Uncovering Viable Microbiome in Anaerobic Sludge Digesters by Propidium Monoazide (PMA)-PCR

  • Jialing Ni
  • Shingo Hatori
  • Yin Wang
  • Yu-You Li
  • Kengo KubotaEmail author


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.


PMA-PCR Anaerobic sludge digester Viable microbiome Residual populations 



We thank Dr. Madan Tandukar of Höganäs Environment Solutions, LLC for reading the manuscript.

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.

Supplementary material

248_2019_1449_MOESM1_ESM.docx (220 kb)
ESM 1 (DOCX 219 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringTohoku UniversitySendaiJapan
  2. 2.Institute of Urban EnvironmentChinese Academy of SciencesXiamenChina

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