Aromatic Hydrocarbon-degrading Bacteria Research Articles

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions.

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg(-1). Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0% in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.

ISOLATION AND DISTRIBUTION OF CRUDE OIL AND POLYCYCLIC AROMATIC HYDROCARBON-DEGRADING BACTERIA FROM POLLUTED HARBOURS IN NORTH JAKARTA

Several harbours in North Jakarta have been polluted by spills of oil and their derivates. We suggest that diverse species of crude oil and polycyclic aromatic hydrocarbon-degrading bacteria  inhabit these harbours. An experiment was undertaken in 2007 to isolate crude oil and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria from oil-polluted harbours, such as Muara Baru, Sunda Kelapa and Tanjung Priok. Sea water and sediment samples were collected twice, in March and April. Crude oil and PAH-degrading bacteria were isolated from enrichment culture of samples in an enrichment medium (SWP), using ONR7a medium with the addition of 5 types of PAH gases or Arabian Light Crude Oil 210 (ALCO 210) onto medium. This study reported that fluoranthene and crude oil-degrading bacteria were the major bacteria isolated from the three polluted harbours. In total, 109 isolates have been collected which can degrade crude oil (29% of total isolates), fluoranthene (33%), fluorene (20%), pyrene (7%), dibenzothiopene (6%), and phenantrene (5 %). Among these isolates, 5 isolates have the capability to degrade 5 types of PAH and ALCO 210. They were Alcanivorax sp. B-1084, Pseudomonas sp. D5-38b, Alcanivorax sp. TE-9, Bacillus sp. L41, Alcanivorax dieselolei strain B-5 clone 1. Culturable bacteria have been isolated mostly from the Sunda Kelapa samples, with fewer in those from Muara Baru and Tanjung Priok, respectively

Sphingobium barthaii sp. nov., a high molecular weight polycyclic aromatic hydrocarbon-degrading bacterium isolated from cattle pasture soil.

A Gram-stain-negative, yellow, rod-shaped bacterium, designated strain KK22(T), was isolated from a microbial consortium that grew on diesel fuel originally recovered from cattle pasture soil. Strain KK22(T) has been studied for its ability to biotransform high molecular weight polycyclic aromatic hydrocarbons. On the basis of 16S rRNA gene sequence phylogeny, strain KK22(T) was affiliated with the genus Sphingobium in the phylum Proteobacteria and was most closely related to Sphingobium fuliginis TKP(T) (99.8%) and less closely related to Sphingobium quisquiliarum P25(T) (97.5%). Results of DNA-DNA hybridization (DDH) revealed relatedness values between strain KK22(T) and strain TKP(T) and between strain KK22(T) and strain P25(T) of 21 ± 4% (reciprocal hybridization, 27 ± 2%) and 15 ± 2% (reciprocal hybridization, 17 ± 1%), respectively. Chemotaxonomic analyses of strain KK22(T) showed that the major respiratory quinone was ubiquinone Q-10, that the polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidyl-N-methylethylethanolamine and sphingoglycolipid, and that C18 : 1ω7c and C14 : 0 2-OH were the main fatty acid and hydroxylated fatty acids, respectively. This strain was unable to reduce nitrate and the genomic DNA G+C content was 64.7 mol%. Based upon the results of the DDH analyses, the fact that strain KK22(T) was motile, and its biochemical and physiological characteristics, strain KK22(T) could be separated from recognized species of the genus Sphingobium. We conclude that strain KK22(T) represents a novel species of this genus for which the name Sphingobium barthaii sp. nov. is proposed; the type strain is KK22(T) ( = DSM 29313(T) = JCM 30309(T)).

Complete Genome Sequence of a Novel Bacterium within the Family Rhodocyclaceae That Degrades Polycyclic Aromatic Hydrocarbons.

A polycyclic aromatic hydrocarbon-degrading bacterium designated strain Ca6, a member of the family Rhodocyclaceae and a representative of the uncharacterized pyrene group 1 (PG1), was isolated and its genome sequenced. The presence of several genes suspected to be associated with PG1 was confirmed, and additional genes for aromatic compound metabolism were detected.

Seasonal assemblages and short-lived blooms in coastal north-west Atlantic Ocean bacterioplankton.

Temperate oceans are inhabited by diverse and temporally dynamic bacterioplankton communities. However, the role of the environment, resources and phytoplankton dynamics in shaping marine bacterioplankton communities at different time scales remains poorly constrained. Here, we combined time series observations (time scales of weeks to years) with molecular analysis of formalin-fixed samples from a coastal inlet of the north-west Atlantic Ocean to show that a combination of temperature, nitrate, small phytoplankton and Synechococcus abundances are best predictors for annual bacterioplankton community variability, explaining 38% of the variation. Using Bayesian mixed modelling, we identified assemblages of co-occurring bacteria associated with different seasonal periods, including the spring bloom (e.g. Polaribacter, Ulvibacter, Alteromonadales and ARCTIC96B-16) and the autumn bloom (e.g. OM42, OM25, OM38 and Arctic96A-1 clades of Alphaproteobacteria, and SAR86, OM60 and SAR92 clades of Gammaproteobacteria). Community variability over spring bloom development was best explained by silicate (32%)--an indication of rapid succession of bacterial taxa in response to diatom biomass--while nanophytoplankton as well as picophytoplankton abundance explained community variability (16-27%) over the transition into and out of the autumn bloom. Moreover, the seasonal structure was punctuated with short-lived blooms of rare bacteria including the KSA-1 clade of Sphingobacteria related to aromatic hydrocarbon-degrading bacteria.

Draft Genome Sequence of Sphingomonas sp. Strain Ant20, Isolated from Oil-Contaminated Soil on Ross Island, Antarctica.

Here, we present the draft genome of Sphingomonas sp. strain Ant20, isolated from oil-polluted soil near Scott Base, Ross Island, Antarctica. The genome of this aromatic hydrocarbon-degrading bacterium provides valuable information on the microbially mediated biodegradation of aromatic compounds in cold-climate systems.

Draft Genome Sequence of the Aromatic Hydrocarbon-Degrading Bacterium Sphingobium sp. Strain Ant17, Isolated from Antarctic Soil

Here, we present the draft genome sequence of Sphingobium sp. strain Ant17, an aromatic hydrocarbon-degrading bacterium that was isolated from Antarctic oil-contaminated soil. An analysis of this genome can lead to insights into the mechanisms of xenobiotic degradation processes at low temperatures and potentially aid in bioremediation applications.

Identification of opsA , a Gene Involved in Solute Stress Mitigation and Survival in Soil, in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.

Draft Genome Sequence of Sphingobium sp. Strain KK22, a High-Molecular-Weight Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Isolated from Cattle Pasture Soil.

Sphingobium sp. strain KK22 was isolated from a bacterial consortium that originated from cattle pasture soil from Texas. Strain KK22 grows on phenanthrene and has been shown to biotransform the high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. The genome of strain KK22 was sequenced to investigate the genes involved in aromatic pollutant biotransformation.

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Stable-isotope probing was previously used to identify bacterial anthracene-degraders in untreated soil from a former manufactured gas plant site. However, subsequent pyrosequence analyses of total bacterial communities and quantification of 16S rRNA genes indicated that relative abundances of the predominant anthracene-degrading bacteria (designated Anthracene Group 1) diminished as a result of biological treatment conditions in lab-scale, aerobic bioreactors. This study identified Alphaproteobacterial anthracene-degrading bacteria in bioreactor-treated soil which were dissimilar to those previously identified. The largest group of sequences was from the Alterythrobacter genus while other groups of sequences were associated with bacteria within the order Rhizobiales and the genus Bradyrhizobium. Conditions in the bioreactor enriched for organisms capable of degrading anthracene which were not the same as those identified as dominant degraders in the untreated soil. Further, these data suggest that identification of polycyclic aromatic hydrocarbon-degrading bacteria in contaminated but untreated soil may be a poor indicator of the most active degraders during biological treatment.

Experimental Study of the Biochemistry Method for Enhancing Oil Recovery in the Oil Reservoir after Polymer Flooding

A delayed cross linked gel profile control agent is used to plug high permeable formations. Also, well nutrient fluid and microbes are injected with 50% of the heavy residual oil, after polymer flooding, in order to improve oil recovery due to the complex environment of oil reservoirs. Four strains of polycyclic aromatic hydrocarbon-degrading bacteria are selected from oilfield produced water with a high efficiency. Two of the four strains, namely BISYX17 and BISYX14, are new. Polycyclic aromatic hydrocarbon-degrading bacteria have high growth activity and they are able to reach a maximum stain concentration after being cultured 4 to 8 days, using phenanthrene as their sole carbon source. They are able to effectively degrade heavy hydrocarbon with a phenanthrene degradation rate of up to 80％, after the sample is cultured for seven days. Strain BISYX7 has the strongest phenanthrene -degrading ability, with a maximum degradation percentage of 89.89％. The strains are capable of producing dioxygenase to open rings of polycyclic aromatic hydrocarbon. The dioxygenase activity, produced by BISYX17, is able to reach 40.2 IU/mg, which is higher than the enzyme activities of a wild strain. This shows the strain has excellent potential to produce enzymes. Enzymes, produced by metabolism, have a direct degradation rate of 68% on crude oil. A core displacement simulation experiment indicates a profile control oil-displacing system is able to improve crude oil recovery efficiency by 17%, after polymer flooding. Thus, the system has excellent application potential for residual oil recovery.

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.

Polycyclic Aromatic Hydrocarbon-degrading Bacteria from Aviation Fuel Spill Site at Ibeno, Nigeria

Polycyclic aromatic hydrocarbon (PAH)–degrading bacteria were isolated from aviation fuel contaminated soil at Inua Eyet Ikot in Ibeno, Nigeria. PAH-degrading bacteria in the contaminated soil were isolated by enrichment culture technique. Isolates with high PAH degrading potential characterized by their extensive growth on PAH-supplemented minimal salt medium were screened for their naphthalene, phenanthrene and chrysene degradability. The screening medium which contained selected PAHs as the sole source of carbon and energy showed that Micrococcus varians AFS-2, Pseudomonas putida AFS-3 and Alcaligenes faecalis AFS-5 exhibited a concentration–dependent growth in all the PAH–compounds tested. There were visible changes in the color of growth medium suggesting the production of different metabolites. Their acclimation to different PAH substrates was also evident as A. faecalis AFS-5 isolated from chrysene grew well on other less complex aromatic compounds. The isolate exhibited best growth (0.44 OD600) when exposed to 10 ppm of chrysene for 5 days and could utilize up to 90 ppm of chrysene. This isolate and others with strong PAH-degrading potentials are recommended for bioremediation of PAHs in aviation fuel-contaminated sites in the tropics.

Identifying polycyclic aromatic hydrocarbon-degrading bacteria in oil-contaminated surface waters at Deepwater Horizon by cultivation, stable isotope probing and pyrosequencing

Polycyclic aromatic hydrocarbons (PAHs) are an important class of chemical pollutants that constitute a major component of total hydrocarbons in crude oils. Based on their poor water solubility, toxicity, persistence and potential to bioaccumulate, these compounds are recognized as high-priority pollutants in the environment and are of significant concern for human health. At oil-contaminated sites, PAH-degrading bacteria perform a critical role in the degradation and ultimate removal of these compounds. In April 2010, enormous quantities of PAHs entered the Gulf of Mexico from the thousands of tons of oil that were released from the ill-fated drilling rig Deepwater Horizon. In the ensuing months after the spill, intense research efforts were devoted to characterizing the microorganisms responsible for degrading the oil, particularly in deep waters where a large oil plume, enriched with aliphatic and low molecular-weight aromatic hydrocarbons, was found in the range of 1,000–1,300 m. PAHs, however, were found mainly confined to surface waters. This paper discusses efforts utilizing DNA-based stable isotope probing, cultivation-based techniques and metagenomics to characterize the bacterial guild associated with PAH degradation in oil-contaminated surface waters at Deepwater Horizon.

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.

Nitratireductor pacificus sp. nov., isolated from a pyrene-degrading consortium

Strain pht-3B(T) was isolated from a pyrene-degrading consortium of an enriched sediment from the Pacific Ocean, collected during the screening of polycyclic aromatic hydrocarbon-degrading bacteria. Cells were Gram-negative, short rods that were motile by means of flagella. Growth was observed at 0-7 % NaCl and 10-41 °C. The isolate was able to reduce nitrate to nitrite, but not to nitrogen. 16S rRNA gene sequence comparisons showed that strain pht-3B(T) was most closely related to Nitratireductor aquibiodomus NL21(T) (97.3 % 16S rRNA gene sequence similarity), N. indicus C115(T) (97.1 %), N. basaltis J3(T) (96.8 %) and N. kimnyeongensis KY 101(T) (96.7 %). DNA-DNA hybridization between strain pht-3B(T) and these reference strains revealed 55, 54, 28 and 42 % DNA-DNA relatedness, respectively. The dominant fatty acids were C(19 : 0)ω8c cyclo (22.6 %) and summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c; 60.4 %). The G+C content of the chromosomal DNA was 63 mol%. These characteristics were in good agreement with those of members of the genus Nitratireductor. According to cell morphology, physiology, fatty acid composition, 16S rRNA gene sequence analysis and DNA-DNA relatedness, the isolate belonged to the genus Nitratireductor but could be readily distinguished from recognized species of the genus. Therefore a novel species is proposed to accommodate strain pht-3B(T), for which the name Nitratireductor pacificus sp. nov. is proposed. The type strain is pht-3B(T) ( = CCTCC AB 209302(T) = LMG 25541(T) = MCCC 1A01024(T)).

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na +, 2Na + and K +), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.

Sphingobium scionense sp. nov., an aromatic hydrocarbon-degrading bacterium isolated from contaminated sawmill soil

This study characterized strain WP01(T), a Gram-staining-negative, rod-shaped, aerobic bacterium isolated from a polycyclic aromatic hydrocarbon-contaminated soil in New Zealand. Strain WP01(T) shared many characteristics of the genus Sphingobium: the predominant respiratory quinone (89 %) was ubiquinone with ten isoprene units (Q-10); the major fatty acids were C(18 : 1)omega7c, C(16 : 1)omega7c, C(16 : 0) and C(14 : 0) 2-OH; spermidine was the major polyamine; the DNA G+C content was 63.8 mol%; and the Sphingobium-specific 16S rRNA signatures were conserved. A point of difference from other species of the genus Sphingobium was that strain WP01(T) reduced nitrate to nitrite. The polar lipid pattern consisted of the predominant compounds diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipids. 16S rRNA gene sequence analysis showed that, amongst the recognized species of the genus Sphingobium, strain WP01(T) was most similar to Sphingobium yanoikuyae GIFU 9882(T) and Sphingobium amiense YT(T) (>97 % 16S rRNA gene sequence similarities). The low DNA-DNA relatedness values between strain WP01(T) and S. yanoikuyae GIFU 9882(T) (46.6 %) and S. amiense DSM 16289(T) (25.6 %) indicated no relatedness at the species level. On the basis of these characteristics, it is concluded that strain WP01(T) should be considered as representing a novel species within the genus Sphingobium, for which the name Sphingobium scionense sp. nov. is proposed. The type strain is WP01(T) (=DSM 19371(T)=ICMP 13533(T)).

Novosphingobium indicum sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from a deep-sea environment

A novel polycyclic aromatic hydrocarbon (PAH)-degrading bacterium, strain H25T, which was isolated from deep-sea water of the Indian Ocean, was studied phenotypically, genotypically and phylogenetically. Strain H25T can utilize several PAHs including phenanthrene and fluoranthene as sole carbon sources. The 16S rRNA gene sequence of strain H25T showed the highest similarity with that of Novosphingobium naphthalenivorans TUT562T (96.3%), and showed lower similarities (92.1-96.0%) with other members of the genus Novosphingobium. The major fatty acids of strain H25T were C14:0 2-OH (3.2%), C16:0 (13.6%), C16:1omega7c (5.2%), C18:0 (13.4%) and C18:1omega7c (57.0%), which accounted for 92.3% of the total fatty acids. It had ubiquinone 10 as the major respiratory quinone and spermidine as the major polyamine. All these characteristics were consistent with those of recognized Novosphingobium species. Results of DNA-DNA hybridization experiments and BOX-PCR fingerprint comparisons also indicate that strain H25T represents a novel Novosphingobium species, for which the name Novosphingobium indicum sp. nov. is proposed. The type strain is H25T (=MCCC 1A01080T=CGMCC 1.6784T=LMG 24713T).

Microbial consortia in mesocosm bioremediation trial using oil sorbents, slow-release fertilizer and bioaugmentation

An experimental prototype oil boom including oil sorbents, slow-release fertilizers and biomass of the marine oil-degrading bacterium, Alcanivorax borkumensis, was applied for sorption and degradation of heavy fuel oil in a 500-L mesocosm experiment. Fingerprinting of DNA and small subunit rRNA samples for microbial activity conducted to study the changes in microbial communities of both the water body and on the oil sorbent surface showed the prevalence of A. borkumensis on the surface of the oil sorbent. Growth of this obligate oil-degrading bacterium on immobilized oil coincided with a 30-fold increase in total respiration. A number of DNA and RNA signatures of aromatic hydrocarbon-degrading bacteria were detected both in samples of water body and on oil sorbent. Ultimately, the heavy fuel oil in this mesocosm study was effectively removed from the water body. This is the first study to successfully investigate the fate of oil-degrading microbial consortia in an experimental prototype for a bioremediation strategy in offshore, coastal or ship-bound oil spill mitigation using a combination of mechanical and biotechnological techniques.