Linking of microorganisms to phenanthrene metabolism in soil by analysis of ¹³C-labeled cell lipids

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [¹⁴C] phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [¹³C] phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The ¹³C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The ¹³C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in ¹³C, suggesting that actinomycetes metabolized phenanthrene in this soil. The ¹³C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess ¹³C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [¹³C] phenanthrene.