Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

Background Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures. Results In this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha, as well as Saccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. Conclusions The eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.

Studies on Bioethanol Production with Thermo Tolerant Yeast Isolates and their Co-Cultures using African Wild Cocoyam as Feedstock

In this work different ways of optimally producing bioethanol at various pH with thermotolerant yeasts and their cocultures using a non-human edible starchy food as feedstock was examined. African wild cocoyam, Xanthosoma roseum, sourced from abandoned farmlands in Obukpa, Nsukka, Nigeria was used as the substrate, while strains of Kluyveromyces marxianus and Pichia stipitis were used to ferment them. First the tubers were gelatinized by boiling under pressure above 100oC before hydrolysis with concentrated H2SO4. The hydrolysates were then fermented at 35oC with the thermotolerant yeasts for five days at different pH. Results obtained showed that gelatinized sample of the substrate gave optimum glucose yield when hydrolysed with 1M H2SO4 for 60 minutes. Kluyveromyces marxianus produced more ethanol than Pichia stipitis at all the four fermentation pH values tested. However, optimum ethanol production was obtained when the two yeast strains were used as coculture at pH 4.5. The peak time for ethanol production was 96 hours for the individual yeast cultures while that of their coculture was 72 hours. The results of the study indicated that wild cocoyam is an excellent feedstock for bioethanol production with many advantages including being non-edible, thereby eliminating concerns for food security, and containing high amount of carbohydrate. The study also revealed that fermenting sugar hydrolysates with a coculture of microorganisms during bioethanol production is a more efficient process than using individual cultures.