基于高通量测序的窖泥原核微生物群落多样性在退化窖池中的空间异质性

Spatial Heterogeneity of Prokaryotic Microbial Community Diversity in Pit Mud from Degraded Cellar for the Production of Strong-flavor Baijiu Revealed by High Throughput Sequencing

采用高通量测序技术结合网络分析及多元统计分析方法,研究退化窖池窖壁上层(PW-T)、中层(PW-M)、下层(PW-U)及窖底(PB)窖泥中原核微生物群落、菌群网络及影响微生物群落的理化因素。结果表明,窖泥微生物群落α及β多样性在退化窖池中均存在明显的空间异质性,且窖池退化是一个自上而下的退化过程。随窖池深度增加,窖泥微生物群落α多样性指数整体呈显著上升趋势,如Shannon指数由大到小依次为PW-U(3.38)>PB(2.51)>PW-M(1.09)>PW-T(0.33)(P<0.05);窖泥优势菌门由单一的厚壁菌门(Firmicutes)(PW-T和PW-M,约99%)增加至Firmicutes、拟杆菌门(Bacteroidetes)、广古菌门(Euryarchaeota)等6个门(PW-U)及PB的4个门。退化窖泥呈现低含水量(平均值32.7%)、低pH值(平均值4.42)及低有效磷(平均值259.27 mg/kg)"三低"的理化特征,其对窖泥微生物群落结构影响较大(冗余分析)。窖泥菌群网络中互呈正相关的微生物主要隶属于不同的纲(占总相关性的78.74%)。同现网络中9个枢纽总含量非常低(0%~2.84%),与窖泥p H值或含水量呈显著或极显著正相关(P<0.05或P<0.01);除互营单胞菌属(Syntrophomonas)外,其他枢纽均与乳酸杆菌属(Lactobacillus)呈极显著负相关(P<0.01)。窖泥p H值与多种理化因子呈显著正、负相关(P<0.05),且与群落Simpson指数呈显著正相关,表明p H值可作为一个综合指标用于窖泥质量的初步判断。本实验较系统地研究了窖泥原核微生物群落多样性在退化窖池中空间分布、微生物之间以及微生物和理化因子之间的相关性,为进一步揭示窖泥的退化机制、防治窖泥退化及人工优质窖泥的制作提供一定的理论支撑。

In this study,high-throughput sequencing technology combined with network analysis and multivariate statistical analysis was used to analyze the prokaryotic microbial community diversity and microbial community networks,as well as the physicochemical factors affecting the microbial community in pit muds from the upper(PW-T),middle(PW-M)and lower(PW-U)layers of the wall,and from the bottom(PB)of a degraded fermentation pit used for the production of strong-flavor Baijiu.The results showed that theαandβdiversity of the pit mud microbial community had obvious spatial heterogeneity in the degraded pit,and the process of degradation could proceed from top to bottom.As the depth of the pit was increased,theαdiversity index of the pit mud microbial community showed a significant upward trend on the whole.Specifically,the Shannon index of microbial diversity followed the decreasing order of PW-U(3.38)>PB(2.51)>PW-M(1.09)>PW-T(0.33)(P<0.05),and there was only one dominant phylum(Firmicutes,about 99%)in PW-T and PW-M,compared to six including Firmicutes,Bacteroidetes,and Euryarchaeota in PW-U and four in PB.All the pit muds had low water content(with an average of 32.7%),low pH(with an average of 4.42),and low available phosphorus content(with an average of 259.27 mg/kg),which had a great impact on the microbial community structure as determined by redundancy analysis(RDA).According to the co-occurrence network analysis,the genera in different classes(interclass)had a high cooccurrence incidence(accounting for 78.74%of the total correlation).The total content of the nine hubs in the co-occurrence network was very low(0%–2.84%),and it had a significantly positive correlation with the pH and water content of pit mud(P<0.05 or P<0.01);all hubs except Syntrophomonas showed a significantly negative correlation with Lactobacillus(P<0.01).The pH of pit mud had a significantly positive or negative correlation with various physicochemical factors(P<0.05)and a significantly positive correlation with Simpson index,which indicates that pH could be used as an indicator to evaluate pit mud quality.This study will provide theoretical support for further uncovering the degradation mechanism of pit mud,preventing the degradation of pit mud,and making high-quality artificial pit mud.