Knowledge Management System Of Institute of process engineering,CAS
|关键词||秸秆丁醇 数字图像 支持向量机 周期蠕动 氧化还原调控|
为解决秸秆丁醇转化率低的难题，论文分别从菌种选育和过程强化入手，基于数字图像技术和支持向量机，建立秸秆丁醇优良菌种的高通量筛选方法，以获得丁醇耐受菌株；并研发丁醇发酵的周期蠕动搅拌方式和氧化还原调控策略，强化菌种的发酵过程。论文取得了如下主要研究结果：（1）基于数字图像分析，建立了还原糖数字图像快速检测方法。基于数字图像分析方法，研究了葡萄糖与二硝基水杨酸显色反应颜色变化规律，发现反应溶液的颜色在Red-Green-Blue空间中的R基色与葡萄糖浓度的相关性为99.8%，优于传统分光光度计法的97.6%。随后，构建神经网络，利用溶液颜色测定葡萄糖浓度，并建立了自动化检测方法。该方法检测范围为0-10 g/L，是传统DNS的3.3倍；检测速度为150样品/15min，是传统DNS的5~10倍。该方法同样适用于木糖、果糖、甘露糖等其他还原糖。（2）运用数字图像分析和支持向量机方法，建立了丁醇高产菌株自动化筛选方法。对丙酮丁醇梭菌发酵数据进行统计分析，确定产溶剂初期（48 h）葡萄糖利用率、溶液pH和菌体氧化还原力可作为丁醇高产菌株的筛选标记，并利用二硝基水杨酸、pH复合指示剂和刃天青实现了筛选标记的可视化；同时，构建支持向量机模型，利用筛选标记的数字图像信息预测菌种的丁醇合成能力，准确率为79.8%，优于人工神经网络的76.4%和非线性拟合的56.3%。基于此，开发了基于数字图像的菌种高通量筛选方法。本方法筛选速率约为“气相法”的250倍，为传统“变色圈法”的100倍。最终，通过筛选得到了一株高产菌株IPE-005，该菌株在葡萄糖基质中总溶剂浓度和丁醇浓度分别为17.5 和10.9 g/L，与出发菌相比分别提高了30.5%和78.6%。数字图像筛选方法同样在红曲筛选上取得了良好效果。（3）系统解析了不同抑制物及其复合物对于菌种IPE-005的抑制作用，并利用代谢通量分析研究了不同抑制物的抑制机理。研究表明丁醇高产菌株IPE-005可耐受5 g/L糠醛、5 g/L 5-羟甲基糠醛、4 g/L 乙酸和2.5 g/L可溶性木质素，分别比出发菌株（Clostridium acetobutylicum ATCC 824）提高了25%，50%，12.5%和47.1%。同时，抑制物存在明显的“低促高抑”现象。对出发菌、耐受菌在抑制物胁迫下的代谢通量进行了统计学对比分析，发现5-羟甲基糠醛、糠醛和可溶性木质素的影响集中在中心碳代谢（磷酸戊糖途径、TCA循环和糖酵解途径），而乙酸的影响分布于中心碳代谢、丙酮酸代谢途径。机理分析表明抑制物胁迫诱发菌体增加糖酵解、TCA和酸合成途径的碳通量，增强能量（ATP）和还原力（NADPH）合成。（4）发明了丁醇发酵周期蠕动搅拌方式，并研究了其强化机理。传统旋涡搅拌方式进行对比研究。发酵试验表明周期蠕动下的菌体量、丁醇产量和总溶剂是传统旋涡搅拌的1.92、2.06和2.04倍，是静置培养下的1.64、1.19和1.41倍。水力学特性分析表明周期蠕动与传统旋涡搅拌在流体类型、剪切力强度、湍流强度和混合效率方面具有明显差异。在相同的旋涡半径下，周期蠕动具有较低的流速（<传统搅拌的60%）、较高的湍流强度（>传统搅拌的10倍）。代谢通量分析表明，6-磷酸葡萄糖、丙酮酸、乙酰CoA，草酰乙酸和α-酮戊二酸是搅拌作用力影响丁醇代谢的关键节点；周期蠕动主要通过三个方面影响菌体的代谢过程：氧化还原平衡状态、能量物质的供应和菌体对于丁醇胁迫的应激反应。(5) 基于细胞氧化还原电位（ORP）对于丁醇合成的重要作用，设计了ORP调控策略，系统解析了ORP调控对于秸秆水解液可发酵性的影响及作用原理。结果表明，当秸秆酶解液溶液ORP控制在-350 mV时，发酵周期缩短了6 h，总溶剂和丁醇的总浓度达到了18.1和10.2 g/L，比未调控组提高了27.5%和34.2%。机理研究表明ORP调控可将酸类合成的代谢流引向丁醇合成，-350 mV条件下胞内的ATP、NADPH/NADPH+和NADH/NAD+比对照组（未施加ORP调控）分别提高了25.1%，81.8%和62.5%，细胞膜的完整度也提高了29%。
To solve the low-yield problem of bio-butanol, in this thesis, novel intensification strategies were carried out from two aspects. Firstly, a novel high-throughput screening method was set up based on digital image processing and support vector machine (SVM) for Clostridium acetobutylicum strain. Then, a new agitation type named Periodic-peristole agitation was invented to enhance the mass transfer in the fermentation process. Lastly, oxidoreduction potential (ORP) controlling strategy was applied to improve the fermentability of enzymatically hydrolyzed steam-exploded corn stover. The main research results obtained are as follows: (1) A rapid detection method for reducing sugars was set up based on digital image processing. By analyzing the color changes between glucose and 3,5-dinitrosalicylic acid (DNS), it was found that the glucose concentrations were highly correlated with the R values of the colors in RGB model with a relative as high as 99.8%, better than that of spectrophotometer (97.6%). The correlation can well be summarized using an artificial neural network model. On this basis, a microtiter plate (96-well plate) platform was set up. Compared to existing methods using a spectrophotometer, the digital color analysis method has a large detection range (0 - 10 g/L1), high accuracy (0.07 g/L) and fast detection rate (150 samples detected within about 15 min). It also shows great promise for use in a variety of reducing sugar measurements such as xylose, fructose and maltose. It formed the basis for strain screening. (2) Digital image processing and support vector machine were applied for the high throughput screening of high-butanol-producing strain. By ming the fermentation data of C. acetobutylicum, the glucose utilization rate, broth pH and cell reducing power at 48 h were determined as the indicators for prediction of butanol producing ablilities. And these indicators can be visualized by color reactions and analyzied with digital image processing. Then a support vector machine model was set up to prediction the butanol production of screened strains with an accuracy of 79.8%, higher than that of artificial neural network (76.4%) and nonline fitting model (56.3%). On this basis , a highly automated screening method was set up, which is fast as 250 folds as high-performance liquid chromatography (HPLC) and 100 folds as the traditional color changing method. Finally, a IPE-005, was obtained. It produced as much as 17.5 g/L solvent and 10.9 g/L butanol with glucose medium, a increase of 30.5% and 78.6%, respectively, compared with the original strain. Digital image processing method also applies to screen other strains. (3) The effects of inhibitors on cell behavors were systemly analyzed to uncover their working mechanisms. It indicated that the strain IPE-005 can tolorate as high as 5.0 g/L furfural, 4.0 g/L 5- hydroxymethylfurfural, 4.0 g/L acetate and 2.5 g/L butyrate, which are 25%, 50%, 12.5% and 47.1 % higher than that of the parent strain, respectively. Statistical analysis on the metabolic flux data indicated that was highly associated with glycolysis, tricarboxylic acid cycle and Pentose Phosphate pathway.(4) A novel agitation type, named Periodic peristole agitation was applied to butanol fermentation with IPE-005 and the enhancement mechanism was studied. Initially, the fermentation performance of periodic peristole agitation was compared with the traditional Rushton impeller and stationary cultivation. Result showed that the biomass, butanol and total solvent in periodic-peristole group (PPG) was enhanced to 1.92 , 2.06 , and 2.4 fold of those in the traditional Rushton impeller group (TIG), as well as 1.64 , 1.19 and 1.41 -fold of those in the stationary group. Hydromechanics analysis indicated that the periodic peristole agitation exhibits significant difference on velocity distribution, shear force, and mixing efficiency from the traditional Rushton impeller agitation. With the same eddy length, PPG owns low flow velocity (<60% of="" that="" and="" high="" turbulence="" intensity="">10 fold of that of TIG). According to metabolism flux analysis, fructose 6-phosphate, pyruvate, acetyl-CoA, oxaloacetate and α-ketoglutarate were determined as the key nodes of cells in response to hydrodynamic mechanical stress. The agitation associated with three issues which resulted in significant changes in cell metabolic behaviors: first, a rebalanced redox status; second, the energy (ATP) acquirement and consumption; third, the tolerance mechanism of the cell for survival of solvent.(5) Oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxificated SECS was controlled at -350mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol ( to 10.2 g/L) respectively. Metabolic flux analysis indicated that flux shifts to butanol production at the expense of acids and hydrogen production. Meanwhile, intracellular concentrations of ATP, NADPH/NADP+ and NADH/NAD+ were increased by 25.1%，81.8% and 62.5%. ORP controlling also resulted in a 29% increase in the cell integrity.
|夏梦雷. 生物数字图像快速检测及发酵过程蠕动强化[D]. 北京. 中国科学院研究生院,2016.|