基于窖泥微生物相对定量与绝对定量联用的窖泥老熟特征分析

目的] 基于传统的相对定量法，在总DNA提取过程中加入内标菌，探索不同老熟程度下窖泥微生物菌群组成特征。方法] 通过添加外源菌丙酮丁醇梭菌（Clostridium acetobutylicum）作为内标菌，利用16S rRNA基因扩增子测序技术，基于相对丰度与绝对丰度解析不同老熟程度窖泥内的菌群结构和理化差异。结果] 相对定量分析表明，随着窖泥老熟，窖泥微生物群落的多样性增加，己酸菌属（Caproiciproducens）、甲烷杆菌属（Methanobacterium）、甲烷八叠球菌属（Methanosarcina）和互营单胞菌属（Syntrophomonas）等菌的相对丰度显著增高。结合绝对丰度分析，老熟窖泥内的生物量分别是新窖泥与趋老熟窖泥的600倍与28倍。不同于相对定量分析，乳杆菌属（Lactobacillus）的绝对丰度在老熟窖泥内并未降低，为新窖泥的20倍。己酸菌属在老窖泥中的绝对丰度为新窖泥的25000倍；其他功能微生物包括甲烷杆菌属、甲烷八叠球菌属、甲烷短杆菌属（Methanobrevibacter）、互营单胞菌属的绝对丰度也随着窖龄的增加而显著增加。菌群结构的绝对定量动态分析表明，新窖泥转变为老熟窖泥的过程是一个菌群组成多样性不断提高、生物量也逐步增加的过程，老窖泥中己酸菌属、互营单胞菌属、多种甲烷菌等微生物绝对量的增加，推测对降低窖泥内的乳酸含量和形成窖泥稳态环境具有重要作用。结论] 绝对定量方法的引入，可观测不同窖龄窖泥微生物生物量的差异，解析不同老熟阶段窖泥功能微生物绝对丰度的动态变化，为窖泥菌群结构和代谢功能的定量化和因果关系研究提供了新的策略。

Profiling the community structure of pit clay during aging process with the combination of relative and absolute quantitative approaches

Objective] Based on the traditional relative quantitative method, the microbial community in pit clay with different cellar ages was investigated by using the absolute quantitative method with addition of an internal standard at the DNA extraction step. Methods] According to the relative and absolute quantitative results, the microbial community and physicochemical factors of pit clay were profiled by using Clostridium acetobutylicum as an internal standard through 16S rRNA gene high-throughput sequencing. Results] With the increase of cellular age, the diversity of microbial community in pit clay increased, and the relative abundances of representative genera such as Caproiciproducens, Methanobacterium, Methanosarcina, and Syntrophomonas significantly increased. By using the absolute quantitative approach, the biomass in the aged pit clay was 600 times and 28 times as much to that in the new pit clay and the aging pit clay, respectively. Compared with the relative quantification, on the contrary, the absolute abundance of Lactobacillus in aged pit clay did not decrease rather than increase during the aging process, with 20 times as that in new pit clay. The absolute abundance of Caproiciproducens in aged pit clay was 25000 times as much to that in new pit clay. Moreover, the absolute abundances of other functional microorganisms such as Methanobacterium, Methanosarcina, Methanobrevibacter and Syntrophomonas also increased across the aging process. The absolute abundance analysis indicates that the aging process of pit clay is a process with increased diversity as well as biomass of microbial community, contributing to lactate depletion and thus maintaining the stability of pit clay microbiota. Conclusion] With the combination of relative and absolute quantitative approaches, the dynamic changes of species members in microbial community were profiled during the pit clay aging process. This study will help to provide a new quantitative and causal correlation analysis method to decipher the composition and function of pit clay microbiome.