Bacterial benz(a)anthracene catabolic networks in contaminated soils and their modulation by other co-occurring HMW-PAHs.

Polycyclic aromatic hydrocarbons (PAHs) are major environmental pollutants in a number of point source contaminated sites, where they are found embedded in complex mixtures containing different polyaromatic compounds. The application of bioremediation technologies is often constrained by unpredictable end-point concentrations enriched in recalcitrant high molecular weight (HMW)-PAHs. The aim of this study was to elucidate the microbial populations and potential interactions involved in the biodegradation of benz(a)anthracene (BaA) in PAH-contaminated soils. The combination of DNA stable isotope probing (DNA-SIP) and shotgun metagenomics of 13C-labeled DNA identified a member of the recently described genus Immundisolibacter as the key BaA-degrading population. Analysis of the corresponding metagenome assembled genome (MAG) revealed a highly conserved and unique genetic organization in this genus, including novel aromatic ring-hydroxylating dioxygenases (RHD). The influence of other HMW-PAHs on BaA degradation was ascertained in soil microcosms spiked with BaA and fluoranthene (FT), pyrene (PY) or chrysene (CHY) in binary mixtures. The co-occurrence of PAHs resulted in a significant delay in the removal of PAHs that were more resistant to biodegradation, and this delay was associated with relevant microbial interactions. Members of Immundisolibacter, associated with the biodegradation of BaA and CHY, were outcompeted by Sphingobium and Mycobacterium, triggered by the presence of FT and PY, respectively. Our findings highlight that interacting microbial populations modulate the fate of PAHs during the biodegradation of contaminant mixtures in soils.

Predictive values of time-dense SARS-CoV-2 wastewater analysis in university campus buildings.

Wastewater-based SARS-CoV-2 surveillance on college campuses has the ability to detect individual clinical COVID-19 cases at the building-level. High concordance of wastewater results and clinical cases has been observed when calculated over a time window of four days or longer and in settings with high incidence of infection. At Duke University, twice a week clinical surveillance of all resident undergraduates was carried out in the spring 2021 semester. We conducted simultaneous wastewater surveillance with daily frequency on selected residence halls to assess wastewater as an early warning tool during times of low transmission with the hope of scaling down clinical test frequency. We evaluated the temporal relationship of the two time-dense data sets, wastewater and clinical, and sought a strategy to achieve the highest wastewater predictive values using the shortest time window to enable timely intervention. There were 11 days with clinical cases in the residence halls (80-120 occupants) under wastewater surveillance with 5 instances of a single clinical case and 3 instances of two clinical cases which also corresponded to a positive wastewater SARS-CoV-2 signal. While the majority (71%) of our wastewater samples were negative for SARS-CoV-2, 29% resulted in at least one positive PCR signal, some of which did not correlate with an identified clinical case. Using a criteria of two consecutive days of positive wastewater signals, we obtained a positive predictive value (PPV) of 75% and a negative predictive value of 87% using a short 2 day time window for agreement. A conventional concordance over a much longer 4 day time window resulted in PPV of only 60%. Our data indicated that daily wastewater collection and using a criteria of two consecutive days of positive wastewater signals was the most predictive approach to timely early warning of COVID-19 cases at the building level.

Complete Genome Sequence of a Novel Hafnia alvei Bacteriophage.

A bacteriophage that is able to infect Hafnia alvei and can also infect two other hosts, Klebsiella pneumoniae and Salmonella enterica, was isolated from wastewater. The complete genome sequence was determined by long-read PacBio sequencing. Based on sequence similarity, the bacteriophage is assigned to the viral genus Kolesnikvirus within the family Myoviridae.

Identifying bioaugmentation candidates for bioremediation of polycyclic aromatic hydrocarbons in contaminated estuarine sediment of the Elizabeth River, VA, USA.

Estuarine sediments near former creosoting facilities along the Elizabeth River (Virginia, USA) are contaminated by polycyclic aromatic hydrocarbons (PAHs). In this study, we interrogated the bacterial community of the Elizabeth River with both culture-based and culture-independent methods to identify potential candidates for bioremediation of these contaminants. DNA-based stable isotope probing (SIP) experiments with phenanthrene and fluoranthene using sediment from the former Republic Creosoting site identified relevant PAH-degrading bacteria within the Azoarcus, Hydrogenophaga, and Croceicoccus genera. Targeted cultivation of PAH-degrading bacteria from the same site recovered 6 PAH-degrading strains, including one strain highly similar to Hydrogenophaga sequences detected in SIP experiments. Other isolates were most similar to organisms within the Novosphingobium, Sphingobium, Stenotrophomonas, and Alcaligenes genera. Lastly, we performed 16S rRNA gene amplicon microbiome analyses of sediment samples from four sites, including Republic Creosoting, with varying concentrations of PAHs. Analysis of these data showed a striking divergence of the microbial community at the highly contaminated Republic Creosoting site from less contaminated sites with the enrichment of several bacterial clades including those affiliated with the Pseudomonas genus. Sequences within the microbiome libraries similar to SIP-derived sequences were generally found at high relative abundance, while the Croceicoccus sequence was present at low to moderate relative abundance. These results suggest that Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation, while Croceicoccus spp. might be good targets for bioaugmentation in these sediments. Furthermore, this study demonstrates the value of culture-based and culture-independent methods in identifying promising bacterial candidates for use in a precision bioremediation scheme. KEY POINTS: • This study highlights the importance of using multiple strategies to identify promising bacterial candidates for use in a precision bioremediation scheme. • We used both selective cultivation techniques and DNA-based stable isotope probing to identify bacterial degraders of prominent PAHs at a historically contaminated site in the Elizabeth River, VA, USA. • Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation in Elizabeth River sediments, while Croceicoccus spp. might be good targets for bioaugmentation.

The role of gut microbial community and metabolomic shifts in adaptive resistance of Atlantic killifish (Fundulus heteroclitus) to polycyclic aromatic hydrocarbons.

Altered gut microbiomes may play a role in rapid evolution to anthropogenic change but remain poorly understood. Atlantic killifish (Fundulus heteroclitus) in the Elizabeth River, VA have evolved resistance to polycyclic aromatic hydrocarbons (PAHs) and provide a unique opportunity to examine the links between shifts in the commensal microbiome and organismal physiology associated with evolved resistance. Here, 16S rRNA sequence libraries derived from fish guts and sediments sampled from a highly PAH contaminated site revealed significant differences collected at similar samples from an uncontaminated site. Phylogenetic groups enriched in the libraries derived from PAH-resistant fish were dissimilar to their associated sediment libraries, suggesting the specific environment within the PAH-resistant fish intestine influence the gut microbiome composition. Gut metabolite analysis revealed shifts between PAH-resistant and non-resistant subpopulations. Notably, PAH-resistant fish exhibited reduced levels of tryptophan and increased levels of sphingolipids. Exposure to PAHs appears to impact several bacterial in the gut microbiome, particularly sphingolipid containing bacteria. Bacterial phylotypes known to include species containing sphingolipids were generally lower in the intestines of fish subpopulations exposed to high concentrations of PAHs, inferring a complex host-microbiome relationship. Overall, killifish microbial community shifts appear to be related to a suppression of overall metabolite level, indicating a potential role of the gut in organismal response to anthropogenic environmental change. These results on microbial and metabolomics shifts are potentially linked to altered bioenergetic phenotype observed in the same PAH-resistant killifish populations in other studies.

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C16:1ω7c/C16:1ω6c, C16:0, and C18:1ω7c, and were consistent with the genus. Genomic G+C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3T as the type strain (=LMG 30136, =DSM 105008).

Nontarget Analysis Reveals a Bacterial Metabolite of Pyrene Implicated in the Genotoxicity of Contaminated Soil after Bioremediation.

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 α,ß-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 µg 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.

Description of Immundisolibacter cernigliae gen. nov., sp. nov., a high-molecular-weight polycyclic aromatic hydrocarbon-degrading bacterium within the class Gammaproteobacteria, and proposal of Immundisolibacterales ord. nov. and Immundisolibacteraceae fam. nov.

The bacterial strain TR3.2T was isolated from aerobic bioreactor-treated soil from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Salisbury, NC, USA. Strain TR3.2T was identified as a member of 'Pyrene Group 2' or 'PG2', a previously uncultivated cluster of organisms associated with the degradation of high-molecular-weight PAHs by stable-isotope probing. Based on its 16S rRNA gene sequence, the strain was classified as a member of the class Gammaproteobacteria but possessed only 90.5 % gene identity to its closest described relative, Methylococcus capsulatus strain Bath. Strain TR3.2T grew on the PAHs pyrene, phenanthrene, anthracene, benz[a]anthracene and fluorene, as well as the azaarene carbazole, and could additionally metabolize a limited number of organic acids. Optimal growth occurred aerobically under mesophilic temperature, neutral pH and low salinity conditions. Strain TR3.2T was catalase and oxidase positive. Predominant fatty acids were C17 : 0 cyclo and C16 : 0. Genomic G+C content of the single chromosome was 67.79 mol% as determined by complete genome sequencing. Due to the high sequence divergence from any cultivated species and its unique physiological properties compared to its closest relatives, strain TR3.2T is proposed as a representative of a novel order, family, genus and species within the class Gammaproteobacteria, for which the name Immundisolibacter cernigliae gen. nov., sp. nov. is proposed. The associated order and family are therefore proposed as Immundisolibacteralesord. nov. and Immundisolibacteraceaefam. nov. The type strain of the species is TR3.2T (=ATCC TSD-58T=DSM 103040T).

Remembering Our Forebears: Albert Jan Kluyver and the Unity of Life.

The Dutch microbiologist/biochemist Albert Jan Kluyver (1888-1956) was an early proponent of the idea of biochemical unity, and how that concept might be demonstrated through the careful study of microbial life. The fundamental relatedness of living systems is an obvious correlate of the theory of evolution, and modern attempts to construct phylogenetic schemes support this relatedness through comparison of genomes. The approach of Kluyver and his scientific descendants predated the tools of modern molecular biology by decades. Kluyver himself is poorly recognized today, yet his influence at the time was profound. Through lens of today however, it has been argued that the focus by Kluyver and others to create taxonomic and phylogenetic schemes using morphology and biochemistry distorted and hindered progress of the discipline of microbiology, because of a perception that the older approaches focused too much on a reductionist worldview. This essay argues that in contrast the careful characterization of fundamental microbial metabolism and physiology by Kluyver made many of the advances of the latter part of the twentieth century possible, by offering a framework which in many respects anticipated our current view of phylogeny, and by directly and indirectly training a generation of scientists who became leaders in the explosive growth of biotechnology.

Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds.

A bacterial strain designated Ca6T was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant in Charlotte, NC, USA, and linked phylogenetically to the family Rhodocyclaceae of the class Betaproteobacteria. Its 16S rRNA gene sequence was highly similar to globally distributed environmental sequences, including those previously designated 'Pyrene Group 1' demonstrated to grow on the PAHs phenanthrene and pyrene by stable-isotope probing. The most closely related described relative was Sulfuritalea hydrogenivorans strain sk43HT (93.6 % 16S rRNA gene sequence identity). In addition to a limited number of organic acids, Ca6T was capable of growth on the monoaromatic compounds benzene and toluene, and the azaarene carbazole, as sole sources of carbon and energy. Growth on the PAHs phenanthrene and pyrene was also confirmed. Optimal growth was observed aerobically under mesophilic temperature, neutral pH and low salinity conditions. Major fatty acids present included summed feature 3 (C16 : 1ω7c or C16 : 1ω6c) and C16 : 0. The DNA G+C content of the single chromosome was 55.14  mol% as determined by complete genome sequencing. Due to its distinct genetic and physiological properties, strain Ca6T is proposed as a member of a novel genus and species within the family Rhodocyclaceae, for which the name Rugosibacter aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the species is Ca6T (=ATCC TSD-59T=DSM 103039T).

Complete Genome Sequence of a Bacterium Representing a Deep Uncultivated Lineage within the Gammaproteobacteria Associated with the Degradation of Polycyclic Aromatic Hydrocarbons.

The bacterial strain TR3.2, representing a novel deeply branching lineage within the Gammaproteobacteria, was isolated and its genome sequenced. This isolate is the first cultivated representative of the previously described "Pyrene Group 2" (PG2) and represents a variety of environmental sequences primarily associated with petrochemical contamination and aromatic hydrocarbon degradation.

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 four- and five-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.

Complete Genome Sequence of a Novel Bacterium within the Family Rhodocyclaceae That Degrades Polycyclic Aromatic Hydrocarbons.

A polycyclic aromatic hydrocarbon-degrading bacterium designated strain Ca6, a member of the family Rhodocyclaceae and a representative of the uncharacterized pyrene group 1 (PG1), was isolated and its genome sequenced. The presence of several genes suspected to be associated with PG1 was confirmed, and additional genes for aromatic compound metabolism were detected.

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil.

Stable-isotope probing was previously used to identify bacterial anthracene-degraders in untreated soil from a former manufactured gas plant site. However, subsequent pyrosequence analyses of total bacterial communities and quantification of 16S rRNA genes indicated that relative abundances of the predominant anthracene-degrading bacteria (designated Anthracene Group 1) diminished as a result of biological treatment conditions in lab-scale, aerobic bioreactors. This study identified Alphaproteobacterial anthracene-degrading bacteria in bioreactor-treated soil which were dissimilar to those previously identified. The largest group of sequences was from the Alterythrobacter genus while other groups of sequences were associated with bacteria within the order Rhizobiales and the genus Bradyrhizobium. Conditions in the bioreactor enriched for organisms capable of degrading anthracene which were not the same as those identified as dominant degraders in the untreated soil. Further, these data suggest that identification of polycyclic aromatic hydrocarbon-degrading bacteria in contaminated but untreated soil may be a poor indicator of the most active degraders during biological treatment.

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP.

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly (13)C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.

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.

Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium.

A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and ß-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

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.