Co-localization of atmospheric H2 oxidation activity and high affinity H2-oxidizing bacteria in non-axenic soil and sterile soil amended with Streptomyces sp. PCB7

Two complementary experimental approaches were utilized to examine the extent to which free soil hydrogenases and H2-oxidizing bacteria contribute to the soil uptake of atmospheric H2. First, high affinity hydrogenase activity and H2-oxidizing bacteria were fractionated in non-axenic soil and axenic soil colonized with the high affinity H2-oxidizing bacterium Streptomyces sp. PCB7. Non-axenic soil was fractionated by buoyant density centrifugation. High affinity H2 oxidation activity measured in individual fractions was proportional to the copy number of hhyL gene, specifying the large subunit of putative high affinity [NiFe]-hydrogenases. 2.5% of the hydrogenase activity was recovered in bacteria-free soil extract. Similarly, sequential centrifugation and wet filtrations of strain PCB7-colonized soil dispersed in solubilization buffer caused a loss of the activity, at a ratio proportional to the number of living cells removed. No abiontic hydrogenase activity was detected in bacteria-free fractions. The second experimental approach was designed to verify whether or not the [NiFe]-hydrogenase of strain PCB7 retains high affinity H2 oxidation activity in soil, under the abiontic state. H2 oxidation rates of crude enzyme extract of strain PCB7 measured under aerobic and anaerobic conditions were indistinguishable, indicating that the high affinity hydrogenase of strain PCB7 is oxygen-tolerant. The hydrogenase activity of sterile soil spiked with as much as 0.14 mg(protein) g(soil-dw)−1 was equivalent to the H2-oxidation activity of only 106–107 CFU of strain PCB7 g(soil-dw)−1. Taken together, our results indicate that high affinity hydrogenase activity is proportional to the abundance of H2-oxidizing bacteria in soil and, that abiontic hydrogenases contribute only a few percent of the total high affinity H2 oxidation activity detected in soil.