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.

Using Environmental Simulations to Test the Release of Hazardous Substances from Polymer-Based Products: Are Realism and Pragmatism Mutually Exclusive Objectives?

The potential release of hazardous substances from polymer-based products is currently in the focus of environmental policy. Environmental simulations are applied to expose such products to selected aging conditions and to investigate release processes. Commonly applied aging exposure types such as solar and UV radiation in combination with water contact, corrosive gases, and soil contact as well as expected general effects on polymers and additional ingredients of polymer-based products are described. The release of substances is based on mass-transfer processes to the material surfaces. Experimental approaches to investigate transport processes that are caused by water contact are presented. For tailoring the tests, relevant aging exposure types and release quantification methods must be combined appropriately. Several studies on the release of hazardous substances such as metals, polyaromatic hydrocarbons, flame retardants, antioxidants, and carbon nanotubes from polymers are summarized exemplarily. Differences between natural and artificial exposure tests are discussed and demonstrated for the release of flame retardants from several polymers and for biocides from paints. Requirements and limitations to apply results from short-term artificial environmental exposure tests to predict long-term environmental behavior of polymers are presented.

Isomer-selective biodegradation of high-molecular-weight azaarenes in PAH-contaminated environmental samples.

Polycyclic aromatic nitrogen heterocycles, or azaarenes, normally co-occur with polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. We recently reported that nontarget analysis using high resolution mass spectrometry of samples from four PAH-contaminated sites revealed a previously unrecognized diversity and abundance of azaarene isomers and their methylated derivatives. Here we evaluated their biodegradability by natural microbial communities from each site in aerobic microcosm incubations under biostimulated conditions. The removal of total quantifiable azaarenes ranged from 15 to 85%, and was related to the initial degree of weathering for each sample. While three-ring azaarenes were readily biodegradable, the five-ring congeners were the most recalcitrant. Microbial-mediated removal of four-ring congeners varied for different isomers, which might be attributed to the position of the nitrogen atom that also influences the physicochemical properties of azaarenes and possibly the susceptibility to transformation by relevant microbial enzymes. The presence of methyl groups also influenced azaarene biodegradability, which decreased with increasing degree of methylation. Several oxidation products of azaarenes were detected, including ketones and dioxygenated derivatives of three- and four-ring compounds. Our results indicate the susceptibility of some azaarenes to bioremediation, while suggesting the potential implications for risk from the persistence of less-biodegradable isomers and the formation of oxidized-azaarene derivatives.

Production and characterisation of a marine Halomonas surface-active exopolymer.

During screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell's 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

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).

Tracing the Biotransformation of Polycyclic Aromatic Hydrocarbons in Contaminated Soil Using Stable Isotope-Assisted Metabolomics.

Biotransformation of organic pollutants may result in the formation of oxidation products more toxic than the parent contaminants. However, to trace and identify those products, and the metabolic pathways involved in their formation, is still challenging within complex environmental samples. We applied stable isotope-assisted metabolomics (SIAM) to PAH-contaminated soil collected from a wood treatment facility. Soil samples were separately spiked with uniformly 13C-labeled fluoranthene, pyrene, or benzo[a]anthracene at a level below that of the native contaminant, and incubated for 1 or 2 weeks under aerobic biostimulated conditions. Combining high-resolution mass spectrometry and automated SIAM workflows, chemical structures of metabolites and metabolic pathways in the soil were proposed. Ring-cleavage products, including previously unreported intermediates such as C11H10O6 and C15H12O5, were detected originating from fluoranthene and benzo[a]anthracene, respectively. Sulfate conjugates of dihydroxy compounds were found as major metabolites of pyrene and benzo[a]anthracene, suggesting the potential role of fungi in their biotransformation in soils. A series of unknown N-containing metabolites were identified from pyrene, but their structural elucidation requires further investigation. Our results suggest that SIAM can be successfully applied to understand the fate of organic pollutants in environmental samples, opening lines of evidence for novel mechanisms of microbial transformation within such complex matrices.

Diversity and Abundance of High-Molecular-Weight Azaarenes in PAH-Contaminated Environmental Samples.

Azaarenes are N-heterocyclic polyaromatic pollutants that co-occur with polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. Despite the known toxicity of some high-molecular-weight azaarenes, their diversity, abundance, and fate in contaminated soils remain to be elucidated. We applied high-resolution mass spectrometry and mass-defect filtering to four PAH-contaminated samples from geographically distant sites and detected 232 azaarene congeners distributed in eight homologous series, including alkylated derivatives and two hitherto unknown series. Four- and five-ring azaarenes were detected among these series, and the most abundant nonalkylated congeners groups (C13H9N, C15H9N, C17H11N, C19H11N, and C21H13N) were quantified. The profiles of congener groups varied among different sites. Three-ring azaarenes presented higher concentrations in unweathered sites, while four- and five-ring azaarenes predominated in weathered sites. Known toxic and carcinogenic azaarenes, such as benzo[c]acridine and dibenzo[a,h]acridine, were detected along with their multiple isomers. Our results highlight a previously unrecognized diversity and abundance of azaarenes in PAH-contaminated sites, with corresponding implications for environmental monitoring and risk assessment.

Identification of polar transformation products and high molecular weight polycyclic aromatic hydrocarbons (PAHs) in contaminated soil following bioremediation.

Bioremediation is a technique commonly used to reduce the toxicity associated with polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. However, the efficacy of bioremedial applications is evaluated based on the removal of a subset of parent (or unsubstituted) PAHs and does not incorporate toxic polar transformation products or the more mutagenic high molecular weight PAHs (MW≥302amu or MW302-PAHs). Previously, an effects-directed analysis approach was used to assess the effect of bioremediation on the toxicity of a coal tar-contaminated soil. Increased genotoxicity and developmental toxicity was measured postbioremedation in the more polar soil extract fractions, as compared to the less polar fractions where the targeted PAHs eluted, and could not be attributed to the 88 target PAHs analyzed for (including selected oxygen-containing PAHs). In this study, comprehensive two-dimensional gas chromatography time-of-flight and liquid chromatography quadrupole time-of-flight mass spectrometry were used to characterize transformation products in the soil extract fractions identified as toxic, previously. Additionally, the degradation of 12MW302-PAHs, picene (MW=278) and coronene (MW=300) were evaluated following bioremediation. Non-targeted analysis resulted in the tentative identification of 10 peaks with increased intensity postbioremediation (based on mass spectral library matching and fragmentation patterns from >5000 candidate peaks in the soil extracts). Several of these compounds contained oxygen, suggesting they would be relatively polar. MW302-PAHs were not significantly degraded during bioremediation, suggesting that the carcinogenic potential associated with these PAHs might remain unchanged. The results of this study suggest that polar transformation products, and MW302-PAHs, should be considered for realistic risk assessment of bioremediated soils.

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).

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.

Enrichment of Fusobacteria in Sea Surface Oil Slicks from the Deepwater Horizon Oil Spill.

The Deepwater Horizon (DWH) oil spill led to rapid microbial community shifts in the Gulf of Mexico, including the formation of unprecedented quantities of marine oil snow (MOS) and of a massive subsurface oil plume. The major taxa that bloomed in sea surface oil slicks during the spill included Cycloclasticus, and to a lesser extent Halomonas, Alteromonas, and Pseudoalteromonas-organisms that grow and degrade oil hydrocarbons aerobically. Here, we show that sea surface oil slicks at DWH contained obligate and facultative anaerobic taxa, including members of the obligate anaerobic phylum Fusobacteria that are commonly found in marine sediment environments. Pyrosequencing analysis revealed that Fusobacteria were strongly selected for when sea surface oil slicks were allowed to develop anaerobically. These organisms have been found in oil-contaminated sediments in the Gulf of Mexico, in deep marine oil reservoirs, and other oil-contaminated sites, suggesting they have putative hydrocarbon-degrading qualities. The occurrence and strong selection for Fusobacteria in a lab-based incubation of a sea surface oil slick sample collected during the spill suggests that these organisms may have become enriched in anaerobic zones of suspended particulates, such as MOS. Whilst the formation and rapid sinking of MOS is recognised as an important mechanism by which a proportion of the Macondo oil had been transported to the sea floor, its role in potentially transporting microorganisms, including oil-degraders, from the upper reaches of the water column to the seafloor should be considered. The presence of Fusobacteria on the sea surface-a highly oxygenated environment-is intriguing, and may be explained by the vertical upsurge of oil that provided a carrier to transport these organisms from anaerobic/micro-aerophilic zones in the oil plume or seabed to the upper reaches of the water column. We also propose that the formation of rapidly-sinking MOS may have re-transported these, and other microbial taxa, to the sediment in the Gulf of Mexico.

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.

Response of the bacterial community associated with a cosmopolitan marine diatom to crude oil shows a preference for the biodegradation of aromatic hydrocarbons.

Emerging evidence shows that hydrocarbonoclastic bacteria (HCB) may be commonly found associated with phytoplankton in the ocean, but the ecology of these bacteria and how they respond to crude oil remains poorly understood. Here, we used a natural diatom-bacterial assemblage to investigate the diversity and response of HCB associated with a cosmopolitan marine diatom, Skeletonema costatum, to crude oil. Pyrosequencing analysis and qPCR revealed a dramatic transition in the diatom-associated bacterial community, defined initially by a short-lived bloom of Methylophaga (putative oil degraders) that was subsequently succeeded by distinct groups of HCB (Marinobacter, Polycyclovorans, Arenibacter, Parvibaculum, Roseobacter clade), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential. Interestingly, these oil-enriched organisms contributed to the apparent and exclusive biodegradation of substituted and non-substituted polycyclic aromatic hydrocarbons (PAHs), thereby suggesting that the HCB community associated with the diatom is tuned to specializing in the degradation of PAHs. Furthermore, the formation of marine oil snow (MOS) in oil-amended incubations was consistent with its formation during the Deepwater Horizon oil spill. This work highlights the phycosphere of phytoplankton as an underexplored biotope in the ocean where HCB may contribute importantly to the biodegradation of hydrocarbon contaminants in marine surface waters.

From Bioavailability Science to Regulation of Organic Chemicals.

The bioavailability of organic chemicals in soil and sediment is an important area of scientific investigation for environmental scientists, although this area of study remains only partially recognized by regulators and industries working in the environmental sector. Regulators have recently started to consider bioavailability within retrospective risk assessment frameworks for organic chemicals; by doing so, realistic decision-making with regard to polluted environments can be achieved, rather than relying on the traditional approach of using total-extractable concentrations. However, implementation remains difficult because scientific developments on bioavailability are not always translated into ready-to-use approaches for regulators. Similarly, bioavailability remains largely unexplored within prospective regulatory frameworks that address the approval and regulation of organic chemicals. This article discusses bioavailability concepts and methods, as well as possible pathways for the implementation of bioavailability into risk assessment and regulation; in addition, this article offers a simple, pragmatic and justifiable approach for use within retrospective and prospective risk assessment.

Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.