Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor

Abstract

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia. Biologically mediated methane formation (methanogenesis)is the largest source of methane on the planet and has long been considered a carbon sink and terminal step in ecological carbon flow (13). However, over the last 25 years there has been compelling evidence suggesting that microbially mediated anaerobic oxidation of methane constitutes another major component of carbon cycling in these environments. Martens and Berner (22), Reeburgh (29), and Barnes and Goldberg (4) first posited the existence of a microbial community in anoxic marine sediments that was responsible for anaerobic oxidation of methane. The net reaction was described as SO 4 2Ϫ ϩ CH 4 3 HCO 3 Ϫ ϩ HS Ϫ ϩ 2H 2 O, where HS Ϫ is hydrogen bisulfide. This net reaction is exergonic at conditions found in situ, yielding ca. 25 kJ per mol of methane oxidized (21).A subsequent field and laboratory study suggested that anaerobic oxidation of methane was mediated by a consortia of archaea and sulfate-reducing bacteria (17). Microbially mediated anaerobic oxidation of methane has been well correlated with characteristic geochemical profiles, namely the concomitant depletion of sulfate and methane in anoxic sediments (12,19,28,38). Molecular phylogenetic analyses combined with 13 C lipid isotopic determinations identified two major groups of methanogen-related archaea (ANME-1 and ANME-2) that apparently incorporate methane-derived carbon into cellular biomass a...

Carbonate-hosted methanotrophy represents an unrecognized methane sink in the deep sea

“…Porous and massive carbonates from active seep sites performed AOM at rates commensurate with active seep sediment [42][43][44] , while sediment and carbonate rates from off-seep, low-activity areas were similar to those reported from continental shelves 10,45,46 . Within the comparative context of FISH-nanoSIMS analysis, active porous carbonate-hosted Archaea-DSS aggregates under anoxic conditions display substantial 15 N incorporation compared with aggregates from unlabelled incubations, confirming significant anabolic activity associated with cells residing in carbonate interiors.…”

“…4), exhibiting mixed or clustered species distributions. When modelling large aggregates (25 mm), Orcutt and Meile 39 found that consortia 43 Hydrate Ridge active seep sediment 32-2,358 Wegener et al 42 North Sea active seep sediment 25-450 Joye et al 70 Gulf of Mexico active seep sediment 121-501 Girguis et al 44 Monterey Bay active seep sediment 82.3 Hansen et al 45 Norsminde Fjord inner shelf sediment 14.3 Hoehler et al 10 Cape Lookout Bight inner shelf sediment 14-18 Reeburgh 46 Skan Bay outer shelf sediment 4.9-9.3 AOM, anaerobic oxidation of methane.…”

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…”

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

“…These long incubation times were necessary due to the slow growth of ANME−SRB consortia (3−7 mo; e.g., refs. [41][42][43], which is attributed to the very low free energy yield of sulfate-coupled AOM (42,44). Subsampling for molecular and geochemical analyses as well as exchange of gaseous headspace and seawater were conducted at regular intervals.…”