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

Many high molecular weight polycyclic aromatic hydrocarbons (PAH) are known or suspected carcinogens and as ubiquitous environmental pollutants, their remediation is necessary to reduce human and environmental health risk. Since PAH are biodegradable, bioremediation offers potential for site clean-up. Bioremediation techniques include in situ methods, or excavation followed by reactor treatment. To determine whether bacterial community diversity depends on treatment method, two incubation conditions were examined by stable isotope probing of pyrene-degrading bacteria in an aged PAHcontaminated soil. Microcosms of continuously mixed soil slurry or static, field-wet soil were spiked with [U-13C] pyrene and incubated in the dark at room temperature for 28 days. Recovered 13C-enriched DNA was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. The minimal diversity observed between DGGE profiles suggests that pyrene-degrading bacterial community diversity may be independent of treatment method, though slurry libraries were slightly more diverse than field wet libraries

Stable-isotope probing-based investigations of polycyclic aromatic hydrocarbon-degrading bacteria in contaminated soil

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic contaminants that are a global environmental problem. These compounds become more recalcitrant to remediation and increase in carcinogenic potential with increasing molecular weight. Engine exhaust and industrial process waste, like that from the sites of former manufactured-gas plants, contain high concentrations of PAHs and are major sources of benzo[a]pyrene contamination in the environment. Bioremediation, the use of microorganisms to remove PAH contamination, is the dominant strategy for removing PAH contamination from soil because many microorganisms can grow on PAHs. Stable-isotope probing (SIP) is a cultivation-independent technique used to identify microorganism able to grow on specific chemicals, such as PAHs. SIP was used to identify bacteria in soil from the site of a former manufactured-gas plant that are capable of degrading naphthalene, phenanthrene, anthracene, pyrene, fluoranthene, or benz[a]anthracene. Group-specific quantitative PCR primers were developed to determine whether the bacteria identified were capable of growth on the respective PAH. SIP with naphthalene, phenanthrene, and fluoranthene selected bacteria previously associated with the degradation of those compounds, and Pigmentiphaga was newly associated with naphthalene, phenanthrene, and anthracene degradation. A group of uncultivated Gammaproteobacteria known as Pyrene Group 2 was newly associated with fluoranthene and benz[a]anthracene degradation, and it was the only group of bacteria associated with pyrene degradation. A group of uncultivated Alphaproteobacteria was the primary anthracene-degrading group and was designated Anthracene Group 1; Herminiimonas was also newly associated with anthracene degradation. In experiments to evaluate the biases associated with using a commercial DNA extraction kit, performing multiple DNA extractions on the same anthracene-enriched soil sample did not affect qualitative results; however, shifts in the relative abundances of anthracene-degrading bacteria were observed between extracts. Since no microorganisms are known to grow on benzo[a]pyrene, a carcinogenic PAH, mineralization experiments and the results of the SIP investigations were used to obtain indirect evidence suggesting that bacteria capable of growth on other PAHs might participate in benzo[a]pyrene metabolism. None of the major SIP-identified bacteria were associated with benzo[a]pyrene mineralization, but members of the genera Cupriavidus, Luteimonas, and Rhizobium may be associated with benzo[a]pyrene mineralization

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 O2/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

Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil

ABSTRACT 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

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

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