Isolation of soil bacteria adapted to degrade humic acid-sorbed phenanthrene

Abstract

The goal of these studies was to determine how sorption by humic acids affected the bioavailability of polynuclear aromatic hydrocarbons (PAHs) to PAH-degrading microbes. Micellar solutions of humic acid were used as sorbents, and phenanthrene was used as a model PAH. Enrichments from PAH-contaminated soils established with nonsorbed phenanthrene yielded a total of 25 different isolates representing a diversity of bacterial phylotypes. In contrast, only three strains of Burkholderia spp. and one strain each of Delftia sp. and Sphingomonas sp. were isolated from enrichments with humic acid-sorbed phenanthrene (HASP). Using [14C]phenanthrene as a radiotracer, we verified that only HASP isolates were capable of mineralizing HASP, a phenotype hence termed "competence." Competence was an all-or-nothing phenotype: noncompetent strains showed no detectable phenanthrene mineralization in HASP cultures, but levels of phenanthrene mineralization effected by competent strains in HASP and NSP cultures were not significantly different. Levels and rates of phenanthrene mineralization exceeded those predicted to be supported solely by the metabolism of phenanthrene in the aqueous phase of HASP cultures. Thus, competent strains were able to directly access phenanthrene sorbed by the humic acids and did not rely on desorption for substrate uptake. To the best of our knowledge, this is the first report of (i) a selective interaction between aerobic bacteria and humic acid molecules and (ii) differential bioavailability to bacteria of PAHs sorbed to a natural biogeopolymer.

FIG. 1.

Equilibrium dialysis analysis of sorption (A) and desorption (B) of phenanthrene by a 2% (wt/vol) humic acid solution. Open circles represent data points. The two broken lines indicate the upper and lower values reported in the literature for the aqueous solubility of phenanthrene. Error bars (filled rectangles) at endpoints represent 95% confidence intervals.

FIG. 2.

Time course of phenanthrene mineralization in NSP (open symbols) and HASP (filled symbols) cultures. Results are shown for Burkholderia sp. strain Eh1-1 (circles), Burkholderia sp. strain Cs1-4 (squares), and Delftia sp. strain Eh2-1 (triangles). All cultures were started with an initial inoculum of 10⁷ CFU except that identified as HASP Eh1-1 (low), which was inoculated with 10⁴ CFU. Bars indicate standard deviations of duplicated measures and do not appear where these were smaller than the symbol plotted.