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

Masashi Hatamoto,Masafumi Kimura,Nobuo Araki,Masato Koizumi,Takafumi Sato,Masanobu Takahashi,Shuji Kawakami,Takashi Yamaguchi,Rajeev Misra

doi:10.1371/journal.pone.0115823

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.

Highlights

Microbial consortia that perform denitrifying anaerobic methane oxidation (DAMO) have been discovered in the last decade [1, 2]
The microorganisms responsible for the DAMO reaction belong to the previously uncultured NC10 phylum and an archaeal group distantly related to anaerobic methanotrophic (ANME) archaea
To select an appropriate seeding for enrichment of DAMO bacteria, three paddy field soil samples were screened with the pmoA gene targeted PCR and phylogenetic analysis assay

Summary

Introduction

Microbial consortia that perform denitrifying anaerobic methane oxidation (DAMO) have been discovered in the last decade [1, 2]. The microorganisms responsible for the DAMO reaction belong to the previously uncultured NC10 phylum and an archaeal group distantly related to anaerobic methanotrophic (ANME) archaea. The complete genome of a bacterium in the phylum NC10, named ‘‘Candidatus Methylomirabilis oxyfera’’, was assembled from metagenomic sequencing of an enrichment culture of DAMO [3]. Using the DAMO reaction, M. oxyfera bypasses the production of nitrous oxide, a canonical intermediate of standard denitrification pathways. The genome sequence of DAMO archaea, named ‘‘Candidatus Methanoperedens nitroreducens’’ was reported, confirming the role of an archaeon in the DAMO reaction [4]. Microorganisms capable of DAMO have great potential for development of a novel denitrification process that could reduce the production of greenhouse gases nitrous oxide and methane

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Results

Conclusion