Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

Abstract

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

FIG. 1

Isolation of PAH-degrading bacteria using the classical liquid enrichment procedure (path A) and using PAH-sorbing membranes (path B).

FIG. 2

Cluster analysis of REP-PCR patterns of the PAH-degrading isolates. The strains that were further characterized are marked with an asterisk.

FIG. 3

Position of the new isolated PAH-degrading Mycobacterium spp. in the distance tree of predominantly fast-growing Mycobacterium species, based on 16S rRNA gene sequence analysis. Bootstrap values are indicated at the corresponding nodes (expressed as percentages). The distance between two species is obtained by summing the lengths of the connecting horizontal branches using the scale at the top. PAH-degrading strains are marked with an asterisk. Accession numbers for the 16S rRNA gene sequences are indicated between brackets.

FIG. 4

Contact angles plotted against the electrophoretic mobilities of several gram-positive and gram-negative isolates, showing the position of the newly isolated PAH degraders. All u values were measured at an ionic strength of 10 mM (or 8.1 mM) and pH 7.0 to 7.2. Contact angles were measured in distilled water or at an ionic strength of 10 mM (29).