title: Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community.
abstract: 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 NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-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 [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]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 [(12)C]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 [(14)C]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.
authors.affiliation: Advanced Wastewater Management Centre, The University of Queensland, St. Lucia 4072, Queensland, Australia.
authors.forename: Maneesha P
authors.initials: MP
authors.lastname: Ginige
authors.forename: Philip
authors.initials: P
authors.lastname: Hugenholtz
authors.forename: Holger
authors.initials: H
authors.lastname: Daims
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
authors.forename: Jürg
authors.initials: J
authors.lastname: Keller
authors.forename: Linda L
authors.initials: LL
authors.lastname: Blackall
mesh_terms: D001345:Autoradiography; D020562:Betaproteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019149:Bioreactors; D002247:Carbon Isotopes / Q000378:metabolism; D017753:Ecosystem; D015723:Gene Library; D017404:In Situ Hybridization, Fluorescence; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism*; D010802:Phylogeny; D012335:RNA, Ribosomal / Q000032:analysis*; D012722:Sewage / Q000382:microbiology*; D014865:Waste Disposal, Fluid
chemical_list: D002247:Carbon Isotopes; D009566:Nitrates; D012335:RNA, Ribosomal; D012722:Sewage; D000432:Methanol