Growth and Population Dynamics of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a Continuous-Flow Bioreactor

Abstract

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes ( ANME-2 ‫؍‬ 0.167 · week ؊1 ), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes ( ANME-1 ‫؍‬ 0.218 · week ؊1 ). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 · week ؊1 , and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.Geochemical studies have shown that the anaerobic oxidation of methane (AOM) in marine sediments is a microbially mediated phenomenon and represents a significant component of carbon cycling in marine environments (4,25,32). Based on field and laboratory experiments and observations using rRNA-targeted fluorescent in situ hybridization, AOM is believed to be mediated by a consortium of archaea and sulfatereducing bacteria (2,5,7,20). Phylogenetic and isotopic analyses have shown that two groups of anaerobic methanotrophic archaea (ANME-1 and ANME-2) cooccur in sediments with sulfate-reducing bacteria and incorporate methane-derived carbon into cellular biomass (19, 30). Genomic and biochemical analyses suggest that microbial AOM may be achieved in part by reversal of the canonical methanogenic pathway (15, 23), and ongoing studies have shown that ANME-1 and ANME-2 methane-oxidizing archaea (or MOA) are distributed throughout the world in anoxic marine sediments where methane and sulfate are available (22,26,31,35,37,39,41).Our understanding of MOA physiology has been advanced by employing both in situ isotopic tracers and serum vial incubations to examine the relation between AOM and sulfate reduction and the effect of physical factors,...

Biogeochemistry of a low-activity cold seep in the Larsen B area, western Weddell Sea, Antarctica

“…Sequences of the Thermoplasmatales were also found (13.6%). Their functioning is also unclear, but they have previously been found to co-occur with ANMEs (Hinrichs et al, 1999;Orphan et al, 2001;Inagaki et al, 2004;Girguis et al, 2005;Knittel et al, 2005;Wegener et al, 2008). Interestingly, no Crenarchaeota were found although they are common members of seep communities (Knittel et al, 2005;Lösekann et al, 2007).…”

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

“…For example, several types of continuous-flow bioreactor systems have been developed and applied for the enrichment and cultivation of archaeal anaerobic methanotrophs (Girguis et al, 2003(Girguis et al, , 2005Meulepas et al, 2009;Zhang et al, 2010). Girguis et al, (2003Girguis et al, ( , 2005) developed a continuous-flow incubation system to mimic the in situ conditions of methane seep marine sediments and reported an increase in the archaeal anaerobic methanotroph population associated with the anaerobic oxidation of methane in the incubation system. Meulepas et al, (2009) also demonstrated that the oxidation of methane rate in a continuous submergedmembrane bioreactor increased exponentially from 0.4 to 286 mmol g dry weight…”

Microbial Ecology of the Dark Ocean above, at, and below the Seafloor

“…There are clearly a few lineages where little is known, and these should be targets for future work. Among these are anaerobic methanotrophs, which have been kept alive in their original sediment for study along with their sulfate-reducing bacterial counterparts (189,402) but never isolated in pure culture. Similarly, microorganisms from deep subsurface oceanic crust have eluded cultivation.…”