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. 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. 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.

中文翻译：

---

使用乳化同时糖化和发酵 (eSSF) 与传统和耐热酵母生产纤维素生物燃料

玉米衍生乙醇的未来扩张引发了对可持续性和与食品行业竞争的担忧。因此，从农业废弃物和专用能源作物中提取的纤维素生物燃料是必要的。迄今为止，缓慢和不完全的糖化以及高昂的酶成本阻碍了纤维素生物燃料的经济可行性，虽然同步糖化和发酵 (SSF) 和使用耐热微生物等方法可以提高产量，但需要进一步改进。纤维素乳液已被证明通过增加酶与纤维素纤维的接触来增强糖化。在这项研究中，我们使用这些乳液开发了一种用于快速高效生产纤维素生物燃料的乳化 SSF (eSSF) 工艺，并对 S. cerevisiae、O. polymorpha 和 K. marxianus 在葡萄糖和纤维素培养基中的乙醇生产进行了直接三向比较不同的温度。在这项工作中，我们表明纤维素乳液在酵母可耐受的温度下快速水解，与微晶纤维素 (MCC) 相比，在第一个小时内的转化率高达 40 倍。为了评估 eSSF 过程的合适条件，我们探索了耐热酵母马克斯克鲁维酵母和 Ogataea polymorpha 以及酿酒酵母的温度上限，并观察了每种酵母在高达 46、50 和 42°C 的强劲发酵，分别。我们表明，eSSF 工艺可在短时间内达到高乙醇滴度，并在低至 30 °C 的温度下产生接近理论的产量。最后，我们通过使用光遗传调节剂在光控 eSSF 中生产先进的生物燃料异丁醇，证明了 eSSF 技术向其他产品的可转移性，从而使效价比 MCC SSF 高出四倍。eSSF 工艺通过在酵母可耐受的温度下提高糖化率来解决纤维素生物燃料生产的主要挑战。这些乳液在低温下的快速水解允许使用非耐热酵母进行发酵，加工时间短，酶负荷低，并且可以将该过程扩展到除乙醇之外的化学品，例如异丁醇。