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利用PCR-DGGE方法分析不同菌剂处理对羊粪沼气发酵微生物种群多样性的影响,了解低温条件下(15℃)羊粪沼气发酵过程中优势细菌群落结构多样性变化规律,从而为北方地区冬季低温沼气发酵促进菌剂的筛选奠定理论基础。通过对不同发酵处理的羊粪沼气反应器中取样,采用溶菌酶法,分别进行沼液中总DNA的提取,总DNA经纯化后采用引物341F-GC和518R进行细菌16S r DNA基因V 3区PCR扩增,扩增产物进行两种胶浓度变性梯度凝胶电泳(DGGE)分离并回收优势条带和变化明显的条带进行扩增、测序及系统进化树的绘制,分析其细菌结构多样性。序列比对的结果表明,羊粪沼气反应器中的优势细菌菌群为:回收的44条带中拟杆菌门(Bacteroidetes)占32%,变形菌门(Proteobacteria)占30%,厚壁菌门(Firmicutes)占36%。其中,在15℃条件下,反应器中细菌优势基因片段更为丰富均匀,存在相对稳定的优势种群,且各处理的优势菌的丰度比对照组中相应菌的丰度更强,存在差异条带,细菌种群变化明显,优势种群亦有所不同,主要有Clostridium sp.,Proteiniphilum sp.,Bacteroidales bacterium,Pseudomonas sp,Advenella sp.等,其中低温下菌剂处理的以梭菌属菌(Clostridium)表现有较高的丰度;而在30℃条件下,主要有Bacteroidales bacterium,Hydrogenophaga sp.,Pseudomonas sp,Clostridiales bacterium等,其中以噬氢菌属(Hydrogenophaga)Hydrogenophaga sp.表现有较高的丰度,条带多样性数量比15℃的要少,添加菌剂处理对细菌种群变化影响不明显。
PCR-DGGE method was used to analyze the effects of different microbial agents on the microbial diversity of sheep manure biogas fermentation and the variation of dominant bacterial community diversity during the biogas fermentation of sheep manure at low temperature (15 ℃) Low temperature biogas fermentation to promote the screening of bacterial agents laid the theoretical foundation. The total DNA in the biogas slurry was extracted by the lysozyme method after the biogas reactors of different mannizations were sampled. The total DNA was purified and then amplified with primers 341F-GC and 518R for the bacterial 16S r DNA gene V 3 region PCR amplification and amplification products were separated by gel concentration denaturing gradient gel electrophoresis (DGGE), and the dominant bands and obvious bands were recovered for amplification, sequencing and phylogenetic tree, and the bacterial diversity . The results of sequence alignment showed that the dominant bacterium flora in sheep manure biogas reactor was: Bacteroidetes accounted for 32%, Proteobacteria accounted for 30%, Bacteroidetes (Firmicutes) accounted for 36%. Among them, at 15 ℃, the bacterial advantage gene fragments in the reactor were more abundant, and there was a relatively stable dominant population, and the dominant bacteria in each treatment had a stronger abundance than the corresponding bacteria in the control group, with differences Bacterialoid bacteria, Pseudomonas sp, Advenella sp. And so on. Among them, Clostridium (Clostridium sp. ) Showed high abundance. However, Bacteroidales bacterium, Hydrogenophaga sp., Pseudomonas sp, Clostridiales bacterium and so on were mainly present at 30 ℃, of which Hydrogenophaga sp. Hydrogenophaga sp. Degree and band diversity were less than those of 15 ℃. The addition of fungicide had no obvious effect on the bacterial population.