Intrinsic bioremediability of an aromatic hydrocarbon-polluted groundwater: diversity of bacterial population and toluene monoxygenase genes

The functional and phylogenetic biodiversity of bacterial communities in a benzene, toluene, ethylbenzene and xylene (BTEX)-polluted groundwater was analysed. To evaluate the feasibility of using an air sparging treatment to enhance bacterial degradative capabilities, the presence of degrading microorganisms was monitored. The amplification of gene fragments corresponding to toluene monooxygenase (tmo), catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and toluene dioxygenase genes in DNA extracted directly from the groundwater samples was associated with the presence of indigenous degrading bacteria. Five months of air injection reduced species diversity in the cultivable community (as calculated by the Shannon-Weaver index), while little change was noted in the degree of biodiversity in the total bacterial community, as characterised by denaturing gradient gel electrophoresis (DGGE) analysis. BTEX-degrading strains belonged to the genera Pseudomonas, Microbacterium, Azoarcus, Mycobacterium and Bradyrhizobium. The degrading capacities of three strains in batch liquid cultures were also studied. In some of these microorganisms different pathways for toluene degradation seemed to operate simultaneously. Pseudomonas strains of the P24 operational taxonomic unit, able to grow only on catechol and not on BTEX, were the most abundant, and were present in the groundwater community at all stages of treatment, as evidenced both by cultivation approaches and by DGGE profiles. The presence of different tmo-like genes in phylogenetically distant strains of Pseudomonas, Mycobacterium and Bradyrhizobium suggested recent horizontal gene transfer in the groundwater.     

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site

Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.     

Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries

Developments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures and to monitor their dynamics under in situ conditions. However, there has been a distinct lack of emphasis on monitoring the functional diversity in the environment. Genes encoding catechol 2,3-dioxygenases (C23O), as key enzymes of various aerobic aromatic degradation pathways, were used as functional targets to assess the catabolic gene diversity in differentially BTEX contaminated environments by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs, and results closely matched with the information retrieved from random sequencing of PCR-DNA clone libraries. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish preselection or enrichment bias and to assess the function of predominant gene polymorphs. C23O abundance in isolates correlated with the levels of BTEX pollution in the soil samples analysed. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralizing phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralize benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. The PCR-SSCP technique is thus a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the functioning of microbial communities.     

Applicability of the functional gene catechol 1,2-dioxygenase as a biomarker in the detection of BTEX-degrading Rhodococcus species

Aims: Catechol 1,2-dioxygenase is a key enzyme in the degradation of monoaromatic pollutants. The detection of this gene is in focus today but recently designed degenerate primers are not always suitable. Rhodococcus species are important members of the bacterial community involved in the degradation of aromatic contaminants and their specific detection could help assess functions and activities in the contaminated environments. To reach this aim, specific PCR primer sets were designed for the detection of Rhodococcus related catechol 1,2-dioxygenase genes. Methods and Results: Primers were tested with genetically well-characterized strains isolated in this study and community DNA samples were used as template for Rhodococcus specific PCR as well. The sequences of the catabolic gene in question were subjected to multiple alignment and a phylogenetic tree was created and compared to a 16S rRNA gene based Rhodococcus tree. A strong coherence was observed between the phylogenetic trees. Conclusions: The results strongly support the opinion that there was no recent lateral gene transfer among Rhodococcus species in the case of catechol 1,2-dioxygenase. Significance and Impact of the Study: In gasoline contaminated environments, aromatic hydrocarbon degrading Rhodococcus populations can be identified based upon the detection and sequence analysis of catechol 1,2-dioxygenase gene.     

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.     

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.     

Biodegradation of Aliphatic and Aromatic Hydrocarbons by Nocardia sp. H17-1

We investigated the biodegradation of hydrocarbon components by Nocardia sp. H17-1 and the catabolic genes involved in the degradation pathways of both aliphatic and aromatic hydrocarbons. After 6 days of incubation, the aliphatic and aromatic fractions separated from Arabian light oil were degraded 99.0 ± 0.1% and 23.8 ± 0.8%, respectively. Detection of the catabolic genes involved in the hydrocarbon degradation indicated that H17-1 possessed the alkB genes for n-alkane biodegradation and catA gene for catechol 1,2-dioxygenase. However, H17-1 had neither the C23O gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity. The investigation of the genes involved in the biodegradation of hydrocarbons supported the low degradation activity of H17-1 on the aromatic fractions.     

PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.     

Proteogenomic Elucidation of the Initial Steps in the Benzene Degradation Pathway of a Novel Halophile,Arhodomonas sp. Strain Rozel,Isolated from a Hypersaline Environment

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

High benzene concentrations can favour Gram-positive bacteria in groundwaters from a contaminated aquifer

Exposure to pollution exerts strong selective pressure on microbial communities, which may affect their potential to adapt to current or future environmental challenges. In this microcosm study, we used DNA fingerprinting based on 16S rRNA genes to document the impact of high concentrations of benzene on two bacterial communities from a benzene-contaminated aquifer situated below a petrochemical plant (SIReN, UK). The two groundwaters harboured distinct aerobic benzene-degrading communities able to metabolize benzene to below detection levels (1 mug L(-1)). A benzene concentration of 100 mg L(-1) caused a major shift from Betaproteobacteria to Actinobacteria, in particular Arthrobacter spp. A similar shift from Betaproteobacteria to Arthrobacter spp. and Rhodococcus erythropolis was observed in minimal medium (MM) inoculated with a third groundwater. These Gram-positive-dominated communities were able to grow on benzene at concentrations up to 600 mg L(-1) in groundwater and up to 1000 mg L(-1) in MM, concentrations that cause significant solvent stress to cellular systems. Therefore, Gram-positive bacteria were better competitors than Gram-negative organisms under experimental conditions of high benzene loads, which suggests that solvent-tolerant Gram-positive bacteria can play a role in the natural attenuation of benzene or the remediation of contaminated sites.     

Determination of chrysene degradation under saline conditions by Fusarium sp. F092, a fungus screened from nature

Sixty-two rotted wood and soil samples were used to screen for chrysene-degrading fungi. A strain of Fusarium, named F092, was identified as most capable of degrading chrysene. F092 was active under saline and nonsaline conditions, breaking down 48% of the chrysene in 30 d. The percentage of chrysene degraded did not change at 35‰ salinity with pH 8.2 in solid and liquid cultures. The degradation under saline conditions increased about 0.6- and 2.1-fold in cultures with polypeptone and Tween80, and 0.03-fold in agitated cultures. F092 secreted nonligninolytic enzymes named 1,2-dioxygenase and 2,3-dioxygenase. The level of 1,2-dioxygenase activity reached 203.5 U L(-1) at 30 d and that of 2,3-dioxygenase activity, 29.7 U L(-1) at 40 d. The degradation pathway was clarified from the intermediates produced; chrysene 1,2-oxide, chrysene trans-1,2-dihydrodiol, 1-hydroxy 2-naphtoic acid, and catechol. F092 is a potential degrader of chrysene for bioremediation.     

邻苯二酚2,3-双加氧酶的结构和功能研究进展

邻苯二酚是所有芳香族化合物降解过程中的重要的中间产物,其降解有邻位和间位裂解两条裂解途径,分别由邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)催化裂解。本综述简要介绍了邻苯二酚2,3-双加氧酶的结构和功能的研究进展。     

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10² to 10³ gene copies, which was lowered to 10⁰ to 10¹ gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.     

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

Microbial community analysis at crude oil‐contaminated soils targeting the 16S ribosomal RNA,xylM, C23O,and bcr genes

Aims: The analyses targeting multiple functional genes were performed on the samples of crude oil‐contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Methods and Results: Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3‐dioxygenase (C23O), and benzoyl‐CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. Conclusions: The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. Significance and Impact of the Study: The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil‐contaminated environments.