| Due to the rising concerns of limited fossil fuel reserves, global environmentaldeterioration, more attention has been recently devoted to develop the renewable and greenenergy sources. Butanol is considered the suitable second generation biofuel as it has manyadvantages such as higher energy content, higher octane number, less corrosive and lowersolubility in water. Therefore, the production of butanol via fermentation process has attractedrenewed interest in recent years. However, butanol toxicity to the current producingmicroorganisms limits accumulation in the fermentation broth, and for this reason thebiobutanol production processes is of low yield, productivity and conversion efficiency. Inthis study, some butanol producing strains were isolated from environmental samples. Someproblems were encountered and resolved in the acetone, butanol and acetone (ABE)fermentation process. The batch and continuous fermentation in fibrous bed bioreactor (FBB)was optimized to enhance butanol production. Furthermore, the mechanisms of butanoltolerant strain to butanol toxicity were also studied. The main findings are summarized asfollows:(1) A total of195butanol producing strains were successfully isolated by “sandwich”isolation method and visual screening model, among which the strain PW12was best in ABEfermentation performance. The strain was identified as Clostridium acetobutylicum by16SrDNA comparison. To obtain higher butanol tolerance strain, atmospheric and roomtemperature plasma (ARTP), serial enrichment and combination of rational selection modelwere used in this study. With this effort, mutant strain SE25was obtained. The tolerance ofthe mutant strain SE25was improved0.75times than that of the wild-type strain PW12.Furthermore, the production of butanol and total solvent were12.85±0.62g·L-1and17.38±0.79g·L-1, respectively, which were49.1%and43.2%higher than that of the wild-typestrain PW12.(2) Among the selected materials, cassava flour was the most suitable for butanolproduction. The reasons for phase shift delay were investigated. One reason was a hugeimbalance between carbon and nitrogen ratio in cassava flour. Another reason was due to theprolonged low pH level and untimely pH recovery. The optimal regulation strategy ofadaptively supplemented corn steep liquor (carbon/nitrogen sources ratio85.34mol·mol-1)and different pH at phase shift stage could resolve these problems. When corn steep liquor(2.5mL·L-1) was added into fermentation medium, the production of butanol and total solventwere14.90±0.73g·L-1and22.70±1.14g·L-1, respectively. The fermentation period wasshortened by about12h through pH regulation strategy. In this condition, the production ofbutanol was16.24±0.70g·L-1, which was14.9%higher than that without pH regulation strategy.(3) Dithiothreitol (DTT), the reducing agent and potassium ferricyanide, the oxidizingagent were the optimal regulators for adjusting the oxidation reduction potential (ORP) offermentation system. When the ORP of fermentation system was controlled at-400mV, thehighest production of butanol was14.32±0.65g·L-1, which was10.8%higher than that ofcontrol group. Tending to reduce the fermentation system and production of hydrogen, andespecially increasing the synthetic efficiency of NADH in solventogenic phase could enhancethe production of butanol. Furthermore, the production of butanol could also be enhancedwhen the level of energy charge and the metabolic intensity of butyric acid loop were reduced.(4) The optimum amount of immobilized carrier for ABE fermentation process byAquamats-AO in FBB was investigated. When the amount of immobilized carrier was8.0g·L-1, the maximum production of butanol and total solvent were obtained. The maximumproductivity of butanol and total solvent were0.24g·L-1·h-1and0.37g·L-1·h-1, respectively,when ABE fermentation was carried out in FBB. The productivity and yield of butaol were26.3%and27.6%higher than that of the traditional fermentation. A butanol concentration of14.00-15.50g·L-1and20.00-23.60g·L-1total solvent were obtained by repeated batchfermentation in FBB. The production of butanol and total solvent were89.54g·L-1and133.16g·L-1, respectively, which were47.4%and18.5%higher than that of the traditionalfermentation. The solvent concentration and the yield of starch decreased with the increase ofthe dilution rate (0.04h-1to0.1h-1) when the effect of dilution rate on the ABE fermentationwas studied. However, the productivity and cell concentration increased in this condition. Theresults indicated that higher dilution rate was beneficial to the growth of cells and the lowerdilution rate was conducive for the accumulation of solvent.(5) The butanol tolerance mechanism of the mutant strain SE25was investigated.Bacterial capsule plays important role during butanol stress. When cells decreased the surfacehydrophobicity, the ability of resistance to organic solvent was increased. During high butanolstress, the membrane permeability increased by77.1%and membrane potential decreased by80.2%for the strain PW12. However, in the same condition, the membrane permeability onlyincreased by11.3%and membrane potential only decreased by9.8%for the mutant strainSE25. Therefore, the cell membranes could maintain better integrity during butanol challengefor the mutant strain SE25. In order to reduce the negative influence under high butanolconcentration, the cells decrease glucose uptake and energy regeneration rate. The proportionof the saturated fat in the total fatty acids increased by22.13%for the mutant strain SE25compared to the wild-type strain PW12. These results indicate that some bacteria resistbutanol toxicity by increasing the saturation of fatty acids, thereby reducing their membranesfluidity. |
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