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 0.14mg₍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.