Phenanthrene-Degrading and Nickel-Resistant Neorhizobium Strain Isolated from Hydrocarbon-Contaminated Rhizosphere of Medicago sativa L

Abstract

Pollutant degradation and heavy-metal resistance may be important features of the rhizobia, making them promising agents for environment cleanup biotechnology. The degradation of phenanthrene, a three-ring polycyclic aromatic hydrocarbon (PAH), by the rhizobial strain Rsf11 isolated from the oil-polluted rhizosphere of alfalfa and the influence of nickel ions on this process were studied. On the basis of whole-genome and polyphasic taxonomy, the bacterium Rsf11 represent a novel species of the genus Neorhizobium, so the name Neorhizobium phenanthreniclasticum sp. nov. was proposed. Analysis of phenanthrene degradation by the Rsf1 strain revealed 1-hydroxy-2-naphthoic acid as the key intermediate and the activity of two enzymes apparently involved in PAH degradation. It was also shown that the nickel resistance of Rsf11 was connected with the extracellular adsorption of metal by EPS. The joint presence of phenanthrene and nickel in the medium reduced the degradation of PAH by the microorganism, apparently due to the inhibition of microbial growth but not due to the inhibition of the activity of the PAH degradation enzymes. Genes potentially involved in PAH catabolism and nickel resistance were discovered in the microorganism studied. N. phenanthreniclasticum strain Rsf11 can be considered as a promising candidate for use in the bioremediation of mixed PAH-heavy-metal contamination.

Keywords: Neorhizobium; PAHs; microbial degradation; nickel resistance; phenanthrene.

Figure 1

ML phylogenetic tree of strain Rsf11 and closely related type strains inferred from 16S rRNA gene sequences under the GTR+GAMMA model. The tree was rooted at the midpoint. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches are support values when larger than 60% from ML (left) and MP (right) bootstrapping. GenBank accession numbers are shown in parentheses. The Rhizobium terrae and Rhizobium populisoli type strains were assigned to Neorhizobium [64] and Rhizobium phenanthrenilyticum type strain to Neorhizobium petrolearium [23,61].

Figure 2

Phylogenetic tree of strain Rsf11 and closely related type strains inferred from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d₅. The numbers near branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 74.4%. The tree was rooted at the midpoint. The Rhizobium terrae and Rhizobium populisoli type strains were assigned to Neorhizobium [64].

Figure 3

Cultural-morphological characteristics of strain Rsf11: (a) 48 h colonies on YMA medium; (b) Gram-negative staining of microbial cells; (c) transmission electron microscopy of a single cell grown on YMA for 48 h; (d) transmission electron microscopy of Rsf11 cell division.

Figure 3

Cultural-morphological characteristics of strain Rsf11: (a) 48 h colonies on YMA medium; (b) Gram-negative staining of microbial cells; (c) transmission electron microscopy of a single cell grown on YMA for 48 h; (d) transmission electron microscopy of Rsf11 cell division.

Figure 4

Degradation of phenanthrene as a sole carbon and energy source and HNA formation during the growth of the Rsf11 strain in mineral medium.

Figure 5

HPLC chromatogram of the ethyl acetate extract of the medium after 7 days’ cultivation of bacterial strain Rsf11 with phenanthrene (0.2 g L⁻¹).

Figure 6

Effect of nickel on growth of the Rsf11 strain in LB medium.

Figure 7

Intracellular accumulation and extracellular adsorption of nickel by Rsf11 cells.

Figure 8

Effect of nickel on growth and phenanthrene degradation by the Rsf11 strain.

Figure 9

The effect of nickel on the activity of PQR and 3,4-PCD involved in the degradation of phenanthrene by the Rsf11 strain.