Enrichment of denitrifying methane-oxidizing microorganisms using up-flow continuous reactors and batch cultures

PLoS One. 2014 Dec 29;9(12):e115823. doi: 10.1371/journal.pone.0115823. eCollection 2014.

Abstract

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L(-1)·day(-1) was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89-91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaea / genetics
  • Archaea / growth & development*
  • Archaea / metabolism
  • Bacterial Proteins / genetics
  • Denitrification*
  • Methane / chemistry
  • Methane / metabolism
  • Methylococcaceae / genetics
  • Methylococcaceae / growth & development*
  • Methylococcaceae / metabolism
  • Nitrites / chemistry
  • Nitrites / metabolism
  • Phylogeny
  • Soil / chemistry
  • Soil Microbiology*

Substances

  • Bacterial Proteins
  • Nitrites
  • Soil
  • Methane

Grants and funding

This study was partially supported by Exploratory Research of Adaptable and Seamless Technology Transfer Program through target-driven R&D program (A-STEP) from Japan Science and Technology Agency, and JSPS KAKENHI Grant Number 25701010, and by a Grant-in-Aid from the Institute for Fermentation, Osaka. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.