Many promising hydrogen technologies utilising hydrogenase enzymes have been slowed by the fact that most hydrogenases are extremely sensitive to O-2. Within the group 1 membrane-bound NiFe hydrogenase, naturally occurring tolerant enzymes do exist, and O-2 tolerance has been largely attributed to changes in iron-sulphur clusters coordinated by different numbers of cysteine residues in the enzyme's small subunit. Indeed, previous work has provided a robust phylogenetic signature of O-2 tolerance [1], which when combined with new sequencing technologies makes bio prospecting in nature a far more viable endeavour. However, making sense of such a vast diversity is still challenging and could be simplified if known species with O-2-tolerant enzymes were annotated with information on metabolism and natural environments. Here, we utilised a bioinformatics approach to compare O-2-tolerant and sensitive membrane-bound NiFe hydrogenases from 177 bacterial species with fully sequenced genomes for differences in their taxonomy, O-2 requirements, and natural environment. Following this, we interrogated a metagenome from lacustrine surface sediment for novel hydrogenases via high-throughput shotgun DNA sequencing using the Illumina (TM) MiSeq platform. We found 44 new NiFe group 1 membrane-bound hydrogenase sequence fragments, five of which segregated with the tolerant group on the phylogenetic tree of the enzyme's small subunit, and four with the large subunit, indicating de novo O-2-tolerant protein sequences that could help engineer more efficient hydrogenases.