低温对沼气菌群产气能力的影响以及产甲烷菌的分离
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摘要
沼气发酵过程是一个由多种微生物联合,交替作用的复杂生化过程。在发酵过程中,不产甲烷细菌和产甲烷菌之间,相互依赖,又互相制约,在发酵过程中处于平衡状态。产甲烷菌是沼气发酵微生物的核心,同时也是自然界碳素循环中厌氧生物链的最后一个成员。甲烷的生物合成是沼气发酵过程的关键步骤,分离鉴定越冬沼气池中占优势的产甲烷菌及研究产甲烷菌群落结构的演替变化,将有利于更好的了解北方冬季条件下产甲烷菌群的活动。这对于有效地控制发酵过程,了解发酵进行的阶段,优化发酵条件,提高产气效率,具有十分重要的意义。
国内对沼气池中产甲烷菌的研究由来已久,但是对低温条件下沼气池中产甲烷菌的研究却不多见。本论文就厌氧发酵过程中产沼气微生物菌群在不同的低温条件下产气能力及厌氧发酵过程中微生物菌群的变化进行了研究,以便为在低温条件下沼气的可控性发酵提供参考依据。并通过传统的Hungate厌氧技术从越冬沼气池中分离出产甲烷菌并进行了初步的鉴定。试验结果表明:
1、通过对比不同温度条件下厌氧发酵的试验发现,在10-30℃温度范围内,产甲烷菌的产气能力随着温度的降低而减小;并且其日产气量的最高峰也随之往后推迟;而且温度在15℃以下时不适宜产甲烷菌的生长。
2、通过对沼气发酵液的pH值测定及其上清液中微生物种类的观察,可以得出,沼气发酵菌群适宜生长的pH值在7.0-8.0之间;并且在沼气发酵过程中,微生物菌群的变化也比较明显,初期以球菌占优势,后期则转为杆菌类为优势菌。
3、通过改进的Hungate厌氧技术从越冬沼气池中分离出一株产甲烷菌。该菌株呈球状,革兰氏染色为阴性,不运动,常单个或成对出现;不形成芽孢,菌落圆形,边缘完整,表面光滑,不透明,淡黄色,在紫外荧光显微镜下,发出蓝绿色荧光。
4、该菌株能利用甲酸钠作为碳源,不能利用甲醇、乙酸钠、三甲胺、丙醇等。该菌株的生长条件温和,在20℃-50℃范围内均可生长,其最适的温度范围为25℃-35℃;其能在pH 5.5-8.0范围内生长产气,最适pH范围为6.0-7.0,高于8.0或低于5.5均不能产气。
The methane fermentation is a process that unites by the many kinds of microorganisms, with replacement complex biochemistry process in it. In the fermentation, the interdependence and restriction of no-produce-methane bacterium and methanogens makes a equilibrium in the fermentation. Methanogen is the core of the methane fermentation. And methanogen is the last member of the anaerobe chain in nature carbon cycle. One of the essential steps of methane fermentative process is the biosynthesis of methane, so the distribution of the superior methanogen species and its customs are widely studied. The study on changes of methanogen in the methane fermentation can make us realize fermentation degree, optimize the fermentation condition and enhance the methane output. And it will be benefited to improve the efficiency of methane fermentation.
There is rare study on the conditions of low temperature in methanogen. This paper studied on the methane production in different low temperature, and microbial flora change during fermentation in order to give a reference for controllability of fermentation, and made preliminary identification of methane bacteria which separate from biogas pool in winter by Hungate anaerobic operation technique. The main conclusions were as follow:
1、Compared different fermentation experiments in different temperature, Between 10-30℃, methane production reduced when the temperature decreased, the pink of daily methane production had delayed at the same time. It was not fit for methanogen growing when the temperature was below 15℃.
2、By monitored pH value and variety of microorganism in the upper liquid of the fermentation, we can conclude that: the methanogen microb flora appropriate pH value is between 7.0-8.0, which is same as most single methanogen, and during fermentation, micro communicate has changed distinctly. At the initial stage methanococcus predominated, but methanobacterium become predominate in end stage.
3、One methanogenic bacteria was isolated by the modified Hungate anaerobic techniques. The strain is curved, Gˉ, non-motile, often single or the doubling appeared, and didn’t form the bud-spore. The colony was circular, smooth, light-yellow, and emitted cyan color fluorescence under the ultraviolet fluorescence microscope.
4、The strain can use formate but not methanol, acetic sodium and trimethylamine as the substrate. The optimal pH is 6.0-7.0, and the optimal temperature is 25-35℃.
国内对沼气池中产甲烷菌的研究由来已久,但是对低温条件下沼气池中产甲烷菌的研究却不多见。本论文就厌氧发酵过程中产沼气微生物菌群在不同的低温条件下产气能力及厌氧发酵过程中微生物菌群的变化进行了研究,以便为在低温条件下沼气的可控性发酵提供参考依据。并通过传统的Hungate厌氧技术从越冬沼气池中分离出产甲烷菌并进行了初步的鉴定。试验结果表明:
1、通过对比不同温度条件下厌氧发酵的试验发现,在10-30℃温度范围内,产甲烷菌的产气能力随着温度的降低而减小;并且其日产气量的最高峰也随之往后推迟;而且温度在15℃以下时不适宜产甲烷菌的生长。
2、通过对沼气发酵液的pH值测定及其上清液中微生物种类的观察,可以得出,沼气发酵菌群适宜生长的pH值在7.0-8.0之间;并且在沼气发酵过程中,微生物菌群的变化也比较明显,初期以球菌占优势,后期则转为杆菌类为优势菌。
3、通过改进的Hungate厌氧技术从越冬沼气池中分离出一株产甲烷菌。该菌株呈球状,革兰氏染色为阴性,不运动,常单个或成对出现;不形成芽孢,菌落圆形,边缘完整,表面光滑,不透明,淡黄色,在紫外荧光显微镜下,发出蓝绿色荧光。
4、该菌株能利用甲酸钠作为碳源,不能利用甲醇、乙酸钠、三甲胺、丙醇等。该菌株的生长条件温和,在20℃-50℃范围内均可生长,其最适的温度范围为25℃-35℃;其能在pH 5.5-8.0范围内生长产气,最适pH范围为6.0-7.0,高于8.0或低于5.5均不能产气。
The methane fermentation is a process that unites by the many kinds of microorganisms, with replacement complex biochemistry process in it. In the fermentation, the interdependence and restriction of no-produce-methane bacterium and methanogens makes a equilibrium in the fermentation. Methanogen is the core of the methane fermentation. And methanogen is the last member of the anaerobe chain in nature carbon cycle. One of the essential steps of methane fermentative process is the biosynthesis of methane, so the distribution of the superior methanogen species and its customs are widely studied. The study on changes of methanogen in the methane fermentation can make us realize fermentation degree, optimize the fermentation condition and enhance the methane output. And it will be benefited to improve the efficiency of methane fermentation.
There is rare study on the conditions of low temperature in methanogen. This paper studied on the methane production in different low temperature, and microbial flora change during fermentation in order to give a reference for controllability of fermentation, and made preliminary identification of methane bacteria which separate from biogas pool in winter by Hungate anaerobic operation technique. The main conclusions were as follow:
1、Compared different fermentation experiments in different temperature, Between 10-30℃, methane production reduced when the temperature decreased, the pink of daily methane production had delayed at the same time. It was not fit for methanogen growing when the temperature was below 15℃.
2、By monitored pH value and variety of microorganism in the upper liquid of the fermentation, we can conclude that: the methanogen microb flora appropriate pH value is between 7.0-8.0, which is same as most single methanogen, and during fermentation, micro communicate has changed distinctly. At the initial stage methanococcus predominated, but methanobacterium become predominate in end stage.
3、One methanogenic bacteria was isolated by the modified Hungate anaerobic techniques. The strain is curved, Gˉ, non-motile, often single or the doubling appeared, and didn’t form the bud-spore. The colony was circular, smooth, light-yellow, and emitted cyan color fluorescence under the ultraviolet fluorescence microscope.
4、The strain can use formate but not methanol, acetic sodium and trimethylamine as the substrate. The optimal pH is 6.0-7.0, and the optimal temperature is 25-35℃.
引文
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