Extraction and characterization of soil hydrogenases oxidizing atmospheric hydrogen

Soils are the largest sink of atmospheric hydrogen contributing about 75% to the total budget. Atmospheric H₂ is assumed to be oxidized in soil by abiontic soil hydrogenases. Extraction of a forest soil with a slightly alkaline (pH 8.5) buffer containing polyethylene glycol (PEG), followed by filtration yielded a bacteria-free extract that oxidized H₂ at ambient concentrations (0.2–2.0 ppmv). Hydrogenase activity was assayed by gas chromatographic analysis of H₂ consumption and by conversion of ³H₂ to tritiated water. Only less than 2% of the original activity was recovered in the extract. Kinetic analysis nevertheless resulted in a biphasic kinetics exhibiting two K_m and V_max values that were similar to those detected in the original soil. In addition, activities of both original soil and soil extract showed similar optima at pH 4–6 and at 30 °C, indicating that representative fractions of soil hydrogenases were recovered in the extract. Precipitation with PEG or ultrafiltration allowed further purification of the activity, albeit only about 20% of that in the crude extract could be recovered in the precipitate or the fraction >100 kDa.