Pyrosequence analysis of bacterial communities in aerobic bioreactors treating polycyclic aromatic hydrocarbon-contaminated soil.
ABSTRACT: Two aerobic, lab-scale, slurry-phase bioreactors were used to examine the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and the associated bacterial communities. The two bioreactors were operated under semi-continuous (draw-and-fill) conditions at a residence time of 35 days, but one was fed weekly and the other monthly. Most of the quantified PAHs, including high-molecular-weight compounds, were removed to a greater extent in the weekly-fed bioreactor, which achieved total PAH removal of 76%. Molecular analyses, including pyrosequencing of 16S rRNA genes, revealed significant shifts in the soil bacterial communities after introduction to the bioreactors and differences in the abundance and types of bacteria in each of the bioreactors. The weekly-fed bioreactor displayed a more stable bacterial community with gradual changes over time, whereas the monthly-fed bioreactor community was less consistent and may have been more strongly influenced by the influx of untreated soil during feeding. Phylogenetic groups containing known PAH-degrading bacteria previously identified through stable-isotope probing of the untreated soil were differentially affected by bioreactor conditions. Sequences from members of the Acidovorax and Sphingomonas genera, as well as the uncultivated "Pyrene Group 2" were abundant in the bioreactors. However, the relative abundances of sequences from the Pseudomonas, Sphingobium, and Pseudoxanthomonas genera, as well as from a group of unclassified anthracene degraders, were much lower in the bioreactors compared to the untreated soil.
Project description:Contaminated soil from a former manufactured-gas plant site was treated in a laboratory-scale bioreactor. Desorbability and biodegradability of 14 polycyclic aromatic hydrocarbons (PAHs) and 4 oxygenated PAHs (oxy-PAHs) were investigated throughout a treatment cycle. Desorbability was determined using a mixed-function sorbent (Oasis<sup>®</sup> HLB) or a hydrophobic sorbent (Tenax<sup>®</sup>) in dialysis tubing suspended in the soil slurry. Toxicity and genotoxicity of the whole soil and the desorbable fractions were determined by DNA damage response analysis with the chicken DT40 B-lymphocyte isogenic cell line and its DNA repair-deficient mutant <i>Rad54</i><sup>-/-</sup>. Biological treatment significantly removed both PAHs and oxy-PAHs, and their desorbability decreased throughout the bioreactor treatment cycle. Collectively, oxy-PAHs were more desorbable and biodegradable than the corresponding PAHs; for example, the oxy-PAH present at the highest concentration, 9,10-anthraquinone, was more desorbable and biodegradable than anthracene. For both PAHs and oxy-PAHs, the percentage removed in the bioreactor significantly exceeded the percentage desorbed from untreated soil, indicating that desorption did not control the extent of biodegradation. Consistent with previous results on the same soil, genotoxicity of the whole soil slightly increased after biological treatment. However, both toxicity and genotoxicity of the desorbable constituents in the soil decreased after treatment, suggesting that any genotoxic constituents that may have formed during treatment were primarily associated with less accessible domains in the soil.
Project description:Quinones and other oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are toxic and/or genotoxic compounds observed to be cocontaminants at PAH-contaminated sites, but their formation and fate in contaminated environmental systems have not been well studied. Anthracene-9,10-dione (anthraquinone) has been found in most PAH-contaminated soils and sediments that have been analyzed for oxy-PAHs. However, little is known about the biodegradation of oxy-PAHs, and no bacterial isolates have been described that are capable of growing on or degrading anthraquinone. PAH-degrading Mycobacterium spp. are the only organisms that have been investigated to date for metabolism of a PAH quinone, 4,5-pyrenequinone. We utilized DNA-based stable-isotope probing (SIP) with [U-(13)C]anthraquinone to identify bacteria associated with anthraquinone degradation in PAH-contaminated soil from a former manufactured-gas plant site both before and after treatment in a laboratory-scale bioreactor. SIP with [U-(13)C]anthracene was also performed to assess whether bacteria capable of growing on anthracene are the same as those identified to grow on anthraquinone. Organisms closely related to Sphingomonas were the most predominant among the organisms associated with anthraquinone degradation in bioreactor-treated soil, while organisms in the genus Phenylobacterium comprised the majority of anthraquinone degraders in the untreated soil. Bacteria associated with anthracene degradation differed from those responsible for anthraquinone degradation. These results suggest that Sphingomonas and Phenylobacterium species are associated with anthraquinone degradation and that anthracene-degrading organisms may not possess mechanisms to grow on anthraquinone.
Project description:Efficacy of bioremediation for soil contaminated with polycyclic aromatic hydrocarbons (PAHs) may be limited by the fractions of soil-bound PAHs that are less accessible to PAH-degrading microorganisms. In previous test-tube-scale work, submicellar doses of nonionic surfactants were screened for their ability to enhance the desorption and biodegradation of residual PAHs in soil after conventional bioremediation in a laboratory-scale, slurry-phase bioreactor. Polyoxyethylene sorbitol hexaoleate (POESH) was the optimum surfactant for enhancing PAH removal, especially the high-molecular weight PAHs. This work extends that concept by treating the effluent from the slurry-phase bioreactor in a second-stage batch reactor, to which POESH was added, for an additional 7 or 12 days. Surfactant amendment removed substantial amounts of the PAHs and oxy-PAHs remaining after conventional slurry-phase bioremediation, including more than 80% of residual 4-ring PAHs. Surfactant-amended treatment decreased soil cytotoxicity, but often increased the genotoxicity of the soil as measured using the DT-40 chicken lymphocyte DNA damage response assay. Potential ecotoxicity, measured using a seed germination assay, was reduced by bioreactor treatment and was reduced further after second-stage treatment with POESH. Of bacteria previously implicated as potential PAH degraders under POESH-amended conditions in a prior study, members of the <i>Terrimonas</i> genus were associated with differences in high-molecular weight PAH removal in the current study. Research using submicellar doses of surfactant as a second-stage treatment step is limited and these findings can inform the design of bioremediation systems at field sites treating soil contaminated with PAHs and other hydrophobic contaminants that have low bioaccessibility.
Project description:Bioremediation is one of the commonly applied remediation strategies at sites contaminated with polycyclic aromatic hydrocarbons (PAHs). However, remediation goals are typically based on removal of the target contaminants rather than on broader measures related to health risks. We investigated changes in the toxicity and genotoxicity of PAH-contaminated soil from a former manufactured-gas plant site before and after two simulated bioremediation processes: a sequencing batch bioreactor system and a continuous-flow column system. Toxicity and genotoxicity of the residues from solvent extracts of the soil were determined by the chicken DT40 B-lymphocyte isogenic cell line and its DNA-repair-deficient mutants. Although both bioremediation processes significantly removed PAHs from the contaminated soil (bioreactor 69% removal, column 84% removal), bioreactor treatment resulted in an increase in toxicity and genotoxicity over the course of a treatment cycle, whereas long-term column treatment resulted in a decrease in toxicity and genotoxicity. However, when screening with a battery of DT40 mutants for genotoxicity profiling, we found that column treatment induced DNA damage types that were not observed in untreated soil. Toxicity and genotoxicity bioassays can supplement chemical analysis-based risk assessment for contaminated soil when evaluating the efficacy of bioremediation.
Project description:A total of five nonionic surfactants (Brij 30, Span 20, Ecosurf EH-3, polyoxyethylene sorbitol hexaoleate, and R-95 rhamnolipid) were evaluated for their ability to enhance PAH desorption and biodegradation in contaminated soil after treatment in an aerobic bioreactor. Surfactant doses corresponded to aqueous-phase concentrations below the critical micelle concentration in the soil-slurry system. The effect of surfactant amendment on soil (geno)toxicity was also evaluated for Brij 30, Span 20, and POESH using the DT40 B-lymphocyte cell line and two of its DNA-repair-deficient mutants. Compared to the results from no-surfactant controls, incubation of the bioreactor-treated soil with all surfactants increased PAH desorption, and all except R-95 substantially increased PAH biodegradation. POESH had the greatest effect, removing 50% of total measured PAHs. Brij 30, Span 20, and POESH were particularly effective at enhancing biodegradation of four- and five-ring PAHs, including five of the seven carcinogenic PAHs, with removals up to 80%. Surfactant amendment also significantly enhanced the removal of alkyl-PAHs. Most treatments significantly increased soil toxicity. Only the no-surfactant control and Brij 30 at the optimum dose significantly decreased soil genotoxicity, as evaluated with either mutant cell line. Overall, these findings have implications for the feasibility of bioremediation to achieve cleanup levels for PAHs in soil.
Project description:Dermal exposure can represent a significant health risk in settings involving potential contact with soil contaminated with polycyclic aromatic hydrocarbons (PAHs). However, there is limited work on the ability of PAHs in contaminated soil to reach the skin surface via desorption from the soil. We evaluated PAH desorption from a field-contaminated soil to a two-dimensional hydrophobic surface (C18 extraction disk) as a measure of potential dermal exposure as a function of soil loading (5-100 mg dry soil cm(-2)), temperature (20-40°C), and soil moisture content (2-40%) over periods up to 16d. The efficacy of bioremediation in removing the most readily desorbable PAH fractions was also evaluated. Desorption kinetics were described well by an empirical two-compartment kinetic model. PAH mass desorbed to the C18 disk kept increasing at soil loadings well above the estimated monolayer coverage, suggesting mechanisms for PAH transport to the surface other than by direct contact. Such mechanisms were reinforced by observations that desorption occurred even with dry or moist glass microfiber filters placed between the C18 disk and the soil. Desorption of all PAHs was substantially reduced at a soil moisture content corresponding to field capacity, suggesting that transport through pore air contributed to PAH transport to the C18 disk. The lower molecular weight PAHs had greater potential to desorb from soil than higher molecular weight PAHs. Biological treatment of the soil in a slurry-phase bioreactor completely eliminated PAH desorption to the C18 disks.
Project description: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.
Project description:Bioremediation is an accepted technology for cleanup of soil contaminated with polycyclic aromatic hydrocarbons (PAHs), but it can increase the genotoxicity of the soil despite removal of the regulated PAHs. Although polar biotransformation products have been implicated as causative genotoxic agents, no specific product has been identified. We pursued a nontarget analytical approach combining effect-directed analysis (EDA) and metabolite profiling to compare extracts of PAH-contaminated soil from a former manufactured-gas plant site before and after treatment in a laboratory-scale aerobic bioreactor. A compound with the composition C15H8O2 and four methylated homologues were shown to accumulate as a result of bioreactor treatment, and the C15H8O2 compound purified from soil extracts was determined to be genotoxic. Its structure was established by nuclear magnetic resonance and mass spectroscopy as a heretofore unidentified ?,?-unsaturated lactone derived from dioxygenation of pyrene at an apical ring, 2H-naphtho[2,1,8-def]chromen-2-one (NCO), which was confirmed by synthesis. The concentration of NCO in the bioreactor was 11 ?g g-1 dry soil, corresponding to 13% of the pyrene removed. It also accumulated in aerobically incubated soil from two additional PAH-contaminated sites and was formed from pyrene by two pyrene-degrading bacterial cultures known to be geographically widespread, underscoring its potential environmental significance.
Project description:We evaluated two nonionic surfactants, one hydrophobic (Brij 30) and one hydrophilic (C(12)E(8)), for their ability to enhance the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil after it had been treated in an aerobic bioreactor. The effects of each surfactant were evaluated at doses corresponding to equilibrium aqueous-phase concentrations well above the surfactant's critical micelle concentration (CMC), slightly above the CMC, and below the CMC. The concentrations of all 3- and 4-ring PAHs were significantly lower in the soil amended with Brij 30 at the two lower doses compared to controls, whereas removal of only the 3-ring PAHs was significantly enhanced at the highest Brij 30 dose. In contrast, C(12)E(8) did not enhance PAH removal at any dose. In the absence of surfactant, <5% of any PAH desorbed from the soil over an 18 day period. Brij 30 addition at the lowest dose significantly increased the desorption of most PAHs, whereas the addition of C(12)E(8) at the lowest dose actually decreased the desorption of all PAHs. These findings suggest that the effects of the two surfactants on PAH biodegradation could be explained by their effects on PAH bioavailability. Overall, this study demonstrates that the properties of the surfactant and its dose relative to the corresponding aqueous-phase concentration are important factors in designing systems for surfactant-enhanced bioremediation of PAH-contaminated soils in which PAH bioavailability is limited.
Project description:The formation of more polar and toxic polycyclic aromatic hydrocarbon (PAH) transformation products is one of the concerns associated with the bioremediation of PAH-contaminated soils. Soil contaminated with coal tar (prebioremediation) from a former manufactured gas plant (MGP) site was treated in a laboratory scale bioreactor (postbioremediation) and extracted using pressurized liquid extraction. The soil extracts were fractionated, based on polarity, and analyzed for 88 PAHs (unsubstituted, oxygenated, nitrated, and heterocyclic PAHs). The PAH concentrations in the soil tested, postbioremediation, were lower than their regulatory maximum allowable concentrations (MACs), with the exception of the higher molecular weight PAHs (BaA, BkF, BbF, BaP, and IcdP), most of which did not undergo significant biodegradation. The soil extract fractions were tested for genotoxicity using the DT40 chicken lymphocyte bioassay and developmental toxicity using the embryonic zebrafish (Danio rerio) bioassay. A statistically significant increase in genotoxicity was measured in the unfractionated soil extract, as well as in four polar soil extract fractions, postbioremediation (p < 0.05). In addition, a statistically significant increase in developmental toxicity was measured in one polar soil extract fraction, postbioremediation (p < 0.05). A series of morphological abnormalities, including peculiar caudal fin malformations and hyperpigmentation in the tail, were measured in several soil extract fractions in embryonic zebrafish, both pre- and postbioremediation. The increased toxicity measured postbioremediation is not likely due to the 88 PAHs measured in this study (including quinones), because most were not present in the toxic polar fractions and/or because their concentrations did not increase postbioremediation. However, the increased toxicity measured postbioremediation is likely due to hydroxylated and carboxylated transformation products of the 3- and 4-ring PAHs (PHE, 1MPHE, 2MPHE, PRY, BaA, and FLA) that were most degraded.