Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the ␣-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the ␥-Proteobacteria group (genus Xanthomonas), the ␣-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the ␥-Proteobacteria group (genus Xathomonas), the ␤-Proteobacteria group (genera Alcaligenes and Achromobacter), and the ␣-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans-or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6-dihydroxy-2,2-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.

The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

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