核诱变产气肠杆菌利用水花生发酵产氢的研究

发布时间：2018-03-11 12:21

  本文选题：氢气　切入点：产气肠杆菌　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：由于化石能源日益减少,氢能作为一种可再生清洁能源吸引了越来越多的关注。微生物发酵制取生物氢气以其环境友好、低能耗特点,因而具有很强应用前景。产气肠杆菌(Enterobacter aerogenes)兼性厌氧,生长速度快,产氢速率高,有望应用于工业生产中。本文研究了核辐射诱变产气肠杆菌利用色圈法筛选优势突变体,通过增强氢酶活性及优化代谢途径提高其产氢能力;将纤维素生物质蒸汽稀酸预处理高效水解后,利用产气肠杆菌突变体发酵提高产氢量。首次利用钴60-γ射线对产气肠杆菌进行核辐照诱变,并利用酸性副产物色圈法筛选高效的产氢突变株。色圈大小代表了细菌代谢产酸多少,色圈越大表明代谢产酸量越多,氢气产量极有可能提高。筛选获得具有较大色圈的突变株E.aerogenes ZJU1,其氢酶活性157.4 mL H2/(g DW·h)明显高于野生菌株的氢酶活性89.8mLH2/(gDW·h)。突变株的糖酵解和丙酮酸代谢增强,甲酸氢气裂解酶、氢酶活性提高,促进了 FHL途径和NADH途径产氢。突变株的氢气产量为301 mLH2/g葡萄糖,比野生株产氢量(166mLH2/g葡萄糖)提高了 81.8%。'突变株的产氢速率峰值为27.2 mLH2/(L·h),比野生菌株的产氢速率峰值(19.3 mL H2/(L·h))提高了 40.9%。突变株发酵产氢过程中的液相副产物乙酸和丁酸产量高于野生株,而乙醇产量降低,这与突变株的产氢能力提高是一致的。通过迭代培养验证了突变株E.aerogenes ZJU1的产氢能力具有遗传稳定性。突变株E.aerogenes ZJU1利用预处理高效水解的水花生促进发酵产氢。微观测试表明:蒸汽稀酸预处理对水花生纤维素结构造成了严重破坏,大量碎片降解并且出现许多裂痕;结晶纤维素减少,非结晶纤维素增加,水花生的结晶度指数由17.6降低至10.8。水花生的还原糖产量在最佳预处理工况下(硫酸浓度1%,蒸汽温度135℃,加热时间15min)达到0.354g/g生物质,酶水解后达到0.575 g/g生物质。产气肠杆菌突变株E.aerogenes ZJU1利用高效水解后的水花生发酵产氢量达到62.2mLH2/g-TVS,比野生菌的发酵产氢量(47.2mLH2/g-TVS)提高了 31.8%。通过优化发酵菌液量得到最大单位产氢速率由1.42mLH2/g-TVS/h提高至4.64mLH2/g-TVS/h,同时氢气产量提高了 42.8%达到89.8mLH2/g-TVS。
[Abstract]:Because of the decreasing of fossil energy, hydrogen energy as a kind of renewable clean energy has attracted more and more attention. Microbial fermentation to produce biological hydrogen is environmentally friendly and low energy consumption. Therefore, Enterobacter aerogenes (Enterobacter aerogenes) facultative anaerobic, rapid growth rate and high hydrogen production rate are expected to be used in industrial production. The hydrogen production ability was improved by enhancing the activity of hydrogen enzyme and optimizing the metabolic pathway. The fermentation of Enterobacter aerogenes mutant was used to increase hydrogen production. Cobalt 60- 纬 -ray was used to mutate Enterobacter aerogenes by nuclear irradiation for the first time. The size of color circle represents the amount of acid produced by bacteria metabolism, and the larger the color circle is, the more the amount of metabolic acid production is. The hydrogen production of E. aerogenes ZJU 1 with large color circle was significantly higher than that of wild strain E. aerogenes ZJU 1. The glycolysis and pyruvate metabolism of the mutant strain were enhanced, and the hydrogen formic acid lyase activity of the mutant strain was significantly higher than that of wild strain E. aerogenes ZJU 1, and the hydrogen formic acid lyase of E. aerogenes ZJU 1 was significantly higher than that of wild strain E. aerogenes ZJU 1. The activity of hydrogenase increased, which promoted hydrogen production by FHL pathway and NADH pathway. The hydrogen production of mutant was 301 mLH2/g glucose. The hydrogen production rate of mutant strain was 27.2 mLH2/(L 路HG, which was higher than that of wild strain (19.3 mLH2/(L 路HG glucose). The liquid phase by-products acetic acid and butyric acid in hydrogen production of mutant strain were higher than that of wild strain (19.3 mLH2/(L 路HG glucose), and the hydrogen production rate of mutant strain was higher than that of wild strain strain (166mLH2 / g glucose). The liquid phase by-products acetic acid and butin in the process of hydrogen production of mutant strain were increased by acetic acid and butyric acid. The acid yield was higher than that of wild plants. And ethanol production is down, The hydrogen production ability of the mutant E. aerogenes ZJU1 was proved to be of genetic stability by iterative culture. The mutant E.aerogenes ZJU1 was used to promote the production of hydrogen by pretreated and highly hydrolyzed peanut. The results showed that the cellulose structure of peanut was seriously damaged by steam dilute acid pretreatment. Large amounts of debris degrade and many cracks occur; crystalline cellulose decreases, amorphous cellulose increases, The crystallinity index of water peanut was reduced from 17.6 to 10.8. Under the optimum pretreatment condition (sulfuric acid concentration 1, steam temperature 135 鈩，

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