Characterization of cultures enriched from acidic polycyclic aromatic hydrocarbon-contaminated soil for growth on pyrene at low pH

Abstract

Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [14C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to Mycobacterium montefiorense, belonging to the slow-growing Mycobacterium sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by Pseudomonas putida and a fast-growing Mycobacterium sp. The M. montefiorense-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant Mycobacterium species in the acidic soils. Our data indicate that a slow-growing Mycobacterium species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.

FIG. 1.

Eubacterial 16S rRNA gene DGGE analysis of the indigenous microbial community in soil samples. Lanes: 1 to 2, soil TM; 3 to 4, soil B3; 5 to 6, soil B7-2; 7 to 8, soil B7-1.

FIG. 2.

Cumulative [¹⁴C]pyrene mineralization curves at pH 3 of the pyrene-utilizing cultures enriched from soil B7-2 at pH 3 and pH 5, enriched from soil B7-1 at pH 7, and enriched from soil TM at pH 7.

FIG. 3.

(A) OD₆₀₀ of the enrichment cultures after 94 days of growth in MM with pyrene as the sole source of carbon and energy at different pHs. The initial OD was <0.01. (B) Growth curves (OD₆₀₀ versus time) at different pHs of the culture enriched at pH 3 from soil B7-2.

FIG. 4.

DGGE analysis of 16S rRNA gene fragments obtained by PCR with eubacterial primers GC40-63f and 518r (lanes 1 to 5) or with Mycobacterium-specific primers MYCO66f and GC40-MYCO600r (lanes 6 to 11) from the enrichment cultures and selected soil samples. Lanes: 1, soil B7-2; 2, culture TM (pH 7); 3, culture B7-2 (pH 3); 4, culture B7-2 (pH 5); 5, culture B7-1 (pH 7); 6, soil B7-1; 7, soil B7-2; 8, culture TM (pH 7); 9, culture B7-2 (pH 3); 10, culture B7-2 (pH 5); 11, culture B7-1 (pH 7). For more details on bands T1 to T9, see Table 1. An oval indicates the position of a band in the soil fingerprints, comigrating with bands T4 and T5 or with bands T7 and T8.