限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度，使发酵进入到产溶剂期后，残留葡萄糖浓度降至接近于0 g/L的水平；（2）在葡萄糖受限的条件下，诱导丙丁梭菌合成分泌糖化酶，分解寡糖，将葡萄糖维持于低浓度，进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时，外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下，适度抑制丁醇生产，有效地利用外添乙酸强化丙酮合成；（3）在外添乙酸的基础上，添加适量酿酒酵母，形成丙丁梭菌/酿酒酵母混合发酵体系，提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平，最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02，并可维持丁醇浓度于10~14 g/L的正常水平，充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。

Control of Acetone Concentration and Acetone/Butanol Ratio in ABE Fermentation by C. acetobutylicum with a Novel Glucose/Acetate Co-substrate System Incorporating Glucose Limitation

A novel strategy for arbitrarily controlling acetone concentration and acetone/butanol ratio in ABE fermentation by C. acetobutylicum was proposed. With this strategy, (1) the residual glucose concentration could reduce to low level close to 0 g/L when ABE fermentation enters the solventogenic phase, by adaptively controlling the initial glucose concentration in the corn-based medium via glucoamylase usage dose, reaction time and temperature regulation; (2) Under the condition of glucose limitation, C. acetobutylicum has the ability to release more glucoamylase for its survival and the secreted glucoamylase could continuously hydrolyze oligosaccharide to maintain glucose at low concentration, and therefore repress the metabolism or synthesis rates of glycolysis and NADH in C. acetobutylicum. At the same time, exogenous addition of acetate creates a glucose/acetate co-substrate environment. The co-substrate system would not deteriorate the energy metabolisms in ABE fermentation, but could adaptively repress butanol synthesis avoiding the early occurrence of butanol inhibition and enhance bio-acetone synthesis by effectively utilizing the exogenously added acetate; (3) On the top of exogenous acetate addition, adaptively adding certain amount of viable S. cerevisiae to form C. acetobutylicum/S. cerevisiae co-culturing system, would enhance C. acetobutylicum tolerant ability against higher butanol concentration environment. The entire system could arbitrarily control acetone concentration and acetone/butanol ratio in the ranges of 5~12 g/L and 0.5~1.0, their maximum values could reach levels of 11.74 g/L and 1.02, while maintaining butanol concentration within normal range of 10~14 g/L, to satisfy different requirements on acetone and butanol products in industrial ABE fermentation.