Conversion of dibenzothiophene by the mushrooms <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and <Emphasis Type="Italic">Coprinellus radians</Emphasis> and their extracellular peroxygenases

The conversion of the heterocycle dibenzothiophene (DBT) by the agaric basidiomycetes Agrocybe aegerita and Coprinellus radians was studied in vivo and in vitro with whole cells and with purified extracellular peroxygenases, respectively. A. aegerita oxidized DBT (110 μM) by 100% within 16 days into eight different metabolites. Among the latter were mainly S-oxidation products (DBT sulfoxide, DBT sulfone) and in lower amounts, ring-hydroxylation compounds (e.g., 2-hydroxy-DBT). C. radians converted about 60% of DBT into DBT sulfoxide and DBT sulfone as the sole metabolites. In vitro tests with purified peroxygenases were performed to compare the product pattern with the metabolites formed in vivo. Using ascorbic acid as radical scavenger, a total of 19 and seven oxygenation products were detected after DBT conversion by the peroxygenases of A. aegerita (AaP) and C. radians (CrP), respectively. Whereas ring hydroxylation was favored over S-oxidation by AaP (again 2-hydroxy-DBT was identified), CrP formed DBT sulfoxide as major product. This finding suggests that fungal peroxygenases can considerably differ in their catalytic properties. Using H₂ ¹⁸O₂, the origin of oxygen was proved to be the peroxide. Based on these results, we propose that extracellular peroxygenases may be involved in the oxidation of heterocycles by fungi also under natural conditions.     

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.     

The Coprophilous Mushroom Coprinus radians Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H₂O₂ concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent K_m values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H₂O₂ were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.     

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

Pyridine as novel substrate for regioselective oxygenation with aromatic peroxygenase from Agrocybe aegerita

Agrocybe aegerita peroxidase (AaP) is a versatile extracellular biocatalyst that can oxygenate aromatic compounds. Here, we report on the selective oxidation of pyridine (PY) yielding pyridine N-oxide as sole product. Using H₂¹⁸O₂ as co-substrate, the origin of oxygen was confirmed to be the peroxide. Therefore, AaP can be regarded as a true peroxygenase transferring one oxygen atom from peroxide to the substrate. To our best knowledge, there are only two types of enzymes oxidizing PY at the nitrogen: bacterial methane monooxygenase and a few P450 monooxygenases. AaP is the first extracellular enzyme and the first peroxidase that catalyzes this reaction, and it converted also substituted PYs into the corresponding N-oxides.     

Competitive metabolism of naphthalene, methylnaphthalenes, and fluorene by phenanthrene-degrading pseudomonads.

Polynuclear aromatic hydrocarbons (PAHs) typically exist as complex mixtures in contaminated soils, yet little is known about the biodegradation of PAHs in mixtures. We have isolated two physiologically diverse bacteria, Pseudomonas stutzeri P-16 and P. saccharophila P-15, from a creosote-contaminated soil by enrichment on phenanthrene as the sole carbon source and studied their ability to metabolize several other two- and three-ring PAHs. Naphthalene, 1-methylnaphthalene, and 2-methylnaphthalene served as growth substrates for both organisms, while fluorene was only cometabolized. We also studied the effects of these compounds on initial rates of phenanthrene uptake in binary mixtures. Lineweaver-Burk analysis of kinetic measurements was used to demonstrate competitive inhibition of phenanthrene uptake by all four compounds, suggesting that multiple PAHs are being transformed by a common enzyme pathway in whole cells. Estimates of the inhibition coefficient, Ki, are reported for each compound. The occurrence of competitive metabolic processes in physiologically diverse organisms suggests that competitive metabolism may be a common phenomenon among PAH-degrading organisms.     

Catabolic Gene Probe Analysis of an Aquifer Microbial Community Degrading Creosote-Related Polycyclic Aromatic and Heterocyclic Compounds

Abstract The biodegradation of a mixture of several creosote-related compounds, p-cresol, phenanthrene, fluorene, and carbazole was examined in columns containing aquifer sands. The aquifer material, itself, had an effect on the migration of the test compounds, with p-cresol being retarded the least, followed by carbazole, then fluorene, and finally phenanthrene. The biodegradation of all the compounds was greatly enhanced by the inclusion of p-cresol (10 ppm) in the substrate mixture. Associated with this enhanced degradation was a 100-fold increase in the total culturable bacterial population, and increases in the xylE- and ndoB-positive bacterial populations of more than three orders of magnitude. The products of these two genes are involved in the degradation of monocyclic and polycyclic aromatic compounds, respectively. In columns that did not receive p-cresol, there was no significant change in either the total culturable bacterial population density or the xylE-positive bacterial population, but there were significant increases of one to two orders of magnitude in the ndoB-positive bacterial populations. The results suggest that the ndoB gene probe can detect bacteria capable of utilizing phenanthrene, carbazole, and possibly fluorene. Received: 26 January 1996; Accepted: 20 June 1996     

Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type—the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago—has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV–Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as “self-sufficient” peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.     

The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.     

Oxygenation Reactions of Various Tricyclic Fused Aromatic Compounds Using Escherichia coli and Streptomyces lividans Transformants Carrying Several Arene Dioxygenase Genes

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and ¹H and ¹³C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-di-hydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.     

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.     

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     

Utilization of solubilized and crystalline mixtures of polycyclic aromatic hydrocarbons by a Mycobacterium sp.

A strain of Mycobacterium, that is able to degrade fluorene, phenanthrene, fluoranthene and pyrene was grown on various mixtures of these substrates. The polycyclic aromatic hydrocarbons (PAH) were provided either as crystals or solubilized by a surfactant. Mixed PAH were degraded simultaneously, but not in parallel, indicating that the degradation pathways were not incompatible. Certain interactions of the substrates were observed. For example, the degradation of solubilized pyrene was delayed in the presence of fluorene and enhanced in the presence of phenanthrene. Fluorene was degraded cometabolically with the other PAH serving as growth substrates, but not as the only source of carbon. The utilization of phenanthrene occurred at the fastest rate and was not affected by the presence of fluorene, pyrene or fluoranthene.     

Degradation of phenanthrene,fluorene and fluoranthene by pure bacterial cultures

Summary Bacterial mixed cultures able to degrade the polycyclic aromatic hydrocarbons (PAH) phenanthrene, fluorene and fluoranthene, were obtained from soil using conventional enrichment techniques. From these mixed cultures three pure strains were isolated:Pseudomonas paucimobilis degrading phenanthrene;P. vesicularis degrading fluorene andAlcaligenes denitrificans degrading fluoranthene. The maximum rates of PAH degradation ranged from 1.0 mg phenanthrene/ml per day to 0.3 mg fluoranthene/ml per day at doubling times of 12 h to 35 h for growth on PAH as sole carbon source. The protein yield during PAH degradation was about 0.25 mg/mg C for all strains. Maximum PAH oxidation rates and optimum specific bacterial growth were obtained near pH 7.0 and 30°C. After growth entered the stationary phase, no dead end-products of PAH degradation could be detected in the culture fluid.     

Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established. Received: 25 May 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Degradation of polycyclic aromatic hydrocarbons by an immobilized mixed bacterial culture

The mixed bacterial culture MK1 was capable of degrading a wide spectrum of aromatic compounds both as free and as immobilized cells. By offering anthracene oil or a defined mixture of phenol, naphthalene, phenanthrene, anthracene and pyrene (in concentrations of 0.1–0.2 mm, respectively) as sources of carbon and energy, a specific degradation pattern correlating with the condensation degree was observed. Regarding the defined mixture of aromatic hydrocarbons, complete metabolism was reached for phenol (0.1 mm) after 1 day, for naphthalene (0.1 mm) after 2 days and for phenanthrene (0.1 mm) after 15 days of cultivation. The conversion of anthracene (0.1 mm) and pyrene (0.1 mm) resulted in minimal residual concentrations, analogous to fluoranthene and pyrene of the anthracene oil (0.1%). Maximal total degradation for the tricyclic compounds dibenzofurane, fluorene, dibenzothiophene, phenanthrene and anthracene of the anthracene oil (0.1%) occurred after 5 days. In general, a significant metabolisation of the tetracyclic aromatic hydrocarbons fluoranthene and pyrene was observed after the degradation of phenol, naphthalene and most of the tricyclic compounds. Doubling the start concentrations of the polycyclic aromatic hydrocarbons effected higher degradation rates. Cell growth occurred simultaneously with the conversion of phenol, naphthalene and the tricyclic compounds. The immobilized cells showed stable growth and, compared to freely suspended cells, the same degradation sequence as well as an equivalent degradation potential — even in a model soil system. Correspondence to: I. Wiesel     

Pyrene Biodegradation by Bacillus spp. Isolated from Coal Tar–Contaminated Soil

Aerobic, mesophilic bacteria from coal tar–contaminated soil were analyzed for pyrene utilization capacity and identified by 16S ribosomal DNA sequencing as members of three genera: Bacillus spp., Pseudomonas sp., and Rhodococcus sp. The soil contained nine different hazardous polyaromatic hydrocarbons (PAHs): benzo[g, h, i]perylene, dibenzo[a, h]anthracene, indeno[1,2,3-c,d]pyrene, pyrene, acenaphthylene, fluorene, phenanthrene, benzo[k]fluoranthene, and benzo[b]fluoranthene. Bacillus spp. (PK-6) MTCC 1005 showed 56.4% utilization of pyrene (C₁₆H₁₀) (50 μg ml^?1) in 4 days, with growth associated biosurfactant activity and resulted in the formation of five new intermediates: phenanthrene (C₁₄H₁₀), 9,10-diphenylphenanthrene (C₂₆H₁₈), 9-methoxyphenanthrene (C₁₅H₁₂O), 5,6,7,8-tetrahydro-1-naphthoic acid (C₁₁H₁₂O₂), and 1,6,7-trimethylnaphthalene (C₁₃H₁₄). The results suggested that Bacillus spp. could be found suitable for practical field application for effective in situ PAH bioremediation.     

Fluorene and Phenanthrene Uptake and Accumulation by Wheat,Alfalfa and Sunflower from the Contaminated Soil

Polycyclic Aromatic Hydrocarbons (PAHs) are diverse organic contaminants released into the environment by both natural and anthropogenic activities. These compounds have negative impacts on plants growth and development. Although there are many reports on their existence in different parts of plant, their uptake and translocation pathways and mechanisms are not well understood yet. This paper highlights the uptake, translocation and accumulation of PAHs by wheat, sunflower and alfalfa through an experimental study under controlled conditions. Seeds were cultivated in a soil containing 50 mg/kg of phenanthrene and fluorene and their concentrations in plants roots and shoots were determined using a gas chromatograph after 7 and 14 days. The results showed that phenanthrene and fluorene concentrations in the treated plants were increased over the time. PAHs bioavailability was time and species dependent and generally, phenanthrene uptake and translocation was faster than that of fluorene, probably due to their higher K_ow. Fluorene tended to accumulate in roots, but phenanthrene was transported to aerial parts of plants.     

Isolation and characterization of novel bacteria degrading polycyclic aromatic hydrocarbons from polluted Greek soils

Three bacterial strains, designated as Wphe1, Sphe1, and Ophe1, were isolated from Greek soils contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste from the wood processing, steel, and oil refinery industries. Wphe1, Sphe1, and Ophe1 were characterized and identified as species of Pseudomonas, Microbacterium, and Paracoccus, respectively, based on Gram staining, biochemical tests, phospholipid analysis, FAME analysis, G+C content and 16S rRNA gene sequence analysis. The results of gas chromatography showed that strain Wphe1 degraded naphthalene, phenanthrene, and m-cresol over a wide temperature range; strain Sphe1 was a degrader of phenanthrene and n-alkanes; most interestingly, strain Ophe1 degraded anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene, as well as cresol compounds and n-alkanes as sole carbon source. This is the first report of a representative of the genus Paracoccus capable of degrading PAHs with such versatility. These three strains may be useful for bioremediation applications.     

The Bioremediation Potential of Hydrocarbonoclastic Bacteria Isolated From a Mangrove Contaminated by Petroleum Hydrocarbons on the Cilacap Coast,Indonesia

The main purpose of the study was to isolate strains of bacteria capable of degrading hydrocarbons from contaminated mangroves and to investigate the ability of the isolated bacteria to degrade total petroleum hydrocarbons (TPH) in a microcosm model of an oily sludge. The potential use of these bacteria strains as environmental clean-up agents was tested by culturing them with six different polyaromatic hydrocarbon (PAH) compounds (phenothiazine, fluorene, fluoranthene, dibenzothiophene, phenanthrene, and pyrene). Six viable and culturable bacteria were isolated, and the 16S rDNA sequence for each was amplified using the primers 9F and 1510R. Sequence results were compared using the National Center for Biotechnology Information (NCBI) BLAST program and, combined with phenotypic and phylogenetic data, were used to identify three strains that belonged to the Bacillus genus and were most closely related (98–99%) to Bacillus aquimaris, Bacillus megaterium, and Bacillus pumilus. The other three strains were closely related (98–100%) to Flexibacteraceae bacterium, Halobacilus trueperi, and Rhodobacteraceae bacterium. Two isolates, BA-PZN and BM-PFFP, which were related to Bacillus aquimaris and Bacillus megaterium, respectively, were further characterized and showed great potential for the removal of more complex hydrocarbon compounds in the oily microcosm model.