Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol
Introduction
Carbon dioxide (CO2) released from the burning of fossil fuels such as petroleum and coal is considered a major contributor to global warming. There is thus a need for alternative, carbon-neutral energy sources. Bioethanol, a renewable fuel, is considered one such alternative fossil fuel (Cardona and Sánchez, 2007). Bioethanol production from various substrates such as corn (Bothast and Schlicher, 2005), wheat (Arifeen et al., 2007), sugar cane juice and molasses (Basso et al., 2008), and woody biomass (Matsushika et al., 2009) has been well-studied.
Highly concentrated bioethanol production requires less volume in fermentation tanks and consumes less distillery energy. Therefore, very high gravity (VHG) technology that can enhance ethanol productivity is attracting interest. VHG technology involves the preparation and fermentation of mash containing highly dissolved solids to yield a high ethanol concentration (Thomas et al., 1993). Because the substrates contain highly concentrated carbohydrates in the VHG fermentation, ethanol stress is imposed on yeasts. Saccharomyces cerevisiae NPO1 produced 11.3% of ethanol from sweet sorghum juice medium containing 24.8% of total sugar (89% theoretical ethanol yield) (Laopaiboon et al., 2009). However, when the total sugar concentration was 33.6%, the ethanol concentration produced was 10.7% (62.3% theoretical ethanol yield), which was almost the same amount as in the case of sorghum juice containing 24.8% of total sugar. Thus, ethanol-tolerant yeasts are needed for efficient fermentation.
It has been reported that extracellular ethanol causes water stress in yeasts (Hallsworth, 1998). Overlapping between osmotolerance and ethanol tolerance has also been suggested by physiological and molecular biological studies (Sharma et al., 1996, Ding et al., 2009). Thus, the potency of osmotolerance may be one of the factors in ethanol tolerance in yeasts.
Additionally, if the temperature is not controlled, the temperature of the fermentation tanks would be raised to above 40 °C by the exothermic reactions of yeasts (Abdel-Fattah et al., 2000). The temperature increase during the fermentation process inhibits ethanol production and leads to a decrease in the final ethanol concentration (Kida et al., 1992). The advantages of rapid fermentation at high temperature using thermotolerant yeast strains would be not only the reduction of cooling costs but also a decreased risk of bacterial contamination (Limtong et al., 2007). At present, industrial ethanol production mostly employs a mesospheric strain S. cerevisiae at a fermentation temperature up to 35 °C. Kluyveromyces marxianus is recognized as a thermotolerant yeast strain capable of growing at 52 °C (Limtong et al., 2007). However, K. marxianus commonly has lower ethanol productivities than S. cerevisiae. On the other hand, S. cerevisiae F111, which can grow at temperatures up to 50 °C, has been isolated and applied to industrial scale fermentation (Abdel-Fattah et al., 2000).
Root and tuber crops generally contain 70–80% water, 16–24% starch and less than 4% protein and lipids (Hoover, 2001). While VHG fermentation technology has been applied to ethanol production from cereal grains, its application to roots and tubers has rarely been reported. The reason why the use of root and tuber mashes for VHG fermentation technology is limited may be because of their high viscosity. Sathaporn et al. (2009) demonstrated that potato mash viscosity could be reduced by pretreatment with a mixed enzyme preparation and SSF of liquefied VHG potato mash at 30 °C using S. cerevisiae NBRC0224 (Seiko et al., 1985). In addition, NBRC0224 has been reported to be suitable for ethanol production from sugar cane molasses (Kuriyama et al., 1985). The objective of this study was to isolate yeast strains more suitable than NBRC0224 for ethanol production in VHG-SSF. Because the viability of NBRC0224 was very low under a high ethanol concentration, NBRC0224 stopped producing ethanol (data not shown). Therefore, there is a need for yeast strains that produce ethanol effectively under a VHG-SSF condition.
In this study, we present the results of a screening of useful yeasts for ethanol synthesis from VHG potato mash. We found several yeasts that produced a high concentration of ethanol from a high concentration of glucose. One yeast strain, S. cerevisiae NFRI3225, was selected as a high-ethanol producing yeast. We also demonstrated the high temperature (37 °C) SSF of VHG potato mash.
Section snippets
Strains and media
The yeast strains (1699 strains) used for screening were obtained from the Microbiological Bank at our institute (NFRI). S. cerevisiae NBRC0224 (Seiko et al., 1985) used as a control strain was obtained from the culture collection of the NITE Biological Resource Center (NBRC; Chiba, Japan). These strains were maintained on YPD plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar).
YPD medium (1% yeast extract, 2% peptone, 2% glucose) was used for pre-cultivation. YPD medium containing
Screening of stress-tolerant yeasts
Because of the relationship between ethanol stress and water stress, we predicted that yeasts producing a high concentration of ethanol would show high-osmolarity tolerance and could produce a high concentration of ethanol from a high concentration of glucose. To obtain such yeasts, we assessed the growth and ethanol productivities of a wide range of yeast strains. We first selected 500 strains from our yeast collection that could grow well in YPD medium containing 30% glucose. Among these
Conclusion
We screened the collection of 1699 yeast strains at our institute and selected S. cerevisiae NFRI3062, NFRI3213, and NFRI3225 as candidates for use in the VHG-SSF process. NFRI3225 was the most useful strain for the VHG-SSF process, and produced 13.7% (w/v) of ethanol from potato mash at 37 °C for 24 h. NFRI3225 also showed excellent ethanol productivity with three kinds of sweet-potato mash. Furthermore, the thermotolerance of NFRI3225 would save cooling energy during the SSF process and heating
Acknowledgements
This work was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Grants Nos. BEC-BC050 and BEC-BA240 for the Rural Biomass Research Project).
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