16S rRNA基因高通量测序分析牛粪发酵细菌多样性

摘要

将养殖粪便进行资源化处理，尤其是将粪便堆肥发酵后变为生物肥料还田，具有重要的经济、社会和生态效益。之前关于细菌在堆肥过程中的研究，大部分采用实验室培养、分离、鉴定的方法，由于受培养方式的限制，仅能分析粪肥中有限的细菌类别。16S rRNA基因作为生物物种的特征核酸序列，被认为是最适于细菌系统发育和分类鉴定研究的指标。本研究使用16S rRNA基因高通量测序技术，分析了牛粪自然发酵与添加益生菌剂发酵过程中细菌种群的多样性变化。结果表明，1）新鲜牛粪、自然发酵1个月、自然发酵6个月的牛粪中细菌种群并没有明显的变化规律，说明自然发酵过程主要依赖于新鲜牛粪中携带的细菌种群；2）添加益生菌发酵后，细菌种群明显不同于不自然发酵过程中的细菌种群，其中变形菌门（Proteobacteria）细菌显著增加，而厚壁菌门（Firmicutes）细菌显著减少，说明益生菌剂能够显著改变堆肥过程中的细菌种群。本研究对于理解牛粪堆肥过程、提高堆肥效果，以及新型堆肥益生菌剂的开发都具有重要意义。%The traditional decentralized animal breeding is gradually being replaced by the large-scale intensive animal production in recent decades in China. While the large number of livestock manure is now becoming the source of environmental pollution, and the limiting factor for the sustainable development of animal husbandry. Resourceful treatments of the livestock manure, especially for return to fields as bio-fertilizer after piling fermentation, have important economic, social and ecological benefits. The previous bacterial studies during the manure fermentation mainly focus on bacterium culture, isolation, and identification. For the limitation of the culture conditions, limited bacteria could be analyzed. 16S rRNA locates on small subunit of prokaryotic ribosomes, including 10 conserved regions and 9 high variable regions, wherein the high variable region has species specificity among bacteria. The 16S rRNA gene was considered to be the characteristic nucleic acid of the bacterium, and it could be used in the bacterial phylogenesis and taxonomic identification studies. In this study, the cattle manure was collected and piled for fermentation. The fresh manure (m0), manure with natural fermentation for 1 month and 6 months (m1, m6), and manure with probiotic-adding fermentation for 3 months (mf) were sampled separately. Total bacterial DNA were isolated from these samples, and the V3 region of the 16S rRNA gene were PCR amplified using the total bacterial DNA as templates. And then the bacterial diversity in these samples were analyzed through high-throughput sequencing of the amplicons. The results indicated that: 1) the average observed species in m0, m1, m6 and mf groups was 1440, 1364, 1239 and 1353, respectively. The sum number of bacteria Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes accounted for more than 70% of the total bacteria number in each group. Statistical analysis showed that there are no differences of the observed species numbers, Shannon index and Chao1 richness estimator between all fermentation groups, suggesting there were high bacterial diversity in the compost pile, and the bacterial diversity didn’t reduced during the fermentation process. 2) the m0、m1 and m6 groups were clustered into a large branch, in which the m1 and m6 groups mixed together and were difficult to be separated by the principal component analysis (PCA) and cluster analysis. The clustering results suggested that the bacteria community structures of the manure were similar after 1 month and 6 months of natural fermentation, and they were dependent on the manure bacteria community structure of the fresh manure. The mf group was clustered into an independent branch relative to the m0, m1 and m6 groups, suggesting that the probiotics added into the manure changed the bacteria community structure significantly. 3) the analysis of the species differences between different fermentation groups showed that, compared with natural fermentation groups (m0, m1 and m6), the proportion of the Proteobacteria significantly increased (P < 0.05) in the probiotic-adding fermentation group (mf), while the Firmicutes decreased significantly (P < 0.05). This study may shine a light on understanding the fermentation process, improving the compost effects, even developing a new probiotics preparation in the future.