沼气发酵复合菌系及其在牛粪厌氧发酵中的应用
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摘要
以自然沼液为接种物,利用限制性培养技术,构建得到1组35℃下遗传稳定、产甲烷量高的沼气发酵复合菌系。利用16S rDNA克隆文库技术研究了该复合菌系的菌种组成多样性,并通过单因素试验和正交试验获得了该复合菌系的最佳培养条件。以该沼气发酵复合菌系为接种物,研究了在20L发酵体系中不同的接种量、物料浓度对沼气发酵的影响。同时,还对牛粪中不同N、P、K浓度对厌氧发酵产气的影响进行了研究。
1、继代培养结果表明,该复合菌系经过26代的培养能够稳定产气,各代间的产气情况和产气量差别不大。以该复合菌系作为接种物,10%的接种量接种于TS为5%的100mL沼气培养装置中,最高日产气量为1.6m3·m-3·d-1,最高日产甲烷量可达1.5 m3·m-3·d-1。通过单因素试验和正交试验得出该复合菌系以牛粪为底物最佳的发酵条件为接种量5%,TS为10%,培养温度35℃,pH自然。
2、利用16S rDNA克隆文库技术对复合菌系菌种组成多样性分析可知,该复合菌系中产甲烷古菌主要由甲烷袋状菌属(Methanoculleus sp.)、甲烷八叠球菌属(Methanosarcina mazeii)、产甲烷古菌(methanogenic archaeon)、广域古菌(Euryarchaeote)组成;细菌主要由消化球菌(Peptococcaceae bacterium)、拟杆菌属(Bacteroides sp.)、真杆菌(eubacterium)、纤维杆菌(Fibrobacteres bacterium)、假单胞菌属(Pseudomonas sp.)、梭菌属(Clostridium sp.)、氨基杆菌(Aminobacterium colombiense)、月形单胞菌属(Selenomonas sp.)及未培养细菌(Uncultured bacterium)组成,说明该复合菌系是由多种功能不同的微生物类群组成且具有很高的多样性。
3、沼气发酵复合菌系在20L发酵体系中的试验结果表明,15%的接种量处理组总产气量最高,且总产甲烷量分别比5%、10%的处理组高37.4%和34.5%。5%的接种处理组的总产气量、总产甲烷量比对照提高3.1%和11.8%。综合考虑甲烷产生的时间、最高含量和总产甲烷量,放大培养的适宜接种量范围为15%。底物不同浓度试验表明TS为10%的底物浓度产气效果最好。
4、初始发酵料液中氮、磷、钾含量对沼气发酵产气影响较大,在底物氮、磷、钾浓度的基础上提高1倍的处理总产气量分别比基础试验组、二倍添加量试验组、三倍添加量试验组高2.4%、78.2%、78.7%,总产甲烷量分别比基础试验组、二倍添加量试验组、三倍添加量试验组高7.3%、96.4%、99.6%,提高2倍和3倍的处理接种后12h后产气,但3d后产气停止,气体中甲烷含量极低。
A microbial community capable of biogas production with genetic stability was enriched from biogas slurry with the method of limited culture at 35℃. The microbial diversity was detected using 16S rDNA clone technology. The optimum cultural conditions of microbial community were investigated with the methods of single factor analysis and orthogonal test analysis. Effects of different quantities of inoculation, materiel concentrations on biogas production were investigated in the fermentation system of 20L. The effects of initial concentration of nitrogen, phosphorus and potassium increased to one, twice and trible times compared with control were researched on biogas production.
The limited culture results showed that biogas production of each subculture was stabile after 26 subcultures, and the maximum daily biogas production and methane production was 1.6 m3·m-3·d-1 and 1.5 m3·m-3·d-1 under the condition of 100 mL volume of fermentation system, 5% total solid and 10% of inoculation of microbial community. The results of single factor analysis and orthogonal test analysis showed that the optimum cultural conditions of microbial community for biogas production using the cattle manure as the material were 5% of inoculation quantity, 10% of concentration of substrate,natural pH and temperature 35℃.
The microbial diversity of microbial community was analyzed using 16S rDNA molecular clone technology. The results showed that archaebacteria group was composed with Methanoculleus sp., Methanosarcina mazeii, methanogenic archaeon, Euryarchaeote. Bacterial group was composed with Peptococcaceae bacterium, Bacteroides sp., Eubacterium, Fibrobacteres bacterium,Pseudomonas sp., Clostridium sp., Aminobacterium colombiense, Selenomonas sp. and uncultured bacterium. Therefore the microbial community was composed of different archaebacteria and eubacteria, it had a high microbial diversity.
The results of experiment of diffrernt inoculum quantities showed that the biogas production of treatment of 15% inoculum (V/V) was the highest, and the total methane production was higher 37.4% and 34.5 % compared with treatments of 5% and 10% inoculum. Total biogas production and total methane production of 5% inoculum treatment were higher 3.1% and 11.8% compared with control. The optimum inoculation quantity of scale-up culture is 15% comprehensively considering the biogas production time, the maximum biogas production level, and the total production level of methane. At the same time, the treatment of 10% TS of substrate concentration was the optimum for biogas production.
The initial concentration nitrogen, phosphorus, and potassium of feed solution significantly effected biogas production. The total biogas production of the treatment that the total content of nitrogen, phosphorus, and potassium were increased one time more than basic concentration was improved 2.4%, 78.2%, 78.7% compared with basic content treatment, two time treatment and three time treatment, respectively. Futhermore, the methane production of one time content treatment was increased 7.3%, 96.4% and 99.6% compared with other treatments, respectively. The some gas was producted in treatments of twice and trible times more than basis nitrogen, phosphorus, and potassium concentration after inoculated 12 hours, but the methane almost was not detected among the gas ingredients.
1、继代培养结果表明,该复合菌系经过26代的培养能够稳定产气,各代间的产气情况和产气量差别不大。以该复合菌系作为接种物,10%的接种量接种于TS为5%的100mL沼气培养装置中,最高日产气量为1.6m3·m-3·d-1,最高日产甲烷量可达1.5 m3·m-3·d-1。通过单因素试验和正交试验得出该复合菌系以牛粪为底物最佳的发酵条件为接种量5%,TS为10%,培养温度35℃,pH自然。
2、利用16S rDNA克隆文库技术对复合菌系菌种组成多样性分析可知,该复合菌系中产甲烷古菌主要由甲烷袋状菌属(Methanoculleus sp.)、甲烷八叠球菌属(Methanosarcina mazeii)、产甲烷古菌(methanogenic archaeon)、广域古菌(Euryarchaeote)组成;细菌主要由消化球菌(Peptococcaceae bacterium)、拟杆菌属(Bacteroides sp.)、真杆菌(eubacterium)、纤维杆菌(Fibrobacteres bacterium)、假单胞菌属(Pseudomonas sp.)、梭菌属(Clostridium sp.)、氨基杆菌(Aminobacterium colombiense)、月形单胞菌属(Selenomonas sp.)及未培养细菌(Uncultured bacterium)组成,说明该复合菌系是由多种功能不同的微生物类群组成且具有很高的多样性。
3、沼气发酵复合菌系在20L发酵体系中的试验结果表明,15%的接种量处理组总产气量最高,且总产甲烷量分别比5%、10%的处理组高37.4%和34.5%。5%的接种处理组的总产气量、总产甲烷量比对照提高3.1%和11.8%。综合考虑甲烷产生的时间、最高含量和总产甲烷量,放大培养的适宜接种量范围为15%。底物不同浓度试验表明TS为10%的底物浓度产气效果最好。
4、初始发酵料液中氮、磷、钾含量对沼气发酵产气影响较大,在底物氮、磷、钾浓度的基础上提高1倍的处理总产气量分别比基础试验组、二倍添加量试验组、三倍添加量试验组高2.4%、78.2%、78.7%,总产甲烷量分别比基础试验组、二倍添加量试验组、三倍添加量试验组高7.3%、96.4%、99.6%,提高2倍和3倍的处理接种后12h后产气,但3d后产气停止,气体中甲烷含量极低。
A microbial community capable of biogas production with genetic stability was enriched from biogas slurry with the method of limited culture at 35℃. The microbial diversity was detected using 16S rDNA clone technology. The optimum cultural conditions of microbial community were investigated with the methods of single factor analysis and orthogonal test analysis. Effects of different quantities of inoculation, materiel concentrations on biogas production were investigated in the fermentation system of 20L. The effects of initial concentration of nitrogen, phosphorus and potassium increased to one, twice and trible times compared with control were researched on biogas production.
The limited culture results showed that biogas production of each subculture was stabile after 26 subcultures, and the maximum daily biogas production and methane production was 1.6 m3·m-3·d-1 and 1.5 m3·m-3·d-1 under the condition of 100 mL volume of fermentation system, 5% total solid and 10% of inoculation of microbial community. The results of single factor analysis and orthogonal test analysis showed that the optimum cultural conditions of microbial community for biogas production using the cattle manure as the material were 5% of inoculation quantity, 10% of concentration of substrate,natural pH and temperature 35℃.
The microbial diversity of microbial community was analyzed using 16S rDNA molecular clone technology. The results showed that archaebacteria group was composed with Methanoculleus sp., Methanosarcina mazeii, methanogenic archaeon, Euryarchaeote. Bacterial group was composed with Peptococcaceae bacterium, Bacteroides sp., Eubacterium, Fibrobacteres bacterium,Pseudomonas sp., Clostridium sp., Aminobacterium colombiense, Selenomonas sp. and uncultured bacterium. Therefore the microbial community was composed of different archaebacteria and eubacteria, it had a high microbial diversity.
The results of experiment of diffrernt inoculum quantities showed that the biogas production of treatment of 15% inoculum (V/V) was the highest, and the total methane production was higher 37.4% and 34.5 % compared with treatments of 5% and 10% inoculum. Total biogas production and total methane production of 5% inoculum treatment were higher 3.1% and 11.8% compared with control. The optimum inoculation quantity of scale-up culture is 15% comprehensively considering the biogas production time, the maximum biogas production level, and the total production level of methane. At the same time, the treatment of 10% TS of substrate concentration was the optimum for biogas production.
The initial concentration nitrogen, phosphorus, and potassium of feed solution significantly effected biogas production. The total biogas production of the treatment that the total content of nitrogen, phosphorus, and potassium were increased one time more than basic concentration was improved 2.4%, 78.2%, 78.7% compared with basic content treatment, two time treatment and three time treatment, respectively. Futhermore, the methane production of one time content treatment was increased 7.3%, 96.4% and 99.6% compared with other treatments, respectively. The some gas was producted in treatments of twice and trible times more than basis nitrogen, phosphorus, and potassium concentration after inoculated 12 hours, but the methane almost was not detected among the gas ingredients.
引文
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