Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1161-1
ORIGINAL PAPER
Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17 Jae Hyung Kim • Jayoung Ryu • In Young Huh • Soon-Kwang Hong • Hyun Ah Kang • Yong Keun Chang
Received: 8 December 2013 / Accepted: 19 February 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A wild-type yeast strain with a good galactoseutilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were preJ. H. Kim J. Ryu I. Y. Huh Y. K. Chang (&) Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea e-mail: [email protected] Present Address: J. Ryu SK Innovation Co., 325, Exporo, Yuseong-gu, Daejeon 305-712, Korea S.-K. Hong Division of Bioscience and Bioinformatics, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido 449-728, Korea H. A. Kang Department of Life Sciences, Chung Ang University, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea
cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L. Keywords Saccharomyces cerevisiae Galactose Ethanol production Fed-batch culture
Introduction The inevitable depletion of fossil fuel reserves causes a great concern of how to satisfy the continuing demand for fuel. In addition, burning of fossil fuel generates carbon dioxide, a major greenhouse gas causing global warming. These problems have been the major concerns for the past decades, stressing the need to find alternate sources that are environmental friendly, sustainable, and renewable. One of the alternatives is biofuel, more specifically bioethanol produced by a microorganism such as Saccharomyces cerevisiae [1]. This particular yeast strain has long been studied due to its good capability to produce ethanol through fermentation. The major carbon sources have been glucose and some disaccharides driven from starch (the first-generation biomass) or cellulosic biomass (the second-generation biomass). Recently, marine algae such as macroalgae (seaweed) and microalgae are drawing attention as the third-generation biomass, as their cultivation on non-arable land requires no encroachment on cropland for food production, and their low lignocellulose content could minimize
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the effort and cost required for pretreatment. Compared with other types of biomass, marine algae are growing very fast (4–6 times of harvest per year is possible in subtropic regions) and easy to cultivate in sea water without using expensive equipments. In addition, the amount of annual CO2 absorption ability of marine algae is 36.7 tons per ha, which is 5–7 times higher than that of wood-based biomass. Among several different types of marine algae, red algae have the highest carbohydrate content, which can be easily converted to biochemicals [2, 3]. The major portion (50–70 %) of red algae is composed of galactan. Red algal galactan, commonly called agar is composed of galactose-based polysaccharides [4, 5]. Galactose can be produced by the hydrolysis of galactan using an acid catalyst or an enzyme. In the case of acid hydrolysis, 5-Hydroxymethylfurfural (5HMF) and levulinic acid (LA) are formed as byproducts [6, 7]. These two compounds are known to inhibit microbial activity of yeast in ethanol fermentation and thus to be removed from the hydrolysate to a non-inhibitory level before fermentation [8–10]. Recently, many research groups reported bioethanol production from galactose as the main carbon source, which had been driven from red algae such as Gelidium amansii [11, 12], Gracilaria verrucosa [13] and Gelidium corneum [14] by various yeast strains [15]. However, unfortunately, the ethanol yield and productivity were found to be significantly lower than those from glucose [16]. Especially, in S. cerevisiae, galactose utilization markedly lags behind glucose consumption due to catabolite repression by glucose [17]. This yeast strain consumes glucose and galactose sequentially showing a diauxic growth pattern, which results in reduced ethanol productivity [18, 19]. A number of studies for the enhancement of galactose utilization by yeast strains have been done. Efforts to improve the performance of S. cerevisiae through metabolic engineering were exerted with no satisfactory results [20–22]. New S. cerevisiae strains with an improved galactose-utilizing ability were screened [23, 24]. However, their efforts were confined to the characterization of the screened strains in the level of flask culture with no performance test in a bioreactor level. In this study, a new strain of S. cerevisiae with a good galactose utilization capability even in the presence of glucose was screened. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. Effects of the carbon source in the seed culture medium on galactose consumption and ethanol production were also investigated. Finally, the inhibitory effects of 5-HMF and LA on cell growth and ethanol production were assessed.
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Materials and methods Strain A yeast strain very efficient in utilizing galactose to produce ethanol was newly isolated from the soil. One gram of soil sample was suspended in 99 mL of saline solution. The sample solution was then diluted by 1 9 107, spread on YPD agar (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L agar) plates and incubated at 30 °C for 3 days. Hundreds of single colonies were established and each of them was cultured in a YPG medium containing 25 g/L galactose instead of 20 g/L glucose for 60 h at 30 °C. One of the strains that produced the greatest amount of ethanol was selected. The selected strain was then identified to be a S. cerevisiae strain by 18s rRNA sequencing. The cell stocks were preserved at -72 °C in 20 % (v/v) glycerol. A small portion of glycerol stock was streaked on freshly prepared YPD agar plates, incubated to grow at 30 °C for 2 days and then stored at 4 °C on the plates. All chemicals were purchased from Sigma-Aldrich Co. (USA) and Difco (USA). Flask cultures For seed culture, a single colony was inoculated into 50 mL Erlenmeyer flask containing 10 mL of a YPD medium containing 20 g/L glucose or a YPG medium containing 20 g/L galactose and cultured at 30 °C and 200 rpm. The seed culture was transferred into 100 mL of YP media with various concentrations of glucose and/or galactose in a 500 mL Erlenmeyer flask. The culture OD600 after the inoculation was 0.2 or 0.5, depending on the sugar concentration in the medium. All the batch cultures were incubated at 30 °C and 200 rpm. Experiments were performed in triplicate. For toxicity test, 0–5 g/L of 5-HMF and 0–10 g/L of LA were added to YPG medium. The initial medium pH was adjusted at 5.5 by ammonia solution. Fed-batch cultures in bioreactor To investigate the performance of the screened strain at an elevated level of ethanol concentration, it was necessary to accumulate ethanol to a high level. For this purpose, fed-batch culture was conducted in a 5.0 L jar fermentor (BioCNS, Korea). The temperature, agitation speed and aeration were controlled at 30 °C, 200 rpm and 1 mL/min, respectively. For the first seed culture, a single colony taken from the stock was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium with 20 g/L glucose and incubated at 30 °C and 200 rpm for 12 h. The culture was transferred into
Bioprocess Biosyst Eng
200 mL of YPD medium for second seed culture and incubated at 30 °C and 200 rpm for 14–16 h. The second seed culture was transferred into the bioreactor. The initial OD600 was 1.0. The fermentation medium consisted of 7.5 g/L yeast extract, 7.5 g/L peptone, 6.0 g/L (NH4)2SO4, 3.0 g/L KH2PO4, 5.0 g/L MgSO47H2O, and 20 g/L galactose (pH 5.5). Initially the bioreactor was operated in batch mode. When the galactose concentration became below 10 g/L, feeding of a concentrated galactose solution (500 g/L) was started. Thereafter intermittent feeding was performed to maintain the galactose concentration in the range of 10–50 g/L. Analytical methods Yeast cell growth was monitored by measuring OD600. The correlation between the cell concentration in g DCW/L and OD600 is: Cell concentration ðg=LÞ ¼ 0:48 OD600 0:06 For dry cell weight measurement, centrifuged cell pallet was washed twice in distilled water, transferred to a preweighed plastic dish, dried in an oven at 80 °C for 24 h, and cooled to the room temperature. The concentration of ethanol was determined using a YSI 2700 Select Biochemistry Analyzer (YSI, Ohio, USA). Glucose and galactose concentrations were analyzed using a high-performance liquid chromatograph (HPLC) (Waters, Milford, USA) equipped with an Aminex HPX87P column (Bio-Rad, Hercules, USA) maintained at 85 °C, and an evaporative light scattering detector (ELSD;
Sedex 75, Sedere, France). Deionized water was used as the eluent at a flow rate of 0.6 mL/min.
Results and discussion Identification of the isolated strain The isolated strain was identified by 18s rRNA analysis using an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, USA), and its phylogenic tree (Fig. 1) was constructed at the Korean Culture Center of Microorganisms (KCCM) (Seoul, Korea). The 18S rRNA sequence of the isolate was very similar to that of S. cerevisiae Z75578 with a sequence homology of 99.5 %, which was calculated based on Gene Bank Data homology search results and phylogenetic analysis. The strain was named as S. cerevisiae KL17 and deposited in KCCM under a deposit No. of KFCC11493P. Cell growth and ethanol production in flask cultures YP media with 20 g/L glucose, 20 g/L galactose, 10 g/L glucose ? 10 g/L galactose, 50 g/L galactose or 100 g/L galactose were used to investigate the basic growth behavior and ethanol production capability of S. cerevisiae KL17 (Fig. 2; Table 1). When the sugar concentration was 20 g/L (Fig. 2a, b), the maximum ethanol concentrations in both cases of glucose and galactose were similar to each other (8.8 and 8.9 g/L, respectively), with an ethanol yield of 0.44 g/g. However,
Fig. 1 Phylogenetic tree of S. cerevisiae KL17 based on 18s rRNA gene sequence
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Fig. 2 Cell growth, galactose and/or glucose consumption and ethanol production of S. cerevisiae KL17 when glucose was used in the seed culture: a 20 g/L glucose, b 20 g/L galactose, c 10 g/L glucose ? 10 g/L galactose, d 50 g/L galactose and e 100 g/L galactose
the sugar consumption rate and ethanol productivity obtained with galactose (2.20 and 0.63 g/L/h, respectively) were significantly lower than those with glucose (3.33 and 0.98 g/L/h, respectively). Such low rate of galactose consumption compared with glucose can be explained by more complicated metabolic pathway of galactose than that of glucose. Galactose metabolism requires more enzymatic
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steps (Leloir pathway) for galactose to enter the glycolytic pathway than glucose metabolism [25, 26]. Although S. cerevisiae KL17 showed somewhat lower sugar consumption rate and ethanol productivity with galactose than those with glucose, such performance with galactose is considered much better than those of most other yeast strains reported to date [20–24]. Bro et al. [20] reported that 15 g/L galactose
Bioprocess Biosyst Eng Table 1 Results of flask cultures: seed culture on glucose
Media
Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
Sugar consumption rate (g/L/h)
Ethanol productivity (g/L/h)
20 g/L glucose
6.22 ± 0.04
8.78 ± 0.00
0.44
3.33 ± 0.00
0.98 ± 0.00
20 g/L galactose
7.83 ± 0.14
8.88 ± 0.40
0.44
2.20 ± 0.00
0.63 ± 0.02
10 g/L glucose ? 10 g/L galactose 50 g/L galactose
7.65 ± 0.09
9.43 ± 0.01
0.47
2.20 ± 0.00
1.05 ± 0.00
15.07 ± 0.99
19.60 ± 0.60
0.39
3.31 ± 0.02
1.15 ± 0.03
100 g/L galactose
27.45 ± 1.40
41.47 ± 0.71
0.41
4.79 ± 0.05
2.07 ± 0.03
was completely consumed in 30 h by a genetically engineered S. cerevisiae, implying that the galactose consumption rate was only about 0.5 g/L/h. Kim et al. [23] reported that 30 g/L galactose was consumed in 12 h by a wild-type S. cerevisiae strain they had screened. Its galactose consumption rate and ethanol production rate were about 2.5 and 0.67 g/L/h, respectively, being comparable to those of S. cerevisiae KL17. However, its ethanol yield of 0.36–0.38 was somewhat lower than that of S. cerevisiae KL17. Another S. cerevisiae strain was reported to consume galactose at a very high rate of about 5 g/L/h. Its ethanol yield ranged 0.36–0.38 [24]. Unfortunately, no data have been reported about the performances of above-mentioned three strains at a bioreactor level. Galactose-utilizing S. cerevisiae strains in general are known to show a diauxic lag period when galactose exists together with glucose in the medium [18]. However, as shown in Fig. 2c for the case with 10 g/L glucose and 10 g/L galactose, S. cerevisiae KL17 could utilize both glucose and galactose, simultaneously although in the very beginning the galactose consumption rate was observed to be lower than that of glucose. Such substrate uptake nature of S. cerevisiae KL17 is advantageous in terms of shortening fermentation time and thus enhancing the ethanol productivity when glucose for some reason is contained in the galactose medium. One good example is an acid hydrolysate of red algae, which contains galactose as the main sugar component with a relatively low concentration of glucose [6, 12, 14]. Another point to notice was that glucose and galactose when mixed together showed a synergic effect of increasing ethanol yield although no plausible explanation was available. To investigate the effects of galactose concentration, flask cultures were carried out at galactose concentrations of 50 and 100 g/L (Fig. 2d, e; Table 1). As expected, the galactose consumption rate and ethanol productivity increased as the galactose concentration was increased. In the case of galactose consumption rate, for example, it increased 2.2-fold as the galactose concentration was increased 5-fold, from 20 to 100 g/L. However, the ethanol yield slightly decreased from 0.44 to about 0.4 as the galactose concentration was increased from 20 to
over 50 g/L, probably due to the inhibitory effect of high ethanol concentration, as well known as product inhibition in ethanol fermentation by yeast. Fed-batch fermentation for ethanol production Fed-batch fermentation was performed in a bioreactor to assess the performance of S. cerevisiae KL17 at an elevated ethanol concentration as commonly the case of commercial production. After the initial period of batch cultivation in which the galactose concentration changed from 20 to below 10 g/L, it was maintained in the range of 10–50 g/L by intermittent feeding of a concentrated galactose solution (500 g/L). Figure 3 shows time profiles of cell, galactose and ethanol concentrations. The maximum ethanol concentration was about 95 g/L at 31.5 h with the final culture volume of 3.5 L, with a volumetric ethanol productivity of 3.03 g/L/h. The total amount of galactose consumed during this period was 856.3 g and the ethanol yield was 0.39 g/g. By performing fed-batch fermentation, the ethanol titer could be increased to about 95 g/L from about 41 g/L in the batch culture with 100 g/L galactose, and the ethanol productivity was increased 1.5-fold. However, the ethanol yield showed practically no difference from that of batch culture. As the ethanol concentration increased over 70 g/L cell growth and ethanol biosynthesis started to be suppressed. They became negligible when the ethanol concentration was higher than 95 g/L. Effects of seed culture medium As shown in Fig. 2d, a significant time lag of about 6 h was observed in the beginning in both cell growth and ethanol production in YPG medium (containing galactose) when the strain had been pre-cultured in YPD medium (containing glucose). This implied that the cells already acclimated to glucose in the seed culture required time to adapt to the new galactose abundant environment [24]. As an effort to shorten the length of time lag and thus to enhance the productivity, the carbon source in the seed was changed to galactose from glucose.
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As shown in Fig. 4 and Table 2, when galactose was used in the seed, galactose in the main culture medium started to be consumed from the very beginning with no significant time lag. As a consequence, 50 g/L galactose was completely consumed in about 11 h, while it took about 15 h when cells were pre-cultured on glucose. A less amount of cells, but more amount of ethanol was formed
than in the case of glucose in the seed. This resulted in improved substrate utilization efficiency and thus ethanol yield as clearly shown in Table 2, which can be a significant advantage in industrial point of view. Another round of fed-batch fermentation was carried out to demonstrate the effects of replacing glucose with galactose in the seed culture, on a bioreactor level. As summarized in Table 3, ethanol concentration reached its maximum value of 97 g/L at 28 h, resulting in a significantly improved ethanol productivity (3.46 g/L/h) compared with that (3.03 g/L/h) in the case when glucose was used in the seed culture. A slightly higher ethanol yield and lower cellular yield were obtained, which was consistent with the results of batch cultures in flask (Table 2). Inhibitory effects of 5-HMF and LA
Fig. 3 Cell growth, galactose consumption and ethanol production of S. cerevisiae KL17 in fed-batch fermentation
Fig. 4 Galactose consumption and ethanol production of S. cerevisiae KL17 when galactose was used in the seed culture: 50 g/L galactose Table 2 Effects of seed culture medium: 50 g/L galactose Seed culture medium
Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
YP ? Glucose
15.1 ± 0.99
19.6 ± 0.60
0.39
YP ? Galactose
12.5 ± 0.24
20.9 ± 0.42
0.42
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As mentioned earlier, crude galactose solution produced from acid hydrolysis of galactan contains 5-HMF and LA as major byproducts, which are known to be inhibitory to the microbial activity of yeast. Table 4 shows adverse effects of 5-HMF and LA on cell growth and ethanol production of S. cerevisiae KL17. Even when the 5-HMF concentration was only 1.0 g/L both cell growth and ethanol were considerably retarded. Over 2 g/L of 5-HMF, they were severely suppressed. Such result with 5-HMF was in consistent with those previously observed by other groups [8, 27, 28]. LA showed much milder inhibitory effects than 5-HMF. Only 6 % of decrease in ethanol production was observed at 10 g/L LA. Thus, it is rational that the detoxification step prior to fermentation should be focused on the removal of 5-HMF only. The detoxification was accomplished using nanofiltration as proposed by our group [29]. In this process, 5-HMF is more easily removed than LA. For the effective removal of LA to a low level, the nanofiltration needs to be operated in a diafiltration mode at the cost of additional efforts. Another negative consequence of such approach is the dilution of the crude galactose solution. However, when LA is no longer a problem as in this study with S. cerevisiae KL17, a simple nanofiltration process with no diafiltration would suffice for detoxification.
Conclusions A wild-type yeast strain highly efficient in producing ethanol from galactose was isolated from the soil and named S. cerevisiae KL17. Its performance was demonstrated on a bioreactor scale in a fed-batch mode with galactose as the main carbon source. A high ethanol productivity of 3.03 g/ L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were precultured on glucose. When the cells were pre-cultured on
Bioprocess Biosyst Eng Table 3 Effects of seed culture medium in fed-batch fermentation Seed culture medium
Cellmax (g/L)
Galactose consumed (g)
Ethanolmax (g/L)
Fermentation time at EtOHmax (h)
Ethanol produced (g)
Galactose consumption rate (g/h)
Ethanol yield (g/g)
Ethanol productivity (g/L/h)
YP ? Glucose
34.5
856.3
95.4
31.5
337.5
27.2
0.39
3.03
YP ? Galactose
30.2
850.8
96.9
28.0
357.4
30.4
0.42
3.46
Table 4 Inhibitory effects of 5-HMF and LA Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
0 (control)
7.70 ± 1.37
8.23 ± 0.29
0.41
–
1 2
7.50 ± 0.09 3.46 ± 0.33
7.32 ± 0.13 4.04 ± 0.11
0.37 0.2
11 51
5
0.78 ± 0.01
1.24 ± 0.04
0.06
85
6.99 ± 0.13
8.35 ± 0.32
0.42
–
5
6.55 ± 0.57
8.11 ± 0.25
0.41
3
10
5.20 ± 0.18
7.84 ± 0.10
0.39
6
Components
Inhibition based on ethanol (%)a
5-HMF (g/L)
Levulinic acid (g/L) 0 (control)
a
Inhibition (%) = [1 - (Ethanolmax/Ethanolmax of control)] 9 100
galactose instead of glucose, fermentation time could be reduced significantly resulting in a significantly improved ethanol productivity of 3.46 g/L/h. However, only slightly increased ethanol concentration and ethanol yield were obtained. The assessment of inhibitory effects of 5-HMF and LA on the activity of S. cerevisiae KL17 clearly showed that only 5-HMF severely inhibited cell growth and ethanol biosynthesis while LA was rather benign. Acknowledgments This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology (ABC-2013-064099).
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ORIGINAL PAPER
Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17 Jae Hyung Kim • Jayoung Ryu • In Young Huh • Soon-Kwang Hong • Hyun Ah Kang • Yong Keun Chang
Received: 8 December 2013 / Accepted: 19 February 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A wild-type yeast strain with a good galactoseutilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were preJ. H. Kim J. Ryu I. Y. Huh Y. K. Chang (&) Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea e-mail: [email protected] Present Address: J. Ryu SK Innovation Co., 325, Exporo, Yuseong-gu, Daejeon 305-712, Korea S.-K. Hong Division of Bioscience and Bioinformatics, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido 449-728, Korea H. A. Kang Department of Life Sciences, Chung Ang University, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea
cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L. Keywords Saccharomyces cerevisiae Galactose Ethanol production Fed-batch culture
Introduction The inevitable depletion of fossil fuel reserves causes a great concern of how to satisfy the continuing demand for fuel. In addition, burning of fossil fuel generates carbon dioxide, a major greenhouse gas causing global warming. These problems have been the major concerns for the past decades, stressing the need to find alternate sources that are environmental friendly, sustainable, and renewable. One of the alternatives is biofuel, more specifically bioethanol produced by a microorganism such as Saccharomyces cerevisiae [1]. This particular yeast strain has long been studied due to its good capability to produce ethanol through fermentation. The major carbon sources have been glucose and some disaccharides driven from starch (the first-generation biomass) or cellulosic biomass (the second-generation biomass). Recently, marine algae such as macroalgae (seaweed) and microalgae are drawing attention as the third-generation biomass, as their cultivation on non-arable land requires no encroachment on cropland for food production, and their low lignocellulose content could minimize
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the effort and cost required for pretreatment. Compared with other types of biomass, marine algae are growing very fast (4–6 times of harvest per year is possible in subtropic regions) and easy to cultivate in sea water without using expensive equipments. In addition, the amount of annual CO2 absorption ability of marine algae is 36.7 tons per ha, which is 5–7 times higher than that of wood-based biomass. Among several different types of marine algae, red algae have the highest carbohydrate content, which can be easily converted to biochemicals [2, 3]. The major portion (50–70 %) of red algae is composed of galactan. Red algal galactan, commonly called agar is composed of galactose-based polysaccharides [4, 5]. Galactose can be produced by the hydrolysis of galactan using an acid catalyst or an enzyme. In the case of acid hydrolysis, 5-Hydroxymethylfurfural (5HMF) and levulinic acid (LA) are formed as byproducts [6, 7]. These two compounds are known to inhibit microbial activity of yeast in ethanol fermentation and thus to be removed from the hydrolysate to a non-inhibitory level before fermentation [8–10]. Recently, many research groups reported bioethanol production from galactose as the main carbon source, which had been driven from red algae such as Gelidium amansii [11, 12], Gracilaria verrucosa [13] and Gelidium corneum [14] by various yeast strains [15]. However, unfortunately, the ethanol yield and productivity were found to be significantly lower than those from glucose [16]. Especially, in S. cerevisiae, galactose utilization markedly lags behind glucose consumption due to catabolite repression by glucose [17]. This yeast strain consumes glucose and galactose sequentially showing a diauxic growth pattern, which results in reduced ethanol productivity [18, 19]. A number of studies for the enhancement of galactose utilization by yeast strains have been done. Efforts to improve the performance of S. cerevisiae through metabolic engineering were exerted with no satisfactory results [20–22]. New S. cerevisiae strains with an improved galactose-utilizing ability were screened [23, 24]. However, their efforts were confined to the characterization of the screened strains in the level of flask culture with no performance test in a bioreactor level. In this study, a new strain of S. cerevisiae with a good galactose utilization capability even in the presence of glucose was screened. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. Effects of the carbon source in the seed culture medium on galactose consumption and ethanol production were also investigated. Finally, the inhibitory effects of 5-HMF and LA on cell growth and ethanol production were assessed.
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Materials and methods Strain A yeast strain very efficient in utilizing galactose to produce ethanol was newly isolated from the soil. One gram of soil sample was suspended in 99 mL of saline solution. The sample solution was then diluted by 1 9 107, spread on YPD agar (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L agar) plates and incubated at 30 °C for 3 days. Hundreds of single colonies were established and each of them was cultured in a YPG medium containing 25 g/L galactose instead of 20 g/L glucose for 60 h at 30 °C. One of the strains that produced the greatest amount of ethanol was selected. The selected strain was then identified to be a S. cerevisiae strain by 18s rRNA sequencing. The cell stocks were preserved at -72 °C in 20 % (v/v) glycerol. A small portion of glycerol stock was streaked on freshly prepared YPD agar plates, incubated to grow at 30 °C for 2 days and then stored at 4 °C on the plates. All chemicals were purchased from Sigma-Aldrich Co. (USA) and Difco (USA). Flask cultures For seed culture, a single colony was inoculated into 50 mL Erlenmeyer flask containing 10 mL of a YPD medium containing 20 g/L glucose or a YPG medium containing 20 g/L galactose and cultured at 30 °C and 200 rpm. The seed culture was transferred into 100 mL of YP media with various concentrations of glucose and/or galactose in a 500 mL Erlenmeyer flask. The culture OD600 after the inoculation was 0.2 or 0.5, depending on the sugar concentration in the medium. All the batch cultures were incubated at 30 °C and 200 rpm. Experiments were performed in triplicate. For toxicity test, 0–5 g/L of 5-HMF and 0–10 g/L of LA were added to YPG medium. The initial medium pH was adjusted at 5.5 by ammonia solution. Fed-batch cultures in bioreactor To investigate the performance of the screened strain at an elevated level of ethanol concentration, it was necessary to accumulate ethanol to a high level. For this purpose, fed-batch culture was conducted in a 5.0 L jar fermentor (BioCNS, Korea). The temperature, agitation speed and aeration were controlled at 30 °C, 200 rpm and 1 mL/min, respectively. For the first seed culture, a single colony taken from the stock was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of YPD medium with 20 g/L glucose and incubated at 30 °C and 200 rpm for 12 h. The culture was transferred into
Bioprocess Biosyst Eng
200 mL of YPD medium for second seed culture and incubated at 30 °C and 200 rpm for 14–16 h. The second seed culture was transferred into the bioreactor. The initial OD600 was 1.0. The fermentation medium consisted of 7.5 g/L yeast extract, 7.5 g/L peptone, 6.0 g/L (NH4)2SO4, 3.0 g/L KH2PO4, 5.0 g/L MgSO47H2O, and 20 g/L galactose (pH 5.5). Initially the bioreactor was operated in batch mode. When the galactose concentration became below 10 g/L, feeding of a concentrated galactose solution (500 g/L) was started. Thereafter intermittent feeding was performed to maintain the galactose concentration in the range of 10–50 g/L. Analytical methods Yeast cell growth was monitored by measuring OD600. The correlation between the cell concentration in g DCW/L and OD600 is: Cell concentration ðg=LÞ ¼ 0:48 OD600 0:06 For dry cell weight measurement, centrifuged cell pallet was washed twice in distilled water, transferred to a preweighed plastic dish, dried in an oven at 80 °C for 24 h, and cooled to the room temperature. The concentration of ethanol was determined using a YSI 2700 Select Biochemistry Analyzer (YSI, Ohio, USA). Glucose and galactose concentrations were analyzed using a high-performance liquid chromatograph (HPLC) (Waters, Milford, USA) equipped with an Aminex HPX87P column (Bio-Rad, Hercules, USA) maintained at 85 °C, and an evaporative light scattering detector (ELSD;
Sedex 75, Sedere, France). Deionized water was used as the eluent at a flow rate of 0.6 mL/min.
Results and discussion Identification of the isolated strain The isolated strain was identified by 18s rRNA analysis using an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, USA), and its phylogenic tree (Fig. 1) was constructed at the Korean Culture Center of Microorganisms (KCCM) (Seoul, Korea). The 18S rRNA sequence of the isolate was very similar to that of S. cerevisiae Z75578 with a sequence homology of 99.5 %, which was calculated based on Gene Bank Data homology search results and phylogenetic analysis. The strain was named as S. cerevisiae KL17 and deposited in KCCM under a deposit No. of KFCC11493P. Cell growth and ethanol production in flask cultures YP media with 20 g/L glucose, 20 g/L galactose, 10 g/L glucose ? 10 g/L galactose, 50 g/L galactose or 100 g/L galactose were used to investigate the basic growth behavior and ethanol production capability of S. cerevisiae KL17 (Fig. 2; Table 1). When the sugar concentration was 20 g/L (Fig. 2a, b), the maximum ethanol concentrations in both cases of glucose and galactose were similar to each other (8.8 and 8.9 g/L, respectively), with an ethanol yield of 0.44 g/g. However,
Fig. 1 Phylogenetic tree of S. cerevisiae KL17 based on 18s rRNA gene sequence
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Fig. 2 Cell growth, galactose and/or glucose consumption and ethanol production of S. cerevisiae KL17 when glucose was used in the seed culture: a 20 g/L glucose, b 20 g/L galactose, c 10 g/L glucose ? 10 g/L galactose, d 50 g/L galactose and e 100 g/L galactose
the sugar consumption rate and ethanol productivity obtained with galactose (2.20 and 0.63 g/L/h, respectively) were significantly lower than those with glucose (3.33 and 0.98 g/L/h, respectively). Such low rate of galactose consumption compared with glucose can be explained by more complicated metabolic pathway of galactose than that of glucose. Galactose metabolism requires more enzymatic
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steps (Leloir pathway) for galactose to enter the glycolytic pathway than glucose metabolism [25, 26]. Although S. cerevisiae KL17 showed somewhat lower sugar consumption rate and ethanol productivity with galactose than those with glucose, such performance with galactose is considered much better than those of most other yeast strains reported to date [20–24]. Bro et al. [20] reported that 15 g/L galactose
Bioprocess Biosyst Eng Table 1 Results of flask cultures: seed culture on glucose
Media
Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
Sugar consumption rate (g/L/h)
Ethanol productivity (g/L/h)
20 g/L glucose
6.22 ± 0.04
8.78 ± 0.00
0.44
3.33 ± 0.00
0.98 ± 0.00
20 g/L galactose
7.83 ± 0.14
8.88 ± 0.40
0.44
2.20 ± 0.00
0.63 ± 0.02
10 g/L glucose ? 10 g/L galactose 50 g/L galactose
7.65 ± 0.09
9.43 ± 0.01
0.47
2.20 ± 0.00
1.05 ± 0.00
15.07 ± 0.99
19.60 ± 0.60
0.39
3.31 ± 0.02
1.15 ± 0.03
100 g/L galactose
27.45 ± 1.40
41.47 ± 0.71
0.41
4.79 ± 0.05
2.07 ± 0.03
was completely consumed in 30 h by a genetically engineered S. cerevisiae, implying that the galactose consumption rate was only about 0.5 g/L/h. Kim et al. [23] reported that 30 g/L galactose was consumed in 12 h by a wild-type S. cerevisiae strain they had screened. Its galactose consumption rate and ethanol production rate were about 2.5 and 0.67 g/L/h, respectively, being comparable to those of S. cerevisiae KL17. However, its ethanol yield of 0.36–0.38 was somewhat lower than that of S. cerevisiae KL17. Another S. cerevisiae strain was reported to consume galactose at a very high rate of about 5 g/L/h. Its ethanol yield ranged 0.36–0.38 [24]. Unfortunately, no data have been reported about the performances of above-mentioned three strains at a bioreactor level. Galactose-utilizing S. cerevisiae strains in general are known to show a diauxic lag period when galactose exists together with glucose in the medium [18]. However, as shown in Fig. 2c for the case with 10 g/L glucose and 10 g/L galactose, S. cerevisiae KL17 could utilize both glucose and galactose, simultaneously although in the very beginning the galactose consumption rate was observed to be lower than that of glucose. Such substrate uptake nature of S. cerevisiae KL17 is advantageous in terms of shortening fermentation time and thus enhancing the ethanol productivity when glucose for some reason is contained in the galactose medium. One good example is an acid hydrolysate of red algae, which contains galactose as the main sugar component with a relatively low concentration of glucose [6, 12, 14]. Another point to notice was that glucose and galactose when mixed together showed a synergic effect of increasing ethanol yield although no plausible explanation was available. To investigate the effects of galactose concentration, flask cultures were carried out at galactose concentrations of 50 and 100 g/L (Fig. 2d, e; Table 1). As expected, the galactose consumption rate and ethanol productivity increased as the galactose concentration was increased. In the case of galactose consumption rate, for example, it increased 2.2-fold as the galactose concentration was increased 5-fold, from 20 to 100 g/L. However, the ethanol yield slightly decreased from 0.44 to about 0.4 as the galactose concentration was increased from 20 to
over 50 g/L, probably due to the inhibitory effect of high ethanol concentration, as well known as product inhibition in ethanol fermentation by yeast. Fed-batch fermentation for ethanol production Fed-batch fermentation was performed in a bioreactor to assess the performance of S. cerevisiae KL17 at an elevated ethanol concentration as commonly the case of commercial production. After the initial period of batch cultivation in which the galactose concentration changed from 20 to below 10 g/L, it was maintained in the range of 10–50 g/L by intermittent feeding of a concentrated galactose solution (500 g/L). Figure 3 shows time profiles of cell, galactose and ethanol concentrations. The maximum ethanol concentration was about 95 g/L at 31.5 h with the final culture volume of 3.5 L, with a volumetric ethanol productivity of 3.03 g/L/h. The total amount of galactose consumed during this period was 856.3 g and the ethanol yield was 0.39 g/g. By performing fed-batch fermentation, the ethanol titer could be increased to about 95 g/L from about 41 g/L in the batch culture with 100 g/L galactose, and the ethanol productivity was increased 1.5-fold. However, the ethanol yield showed practically no difference from that of batch culture. As the ethanol concentration increased over 70 g/L cell growth and ethanol biosynthesis started to be suppressed. They became negligible when the ethanol concentration was higher than 95 g/L. Effects of seed culture medium As shown in Fig. 2d, a significant time lag of about 6 h was observed in the beginning in both cell growth and ethanol production in YPG medium (containing galactose) when the strain had been pre-cultured in YPD medium (containing glucose). This implied that the cells already acclimated to glucose in the seed culture required time to adapt to the new galactose abundant environment [24]. As an effort to shorten the length of time lag and thus to enhance the productivity, the carbon source in the seed was changed to galactose from glucose.
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As shown in Fig. 4 and Table 2, when galactose was used in the seed, galactose in the main culture medium started to be consumed from the very beginning with no significant time lag. As a consequence, 50 g/L galactose was completely consumed in about 11 h, while it took about 15 h when cells were pre-cultured on glucose. A less amount of cells, but more amount of ethanol was formed
than in the case of glucose in the seed. This resulted in improved substrate utilization efficiency and thus ethanol yield as clearly shown in Table 2, which can be a significant advantage in industrial point of view. Another round of fed-batch fermentation was carried out to demonstrate the effects of replacing glucose with galactose in the seed culture, on a bioreactor level. As summarized in Table 3, ethanol concentration reached its maximum value of 97 g/L at 28 h, resulting in a significantly improved ethanol productivity (3.46 g/L/h) compared with that (3.03 g/L/h) in the case when glucose was used in the seed culture. A slightly higher ethanol yield and lower cellular yield were obtained, which was consistent with the results of batch cultures in flask (Table 2). Inhibitory effects of 5-HMF and LA
Fig. 3 Cell growth, galactose consumption and ethanol production of S. cerevisiae KL17 in fed-batch fermentation
Fig. 4 Galactose consumption and ethanol production of S. cerevisiae KL17 when galactose was used in the seed culture: 50 g/L galactose Table 2 Effects of seed culture medium: 50 g/L galactose Seed culture medium
Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
YP ? Glucose
15.1 ± 0.99
19.6 ± 0.60
0.39
YP ? Galactose
12.5 ± 0.24
20.9 ± 0.42
0.42
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As mentioned earlier, crude galactose solution produced from acid hydrolysis of galactan contains 5-HMF and LA as major byproducts, which are known to be inhibitory to the microbial activity of yeast. Table 4 shows adverse effects of 5-HMF and LA on cell growth and ethanol production of S. cerevisiae KL17. Even when the 5-HMF concentration was only 1.0 g/L both cell growth and ethanol were considerably retarded. Over 2 g/L of 5-HMF, they were severely suppressed. Such result with 5-HMF was in consistent with those previously observed by other groups [8, 27, 28]. LA showed much milder inhibitory effects than 5-HMF. Only 6 % of decrease in ethanol production was observed at 10 g/L LA. Thus, it is rational that the detoxification step prior to fermentation should be focused on the removal of 5-HMF only. The detoxification was accomplished using nanofiltration as proposed by our group [29]. In this process, 5-HMF is more easily removed than LA. For the effective removal of LA to a low level, the nanofiltration needs to be operated in a diafiltration mode at the cost of additional efforts. Another negative consequence of such approach is the dilution of the crude galactose solution. However, when LA is no longer a problem as in this study with S. cerevisiae KL17, a simple nanofiltration process with no diafiltration would suffice for detoxification.
Conclusions A wild-type yeast strain highly efficient in producing ethanol from galactose was isolated from the soil and named S. cerevisiae KL17. Its performance was demonstrated on a bioreactor scale in a fed-batch mode with galactose as the main carbon source. A high ethanol productivity of 3.03 g/ L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were precultured on glucose. When the cells were pre-cultured on
Bioprocess Biosyst Eng Table 3 Effects of seed culture medium in fed-batch fermentation Seed culture medium
Cellmax (g/L)
Galactose consumed (g)
Ethanolmax (g/L)
Fermentation time at EtOHmax (h)
Ethanol produced (g)
Galactose consumption rate (g/h)
Ethanol yield (g/g)
Ethanol productivity (g/L/h)
YP ? Glucose
34.5
856.3
95.4
31.5
337.5
27.2
0.39
3.03
YP ? Galactose
30.2
850.8
96.9
28.0
357.4
30.4
0.42
3.46
Table 4 Inhibitory effects of 5-HMF and LA Cellmax (g/L)
Ethanolmax (g/L)
Ethanol yield (g/g)
0 (control)
7.70 ± 1.37
8.23 ± 0.29
0.41
–
1 2
7.50 ± 0.09 3.46 ± 0.33
7.32 ± 0.13 4.04 ± 0.11
0.37 0.2
11 51
5
0.78 ± 0.01
1.24 ± 0.04
0.06
85
6.99 ± 0.13
8.35 ± 0.32
0.42
–
5
6.55 ± 0.57
8.11 ± 0.25
0.41
3
10
5.20 ± 0.18
7.84 ± 0.10
0.39
6
Components
Inhibition based on ethanol (%)a
5-HMF (g/L)
Levulinic acid (g/L) 0 (control)
a
Inhibition (%) = [1 - (Ethanolmax/Ethanolmax of control)] 9 100
galactose instead of glucose, fermentation time could be reduced significantly resulting in a significantly improved ethanol productivity of 3.46 g/L/h. However, only slightly increased ethanol concentration and ethanol yield were obtained. The assessment of inhibitory effects of 5-HMF and LA on the activity of S. cerevisiae KL17 clearly showed that only 5-HMF severely inhibited cell growth and ethanol biosynthesis while LA was rather benign. Acknowledgments This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology (ABC-2013-064099).
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