| Fermentation pit mud(FPM) harbors a large number of culturable and unculturable microbes, the community structure and diversity of which reflects the FPM quality and determines the quality of Chinese strong-flavor liquor(CSAL). Furthermore, the obligate anaerobic Clostridia and caproic acid-producing bacteria have been regarded as the important functional microbial groups in FPM. However, there is no systematic study on the analysis of communities of Clostridia and caproic acid-producing bacteria, and the relationships between FPM quality and microbial community. Therefore, it will contribute to understanding the mechanism of CSAL production and improving the CSAL quality fundamentally by comprehensively analyzing the structure and diversity of above microbial community and relationships. Synchronously, it will be helpful for upgrading traditional technologies and achieving sustainable development in CSAL production.In this work, FPM was selected for the analysis of the microbial community structure and diversity, relationships among microbial community members and indentification of functional microbes, which are core issues in microbiology. Traditional culturable and multiple molecular ecological techniques combined with newly developed methods and strategies, were used to analyze qualitatively and quantitatively the community structure and diversity of functional microbes and microbes in FPMs with different quality grades. Simultaneously, multiple bioinformatics methods have been reasonably applied to clarify the relationships among microbial community, environmental conditions and pit-mud quality. The main results of this study are as follows:(1) The microbial community structure and diversity in FPMs with different quality grades was investigated by using Illumina Miseq sequencing based on V4 variable region of microbial 16 S rRNA gene, and a total of 225 genera assigned into 145 orders under 33 phyla were detected in all samples. The results of microbial community α-diversity showed that Shannon and Chao 1 indices were significantly correlated with FPM quality. The results of microbial community β-diversity showed that the microbial community was closely correlated with FPM quality. The dominant phyla Firmicutes was replaced by Firmicutes, Euryarchaeota and Bacteroidetes with FPM quality-improving, and the number of core genera increased from 2 to 15 genera, which were mainly assigned into four classes including Clostridia, Bacteroidia, Methanobacteria, Methanomicrobia, especially Clostridia(7 genera).(2) Network analysis revealed that microbial co-correlation was mainly occoured among different phyla, especially among Firmicutes, Euryarchaeota, Bacteroidetes and Proteobacteria. About 77% hubs of co-occurrence map were members of four classes including Clostridia, Bacteroidia, Methanobacteria and Methanomicrobia that mainly inhabited normal and high-quality FPMs, while three lactic acid bacteria(LAB) including Lactobacillus, Pediococcus and Streptococcus that mainly dominated in degraded FPMs, were identified as hubs of negative correlation map, which showed significant negative correlation with 11 co-occurrence hubs. Therefore, the co-occurrence hubs will be decline and even disappear when LAB rapidly increased, which may destroy this stability and lead to FPM degradation. The integrated parameter pH showed the highest correlation with Shannon diversity index, which tended to be higher and then stable at near-neutral pHs with increasing FPM pH. The result of redundancy analysis(RDA) revealed that biogeochemical attributes including dissolved organic carbon(DOC), NH4+, pH, available phosphorus(AP) and lactate were significantly correlated with microbial community, especially DOC, while total nitrogen(TN) and moisture showed no significant correlation with microbial community.(3) The method for rapid analyzing the clostridial community structure and diversity was developed, the key step of which was the design of clostridial universal and specific primers. A Clostridia-specific primer set SJ-F and SJ-R, based on the available 16 S rRNA gene sequences from ribosomal database project(RDP), was successfully designed, which targeted V4 and V5 variable regions of 16 S rRNA gene. 98.6% of theoretical amplicons of this pair of primers belonged to class Clostridia and 19 clostridial families and unclassified_Clostridia with different coverage rates could be amplified. This primer set was applied to analyze the clostridial community in a complex ecosystem(i.e., FPM) using denaturing gradient gel electrophoresis(DGGE), and seven phylogenetic clusters within Clostridia were detected. Compared with the well-accepted bacterial universal primer pair P3 / P2, it is more sensitive and accurate to analyze clostridial community by using primer set SJ-F / SJ-R. Therefore, this study could provide a good alternative to investigate the clostridial diversity and monitor their population dynamics rapidly and efficiently in various anaerobic environments and fermentation systems.(4) The clostridial community structure and diversity of FPMs with different used ages was investigated by using Illumina Miseq sequencing combined with primer set SJ-F/SJ-R. A total of 20 clostridial families containing 40 genera, and 12 families including 21 genera in FPM ecosystem were reported for the first time, and the main operation classification units(OTUs) were clustered into 16 phylogenetic clusters within Clostridia, which could expand our understanding of phylogenetic status of an enormous number of uncultured Clostridia in this ecosystem. Clostridiaceae and Tissierellaceae dominated in all samples, and Lachnospiraceae and Mogibacteriaceae in new(1-year) sample were more abundant than old(≥30 years) samples, while abundances of Anaerobrancaceae and Eubacteriaceae in new sample were lower than old samples. Real-time quantitative PCR(qPCR) assay revealed that the contents of Clostridia(2.06×108-1.62×109 16 S rRNA gene copies·g-1 FPM) in old FPMs were 10-100 times higher than new FPM.(5) Clostridium kluyveri was identified as a main caproic acid-producing species of Clostridia in different spatial and temporal FPMs by comparing the microbial data obtained from culture-dependent and-independent approaches combined with data of their corresponding volatile metabolites. A representative strain C. kluyveri N6 can produce various substance including fatty acids: butyric, caproic and octanoic acids; alcohols: 1-butanol, 1-hexanol and 1-octanol; ethyl esters: ethyl butyrate, ethyl caproate and ethyl octanoate. |
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