Bioresource Technology 98 (2007) 33673374

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus
Savitree Limtong *, Chutima Sringiew, Wichien Yongmanitchai
Department of Microbiology, Faculty of Science, Kasetsart University, 50 Paholyothin Road, Bangkok 10900, Thailand Received 13 July 2006; received in revised form 5 October 2006; accepted 19 October 2006 Available online 29 May 2007

Abstract Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 C, produced high concentrations of ethanol at both 40 and 45 C. Ethanol production by this strain in shaking ask cultivation in sugar cane juice media at 37 C was highest in a medium containing 22% total sugars, 0.05% (NH4)2SO4, 0.05% KH2PO4, and 0.15% MgSO4 7H2O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5 l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a nal ethanol concentration of 6.43% (w/v), a productivity of 1.3 g/l/h and a yield of 57.1% of theoretical yield. 2007 Elsevier Ltd. All rights reserved.
Keywords: Ethanol production; Sugar cane juice; Thermotolerant yeast; Kluyveromyces marxianus

1. Introduction Sugar cane (Saccharum ocinarum) is a high biomass tropical crop. It contains 1217% total sugars, of which 90% is sucrose and 10% is glucose and/or fructose (Wheals et al., 1999). Sugar cane juice normally has sucient organic nutrients and minerals that make it suitable for ethanol production by fermentation with Saccharomyces cerevisiae. Brazil, the largest fuel ethanol producer in the world, is using sugar cane juice (Moreira, 2000) and/or molasses as substrates (Moreira, 2000; Wheals et al., 1999). Thailand is a tropical country and sugar cane is cultivated in various parts of the country, but the entire crop is used in sugar production. So far, ethanol production in Thailand has used molasses as the substrate; however, this carbon source is becoming limited due to the needs of many fermentation industries. Therefore, an alternative
*

Corresponding author. Tel.: +66 2 5625444; fax: +66 2 5792081. E-mail address: fscistl@ku.ac.th (S. Limtong).

renewable carbon source is required to sustain ethanol production in Thailand, and sugar cane juice seems the most promising one. Ethanol fermentation at high temperature is a key requirement for eective ethanol production in tropical counties where average day-time temperatures are usually high throughout the year. The advantages of rapid fermentation at high temperature are not only decreased risk of contamination but also reduction in cooling costs. To achieve high temperature fermentation it is necessary to use an ecient yeast strain that can tolerate high temperature. At present industrial ethanol production employs a mesophilic strain of S. cerevisiae. Although there have been numerous reports of potential applications of thermotolerant yeast strains in industrial ethanol production, in only a few have been concerned with S. cerevisiae (Kida et al., 1992; Morimura et al., 1997; Kiran Sree et al., 2000; Sridhar et al., 2002); most of them focused on to Kluyveromyces marxianus (Banat et al., 1992, 1995; Gough and McHale, 1998; Kourkoutas et al., 2002; Riordan et al.,

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.10.044

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1995; Zafar and Owais, 2006). However, ligno-cellulosic hydrolysates were used as raw materials for the fermentation. The objective of the present study was to select a yeast strain capable of ethanol fermentation of sugar cane juice at elevated temperature and to investigate the optimal conditions for ethanol production by this strain. 2. Methods 2.1. Isolation and selection of thermotolerant yeast strains Yeasts were isolated from soil and water samples from sugar cane plantations and sugar factories in four provinces, namely Phra Nakhon Si Ayutthaya, Ratchaburi, Suphanburi and Uthaithani, Thailand. Isolation was carried out at 35 C by an enrichment technique using sugar juice media containing sugar cane juice (5% or 8% total sugars), 0.05% (NH4)2SO4 and 4% (v/v) ethanol and with (pH 4.5) and without pH adjustment (pH 3.8). After inoculation, cultures were incubated for 3 days in a rotary shaker (Gallenkamp Orbital Incubator, Leicester, UK) at a predetermined temperature with shaking speed of 170 rpm. Enriched cultures were then streaked on agar plates containing the same medium and inoculated at 35 C. Puried yeast cultures were kept on YPD agar slants (1% yeast extract, 2% peptone, 2% glucose and 2% agar) and stored at 8 C. Thermotolerant yeast strains were selected based on their growth performances at 40 and 45 C in sugar cane juice broth containing 8% total sugars and 0.05% (NH4)2SO4 and supplemented with 4% (v/v) ethanol. Successful cultures were collected and screened further for their ethanol production eciency at elevated temperature. 2.2. Screening of thermotolerant yeasts for ethanol production at high temperature Screening for high ethanol production was conducted at 40 and 45 C in 250 ml Erlenmeyer asks containing 100 ml of a basal sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 18% total sugars and 0.05% (NH4)2SO4 and with pH adjustment to 4.5 with 1 N HCl. Inocula were prepared by transferring one loop full of 24 h culture grown on a slant of YPD agar to an Erlenmeyer ask containing 50 ml of a sugar cane juice medium as above but with only 2% total sugars. After incubation on a rotary shaker at 2528 C for 24 h, the inocula were transferred at the rate of 5% to the screening medium, followed by incubation on a rotary shaker at 40 and 45 C. 2.3. Identication of the selected thermotolerant yeast strain The selected yeast strain was morphologically, physiologically and biochemically characterized by standard methods described by Yarrow (1998). Assimilation of nitrogen compounds was examined on solid media with

starved inocula following the method of Nakase and Suzuki (1986). Growth at various temperatures was determined by cultivation on YM agar and in YM broth using an incubator and a water bath, respectively. Identication and phylogenetic analysis of the yeast strain based on comparative analysis of the sequence of the D1/D2 domain of the large-subunit (LSU) rDNA was carried out. The sequencing of the D1/D2 domain of the LSU rDNA was carried out from PCR products of genomic DNA fragments that were extracted from yeast cells by the method of Lachance et al. (1999) with slight modications. The D1/D2 domain of the LSU rDNA was amplied by PCR with forward primer NL-1 and reverse primer NL-4 (ODonnell, 1993). The PCR product was checked by agarose gel electrophoresis, puried using QIA Quick Purication Kit (Qiagen, Ontario, USA) and cycle-sequenced using the ABI BigDye Terminator Cycle Sequencing Kit Version. 3.1 (Applied Biosystems, California, USA) with the external primers NL-1 and NL-4 (Kurtzman and Robnett, 1998). The sequences were determined with an ABI PRISM 3100 automated DNA sequencer (Applied Biosystems, California, USA) according to the instructions of the manufacturer. The sequences were compared pairwise using the BLAST homology search (Altschul et al., 1990). 2.4. Optimization of ethanol fermentation in Erlenmeyer ask Ethanol fermentations in 500 ml Erlenmeyer asks were performed in triplicate using 200 ml basal sugar cane juice medium. Inoculum size was 5%. Flasks were inculcated on a reciprocating water bath shaker (Model R76, New Brunswick Scientic, New Jersey, USA) at predetermined temperatures with 110 strokes/min. Study of the eect of temperature on ethanol fermentation was carried out at 30, 37, 40 and 45 C in the basal sugar cane juice medium. Eects of nutrient composition, including sugar concentrations, and sources of nitrogen, phosphate and magnesium, were also studied at 37 and 40 C. The sugar concentrations were 16%, 18%, 20%, 22% and 24%. (NH4)2SO4 was used as a supplementary nitrogen source at 0, 0.05%, 0.07% and 0.1%. KH2PO4 at 0, 0.025%, 0.05% and 0.10% was used to determine the requirement for additional phosphate in the sugar cane juice. MgSO4 7H2O at 0, 0.05%, 0.1%, 0.15%, 0.2% or 0.3% was also added to study the eect of magnesium. Sugar cane juice media with pHs were of 4, 4.5, 5 and 5.5 were used to study the eect of pH on ethanol fermentation. 2.5. Ethanol fermentation in a jar fermenter Batch fermentations were carried out in a 5 l jar fermenter (Microferm 114, New Brunswick, New Jersey, USA) with a 3 l working volume, 5% inoculum size and 37 C incubation temperature. A sugar cane juice medium composed of the optimal concentrations of sugars, (NH4)2SO4,

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KH2PO4 and MgSO4 7H2O, and the optimal pH level, as determined in the shaking ask experiments was employed. Four conditions of batch fermentation were studied, i.e., (1) agitation at 300 rpm, (2) agitation at 300 rpm with aeration at 0.2 vvm, (3) agitation at 300 rpm throughout the fermentation with aeration at 0.2 vvm for the rst 12 h and (4) agitation at 300 rpm with aeration at 0.2 vvm for the rst 12 h, followed by only agitation at 150 rpm. During the course of fermentation, samples were taken at 4 or 6 h intervals. 2.6. Analysis of fermentation parameters Yeast growth was determined by measuring optical density at 660 nm with a spectrophotometer (Spectrophotometer 258, Corning, New York, USA) after washing twice with 0.1 N HCl and resuspending in 0.1 M Na2-EDTA. A direct viable cell count was carried out using a hemacytometer after staining with 0.1% methylene blue (Borzani and Vario, 1958). Ethanol concentration was analyzed with a gas chromatograph (Shimadzu GC-9A, Shimadzu, Kyoto, Japan) with a ame ionization detector using a glass column packed with PEG-20M (Shimadzu, Kyoto, Japan). Ethanol productivity, dened as grams of ethanol per liter per hour, was calculated from the nal ethanol concentration. Ethanol yield was calculated based on initial sugar concentration and reported as percentage of theoretical yield. Total sugars as the sum of sucrose, glucose and fructose were quantied with HPLC (Agilent 1100, Agilent Technologies, Palo Alto, USA) with RI detector and a sugar column (ULTRON PS-80, Shinwa Chemical Industries, Kyoto, Japan). Concentrations of the three sugars (sucrose, glucose and fructose) were combined and reported as sugar concentration. 3. Results and discussion 3.1. Isolation and selection of thermotolerant yeast strains Of various techniques to obtain yeasts capable of growing and producing ethanol at high temperature, Banat et al. (1998) stated that isolation from nature was the most successful technique. Therefore, in this work soil samples from natural habitat associated with sugar cane were enriched in sugar cane juice supplemented with 4% (w/v) ethanol and incubated at 35 C. A total of 72 yeast strains were obtained. Among these, 37 strains grew satisfactorily even at 45 C while 18 strains grew only up to 40 C. All of the strains were then subjected to further screening at various high temperature levels. Though Banat et al. (1992) used higher temperatures (4550 C) for enrichment isolation and obtained several thermophilic yeast strains capable of growth at 52 C and ethanol production at 4550 C, a lower temperature, 35 C, was used in this study. In order to obtain yeast strains that tolerated both high temperature and ethanol, 4% (w/v) ethanol was added to the medium,

for the eects of high temperature have been often exacerbated by ethanol concentrations above 3% (Van Uden, 1984). 3.2. Screening of thermotolerant yeasts for ethanol fermentation at high temperature Fifty ve yeast strains from the previous selection scheme were screened for their fermentation abilities in an ethanol production medium in which the sugar content was 18%. The investigation was carried out at 37 C, which is a temperature commonly attained during fermentation in a tropical country like Thailand. However, during certain periods, the temperature may easily reach 40 C due to exothermic metabolic reactions of yeasts during active growth. The results were that four strains, namely DMKU 3-1042, DMKU 3-118, DMKU 3-p106 and DMKU 3-p1042, produced high ethanol concentrations at both 37 and 40 C after 72 h fermentation. At 37 C, the strains DMKU 3-1042, DMKU 3-118, DMKU 3-p106 and DMKU 3-p1042 produced 6.78%, 6.57%, 6.50% and 6.36% (w/v) ethanol, respectively, while at 40 C the maximal ethanol concentrations obtained were slightly lower, viz., 6.17%, 5.58%, 5.14% and 6.33% (w/v), respectively. The remaining 51 strains produced ethanol at relatively lower concentrations, in the range of 0.624.55% (w/v), at 37 C and 0.413.45% (w/v) at 40 C (data not shown). Successful works on selection of yeasts for their ability to ferment ethanol at high temperatures have been reported by several investigators. However, sugar cane juice was not directly used as the substrate for selection. For example, DAmore et al. (1989) used glucose for selection and derived a strain of S. diastaticus that produced 7% (w/v) ethanol from 20% glucose. Ballesterose et al. (1991) examined the ability of Saccharomyces, Candida and Kluyveromyces to ferment glucose at temperatures above 40 C and reported that K. marxianus L.G. produced 3.76% (w/ v) ethanol at 42 C on a glucose medium. Banat et al. (1992) isolated K. marxianus IMB3 and four other strains by enrichment at 45 and 50 C. These strains produced high concentrations of ethanol from glucose at 4550 C and from supplemented molasses at 37 and 40 C. Later on, Banat and Marchant (1995) reported that all of these strains were able to produce ethanol from a wide range of substrates at 45 C. 3.3. Optimization of ethanol fermentation in Erlenmeyer ask The eect of temperature on ethanol fermentation in a basal sugar cane juice medium by the four strains, DMKU 3-1042, DMKU 3-118, DMKU 3-p106 and DMKU 3p1042, was investigated. Maximal ethanol concentrations produced by these four strains at 30 and 37 C were in the range of 7.137.6% (w/v) (Fig. 1). The productivities of DMKU 3-118 and DMKU 3-1042 were almost the same at 30 and 37 C while those of DMKU 3-p106 and DMKU

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Fig. 1. Ethanol production by DMKU 3-p1042 (s), DMKU 3-1042 (d), DMKU 3-p106 (h) and DMKU 3-118 (j) in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 18% total sugars and 0.05% (NH4)2SO4 with pH 4.5 by reciprocating ask cultivation at 30, 37, 40 and 45 C.

3-1042 at 37 C were higher than at 30 C. At 40 C, after 48 h of fermentation, DMKU 3-1042 produced highest concentration of ethanol (7.23% w/v) among the four, and this was only slightly lower than that obtained at 37 C (7.43% w/v); this was reected in its productivity at 40 C (1.51 g/l/h), which was higher than at 37 C (1.03 g/l/h). The other three strains produced ethanol at concentrations of 6.436.85% (w/v). However, increasing the fermentation temperature of these four strains from 40 C to 45 C resulted in drastically decreased maximal ethanol concentrations and productivities (Table 1). At 45 C, the highest ethanol concentration (4.93% w/v) was obtained by DMKU 3-1042. Therefore, the strain DMKU 3-1042 was selected for further studies. Study of ethanol fermentation by the strain DMKU 31042 of a sugar cane juice medium composed of sugar cane

juice supplemented with sucrose to 1624% sugars revealed that increasing the sugar concentration resulted in an increase in the nal ethanol concentration but only up to 22% total sugars at both 37 C and 40 C (Table 2). That sugar concentrations higher than 22% resulted in a decrease in ethanol production might be attributed to various factors including high osmotic pressure and high temperature (Grubb and Mawson, 1993). At 37 C, the highest ethanol concentration (8.29%, w/v) and productivity (1.54 g/l/h) were obtained in a medium composed of 22% sugars after 54 h. It should be noted that the maximal ethanol concentrations were produced in media containing 22% sugars at both 37 C and 40 C. At 37 C, the fermentation yields were not much dierent in media containing 16%, 18%, 20% and 22% sugars, viz., 76.7%, 75.4%, 76.2% and 73.9% of theoretical yield, respectively. At

Table 1 Ethanol fermentation by the four isolates in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 8% sugars, 0.05% (NH4)2SO4 and pH 4.5 at various temperatures Strain 30 C Ethanol % (w/v) DMKU 3-p106 DMKU 3-118 DMKU 3-1042 DMKU 3-p1402 7.60 7.48 7.21 7.13 (72 h) (72 h) (72 h) (72 h) Productivity g/l/h 1.06 1.04 1.00 0.99 37 C Ethanol % (w/v) 7.56 7.42 7.43 7.39 (48 h) (72 h) (72 h) (48 h) Productivity g/l/h 1.57 1.03 1.03 1.54 40 C Ethanol % (w/v) 6.43 6.78 7.23 6.84 (42 h) (48 h) (48 h) (48 h) Productivity g/l/h 1.51 1.41 1.51 1.43 45 C Ethanol % (w/v) 4.74 4.78 4.93 4.59 (48 h) (36 h) (42 h) (48 h) Productivity g/l/h 0.99 1.33 1.17 0.96

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Table 2 Ethanol production by DMKU 3-1042 in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose with various nutrients and pHs by reciprocating ask cultivation at 37 C and 40 C Condition At 37 C Ethanol % (w/v) Sugars (%) 16 18 20 22 24 6.26 6.92 7.77 8.29 7.51 (42 h) (54 h) (54 h) (54 h) (60 h) (72 h) (54 h) (72 h) (72 h) Productivity (g/l/h) 1.49 1.28 1.44 1.54 1.25 0.95 1.36 1.08 1.04 0.96 0.99 1.06 1.05 1.02 1.01 1.00 1.03 0.99 0.96 0.76 0.80 1.45 1.42 % of theoretical yield 76.7 75.4 76.2 73.9 61.4 61.0 65.5 69.5 66.5 61.7 63.5 68.1 67.3 65.2 64.6 64.5 65.9 63.4 61.9 64.7 68.5 77.5 75.8 At 40 C Ethanol % (w/v) 5.95 6.03 6.79 6.88 6.54 6.22 6.60 6.27 6.41 6.45 6.02 6.33 6.12 6.08 6.11 6.11 6.28 6.08 6.04 5.94 5.78 6.57 6.78 (48 h) (48 h) (48 h) (54 h) (48 h) (54 h) (48 h) (60 h) (60 h) (60 h) (60 h) (72 h) (54 h) (72 h) (54 h) (60 h) (60 h) (60 h) (72 h) (84 h) (96 h) (60 h) (60 h) Productivity (g/l/h) 1.42 1.26 1.42 1.27 1.36 1.15 1.37 1.05 1.07 1.07 1.00 0.88 1.13 0.84 1.13 1.02 1.05 1.01 0.84 0.71 0.60 1.09 1.13 % of theoretical yield 72.6 65.7 66.5 61.3 53.4 55.4 58.8 55.9 57.2 57.5 53.7 56.4 54.6 54.22 54.50 54.43 55.98 54.22 53.87 52.9 51.5 58.5 60.4

(NH4)2SO4 (%) 0 6.85 0.05 7.35 0.07 7.80 0.10 7.46

KH2PO4 (%) 0 6.92 (72 h) 0.025 7.12 (72 h) 0.05 7.65 (72 h) 0.10 7.55 (72 h) MgSO4 7H2O (%) 0 7.31 (72 h) 0.05 7.25 (72 h) 0.10 7.24 (72 h) 0.15 7.39 (72 h) 0.20 7.12 (72 h) 0.30 6.94 (72 h) pH pH pH pH pH 4.0 4.5 5.0 5.5 7.26 7.68 8.70 8.50 (96 h) (96 h) (60 h) (60 h)

40 C, an ethanol concentration of 6.88% (w/v) was obtained in a medium containing 22% sugars at the same fermentation time as at 37 C. However, the highest productivity (1.42 g/l/h), and yield (72.6% of theoretical yield) were attained with a medium containing 16% sugars at 40 C. The eect of (NH4)2SO4 as a nitrogen source at a concentration of 00.1% on ethanol fermentation of DMKU 3-1042 was determined in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 22% total sugars and adjusted to pH 4.5. At 37 C, the highest ethanol concentration, 7.8% (w/v), was obtained with 0.07% (NH4)2SO4 supplementation (Table 2); the productivity was 1.08 g/l/h and the yield was the highest: 69.5% of theoretical yield. However, the highest productivity, 1.36 g/l/l, was attained when the medium was supplemented with 0.05% (NH4)2SO4; the maximal ethanol concentration was 7.35% (w/v) and the yield was 65.5% of theoretical yield. In sugar cane juice media without supplementation with a nitrogen source, the ethanol concentration (6.85% w/v), productivity (0.95 g/l/h) and ethanol yield (61.0% of theoretical yield) observed were lower. At 40 C, the ethanol concentrations between 6.226.66% (w/v) were obtained in the medium without and with sup-

plementation with a nitrogen source at various concentrations; these concentrations were signicantly lower than those obtained at 37 C. The productivity in the medium supplemented with 0.05% (NH4)2SO4 was 1.37 g/l/h, which was the highest among all treatments, while in the medium without nitrogen supplementation, productivity was only 1.15 g/l/h; in media supplemented with 0.07% and 0.1% (NH4)2SO4, the productivities were 1.05 and 1.07 g/l/h, respectively. Fermentation yield obtained in media with and without supplementation with a nitrogen source were in the range of 55.458.8% of theoretical yield. Hence, (NH4)2SO4 at 0.05% was chosen for further study since it provided the highest productivity at both temperatures with not much dierence in maximal ethanol concentrations and yields. The eect of a potassium source was studied using KH2PO4 at 00.10% added to a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 22% total sugars and 0.05% (NH4)2SO4 and having a pH of 4.5. At 37 C, the highest ethanol concentration, productivity and yield: 7.65% (w/v), 1.06 g/l/h and 68.1% of theoretical yield, respectively, were attained by fermentation in a medium supplemented with 0.05% KH2PO4. In contrast, at 40 C, there was little dierence in ethanol

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production in media without and with KH2PO4 supplementation; the highest ethanol concentration, 6.45% (w/ v), was obtained in a medium without KH2PO4 supplementation (Table 2). The eect of a magnesium source on ethanol fermentation was studied using MgSO4 7H2O at concentrations of 00.3% in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 22% total sugars, 0.05% (NH4)2SO4, 0.05% KH2PO4 and having a pH of 4.5. At 37 C, an ethanol concentration of 7.39% (w/v), a productivity of 1.03 g/l/h and a yield of 65.9% of theoretical yield were obtained when the medium was supplement with 0.15% MgSO4 7H2O; these values were similar to those obtained in a medium without MgSO4 7H2O supplementation (7.31% w/v, 1.02 g/l/h and 65.2% of theoretical yield, respectively) (Table 2). At 40 C, the maximal ethanol concentrations were in the range of 6.046.28% (w/v) while the productivity and yields obtained from the various concentrations of MgSO4 7H2O were only slightly dierent (Table 2). The eect of the initial pH of the medium was investigated, and it was found that at 37 C, the highest ethanol concentration (8.7% w/v) (Table 2), productivity (1.45 g/l/h) and yield (77.5% of theoretical yield) were obtained when the fermentation was carried out at pH 5; these values were only slightly higher than those obtained from fermentation at pH 5.5, while the lowest values were obtained from fermentation at pH 4.0. Ethanol fermentation carried out at 40 C provided the similar results; fermentations at pH 5.5 and 5 gave better results than those at pH 4 and 4.5 (Table 2). Growth of the strain DMKU 3-1042 in all the optimization studies was much lower than that of S. cerevisiae Sc90, a strain commonly used in industrial ethanol production in Thailand (data not shown). The maximal growth determined as optical density at 660 nm of the strain DMKU 3-1042 in all cases was less than 12, and there was not much dierence in growth between the fermentation temperatures of 37 C and 40 C (data not shown). Ethanol fermentation of the strain DMKU 3-1042 by shaking ask cultivation (110 strokes/min) at 37 C in a sugar cane juice medium was optimum when the medium composed of sugar cane juice supplemented with sucrose to 22% total sugar, 0.05% (NH4)2SO4, 0.05% KH2PO4, and 0.15% MgSO4 7H2O and having a pH of 5.0. These optimal nutrient and pH conditions gave a maximal ethanol concentration of 8.7% (w/v) (Fig. 2), a productivity of 1.45 g/l/h, and a yield 77.5% of theoretical yield. Growth measured by OD at this temperature and under these conditions was 11.42 at 54 h and 14.33 after 72 h (Fig. 2). At 40 C, a maximal ethanol concentration of 6.78% (w/v) (Fig. 2), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained in a medium of the same composition except that the pH of the medium was 5.5. Growth as determined by OD was 13.55 at 72 h of fermentation at 40 C under optimal conditions (Fig. 2).

Ethanol (% w/v)

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Fig. 2. Ethanol production (s, h) and growth (d, j) by DMKU 3-1042 in a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 22% total sugars, 0.07% (NH4)2SO4, 0.05% KH2PO4 and 0.15% MgSO4 7H2O with pHs of 5 and 5.5 by reciprocating ask cultivation at 37 C (s, d) and 40 C (h, j), respectively.

3.4. Ethanol production by batch fermentation in a sugar cane juice medium in a jar fermenter Studies on ethanol production at 37 C in a 5 l jar fermenter using 3 l of a sugar cane juice medium composed of sugar cane juice supplemented with sucrose to 22% total sugars, 0.05% (NH4)2SO4, 0.05% KH2PO4, 0.15% MgSO4 7H2O and having a pH of 5.0 were conducted under various fermentation conditions including (1) agitation at 300 rpm, (2) agitation at 300 rpm with aeration at 0.2 vvm, (3) agitation at 300 rpm throughout the fermentation with aeration at 0.2 vvm for the rst 12 h and (4) agitation at 300 rpm with aeration at 0.2 vvm for the rst 12 h, followed by only agitation at 150 rpm. The highest ethanol concentration, viz., 6.43% (w/v) at 48 h, equivalent to a productivity of 1.3 g/l/h and 57.1% of theoretical yield, was achieved under Condition (2). On the other hand, the lowest ethanol concentration, which was 4.96% (w/v) at 60 h, or a productivity of 0.81 g/l/h and a yield 44% of theoretical yield, was obtained when Condition (1) was applied. Conditions (3) and (4) yielded slightly higher ethanol concentrations than (1), viz. 6.17% (w/v) at 54 h and 6.03% (w/v) at 60 h, respectively, with similar productivities and percentages of theoretical yields. Although Condition (2) gave the highest growth and although rapid growth was observed in the rst 24 h the maximal growth measured as optical density was only 12.73. Surprisingly, after 24 h, the viable cell count determined by methylene blue staining decreased rapidly, and no viable cells could be detected at 54 h. Condition (1) batch fermentation yielded the lowest growth among all conditions with OD at 5.83, and it consequently produced the lowest ethanol concentration, while (3) and (4) gave maximal growths as determined by OD of only 8.92 at 36 h and 8.10 at 44 h, respectively. It was, therefore, quite obvious that the low yeast cell density in these batch fermentations resulted in unsatisfactorily low ethanol production. Banat et al. (1998) indicated that K. marxinus did

OD (660 nm)

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not show a high growth rate under anaerobic conditions, which was a common characteristic of S. cerevisiae. Therefore, low growth of the strain DMKU 3-1042 used in this study might have been caused by insucient aeration of the system. In addition, DAmore et al. (1989) reported that increasing the cell density resulted in an increase in ethanol production at high temperature. Hence, in order to address this problem, increasing the initial cell concentration together with increasing the aeration rate might be necessary. Sugar utilization further conrmed the low ethanol production and yeast cell growth. Condition (2), where the highest ethanol production and growth were obtained, gave the lowest sugar concentration remaining at the end of fermentation: 7.05%. On the other hand, under Condition (1), with the lowest ethanol production and growth, the highest concentration of remaining sugar (10.35%) was observed. For Condition (3) and (4), the concentrations of remaining sugars were 9.24% and 7.93%, respectively.

References
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3.5. Identication of the selected thermotolerant yeast strains Identication of the strain DMKU 3-1042 following a conventional taxonomic approach was made by comparing its morphological, biochemical and physiological characteristics (data not shown) to a taxonomic key of Kurtzman and Fell (1998). The strain DMKU 3-1042 was revealed to be K. marxianus. In molecular taxonomic studies on nucleotide sequences of D1/D2 domain of LSU rDNA showed that the sequences of the strain DMKU 3-1042 and the type strain of K. marxianus were identical. Therefore, the strain DMKU 3-1042 was identied as K. marxianus.

4. Conclusion The results of this study demonstrated that the newly isolated K. marxianus strain DMKU 3-1042 was an eective strain that could be employed for ethanol production at elevated temperature when sugar cane juice was used as a raw material. It produced 8.7% (w/v) ethanol at 37 C and 6.78% (w/v) at 40 C from sugar cane juice medium composed of 22% total sugars by shaking ask cultivation. Though it was quite obvious that low yeast cells density in the batch fermentations in the 5 l jar fermenter resulted in unsatisfactorily low ethanol production, increasing the initial cell concentration together with increasing the aeration rate might be employed to remedy this problem.

Acknowledgement This work was supported in part by a research grant from Graduate school, Kasetsart University, Bangkok Thailand.

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