Association of Growth Substrates and Bacterial Genera with Benzo[a]pyrene Mineralization in Contaminated Soil.

Benzo[a]pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon (PAH) that is not known to be a bacterial growth substrate. Organisms capable of cometabolizing BaP in complex field-contaminated systems have not previously been identified. We evaluated BaP mineralization by a bacterial community from a bioreactor treating PAH-contaminated soil during coincubation with or after pre-enrichment on various PAHs as growth substrates. Pyrosequence libraries of 16S rRNA genes were used to identify bacteria that were enriched on the added growth substrate as a means of associating specific organisms with BaP mineralization. Coincubating the bioreactor-treated soil with naphthalene, phenanthrene, or pyrene inhibited BaP mineralization, whereas pre-enriching the soil on the same three PAHs enhanced BaP mineralization. Combined, these results suggest that bacteria in the bioreactor community that are capable of growing on naphthalene, phenanthrene, and/or pyrene can metabolize BaP, with coincubation competitively inhibiting BaP metabolism. Anthracene, fluoranthene, and benz[a]anthracene had little effect on BaP mineralization compared to incubations without an added growth substrate under either coincubation or pre-enrichment conditions. Substantial increases in relative abundance after pre-enrichment with phenanthrene, naphthalene, or pyrene, but not the other PAHs, suggest that members of the genera Cupriavidus and Luteimonas may have been associated with BaP mineralization.

Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil.

Barcoded amplicon pyrosequencing was used to generate libraries of partial 16S rRNA genes from two columns designed to simulate in situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) in weathered, contaminated soil. Both columns received a continuous flow of artificial groundwater but one of the columns additionally tested the impact of biostimulation with oxygen and inorganic nutrients on indigenous soil bacterial communities. The penetration of oxygen to previously anoxic regions of the columns resulted in the most significant community changes. PAH-degrading bacteria previously determined by stable-isotope probing (SIP) of the untreated soil generally responded negatively to the treatment conditions, with only members of the Acidovorax and a group of uncharacterized PAH-degrading Gammaproteobacteria maintaining a significant presence in the columns. Additional groups of sequences associated with the Betaproteobacterial family Rhodocyclaceae (including those associated with PAH degradation in other soils), and the Thiobacillus, Thermomonas, and Bradyrhizobium genera were also present in high abundance in the biostimulated column. Similar community responses were previously observed during biostimulated ex situ treatment of the same soil in aerobic, slurry-phase bioreactors. While the low relative abundance of many SIP-determined groups in the column libraries may be a reflection of the slow removal of PAHs in that system, the similar response of known PAH degraders in a higher-rate bioreactor system suggests that alternative PAH-degrading bacteria, unidentified by SIP of the untreated soil, may also be enriched in engineered systems.

Long-term simulation of in situ biostimulation of polycyclic aromatic hydrocarbon-contaminated soil.

A continuous-flow column study was conducted to evaluate the long-term effects of in situ biostimulation on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil from a manufactured gas plant site. Simulated groundwater amended with oxygen and inorganic nutrients was introduced into one column, while a second column receiving unamended groundwater served as a control. PAH and dissolved oxygen (DO) concentrations, as well as microbial community profiles, were monitored along the column length immediately before and at selected intervals up to 534 days after biostimulation commenced. Biostimulation resulted in significantly greater PAH removal than in the control condition (73% of total measured PAHs vs. 34%, respectively), with dissolution accounting for a minor amount of the total mass loss (~6%) in both columns. Dissolution was most significant for naphthalene, acenaphthene, and fluorene, accounting for >20% of the total mass removed for each. A known group of PAH-degrading bacteria, 'Pyrene Group 2' (PG2), was identified as a dominant member of the microbial community and responded favorably to biostimulation. Spatial and temporal variations in soil PAH concentration and PG2 abundance were strongly correlated to DO advancement, although there appeared to be transport of PG2 organisms ahead of the oxygen front. At an estimated oxygen demand of 6.2 mg O(2)/g dry soil and a porewater velocity of 0.8 m/day, it took between 374 and 466 days for oxygen breakthrough from the 1-m soil bed in the biostimulated column. This study demonstrated that the presence of oxygen was the limiting factor in PAH removal, as opposed to the abundance and/or activity of PAH-degrading bacteria once oxygen reached a previously anoxic zone.

Stable-isotope probing of the polycyclic aromatic hydrocarbon-degrading bacterial guild in a contaminated soil.

The bacteria responsible for the degradation of naphthalene, phenanthrene, pyrene, fluoranthene or benz[a]anthracene in a polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated by DNA-based stable-isotope probing (SIP). Clone libraries of 16S rRNA genes were generated from the (13) C-enriched ('heavy') DNA recovered from each SIP experiment, and quantitative PCR primers targeting the 16S rRNA gene were developed to measure the abundances of many of the SIP-identified sequences. Clone libraries from the SIP experiments with naphthalene, phenanthrene and fluoranthene primarily contained sequences related to bacteria previously associated with the degradation of those compounds. However, Pigmentiphaga-related sequences were newly associated with naphthalene and phenanthrene degradation, and sequences from a group of uncultivated γ-Proteobacteria known as Pyrene Group 2 were newly associated with fluoranthene and benz[a]anthracene degradation. Pyrene Group 2-related sequences were the only sequences recovered from the clone library generated from SIP with pyrene, and they were 82% of the sequences recovered from the clone library generated from SIP with benz[a]anthracene. In time-course experiments with each substrate in unlabelled form, the abundance of each of the measured groups increased in response to the corresponding substrate. These results provide a comprehensive description of the microbial ecology of a PAH-contaminated soil as it relates to the biodegradation of PAHs from two to four rings, and they underscore that bacteria in Pyrene Group 2 are well-suited for the degradation of four-ring PAHs.

Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

Effect of incubation conditions on the enrichment of pyrene-degrading bacteria identified by stable-isotope probing in an aged, PAH-contaminated soil.

To determine whether the diversity of pyrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon-contaminated soil is affected by the addition of inorganic nutrients or by slurrying the soil, various incubation conditions (all including phosphate buffer) were examined by mineralization studies and stable-isotope probing (SIP). The addition of nitrogen to either continuously mixed slurry or static field-wet soil incubations increased the rate and extent of mineralization of [(14)C]pyrene, with the most rapid mineralization observed in slurried, nitrogen-amended soil. Microcosms of slurry and static field-wet soil amended with nitrogen were also examined by SIP with [U-(13)C]pyrene. Recovered (13)C-enriched deoxyribonucleic acid (DNA) was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S ribosomal ribonucleic acid (rRNA) gene clone libraries. DGGE profiles of (13)C-enriched DNA fractions from both incubation conditions were similar, suggesting that pyrene-degrading bacterial community diversity may be independent of treatment method. The vast majority (67 of 71) of the partial sequences recovered from clone libraries were greater than or equal to 97% similar to one another, 98% similar to sequences of pyrene-degrading bacteria previously detected by SIP with pyrene in different soil, and only 89% similar to the closest cultivated genus. All of the sequences recovered from the field-wet incubation and most of the sequences recovered from the slurry incubation were in this clade. Of the four sequences from slurry incubations not within this clade, three possessed greater than 99% similarity to the 16S rRNA gene sequences of phylogenetically dissimilar Caulobacter spp.