Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge

Geets, Joke, De Cooman, Michaël, Wittebolle, Lieven, Heylen, Kim, Vanparys, Bram, De Vos, Paul, Verstraete, William and Boon, Nico (2007) Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Applied Microbiology and Biotechnology, 75 1: 211-221. doi:10.1007/s00253-006-0805-8


Author Geets, Joke
De Cooman, Michaël
Wittebolle, Lieven
Heylen, Kim
Vanparys, Bram
De Vos, Paul
Verstraete, William
Boon, Nico
Title Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge
Journal name Applied Microbiology and Biotechnology   Check publisher's open access policy
ISSN 0175-7598
Publication date 2007-05
Sub-type Article (original research)
DOI 10.1007/s00253-006-0805-8
Volume 75
Issue 1
Start page 211
End page 221
Total pages 12
Place of publication Berlin
Publisher Springer
Language eng
Subject 100204 Environmental Biotechnology Diagnostics (incl. Biosensors)
090409 Wastewater Treatment Processes
Abstract In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation (R 2 > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 106 down to 104 ml−1, whereas nitrite oxidizers were less abundant (103–101 order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 108–105. When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples
Keyword SYBR Green I real-time PCR
16S rRNA gene
amoA
Nitrite oxidoreductase
nosZ
nirS
nirK
Activated sludge
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Advanced Water Management Centre Publications
 
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