Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment

Ochrobactrum sp. N1, Brevibacillus parabrevis N2, B. parabrevis N3 and B. parabrevis N4 were selected when preparing a mixed bacterial consortium based on the efficiency of crude oil utilization. A crude oil degradation rate of the N-series microbial consortium reached upwards of 79% at a temperature of 25 °C in a 3.0% NaCl solution in the shake flask trial. In the mesocosm experiment, a specially designed device was used to simulate the marine environment. The internal tank size was 1.5 m (L)×0.8 m (W)×0.7 m (H). The microbial growth conditions, nutrient utilization and environmental factors were thoroughly investigated. Over 51.1% of the crude oil was effectively removed from the simulated water body. The escalation process (from flask trials to the mesocosm experiment), which sought to represent removal under conditions more similar to the field, proved the high efficiency of using N-series bacteria in crude oil degradation.     

Biodegradation of Crude Oil by Individual Bacterial Strains and a Mixed Bacterial Consortium Isolated from Hydrocarbon Contaminated Areas

A preliminary study was undertaken to determine the optimal conditions for the biodegradation of a crude oil. Among 57 oil‐degrading bacterial cultures isolated from oil‐contaminated soil samples, Bacillus sp. IOS1‐7, Corynebacterium sp. BPS2‐6, Pseudomonas sp. HPS2‐5, and Pseudomonas sp. BPS1‐8 were selected for the study based on the efficiency of crude oil utilization. Along with the selected individual strains, a mixed bacterial consortium prepared using the above strains was also used for degradation studies. The mixed bacterial consortium showed more growth and degradation than did individual strains. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 77% of the crude oil. This was followed by 69% by Pseudomonas sp. BPS1‐8, 64% by Bacillus sp. IOS1‐7, 45% by Pseudomonas sp. HPS2‐5, and 41% by Corynebacterium sp. BPS2‐6. The percentage of degradation by the mixed bacterial consortium decreased from 77 to 45% as the concentration of crude oil was increased from 1 to 12%. Temperature of 35°C and pH 7 were found to be optimum for maximum degradation.     

Depth-related influences on biodegradation rates of phenanthrene in polluted marine sediments of Puget Sound, WA

A whole-core injection method was used to determine depth-related rates of microbial mineralization of (14)C-phenanthrene added to both contaminated and clean marine sediments of Puget Sound, WA. For 26-day incubations under micro-aerobic conditions, conversions of (14)C-phenanthrene to (14)CO(2) in heavily PAH-contaminated sediments from two sites in Eagle Harbor were much higher (up to 30%) than those in clean sediments from nearby Blakely Harbor (<3%). The averaged (14)C-phenanthrene degradation rates in the surface sediment horizons (0-3 cm) were more rapid (2-3 times) than in the deeper sediment horizons examined (>6 cm), especially in the most PAH polluted EH9 site. Differences in mineralization were associated with properties of the sediments as a function of sediment depth, including grain-size distribution, PAH concentration, total organic matter and total bacterial abundance. When strictly anaerobic incubations (in N(2)/H(2)/CO(2) atmosphere) were used, the phenanthrene biodegradation rates at all sediment depths were two times slower than under micro-aerobic conditions, with methanogenesis observed after 24 days. The main rate-limiting factor for phenanthrene degradation under anaerobic conditions appeared to be the availability of suitable electron acceptors. Addition of calcium sulfate enhanced the first order rate coefficient (k(1) increased from 0.003 to 0.006 day(-1)), whereas addition of soluble nitrate, even at very low concentration (<0.5 mM), inhibited mineralization. Long-term storage of heavily polluted Eagle Harbor sediment as intact cores under micro-aerobic conditions also appeared to enhance anaerobic biodegradation rates (k(1) up to 0.11 day(-1)).     

Effect of Biofuels on Biodegradation of Benzene and Toluene at Gasoline Spill Sites

The risk that benzene and toluene from spills of gasoline will impact drinking water wells is largely controlled by the natural anaerobic biodegradation of benzene and toluene. Benzene and toluene, as well as ethanol and other biofuels, are degraded under anaerobic conditions to the same pool of degradation products. Biodegradation of biofuels may produce concentrations of degradation products that make the thermodynamics for degradation of benzene and toluene infeasible under methanogenic conditions and produce larger plumes of benzene and toluene. This study evaluated the concentrations of fuel alcohols that are necessary to inhibit the anaerobic degradation of benzene and toluene under methanogenic conditions. At two ethanol spill sites, concentrations of ethanol greater ≥42 mg/L inhibited the anaerobic degradation of toluene. The pH and concentrations of acetate, dissolved inorganic carbon, and molecular hydrogen were used to calculate the Gibbs free energy for the biodegradation of toluene. In general, the anaerobic biodegradation of toluene was not thermodynamically feasible in water with ≥42 mg/L ethanol. In a microcosm study, when the concentrations of ethanol were ≥14 mg/L or the concentrations of n‐butanol were ≥16 mg/L, the biodegradation of the alcohols consistently produced concentrations of hydrogen, dissolved inorganic carbon, and acetate that would preclude natural anaerobic biodegradation of benzene and toluene by syntrophic organisms. In contrast, iso‐butanol and n‐propanol only occasionally produced conditions that would preclude the biodegradation of benzene and toluene.     

Review of Abiotic Degradation of Chlorinated Solvents by Reactive Iron Minerals in Aquifers

Abiotic degradation of chlorinated solvents by reactive iron minerals such as iron sulfides, magnetite, green rust, and other Fe(II)‐containing minerals has been observed in both laboratory and field studies. These reactive iron minerals form under iron‐ and sulfate‐reducing conditions which are commonly found in permeable reactive barriers (PRBs), enhanced reductive dechlorination (ERD) treatment locations, landfills, and aquifers that are chemically reducing. The objective of this review is to synthesize current understanding of abiotic degradation of chlorinated solvents by reactive iron minerals, with special focus on how abiotic processes relate to groundwater remediation. Degradation of chlorinated solvents by reactive minerals can proceed through reductive elimination, hydrogenolysis, dehydrohalogenation, and hydrolysis reactions. Degradation products of abiotic reactions depend on degradation pathways and parent compounds. Some degradation products (e.g., acetylene) have the potential to serve as a signature product for demonstrating abiotic reactions. Laboratory and field studies show that various minerals have a range of reactivity toward chlorinated solvents. A general trend of mineral reactivity for degradation of chlorinated solvents can be approximated as follows: disordered FeS > FeS > Fe(0) > FeS₂ > sorbed Fe²⁺ > green rust = magnetite > biotite = vermiculite. Reaction kinetics are also influenced by factors such as pH, natural organic matter (NOM), coexisting metal ions, and sulfide concentration in the system. In practice, abiotic reactions can be engineered to stimulate reactive mineral formation for groundwater remediation. Under appropriate site geochemical conditions, abiotic reactions can occur naturally, and can be incorporated into remedial strategies such as monitored natural attenuation.     

Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp. VA1

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the environment and are derived from both man-made and natural resources. The present study is focused on the degradation of PAHs by a halotolerant bacterial strain under saline conditions. The bacterial strain VA1 was isolated from a PAH-degrading consortium that was enriched from marine water samples that were collected from different sites at Chennai, India. In the present study, a clearing zone formed on PAH-amended mineral salt agar media confirmed the utilization of PAH by the bacterial strain VA1. The results show that the strain VA1 was able to degrade anthracene (88%), phenanthrene (98%), naphthalene (90%), fluorene (97%), pyrene (84%), benzo(k)fluoranthene (57%) and benzo(e)pyrene (50%) at a 30 g/L NaCl concentration. The present study reveals that the VA1 strain was able to degrade PAHs in petroleum wastewater under saline conditions. The promising PAH-degrading halotolerant bacterial strain, VA1, was identified as Ochrobactrum sp. using biochemical and molecular techniques.     

Degradation of Reactive Brilliant Red X‐3B Dye by Microwave Electrodeless UV Irradiation

Degradations of reactive brilliant red X‐3B solution by both conventional UV irradiation and microwave electrodeless UV irradiation were investigated. Degradation processes were studied by UV–VIS spectrophotometry, total organic carbon (TOC), high performance capillary electrophoresis (HPCE), conductivity, pH value, and ion chromatography. The results of color removal (%) and TOC removal (%) showed that the degradation by microwave electrodeless UV irradiation was more effective than by conventional UV irradiation. The results of UV–VIS absorption spectra and HPCE analyses indicated that the degradation of reactive brilliant red X‐3B was occurred at the conjugation system first, the benzene ring and the naphthalene ring later. The reactive brilliant red X‐3B was cleaved into some new small compounds and eventually most of the organic substances were mineralized to CO₂ and H₂O. The results of the conductivity analysis suggested that the degradation has mainly occurred in the first 40 min of reaction. The pH value of reactive brilliant red X‐3B solution was decreased first and then was increased. The results of inorganic anions analysis hinted that many of the N, Cl, and S elements from reactive brilliant red X‐3B were still attached in organic molecules.     

Reduktive Dehalogenierung von Chlorkohlenwasserstoffen während der anaeroben Stabilisierung von Hausmüll

Reductive Dehalogenation of Chlorinated Hydrocarbons during Anaerobic Stabilization of Municipal Wastes During sequential anaerobic digestion of municipal wastes, distinct biogeochemical phases exist which show different capabilities to transform halogenated hydrocarbons. Chlorophenols, tetrachloroethylene, and chloroanilines codisposed together with organic-rich waste substrates are reductively dehalogenated during methanogenic conditions. Lindane is degraded during acidogenesis as well as during methanogenesis. However, degradation in methanogenic leachates is faster by a factor of 10. The poor transformation potential during acidogenesis compared to subsequent transient methanogenic and stabile methanogenic phases cannot be explained by inadequate acclimation of prevailing microorganisms to the codisposed organochlorines. Thus, observed transformation capabilities are a pertinent feature of methanogenic leachates, probably due to prevailing low redox potential and/or presence of suitable microbial activities (not necessarily methanogenis). Dehalogenation of 2,3,4,6-tetrachlorophenol as a model compound is hampered in methanogenic leachate by addition of a surplus of sulfate and is completely suppressed by addition of molybdate which selectively inhibits sulfate reducing microorganisms. Competition for common electron donators (e.g. H₂) is discussed as an explanation of these results. The results point to sulfate reducing microorganisms being involved in reductive dehalogenation of chlorophenols.     

Long-term changes in ground water chemistry at a phytoremediation demonstration site

A field-scale demonstration project was conducted to evaluate the capability of eastern cottonwood trees (Populus deltoides) to attenuate trichloroethene (TCE) contamination of ground water. By the middle of the sixth growing season, trees planted where depth to water was <3 m delivered enough dissolved organic carbon to the underlying aquifer to lower dissolved oxygen concentrations, to create iron-reducing conditions along the plume centerline and sulfate-reducing or methanogenic conditions in localized areas, and to initiate in situ reductive dechlorination of TCE. Apparent biodegradation rate constants for TCE along the centerline of the plume beneath the phytoremediation system increased from 0.0002/d to 0.02/d during the first six growing seasons. The corresponding increase in natural attenuation capacity of the aquifer along the plume centerline, from 0.0004/m to 0.024/m, is associated with a potential decrease in plume-stabilization distance from 9680 to 160 m. Demonstration results provide insight into the amount of vegetation and time that may be needed to achieve cleanup objectives at the field scale.     

Role of a moderately halophilic bacterial consortium in the biodegradation of polyaromatic hydrocarbons

Polycyclic aromatic hydrocarbons are ubiquitous pollutants in the environment, and most high molecular weight PAHs cause mutagenic, teratogenic and potentially carcinogenic effects. While several strains have been identified that degrade PAHs, the present study is focused on the degradation of PAHs in a marine environment by a moderately halophilic bacterial consortium. The bacterial consortium was isolated from a mixture of marine water samples collected from seven different sites in Chennai, India. The low molecular weight (LMW) PAHs phenanthrene and fluorine, and the high molecular weight (HMW) PAHs pyrene and benzo(e)pyrene were selected for the degradation study. The consortium metabolized both LMW and HMW PAHs. The consortium was also able to degrade PAHs present in crude oil-contaminated saline wastewater. The bacterial consortium was able to degrade 80% of HMW PAHs and 100% of LMW PAHs in the saline wastewater. The strains present in the consortium were identified as Ochrobactrum sp., Enterobacter cloacae and Stenotrophomonas maltophilia. This study reveals that these bacteria have the potential to degrade different PAHs in saline wastewater.     

Bioremediation of petroleum hydrocarbons in anoxic marine sediments: Consequences on the speciation of heavy metals

We investigated the effects of biostimulation and bioagumentation strategies applied to harbor sediments displaying reducing conditions and high concentrations of petroleum hydrocarbons and heavy metals. We compared the microbial efficiency of hydrocarbon removal from sediments maintained for 60 days in anoxic conditions and inoculated with acetate, sulfate-reducing bacterial strains and acetate and sulfate-reducing bacteria. All treatments determined a significant increase in the microbial growth and significant decreases of hydrocarbon contents and of redox potential values. The addition of sulfate-reducing bacterial strains to the sediment was the most efficient treatment for the hydrocarbon removal. In all experiments, significant changes of the heavy metals’ phase repartition were observed. The results reported here suggest that the biodegradation of petroleum hydrocarbons in anoxic marine sediments may be enhanced by stimulating microbial anaerobic metabolism, but care should be applied to monitor the potential changes in the mobility and bioavailability of heavy metals induced by bio-treatments.     

Mercury and lead tolerance in hypersaline sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) HSR1, HSR4, and HSR14 isolated from the salt pans of Goa grew best at 90-100/1000 salinity on substrates like formate, acetate, lactate, butyrate, ethanol and benzoate. They were gram negative, non-sporulating, non-motile rods lacking in desulfoviridin and cytochromes. Examination of these isolates for heavy metal tolerance and response studies in terms of growth and sulfate-reducing activity (SRA) were carried out using HgCl2 and Pb(NO3)2 at final concentration of 50, 100, and 200 and 100, 200 and 500 microg ml(-1) respectively. With Hg, HSR1 showed approximately 80% of the control's growth at 100 and 200 microg ml(-1) but SRA reached only 60% of the control values at the end of 14 days. HSR14 could reach >100% of the control's growth at 200 microg ml(-1) but the SRA reached only up to 60% of the control without metal at 100 microg ml(-1). Though the concentration of Pb was double that of Hg, HSR4 could grow and respire better than the control, the growth being stimulated by 160% and respiration by 170% in the presence of 500 microg ml(-1) of Pb(NO3)2. It is probable that some hypersaline SRB are more tolerant to heavy metals than the mesohaline counterparts and could be more effectively used for precipitating these metals in bioremediatory measures. Further examination of their responses to varied concentration of metals under different salinities would indicate their range of applicability.     

Abbau von Monochlornitrobenzolen durch Pseudomonas acidovorans CA50

Degradation of Monochloronitrobenzenes by Pseudomonas acidovorans CA50 Pseudomonas acidovorans strain CA50 was used for degradation experiments with monochloronitrobenzenes in aerobic batch culture. The monochloronitrobenzenes were reduced to the corresponding monochloroanilines. The reduction only occurred with an additional carbon and nitrogen source. Chlorocatechols were found to be present. 3-Chlorocatechol accumulated in the presence of 2-chloroaniline, whereas 4-chlorocatechol was an intermediate metabolite of 3- and 4-chloroaniline. Contrary to the degradation of monochloronitrobenzenes, Pseudomonas acidovorans strain CA50 used the monochloroanilines as a sole source of carbon, energy, and nitrogen for growth. The oxidation of monochloroanilines was not repressed by the additional substrates. 2-Chloronitrobenzene was degraded with the lowest rate because of the low turnover of the intermediate metabolite 2-chloroaniline. 3-Chloronitrobenzene was completely degraded also in a mixture. A complete degradation of 4-chloronitrobenzene was achieved only when it was the sole chloronitrobenzene. The results suggest that a dechlorination and mineralization of monochlornitrobenzenes is possible, but for a final proof, further investigations will be necessary.     

Anaerobic biodegradation of TCE in laboratory columns of fractured saprolite

An experiment was conducted to determine if biodegradation of trichloroethylene (TCE) can occur in previously uncontaminated ground water in saturated fractured saprolite (highly weathered material derived from sedimentary rocks). Two undisturbed columns (0.23 m diameter by 0.25 m long) of fractured saprolite were collected from approximately 2 m depth at an uncontaminated site on the Oak Ridge Reservation, Oak Ridge, Tennessee. Natural, uncontaminated ground water from the site, which was degassed and spiked with dissolved phase TCE, was continuously pumped through one column containing the natural microbial communities (the biotic column). In a second column, the microorganisms were inhibited and the dissolved phase TCE was added under aerobic conditions (dissolved oxygen conditions > 2 ppm). In effluent from the biotic column, reducing conditions rapidly developed and evidence of anaerobic biodegradation of TCE, by the production of cDCE, first appeared approximately 31 days after addition of TCE. Reductive dechlorination of TCE occurred after iron-reducing conditions were established and about the same time that sulfate reduction began. There was no evidence of methanogenesis. Analyses using polymerase chain reaction with specific primers sets detected the bacteria Geothrix, Geobacter, and Desulfococcus-Desulfonema-Desulfosarcina in the effluent of the biotic column, but no methanogens. The presence of these bacteria is consistent with iron- and sulfate-reducing conditions. In the inhibited column, there were no indicators of TCE degradation. Natural organic matter that occurs in the saprolite and ground water at the site is the most likely primary electron donor for supporting reductive dechlorination of TCE. The relatively rapid appearance of indicators of TCE dechlorination suggests that these processes may occur even in settings where low oxygen conditions occur seasonally due to changes in the water table.     

PAH-degrading microbial consortium and its pyrene-degrading plasmids from mangrove sediment samples in Huian, China

PAH-degrading microbial consortium and its pyrene-degrading plasmids were enriched from the sediment samples of Huian mangroves. The consortium YL showed degrading abilities of 92.1%, 87.6%, 92.3%, and 95.8% for pyrene, fluoranthene, phenanthrene, and fluoene at 50 mg l(-1) after 21 days incubation, respectively. The dynamics of pH changes in the cultures was consistent with that of PAH concentration change. Bacillus cereus Py5 and Bacillus megaterium Py6 were isolated from the consortium and observed consuming 65.8% and 33.7% of pyrene (50 mg l(-1)) within three weeks, respectively. The enriched Escherichia coli DH5alpha cells containing the plasmids of YL were demonstrated to degrade 85.7% of the original pyrene concentration at the 21st day.     

Effect of sediment pH and redox conditions on degradation of benzo(a)pyrene

A study was conducted using an estuarine sediment maintained under controlled pH and redox potentials to determine the effect of these parameters on the degradation of benzo(a)pyrene. Degradation was strongly influenced by sediment pH and redox potential. Highest degradation rates occurred in sediment maintained at pH 8.0 and the lowest at pH 5.0. At all pH values degradation of benzo(a)pyrene increased with increasing redox potential. Up to a hundredfold difference in degradation rates could be attributed to changes in sediment pH and redox potential.     

Methane production from rice straw carbon in five different methanogenic rice soils:rates,quantities and microbial communities

The input of organic substances(e.g.,rice straw)in rice field soils usually stimulates the production and emission of the greenhouse gas methane(CH4).However,the amount of CH4 derived from the applied rice straw,as well as the response of bacterial and archaeal communities during the methanogenic phase,are poorly understood for different rice field soils.In this study,samples of five different rice soils were amended with 13^C-labeled rice straw(RS)under methanogenic conditions.Immediately after RS addition,the RS-derived CH4 production rates were higher in soils(Uruguay,Fuyang)that possessed a stronger inherent CH4 production potential compared with other soils with lower inherent potentials(Changsha,the Philippines,Vercelli).However,soils with higher inherent potential did not necessarily produce higher amounts of CH4 from the RS applied,or vice versa.Quantitative PCR showed copy numbers of both bacteria and methanogens increased in straw-amended soils.High-throughput sequencing of 16 S rRNA genes showed distinct bacterial communities among the unamended soil samples,which also changed differently in response to RS addition.Nevertheless,RS addition generally resulted in all the rice field soils in a relative increase of primary fermenters belonging to Anaerolineaceae and Ruminococcaceae.Meanwhile,RS addition also generally resulted in a relative increase of Methanosarcinaceae and/or Methanocellaceae.Our results suggest that after RS addition the total amounts of RSderived CH4 are distinct in different rice field soils under methanogenic conditions.Meanwhile,there are potential core bacterial populations that are often involved in primary fermentation of RS under methanogenic conditions.     

Biodegradation by Co‐inoculated Bacteria and Fungi Alleviates Adverse Effects of Red‐S3B on Growth and Nitrogen Uptake of Wheat

Textile wastewater contains huge quantities of nitrogen (N)‐containing azo‐dyes. Irrigation of crops with such wastewater adds toxic dyes into our healthy soils. One of the ways to prevent their entry to soils could be these waters after the dyes' biodegradation. Therefore, the present study was conducted to evaluate the impact of textile dyes on wheat growth, dye degradation efficiency of bacteria‐fungi consortium, and alleviation of dye toxicity in wheat by treatment with microbial consortium. Among dyes, Red‐S3B (3.19% N) was found to be the most toxic to germination and growth of seven‐day‐old wheat seedlings. Shewanella sp. NIAB‐BM15 and Aspergillus terreus NIAB‐FM10 were found to be efficient degraders of Red‐S3B. Their consortium completely decolorized 500 mg L^?1 Red‐S3B within 4 h. Irrigation with Red‐S3B‐contaminated water after treatment with developed consortium increased root length, shoot length, root biomass, and shoot biomass of 30‐day‐old wheat seedlings by 47, 18, 6, and 25%, respectively, than untreated water. Moreover, irrigation after microbial treatment of dye‐contaminated water resulted in 20 and 51% increase in shoot N content and N uptake, respectively, than untreated water. Thus, co‐inoculation of bacteria and fungi could be a useful bioremediation strategy for the treatment of azo‐dye‐polluted water.     

Development of longitudinal profiles of alluvial channels in response to base-level lowering

Degradation of alluvial channels in cohesive sediments was studied in 15 m and 20 m long flumes with a slope of 0°01 cm/cm. Degradation was initiated by lowering base level to a fixed position, and the development of the longitudinal profile of the channel is analysed through a model formulated as a heat (diffusion) equation. It is based on the equation of sediment continuity, combined with an assumption regarding sediment transport, namely that sediment discharge is linearly proportional to the channel slope. In accordance with the boundary and initial conditions imposed by the experimental setup and procedure, the basic equation is amenable to an analytical solution, which defines bed elevation at any distance and time, as a function of the amount of base-level lowering and a ‘diffusion’ coefficient. Additional problems arising from bank erosion and channel armouring are also treated successfully within the framework of the same model. The results show that in homogeneous alluvial sediments, not subject to armouring, the ultimate result of base-level lowering by a certain amount is degradation all along the channel by the same amount. The main impact of erosion is felt in the early stages after initiation of the process, and mainly near the mouth. The rate of degradation at any station along the channel reaches a peak and then slowly decreases with time, and the peak rate is attenuated with distance from the outlet. The model permits the prediction of intermediate stages of profile development at any distance from the outlet and at different times.     

Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE

A microbial consortium was obtained by enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of a mixture of polycyclic aromatic hydrocarbons enrichment strategy. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial composition and community dynamic changes based on PCR amplification of 16S rRNA genes during batch culture enrichment. Using the spray-plate method, three bacteria, designated as BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequence analysis showed that BL01, BL02 and BL03 were phylogenetically close to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens, respectively. The degradation of benzo(a)pyrene (BaP), a model high-molecular-weight polycyclic aromatic hydrocarbon (HMW PAH) compound was investigated using individual isolates, a mixture of the three isolates, and the microbial consortium (BL) originally isolated from the oil port sea water. Results showed that the order of degradative ability was BL > the mixture of the three isolates > individual isolates. BL degraded 44.07% of the 10 ppm BaP after 14 days incubation, which showed the highest capability for HMW PAH compound degradation.Our results revealed that this high selective pressure strategy was feasible and effective in enriching the HMW PAH-degraders from the original sea water samples.