Isolation and characterization of H₂ oxidizing bacteria in H₂ treated soil and soil adjacent to Hup⁻ soybean nodules

Abstract

Previous studies have shown that the hydrogen (H₂) gas evolved from legume nodules inoculated by hydrogen releasing (Hup⁻) rhizobium strains contributes to growth promotion of subsequent crop plants by altering the populations of soil H₂ oxidizing bacteria; this may be responsible for the major beneficial effect of legumes in crop rotation. To further study the plant growth promoting bacteria in H₂ treated soil and soil adjacent to the Hup⁻ legume nodules, the H₂ oxidizing bacteria were isolated from H₂ treated soil and soil adjacent to Hup⁻ soybean nodules grown in greenhouse and field. A much higher bacterial density was found in soils with a higher H₂ uptake rate. Twenty isolates of H₂ oxidizing bacteria were obtained. They are slow growing bacteria on mineral salt medium incubated under H₂- enriched air. All H₂ oxidizing bacterial strains exhibited one high K[subscript m] for H₂ at 1000ppm. The isolates of H₂ oxidizing bacteria were identified as Variovorax paradoxus, Flavobacterium johnsonae and Burkholderia sordidicola by conventional microbiological tests and 16S rRNA gene sequence analysis. All isolated H₂ oxidizing bacteria increased the root elongation of spring wheat seedlings ranging from 21% to 254% compared to controls. Seventeen of them had a significantly positive effect on root elongation (p<0.005). The strains of V. paradoxus found in both H₂ treated soil and soil adjacent to Hup⁻ soybean nodules had the highest direct plant growth effect. The results confirm the hypothesis that the microorganisms responsible for the oxidation of H₂ released by Hup⁻ legume nodules are bacteria and they act as plant growth promoting rhizobacteria (PGPR).