产溶剂梭菌兼养发酵转化富CO炼钢尾气产乙醇的碳代谢流调控机制研究

Steel mill waste gas is rich of carbon monoxide (CO), which is considered as an air pollutant and a low-value resource. CO can be utilized as carbon and energy source by solventogenic Clostridia and metabolically converted to produce ethanol via the Wood-Ljungdahl Pathway (WLP) in autotrophy. Such a CO-to-ethanol biological process is characterized as being highly efficient, low-carbon-emission and environmentally friendly, which can be potentially applied for valorization of steel mill waste gas. However, ethanol productivity is still too low by this technology, which is significantly limited by gas-to-liquid mass transfer efficiency and low net ATP yield with WLP metabolism. To overcome the above key bottlenecks, this proposed research aims to develop a high-ATP-yield “mixtrophy” fermentation process coupling WLP with EMP glycolysis, so that the ethanol production can be enhanced. The most commonly reported solventogenic clostridia, Clostridium ljungdahlii will be employed as the microbial host to convert CO for ethanol production. First, we will investigate the effect of gas-to-liquid mass transfer on CO-derived ethanol production in such a mixtrophy, in order to determine the threshold of mass-transfer-limited regime. Second, based on the threshold point, we will explore the mixtrophy fermentation using physiological, metabolomics, transcriptomics and proteomics approaches for C. ljungdahlii steady-state chemostat cultures grown on fructose and synthetic steel mill waste gas (CO/CO2) under various gas-liquid mass transfer conditions, in order to elucidate the regulatory mechanism. Furthermore, based on the “omics” results and sequencing information, a genome-scale metabolic model (GEM) will be developed. Third, by developing 13C isotopically nonstationary metabolic flux analysis (INST-MFA) method, we will characterize the carbon flux and curate the GEM. By adopting such a systems-level comprehensive approach, we will determine the key metabolic pathway for ethanol production in C. ljungdahlii mixtrophy and will decipher the regulatory mechanism. Overall, the proposed research will advance our fundamental understanding of solventogenic Clostridia mixtrophy for enhanced ethanol production. Ultimately, this study will contributes to minimize pollution caused by steel mill waste gases, to promote valorization of the low-value CO-rich waste gases and to advance the “non-food feedstock-derived” bioethanol economy.

炼钢尾气富含一氧化碳CO，具有污染源和资源双重属性。利用产溶剂梭菌自养代谢转化其中CO生成燃料乙醇，是一种高效、环境友好的尾气资源化途径。然而，受气液传质及代谢ATP产量过低的限制，该技术的乙醇产率仍较低。为突破瓶颈，本项目拟通过自养代谢耦合糖酵解途径，构建高ATP产出的兼养发酵体系以转化富CO炼钢尾气，明晰其代谢特征及调控机制，从而提高乙醇产量。选用扬氏梭菌为细胞工厂，首先研究兼养发酵体系中传质对CO合成乙醇的影响，确定传质限制的阈值；根据阈值，采用多组学手段揭示不同传质条件下的细胞代谢规律；最后，建立非稳态代谢通量分析方法解析碳代谢流特征，构建基因组尺度代谢网络模型，明确兼养发酵体系中乙醇合成的关键代谢通路，揭示其调控机制，为高效生物转化CO制乙醇提供理论依据。此研究对钢铁工业尾气的污染控制、低值资源CO的有效利用、以及我国“非粮食”生物燃料乙醇产业的发展具有重要的科学意义和应用价值。

产溶剂梭菌的非光合厌氧固碳产乙醇是极具潜力的二氧化碳资源化生物技术，但受Wood-Ljungdahl途径（WLP）的ATP产能限制，乙醇产率偏低。本课题选用扬氏梭菌C. ljungdahlii为模式微生物，采用兼养发酵策略突破ATP限制，首先比选了不同有机碳源和还原力作用对扬氏梭菌兼养发酵过程的细胞生长、底物利用、碳回收效率、能量分配和代谢产物分布影响，发现果糖为最佳有机碳源，且H2补充和硝酸盐呼吸耦合作用对乙醇产率均有明显提高。发展了基于13C标记果糖底物平行示踪的13C-MFA分析方法，构建了扬氏梭菌在同步代谢果糖+CO2/H2条件下的代谢网络模型，结果表明有72.4%的乙酰辅酶A（中心代谢的重要中间代谢产物）碳通量仍由固定CO2的WLP合成，明确了兼养发酵过程中固碳途径的定量贡献；此外，基于该代谢模型的能量平衡计算结果显示，兼养发酵模式下果糖的糖酵解途径也为体系提供了足量的还原力和氧化还原辅因子，体系中的ATP、NADH和还原性铁氧还蛋白分别提高了28%、28%和25%。然后，在CSTR反应器中进行了为期190天的连续发酵，通过对比不同气液传质条件下扬氏梭菌在兼养和自养模式下的产乙醇效能，确定了兼养发酵固定CO2的气体传质限制阈值并解析了碳代谢特征，结果表明在高传质条件下，乙醇主要通过乙醛:铁氧还蛋白氧化还原酶（AOR）的作用还原乙酸来产生，进一步阐明了兼养发酵模式下气液传质对合成乙醇代谢通路的调控机制。最后，探究了实际工业尾气固碳转化过程中常见的氧胁迫对扬氏梭菌在自养和兼养发酵模式下的代谢响应，发现了微氧环境（顶空5-10% O2）对产乙醇的促进作用（57%），且兼养环境中扬氏梭菌的细胞密度更高，这意味着更高的氧气耐受度和代谢韧性。

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