrolysis and fermentation rates were equal. Several studies have been conducted using thermotolerant yeast strains in SSF processes at temperatures greater than 378C, but none of the strains used in these studies were observed to perform well in SSF at 458C. Kadam and Schmidt (1997) used Candida acidothermophilum in an SSF process using dilute-acid pretreated poplar and a cellulase loading of 25 FPU/g cellulose. They found that C. acidothermophilum produced more ethanol at a faster rate at 40 and 428C than did S. cerevisiae D5A at 378C. They also observed that 458C is not a feasible temperature for SSF with C. acidothermophilum. Krishna et al. (2001) used K. fragilis in an SSF process using Solka floc, Antigonum leptopus leaves, and sugar cane leaves and a cellulase loading was 40 FPU/g substrate. They compared the ethanol concentration produced by K. fragilis at 438C with the ethanol concentration produced by S. cerevisiae at 408C. K. fragilis produced more ethanol than S. cerevisiae; however, S. cerevisiae ethanol yields may have been depressed due to the high SSF temperature used (408C). Ethanol yields could not be calculated due to lack of cellulose composition data (Krishna et al., 2001). Lark et al. (1997) used K. marxianus (ATCC 36907) to produce ethanol from recycled paper sludge using SSF and a cellulase loading of 14 FPU/g cellulose. They observed that 40 h ethanol yields from SSF of recycled paper sludge increased as SSF temperature increased from 25 to 438C. Bollok et al. (2000) used K. marxianus Y.00243 in an SSF process at 428C using steam pretreated spruce and a cellulase loading of 37 FPU/g cellulose. They observed cessation of ethanol production after 21 h and accumulation of glucose when an initial pH of 5.4 and no pH regulation was used. When pH was regulated at 4.98, ethanol yield was 39% of that achieved without pH regulation and an initial pH of 5.4. Ballesteros et al. (1993) used temperature adaptation, UV irradiation and ethylmethanesulfonate (EMS) mutagenesis to produce K. marxianus mutants capable of fermenting glucose at 498C. When the mutant K. marxianus LG C-22 was used in

Table III. Ethanol concentrations and specific ethanol productivities (qp) for SSF of Kanlow switchgrass pretreated with hot water at 2008C and 10 min using K. marxianus IMB4 (initial cell concentration ¼ 0.14 g/L) and S. cerevisiae D5A (initial cell concentration ¼ 0.20 g/L). Yeast

Temperature (8C)

Initial SSF pH

Ethanol concentration at 72 h (g/L)a

qp (g ethanol/g cells/h)b

IMB4 IMB4 IMB4 IMB4 D5A

37 41 45 45 37

4.8 4.8 4.8 5.5 4.8

12.3 14.8 15.8 16.6 14.0

1.22 1.47 1.57 1.65 0.97

Values for IMB4 at 458C and pH 5.5 were means of two runs; others are means of three runs. a Ethanol concentration does not include ethanol produced from sugars in enzyme preparation or yeast extract. b Based on initial cell concentration.

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SSF of 10% Solka-Floc media, ethanol production and glucose consumption was lower at 458C than at 428C, and glucose fermentation ceased at 78 h for 458C and 100 h for 428C. These results are similar to ours when using pH 4.8 buffer (Figs. 1 and 2). The strain K. marxianus CECT 10875 has been used in several SSF experiments at 428C with olive oil extraction residue, poplar wood, recycled paper, eucalyptus wood, sweet sorghum bagasse, wheat straw and Brassica carinata residue (Ballesteros et al., 2001, 2002a,b, 2004; Negro et al., 2003). The maximum SSF ethanol yields reported using K. marxianus CECT 10875 at 428C were 80% theoretical using hot water pretreated olive oil residue in a batch reactor and recycled paper in a fed batch reactor (Ballesteros et al., 2002a,b). The enzyme loading for both experiments was 15 FPU/g substrate. K. marxianus CECT 10875 has not been compared to S. cerevisiae in terms of ethanol yield on similar substrate in published reports. K. marxianus IMB4 compares favorably with other thermotolerant yeast strains in terms of ethanol yield and the temperature that may be used. Unlike the strains previously described, ethanol yields were similar at 41 and 458C, indicating IMB4 may have greater thermotolerance than other yeasts. Also, IMB4 ethanol yields at 458C using pH 5.5 buffer were greater than a control of S. cerevisiae D5A for the first 96 h of SSF, and the maximum ethanol yield from IMB4 at 458C, pH 5.5 was the same as the maximum ethanol yield from the control, but achieved 72 h earlier than the control. Also, the cellulase loading used in our study (15 FPU/g glucan) is less than or equal to those used in the studies previously described, indicating that lower enzyme loadings may be required with IMB4 than other thermotolerant yeasts. However, it should be noted that it is difficult to compare previous studies with this study since we know of no previous reports that used switchgrass as a substrate for SSF by thermotolerant yeast. Nevertheless, K. marxianus IMB4 is a promising candidate for SSF of cellulose due to its thermotolerance, its ability to produce ethanol at temperatures greater than other thermotolerant yeast, and its greater productivity than S. cerevisiae, the most commonly used yeast for ethanol production.

Conclusion Hydrothermolysis of switchgrass at 2008C for 10 min dissolved 43% of switchgrass dry matter and 0.23 g/L HMF and 0.87 g/L furfural concentration were measured in the prehydrolyzate. Ethanol yield from SSF using K. marxianus IMB4 at 458C was greater than a control SSF using S. cerevisiae D5A at 72 h. Glucose fermentation by IMB4 at 41 and 458C ceased after 96 and 72 h, respectively, which was before completion of cellulose hydrolysis. IMB4 at 378C and the control fermented glucose as quickly as it was produced throughout SSF. When SSF was done using IMB4 at 458C and a pH 5.5 buffer instead of a pH 4.8 buffer, glucose fermentation continued for 24 more hours than when the

pH 4.8 buffer was used and ethanol yield was greater than the control at 96 h. When nutrient concentration was tripled, ethanol yield with IMB4 at 458C decreased. Specific ethanol productivity at 72 h was greater for IMB4 than the control. Maintaining pH above pKa of acetic acid improved IMB4’s ethanol yield. K. marxianus IMB4 compares favorably with other thermotolerant yeasts researched in literature in terms of its thermotolerance and its ability to produce ethanol at 458C. This manuscript was approved and supported by the Oklahoma Agricultural Experiment Station. The authors would like to thank Dr. Nurhan Dunford and Mr. Robert Ingraham for their assistance with analytical procedures.

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