Department of Biomass Science & Conversion Technologies, Sandia National Laboratories, Livermore, CA, 94550, USA.
Department of Systems Biology, Sandia National Laboratories, Livermore, CA, 94550, USA.
Microb Cell Fact. 2017 Nov 9;16(1):192. doi: 10.1186/s12934-017-0804-8.
First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers' grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate 'one-pot' bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains.
The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn't compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer.
The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.
第一代生物乙醇生产利用玉米的淀粉部分，约占谷物无灰干重的 60%。这项技术的扩大应用导致了大量的酒糟可溶物（DGS）副产物，富含纤维素多糖和蛋白质。据推测，DGS 将很快被用于动物饲料应用，但基于产品流的不一致性和其他与物流相关的风险，特别是产毒污染物，这种情况并未出现。因此，高效利用 DGS 生产石油替代产品将显著提高已建立的淀粉生物乙醇工艺的技术经济可行性和净能量回报。在这项研究中，我们通过开发包含两种工程大肠杆菌生物催化剂菌株的微生物联合体，展示了从 DGS 水解物的蛋白质和碳水化合物部分“一锅法”转化为 C4 和 C5 杂醇的方法。
碳水化合物转化菌株 E. coli BLF2 是由野生型大肠杆菌菌株 B 构建的，表现出从己糖和戊糖生产杂醇的能力得到提高。E. coli BLF2 从葡萄糖或木糖合成培养基中生产高达 12g/L 的杂醇。第二株大肠杆菌 AY3 专用于利用水解物中的蛋白质生产混合 C4 和 C5 醇。为了使共培养物的转化率最大化，优化了两种菌株的接种比例。E. coli BLF2 和 AY3 的接种比例为 1:1.5 的共培养物从 DGS 水解物中达到了高达 10.3g/L 的总杂醇最高产量。该工程大肠杆菌共培养系统同样适用于从其他生物质来源生产生物燃料，包括藻类水解物。此外，通过定量 PCR 分析揭示的共培养物种群动态表明，尽管两种菌株的生长速度存在差异，但共培养并不影响每种菌株的生长。q-PCR 分析还表明，发酵时两种菌株的初始接种比例适当对实现平衡的共培养物种群很重要，这会导致更高的总燃料产量。
高效地将 DGS 水解物转化为杂醇将显著提高第一代生物乙醇工艺的可行性。这里开发的碳水化合物和蛋白质综合转化平台适用于富含糖和蛋白质的各种生物质原料的生物转化。
