浓香型白酒窖泥中互营细菌与梭菌互作机制及其功能解析

Unveiling species interaction networks is fundamental to understanding microbial community stability and ecosystem functioning. Clostridia is the predominant class in pit mud of strong-flavor baijiu (SFB), and it is the major producers of butyric acid and hexanoic acid, which pertain to the key aroma contributors of SFB. Amongst Clostridia, Clostridium genus plays a pivotal role in producing short-chain fatty acids (SCFA), however, the capacity of SCFA-biosynthesis is species-specific. Previous researches showed some species could produce SCFA merely when co-cultured with other microorganisms. We also identified the synergistic interactions of Clostridium and syntrophic bacteria, resulting in significant enhancement of SCFA production, especially butyric acid and hexanoic acid. Nevertheless, the underlying mechanisms causing these synergistic effects are not well understood. Here, the interactions of syntrophic bacteria and Clostridium scattered in different species were investigated through microbial cultivation, multi-omics and other approaches. Comparative analysis of their growth, metabolic and morphological characteristics was performed between mono- and co-culture. Subsequently, gene expression levels were analyzed by RNA-Seq to reveal the divergent SCFA biosynthetic genes and metabolism pathways in Clostridium versus the co-culture system. We will also explore the in situ effects of their interactions in shaping the microbial community structure and influencing their metabolic functions in pit mud by metatranscriptomics analysis. This project provides a scientific basis to better understand the importance of microbial interactions in regulating the structure and function of brewing microbiota, which is indispensable for instructing the preparation, maintenance and quality improvement of artificial pit mud.

白酒窖泥微生物相互作用是保障其群落结构稳定和功能发挥的重要基础。梭菌纲（Clostridia）是浓香型白酒窖泥菌群中的主体微生物，亦是浓香白酒典型呈味物质丁酸和己酸的重要产生菌。其中的梭菌（Clostridium）产脂肪酸能力具有种特异性，已有研究显示部分梭菌只有与其它菌共栖生长才产酸。申请人也发现有些低产或几乎不产酸的梭菌在与互营细菌共培养时，它们的产酸量显著提高。然而，上述共栖互作产酸现象的机理尚不清楚。本项目拟采用共培养结合多组学分析等技术考察不同种梭菌与互营细菌的相互作用关系类型；解析梭菌与互营细菌相互作用对基因表达与代谢通路的调控，以及对产酸的影响；探究梭菌与互营细菌相互作用对窖泥菌群结构演替与重要产酸功能的影响，以及原位条件下两者的互作关系。本项目可为更好地理解微生物互作在调控酿造菌群结构与功能中的关键作用提供科学依据，为人工窖泥养护与老熟等技艺的理性改进奠定理论基础。

物种间的交互作用对微生物群落的结构、组装与演替以及生态功能有着重要影响。浓香型白酒以泥窖池为发酵容器，窖泥菌群在其典型风味物质前体（丁酸、己酸等）形成中发挥着关键作用。互营细菌（如互营球菌、互营单胞菌）和梭菌属微生物是窖泥中的优势功能菌，它们的代谢活动和丁酸、己酸等脂肪酸的产生密切相关。前期研究发现互营球菌与梭菌共培养影响脂肪酸的积累，因而，本项目针对它们的交互作用开展研究。以分离自窖泥的互营球菌（Novisyntrophococcus fermenticellae JN500902, N.902）和梭菌属（Clostridium）不同种菌株为研究对象，首先对比了互营球菌和梭菌纯培养与共培养间生长与代谢特征的差异，发现N.902和不同梭菌属微生物之间的互作类型表现出种水平的多样性；为充分了解梭菌属微生物的代谢特征，我们对30株分离自窖泥的梭菌和100株分离自其他环境（如肠道、土壤）的梭菌进行了比较基因组分析，结果表明，大多数窖泥梭菌菌株具有代谢乙醇、乳酸和淀粉合成脂肪酸的良好潜力，进一步地围绕明确互作关系类型的8株菌进行了短链脂肪酸代谢途径构建；选取共生促生长和产酸的组合，即N.902和老窖梭菌（Clostridium fermenticellae JN500901, C.901），通过转录组学等手段阐释了它们之间的互作机制，即C.901产生的甲酸能被N.902通过Wood-Ljungdahl途径所利用，促进其乙酸的产生，乙酸又进一步被C.901通过逆β-氧化途径利用，从而促进了丁酸和己酸的积累；最后通过研究不同碳源对窖泥微生物群落的影响，确定了窖泥培养条件，将N.902和不同梭菌属微生物进行两两或多菌组合强化至窖泥培养体系，探究了互营球菌与梭菌共栖对窖泥菌群结构和功能的调控作用。本研究有助于加深对窖泥中功能微生物间复杂互作关系的科学认识，同时也为人工理性调控窖泥菌群，实现“人工新窖老熟”奠定理论基础。