Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan.
Biotechnol Biofuels. 2013 Dec 21;6(1):184. doi: 10.1186/1754-6834-6-184.
Cellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition.
This work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose (617 mM initial glucose equivalents), we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition.
Our process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of C. thermocellum supplemented with a thermostable beta-glucosidase and cultured under a high cellulose load. We named this approach biological simultaneous enzyme production and saccharification (BSES). BSES may resolve a significant barrier to economical production by providing a platform for production of fermentable sugars with reduced enzyme amounts.
纤维素酶仍然是木质纤维素水解过程中主要成本之一。产热梭菌是一种厌氧、嗜热、产纤维素的细菌，它能产生能有效降解植物细胞壁的细胞外酶。然而，终产物纤维二糖会抑制降解。为了最大限度地提高产热梭菌的纤维素酶活力，消除这种终产物抑制是很重要的。
本工作描述了一种生物糖化系统，该系统在水解木质纤维素生物质后可产生葡萄糖。该系统由补充了耐热β-葡萄糖苷酶的产热梭菌培养物组成。这种方法不需要任何纤维素酶和半纤维素酶的补充。当产热梭菌 S14 菌株与 Thermoanaerobacter brockii 的β-葡萄糖苷酶（CglT，活性为 30 U/g 纤维素）一起培养在含有 100 g/L 纤维素（617 mM 初始葡萄糖当量）的培养基中时，我们不仅观察到纤维素的高降解，而且在培养液中积累了 426 mM 的葡萄糖。相比之下，没有 CglT 的培养物，或含有耐热β-葡萄糖苷酶的培养物，不能有效地水解纤维素，并且积累了高浓度的葡萄糖。葡萄糖的生产需要超过 10 g/L 的纤维素负荷。当用碱预处理的水稻秸秆作为木质纤维素生物质，其中含有 100 g/L 的葡聚糖时，大约 72%的葡聚糖被糖化，葡萄糖在培养液中积累到 446 mM。含有葡萄糖的水解物浆液通过添加酿酒酵母直接发酵到 694 mM 的乙醇，理论产率为 85%，没有任何抑制。
我们的工艺是首例从木质纤维素生物质中进行生物糖化，仅生产和积累葡萄糖。成功的关键是使用产热梭菌补充耐热β-葡萄糖苷酶，并在高纤维素负荷下培养。我们将这种方法命名为生物同步酶生产和糖化（BSES）。BSES 可能通过提供一种用较少酶生产可发酵糖的平台，解决经济生产的一个重大障碍。
