Enhanced xylose fermentation and hydrolysate inhibitor tolerance of Scheffersomyces shehatae for efficient ethanol production from non-detoxified lignocellulosic hydrolysate
© The Author(s) 2016
Received: 23 January 2016
Accepted: 29 June 2016
Published: 11 July 2016
Effective conversion of xylose into ethanol is important for lignocellulosic ethanol production. In the present study, UV-C mutagenesis was used to improve the efficiency of xylose fermentation. The mutated Scheffersomyces shehatae strain TTC79 fermented glucose as efficiently and xylose more efficiently, producing a higher ethanol concentration than the wild-type. A maximum ethanol concentration of 29.04 g/L was produced from 71.31 g/L xylose, which was 58.95 % higher than that of the wild-type. This mutant also displayed significantly improved hydrolysate inhibitors tolerance and increased ethanol production from non-detoxified lignocellulosic hydrolysates. The ethanol yield, productivity and theoretical yield by TTC79 from sugarcane bagasse hydrolysate were 0.46 g/g, 0.20 g/L/h and 90.61 %, respectively, while the corresponding values for the wild-type were 0.20 g/g, 0.04 g/L/h and 39.20 %, respectively. These results demonstrate that S. shehatae TTC79 is a useful non-recombinant strain, combining efficient xylose consumption and high inhibitor tolerance, with potential for application in ethanol production from lignocellulose hydrolysates.
KeywordsEthanol Scheffersomyces shehatae Lignocellulose Hydrolysate inhibitor UV-mutagenesis
With the increased interests in alternative energy, lignocellulosic biomass is attracting considerable attention as a potential low-cost feedstock for ethanol production. Lignocellulosic biomass is mainly composed of cellulose and hemicellulose. Cellulose is a linear polymer of glucose units linked by β-1-4-glycosidic bonds, whereas hemicellulose is a branched chain of pentoses (xylose and arabinose) and hexoses (glucose, mannose and galactose) (Zaldivar et al. 2001).
Xylose is the second most abundant fermentable sugar in lignocellulosic materials after glucose. Efficient conversion of xylose into ethanol is therefore important for yeast strains used in lignocellulosic ethanol production. Saccharomyces cerevisiae is the best-known microorganism used for industrial ethanol fermentation, but this yeast does not naturally ferment pentose sugars to ethanol (Matsushika et al. 2009). Several non-Saccharomyces yeasts, such as Scheffersomyces shehatae (Syn. Candida shehatae), Scheffersomyces stipitis (Syn. Pichia stipitis) and Pachysolen tannophilus, have been found to ferment both glucose and xylose to ethanol and have been investigated for applications in ethanol production (Bajwa et al. 2010; Cheng et al. 2007; Martiniano et al. 2013). S. shehatae is one good candidate for sugar mixture fermentation. It is well known that this yeast is Crabtree negative which requires oxygen for growth and produces ethanol under oxygen limited conditions (Hahn-Hägerdal et al. 2006; Tanimura et al. 2015). Nevertheless, a few strains of this yeast, such as S. shehatae JCM 18690, have been reported as Crabtree positive (Tanimura et al. 2015). S. shehatae showed high performances in terms of yield and productivity using synthetic media (Hickert et al. 2013; Li et al. 2012). However, ethanol production from lignocellulosic residues by S. shehatae and other xylose-fermenting yeasts result in a relatively low ethanol yield and productivity. In addition, these yeasts are also sensitive to breakdown compounds in the hydrolysate, such as weak acids, furan derivatives and phenolic compounds which have inhibitory effects on microbial growth and fermentation (Georgieva et al. 2008; Lohmeier-Vogel et al. 1998; Zhang et al. 2011). Consequently, a considerable amount of research has focused on xylose-fermenting yeasts that show high substrate consumption rates and can yield a large amount of ethanol from lignocellulosic biomass such that it would be beneficial to commercial ethanol production. Johannsen et al. (1985) attempted to generate polyploid strains of S. shehatae by protoplast fusion. Increasing the level of ploidy from the haploid to the diploid, triploid and tetraploid levels of the fusants resulted in improvement in ethanol production rate from xylose. Li et al. (2012) attempted to improve ethanol production of xylose-fermenting S. shehatae ATCC 22984 by UV-mutagenesis. The mutant, Cs3512, showed better fermentation of xylose and mixtures of xylose and glucose. It also showed potential in simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw achieving 77 % of the theoretical yield. Also using UV-mutagenesis, Hughes et al. (2012) obtained mutant of S. stipitis with increased ethanol production and anaerobic growth on lignocellulosic hydrolysate. Pereira et al. (2015) was able to obtain a mutant of S. stipitis adapted to hardwood spent sulfite liquor with improved ethanol yield and tolerance to inhibitors. Huang et al. (2009) also obtained an adapted strain of S. stipitis with increased ethanol production from rice straw hydrolysate and enhanced inhibitor tolerance.
In this study, we attempted to improve the ethanol production ability from xylose of S. shehatae 43CS using UV-mutagenesis followed by selection of mutants having increased ethanol production from xylose using 2,3,5-triphenyltetrazolium chloride (TTC) screening. The selected mutant was characterized and compared with the wild-type, S. shehatae 43CS, for its fermentative ability in both synthetic media and in non-detoxified biomass hydrolysate. Additionally, its ability to tolerate inhibitory compounds in lignocellulosic hydrolysate was also investigated.
Results and discussion
UV-mutagenesis and selection of improved xylose-fermenting mutants
Ethanol production of mutants and the wild-type in YPX medium containing 50 g/L xylose at 30 °C for 48 h
Residual xylose (g/L)
Ethanol yield (% of theoretical yield)1
8.80 ± 0.14c
9.92 ± 0.38b
17.12 ± 0.12a
0.16 ± 0.00d
12.32 ± 0.12b
3.52 ± 0.28c
6.08 ± 0.16d
10.72 ± 0.32a
Fermentation characterization of S. shehatae TTC79 in synthetic medium
Xylose was utilized and fermented to ethanol by TTC79 and the wild-type at a slower rate than glucose (Fig. 1b). This pentose sugar was almost completely consumed by TTC79 within 48 h, while the wild-type consumed only 38.06 g/L of the xylose within 72 h. TTC79 produced ethanol from xylose more rapidly and at a higher yield than the wild-type. The maximum ethanol production of 29.04 g/L was obtained for TTC79 at 48 h and that for the wild-type was 11.92 g/L at 72 h. Naturally, xylose-fermenting yeasts, including S. shehatae, have been reported to ferment xylose to ethanol and xylitol (Buhner and Agbleror 2004; Li et al. 2012). In this study, xylitol accumulation was observed at very low concentration values by TTC79 (2.35 g/L) and the wild-type (<0.20 g/L) at 72 h (data not shown). With regard to xylose consumption and fermentation of TTC79, these results suggested that higher ethanol production and xylitol production by TTC79 was due to increased efficient xylose consumption.
Under the mixture of glucose and xylose, glucose repression on xylose uptake is a very common among xylose-fermenting yeasts (Bajwa et al. 2010; Lebeau et al. 2007). In this study, glucose underwent fast depletion within the first 24 h by TTC79 and the wild-type. Xylose consumption occurred simultaneously to glucose consumption, and then xylose was rapidly consumed after glucose depletion. This pentose sugar was almost completely consumed by TTC79 within 60 h, while the wild-type consumed only 24.78 g/L of the xylose within 72 h. The maximum ethanol production of 39.84 g/L was obtained at 60 h by TTC79, whereas 17.12 g/L ethanol was obtained by the wild-type at 72 h. The xylitol production during fermentation was very low, only 0.85 g/L was observed by TTC79 at 72 h (data not shown). The results in this study clearly demonstrated that TTC79 increased efficient xylose consumption while maintaining high glucose consumption ability, leading to improved ethanol production from the glucose-xylose mixture.
Growth tolerance of S. shehatae TTC79 in the presence of acetic acid, furfural and 5-hydroxymethy furfural (HMF)
Generally, yeast cell growth was inhibited at an acetic acid concentration of around 2.00–5.00 g/L (Bajwa et al. 2009, 2010; Larsson et al. 1999). Furfural and HMF are the inhibitors produced from pentose and hexose sugars degraded during acid hydrolysis. It was found that 0.90–2.00 g/L furfural in hydrolysate was able to reduce fermentation rate and/or stop yeast growth (Agbogbo and Wenger 2007; Bajwa et al. 2010; Huang et al. 2009). HMF concentrations of 0.50 g/L or higher have been reported to inhibit yeast growth (Agbogbo and Wenger 2006; Bajwa et al. 2010). It has been reported that pentose-fermenting yeasts including S. shehatae are susceptible to the inhibitors generated during the diluted acid pretreatment of plant biomass (Huang et al. 2009; Lohmeier-Vogel et al. 1998). According to the cell viability of S. shehatae TTC79 in the presence of individual inhibitors, it was evident that TTC79 exhibited enhanced tolerance to the inhibitors in lignocellulosic hydrolysate compared to the wild-type. Efficient xylose fermentation and tolerance of toxic compounds are polygenic traits arising via complex mechanisms (Demeke et al. 2013; Wang et al. 2014; Zhao and Bai 2009). Improved understanding of the intracellular responses and mechanisms of TTC79 to inhibitory compounds and the synergistic effect of these inhibitors on yeast cell metabolism during lignocellulosic ethanol production will enable superior strains for efficient lignocellulosic ethanol production to be developed.
Fermentation characterization of S. shehatae TTC79 in non-detoxified hydrolysate
Ethanol production by TTC79 and the wild-type from non-detoxified sugarcane bagasse hydrolysates1 at 30 °C
Maximum ethanol concentration (g/L)
12.15 ± 1.57a
2.64 ± 0.09b
Ethanol yield2 (gp/gs)
0.46 ± 0.06a
0.20 ± 0.06b
Theoretical yield3 (%)
90.61 ± 0.58a
39.20 ± 0.51b
Fermentation time4 (h)
Ethanol productivity (g/L/h)
0.20 ± 1.55a
0.04 ± 0.01b
Generally, several wild-type and mutant of xylose-fermenting yeast strains have been reported to ferment xylose with satisfactory yield in detoxified hydrolysates. Martiniano et al. (2013) found ethanol yield, 0.30 g/g, and ethanol productivity, 0.15 g/L/h, from S. shehatae CGS8BY using sugarcane bagasse hydrolysate detoxification by activated charcoal. Cheng et al. (2007) reported that the ethanol yield and ethanol productivity of 0.35 g/g and 0.59 g/L/h using the detoxified sugarcane bagasse hydrolysate by P. tannophilus DW06 DSM3651. Huang et al. (2009) reported the ethanol yield using S. stipitis BCRC21777 and the adapted S. stipitis with detoxified rice straw achieved 0.40 g/g and 0.44 g/g, respectively. However, some studies have reported on the efficient ability of yeast strains to produce ethanol from non-detoxified hydrolysate. Agbogbo et al. (2008) reported the ethanol yields, 0.38–0.42 g/g, from S. stipitis CBS6054 using corn stalk without detoxification. Huang et al. (2009) obtained the adapted strain of S. stipitis with high ethanol yield, 0.44 g/g, by fermenting non-detoxified rice straw hydrolysate. Wan et al. (2012) obtained ethanol yield of 0.43 g/g, corresponding to 85.10 % of the theoretical yield from cocultures of S. cerevisiae Y5 and S. stipitis CBS6054. Although, it is difficult to directly compare the results of ethanol production between different studies, it is still useful to display the competitiveness of this yeast strain, S. shehatae TTC79, for lignocellulosic ethanol production.
Most lignocellulosic biomass feedstock contains a significant amount of xylan that is converted to xylose by hydrolysis. High consumption and fermentation of pentose sugars present in lignocellulosic biomass is an important factor to make ethanol production commercially feasible. In the present study, the increased xylose fermentation yeast strain was successfully obtained through UV-C mutagenesis. The S. shehatae TTC79 mutant exhibited excellent xylose consumption and fermentation both with xylose alone and with sugars mixture. This mutant also showed high resistance to lignocellulosic inhibitors along with high ethanol yield from dilute-acid lignocellulosic hydrolysate without the need for detoxification. These results demonstrate that S. shehatae TTC79 is one of the most efficient non-recombinant strains for lignocellulosic ethanol production described to date.
The stock culture of S. shehatae 43CS from our laboratory culture collection was maintained on YPX agar (10 g/L yeast extract, 20 g/L peptone, 2 g/L xylose) at 4 °C.
UV-C mutagenesis and mutant selection
UV-mutagenesis was carried out according to Thammasittirong et al. (2013) except that yeast cell suspension was spread on YPX medium. Following UV-treatment, the grown colonies were covered with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, St. Louis, USA) agar containing 0.5 g/L xylose, 10 g/L agar and 0.05 g/L TTC (Li et al. 2012). After solidification, the TTC agar-covered plates were incubated at 30 °C for 2 h. The red colonies were selected for xylose fermentation evaluation. The mutant selection experiment was performed in two steps. First, a loopfull of 24 h YPX-grown culture of each mutant was inoculated in 5 mL YPX medium in a test tube containing a Durham tube and incubated at 30 °C for 10 days. Those strains showing high accumulation of CO2 gas in the Durham tubes were selected for screening of mutant strains with high ethanol production ability. YPX medium containing 50 g/L xylose was inoculated with overnight YPX cultures to achieve a cell density of 5 × 105 cells/mL. The cultures in Erlenmeyer flasks plugged with cotton were incubated at 30 °C in a shaking incubator under oxygen limited condition, 100 rpm, for 48 h. The mutant that showed the best xylose fermentation ability was selected for further studies.
Fermentation of sugars in synthetic medium
The selected mutant and wild-type were investigated for their abilities to utilize and ferment glucose (80 g/L) and xylose (80 g/L) individually and 20 g/L glucose/60 g/L xylose mixture. The 24 h pre-cultivated yeast cells in YPX medium were inoculated into 100 mL synthetic medium containing 10 g/L yeast extract, 20 g/L peptone and sugar concentrations as described above in 250 mL Erlenmeyer flasks plugged with cotton. The initial cell concentration was adjusted to cell density of 5 × 105 cells/mL. Fermentations were performed for 72 h at 30 °C in a shaking incubator under oxygen limited condition, 100 rpm. Fermentation samples were withdrawn every 12 h for measurement of cell concentrations, sugar and ethanol analysis. All experiments were performed in three independent experiments.
Determination of inhibitors tolerance
Yeasts were inoculated in 65 mL YPX medium containing 5.25 g/L acetic acid, 1.75 g/L furfural and 1.30 g/L HMF individually to achieve an initial cell density of 1 × 107 cells/mL. The cultures were incubated at 30 °C with shaking at 100 rpm for 72 h. The appropriate dilutions of each culture were taken for measurement of viable cells using a NucleoCounter YC-100 automated cell counter unit (Chemometec, Inc., Allerød, Denmark).
Preparation of sugarcane bagasse hydrolysate by dilute-acid hydrolysis
The sun-dried chopped sugarcane bagasse was milled to a particle size 3–5 mm and dried at 60 °C for 24 h. The oven-dried milled bagasse was soaked in 1 % H2SO4, in a solid–liquid proportion of 1:10, at ambient temperature for 30 min. Acid hydrolysis was performed at 121 °C for 30 min. The hydrolysate was separated from the bagasse solid fraction by filtration. The hydrolysate was neutralized with CaO to pH 5.5 and then centrifuged at 5000×g for 5 min to remove the solid. The precipitate formed was removed by vacuum filtration. The hydrolysate was supplemented with 5 g/L KH2PO4, 2 g/L (NH4)2SO4, 0.2 g/L MgSO4·7H2O, 1 g/L peptone, 5 g/L yeast extract and finally the pH of hydrolysate was adjusted to 5.5 and autoclaved at 110 °C for 15 min. Sugars and hydrolysate inhibitors in the hydrolysate were analyzed by high-performance liquid chromatography (HPLC).
Fermentation of the non-detoxified sugarcane bagasse hydrolysate
The 24 h pre-cultivated yeast cells in YPX medium were inoculated into hydrolysate medium with cells initially adjusted to cell density of 1 × 107 cells/mL. Fermentations were carried out at 30 °C as described above. Fermentation samples were taken every 12 h for determining ethanol concentration and sugar concentration in the culture. All experiments were performed in three independent experiments.
The ethanol and sugar concentrations were analyzed by Waters 600E HPLC system (Waters Inc., Milford, USA) using a sugar pak I column at 85 °C and a refractive index detector. The mobile phase was deionized water at a flow rate of 0.5 mL/min. Furfural (Sigma-Aldrich, St. Louis, USA), HMF (Sigma-Aldrich, St. Louis, USA) and acetic acid (Merck, Darmstadt, Germany) were separated on C18 column at 25 °C and UV detector. Furfural and HMF were eluted with 20 % acetonitrile in deionized water (80 %) at a flow rate of 1.1 mL/min. Acetic acid was eluted with 1 % acetonitrile in 0.05 M KH2PO4 (99 %) at a flow rate of 1.0 mL/min.
SN-RT conceived and designed the experiments. SS, TC and YK performed the experiments. SN-RT, MS, AT, SS, AE, IR and KW analyzed the data. SN-RT and AT drafted the manuscript. AE, IR and KW edited the manuscript. All authors read and approved the final manuscript.
This research was financially supported by Energy Conservation Promotion Fund (ENCON Fund), Thailand and partially supported by Microbial Biotechnology Unit and Department of Microbiology (Grant Year 2015), Faculty of Liberal Arts and Science, Kasetsart University, Thailand. KW, IR and AE were supported by the UK BBSRC Institute Strategic Programme ‘Food and Health’ (Grant Number BB/J004545/1).
The authors declare that they have no competing interests.
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