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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.

SUBMITTER: Ginige MP

PROVIDER: S-EPMC321253 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community.

Ginige Maneesha P MP Hugenholtz Philip P Daims Holger H Wagner Michael M Keller Jürg J Blackall Linda L LL

Applied and environmental microbiology 20040101 1

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) ...[more]

PMID: 14711691

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Project description:BackgroundThe biofilm of a methanol-fed, marine denitrification system is composed of a multi-species microbial community, among which Hyphomicrobium nitrativorans and Methylophaga nitratireducenticrescens are the principal bacteria involved in the denitrifying activities. To assess its resilience to environmental changes, the biofilm was cultivated in artificial seawater (ASW) under anoxic conditions and exposed to a range of specific environmental conditions. We previously reported the impact of these changes on the denitrifying activities and the co-occurrence of H. nitrativorans strain NL23 and M. nitratireducenticrescens in the biofilm cultures. Here, we report the impact of these changes on the dynamics of the overall microbial community of the denitrifying biofilm.MethodsThe original biofilm (OB) taken from the denitrification system was cultivated in ASW under anoxic conditions with a range of NaCl concentrations, and with four combinations of nitrate/methanol concentrations and temperatures. The OB was also cultivated in the commercial Instant Ocean seawater (IO). The bacterial diversity of the biofilm cultures and the OB was determined by 16S ribosomal RNA gene sequences. Culture approach was used to isolate other denitrifying bacteria from the biofilm cultures. The metatranscriptomes of selected biofilm cultures were derived, along with the transcriptomes of planktonic pure cultures of H. nitrativorans strain NL23 and M. nitratireducenticrescens strain GP59.ResultsHigh proportions of M. nitratireducenticrescens occurred in the biofilm cultures. H. nitrativorans strain NL23 was found in high proportion in the OB, but was absent in the biofilm cultures cultivated in the ASW medium at 2.75% NaCl. It was found however in low proportions in the biofilm cultures cultivated in the ASW medium at 0-1% NaCl and in the IO biofilm cultures. Denitrifying bacterial isolates affiliated to Marinobacter spp. and Paracoccus spp. were isolated. Up regulation of the denitrification genes of strains GP59 and NL23 occurred in the biofilm cultures compared to the planktonic pure cultures. Denitrifying bacteria affiliated to the Stappia spp. were metabolically active in the biofilm cultures.ConclusionsThese results illustrate the dynamics of the microbial community in the denitrifying biofilm cultures in adapting to different environmental conditions. The NaCl concentration is an important factor affecting the microbial community in the biofilm cultures. Up regulation of the denitrification genes of M. nitratireducenticrescens strain GP59 and H. nitrativorans strain NL23 in the biofilm cultures suggests different mechanisms of regulation of the denitrification pathway in the biofilm. Other denitrifying heterotrophic bacteria are present in low proportions, suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments.

Dataset Information

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community.

Publications

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community.

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