Abstract
BackgroundFuture 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry
To reduce yeast ethanol consumption, we conducted the 2% cellulose fermentations without aeration (Fig. 5). These results show that the emulsified SSF (eSSF) process increases ethanol titers and productivities relative to microcrystalline cellulose at concentrations equivalent to at least 2% glucose, which raises the possibility of using this method to produce other valuable chemicals from cellulose with engineered strains of S. cerevisiae
In this study, we report both a three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus at different temperatures as well as the effect that the eSSF technology has on productivity
Summary
Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Lignocellulosic biomass is an attractive alternative feedstock to address these problems, as cellulose is the most abundant organic polymer found in nature, which can be obtained from agricultural waste and energy crops grown on land not suitable for food production [4,5,6]. Because of these advantages, shifting biofuel processing from corn starch to cellulose opens the opportunity to expand production and replace a larger portion of fossil fuels with renewable energy while avoiding the negative environmental and financial impacts of using corn. There is much need for novel ways to improve enzymatic hydrolysis rates and conversion in cellulosic biofuel processes
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