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Applied and Environmental Microbiology, January 2004, p. 588-596, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.588-596.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

Maneesha P. Ginige,1 Philip Hugenholtz,2 Holger Daims,3 Michael Wagner,3 Jürg Keller,1 and Linda L. Blackall1*

Advanced Wastewater Management Centre, The University of Queensland, St. Lucia 4072, Queensland, Australia,1 Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720-3110,2 Abteilung für Mikrobielle Ökologie, Institut für Ökologie und Naturschutz, Universität Wien, 1090 Vienna, Austria3

Received 28 May 2003/ Accepted 26 September 2003

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3--N mg of mixed-liquor volatile suspended solids (MLVSS)-1 h-1 to a steady-state value of 0.06 mg of NO3--N mg of MLVSS-1 h-1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

* Corresponding author. Mailing address: Advanced Wastewater Management Centre, The University of Queensland, St. Lucia 4072, Queensland, Australia. Phone: 61 7 33654645. Fax: 61 7 33654726. E-mail: blackall{at}awmc.uq.edu.au.

Applied and Environmental Microbiology, January 2004, p. 588-596, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.588-596.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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