Bacterial Degraders of Polycyclic Aromatic Hydrocarbons Isolated from Salt-Contaminated Soils and Bottom Sediments in Salt Mining Areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical- and salt-producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1–1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.     

Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+--a degrader of polycyclic aromatic hydrocarbons

Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l.     

Degradation of phenanthrene by mutant strains--naphthalene degraders

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-borne genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

PAH utilization by Pseudomonas rhodesiae KK1 isolated from a former manufactured-gas plant site

Pseudomonas rhodesiae KK1 was isolated from a former manufactured-gas plant site, due to its ability to grow rapidly in a mixture of polycyclic aromatic hydrocarbons (PAHs). Radiorespirometric analysis revealed that strain KK1 was found to be able to mineralize anthracene, naphthalene and phenanthrene. Notably, phenanthrene-grown cells were able to mineralize anthracene much more rapidly than naphthalene-grown cells. Comparative analysis of amino acid sequences from 17 randomly selected dioxygenases capable of hydroxylating unactivated aromatic nuclei indicated that the enzymes for catabolism of PAHs, such as naphthalene and phenanthrene, might exist redundantly in strain KK1. Northern hybridization for cells grown on naphthalene or phenanthrene, using the putative naphthalene or phenanthrene dioxygenase gene fragment as a probe, suggested that the enzyme for naphthalene catabolism might share some homology in deduced amino acid sequences with phenanthrene dioxygenases. Also, it was found that three lipids (17:0 cyclo, 18:1 omega7c, 19:0 cyclo) increased in response to both naphthalene and phenanthrene, while the shift of other lipids varied from substrate to substrate.     

Bacterial metabolism of hydroxylated biphenyls.

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Bacterial metabolism of hydroxylated biphenyls

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Biodegradation of Phenanthrene by a Bacillus Species

A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp. The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates. The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 1 December 1999 / Accepted: 5 January 2000     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Preliminary study on relationships among strains forming a bacterial community selected on naphthalene from a marine sediment

Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source. The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains. The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains. Regulation of naphthalene metabolism was studied for the two primary strains. They oxidized naphthalene into catechol, which was degraded only by the meta pathway. For Pseudomonas Lav. 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive. For Moraxella Lav. 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible. The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size. In the bacterial community culture medium, Moraxella Lav. 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav. 4. The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.     

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis.

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.     

Degradation of fluoranthene, pyrene, benz[a]anthracene and dibenz[a,h]anthracene by Burkholderia cepacia

Microbiological analysis of soils from a polycyclic aromatic hydrocarbon (PAH)-contaminated site resulted in the enrichment of five microbial communities capable of utilizing pyrene as a sole carbon and energy source. Communities 4 and 5 rapidly degraded a number of different PAH compounds. Three pure cultures were isolated from community 5 using a spray plate method with pyrene as the sole carbon source. The cultures were identified as strains of Burkholderia ( Pseudomonas ) cepacia on the basis of biochemical and growth tests. The pure cultures (VUN 10 001, VUN 10 002 and VUN 10 003) were capable of degrading fluorene, phenanthrene and pyrene (100 mg l⁻¹) to undetectable levels within 7–10 d in standard serum bottle cultures. Pyrene degradation was observed at concentrations up to 1000 mg l⁻¹. The three isolates were also able to degrade other PAHs including fluoranthene, benz[ a ]anthracene and dibenz[ a , h ]anthracene as sole carbon and energy sources. Stimulation of dibenz[ a , h ]anthracene and benzo[ a ]pyrene degradation was achieved by the addition of small quantities of phenanthrene to cultures containing these compounds. Substrate utilization tests revealed that these micro-organisms could also grow on n -alkanes, chlorinated- and nitro-aromatic compounds.     

Degradation of polycyclic aromatic hydrocarbons by a newly isolated dibenzofuran-utilizing<Emphasis Type="Italic"> Janibacter</Emphasis> sp. strain YY-1

The dibenzofuran (DF)-utilizing bacterium strain YY-1 was newly isolated from soil. The isolate was identified as Janibacter sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various physiological characteristics. In addition to DF, strain YY-1 could grow on fluorene and dibenzothiophene as sole sources of carbon and energy. It was also able to cometabolize a variety of polycyclic aromatic hydrocarbons including dibenzo-p-dioxin, phenanthrene, and anthracene. The major metabolites formed from DF, biphenyl, dibenzothiophene, and naphthalene were identified by using gas chromatography-mass spectrometry as 2,3,2-trihydroxybiphenyl, biphenyl-dihydrodiol, dibenzothiophene 5-oxide, and coumarin, respectively. These results indicate that strain YY-1 can catalyze angular dioxygenation, lateral dioxygenation, and sulfoxidation.     

Biodegradation of crude oil and pure hydrocarbons by extreme halophilic archaea from hypersaline coasts of the Arabian Gulf

Two extreme halophilic Haloferax strains and one strain each of Halobacterium and Halococcus were isolated from a hypersaline coastal area of the Arabian Gulf on a mineral salt medium with crude oil vapor as a sole source of carbon and energy. These archaea needed at least 1 M NaCl for growth in culture, and grew best in the presence of 4 M NaCl or more. Optimum growth temperatures lied between 40 and 45oC. The four archaea were resistant to the antibiotics chloramphenicol, cycloheximide, nalidixic acid, penicillin, streptomycin and tetracycline. The strains could grow on a wide scope of aliphatic and aromatic (both mono-and polynuclear) hydrocarbons, as sole sources of carbon and energy. Quantitative measurements revealed that these extreme halophilic prokaryotes could biodegrade crude oil (13–47%, depending on the strain and medium salinity), n-octadecane (28–67%) and phenanthrene (13–30%) in culture after 3 weeks of incubation. The rates of biodegradation by all strains were enhanced with increasing NaCl concentration in the medium. Optimal concentration was 3 M NaCl, but even with 4 M NaCl the hydrocarbon-biodegradation rates were higher than with 1 and 2 M NaCl. It was concluded that these archaea could contribute to self-cleaning and bioremediation of oil-polluted hypersaline environments.     

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.     

Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulf coasts

Two halophilic, hydrocarbonoclastics bacteria, Marinobacter sedimentarum and M. flavimaris, with diazotrophic potential occured in hypersaline waters and soils in southern and northern coasts of Kuwait. Their numbers were in the magnitude of 10³ colony forming units g^?1. The ambient salinity in the hypersaline environments was between 3.2 and 3.5 M NaCl. The partial 16S rRNA gene sequences of the two strains showed, respectively, 99 and 100 % similarities to the sequences in the GenBank. The two strains failed to grow in the absence of NaCl, exhibited best growth and hydrocarbon biodegradation in the presence of 1 to 1.5 M NaCl, and still grew and maintained their hydrocarbonoclastic activity at salinities up to 5 M NaCl. Both species utilized Tween 80, a wide range of individual aliphatic hydrocarbons (C₉–C₄₀) and the aromatics benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole sources of carbon and energy. Experimental evidence was provided for their nitrogen-fixation potential. The two halophilic Marinobacter strains successfully mineralized crude oil in nutrient media as well as in hypersaline soil and water microcosms without the use of any nitrogen fertilizers.     

Isolation and Characterization of a Mycobacterium Species Capable of Degrading Three- and Four-Ring Aromatic and Aliphatic Hydrocarbons

Mycobacterium sp. strain CH1 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated freshwater sediments and identified by analysis of 16S rDNA sequences. Strain CH1 was capable of mineralizing three- and four-ring PAHs including phenanthrene, pyrene, and fluoranthene. In addition, strain CH1 could utilize phenanthrene or pyrene as a sole carbon and energy source. A lag phase of at least 3 days was observed during pyrene mineralization. This lag phase decreased to less than 1 day when strain CH1 was grown in the presence of phenanthrene or fluoranthene. Strain CH1 also was capable of using a wide range of alkanes as sole carbon and energy sources. No DNA hybridization was detected with the nahAc gene probe, indicating that enzymes involved in PAH metabolism are not related to the well-characterized naphthalene dioxygenase gene. DNA hybridization was not detected when the alkB gene from Pseudomonas oleovorans was used under high-stringency conditions. However, there was slight but detectable hybridization under low-stringency conditions. This suggests a distant relationship between genes involved in alkane oxidation.