Surfactant-induced bacterial community changes correlated with increased polycyclic aromatic hydrocarbon degradation in contaminated soil

Bioremediation as a method for removing polycyclic aromatic hydrocarbons (PAHs) from contaminated environments has been criticized for poor removal of potentially carcinogenic but less bioavailable high-molecular-weight (HMW) compounds. As a partial remedy to this constraint, we studied surfactant addition at sub-micellar concentrations to contaminated soil to enhance the biodegradation of PAHs remaining after conventional aerobic bioremediation. We demonstrated increased removal of 4- and 5-ring PAHs using two nonionic surfactants, polyoxyethylene(4)lauryl ether (Brij 30) and polyoxyethylene sorbitol hexaoleate (POESH), and analyzed bacterial community shifts associated with those conditions. Eight groups of abundant bacteria were implicated as potentially being involved in increased HMW PAH removal. A group of unclassified Alphaproteobacteria and members of the Phenylobacterium genus in particular showed significantly increased relative abundance in the two conditions exhibiting increased PAH removal. Other implicated groups included members of the Sediminibacterium, Terrimonas, Acidovorax, and Luteimonas genera, as well as uncharacterized organisms within the families Chitinophagaceae and Bradyrhizobiaceae. Targeted isolation identified a subset of the community likely using the surfactants as a growth substrate but few of the isolates exhibited PAH-degradation capability. Isolates recovered from the Acidovorax and uncharacterized Bradyrhizobiaceae groups suggest the abundance of those groups may have been attributable to growth on surfactants. Understanding the specific bacteria responsible for HMW PAH removal in natural and engineered systems and their response to stimuli such as surfactant amendment may improve bioremediation efficacy during treatment of contaminated environmental media

Improving Polycyclic Aromatic Hydrocarbon Biodegradation in Contaminated Soil Through Low-Level Surfactant Addition After Conventional Bioremediation

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 Terrimonas 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

Bioavailability of (Geno)toxic Contaminants in Polycyclic Aromatic Hydrocarbon–Contaminated Soil Before and After Biological Treatment

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® HLB) or a hydrophobic sorbent (Tenax®) 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 Rad54−/−. 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

Identification of Anthraquinone-Degrading Bacteria in Soil Contaminated with Polycyclic Aromatic Hydrocarbons

ABSTRACT 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

Identification of Anthraquinone-Degrading Bacteria in Soil Contaminated with Polycyclic Aromatic Hydrocarbons

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

Screening Nonionic Surfactants for Enhanced Biodegradation of Polycyclic Aromatic Hydrocarbons Remaining in Soil After Conventional Biological Treatment

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

Bioavailability of (Geno)toxic Contaminants in Polycyclic Aromatic Hydrocarbon–Contaminated Soil Before and After Biological Treatment

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(®) HLB) or a hydrophobic sorbent (Tenax(®)) 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 Rad54(−/−). 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

Improving Polycyclic Aromatic Hydrocarbon Biodegradation in Contaminated Soil Through Low-Level Surfactant Addition After Conventional Bioremediation

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 Terrimonas 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

Surfactant-induced bacterial community changes correlated with increased polycyclic aromatic hydrocarbon degradation in contaminated soil

Bioremediation as a method for removing polycyclic aromatic hydrocarbons (PAHs) from contaminated environments has been criticized for poor removal of potentially carcinogenic but less bioavailable high-molecular-weight (HMW) compounds. As a partial remedy to this constraint, we studied surfactant addition at sub-micellar concentrations to contaminated soil to enhance the biodegradation of PAHs remaining after conventional aerobic bioremediation. We demonstrated increased removal of 4- and 5-ring PAHs using two nonionic surfactants, polyoxyethylene(4)lauryl ether (Brij 30) and polyoxyethylene sorbitol hexaoleate (POESH), and analyzed bacterial community shifts associated with those conditions. Eight groups of abundant bacteria were implicated as potentially being involved in increased HMW PAH removal. A group of unclassified Alphaproteobacteria and members of the Phenylobacterium genus in particular showed significantly increased relative abundance in the two conditions exhibiting increased PAH removal. Other implicated groups included members of the Sediminibacterium, Terrimonas, Acidovorax, and Luteimonas genera, as well as uncharacterized organisms within the families Chitinophagaceae and Bradyrhizobiaceae. Targeted isolation identified a subset of the community likely using the surfactants as a growth substrate but few of the isolates exhibited PAH-degradation capability. Isolates recovered from the Acidovorax and uncharacterized Bradyrhizobiaceae groups suggest the abundance of those groups may have been attributable to growth on surfactants. Understanding the specific bacteria responsible for HMW PAH removal in natural and engineered systems and their response to stimuli such as surfactant amendment may improve bioremediation efficacy during treatment of contaminated environmental media