| To solve the low-yield problem of bio-butanol, in this thesis, novel intensification strategies were carried out from strain breeding and process intensification aspects. 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 and oxidoreduction potential (ORP) controlling strategy were applied to intensify the fermentation process.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 reaction 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 high butanol producing strain, 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 the strain tolerance 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 of TIG) and high turbulence intensity (> 10 fold of that of TIG). According to metabolism flux analysis, fructose 6- phos-phate, 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. |
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