溶氧对芽孢杆菌（Bacillus）C2丙酮丁醇发酵的影响机制研究

Acetone-Butanol-Ethanol (ABE) fermentation is one of the oldest and largest biotechnological processes ever known, ranking second in scale next to ethanol fermentation. Because of the potential application of butanol as an advanced biofuel, ABE fermentation has been a research hotspot once again in microbial and energy engineering fields recently. A high yield of butanol producing Bacillus strain named C2 has been isolated by our research group and this found breaks the limits that all of the ABE producing strains are obligate anaerobic Clostridium. We found that Bacillus C2 could grow better and produce more ABE in facultative anaerobic condition than that in obligated anaerobic condition. Literature reported that micro oxygen can enhance the production of ABE and shift the central carbon flux from acetone to ethanol during Clostridium acetobutylicum ABE fermentation. While how dissolved oxygen affects the ABE fermentation of C2 remains unclear. In this research, fermentation kinetics and metabolic flux analysis will be researched to illustrate the influences of dissolved oxygen on Bacillus sp. C2 growth and ABE fermentation. In order to reveal the mechanisms of this influences, the accumulation amount of intracellular energy [ATP] and reducing power [NAD(P)H] with different concentration of dissolved oxygen will be determined and the differentially expressed proteins will be screened by two-dimensional gel electrophoresis and mass spectrometry in proteomics research. The results will provide theoretical basis for engineering strain rational construction and process engineering directional regulation of the ABE fermentation.

丙酮丁醇发酵是仅次于乙醇发酵的第二大传统发酵，近年来，因其主产物丁醇作为燃料的优势，成为微生物和能源工程领域研究的热点。课题组筛选得到一株高产丁醇的芽孢杆菌（Bacillus）C2菌株，突破了丙酮丁醇发酵均为专性厌氧梭菌（Clostridium）的局限。前期研究发现兼性厌氧有利于C2菌体生长和产物生成，文献资料报道微量供氧能改变丙酮丁醇梭菌发酵的碳源流流向，提高发酵产量，但溶氧对C2菌株丙酮丁醇发酵的影响规律及影响机制尚不清楚。本项目拟通过构建不同溶氧条件下的发酵动力学方程，结合代谢通量分析，阐明溶氧浓度对C2菌体生长和发酵的影响规律；通过分析不同溶氧浓度下菌体细胞内能量[ATP]和还原力[NAD(P)H]积累情况及全细胞蛋白表达谱的差异，揭示氧气对芽孢杆菌C2菌株丙酮丁醇发酵的影响机制，为丙酮丁醇发酵工程菌株的理性构建和发酵过程工程定向调控提供理论依据。

随着石化资源的日益耗竭和环境污染的日益严重，利用可再生资源生产生物基燃料和生物基化学品受到了社会的广泛关注。与乙醇相比，丁醇具有能量密度和燃烧值高、蒸气压低、可在现有石油管道中运输等优点，已被列为仅次于乙醇的第二代生物燃料。本项目以课题组分离筛选得到的产丁醇芽孢杆菌（Bacillus sp.）C2为研究对象，研究了不同溶氧浓度对丙酮丁醇发酵的影响。在40 L发酵罐内，通过定期通入空气和氮气控制发酵液的溶氧浓度分别为4.90-6.79 mg/L和0.3-0.7 mg/L进行兼性厌氧和专性厌氧发酵。兼性厌氧条件下丙酮丁醇发酵周期为60 h，比专性厌氧发酵缩短了36 h；丁醇和总溶剂的产量分别为10.644和15.771 g/L，比专性厌氧条件下分别高5.0%和6.4%。构建了兼性厌氧和专性厌氧条件下丙酮丁醇发酵的菌体生长动力学、溶剂生成动力学和底物消耗动力学模型。代谢通量分析表明，兼性厌氧产酸期乙酸分支途径流量相对较高，有利于ATP的产生和菌体的快速生长；产溶剂期兼性厌氧条件下乙酸分支途径、丁酸分支途径、丙酮分支途径以及丁酸吸收的闭合回路分支途径碳源流所占比列升高，从而导致发酵产物中丙酮的比例升高。与专性厌氧相比，兼性厌氧条件下微生物细胞内ATP含量提高0.35-5.99 fmol/cell (mg)；NADH/NAD+的总量和比例分别提高3.7-22.6%和2.7-13.3%；产溶剂关键酶乙酸激酶、丁酸激酶、乙醇脱氢酶、丁醛脱氢酶、丁醇脱氢酶和辅酶A转移酶分别提高了9.44-23.09%、9.18-51.48%、12.53-63.20%，4.42-22.49%、17.08-72.93%和21.84-72.68%。能量、还原力及关键酶活的提高有利于微生物菌体的快速生长和产物的快速生成，从而提高了产物的生成速率和产量，缩短了发酵周期。蛋白双向电泳结果表明，专性厌氧和兼性厌氧条件下蛋白表达谱和表达量存在显著差异。该研究揭示了氧气对芽孢杆菌C2菌株丙酮丁醇发酵的影响机制，为丙酮丁醇发酵工程菌株的理性构建和发酵过程工程定向调控提供了理论依据。