Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H2 oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.

18 19 Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 20 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in 21 aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on 22 Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to 23 recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in 24 various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in 25 bacterial metabolism. Here we investigated the role of this enzyme in three species from 26 different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans 27 (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and 28 Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed 29 that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated 30 during exponential growth compared to stationary phase, in contrast to the profile of the 31 persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays 32confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and 33 oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported 34 mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, 35 we used phylogenomic analyses to show that this hydrogenase is widely distributed and 36 is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric 37 model of the physiological role of atmospheric H2 oxidation and extends this process to 38 two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have 39 broader relevance for understanding how bacteria conserve energy in different 40 environments and control the biogeochemical cycling of atmospheric trace gases. 41 42 43 Aerobic bacteria mediate the biogeochemically and ecologically important process of 44 atmospheric hydrogen (H2) oxidation [1]. Terrestrial bacteria constitute the largest sink 45 of this gas and mediate the net consumption of approximately 70 million tonnes of 46 atmospheric H2 per year [2, 3]. The energy derived from this process appears to be 47 critical for sustaining the productivity and biodiversity of ecosystems with low organic 48 carbon inputs [4-9]. Atmospheric H2 oxidation is thought to be primarily mediated by 49 group 1h [NiFe]-hydrogenases, a specialised oxygen-tolerant, high-affinity class of 50 hydrogenases [4, 10-13]. To date, aerobic heterotrophic bacteria from four distinct 51 bacterial phyla, the Actinobacteriota [10, 12, 14, 15], Acidobacteriota [16, 17], 52 Chloroflexota [18], and Verrucomicrobiota [19], have been experimentally shown to 53 consume atmospheric H2 using this enzyme. This process has been primarily linked 54 to energy conservation during persistence. Reflecting this, the expression and activity 55 of the group 1h hydrogenase is induced by carbon starvation across a wide...

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