Effect of model sorptive phases on phenanthrene biodegradation: molecular analysis of enrichments and isolates suggests selection based on bioavailability

Abstract

Reduced bioavailability of nonpolar contaminants due to sorption to natural organic matter is an important factor controlling biodegradation of pollutants in the environment. We established enrichment cultures in which solid organic phases were used to reduce phenanthrene bioavailability to different degrees (R. J. Grosser, M. Friedrich, D. M. Ward, and W. P. Inskeep, Appl. Environ. Microbiol. 66:2695-2702, 2000). Bacteria enriched and isolated from contaminated soils under these conditions were analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S ribosomal DNA segments. Compared to DGGE patterns obtained with enrichment cultures containing sand or no sorptive solid phase, different DGGE patterns were obtained with enrichment cultures containing phenanthrene sorbed to beads of Amberlite IRC-50 (AMB), a weak cation-exchange resin, and especially Biobead SM7 (SM7), a polyacrylic resin that sorbed phenanthrene more strongly. SM7 enrichments selected for mycobacterial phenanthrene mineralizers, whereas AMB enrichments selected for a Burkholderia sp. that degrades phenanthrene. Identical mycobacterial and Burkholderia 16S rRNA sequence segments were found in SM7 and AMB enrichment cultures inoculated with contaminated soil from two geographically distant sites. Other closely related Burkholderia sp. populations, some of which utilized phenanthrene, were detected in sand and control enrichment cultures. Our results are consistent with the hypothesis that different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted to different phenanthrene bioavailabilities.

FIG. 1

DGGE analysis of PCR-amplified 16S rRNA gene segments from replicates (labeled a and b) after sequential transfers (numbers above the lanes) of enrichment cultures inoculated with Dover, Ohio, soil. (A) Control. (B) Sand. (C) AMB. (D) SM7. The band numbers correspond to those in Tables 1 and 2. The dashed lines are included to help visualize comigrating bands. hd, heteroduplex bands.

FIG. 2

Comparison of the DGGE profiles of PCR-amplified 16S rDNA segments from isolates to those obtained in the enrichment cultures from which the isolates were cultivated. The band numbers correspond to those in Fig. 1 and Tables 1 and 2. The dashed lines indicate possible comigration. The positions of bands whose numbers are highlighted and italicized were inferred based on comigration with bands in earlier transfer preparations that were actually sequenced (Fig. 1).

FIG. 3

Comparison of DGGE profiles of PCR-amplified 16S rRNA gene segments from stabilized AMB (A) and SM7 (B) enrichment cultures inoculated with soil from Dover, Ohio, or Libby, Mont. The band numbers correspond to those in Tables 1 and 2.