Microbial Ecology

, Volume 67, Issue 4, pp 829–836 | Cite as

Detecting the Nonviable and Heat-Tolerant Bacteria in Activated Sludge by Minimizing DNA from Dead Cells

  • Feng Guo
  • Tong ZhangEmail author
Environmental Microbiology


Propidium monoazide (PMA) has been used to determine viable microorganisms for clinical and environmental samples since selected naked DNA which was covalently cross-linked by this dye could not be PCR-amplified. In this study, we applied PMA to the activated sludge samples composed of complex bacterial populations to investigate the viability of human fecal bacteria and to determine the heat-tolerant bacteria by high-throughput sequencing of 16S ribosomal DNA (rDNA) V3 region. The methodological evaluation suggested the validity, and about 2–3 magnitude signals decreasing from the stained DNA were observed. However, the nest PCR, which was previously conducted to further minimize signals from dead cells, seemed not suitable perhaps due to the limitation of the primers. On one hand, for typical human fecal bacteria, less than half of them were viable, and most genera exhibited the similar viable percentages. It was interesting that many “unclassified bacteria” showed low viability, implying their sensitivity to environmental change. On the other hand, after heating at 60 °C for 4 h, the bacteria with high survival rate in activated sludge samples included those reported thermophiles or heat-tolerant lineages, such as Anoxybacillus and diverse species in Actinobacteria, and some novel ones, such as Gp16 subdivision in Acidobacteria. In summary, our results took a glance at the fate of fecal bacteria during sewage treatment and established an example for identifying tolerant species to lethal shocks in a complex community.


Activate Sludge Actinobacteria Aeration Tank Fecal Bacterium Activate Sludge Sample 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank the financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (project no. HKU7190/12E). Dr. Feng Guo wants to thank the postdoctoral fellowship from the University of Hong Kong.

Supplementary material

248_2014_389_MOESM1_ESM.docx (1.9 mb)
ESM 1 (DOCX 1904 kb)


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Environmental Biotechnology Laboratory, Department of Civil EngineeringThe University of Hong KongHong KongChina

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