Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Abstract:Mud volcanism is an important natural source of the greenhouse gas methane to hydrosphere and atmosphere 1,2 . Recent investigations show that the number of active submarine mud volcanoes may be much higher than anticipated (eg. 3-5), and that gas emitted from deep-sea seeps may reach the upper mixed ocean [6][7][8] . Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release is unknown 9 . Another uncertainty is the efficiency of methane oxidizing microorganisms in methane removal. Here we investigated the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72°N, 14°44'E; 1250 m water depth), to provide quantitative estimates of in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities; aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a novel clade of archaea (ANME-3) associated with bacterial mats. We found that upward flow of sulphate-and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to <40% of the total flux.The HMMV (Fig. 1), a circular structure of 1 km diameter and <10 m elevation above the adjacent seafloor, has been studied since the 1990s as a typical example of an active mud volcano 9 . Its formation may have coincided with a submarine landslide during the late Pleistocene, 330-200 ka before present 10 . Today, fluids, gas and muds rise from 2-3 km depth through a conduit below the HMMV 11,10 . The emitted gas is of a mixed microbial/thermogenic origin and consists of >99% CH4 with a δ 13 C-isotope signature of -60‰ 12,13 . The rising fluids are depleted in sulphate, chloride and magnesium caused by subsurface clay dewatering 11 . Investigation of the HMMV with RV POLARSTERN and ROV VICTOR 6000 in 2003 showed extensive outcroppings 1 2006-01-01028b_Boetius_MS 3 of fresh subsurface muds associated with steep thermal gradients 14 , gas and fluid vents, and a large gas plume reaching the mixed upper water column above the HMMV 12,8 .Seafloor videography in combination with geochemical measurements provided in situ estimates of gas flux 8 , fluid flow 15 and habitat distribution 16 . We focused on the three main concentric habitats above the gassy muds (Fig. 2): the centre of HMMV, which was devoid of epifauna; thiotrophic bacterial mats dominated by a Beggiatoa species; and surrounding fields of siboglinid tubeworms. Gas concentrations in sediments and bottom water were elevated in all three habitats (Tab. (Fig. 3a). Only minor amounts of methanotroph lipids (<0.1 µg gdw -1 ) and very low cell numbers (~10 7 cells cm -3 ) were found below 5 cm sediment depth ( Fig. 3a3-3a4). ANME cells were not microscopically detectable in the centre cores using all know...

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Aerobic biofilms were found to have a complex structure consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. The oxygen distribution was strongly correlated with these strutures. The voids facilitated oxygen transport from the bulk liquid through the biofilm, supplying approximately 50% of the total oxygen consumed by the cells. The mass transport rate from the bulk liquid is influenced by the biofilm structure; the observed exchange surface of the biofilm is twice that calculated for a simple planar geometry. The oxygen diffusion occurred in the direction normal to the cluster surfaces, the horizontal and vertical components of the oxygen gradients were of equal importance. Consequently, for calculations of mass transfer rates a three-dimensional model is necessary. These findings imply that to accurately describe biofilm activity, the relation between the arrangement of structural components and mass transfer must be undrstood. (c) 1994 John Wiley & Sons, Inc.

Håkon Mosby Mud Volcano (HMMV, SW Barents Sea slope, 1280 m) is one of the numerous cold methane-venting seeps existing along the continental margins. Analyses of videoguided core samples revealed extreme differences in the diversity and density of the metazoan meiobenthic communities associated with the different sub-habitats (centre, microbial mats, Pogonophora field, outer rim) of this mud volcano. Diversity was lowest in the sulphidic, microbial mat sediments that supported the highest standing stock, with unusually high densities (11 000 ind. 10 cm -2 ) of 1 nematode species related to Geomonhystera disjuncta. Stable carbon isotope analyses revealed that this nematode species was thriving on chemosynthetically derived food sources in these sediments. Ovoviviparous reproduction has been identified as an important adaptation of parents securing the survival and development of their brood in this toxic environment. The proliferation of this single species in exclusive association with free-living, sulphide-oxidising bacteria (Beggiatoa) indicates that its dominance is strongly related to trophic specialisation, evidently uncommon among the meiofauna. This chemoautotrophic association was replaced by copepods in the bare, sulphidefree sediments of the volcano's centre, dominated by aerobic methane oxidation as the chemosynthetic process. Copepods and nauplii reached maximum densities and dominance in the volcano's centre (500 ind. 10 cm -2 ). Their strongly depleted carbon isotope signatures indicated a trophic link with methane-derived carbon. This proliferation of only selected meiobenthic species supported by chemosynthetically derived carbon suggests that, in addition to the sediment geochemistry, the associated reduced meiobenthic diversity may equally be related to the trophic resource specificity in HMMV sub-habitats.