Mycobacterium diversity and pyrene mineralization in petroleum-contaminated soils

Abstract

Degradative strains of fast-growing Mycobacterium spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using Mycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacterium community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer Mycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacterium community. Mineralization studies using [(14)C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.

FIG. 1

TGGE separation of the 16S rDNAs of fast-growing mycobacteria in eight soil samples. Lane 135, Mycobacterium sp. strain 135; lanes AC, CC, JC, and KC, less contaminated soil samples AC, CC, JC, and KC, respectively; lanes AT, CT, JT, and KT, heavily contaminated soil samples AT, CT, JT, and KT, respectively. TGGE bands 1, 3, 4, 9, 24, 10, 11, 12, 13, 14, 15, 17, 18, 19, 22, and 23 correspond to bands AC-1, AT-3, CC-4, CT-9, CT24, CT-10, CT-11, CT-12, JC-13, JC-14, JT-15, KC-17, KC-18, KT-19, KT-22, and KT-23, respectively. Electrophoresis was carried out with a temperature gradient of 55 to 65°C for 16 h 40 min in a 9 M urea–6% gel.

FIG. 2

Relationship between PAH concentration and the number of TGGE bands recovered from individual soils. Data points represent the eight soils, including heavily contaminated and less contaminated soils.

FIG. 3

Neighbor-joining tree for sequences of 17 excised TGGE bands, strains of known Mycobacterium spp., and N. farcinica used as an outgroup. The scale bar corresponds to 0.01 estimated nucleotide substitution per sequence position. The 17 sequenced bands (GenBank accession numbers) are as follows: AC-1 (AF294742), AT-3 (AF220427), CC-4 (AF330695), CT-9 (AF294743), CT-10 (AF294744), CT-11 (AF220428), CT-12 (AF220429), CT-24 (AF294745), CT-25 (AF294746), JC-13 (AF294747), JC-14 (AF294748), JT-15 (AF220430), KT-19 (AF220431), KT-22 (AF220432), KT-23 (AF220433), KT-26 (AF294749), and KT-27 (AF294750). The 11 Mycobacterium spp. and the outgroup (accession numbers) are as follows: M. fluoroanthenivorans (AJ276274), Mycobacterium sp. strain LB501T (AJ245702), Mycobacterium sp. strain CH-1 (AF054278), Mycobacterium sp. strain RJGII-135 (U30661), M. flavescens (X52932), M. monacense (AF107039), Mycobacterium sp. strain U46146 (U46146), Mycobacterium sp. strain PYR-1 (U30662), M. austroafricanum (X93182), M. chlorophenolicum (X81926), Mycobacterium sp. strain TA5 (AB028483), and N. farcinica (X91041).

FIG. 4

Pyrene mineralization curves and TGGE analysis of contaminated soils. (A, C, E, and G) Pyrene mineralization curves. AT, CT, JT, and KT are soils amended with pyrene only. AT+135, CT+135, JT+135, and KT+135 are soils amended with pyrene and Mycobacterium sp. strain 135. Ster AT+135, Ster CT+135, Ster JT+135, and Ster KT+135 are sterilized soils amended with pyrene and strain 135. Soils were sampled for TGGE analysis at days 4, 26, and 80 (asterisks). Error bars represent standard errors from three replicates. (B, D, F, and H) TGGE analysis. Lanes AT, CT, JT, and KT, unamended soil at day 0; lanes, 4, 26, and 80, pyrene-amended soils extracted at days 4, 26, and 80, respectively. Bands CT-25, KT-26, and KT-27 were excised for sequencing and phylogenetic analysis. Electrophoresis was carried out with a temperature gradient of 55 to 65°C for 16 h 40 min in a 9 M urea–6% gel.