Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

Hydrogen sulfide (H2S) is produced in a wide range of anoxic environments where sulfate (SO42−) reduction is coupled to decomposition of organic matter. In the same environments, methane (CH4) is the end product of an anaerobic food chain and both H2S and CH4 diffuse upwards into oxic zones where aerobic microorganisms can utilize these gases. Methane-oxidizing bacteria are known to oxidize a major part of the produced CH4 in these ecosystems, mitigating the emissions of this potent greenhouse gas to the atmosphere. However, how methanotrophy is affected by toxic H2S is largely unexplored. Here, we show that a single microorganism can oxidize CH4 and H2S simultaneously. By oxidizing H2S, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV can alleviate the inhibitory effects on CH4 oxidation. In response to H2S, strain SolV upregulated a type III sulfide:quinone oxidoreductase (SQR) and a sulfide-insensitive ba3-type terminal oxidase to dissipate the reducing equivalents derived from H2S oxidation. Through extensive chemostat cultivation of M. fumariolicum SolV we demonstrate that it converts high loads of H2S to elemental sulfur (S0). Moreover, we show chemolithoautotrophy by tracing 13CO2 fixation into new biomass by using H2S as sole energy source. Molecular surveys revealed several putative SQR sequences in a range of proteobacterial methanotrophs from various environments, suggesting that H2S detoxification is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in new ways.

Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH3SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H2S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 μM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments.

The trace amounts (0.53 ppmv) of atmospheric hydrogen gas (H2) can be utilized by microorganisms to persist during dormancy. This process is catalyzed by certain Actinobacteria, Acidobacteria, and Chloroflexi, and is estimated to convert 75 × 1012 g H2 annually, which is half of the total atmospheric H2. This rapid atmospheric H2 turnover is hypothesized to be catalyzed by high-affinity [NiFe] hydrogenases. However, apparent high-affinity H2 oxidation has only been shown in whole cells, rather than for the purified enzyme. Here, we show that the membrane-associated hydrogenase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV possesses a high apparent affinity (Km(app) = 140 nM) for H2 and that methanotrophs can oxidize subatmospheric H2. Our findings add to the evidence that the group 1h [NiFe] hydrogenase is accountable for atmospheric H2 oxidation and that it therefore could be a strong controlling factor in the global H2 cycle. We show that the isolated enzyme possesses a lower affinity (Km = 300 nM) for H2 than the membrane-associated enzyme. Hence, the membrane association seems essential for a high affinity for H2. The enzyme is extremely thermostable and remains folded up to 95 °C. Strain SolV is the only known organism in which the group 1h [NiFe] hydrogenase is responsible for rapid growth on H2 as sole energy source as well as oxidation of subatmospheric H2. The ability to conserve energy from H2 could increase fitness of verrucomicrobial methanotrophs in geothermal ecosystems with varying CH4 fluxes. We propose that H2 oxidation can enhance growth of methanotrophs in aerated methane-driven ecosystems. Group 1h [NiFe] hydrogenases could therefore contribute to mitigation of global warming, since CH4 is an important and extremely potent greenhouse gas.

Emissions of the strong greenhouse gas methane (CH4) to the atmosphere are mitigated by methanotrophic microorganisms. Methanotrophs found in extremely acidic geothermal systems belong to the phylum Verrucomicrobia. Thermophilic verrucomicrobial methanotrophs from the genus Methylacidiphilum can grow autotrophically on hydrogen gas (H2), but it is unknown whether this also holds for their mesophilic counterparts from the genus Methylacidimicrobium. To determine this, we examined H2 consumption and CO2 fixation by the mesophilic verrucomicrobial methanotroph Methylacidimicrobium tartarophylax 4AC. We found that strain 4AC grows autotrophically on H2 with a maximum growth rate of 0.0048 h–1 and a yield of 2.1 g dry weight⋅mol H2–1, which is about 12 and 41% compared to the growth rate and yield on methane, respectively. The genome of strain 4AC only encodes for an oxygen-sensitive group 1b [NiFe] hydrogenase and H2 is respired only when oxygen concentrations are below 40 μM. Phylogenetic analysis and genomic comparison of methanotrophs revealed diverse [NiFe] hydrogenases, presumably with varying oxygen sensitivity and affinity for H2, which could drive niche differentiation. Our results show that both thermophilic and mesophilic verrucomicrobial methanotrophs can grow as autotrophs on H2 as a sole energy source. Our results suggest that verrucomicrobial methanotrophs are particularly well-equipped to thrive in hostile volcanic ecosystems, since they can consume H2 as additional energy source.