Abstract
Functional assessment of hydrogenase activity in native bacterial systems is often complicated by the requirements associated with enzyme purification, heterologous expression, and cofactor maturation. Here, we present a standardized gas chromatography‐based workflow for the direct assessment of hydrogenase activity in crude membrane and lysate fractions without enzyme purification or genetic manipulation. The workflow was applied to a defined panel of phylogenetically distinct bacterial strains. Hydrogen oxidation activity was detected in
Shewanella acanthi
and
Insolitispirillum peregrinum
.
Burkholderia plantarii
exhibited low but reproducible hydrogen oxidation activity despite the absence of a clear hydrogenase candidate in the annotation‐based search applied here. This observation prompted further screening of
Paraburkholderia mimosarum
,
P. xenovorans
,
P. kururiensis
,
P. phenoliruptrix
, and
P. phymatum
, revealing hydrogen oxidation activity across these related strains. Among the initially investigated nonmodel organisms,
I. peregrinum
exhibited the highest hydrogen oxidation activity and showed prolonged activity, partial oxygen tolerance, and substantial activity at elevated temperatures. Hydrogen production was consistently lower than hydrogen oxidation under the applied assay conditions. Overall, the workflow provides a practical approach for comparative screening of hydrogen‐converting activity in native bacterial fractions and can support the selection of candidate systems for further biochemical characterization.