Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE (3.3 μM), 1,1-DCE (1.25 μM), and chloroform (6.3 μM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min · mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl⁻ atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.     

Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by "Dehalococcoides ethenogenes" 195.

"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response

Pure cultures of ammonia-oxidizing bacteria, Nitrosomonas europaea, were exposed to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), chloroform (CF), 1,2-dichloroethane (1,2-DCA), or carbon tetrachloride (CT), in the presence of ammonia, in a quasi-steady-state bioreactor. Estimates of enzyme kinetics constants, solvent inactivation constants, and culture recovery constants were obtained by simultaneously fitting three model curves to experimental data using nonlinear optimization techniques and an enzyme kinetics model, referred to as the inhibition, inactivation, and recovery (IIR) model, that accounts for inhibition of ammonia oxidation by the solvent, enzyme inactivation by solvent product toxicity, and respondent synthesis of new enzyme (recovery). Results showed relative enzyme affinities for ammonia monooxygenase (AMO) of 1,1-DCE approximately TCE > CT > NH(3) > CF > 1,2-DCA. Relative maximum specific substrate transformation rates were NH(3) > 1,2-DCA > CF > TCE approximately 1,1-DCE > CT (=0). The TCE, CF, and 1,1-DCE inactivated the cells, with 1,1-DCE being about three times more potent than TCE or CF. Under the conditions of these experiments, inactivating injuries caused by TCE and 1,1-DCE appeared limited primarily to the AMO enzyme, but injuries caused by CF appeared to be more generalized. The CT was not oxidized by N. europaea while 1,2-DCA was oxidized quite readily and showed no inactivation effects. Recovery capabilities were demonstrated with all solvents except CF. A method for estimating protein yield, the relationship between the transformation capacity model and the IIR model, and a condition necessary for sustainable cometabolic treatment of inactivating substrates are presented. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 520-534, 1997.     

Reductive Dechlorination of Chlorinated Ethenes and 1,2-Dichloroethane by “Dehalococcoides ethenogenes” 195

“Dehalococcoides ethenogenes” 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity. Received: 17 June 1999 / Accepted: 3 September 1999     

Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1

A recombinant strain of Escherichia coli (JM109/pBZ1260) expressing constitutively toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 degraded binary mixtures (100 microM each) of tetrachloroethylene (PCE) with either trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), or vinyl chloride (VC). PCE degradation was 8-20% for these binary mixtures, while TCE and trans-DCE with PCE were degraded at 19%, 1,1-DCE at 37%, cis-DCE at 97%, and VC at 27%. The host P. stutzeri OXI was also found to degrade binary mixtures of PCE/TCE, PCE/cis-DCE, and PCE/VC when induced with toluene. Degradation of quaternary mixtures of PCE/TCE/trans-DCE/VC and PCE/TCE/cis-DCE/VC by JM109/pBZ1260 were also investigated as well as mixtures of PCE/TCE/trans-DCE/1,1-DCE/cis-DCE/VC; when all the chlorinated compounds were present, the best degradation occurred with 24-51% removal of each. For these degradation reactions, 39-85% of the stoichiometric chloride expected from complete degradation of the chlorinated ethenes was detected. The time course of PCE/TCE/1,1-DCE degradation was also measured for a mixture of 8, 17, and 6 microM, respectively; initial degradation rates were 0.015, 0.023. and 0.029 nmol/min x mg protein, respectively. This indicates that for the first time an aerobic enzyme can degrade mixtures of all chlorinated ethenes, including the once--so it was believed-completely recalcitrant PCE.     

Microbial community structure and trichloroethylene degradation in groundwater

Trichloroethylene (TCE) is a prevalent contaminant of groundwater that can be cometabolically degraded by indigenous microbes. Groundwater contaminated with TCE from a US Department of Energy site in Ohio was used to characterize the site-specific impact of phenol on the indigenous bacterial community for use as a possible remedial strategy. Incubations of 14C-TCE-spiked groundwater amended with phenol showed increased TCE mineralization compared with unamended groundwater. Community structure was determined using DNA directly extracted from groundwater samples. This DNA was then analyzed by amplified ribosomal DNA restriction analysis. Unique restriction fragment length polymorphisms defined operational taxonomic units that were sequenced to determine phylogeny. DNA sequence data indicated that known TCE-degrading bacteria including relatives of Variovorax and Burkholderia were present in site water. Diversity of the amplified microbial rDNA clone library was lower in phenol-amended communities than in unamended groundwater (i.e., having Shannon-Weaver diversity indices of 2.0 and 2.2, respectively). Microbial activity was higher in phenol-amended ground water as determined by measuring the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride. Thus phenol amendments to groundwater correlated with increased TCE mineralization, a decrease in diversity of the amplified microbial rDNA clone library, and increased microbial activity.     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Microbial Population Changes during Bioremediation of an Experimental Oil Spill

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the α-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. α-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.     

Microbial population changes during bioremediation of an experimental oil spill.

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the alpha-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. alpha-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.     

Enhanced biotransformation of TCE using plant terpenoids in contaminated groundwater

Aims:  To examine plant terpenoids as inducers of TCE (trichloroethylene) biotransformation by an indigenous microbial community originating from a plume of TCE-contaminated groundwater.
Methods and Results:  One-litre microcosms of groundwater were spiked with 100 μmol 1⁻¹ of TCE and amended weekly for 16 weeks with 20 μl 1⁻¹ of the following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-(−) carvone, farnesol, cumene. Yeast extract-amended and unamended control treatments were also prepared. The addition of R-carvone and S-carvone, linalool and cumene resulted in the biotransformation of upwards of 88% of the TCE, significantly more than the unamendment control (61%). The aforementioned group of terpenes also significantly ( P  < 0·05) allowed more TCE to be degraded than the remaining two terpenes (farnesol and pulegone), and the yeast extract treatment which biotransformed 74–75% of the TCE. The microbial community profile was monitored by denaturing gradient gel electrophoresis and demonstrated much greater similarities between the microbial communities in terpene-amended treatments than in the yeast extract or unamended controls.
Conclusions:  TCE biotransformation can be significantly enhanced through the addition of selected plant terpenoids.
Significance and Impact of the Study:  Plant terpenoid and nutrient supplementation to groundwater might provide an environmentally benign means of enhancing the rate of in situ TCE bioremediation.     

Biological Reduction of Trichloroethene Supported by Fe(0)

An anaerobic culture reductively transformed trichloroethene (TCE) in an aqueous medium containing elemental iron as the sole electron source. The TCE disappearance rate was enhanced and the product distribution was markedly altered when the culture was present. In abiotic samples containing Fe(0) but no culture, 11 µmol TCE (equivalent to an aqueous concentration of 260 µM) disappeared over a period of 39 days, with ethene and ethane as the major reduction products. Small amounts of cis-dichloroethene (cis-DCE), 1,1-DCE, and vinyl chloride (VC) also were detected. When the culture was incubated with TCE and Fe(0), the same amount of TCE was transformed in less than 2 weeks. The major products after 39 days were VC, ethene, and ethane. VC accounted for 65% of the initial TCE and appeared to be reduced further to ethene at slow rates. The significant VC production in the culture-amended samples indicates that most TCE was transformed microbially rather than chemically. The data indicate that abiotic and biological reduction of chlorinated ethenes can be coupled to enhance treatment efficiency. The results also suggest that microbial dechlorination within and downgradient from iron walls is potentially important for evaluating the long-term performance of permeable iron barriers.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Phenol- and Toluene-Degrading Microbial Populations from an Aquifer in Which Successful Trichloroethene Cometabolism Occurred

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol- or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol- and toluene-degrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol- and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.     

Effect of Chemical Oxidation on Subsurface Microbiology and Trichloroethene (TCE) Biodegradation

Research was conducted to determine the effect of chemical oxidation on subsurface microbiology and cometabolic biodegradation capacity in a trichloroethene (TCE)/perchloroethene (PCE)-contaminated aquifer previously treated with Fenton's reagent. Groundwater pH declined from 5 to 2.4 immediately after the treatment, and subsequently rose to a range of 3.4 to 4.0 after 17 months. Limited microbial growth and TCE degradation were detected in the treated zone (pH 3.37 and TCE 5 to 21 mg/L) with carbon addition (i.e., methane and phenol). Methane addition resulted in the enrichment of yeast and fungi in microcosms at low pH. In contrast, methane addition to groundwater from the control well (pH 4.9 and TCE ca. 0.7 mg/L) stimulated methanotrophic growth, indicated by methane consumption, fluorescent antibody analysis, phospholipid-based markers, and rDNA probes. TCE degradation was measured in the control microcosms, but only when phenol was added. Although higher TCE concentrations in the treated zone might have inhibited TCE cometabolism, these results also indicate that low groundwater pH resulting from the chemical oxidation process (pH 3.37 versus 4.9) inhibited TCE degradation. Methanotrophic growth and TCE biodegradation may be possible as pH increases both in the treated zone and at the leading edge of plume, as long as the local soil is able to buffer the groundwater pH. Moreover, the Fenton's reagent process could be designed to operate at a higher pH (e.g., ≥ 4.5) and/or lower hydrogen peroxide concentration to minimize detrimental effects, providing an optimal environment to couple advanced oxidation processes with bioremediation technologies.     

Stable isotope probing in the metagenomics era: A bridge towards improved bioremediation

Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.     

Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.