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Multi-phase extraction (MPE) is commonly used at petroleum-contaminated sites to volatilize and recover hydrocarbons from the vadose and saturated zones in contaminant source areas. Although primarily a physical treatment technology, the induced subsurface air flow can potentially increase oxygen supply and promote aerobic biodegradation of benzene, toluene, ethylbenzene, and xylenes (BTEX), the contaminants of concern at gasoline-contaminated sites. In this study, real-time PCR enumeration of aromatic oxygenase genes and PCR-DGGE profiles were used to elucidate the impact of MPE operation on the aquifer microbial community structure and function at a gasoline-contaminated site. Prior to system activation, ring-hydroxylating toluene monooxygenase (RMO) and naphthalene dioxygenase (NAH) gene copies were on the order of 10(6) to 10(10)copies L(-1) in groundwater samples obtained from BTEX-impacted wells. Aromatic oxygenase genes were not detected in groundwater samples obtained during continuous MPE indicating decreased populations of BTEX-utilizing bacteria. During periods of pulsed MPE, total aromatic oxygenase gene copies were not significantly different than prior to system activation, however, shifts in aromatic catabolic genotypes were noted. The consistent detection of RMO, NAH, and phenol hydroxylase (PHE), which catabolizes further oxidation of hydroxylated BTEX metabolites indicated the potential for aerobic biodegradation of dissolved BTEX during pulsed MPE.

Subsurface injection of oxygen-releasing materials (ORMs) is frequently performed at petroleum-contaminated sites to stimulate aerobic bioremediation of benzene, toluene, ethylbenzene, and xylenes (BTEX). In this study, qPCR enumeration of aromatic oxygenase genes and PCR-DGGE profiles of bacterial 16S rRNA genes were combined with groundwater monitoring to determine the impact of ORM injection on BTEX bioremediation at a gasoline-contaminated site. Prior to injection, BTEX concentrations were greater than 3 mg/L and DO levels were typically lessthan 2 mg/L, butphenol hydroxylase (PHE) and ring-hydroxylating toluene monooxygenase (RMO) genes were detected in impacted wells indicating the potential for aerobic BTEX biodegradation. Following injection, DO increased, BTEX concentrations decreased substantially, and PHE and RMO genes copies increased by 1-3 orders of magnitude. In addition, naphthalene dioxygenase (NAH) and xylene monooxygenase (TOL) genes were intermittently detected during periods of increased DO. Following depletion of the ORM, DO decreased, BTEX concentrations rebounded, and oxygenase genes were no longer detected. Temporal changes in PCR-DGGE microbial community profiles reflected the dynamic changes in subsurface conditions. Overall, the combination of chemical and geochemical analyses with quantification of aromatic oxygenase genes demonstrated that injection stimulated BTEX biodegradation until the ORM was depleted.

Monitoring groundwater benzene, toluene, ethylbenzene, and xylene (BTEX) concentrations is the typical method to assess monitored natural attenuation (MNA) and bioremediation as corrective actions at gasoline-contaminated sites. Conclusive demonstration of bioremediation, however, relies on converging lines of chemical and biological evidence to support a decision. In this study, real-time PCR quantification of aromatic oxygenase genes was used to evaluate the feasibility of MNA at two gasoline-impacted sites. Phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) genes were routinely detected in BTEX-impacted wells. Aromatic oxygenase genes were not detected in sentinel wells outside the plume indicating that elevated levels of oxygenase genes corresponded to petroleum hydrocarbon contamination. Total aromatic oxygenase gene copy numbers detected in impacted wells were on the order of 10(6)-10(9)copies L(-1). PHE, RMO, NAH, TOD, and BPH4 gene copies positively correlated to total BTEX concentration. Mann-Kendall analysis of benzene concentrations was used to evaluate the status of the dissolved BTEX plume. The combination of trend analysis of contaminant concentrations with quantification of aromatic oxygenase genes was used to assess the feasibility of MNA as corrective measures at both sites.

The objective of this work was to test a molecular genetic method for in situ monitoring of aerobic benzene, toluene, and xylene (BTX) biodegrading microorganisms. Catechol 2,3-dioxygenase (C23DO) genes occur in bacteria that biodegrade benzene, toluene, xylenes, phenol, biphenyl, and naphthalene. A competitive quantitative polymerase chain reaction (QC-PCR) technique using a single set of primers specific for an entire subfamily of C23DO genes was recently developed. To determine whether bacteria containing these C23DO genes actually exist in environments contaminated by BTX, aerobic microcosms containing previously uncontaminated soil were amended with different aromatic hydrocarbons and DNA extracts were analyzed by QC-PCR for C23DO genes. Anaerobic microcosms were established to confirm that oxygen was also necessary for the enrichment of C23DO genes. Field testing was done at two sites undergoing monitored natural attenuation. In microcosm experiments naphthalene, m-xylene, and p-xylene strongly enriched for C23DO genes while benzene, toluene, and o-xylene produced only transient, weakly detectable genes. In the field study, C23DO genes were detected in groundwater samples contaminated with either xylenes or naphthalene. The results of this study demonstrated that molecular genetic techniques can provide an accurate and rapid method to detect microorganisms capable of aromatic hydrocarbon biodegradation. Such a technique would be useful for monitoring the effectiveness of aeration technologies and for documenting microbial processes for monitored natural attenuation.

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5 degrees C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 10(2) copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

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.

The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined. The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III. Expression studies were carried out with Alcaligenes sp. strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60. BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite. These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently. These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite. Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp. BR60. The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains. o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins. These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).

Horizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions. The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients. The phylogenetic distribution of cbaAB genes for chlorobenzoate 3,4-(4,5)-dioxygenase, which are carried within Tn5271 on the IncP beta plasmid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed. The latter included isolates from surface water, groundwater and bioremediation reactor samples. The cbaAB genes have become integrated, through interspecific transfer, primarily into species of the beta Proteobacteria (44/48 isolates). Only four isolates, identified as Pseudomonas fluorescens (3/48) and Xanthomonas maltophilia (1/48), belonged to the gamma Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of beta and gamma Proteobacteria in the laboratory. The natural host range correlated with the distribution of the meta-ring-fission pathway for metabolism of protocatechuates formed when the cbaAB genes were expressed (45/48 isolates). We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conserved meta ring-fission pathway. The hypothesis was tested by transferring a plasmid construct containing the cbaAB genes into type strains representative of the beta and gamma Proteobacteria. The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pathways is discussed.

The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated cbaAB, from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expression in vivo to show that the cbaA and cbaB genes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of the cbaAB genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features. This lineage includes phthalate 4,5-dioxygenase (pht23), and based on the available NH3-terminal sequence of component A, also includes 4-sulphobenzoate 3,4-dioxygenase. Vanillate demethylase, encoded by the vanAB genes and formally a monooxygenase enzyme catalysing an oxidative demethylation, is also included in this lineage. The terminal chlorobenzoate dioxygenase (CbaA) component is characterized by a conserved Rieske-type [2Fe-2S]R ligand centre. The reductase component (CbaB) contains a plant-type ferredoxin [2Fe-2S]Fd, FMN-isoalloxazine and NAD-ribose-binding domains and the orientation of these domains is conserved in all known class IA reductases. These results support the hypothesis that alternative fusions of the electron transfer modules of the reductases arose early in the divergence of oxygenase systems. The over-riding evolutionary constraint acting on the divergence of the class IA oxygenases would appear to be the requirement for a carboxyl group para to the site of oxygen insertion into the aromatic ring.

Growth on 3-chlorobenzoate was found to induce the enzymes of the protocatechuate meta ring fission pathway in Alcaligenes sp. strain BR60. The chlorobenzoate catabolic genes, designated cba, were localized to a 3.7-kb NotI-EcoRI fragment within the nonrepeated region of the composite transposon Tn5271. The cba genes were cloned onto two broad-host-range vectors and expressed in Escherichia coli and Alcaligenes sp. strain BR6024. In E. coli, expression of the cba genes with the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible tac promoter of the IncQ vector pMMB66HE resulted in the production of protocatechuate and chlorodihydroxybenzoate metabolites of 3-chlorobenzoate. Expression of this construct in one orientation resulted in the formation of two polypeptides 51 and 42 kDa in size. This result was confirmed by subcloning into pGEM3Zf and then incorporating L-35S-methionine into newly synthesized proteins, using the thermally regulated T7 polymerase-promoter system. Introduction of the NotI-EcoRI fragment into Alcaligenes sp. strain BR6024 (Cba-P), using the IncP broad-host-range, mobilizable plasmid pBW13, restored the 3-chlorobenzoate-degradative phenotype and resulted in the accumulation of protocatechuate and chlorodihydroxybenzoate intermediates. The data indicate that a two-component dioxygenase specified by Tn5271 oxidizes 3-chlorobenzoate at the 3,4- or 4,5-positions. This activity extends the range of pathways for chloroaromatic compounds known to be functional in the environment. The new pathway avoids the toxicity attributed to the accumulation of chlorocatechol metabolites in bacteria degrading chlorobenzoates.