TY - JOUR
T1 - Uncovering Viable Microbiome in Anaerobic Sludge Digesters by Propidium Monoazide (PMA)-PCR
AU - Ni, Jialing
AU - Hatori, Shingo
AU - Wang, Yin
AU - Li, Yu You
AU - Kubota, Kengo
N1 - Funding Information:
The first author JN was supported by the Inter-Graduate School Doctoral Degree Program on Science for Global Safety of Tohoku University. This research was supported by the Strategic International Collaborative Research Program of the Japan Science and Technology Agency, by the Gesuido Academic Incubation to Advanced Project of the Ministry of Land, Infrastructure, Transport and Tourism and by Grant-in-Aid for Scientific Research (B) (KAKENHI Grant Number JP18H01564) from Japan Society for the Promotion of Science.
Publisher Copyright:
© 2019, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5–30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.
AB - Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5–30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.
KW - Anaerobic sludge digester
KW - PMA-PCR
KW - Residual populations
KW - Viable microbiome
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U2 - 10.1007/s00248-019-01449-w
DO - 10.1007/s00248-019-01449-w
M3 - Article
C2 - 31701171
AN - SCOPUS:85075171565
VL - 79
SP - 925
EP - 932
JO - Microbial Ecology
JF - Microbial Ecology
SN - 0095-3628
IS - 4
ER -