Microbially Mediated Formation of Benzonaphthothiophenes from Benzo[b]thiophenes

Studies of the microbial metabolism of benzo[b]thiophene (molecular weight 134) by three Pseudomonas isolates showed the formation of benzothiophene sulfoxide, benzothiophene sulfone, and a sulfur-containing metabolite with a molecular weight of 234. Desulfurization of the high-molecular-weight product with nickel boride gave 1-phenylnaphthalene, indicating that the metabolite was benzo[b]naphtho[1,2-d]thiophene. Similarly, the isolates were capable of producing the analogous dimethyl-substituted benzonaphthothiophenes from methylbenzothiophenes that had the methyl substitution on the benzene ring. The formation of benzo[b] naphtho[1,2-d]thiophene was also observed when a petroleum-degrading mixed culture was incubated with benzothiophene-supplemented Prudhoe Bay crude oil. Investigations into the mechanism of formation of these high-molecular-weight compounds showed that they resulted from an abiotic, Diels-Alder-type condensation of two molecules of the sulfoxide, which were microbially produced from the respective benzothiophene, with the subsequent loss of two atoms of hydrogen and oxygen and one atom of sulfur. The condensation products also formed from the sulfoxides of benzothiophene and methylbenzothiophenes when the sulfoxides were enzymatically synthesized by oxidation of the benzothiophene with horse heart cytochrome c and H₂O₂.     

Biodegradation of benzothiophene sulfones by a filamentous bacterium.

Previous studies showed that benzothiophene and 3- and 5-methylbenzothiophenes are oxidized by some bacteria to yield their corresponding sulfones, which were not subsequently degraded. In this study, a filamentous bacterium was isolated, which grew on each of these three sulfones as its sole carbon, sulfur, and energy source. Based on 16S rRNA gene sequencing and scanning electron microscopy, the isolate was found to belong to the genus Pseudonocardia and assigned the strain designation DB1. Benzothiophene sulfone and 3-methylbenzothiophene sulfone were more readily biodegraded than 5-methylbenzothiophene sulfone, and growth on these three compounds resulted in the release of 57, 62, and 28% of the substrate carbon as CO2, respectively. The thiophene ring was also cleaved, and between 44 and 88% of the sulfur from the consumed substrate was found as sulfate and (or) sulfite. Strain DB1 grew on benzoate, dibenzothiophene sulfone, and hexadecanoic acid, but it could not grow on benzofuran, dibenzothiophene, dibenzothiophene sulfoxide, hexadecane, indole, naphthalene, phenol, 2-sulfobenzoic acid, sulfolane, benzothiophene, or 3- or 5-methylbenzothiophenes. In addition, it did not oxidize the latter three compounds to their sulfones.     

Sulfur from benzothiophene and alkylbenzothiophenes supports growth of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. strain JVH1

Rhodococcus sp. strain JVH1 was previously reported to use a number of compounds with aliphatic sulfide bridges as sulfur sources for growth. We have shown that although JVH1 does not use the three-ring thiophenic sulfur compound dibenzothiophene, this strain can use the two-ring compound benzothiophene as its sole sulfur source, resulting in growth of the culture and loss of benzothiophene. Addition of inorganic sulfate to the medium reduced the conversion of benzothiophene, indicating that benzothiophene metabolism is repressed by sulfate and that benzothiophene is therefore used specifically as a sulfur source. JVH1 also used all six isomers of methylbenzothiophene and two dimethylbenzothiophene isomers as sulfur sources for growth. Metabolites identified from benzothiophene and some methylbenzothiophenes were consistent with published pathways for benzothiophene biodesulfurization. Products retaining the sulfur atom were sulfones and sultines, the sultines being formed from phenolic sulfinates under acidic extraction conditions. With 2-methylbenzothiophene, the final desulfurized product was 2-methylbenzofuran, formed by dehydration of 3-(o-hydroxyphenyl) propanone under acidic extraction conditions and indicating an oxygenative desulfination reaction. With 3-methylbenzothiophene, the final desulfurized product was 2-isopropenylphenol, indicating a hydrolytic desulfination reaction. JVH1 is the first microorganism reported to use all six isomers of methylbenzothiophene, as well as some dimethylbenzothiophene isomers, as sole sulfur sources. JVH1 therefore possesses broader sulfur extraction abilities than previously reported, including not only sulfidic compounds but also some thiophenic species.     

Desulfurization of Benzothiophene by the Gram-Negative Bacterium, Sinorhizobium sp. KT55

Sinorhizobium sp. KT55 was the first Gram-negative isolate to be capable of utilizing benzothiophene as the sole source of sulfur. By GC-MS analysis of metabolites of benzothiophene by this strain, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide and o-hydroxystyrene were detected, suggesting that the benzothiophene desulfurization pathway of this strain is benzothiophene → benzothiophene sulfoxide → benzothiophene sulfone → benzo[e][1,2]oxathiin S-oxide →o-hydroxystyrene. Desulfurization activity of this strain was significantly repressed by methionine, cysteine, sulfate, dimethyl sulfoxide, and Casamino acids. Received: 5 January 2001/Accepted: 6 February 2001     

Selectivity among organic sulfur compounds in one- and two-liquid-phase cultures of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. strain JVH1

The selectivity of Rhodococcus sp. strain JVH1 among selected sulfidic and thiophenic compounds was investigated in both single-liquid-phase (aqueous) cultures and in two-liquid-phase cultures, where the sulfur compounds were dissolved in 2,2,4,4,6,8,8-heptamethylnonane as the immiscible organic carrier phase. In the single-liquid-phase cultures, Rhodococcus sp. strain JVH1 showed a preference for benzyl sulfide over both 1,4-dithiane and benzothiophene. An increased lag was observed in the degradation of benzyl sulfone and benzothiophene sulfone when both compounds were present. These results were consistent with a competitive inhibition mechanism, affecting both sulfur oxidation and carbon–sulfur bond cleavage. In the two-liquid-phase cultures, the effect of partitioning between the two liquid phases dominated the desulfurization activity of the culture. This partitioning resulted in an apparent absence of selectivity, as well as decreases in lag time, extent of degradation, and time to completion of degradation. Desulfurization activity also depended on the growth phase of the cultures. Mass transfer rate limitations were not observed at the low degradation rates of 0.02 mmol day^-1 l⁻¹. Owing to the importance of partitioning, Rhodococcus sp. strain JVH1 is predicted to show nonselective activity towards the sulfur species in a whole crude oil.     

Use of a novel fluorinated organosulfur compound to isolate bacteria capable of carbon-sulfur bond cleavage

The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C-S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C-S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound, bis-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C-S bonds. A unique bacterial strain, Rhodococcus sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C-S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including Rhodococcus sp. strain IGTS8, were all unable to cleave the C-S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C-S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.     

Effects of Petroleum Hydrocarbons on Plant Litter Microbiota in an Arctic Lake

The effects of petroleum hydrocarbons on the microbial community associated with decomposing Carex leaf litter colonized in Toolik Lake, Alaska, were examined. Microbial metabolic activity, measured as the rate of acetate incorporation into lipid, did not vary significantly from controls over a 12-h period after exposure of colonized Carex litter to 3.0 ml of Prudhoe Bay crude oil, diesel fuel, or toluene per liter. ATP levels of the microbiota became elevated within 2 h after the exposure of the litter to diesel fuel or toluene, but returned to control levels within 4 to 8 h. ATP levels of samples exposed to Prudhoe Bay crude oil did not vary from control levels. Mineralization of specifically labeled ¹⁴C-[lignin]-lignocellulose and ¹⁴C-[cellulose]-lignocellulose by Toolik Lake sediments, after the addition of 2% (vol/vol) Prudhoe Bay crude oil, motor oil, diesel fuel, gasoline, n-hexane, or toluene, was examined after 21 days of incubation at 10°C. Diesel fuel, motor oil, gasoline, and toluene inhibited ¹⁴C-[lignin]-lignocellulose mineralization by 58, 67, 67, and 86%, respectively. Hexane-treated samples displayed an increase in the rate of ¹⁴C-[lignin]-lignocellulose mineralization of 33%. ¹⁴C-[cellulose]-lignocellulose mineralization was inhibited by the addition of motor oil or toluene by 27 and 64%, respectively, whereas diesel fuel-treated samples showed a 17% increase in mineralization rate. Mineralization of the labeled lignin component of lignocellulose appeared to be more sensitive to hydrocarbon perturbations than was the labeled cellulose component.     

Effect of inoculation on the biodegradation of wathered Prudhoe Bay crude oil

Summary Enrichment cultures from oil-contaminated beach material from Prince William Sound, Alaska, generated both a mixed bacterial community of indigenous, oil-degrading marine microorganisms and a pure culture oil-degrader, strain EI2V. The mixed and axenic cultures were used in comparative shake flask studies of inoculation on biodegradation of Prudhoe Bay crude oil. Within 12 h following inoculation of homogenized, oiled beach material with the mixed culture, total CO₂ production was increased 2-fold relative to a noninoculated control. Moreover, measurements of phenanthrene degradation (as determined by the release of¹⁴CO₂ from [9-¹⁴C]phenanthrene) showed a 2-or 3-fold greater degradation when inoculated with either strain EI2V or with the mixed culture, respectively. However, as medium was replaced by a simulated tidal cycle, the observed stimulation of CO₂ production decreased, and the addition of strain EI2V had no greater effect on total CO₂ production than the addition of inorganic nutrients alone. Chemical analysis of oil recovered after 7 days incubation also suggested that, while these cultures are capable of efficient biodegradation of Prudhoe Bay crude in liquid culture, inoculation of beach material with high numbers of these microorganisms had little effect on the rate and extent of biodegradation of weathered crude oil. Overall, the sustained stimulatory effect was no greater than that observed with the addition of inorganic nutrients alone.     

Bacterial Transformations of 1,2,3,4-Tetrahydrodibenzothiophene and Dibenzothiophene

The transformations of 1,2,3,4-tetrahydrodibenzothiophene (THDBT) were investigated with pure cultures of hydrocarbon-degrading bacteria. Metabolites were extracted from cultures with dichloromethane (DCM) and analyzed by gas chromatography (GC) with flame photometric, mass, and Fourier transform infrared detectors. Three 1-methylnaphthalene (1-MN)-utilizing Pseudomonas strains oxidized the sulfur atom of THDBT to give the sulfoxide and sulfone. They also degraded the benzene ring to yield 3-hydroxy-2-formyl-4,5,6,7-tetrahydrobenzothiophene. A cell suspension of a cyclohexane-degrading bacterium oxidized the alicyclic ring to give a hydroxy-substituted THDBT and a ketone, and it oxidized the aromatic ring to give a phenol, but no ring cleavage products were detected. GC analyses with an atomic emission detector, using the sulfur-selective mode, were used to quantify the transformation products from THDBT and dibenzothiophene (DBT). The cyclohexane degrader oxidized 19% of the THDBT to three metabolites. The cometabolism of THDBT and DBT by the three 1-MN-grown Pseudomonas strains resulted in a much greater depletion of the condensed thiophenes than could be accounted for in the metabolites detected by GC analysis, but there was no evidence of sulfate release from DBT. These 1-MN-grown strains transiently accumulated 3-hydroxy-2-formylbenzothiophene (HFBT) from DBT, but it was subsequently degraded. On the other hand, Pseudomonas strain BT1d, which was maintained on DBT as a sole carbon source, accumulated 52% of the sulfur from DBT as HFBT over 7 days, and, in total, 82% of the sulfur from DBT was accounted for by the GC method used. Lyophilization of cultures grown on 1-MN with DBT and methyl esterification of the residues gave improved recoveries of total sulfur over that obtained by DCM extraction and GC analysis. This suggested that the further degradation of HFBT by these cultures leads to the formation of organosulfur compounds that are too polar to be extracted with DCM. We believe that this is the first attempt to quantify the products of DBT degradation by the so-called Kodama pathway.     

Emulsifier of Arthrobacter RAG-1: specificity of hydrocarbon substrate.

The purified extracellular emulsifying factor produced by Arthrobacter RAG-1 (EF-RAG) emulsified light petroleum oil, diesel oil, and a variety of crude oils and gas oils. Although kerosine and gasoline were emulsified poorly by EF-RAG, they were converted into good substrates for emulsification by addition of aromatic compounds, such as 2-methylnaphthalene. Neither aromatic nor aliphatic fractions of crude oil were emulsified by EF-RAG; however, mixtures containing both fractions were emulsified. Pure aliphatic or aromatic hydrocarbons were emulsified poorly by EF-RAG. Binary mixtures containing an aliphatic and an aromatic hydrocarbon, however, were excellent substrates for EF-RAG-induced emulsification. Of a variety of alkylcyclohexane and alkylbenzene derivatives tested, only hexyl- or heptylbenzene and octyl- or decylcyclohexane were effectively emulsified by EF-RAG. These data indicate that for EF-RAG to induce emulsification of hydrocarbons in water, the hydrocarbon substrate must contain both aliphatic and cyclic components. With binary mixtures of methylnaphthalene and hexadecane, maximum emulsion was obtained with 25% hexadecane.     

Emulsifier of Arthrobacter RAG-1: specificity of hydrocarbon substrate.

The purified extracellular emulsifying factor produced by Arthrobacter RAG-1 (EF-RAG) emulsified light petroleum oil, diesel oil, and a variety of crude oils and gas oils. Although kerosine and gasoline were emulsified poorly by EF-RAG, they were converted into good substrates for emulsification by addition of aromatic compounds, such as 2-methylnaphthalene. Neither aromatic nor aliphatic fractions of crude oil were emulsified by EF-RAG; however, mixtures containing both fractions were emulsified. Pure aliphatic or aromatic hydrocarbons were emulsified poorly by EF-RAG. Binary mixtures containing an aliphatic and an aromatic hydrocarbon, however, were excellent substrates for EF-RAG-induced emulsification. Of a variety of alkylcyclohexane and alkylbenzene derivatives tested, only hexyl- or heptylbenzene and octyl- or decylcyclohexane were effectively emulsified by EF-RAG. These data indicate that for EF-RAG to induce emulsification of hydrocarbons in water, the hydrocarbon substrate must contain both aliphatic and cyclic components. With binary mixtures of methylnaphthalene and hexadecane, maximum emulsion was obtained with 25% hexadecane.     

Effects of creosote compounds on the aerobic bio-degradation of benzene

The inhibitory effect of creosote compounds on the aerobic degradation of benzene was studied in microcosm experiments. A total removal of benzene was observed after twelve days of incubation in microcosms where no inhibition was observed. Thiophene and benzothiophene, two heterocyclic aromatic compounds containing sulfur (S-compounds), had a significant inhibitory effect on the degradation of benzene, but also an inhibitory effect of benzofuran (an O-compound) and 1-methylpyrrole (a N-compound) could be observed, although the effect was weaker. The NSO-compounds also had an inhibitory effect on the degradation of p-xylene, o-xylene, and naphthalene, while they only had a weak influence on the degradation of 1-methylnaphthalene, o-cresol and 2,4-dimethylphenol. The phenolic compounds seemed to have a weak stimulating effect on the degradation of benzene whereas the monoaromatic hydrocarbons and the naphthalenes had no significant influence on the benzene degradation. The inhibitory effect of the NSO-compounds on the aerobic degradation of benzene could be identified as three different phenomena. The lag phase increased, the degradation rate decreased, and a residual concentration of benzene was observed in microcosms when NSO-compounds were present. The results show that NSO-compounds can have a potential inhibitory effect on the degradation of many creosote compounds, and that inhibitory effects in mixtures can be important for the degradation of different compounds.Abbreviations ben benzene - bf benzofuran - bt benzothiophene - dmp 2,4-dimethylphenol - GC gas chromatograph - ind indole - mnap 1-methylnaphthalene - MAHs monoaromatic hydrocarbons - mp 1-methylpyrrole - nap naphthalene - NSO-compounds heterocyclic aromatic compounds containing nitrogen, sulphur or oxygen - o-cre o-cresol - o-xyl o-xylene - PAHs polyaromatic hydrocarbons - phe phenol - p-xyl p-xylene - pyr pyrrole - thi thiophene - qui quinoline     

Benzo[b]thiophene desulfurization by Gordonia rubropertinctus strain T08

A benzothiophene-desulfurizing bacterium which has a novel desulfurization pathway was isolated and identified as Gordonia rubropertinctus strain T08. Gas chromatography/mass spectroscopy analysis of the ethyl acetate extract of the culture broth detected benzothiophene sulfoxide, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide (BT-sultine), benzo[e][1,2]oxathiin S,S-dioxide (BT-sultone), o-hydroxystyrene, and 2-coumaranone, but not 2-(2'-hydroxyphenyl)ethan-1-al, which has been reported to be a desulfurized product of mesophilic nocardioforms.     

Novel Reactivity of Dibenzothiophene Monooxygenase from Bacillus subtilis WU-S2B

Dibenzothiophene monooxygenase (BdsC) from Bacillus subtilis WU-S2B utilized aromatic compounds not having sulfur atoms as substrates. It acted on indole and its derivatives to form indigoid pigments, and also utilized indoline and phenoxazine. In addition, BdsC exhibited activity toward benzothiophene (BT) derivatives but not BT, suggesting that it shows wide reactivity toward aromatic compounds.     

Mechanisms of petroleum hydrocarbon toxicity: destruction of liver microsomal and mitochondrial calcium pump activities by a Prudhoe Bay crude oil

Administration of Prudhoe Bay crude oil (PBCO) to rats resulted in an abrupt drop in liver mitochondrial and microsomal ATP-dependent calcium uptake activity. Also, in vitro incubations of either mitochondria or microsomes in the presence of a dimethyl sulfoxide (DMSO) extract of PBCO resulted in a dose-dependent inhibition of calcium influx. The release of calcium from calcium-loaded mitochondria and microsomes was also observed in the presence of the PBCO extract. At concentrations which effect calcium sequestration, the PBCO extract produced swelling of mitochondria. Microsomal ATPase activity in the presence or absence of calcium was unaffected by PBCO. The results indicate that increased permeability of the membranes to calcium is a contributory factor in the inhibition of calcium uptake by PBCO.     

Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Effect of a Prudhoe Bay crude oil on hepatic and placental drug metabolism in rats

Administration of a Prudhoe Bay crude oil (PBCO) to pregnant rats resulted in induction of hepatic microsomal P-450 levels and various monooxygenases in a dose-dependent manner. The activities of aniline hydroxylase, benzo[a]pyrene hydroxylase, aminopyrine-N-demethylase, ethoxyresorufin-O-deethylase, and pentoxyresorufin-O-depentylase were increased 2-3-fold, 12-15-fold, 1.4-1.8-fold, 20-24-fold, and 6-8-fold, respectively, on gestation day 18, when a single dose of PBCO (5-10 mL/kg body weight, p.o.) had been administered 24 h earlier. Glutathione-S-transferase, UDPG transferase, and DT-diaphorase activities were also increased; however, maximum induction was noticed when crude oil was given 72 h earlier. Repeated exposure (day 6-day 17, daily) of crude oil at lower levels was able to produce similar induction patterns in enzyme systems at day 18 of gestation. The xenobiotic-metabolizing enzyme systems were also induced transplacentally: treatment of pregnant rats with PBCO induced both placental and fetal hepatic enzyme systems. Liver microsomal P-450 contents, benzo[a]pyrene hydroxylase, and ethoxyresorufin-O-deethylase activities were increased 2-fold, 2-3-fold, and 10-12-fold, respectively in 18-day-old fetuses. Similar trends were noticed in placenta. Activities of phase II enzymes such as glutathione-S-transferase, UDPG transferase, and DT-diaphorase were also significantly elevated. It is suggested that crude oil induces maternal hepatic drug metabolism and that some of its constituents (mainly aromatic hydrocarbons) and (or) their metabolites pass through the placenta and thus induce drug-metabolizing enzymes transplacentally. The practical importance of the results in relation to human and environmental health is also discussed.     

Hydrocarbon emulsification and enhanced crude oil degradation by lauroyl glucose ester

Enzymatically synthesized lauroyl glucose emulsified different hydrophobic substrates when assayed spectrophotometrically. Stable emulsions were formed with triglycerides as well as with hydrocarbons. There was a linear relation between the concentration of lauroyl glucose (50-450 microg) and emulsification activity under the assay conditions when tested with aromatic and aliphatic hydrocarbons. This sugar ester was able to emulsify the aromatic hydrocarbons benzene, toluene and xylene. Long chain alkanes (n-decane and n-hexadecane) as well as brominated long chain alkanes (1-bromodecane and 1-bromohexadecane) were efficiently emulsified. The effect of lauroyl glucose ester on degradation of crude oil by a known oil-degrading Rhodococcus species was also investigated. The culture showed enhanced degradation of crude oil when lauroyl glucose ester was used as an emulsifier. It degraded 70% of the aliphatic fraction of Bombay High crude oil in the presence of the sugar ester at a concentration of 200mg l(-1) as compared to 50% without the emulsifier.     

Anaerobic degradation of 1-methylnaphthalene by a member of the <Emphasis Type="Italic">Thermoanaerobacteraceae</Emphasis> contained in an iron-reducing enrichment culture

An anaerobic culture (1MN) was enriched with 1-methylnaphthalene as sole source of carbon and electrons and Fe(OH)₃ as electron acceptor. 1-Naphthoic acid was produced as a metabolite during growth with 1-methylnaphthalene while 2-naphthoic acid was detected with naphthalene and 2-methylnaphthalene. This indicates that the degradation pathway of 1-methylnaphthalene might differ from naphthalene and 2-methylnaphthalene degradation in sulfate reducers. Terminal restriction fragment length polymorphism and pyrosequencing revealed that the culture is mainly composed of two bacteria related to uncultured Gram-positive Thermoanaerobacteraceae and uncultured gram-negative Desulfobulbaceae. Stable isotope probing showed that a ¹³C-carbon label from ¹³C₁₀-naphthalene as growth substrate was mostly incorporated by the Thermoanaerobacteraceae. The presence of putative genes involved in naphthalene degradation in the genome of this organism was confirmed via assembly-based metagenomics and supports that it is the naphthalene-degrading bacterium in the culture. Thermoanaerobacteraceae have previously been detected in oil sludge under thermophilic conditions, but have not been shown to degrade hydrocarbons so far. The second member of the community belongs to the Desulfobulbaceae and has high sequence similarity to uncultured bacteria from contaminated sites including recently proposed groundwater cable bacteria. We suggest that the gram-positive Thermoanaerobacteraceae degrade polycyclic aromatic hydrocarbons while the Desulfobacterales are mainly responsible for Fe(III) reduction.     

A Perspective on the Toxicity of Low Concentrations of Petroleum-Derived Polycyclic Aromatic Hydrocarbons to Early Life Stages of Herring and Salmon

This article presents a critical review of two groups of studies that reported adverse effects to salmon and herring eggs and fry from exposure to 1 μg/L or less of aqueous total polycyclic aromatic hydrocarbons (TPAH), as weathered oil, and a more toxic aqueous extract of “very weathered oil.” Exposure media were prepared by continuously flowing water up through vertical columns containing gravel oiled at different concentrations of Prudhoe Bay crude oil. Uncontrolled variables associated with the use of the oiled gravel columns included time- and treatment-dependent variations in the PAH concentration and composition in the exposure water, unexplored toxicity from other oil constituents/degradation products, potential toxicity from bacterial and fungal activity, oil droplets as a potential contaminant source, inherent differences between control and exposed embryo populations, and water flow rate differences. Based on a review of the evidence from published project reports, peer-reviewed publications, chemistry data in a public database, and unpublished reports and laboratory records, the reviewed studies did not establish consistent dose (concentration) response or causality and thus do not demonstrate that dissolved PAH alone from the weathered oil resulted in the claimed effects on fish embryos at low μg/L TPAH concentrations. Accordingly, these studies should not be relied on for management decision-making, when assessing the risk of very low–level PAH exposures to early life stages of fish.