产氢产乙酸互营共培养体的选育及其应用基础研究
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摘要
传统观点认为,产甲烷作用是厌氧消化过程的限速步骤。然而,前期研究发现,产氢产乙酸菌群的生态幅要比产甲烷菌更加狭窄,对环境的变化更加敏感,其代谢强度直接决定了产甲烷菌群的增殖和代谢能力,因此提出了“产氢产乙酸菌群的产氢产乙酸作用是厌氧消化的第一限速步骤”这一学术观点。产氢产乙酸菌的分离纯化非常困难,其研究进展缓慢,目前获得的纯培养物只有少数几个。种子资源的匮乏,极大限制了基于强化产氢产乙酸作用的高效厌氧生物处理技术的研发。基于以上认识和研究现状,本论文开展了产氢产乙酸互营共培养体的分离筛选工作,并以此为基础,对所筛选的共培养体进行了生理生态特性研究,并进一步探索了以其强化厌氧生物处理系统效能的可行性。
论文利用基质选择作用,通过选择培养基的传代培养和代时控制以及培养条件的优化,最终选育到7号和8号两个产氢产乙酸菌互营共培养体。在含有丙酸钠和丁酸钠各10 g/L的培养基中,经过30 d的培养,7号和8号互营共培养体的乙酸产量分别达到2485 mg/L和3362 mg/L,产氢率分别达到549.06 mL/L-culture和456.57 mL/L-culture。
通过PCR-DGGE分子检测手段,分析了7号和8号两个互营共培养体的种群结构。在PCR-DGGE图谱中,7号培养物显示出4个条带,8号则呈现出5个条带,而且两个共培养体中有重叠条带,说明这两个培养物均为微生物的混合培养物。将扩增得到的所有16SrDNA序列测序,并构建了系统发育树,发现两个共培养体中都包含有专性产氢产乙酸菌Syntrophospora bryantiii和专性的互营产乙酸菌Desulfotomaculum sp.,除此之外还有伴生菌,如能利用甲酸盐和H2/CO2的Uncultured bacterium和Sedimentibacter sp.等。
生态因子对7号和8号两个互营共培养体的生长和产氢产乙酸特性研究表明,以丁酸作为碳源(10 g/L)、胰蛋白胨和酵母膏作为氮源时,7号共培养体在35℃、初始pH 7的条件下,具有最佳的生长和产氢产乙酸能力;8号共培养体则在45℃、初始pH 7.5时具有最大的生长和产氢产乙酸能力。
摇瓶发酵试验结果表明,7号和8号两个互营共培养体是开发厌氧发酵系统生物强化技术的良好种子资源。取自发酵制氢反应系统的污泥,在投加7号共培养体后,其氢气产量和氢气产生速率分别可提高1.15倍和1.24倍;取自厌氧废水处理系统的污泥,在投加8号共培养体后,其底物转化率和甲烷产生速率可分别提高1.35倍和1.76倍。初步证明了以产氢产乙酸菌互营共培养体强化厌氧生物处理系统效能的可行性。
Among the bacteria colony in the anaerobic digestion system, hydrogen-producing acetogens (HPA) connect acidogenic fermentation bacteria with methanogens in functional niche. But HPA is quite difficult to be isolated in purity and ampliative culture by reason of the accrete characteristic.In order to research into its physiological and ecological characteristics and to lay the scientific basis for the development of highly efficient anaerobic biological treatment technology,the syntrophic acetogen cocultures were enriched and Selective bred by the Selective medium which is mixed by the Propionic acid and butyric acid from the anaerobic activated sludge with methanogens. Cultured in selective medium with propionic acid and butyric acid as carbon source for 30 days, the acetic acid yields for 7th and 8th were 2485 mg/L and 3326 mg/L with a hydrogen yield of 549.06 mL/L-culture and 456.57 mL/L-culture respectively.
The population structures of two co-cultures were made clear by polymerase chain reaction - denaturing gradient gel electrophoreses (PCR-DGGE) analysis. Four bandings were showed in the DGGE profile of 7th and five in 8th profile, indicating that two or more bacteria existed in 7th and 8th. The 16S rDNA sequences of all the clones from the 16 bandings showed that both 7th and 8th were new syntrophic acetogen cocultures with non-methanogens as the associated bacteria. The associated bacteria are uncultured bacterium Syntrophospora bryantiii and Desulfotomaculum sp., which could utilize the formate and the H2/CO2. There were no CH4 or H2S being evolved in both culture systems, while hydrogen and acetic acid were detected.
The influence of ecological factors on the growth and metabolism of the two co-cultures was investigated farther more. With butyrate as carbon source (10g/L), tryptone and the yeast extract as nitrogen source, 7th could present an excellent performance in growth and metabolism at 45℃with a initial pH 8, while 8th with a initial pH 7.
According to the reaseach of the physiology and ecology characters of the two co-cultures and the ability to producing hydrogen and acetic acid, Then the 7th and 8th co-cultures were separately put into the fermentation hydrogen production system and wastewater treatment system, The result of wave-bottled ferment examination prove that the two co-cultures are favorable seminal bacteria to expand bioaugmentation of anaerobic ferment system..The muddily mud from anaerobic ferment system by enhancing the 7th co-culture.The results show that the efficiency of hydric yield and speed were improved by enhancing the 8th co-culture with 1.15 and 1.24 times while improved 1.35 and 1.76 times in anaerobic organic wastewater treatment by enhancing the 8th co-culture.
论文利用基质选择作用,通过选择培养基的传代培养和代时控制以及培养条件的优化,最终选育到7号和8号两个产氢产乙酸菌互营共培养体。在含有丙酸钠和丁酸钠各10 g/L的培养基中,经过30 d的培养,7号和8号互营共培养体的乙酸产量分别达到2485 mg/L和3362 mg/L,产氢率分别达到549.06 mL/L-culture和456.57 mL/L-culture。
通过PCR-DGGE分子检测手段,分析了7号和8号两个互营共培养体的种群结构。在PCR-DGGE图谱中,7号培养物显示出4个条带,8号则呈现出5个条带,而且两个共培养体中有重叠条带,说明这两个培养物均为微生物的混合培养物。将扩增得到的所有16SrDNA序列测序,并构建了系统发育树,发现两个共培养体中都包含有专性产氢产乙酸菌Syntrophospora bryantiii和专性的互营产乙酸菌Desulfotomaculum sp.,除此之外还有伴生菌,如能利用甲酸盐和H2/CO2的Uncultured bacterium和Sedimentibacter sp.等。
生态因子对7号和8号两个互营共培养体的生长和产氢产乙酸特性研究表明,以丁酸作为碳源(10 g/L)、胰蛋白胨和酵母膏作为氮源时,7号共培养体在35℃、初始pH 7的条件下,具有最佳的生长和产氢产乙酸能力;8号共培养体则在45℃、初始pH 7.5时具有最大的生长和产氢产乙酸能力。
摇瓶发酵试验结果表明,7号和8号两个互营共培养体是开发厌氧发酵系统生物强化技术的良好种子资源。取自发酵制氢反应系统的污泥,在投加7号共培养体后,其氢气产量和氢气产生速率分别可提高1.15倍和1.24倍;取自厌氧废水处理系统的污泥,在投加8号共培养体后,其底物转化率和甲烷产生速率可分别提高1.35倍和1.76倍。初步证明了以产氢产乙酸菌互营共培养体强化厌氧生物处理系统效能的可行性。
Among the bacteria colony in the anaerobic digestion system, hydrogen-producing acetogens (HPA) connect acidogenic fermentation bacteria with methanogens in functional niche. But HPA is quite difficult to be isolated in purity and ampliative culture by reason of the accrete characteristic.In order to research into its physiological and ecological characteristics and to lay the scientific basis for the development of highly efficient anaerobic biological treatment technology,the syntrophic acetogen cocultures were enriched and Selective bred by the Selective medium which is mixed by the Propionic acid and butyric acid from the anaerobic activated sludge with methanogens. Cultured in selective medium with propionic acid and butyric acid as carbon source for 30 days, the acetic acid yields for 7th and 8th were 2485 mg/L and 3326 mg/L with a hydrogen yield of 549.06 mL/L-culture and 456.57 mL/L-culture respectively.
The population structures of two co-cultures were made clear by polymerase chain reaction - denaturing gradient gel electrophoreses (PCR-DGGE) analysis. Four bandings were showed in the DGGE profile of 7th and five in 8th profile, indicating that two or more bacteria existed in 7th and 8th. The 16S rDNA sequences of all the clones from the 16 bandings showed that both 7th and 8th were new syntrophic acetogen cocultures with non-methanogens as the associated bacteria. The associated bacteria are uncultured bacterium Syntrophospora bryantiii and Desulfotomaculum sp., which could utilize the formate and the H2/CO2. There were no CH4 or H2S being evolved in both culture systems, while hydrogen and acetic acid were detected.
The influence of ecological factors on the growth and metabolism of the two co-cultures was investigated farther more. With butyrate as carbon source (10g/L), tryptone and the yeast extract as nitrogen source, 7th could present an excellent performance in growth and metabolism at 45℃with a initial pH 8, while 8th with a initial pH 7.
According to the reaseach of the physiology and ecology characters of the two co-cultures and the ability to producing hydrogen and acetic acid, Then the 7th and 8th co-cultures were separately put into the fermentation hydrogen production system and wastewater treatment system, The result of wave-bottled ferment examination prove that the two co-cultures are favorable seminal bacteria to expand bioaugmentation of anaerobic ferment system..The muddily mud from anaerobic ferment system by enhancing the 7th co-culture.The results show that the efficiency of hydric yield and speed were improved by enhancing the 8th co-culture with 1.15 and 1.24 times while improved 1.35 and 1.76 times in anaerobic organic wastewater treatment by enhancing the 8th co-culture.
引文
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