Impact of irradiation and polycyclic aromatic hydrocarbon spiking on microbial populations in marine sediment for future aging and biodegradability studies

Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria: Vibrio, Marinobacter or Cycloclasticus, Pseudoalteromonas, Marinomonas, and HALOMONAS: This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize 14C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.     

Indigenous PAH-degrading bacteria from oil-polluted sediments in Caleta Cordova,Patagonia Argentina

Indigenous bacteria with the capability to degrade polycyclic aromatic hydrocarbons (PAH) were isolated from polluted sediment samples recovered from Caleta Cordova by using selective enrichment cultures supplemented with phenanthrene. Bacterial communities were evaluated by denaturing gradient gel electrophoresis (DGGE) in order to detect changes along enrichment culture and relationships with the representative strains subsequently isolated. Members of these communities included marine bacteria such as Lutibacter, Polaribacter, Arcobacter and Olleya, whose degradation pathway of PAH has not been studied yet. However, isolated bacteria obtained from this enrichment comprised the genus Pseudomonas, Marinobacter, Salinibacterium and Brevibacterium. The ability of isolates to grow and degrade naphthalene, phenanthrene and pyrene was demonstrated by detection of the residual substrate by HPLC. Archetypical naphthalene and catechol dioxygenase genes were found in two isolates belonging to genus Pseudomonas (Pseudomonas monteilii P26 and Pseudomonas xanthomarina N12), suggesting biodegradation potential in these sediments. The successful bacterial isolation with the ability to degrade PAH in pure culture suggest the possibility to study and further consider strategies like growth stimulation in situ, in order to increase the intrinsic bioremediation opportunities in the polluted Caleta Cordova harbor.     

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas , Halomonas , Marinobacter , Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus , Alteromonas , Thalassospira , Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.     

Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH)

The use of a plate screening technique allowed the direct isolation and quantification of polycylic aromatic hydrocarbon (PAH)-degrading bacteria from different soil sites. Bacteria that were able to grow on anthracene, phenanthrene, fluoranthene or pyrene as a sole carbon source were found with numbers between 10³ and 10⁵ colony-forming units (cfu)/g of soil dry weight, but only in samples that originated from PAH-contaminated sites. No isolates were found that could grow on perylene, triphenylene, benzo(a)pyrene or chrysene as sole carbon source. Bacteria that had been selected on the same PAH substrate showed a related degradation pattern for both other PAH and oil compounds and carbohydrate substrates even if they had been collected at distant soil sites. Based on these findings the isolates could be clustered into four different catabolic and taxonomic similarity groups. Taxonomic determination of representative isolates suggested that nocardioform actinomycetes of the genera Mycobacterium, Rhodococcus and Gordona represented a major part of the soil microflora able to mineralize PAH. Three new isolates able to grow on anthracene, pyrene or fluoranthene as the sole carbon source, respectively, have been isolated and identified (Sphingomonas paucimobilis BA2, Gordona sp. BP9, Mycobacterium sp. VF1). The ubiquitous presence of a potent and versatile mineralizing microflora in PAH-contaminated soils indicated that the microflora is not the limiting factor for the degradation of PAH with up to four rings.     

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-¹³C]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-¹³C]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.     

Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [¹³C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.     

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

The catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation in oil-contaminated soil

Rhizoremediation is a potential technique for polycyclic aromatic hydrocarbon (PAH) remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable-isotope-probing coupled with metagenomics and molecular ecological network analyses were first used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of ¹³C-DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non-rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH-contaminated sites.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Bacterial Diversity of a Consortium Degrading High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in a Two-Liquid Phase Biosystem

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil–water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one—IAFILS9 affiliated to Novosphingobium pentaromativorans—was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species–genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.     

DIFFERENCES IN PAH DESORPTION AND SEDIMENT ORGANIC MATTER COMPOSITION BETWEEN NON-VEGETATED AND RECENTLY VEGETATED FUEL-OILED SEDIMENTS

We assessed the desorption behavior of pyrene, chrysene, phenanthrene, and tri-alkylated (C₃) phenanthrene/anthracenes for non-vegetated and recently vegetated (< 2 yrs) fuel-oiled sediments collected from the Indiana Harbor Canal (IHC), Gary, IN. Bulk sediment and humin were analyzed for PAH concentrations, organic matter composition, and PAH desorption behavior. PAH desorption isotherms and kinetics were determined using batch aqueous extractions and a two compartment, first-order kinetic model. Vegetated sediments contained more plant carbon and were more nonpolar and less oxidized than non-vegetated sediments. Desorption kinetics indicated that PAH desorption was primarily controlled by a slow PAH-desorbing fraction (F₂) of IHC sediments. However, in vegetated sediments, particularly humin, PAH release from a faster PAH-desorbing fraction (F₁) increased as did the rates (k₂) of PAH desorption from the dominant slow PAH-desorbing fraction (F₂). We propose that vegetation provides aliphatic, nonpolar carbon to IHC sediments that facilitates more rapid PAH desorption from bulk sediment and humin.     

The direct role of aerobic heterotrophic bacteria associated with cyanobacteria in the degradation of oil compounds

This study aimed at evaluating the role of cyanobacteria and their associated aerobic heterotrophic bacteria in biodegradation of petroleum compounds. We investigated the potential of ten non-axenic typical mat-forming cyanobacterial strains to degrade phenanthrene, pristane, n-octadecane, and dibenzothiophene. Five strains (Aphanothece halophyletica, Dactyolococcopsis salina, Halothece strain EPUS, Oscillatoria strain OSC, and Synechocystis strain UNIGA) were able to degrade n-alkanes. In case of the other five strains (Microcoleus chthonoplastes, Oscillatoria sp. MPI 95 OS 01, Halothece strain EPUG, Halomicronema exentricum, and Phormidium strain UNITF) alkanes were not significantly affected. Moderate changes in the concentration of the aromatic compounds were observed for three isolates only. In follow-up experiments with Oscillatoria strain OSC, we demonstrated that the cyanobacteria-associated aerobic heterotrophic bacteria were responsible for the observed biodegradation. The cyanobacteria themselves apparently do not degrade petroleum compounds, but more likely play a significant, indirect role in biodegradation by supporting the growth and activity of the actual degraders.     

Stable isotope probing identifies anthracene degraders under methanogenic conditions

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in groundwater. The remediation of PAH-contaminated groundwater often involves anaerobic biodegradation. The knowledge about the microorganisms responsible for PAH degradation in anaerobic subsurface environment is still lacking. DNA-based stable isotope probing (SIP) was applied to discover the microorganisms responsible for anaerobic anthracene degradation within microcosms inoculated with aquifer sediment from landfill leachate-contaminated site. Three phylotypes were identified as the degraders, all falling within the phylum Proteobacteria. Two anthracene degraders were classified within the genera Methylibium and Legionella, while another one was an unclassified Rhizobiales species. They all were first linked to PAH degradation. These findings also provide an illustration of the utility of SIP to discover the roles of uncultured microorganisms in PAH-degrading processes.     

Phenanthrene toxicity and dissipation in rhizosphere of grassland plants (Lolium perenne L. and Trifolium pratense L.) in three spiked soils

Rhizodegradation is a technique involving plants that offers interesting potential to enhance biodegradation of persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). Nevertheless, the behaviour of PAHs in plant rhizosphere, including micro-organisms and the physico-chemical soil properties, still needs to be clarified. The present work proposes to study the toxicity and the dissipation of phenanthrene in three artificially contaminated soils (1 g kg^-1 DW). Experiments were carried out after 2 months of soil aging. They consisted in using different systems with two plant species (Ryegrass—Lolium perenne L. var. Prana and red clover—Trifolium pratense L. var. fourragère Caillard), three kinds of soils (a silty-clay-loam soil “La Bouzule”, a coarse sandy-loam soil “Chenevières” and a fine sandy-loam soil “Maconcourt”). Phenanthrene was quantified by HPLC in the beginning (T ₀) and the end of the experiments (30 days). Plant biomass, microbial communities including mycorrhizal fungi, Rhizobium and PAH degraders were also recorded. Generally phenanthrene contamination did not affect plant biomass. Only the red clover biomass was enhanced in Chenevières and La Bouzule polluted soils. A stimulation of Rhizobium red clover colonisation was quantified in spiked soils whereas a drastic negative phenanthrene effect on the mycorrhization of ryegrass and red clover was recorded. The number of PAH degraders was stimulated by the presence of phenanthrene in all tested soils. Both in ryegrass and red clover planted soils, the highest phenanthrene dissipation due to the rhizosphere was measured in La Bouzule soils. On the contrary, in non-planted soils, La Bouzule soils had also the lowest pollutant dissipation. Thus, in rhizospheric and non-rhizospheric soils the phenanthrene dissipation was found to depend on soil clay content.     

Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential

A laboratory experiment was conducted to identify key hydrocarbon degraders from a marine oil spill sample (Prestige fuel oil), to ascertain their role in the degradation of different hydrocarbons, and to assess their biodegradation potential for this complex heavy oil. After a 17-month enrichment in weathered fuel, the bacterial community, initially consisting mainly of Methylophaga species, underwent a major selective pressure in favor of obligate hydrocarbonoclastic microorganisms, such as Alcanivorax and Marinobacter spp. and other hydrocarbon-degrading taxa (Thalassospira and Alcaligenes), and showed strong biodegradation potential. This ranged from >99% for all low- and medium-molecular-weight alkanes (C₁₅–C₂₇) and polycyclic aromatic hydrocarbons (C₀- to C₂- naphthalene, anthracene, phenanthrene, dibenzothiophene, and carbazole), to 75–98% for higher molecular-weight alkanes (C₂₈–C₄₀) and to 55–80% for the C₃ derivatives of tricyclic and tetracyclic polycyclic aromatic hydrocarbons (PAHs) (e.g., C₃-chrysenes), in 60 days. The numbers of total heterotrophs and of n-alkane-, aliphatic-, and PAH degraders, as well as the structures of these populations, were monitored throughout the biodegradation process. The salinity of the counting medium affects the counts of PAH degraders, while the carbon source (n-hexadecane vs. a mixture of aliphatic hydrocarbons) is a key factor when counting aliphatic degraders. These limitations notwithstanding, some bacterial genera associated with hydrocarbon degradation (mainly belonging to α- and γ-Proteobacteria, including the hydrocarbonoclastic Alcanivorax and Marinobacter) were identified. We conclude that Thalassospira and Roseobacter contribute to the degradation of aliphatic hydrocarbons, whereas Mesorhizobium and Muricauda participate in the degradation of PAHs.     

Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 μg/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20°C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7°C, 53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions, naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7°C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7°C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations, including members of the genera Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Polycyclic Aromatic Hydrocarbon Degradation of Phytoplankton-Associated Arenibacter spp. and Description of Arenibacter algicola sp. nov., an Aromatic Hydrocarbon-Degrading Bacterium

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409^T) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409^T represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409^T emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton.     

Isolation of Marine Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Cycloclasticus Strains from the Gulf of Mexico and Comparison of Their PAH Degradation Ability with That of Puget Sound Cycloclasticus Strains

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10² to 10⁶ cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically. Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.