Biodegradation of Trichloroethylene and Its Anaerobic Daughter Products in Freshwater Wetland Sediments

The wide range of redox conditions and diversity of microbial populations in organic-rich wetland sediments could enhance biodegradation of chlorinated solvents. To evaluate potential biodegradation rates of trichloroethylene (TCE) and its anaerobic daughter products (cis-1,2-dichloroethylene; trans-1,2-dichloroethylene; and vinyl chloride), laboratory microcosms were prepared under methanogenic, sulfate-reducing, and aerobic conditions using sediment and groundwater from a freshwater wetland that is a discharge area for a TCE contaminant plume. Under methanogenic conditions, biodegradation rates of TCE were extremely rapid at 0.30 to 0.37 d^-1 (half-life of about 2 days). Although the TCE biodegradation rate was slower under sulfate-reducing conditions (0.032 d^-1) than under methanogenic conditions, the rate was still two orders of magnitude higher than those reported in the literature for microcosms constructed with sandy aquifer sediments. In the aerobic microcosm experiments, biodegradation occurred only if methane consumption occurred, indicating that methanotrophs were involved. Comparison of laboratory-measured rates indicates that production of the 1,2-dichloroethylene isomers and vinyl chloride by anaerobic TCE biodegradation could be balanced by their consumption through aerobic degradation where methanotrophs are active in wetland sediment. TCE degradation rates estimated using field data (0.009 to 0.016 d^-1) agree with the laboratory-measured rates within a factor of 3 to 22, supporting the feasibility of natural attenuation as a remediation method for contaminated groundwater discharging in this wetland and other similar environments.     

Natural Attenuation of Trichloroethylene in Rhizosphere Soils at the Savannah River Site

Extensive trichloroethylene (TCE) groundwater contamination has resulted from discharges to a former seepage basin in the A/M Area at the Department of Energy's Savannah River Site. The direction of groundwater flow has been determined and a seep line where the contaminated groundwater is estimated to emerge as surface water has been identified in a region of the Southern Sector of the A/M Area. This study was undertaken to estimate the potential of four rhizosphere soils along the seep line to naturally attenuate TCE. Microcosms were setup to evaluate both biotic and abiotic attenuation of TCE. Results demonstrated that sorption to soil was the dominant mechanism during the first week of incubation, with as much as 90% of the TCE removed from the aqueous phase. Linear partitioning coefficients (K_d) ranged from 0.83 to 7.4?mL/g, while organic carbon partition coefficients (K_oc) ranged from 72 to 180?mL/gC. Diffu-sional losses from the microcosms appeared to be a dominant fate mechanism during the remainder of the experiment, as indicated by results from the water controls. A limited amount of TCE biodegradation was observed, and attempts to stimulate TCE biodegradation by either methanotrophic or methanogenic activity through amendments with methane, oxygen, and methanol were unsuccessful. The appearance of cis-1,2-dichloroethylene (c-DCE), and trans-1,2-dichloroethylene (t-DCE) confirmed the potential for anaerobic reductive dechlorination. However, these daughter products represented less than 5% of the initial TCE added. The sorption results indicate that natural attenuation may represent a viable remediation option for the TCE plume as it passes through the rhizosphere.     

Potential for cometabolic biodegradation of 1,4-dioxane in aquifers with methane or ethane as primary substrates

The objective of this research was to evaluate the potential for two gases, methane and ethane, to stimulate the biological degradation of 1,4-dioxane (1,4-D) in groundwater aquifers via aerobic cometabolism. Experiments with aquifer microcosms, enrichment cultures from aquifers, mesophilic pure cultures, and purified enzyme (soluble methane monooxygenase; sMMO) were conducted. During an aquifer microcosm study, ethane was observed to stimulate the aerobic biodegradation of 1,4-D. An ethane-oxidizing enrichment culture from these samples, and a pure culture capable of growing on ethane (Mycobacterium sphagni ENV482) that was isolated from a different aquifer also biodegraded 1,4-D. Unlike ethane, methane was not observed to appreciably stimulate the biodegradation of 1,4-D in aquifer microcosms or in methane-oxidizing mixed cultures enriched from two different aquifers. Three different pure cultures of mesophilic methanotrophs also did not degrade 1,4-D, although each rapidly oxidized 1,1,2-trichloroethene (TCE). Subsequent studies showed that 1,4-D is not a substrate for purified sMMO enzyme from Methylosinus trichosporium OB3b, at least not at the concentrations evaluated, which significantly exceeded those typically observed at contaminated sites. Thus, our data indicate that ethane, which is a common daughter product of the biotic or abiotic reductive dechlorination of chlorinated ethanes and ethenes, may serve as a substrate to enhance 1,4-D degradation in aquifers, particularly in zones where these products mix with aerobic groundwater. It may also be possible to stimulate 1,4-D biodegradation in an aerobic aquifer through addition of ethane gas. Conversely, our results suggest that methane may have limited importance in natural attenuation or for enhancing biodegradation of 1,4-D in groundwater environments.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

Aerobic cometabolic degradation of trichloroethene by methane and ammonia oxidizing microorganisms naturally associated with <Emphasis Type="Italic">Carex comosa</Emphasis> roots

The degradation potential of trichloroethene by the aerobic methane- and ammonia-oxidizing microorganisms naturally associated with wetland plant (Carex comosa) roots was examined in this study. In bench-scale microcosm experiments with washed (soil free) Carex comosa roots, the activity of root-associated methane- and ammonia-oxidizing microorganisms, which were naturally present on the root surface and/or embedded within the roots, was investigated. Significant methane and ammonia oxidation were observed reproducibly in batch reactors with washed roots incubated in growth media, where methane oxidation developed faster (2 weeks) compared to ammonia oxidation (4 weeks) in live microcosms. After enrichment, the methane oxidizers demonstrated their ability to degrade 150 μg l⁻¹ TCE effectively at 1.9 mg l⁻¹ of aqueous CH₄. In contrast, ammonia oxidizers showed a rapid and complete inhibition of ammonia oxidation with 150 μg l⁻¹ TCE at 20 mg l⁻¹ of NH₄ ⁺-N, which may be attributed to greater sensitivity of ammonia oxidizers to TCE or its degradation product. No such inhibitory effect of TCE degradation was detected on methane oxidation at the above experimental conditions. The results presented here suggest that microorganisms associated with wetland plant roots can assist in the natural attenuation of TCE in contaminated aquatic environments.     

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture

Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.     

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture.

Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.     

Homolytic carbon to phosphorus bond scission of some phosphonates catalyzed by bacterial carbon-phosphorus lyase

The production of volatile degradation products of phosphonates was monitored to investigate the mechanism involved in the biodegradation of propylphosphonic acid and phenylphosphonic acid byRhizobium sp MMM101a. The biodegradation of propylphosphonic acid gave rise to the production, in decreasing order, of propane, methane, ethane, 1-butene, propene, isobutene, butane and ethene. The formation of these degradation products was strongly reduced by adding catalase to the growing cultures indicating the involvement of peroxides in the biodegradation mechanism. OH⁰ radical scavengers did not reduce the rate of biodegradation, and therefore these radicals appear not to be involved. Addition of ascorbate, a known hydroxylating agent in biological systems, increased the amount of biodegradation products. The involvement of iron in the degradation was indicated and was optimal at a concentration of 950 µM. This suggests the involvement of a metalloenzyme involving iron and peroxide. The decomposition of phenylphosphonic acid yielded benzene and biphenyl. No phenol could be detected, again suggesting that OH⁰ radicals were not involved in the biodegradation. The presence of deuterated benzene did not result in the occurrence of biphenyl consisting of one nondeuterated and a deuterated ring, which is chemically more likely. It therefore appears that the degradation of the phosphonates occurs on a multicentered enzyme. The diversity of the products generated by this bacterium from phosphonates, many of them due to rearrangement of the carbon moiety of the substrate molecule, suggests an overall involvement of superoxide radicals in the homolytic carbon to phosphorus bond scission.     

Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.     

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h^-1 mg of protein ^-1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h^-1 mg of protein^-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K_m, 70 ± 4 μM) and TCE (K_m, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.     

Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

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.     

Sulfate reduction and trichloroethylene biodegradation by a marine microbial community from hydrothermal vents sediments

Sulfate reduction (SR) and trichloroethylene (TCE) biodegradation at two different temperatures (37 and 70 °C) were investigated in enrichment cultures prepared with two different samples of sediments collected from hydrothermal vents. The unadapted sediments were incubated with sulfate (4 g L⁻¹) as the electron acceptor before TCE addition to enrich them in biomass and to establish a constant sulfate reduction (SR, 87% sulfate conversion and specific H₂S concentration of 90.81 ± 8.19 mg H₂S g VSS⁻¹), afterwards TCE was added at an initial concentration of 300 ??mol L⁻¹. The best results for TCE biodegradation were obtained at 37 °C. At this temperature, SR was up to 92%, whereas TCE biodegradation reached 75% and ethane was detected as the main degradation product. Under thermophilic conditions (70 °C) TCE biodegradation reached up to approximately 60% and the SR was 30% in 30 days of incubation with the chlorinated solvent. Along with these results, the 16S rDNA analysis from samples at 37 °C showed the presence of bacteria belonging to the genera: Clostridium, Bacillus and Desulfuromonas. The overall results on TCE degradation and SR suggest that cometabolic TCE degradation is carried out by sulfate or sulfur reducers and fermentative bacteria at mesophilic conditions.     

Development of an attached-growth process for the on-site bioremediation of an aquifer polluted by chlorinated solvents

A procedure for the design of an aerobic cometabolic process for the on-site degradation of chlorinated solvents in a packed bed reactor was developed using groundwater from an aquifer contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). The work led to the selection of butane among five tested growth substrates, and to the development and characterization from the site’s indigenous biomass of a suspended-cell consortium capable to degrade TCE (first order constant: 96 L g _protein ^–1  day^–1 at 30 °C and 4.3 L g _protein ^–1  day^–1 at 15 °C) with a 90 % mineralization of the organic chlorine. The consortium immobilization had strong effects on the butane and TCE degradation rates. The microbial community structure was slightly changed by a temperature shift from 30 to 15 °C, but remarkably affected by biomass adhesion. Given the higher TCE normalized degradation rate (0.59 day^–1 at 15 °C) and attached biomass concentration (0.13 g_protein L _bioreactor ^–1 at 15 °C) attained, the porous ceramic carrier Biomax was selected as the best option for the packed bed reactor process. The low TeCA degradation rate exhibited by the developed consortium suggested the inclusion of a chemical pre-treatment based on the TeCA to TCE conversion via β-elimination, a very fast reaction at alkaline pH. To the best of the authors’ knowledge, this represents the first attempt to develop a procedure for the development of a packed bed reactor process for the aerobic cometabolism of chlorinated solvents.     

Effects of copper on expression of methane monooxygenases,trichloroethylene degradation,and community structure in methanotrophic consortia

Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu²⁺) concentrations. The results demonstrated that an increase in Cu²⁺ concentration from 0 to 15 μM altered the specific first‐order rate constant k_1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu²⁺ concentration of 0.03 μM. Notably, sMMO‐specific activity ranged from 74.41 nmol/(mg_cell h) in 15 μM Cu²⁺ to 654.99 nmol/(mg_cell h) in 0.03 μM Cu²⁺, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu²⁺ concentrations, indicating a special regulatory role for Cu²⁺ in MC. The results of MiSeq pyrosequencing indicated that higher Cu²⁺ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper‐mediated regulatory mechanisms in MC.     

Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Trichloroethylene biodegradation by mesophilic and psychrophilic ammonia oxidizers and methanotrophs in groundwater microcosms.

This study investigated the efficiency of methane and ammonium for stimulating trichloroethylene (TCE) biodegradation in groundwater microcosms (flasks and batch exchange columns) at a psychrophilic temperature (12 degrees C) typical of shallow aquifers in the northern United States or a mesophilic temperature (24 degrees C) representative of most laboratory experiments. After 140 days, TCE biodegradation rates by ammonia oxidizers and methanotrophs in mesophilic flask microcosms were similar (8 to 10 nmol day-1), but [14C]TCE mineralization (biodegradation to 14CO2) by ammonia oxidizers was significantly greater than that by methanotrophs (63 versus 53%). Under psychrophilic conditions, [14C]TCE mineralization in flask systems by ammonia oxidizers and methanotrophs was reduced to 12 and 5%, respectively. In mesophilic batch exchange columns, average TCE biodegradation rates for methanotrophs (900 nmol liter-1 day-1) were not significantly different from those of ammonia oxidizers (775 nmol liter-1 day-1). Psychrophilic TCE biodegradation rates in the columns were similar with both biostimulants and averaged 145 nmol liter-1 day-1. Methanotroph biostimulation was most adversely affected by low temperatures. At 12 degrees C, the biodegradation efficiencies (TCE degradation normalized to microbial activity) of methanotrophs and ammonia oxidizers decreased by factors of 2.6 and 1.6, respectively, relative to their biodegradation efficiencies at 24 degrees C. Collectively, these experiments demonstrated that in situ bioremediation of TCE is feasible at the psychrophilic temperatures common in surficial aquifers in the northern United States and that for such applications biostimulation of ammonia oxidizers could be more effective than has been previously reported.     

Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene