Biosci. Biotechnol. Biochem., 77 (7), 1505–1510, 2013
Bioethanol Production from Lignocellulosic Biomass by a Novel Kluyveromyces marxianus Strain Tetsuya G OSHIMA,1; * Masaharu T SUJI,1; * Hiroyuki I NOUE,1 Shinichi Y ANO,1 Tamotsu H OSHINO,1;2 and Akinori M ATSUSHIKA1; y 1
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashi-hiroshima, Hiroshima 739-0046, Japan 2 Graduate School of Life Science, Hokkaido University, N10W8 Kita-ku, Sapporo, Hokkaido 060-0810, Japan Received March 4, 2013; Accepted April 15, 2013; Online Publication, July 7, 2013 [doi:10.1271/bbb.130173]
The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K. marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 C. Reference strains K. marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation. Key words:
thermotolerant yeast; sugar utilization; ethanol production; lignocellulose
Bioethanol production from plant biomass has recently received considerable attention with respect to alleviating global warming and decreasing the global demand for non-renewable fossil fuels. Lignocellulosic biomasses, such as agricultural waste and woods, are being regarded as potential renewable sources of ethanol production, because they contain large quantities of potentially fermentable sugars. The main structural components of lignocellulosic biomass are cellulose, hemicelluloses, and lignin. Hexose sugars, including glucose obtained from cellulose and hemicellulose by chemical or enzymatic hydrolysis (saccharification), can serve as raw materials in producing ethanol by fermentation.1) The bioconversion of lignocellulose to ethanol involves several processes, including pretreatment to soften hard lignocellulose structures followed by enzymatic digestion and ethanol fermentation from sugars derived from the lignocellulose. Since these complex processes can increase ethanol production, various approaches can be taken to realize more cost-effective generation from several biomass sources. Producing ethanol at high temperature would significantly reduce the costs associated with cooling.2) One of y
the most effective ethanol-producing microorganisms, Saccharomyces cerevisiae, has several advantages, including high ethanol productivity, high ethanol tolerance, and tolerance of inhibitory compounds in lignocellulosic biomass hydrolysates,3) but the optimal temperature range for S. cerevisiae growth is 25– 35 C. If fermentation can proceed at higher temperatures, such as 40–50 C, large-scale commercial ethanol production should become far more economical. Moreover, fermentation at higher temperatures might confer several benefits, including reduced risk of contamination from other microorganisms and suitability for practice in tropical countries.2) Kluyveromyces marxianus is a generally recognized as safe (GRAS) yeast, like S. cerevisiae, that is abundant in fermented foods. It has a high growth rate, it can survive at 52 C,4) and its fermentation efficiency is similar to that of S. cerevisiae at 30 C.5) K. marxianus can ferment ethanol efficiently at temperatures of 38–45 C, and it can metabolize several economically relevant industrial substrates.5) Thus, it offers promise as an alternative to S. cerevisiae for bioethanol production. The present study focused on high-temperature fermentation with a novel K. marxianus DMB1 strain. We evaluated the thermotolerance, ethanol productivity, and ability of strain DMB1 to ferment glucose in both complex medium and lignocellulosic hydrolysates during fermentation at high temperatures.
Materials and Methods Yeast strains and media. Standard methods for yeasts and media were followed as described previously.6) S. cerevisiae BY4743 (Open Biosystems, Huntsville, AL), widely used in genetics research, and K. marxianus NBRC1777 (NITE Biological Resource Center, Chiba, Japan), a yeast species with outstanding thermotolerance,7) served as reference strains for K. marxianus DMB1. We isolated DMB1 and determined the details of the strain. Yeast strains were grown in yeast peptone dextrose (YPD) medium (10 g/L of yeast extract, 20 g/L of peptone, and 20 g/L of glucose) and in synthetic minimal medium (6.7 g/L of yeast nitrogen base without amino acids and 20 g/L of glucose) supplemented with appropriate amino acids (SCD medium). Cell growth was monitored by the absorbance at 600 nm with a BioSpectrometer (Eppendorf, Hamburg, Germany).
To whom correspondence should be addressed. Tel: +81-82-420-8289; Fax: +81-82-420-8291; E-mail:
[email protected] These authors equally contributed to this work. Abbreviations: DCW, dry-cell weight; HPLC, high-performance liquid chromatography; ITS, intergenic spacer region; SSF, simultaneous saccharification and fermentation *
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Phylogenetic analysis. The novel DMB1 strain was analyzed phylogenetically by sequencing the intergenic spacer region (ITS), including the 5.8S rRNA and D1/D2 domain of 26S rRNA. Yeast cells were harvested from 24 h cultures, and DNA was extracted with ISOPLANT II kits (Wako Pure Chemical Industries, Osaka, Japan), as described by the manufacturer. Extracted DNA was amplified by PCR with KOD-plus DNA polymerase (Toyobo, Osaka, Japan). The ITS region was amplified with primers ITS1 (50 -TCC GTA GGT GAA CCT GCG G) and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC). The D1/D2 domain was amplified with primers NL1 (50 -GCA TAT CAA TAA GCG GAG GAA AAG) and NL4 (50 -GGT CCG TGT TTC AAG ACG G). The amplified DNA was sequenced at Macrogen Japan (Tokyo). The ITS region and the D1/D2 domain sequences of yeast strain DMB1 have been deposited in the DNA Data Bank of Japan (DDBJ) (accession nos. AB771424 and AB771425). Sequences were aligned by CLUSTAL W (http://clustalw.ddbj.nig.ac.jp/), and were manually corrected. Phylogenetic analysis proceeded with MEGA software version 5.1,8) with neighbor-joining analysis of the ITS region containing 5.8S rRNA and maximum parsimony analysis of the D1/D2 domain of 26S rRNA. Bootstrap analysis included 1,000 replicates. Growth experiments. Growth experiments were followed as described previously.9) Strains were shaken at 120 rpm overnight at 30 C in 5 mL of SCD medium and diluted to an OD600 of 0.5 for spot assays. Serial 3-fold dilutions were spotted onto plates and grown for 1 d at the indicated temperatures. Lignocellulosic hydrolysates. Japanese cedar and eucalyptus chips were mechanochemically processed as described previously,10,11) and then 20% (w/v) each was hydrolyzed with 6 FPU/g of Acremonium cellulase (Meiji Seika Pharma, Tokyo) and 20 mL/g of Optimash BG (Genencor International, Rochester, NY) in 0.02 M citrate buffer (pH 5.0) at 120 rpm at 37 C. After 120 h of incubation, the saccharified samples were separated by centrifugation at 3;000 g for 10 min at 4 C. Glucose concentrations were measured in the supernatants after passage through a 0.2-mm filter (Merck Millipore, Billerica, MA). Fermentation was analyzed in hydrolysates supplemented with yeast extract (10 g/L). The Japanese cedar- and eucalyptus-based hydrolysates contained glucose, 50.3 and 57.4; mannose, 6.7 and 0.65; galactose, 1.8 and 1.2; xylose, 8.9 and 13.9; and arabinose, 1.6 and 0.36 g/L respectively. Fermentation. Anaerobic fermentation experiments proceeded, and the dry-cell weights (DCW) of the yeast strains were calculated as described previously.6) The initial cell density in the fermentation medium was adjusted to an optical density of 10 at 600 nm, which corresponds to approximately 2.4 g of DCW/L. All experiments were repeated at least 3 times. Analysis of substrates and fermentation products. Monosaccharides and alcohols were measured by high-performance liquid chromatography (HPLC) equipped with a refractive index detector (RI-2031Plus; Jasco, Tokyo). Ethanol, glucose, xylose, xylitol, glycerol, and acetic acid in the YPD medium were separated with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). The HPLC equipment was operated at 65 C with 8 mM H2 SO4 at a flow rate of 0.6 mL/min as mobile phase. The ethanol, glucose, xylose, mannose, galactose, arabinose, and glycerol in lignocellulosic hydrolysate were separated with an Aminex HPX-87P column (Bio-Rad). The HPLC system was operated at 80 C with sterile water (flow rate, 0.6 mL/min) as mobile phase.
Results Kluyveromyces marxianus strain DMB1 isolated from hydrolysate of bagasse Inoue and collaborators have evaluated the effectiveness of ball milling and wet disk milling in treating sugarcane bagasse and straw, which were kindly supplied by Usina Itaruma˜ (State of Goia´s, Brazil).12) In this process, a yeast-like microorganism was isolated from hydrolysates of the sugarcane bagasse as a
contaminate, and was named strain DMB1. Findings from physiological assays, cell morphological analysis, and as to the ability to assimilate sugar indicated that the microorganism was the yeast K. marxianus. We determined the nucleotide sequence of the ITS region and the D1/D2 domain of the ribosomal RNA to confirm the species. A phylogenetic analysis of the ITS region and D1/D2 domain grouped yeast strain DMB1 within the K. marxianus NBRC1777 clade (Fig. 1A and B). A comparison of the ITS region, containing 5.8S rRNA and the D1/D2 domain, as between strains DMB1 and NBRC1777, confirmed an absence of variations. DMB1 was more thermotolerant than NBRC1777 K. marxianus is a thermotolerant yeast.4,5) We compared thermotolerance among serial dilutions of K. marxianus strains DMB1 and NBRC1777 and S. cerevisiae BY4743 incubated on YPD plates at various temperatures. Both K. marxianus strain DMB1 and strain NBRC1777 survived above 40 C, whereas BY4743 did not (Fig. 2A), and DMB1 grew at a maximum temperature of 48 C, whereas NBRC1777 did not (Fig. 2A). To confirm these findings, we calculated specific growth rates at various temperatures in YPD liquid cultures. At a moderate temperature of 30 C, the specific growth rates of K. marxianus strains DMB1 and NBRC1777 were more rapid than that of S. cerevisiae BY4743 (0.761 vs. 0.873 and 0.576 h1 , Fig. 2B), and the maximum growth rates of DMB1 and NBRC1777 at 42 C were 0.939 and 1.061 h1 respectively. At 48 C, the growth rate of DMB1 was faster than that of NBRC1777 (0.244 vs. 0.038 h1 , Fig. 2B). These results indicate that DMB1 has better thermotolerance than NBRC1777. We investigated the capacity of DMB1, NBRC1777, and BY4743 to produce ethanol during fermentation at 48 C. Consistently with the results of the aerobic growth experiments, only DMB1 proliferated well during fermentation (data not shown). It converted glucose into ethanol at 48 C while BY4743 and NBRC1777 produced minuscule amounts (Fig. 2C and Table 1). Ethanol yield by DMB1 increased slightly at 48 C, as compared to 30 C (0.467 vs. 0.415 g/g) (Table 1), perhaps due to decreased production of by-products, including acetic acid and glycerol (data not shown). Thus DMB1 among the three strains fermented ethanol at high temperature most efficiently, suggesting that it is more commercially viable than NBRC1777. Strain DMB1 can utilize a variety of carbon sources, including sorbitol Lignocellulosic hydrolysates generally contain hexoses, including glucose, mannose, and galactose, and pentoses, including xylose and arabinose. A yeast should be able to ferment ethanol from various carbon sources to produce bioethanol economically from a lignocellulosic biomass. Unlike S. cerevisiae, K. marxianus strains can utilize various sugars, including pentoses, xylose, and arabinose.5) We determined the potential of DMB1 to utilize sugars in lignocellulosic hydrolysates by spot assays and synthetic complete plates supplemented with 20 g/L of various carbon sources (glucose, galactose, fructose, xylose, xylitol, xylulose, arabinose,
K. marxianus Produces Ethanol from Lignocellulosic Biomass
A
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Kluyveromyces marxianus DMB1(AB771424) Kluyveromyces marxianus CECT1123 (AJ401692) Kluyveromyces marxianus CECT10668 (AJ401699) Kluyveromyces marxianus NBRC1777 (AB771426) 79 Kluyveromyces lactis CECT1122 (AJ401703) 100 Kluyveromyces lactis CECT10390 (AJ401709) Kluyveromyces dobzhanskii NRLL Y-1974 (AY046215) 100 79 Kluyveromyces wickerhamii NRLL Y-8286 (AY046212) Kluyveromyces aestuarii YB-4510 (AY046210) 78 Kluyveromyces nonfermentans NRLL Y-27343 (AY046211) Kluyveromyces thermotolerans CBS6924 (AJ229073) 100 Kluyveromyces waltii NRLL Y-8285 (AY046208) Kluyveromyces polysporus CBS2163 (AJ229076) Kluyveromyces blattae NRLL Y-10934 (AY046175) Kluyveromyces sinensis NRLL Y-27222 (AY046167) Kluyveromyces africanus NRLL Y-8276 (AY046155) Kluyveromyces hubeiensis AS 2.1536 (AH013031) Kluyveromyces lodderae NRLL Y-8280 (AY046160) Kluyveromyces piceae NRLL Y-17977 (AY046159) Kluyveromyces delphensis NRLL Y-2379 (AY046166) Kluyveromyces bacillisporus NRLL Y-17846 (AY046195) Aspergillus oryzae XJ28 (AB470911) 92
95
70
83 99 57
B
0.05
Kluyveromyces aestuarii YB-4510 (AF399808) Kluyveromyces nonfermentans NRLL Y-27343 (AF399809) 99 Kluyveromyces wickerhamii NRLL Y-8286 (AF399810) 76 95 Kluyveromyces dobzhanskii NRLL Y-1974 (AF399813) Kluyveromyces lactis NRLL Y-8279 (AF399811) Kluyveromyces thermotolerans NRLL Y-8284 (AF399805) Y Kluyveromyces bacillisporus NRLL Y-17846 AF399793 Kluyveromyces polysporus NRRL Y-8283 (AY048169) 80 Kluyveromyces africanus NRRL Y-8276 (AY048159) 79 Kluyveromyces piceae NRLL Y-17977 (AF399767) 89 Kluyveromyces lodderae NRRL Y-8280 (AY048161) Kluyveromyces delphensis NRLL Y-2379 (AF399772) Kluyveromyces blattae NRRL Y-10934 (AY048165) Kluyveromyces lactis NRLL Y-8278 (U94919) 99 Kluyveromyces marxianus NBRC1777 (AB771427) Kluyveromyces marxianus DMB1 (AB771425) 99 Kluyveromyces marxianus Y-12 (JF715180) 70 Kluyveromyces siamensis RS8 (AB330824) 75 Kluyveromyces waltii NRLL Y-8285 (U69582) Kluyveromyces hubeiensis AS 2.1536 (AH013031) Kluyveromyces sinensis NRLL Y-27222 (AF398484) Aspergillus oryzae MN (GQ382275) 99 56
100
99
50
Fig. 1. Phylogenetic Trees of K. marxianus DMB1 and Related Species. A, Neighbor-joining tree of the ITS region containing the 5.8S rRNA gene sequence. Bootstrap values (%) from 1,000 replicates are shown on branches. Aspergillus oryzae XJ28 served as out group. DMB1 is highlighted in bold type. B, Maximal parsimony analysis of the D1/D2 domain of the 26S rRNA gene sequence. Bootstrap values (%) from 1,000 replicates are shown on branches. Aspergillus oryzae MN served as out group.
mannose, maltose, sucrose, glycerol, and sorbitol) at 30 C (Fig. 3 and Table 2). DMB1 utilized not only hexoses and pentoses but also sorbitol for growth, whereas BY4743 and NBRC1777 did not assimilate sorbitol (Fig. 3 and Table 2). The fact that DMB1 can digest pentoses, including xylose and arabinose is consistent with the finding that other K. marxianus strains utilize pentoses by assimilation under aerobic conditions.5) Accordingly, DMB1 appeared to have effectively metabolic pathways to be able to utilize such a wide range of substrates. We performed fermentation experiments using DMB1 together with BY4743 and NBRC1777 to examine DMB1 sugar utilization under anaerobic conditions. One report indicated that K. marxianus strains cannot efficiently ferment xylose to ethanol under anaerobic conditions.13) Consistently with other K. marxianus strains, DMB1 did not produce ethanol from xylose, xylitol, or arabinose under anaerobic conditions (Table 2). Moreover, DMB1 did not convert sorbitol to ethanol by fermentation. Thus although sugars such as xylose, xylitol, arabinose, glycerol, and sorbitol were
assimilated and metabolized by DMB1, they were apparently non-fermentable carbon sources for this strain. DMB1 produced ethanol efficiently from Japanese cedar-based lignocellulosic biomass at high temperature A considerable fraction of the hydrolysates obtained from lignocellulosic biomass comprises toxic compounds such as furfural, HMF, levulinic acid, and formic acid, together with phenolic compounds derived from degraded soluble lignin during pretreatment. Based on this information, we compared the abilities of the K. marxianus and the S. cerevisiae strain to ferment hydrolysates of Japanese cedar and eucalyptus. Thermotolerant yeast strains DMB1 and NBRC1777 produced ethanol from Japanese cedar hydrolysate at 42 C, but not at 45 C (Table 1). The specific ethanol production rate at 42 C was higher for DMB1 than for NBRC1777 or BY4743, 0.613 vs. 0.403 and 0.348 (g/gDCW/h). Both K. marxianus strains, DMB1 and NBRC1777, produced ethanol from eucalyptus hydro-
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T. GOSHIMA et al. Table 1. Specific Glucose Consumption Rates, Specific Ethanol Production Rates, and Ethanol Yield by Fermentation
Fermentation medium
Temperature ( C)
YPD
30
48
Japanese cedar
30
42
45
Eucalyptus
30
42
45
Strain
Specific glucose consumption ratea (g/g DCW/h)
Specific ethanol production rateb (g/g DCW/h)
Concentration of ethanol (g/L)
Ethanol yieldc (g/g)
S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1 S. cerevisiae BY4743 K. marxianus NBRC1777 K. marxianus DMB1
1:366 0:069 1:366 0:092 0:991 0:018 0:035 0:011 0:280 0:015 1:566 0:015 1:311 0:001 1:089 0:002 0:768 0:006 0:665 0:006 0:563 0:009 0:580 0:044 0:353 0:022 0:210 0:006 0:506 0:105 2:076 0:026 0:173 0:058 0:255 0:045 0:363 0:118 0:055 0:008 0:229 0:031 0:151 0:043 0:031 0:020 0:189 0:012
0:702 0:018 0:619 0:045 0:426 0:010 0:017 0:007 0:130 0:025 0:734 0:023 0:665 0:006 0:563 0:009 0:580 0:044 0:348 0:008 0:403 0:064 0:613 0:109 0:039 0:001 0:022 0:002 0:046 0:006 0:859 0:026 0:090 0:028 0:242 0:049 0:211 0:016 0:055 0:020 0:257 0:002 0:110 0:025 ND 0:053 0:020
17:85 0:08 17:10 0:05 16:04 0:01 1:41 0:36 2:58 0:40 18:00 0:20 20:34 0:11 20:11 0:32 19:78 0:04 26:87 0:25 26:46 0:35 25:98 0:21 13:43 0:47 6:79 0:09 13:40 0:19 24:09 0:99 23:53 0:01 23:74 0:59 10:22 2:64 1:48 0:07 8:92 0:77 4:31 0:79 ND 2:47 0:344
0:457 0:002 0:442 0:002 0:415 0:002 0:219 0:020 0:337 0:014 0:467 0:002 0:445 0:007 0:439 0:001 0:455 0:007 0:551 0:004 0:533 0:005 0:602 0:094 0:488 0:053 0:444 0:015 0:356 0:016 0:401 0:017 0:413 0:001 0:411 0:012 0:492 0:066 0:220 0:019 0:491 0:015 0:425 0:016 ND 0:342 0:058
Values are averages of three independent experiments standard deviation. ND, not detected. a Expressed as grams of glucose per gram of dry weight of cells per hour. b Expressed as grams of ethanol per gram of dry weight of cells per hour. c Expressed as grams of produced ethanol per gram of total consumed sugar after 72 h of fermentation.
lysates very slowly at 30 C, whereas production by S. cerevisiae BY4743 was normal. The specific ethanol production rates of DMB1, NBRC1777, and BY4743 were 0.242, 0.090, and 0.859 g/gDCW/h respectively.
Discussion We isolated novel thermotolerant yeast K. marxianus strain DMB1 from bagasse hydrolysates. It proliferated under both aerobic and anaerobic conditions at 48 C and produced ethanol efficiently by fermentation (Fig. 2 and Table 1), whereas thermotolerant K. marxianus strain NBRC1777 did not.7) Moreover, the growth rates of other K. marxianus strains are significantly lower at 45 C under anaerobic conditions.13) Hence we speculate that DMB1 is a suitable microorganism for bioethanol production at higher temperatures and that it can increase the efficiency of ethanol fermentation. In general, K. marxianus can utilize a variety of industrial substrates, including cellobiose, xylose, arabinose, and lactose, but not sorbitol.5) On the other hand, DMB1 utilized sorbitol as well as pentoses including xylose and arabinose, but did not convert them into ethanol by fermentation (Fig. 3 and Table 2). Both DMB1 and NBRC1777 produced ethanol from lignocellulosic hydrolysates of Japanese cedar at 42 C (Table 1), but grew poorly and produced very small amounts of ethanol from eucalyptus-based hydrolysates even at 30 C (Table 1). These results suggest that eucalyptus-based hydrolysates contain far more fermentation inhibitors than Japanese cedar, and that K. marxianus has lower tolerance of fermentation inhibitors than S. cerevisiae. Hence the tolerance of DMB1 of fermen-
tation inhibitors should be improved for large-scale bioethanol production. Notably, S. cerevisiae BY4743 produced ethanol from hydrolysates of eucalyptus at 30 C and from Japanese cedar at 42 C (Table 1). A recent study found that S. cerevisiae BY4742 can produce ethanol at 45 C.14) Various potential strategies including simultaneous saccharification and fermentation (SSF) can be used to produce ethanol from biomasses more cost effectively. One of the major drawbacks of the SSF process lies in the optimal temperatures required for saccharification and fermentation. Saccharification with cellulolytic enzymes is most effective at about 50 C, whereas the optimal temperature for most microbes to produce ethanol by fermentation is 25–35 C. Accordingly, high-temperature fermentation is in demand for SSF processes.2) In practice, SSF has proceeded at 45 C with switchgrass and K. marxianus.15,16) Here, we found that DMB1 might be a better strain for the high temperature SSF process, because it can produce ethanol at 48 C. Since the pentose sugar xylose comprises a considerable fraction of lignocellulosic biomass hydrolysates, the conversion of a biomass to ethanol should utilize xylose to be economical. S. cerevisiae cannot metabolize pentoses, including xylose, in nature. To improve ethanol production by xylose-utilizing recombinant yeast strains, various genetically engineered S. cerevisiae strains have been investigated.17) We and others have found that K. marxianus strains cannot convert xylose to ethanol, despite being able to utilize xylose for growth, but the K. marxianus genome harbors genes encoding the enzymes required for xylose metabolism, xylose reductase (KmXYL1), xylitol dehydrogenase
K. marxianus Produces Ethanol from Lignocellulosic Biomass
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Table 2. Carbon Source Utilization by Yeast Strains under Aerobic and Anaerobic Conditions at 30 C
Sugar
b
Fermentationb
OD600
Ethanol production (g/L)
S. cerevisiae
K. marxianus
S. cerevisiae
K. marxianus
BY4743
NBRC1777
DMB1
BY4743
NBRC1777
DMB1
11.3 9.8 11.1 0.1 0.1 0.8 0.1 11.6 1.2 12.8 0.4 0.1
13.7 12.4 13.9 5.6 3.8 3.9 2.9 14.5 1.5 13.6 2.0 0.1
16.4 15.1 16.5 7.9 3.9 5.9 3.8 16.9 1.8 17.0 1.2 4.7
20.7 20.7 19.9 ND ND ND ND 19.8 ND 20.8 ND ND
20.3 20.2 18.2 ND ND 1.6 ND 18.0 ND 20.7 ND ND
20.8 8.7 17.2 ND ND 1.0 ND 17.9 ND 20.3 ND ND
Glucose Galactose Fructose Xylose Xylitol Xylulose Arabinose Mannose Maltose Sucrose Glycerol Sorbitol a
Growtha
Aerobic growth after 24 h for an initial OD600 of 0.04. Anaerobic ethanol production from 45 g/L of carbon source after 72 h of fermentation. ND, not detected.
A
30 37 40 42 45 47.5 48 49 50 (°C) BY4743
Glc Fru Gal Man Suc
Mal Ara Xlo
Xli
Xlu Gly Sor
BY4743
NBRC1777
DMB1
DMB1 NBRC1777
Specific growth rate µ (h-1)
B
BY4743 1.2
NBRC1777
1 0.8 0.6 0.4 0.2 0 30
35 40 45 Temperature (°C)
50
Glucose (g/L)
40
Ethanol (g/L)
C
DMB1
Fig. 3. Carbon Source Utilization of DMB1 under Aerobic Conditions. The ability of yeast strains, BY4743, NBRC1777, and DMB1 to utilize sugars was assessed using spots on synthetic complete plates containing sole carbon sources at an initial cell concentration of approximately OD600 ¼ 0:06 after incubation for 24 h at 30 C. Ara, arabinose; Fru, fructose; Gal, galactose; Glc, glucose; Gly, glycerol; Mal, maltose; Man, mannose; Sor, sorbitol; Suc, sucrose; Xli, xylitol; Xlo, xylose; Xlu, xylulose.
modify xylose metabolism. We plan to construct a recombinant K. marxianus DMB1 with xylose-fermenting ability by the same strategy as for S. cerevisiae.
30 20 10
Acknowledgment
0 25 20 15 10 5 0 0
24
48
72
Time (h)
Fig. 2. Growth and Ethanol Fermentation by DMB1 at High Temperature. A, Thermotolerance of yeast strains S. cerevisiae BY4743 as well as K. marxianus NBRC1777 and DMB1 assessed as spots on YPD plates at an initial cell concentration of approximately OD600 ¼ 0:06 incubated for 24 h at the indicated temperatures. B, Specific growth rates of BY4743, NBRC1777, and DMB in YPD liquid media at various temperatures monitored as the absorbance at 600 nm. C, Yeast strains BY4743, NBRC1777, and DMB1 were inoculated into YPD medium containing 40 g/L of glucose and incubated at 48 C under anaerobic conditions. Fermentation was started with an initial cell density of about 10 at OD600 .
(KmXYL2), and xylulokinase (KmXKS1).18–20) DMB1 efficiently converted ethanol from xylulose (Table 2), suggesting that it has higher xylulokinase activity than S. cerevisiae and is thus the ideal host strain in which to
We thank Dr. Tomoaki Minowa (BRRC, AIST) for generously providing mechanochemically treated Japanese cedar and Eucalyptus chips, and Ms. Kanako Negi for excellent technical assistance. We declare that we have no competing financial interests.
References 1) 2) 3) 4) 5)
6)
7)
Hahn-Ha¨gerdal B, Galbe M, Gorwa-Grauslund MF, Lide´n G, and Zacchi G, Trends Biotechnol., 24, 549–556 (2006). Hasunuma T and Kondo A, Process Biochem., 47, 1287–1294 (2012). Olsson L and Nielsen J, Enzyme Microb. Technol., 26, 785–792 (2000). Banat IM and Marchant R, World J. Microbiol. Biotechnol., 11, 304–306 (1995). Nonklang S, Abdel-Banat BM, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, and Akada R, Appl. Environ. Microbiol., 74, 7514–7521 (2008). Matsushika A, Watanabe S, Kodaki T, Makino K, Inoue H, Murakami K, Takimura O, and Sawayama S, Appl. Microbiol. Biotechnol., 81, 243–255 (2008). Hong J, Wang Y, Kumagai H, and Tamaki H, J. Biotechnol., 130, 114–123 (2007).
1510 8) 9) 10)
11) 12) 13)
T. GOSHIMA et al. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, and Kumar S, Mol. Biol. Evol., 28, 2731–2739 (2011). Matsushika A, Inoue H, Murakami K, Takimura O, and Sawayama S, Bioresour. Technol., 100, 2392–2398 (2009). Endo T, Tanaka N, Sakai M, Teramoto Y, and Lee SH, The Third Biomass-Asia Workshop, Tokyo and Tsukuba, November 15–17, 2006, p. 105. Inoue H, Yano S, Endo T, Sasaki T, and Sawayama S, Biotechnol. Biofuels, 1, 2 (2008). da Silva AS, Inoue H, Endo T, Yano S, and Bon EP, Bioresour. Technol., 101, 7402–7409 (2010). Rodrussamee N, Lertwattanasakul N, Hirata K, Suprayogi, Limtong S, Kosaka T, and Yamada M, Appl. Microbiol. Biotechnol., 90, 1573–1586 (2011).
14) 15) 16) 17) 18) 19) 20)
Lin Y, Zhang W, Li C, Sakakibara K, Tanaka S, and Kong H, Biomass Bioenergy, 47, 395–401 (2012). Ballesteros M, Oliva JM, Negro MJ, Manzanares P, and Ballesteros I, Process Biochem., 39, 1843–1848 (2004). Pessani NK, Atiyeh HK, Wilkins MR, Bellmer DD, and Banat IM, Bioresour. Techonol., 102, 10618–10624 (2011). Matsushika A, Inoue H, Kodaki T, and Sawayama S, Appl. Microbiol. Biotechnol., 84, 37–53 (2009). Wang R, Zhang L, Wang D, Gao X, and Hong J, J. Ind. Microbiol. Biotechnol., 38, 1739–1746 (2011). Zhang B, Zhang L, Wang D, Gao X, and Hong J, J. Ind. Microbiol. Biotechnol., 38, 2001–2010 (2011). Li L, Zhang L, Whang D, Gao X, Tamaki H, Kumagai H, and Hong J, Mol. Biotechnol., 53, 159–169 (2013).
