Abstract
Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strong-flavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 μM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.
Highlights
Traditional isolation and culture technology to study structure and diversity of microbial community have several limitations, as most microorganisms are not yet culturable (Rondon et al, 2000)
We investigated the effectiveness of removal of DNA from non-viable bacteria by propidium monoazide (PMA) and evaluated the optimal PMA concentration
We used 20-year pit mud (PM) for the concentration optimization because we assumed that an optimal PMA concentration at which DNA could be removed from non-viable bacteria for 20-year PM was applicable for 5-year PM
Summary
Traditional isolation and culture technology to study structure and diversity of microbial community have several limitations, as most microorganisms are not yet culturable (Rondon et al, 2000). DNA-based molecular technologies, such as amplification sequencing and metagenomic sequencing, have been widely applied (Carini et al, 2017). These methods have ignored DNA from non-viable microbes in the total DNA extraction. Non-viable microbes are defined as dead microbial cells with damaged membranes. It is necessary to investigate the effect of non-viable bacteria on the analysis of the microbial community by DNA-based molecular technologies. Non-viable microbes were reported to affect the estimation of microbial community diversity in meconium (Stinson et al, 2019), clinical feces (Young et al, 2017), topsoil (Carini et al, 2017), rice wine (Lv et al, 2016), and cheese (Erkus et al, 2016), but not that in the samples of groundwater (Lopez-Fernandez et al, 2018) or soil battery (Gustave et al, 2019)
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