Abstract
Evidence of aerobic methane oxidation coupled to denitrification has been provided for different freshwater environments, whereas the significance of this process for the marine realm has not been adequately investigated. The goal of this study was to investigate the methane-related reduction of nitrate/nitrite in a marine environment (salinity 8.5). A water sample was collected from the oxic-anoxic transition zone of the Gotland Deep (central Baltic Sea) and the microorganisms contained therein were cultivated in a bioreactor under hypoxic conditions (0.5 µM O2). To enrich the microorganisms involved in the coupled process the bioreactor was continuously sparged with methane as the sole energy and carbon source and simultaneously supplied with a nutrient solution rich in nitrate and nitrite. The bioreactor experiment showed a relationship between the turnover of methane and the concomitant concentration decrease of nitrite and nitrate at the early stage of the experiment. This relationship indicates the role of methanotrophs, which may support heterotrophic denitrifiers by the release of organic compounds as an energy source. Besides, a mixture of uncultured microorganisms, aerobic methanotrophic and heterotrophic denitrifying bacteria were identified in the enrichment culture. Microbial incorporation of nitrite and methane was proven on the cellular and gene levels via 15NO2- / 13CH4 incubation experiments and subsequent analyses with nano secondary ion mass spectrometry (NanoSIMS) and stable isotope probing (SIP). The NanoSIMS showed the incorporation of 15N in almost all the bacteria and in 9% of those there was a concomitant enrichment in 13C. The relatively low abundance of methane-consuming bacteria in the bioreactor was further reflected in specific fatty acids indicative for type I methanotrophic bacteria. Based on pmoA gene analyses, this bacterium is different from the one that was identified as the only key player of methane oxidation in previous studies in the Gotland Deep, indicating the existence of other subordinate methanotrophic bacteria at that site. The results provide the first indications for the predisposition of a methane-related reduction of nitrate/nitrite under hypoxic conditions in the marine realm, supporting the assumption of an interaction between methanotrophic and denitrifying bacteria which hitherto has only been described for fresh water environments.
Highlights
Apart from the classical microbial processes that oxidize methane via electron acceptors such as oxygen and sulfate, various alternative pathways have been identified in different aquatic systems over the last few decades (Modin et al, 2007; Reeburgh, 2007)
Because of the limitations of the flowcytometrical method, which does not differentiate between single cells and cell aggregates, an evaluation of the number of single cells is only assessable at the beginning of the experiment before the formation of particles
Our observations during the bioreactor experiment showed an obvious decrease in nitrite and nitrate concentrations from week 12 to 18 (Figure 4, II) that was followed by an increase in methane oxidation rates between week 7 and 13
Summary
Apart from the classical microbial processes that oxidize methane via electron acceptors such as oxygen and sulfate, various alternative pathways have been identified in different aquatic systems over the last few decades (Modin et al, 2007; Reeburgh, 2007). The first evidence for anaerobic methane oxidation coupled to denitrification was shown in a bioreactor containing acetate degrading and denitrifying organisms from fresh water environments (Islas-Lima et al, 2004). Sludge from this bioreactor was exposed to methane, which led to a decrease in nitrate concentration as well as an increase in dinitrogen production, with methane as the sole electron donor. Further bioreactor studies disproved the involvement of archaea and identified a single bacterium responsible for this process This bacterium named “Ca. Methylomirabilis oxyfera” uses internally generated oxygen for the consumption of methane (Ettwig et al, 2010). Specific labeling experiments with “Ca. Methylomirabilis oxyfera” identified the incorporation of carbon originating from bicarbonate instead of methane, providing evidence of a carbon dioxide fixing organism as well as of the role of methane as the dominating energy source in the process (Rasigraf et al, 2014)
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