Hoffman Shannon M, Alvarez Maria, Alfassi Gilad, Rein Dmitry M, Garcia-Echauri Sergio, Cohen Yachin, Avalos José L
Department of Chemical and Biological Engineering, Hoyt Laboratory, Princeton University, 101 Hoyt Laboratory, William Street, Princeton, NJ, 08544, USA.
Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain.
Biotechnol Biofuels. 2021 Jul 17;14(1):157. doi: 10.1186/s13068-021-02008-7.
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.
未来玉米衍生乙醇的扩张引发了对可持续性以及与食品工业竞争的担忧。因此，源自农业废弃物和专用能源作物的纤维素生物燃料是必要的。迄今为止，缓慢且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，虽然诸如同步糖化发酵（SSF）和使用耐热微生物等方法可以提高产量，但仍需要进一步改进。纤维素乳液已被证明可通过增加酶与纤维素纤维的接触来提高糖化效率。在本研究中，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同步糖化发酵（eSSF）工艺，并在不同温度下的葡萄糖和纤维素培养基中，对酿酒酵母、多形汉逊酵母和马克斯克鲁维酵母的乙醇产量进行了直接的三方比较。
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能快速水解，与微晶纤维素（MCC）相比，在第一个小时内转化率高达40倍。为了评估eSSF工艺的合适条件，我们探索了耐热酵母马克斯克鲁维酵母、多形奥默酵母以及酿酒酵母的温度上限，并分别观察到每种酵母在高达46℃、50℃和42℃时都能进行强劲发酵。我们表明eSSF工艺在短处理时间内可达到高乙醇滴度，并且在低至30℃的温度下能产生接近理论产量的乙醇。最后，我们通过使用光遗传学调控因子在光控eSSF中生产高级生物燃料异丁醇，证明了eSSF技术对其他产品的可转移性，相对于MCC SSF，异丁醇滴度提高了四倍。
eSSF工艺通过在酵母可耐受的温度下提高糖化速率，解决了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、缩短处理时间、降低酶负载，并使得将该工艺扩展到乙醇以外的化学品（如异丁醇）成为可能。这种可转移性将eSSF工艺确立为一个可持续生产生物燃料和化学品的整体平台。
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