Page 239 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Genome sequencing and analysis of aerobic methanotrophs
To confirm the identity of the methanotrophs, the culture was sequenced using Illumina HiSeq technology. Sequence analysis was performed using consensus binning, annotation, and de novo assembly based on a 16S rRNA gene identification approach. The culture appeared to contain two methanotrophic species. The first bin was assigned to Methylosinus (3.79 Mbp, GC-content 65.2%, N50 = 96,528 bp, 55 contigs) and had 100% completeness, 0.31% contamination, and no strain heterogeneity. The second bin was identified as Methylocystis (4.36 Mbp, GC-content 62.5%, N50 = 136,571 bp, 55 contigs) with 99.7% completeness, 0.32% contamination, and no strain heterogeneity. De novo assembly of SILVA database mapped reads resulted in two 16S rRNA gene contigs. Contig 1 (842 bp) showed 100% nucleotide sequence identity (E-value 0.00, bitscore 1,350) with the 16S rRNA gene of Methylosinus sporium strain NCIMB 11126 as ordered from the DSMZ culture collection. Contig 2 (1,431 bp) showed 98.5% nucleotide sequence identity (E-value 0.00, bitscore 2,328) to the 16S rRNA gene of Methylocystis rosea strain SV97.
Discussion
Here, we present a proof of concept for the co-cultivation of anaerobic methanogenic archaea and aerobic methanotrophic bacteria in an oxygen-limited bioreactor. Only few studies have previously investigated co-cultivation of methanogens and methanotrophs (Gerritse and Gottschal 1993; Shen, Miguez and Bourque 1996; Miguez et al. 1999). These studies showed that co-cultivation of methanogens and methanotrophs is possible, but they did not provide in- depth data on species interactions and system operation. With the present study, we provide a method to study interspecies interactions between methane cycle microorganisms under oxygen limitation. FISH micrographs showed a tight spatial organization between the methanogenic sarcina clusters and layers of aerobic methanotrophic cells. This confirmed our hypothesis that methanotrophs profited from the production of methane from acetate by the methanogens and were therefore closely associated with the methanogens (Fig. 5). Methanogens are most likely protected against oxygen by methanotrophs (Fig. 5).
Genome sequencing of the methanotrophic culture indicated presence of both a Methylosinus and a Methylocystis species. Both species are members of the
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