摘要
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。
Sheep manure biogas fermentation under low temperatue（ 15℃ ） was treated with different bacteria agent, and bacteria diversity was analyzed by method of two gel concentration PCR-DGGE. Taking the samples from different treated sheep manure biogas fermentation reactors, and the total DNA in biogas slurry were extracted by lysozyme method. After purification, the total DNA were processed for 16S rDNA PCR amplification by primers 341F-GC and 518R. The amplified products were separated by PCR-DGGE method with two level gel concentration, and dominant bands were recovered for amplifing, sequencing and bacterial diversity analyzing. The results showed that the dominant bacteria in sheep manure biogas reactor were Bacteroidetes which accounted for 32% of totally recovered 44 bands, Proteobacteria accounted for 30%, Firmicutes accounted for 36%. Under the condition of 15 ℃, the gene fragments for dominant bacteria in the reactor were more abundant; the bacterial populations were significantly different for different treatment, the main dominant bacteria were Glostridium sp. , Proteiniphilum sp., Bacteroidales bacterium, Pseudomonas sp, Advenella sp; among which the Clostrldium bacteria showed higher abundance. Under the condition of 30 ℃, the dominant bacteria were Bacteroidales bacterium, Hydrogenophaga sp. , Pseudomonas sp. , Clostridiales bacterium; and the Hydrogenophaga sp. showed higher abundance; the band diversity nmnber less than that under 15 ℃. And addition of bacterial agent did not change the bacterial population significantly.
出处
《中国沼气》
北大核心
2014年第6期9-15,共7页
China Biogas
基金
国家自然科学基金项目(31260016)
新疆维吾尔自治区重大专项(201130104)
新疆维吾尔自治区高技术研究发展计划项目(201010104)
