发酵产氢菌与好氧菌呼吸互作强化有氧产氢

发布时间：2018-06-24 17:05

本文选题：有氧产氢 + 产乙醇杆菌　；参考：《哈尔滨工业大学》2017年硕士论文

【摘要】：目前生物制氢技术已经走上工业化的道路,但仍然有很多亟待解决的问题。哈尔滨产乙醇杆菌YUAN-3(Ethanoligenens harbinense YUAN-3)是一株高效产氢菌种,实验室中需要煮沸曝气步骤,以去除环境中的氧气,而此种方法具有昂贵、造成二次污染、使用困难等缺点,特别是考虑厌氧菌培养扩大化时,利用种间呼吸互作实现厌氧菌培养是值得考虑的新途径。细菌之间的互作广泛存在且多种多样,本文中,这种种间作用被共同培养兼性好氧细菌铜绿假单胞菌PAO1和严格厌氧细菌哈尔滨产乙醇杆菌YUAN-3充分诠释,我们用特定的培养基实现了在有氧条件下的生物产氢,这在之前是被认为不可能实现的。在序批培养的产氢实验中,首先考察两种细菌在未经厌氧处理的培养基中共同培养制取氢气是否可行。在单独培养的条件下,存在和不存在L-半胱氨酸,在培养的300小时之内不能产生氢气,而共培养的系统内接种后即可开始产生气体,且加入L-半胱氨酸的培养基表现出更高的氢气产率和产氢速率。在后续实验中,我们选用0 m M、5 m M、10 m M、15 mM的L-半胱氨酸浓度,其中5 m M的L-半胱氨酸获得了最高的氢气产率,达到1.05 mol-氢气/mol-葡萄糖,而更高的浓度得到了更快的产氢速率和细胞干重,凝集性也被增强,表明适量的L-半胱氨酸可以促进氢气的产生,而过量则对细菌生长的促进更为明显。底物的浓度和类型也对产氢有明显影响,考察了不同底物浓度下的共培养产氢效能,其中15 g/L的葡萄糖获得了较为完整的产气曲线且氢气产率也达到最高,为1.04 mol-氢气/mol-葡萄糖。在5 m M L-半胱氨酸和15 g/L葡萄糖的条件下对形成的菌株进行连续培养,即将培养基中形成的球形菌胶团继续接种到新的培养基中,以厌氧处理的YUAN-3单独培养作为对照。虽然传统厌氧培养方法获得了更高的产氢速率,但是共培养的方法获得了更高的氢气产率,达到1.11 mol-氢气/mol-葡萄糖。考虑到两种细菌都可以葡萄糖作为碳源,本实验利用不同二糖作为底物进行共培养,很大程度上减少了竞争作用,最大限度发挥协同作用,其中以蔗糖作为底物的体系取得了最好效果,产氢速率在5m M半胱氨酸,15g/L蔗糖的条件下达到了73 m L/L-培养基·h,氢气产率达到了2.58 mol-氢气/mol-蔗糖。
[Abstract]:At present, biological hydrogen production technology has embarked on the road of industrialization, but there are still many problems to be solved. Harbin ethanobacter harbinense YUAN-3 is a highly efficient hydrogen producing strain. It needs boiling aeration in laboratory to remove oxygen from the environment. This method has the disadvantages of high cost, secondary pollution and difficulty in use. Especially considering the expansion of anaerobic culture, it is a new way to realize anaerobic culture by means of interspecific respiratory interaction. The interaction between bacteria is widespread and varied. In this paper, these interactions have been fully interpreted by co-cultured facultative bacteria Pseudomonas aeruginosa PAO1 and strict anaerobic bacteria Harbin ethanol producing bacilli YUAN-3. We used a specific medium to produce hydrogen from organisms under aerobic conditions, which was previously considered impossible. In the experiment of hydrogen production by sequencing batch culture, the feasibility of producing hydrogen by co-culture of two bacteria in unanaerobic medium was first investigated. The presence and absence of L-cysteine could not produce hydrogen gas within 300 hours of culture, but the co-cultured system could produce gas after inoculation. And the medium added L-cysteine showed higher hydrogen yield and hydrogen production rate. In the subsequent experiments, we selected the concentration of L- cysteine of 0 m MU, 5 m M, 10 m M, 15 mm, of which 5 m M L- cysteine obtained the highest hydrogen yield, reaching 1.05 mol-hydrogen / mol-glucose, and the concentration of L-cysteine was 1.05 mol 路mol ~ (-1) 路L ~ (-1) 路L ~ (-1). Higher concentration of L- cysteine increased hydrogen production rate, cell dry weight and agglutination, which indicated that L- cysteine could promote hydrogen production, while excessive concentration could promote bacteria growth more obviously. The concentration and type of substrate also had a significant effect on hydrogen production. The hydrogen production efficiency of co-culture at different substrate concentrations was investigated. Among them, 15 g / L glucose obtained a complete gas production curve and the hydrogen yield reached the highest. 1.04 mol-hydrogen / mol-glucose. Under the condition of 5mm L- cysteine and 15g / L glucose, the strains were cultured continuously. The micelles formed in the culture medium were inoculated into a new medium, and the anaerobic YUAN-3 was used as the control. Although the traditional anaerobic culture method achieved a higher hydrogen production rate, the co-culture method obtained a higher hydrogen yield of 1.11 mol-hydrogen / mol-glucose. Considering that both bacteria can use glucose as carbon source, we used different disaccharides as substrate for co-culture, which greatly reduced the competitive effect and maximized the synergistic effect. The best effect was obtained by using sucrose as substrate. The hydrogen production rate reached 73 mL 路L ~ (- 1) h under the condition of 5m M cysteine and 15 g / L sucrose, and the hydrogen yield reached 2.58 mol 路h ~ (-1) 路mol ~ (-1) sucrose.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ116.2;TQ920.6

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