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

Abstract

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.

Keywords: Biofuels; Biomass pretreatment; Cellulose; Emulsions; Ethanol; Isobutanol; Kluyveromyces marxianus; Ogataea polymorpha; Optogenetics; SSF; Saccharomyces cerevisiae; Thermotolerant.

Fig. 1

Comparison of cellulosic emulsion and microcrystalline cellulose hydrolysis kinetics. The conversion of cellulose was compared using a 0.6% cellulose emulsion and microcrystalline cellulose (MCC) at four temperatures and an enzyme load of 53 FPU/g substrate. Data is shown as the mean values and error bars represent the standard deviation of three replicates

Fig. 2

Simultaneous saccharification and fermentation (SSF) process schematic for cellulosic biofuel production. In both the a microcrystalline cellulose SSF (mcSSF) and b emulsified SSF (eSSF) processes, cellulase enzymes degrade cellulose into glucose, which is simultaneously metabolized by yeast into ethanol (or other chemicals). a In the mcSSF process, the untreated cellulose maintains a microcrystalline structure, which is more difficult for enzymes to hydrolyze. b In the eSSF process, the cellulose fibers coat the surface of oily droplets in the emulsion, providing better access to enzymes [45], and thus easier hydrolysis

Fig. 3

Three-way comparison of ethanol production from glucose by thermotolerant yeasts and S. cerevisiae. Ethanol production throughout time is shown for a K. marxianus, b O. polymorpha, and c S. cerevisiae, with 2% glucose provided as the carbon source. All data is shown as mean values and error bars represent the standard deviation of three independent replicates exposed to the same conditions

Fig. 4

Ethanol production using eSSF or mcSSF processes. Ethanol titers obtained from the mcSSF and eSSF processes show that higher ethanol production is consistently achieved from cellulosic emulsions than from microcrystalline cellulose. The highest ethanol concentrations reached with K. marxianus, O. polymorpha, and S. cerevisiae using a 0.6% cellulose emulsion (eSSF) or microcrystalline cellulose (mcSSF) are depicted. The times when maximum ethanol titer are reached for each yeast are shown. An enzyme load of 53 FPU/g cellulose was used for all yeasts, and data is shown as mean values and error bars represent the standard deviation of three independent replicates

Fig. 5

Ethanol production in S. cerevisiae using an increased cellulose load in the eSSF or mcSSF processes. Ethanol titers and percent theoretical yield obtained from S. cerevisiae in eSSF or mcSSF processes using a 2% cellulose content at both a 30 °C and b 42 °C with an enzyme load of 53 FPU/g cellulose. At this enzyme load, the eSSF process reaches at least 86% of the theoretical yield within 24 h at both temperatures. The eSSF and mcSSF processes are also compared at a lower enzyme load of 26 FPU/g cellulose at 30 °C (c), with the eSSF process exceeding the mcSSF titers by a factor of two after 36 h. Data is shown as mean values and error bars represent the standard deviation of three independent replicates

Fig. 6

Cellulosic isobutanol production using an optogenetically controlled eSSF process. a Schematic of the optogenetically controlled isobutanol and ethanol biosynthetic pathways, which compete for pyruvate from glycolysis. The enzymes which catalyse the first steps of each of these pathways are placed under optogenetic control (see “Methods”), with PDC1 of the ethanol pathway expressed in the light, and ILV2 of the isobutanol pathway expressed in the dark. The isobutanol pathway is localized to the mitochondria, whereas ethanol biosynthesis occurs in the cytosol. b Isobutanol titers recorded in eSSF and mcSSF experiments at 30 °C, using the optogenetic S. cerevisiae strain YEZ546-2 and different enzyme loads. All tests used either a 2% cellulose emulsion or 2% microcrystalline cellulose mixture and were switched from the light-driven growth phase to the darkness-induced production phase at the same cell density. Mean values are shown, and error bars represent the standard deviation of three independent replicates