Isolation, characterization and symbiotic efficiency of nitrogen-fixing and heavy metal-tolerant bacteria from a Coalmine Wasteland

Abstract

Areas affected by coal mining can be recovered by revegetation with leguminous plants associated with nitrogen-fixing bacteria. This study addressed the isolation and characterization of native nitrogen-fixing bacteria from coalmine wasteland under different vegetation restoration approaches using Macroptilium atropurpureum (DC) Urb and Vicia sativa L. as trap plants. The bacteria were characterized and identified on the basis of 16S rRNA sequences. Additionally, nitrogen-fixing strains were characterized for tolerance to high heavy metal and low pH levels, as well as for their effect on growth, nodulation, and symbiotic efficiency of M. atropurpureum and V. sativa. Soil samples were taken from the rhizosphere of eight areas, between 6 and 20 years under vegetation restoration, in the coal mining area of Candiota, RS-Brazil. The following properties were evaluated: colony characterization on solid “79” culture medium; pH (3.0-9.0) and heavy metal (Cr, Cd, Zn, Cu, and Ni) tolerance; partial sequencing of 16S rRNA gene; presence of nodA and nifH genes and symbiotic efficiency. A total of 115 isolates, i.e., 77 from M. atropurpureum and 38 from V. sativa, were obtained. The tolerance of these isolates is high for a wide range of pH levels and heavy metal contents, and 18 among them were selected for symbiotic efficiency and 16S rRNA sequencing. Inoculated with M. atropurpureum, the strains UFSM-B53, UFSM-B64, and UFSM-B74 had high symbiotic efficiency. The nitrogen-fixing bacteria were classified in the genera Rhizobium, Bradyrhizobium, and Burkholderia. The results indicate the potential of these native rhizospheric bacterial strains as inoculants and biofertilizers for legume species under pH and heavy metal stress in coal mining degraded areas in Southern Brazil.