Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophic cultures from a groundwater aquifer.

Trichloroethylene (TCE)-transforming aquifer methanotrophs were evaluated for the influence of TCE oxidation toxicity and the effect of reductant availability on TCE transformation rates during methane starvation. TCE oxidation at relatively low (6 mg liter-1) TCE concentrations significantly reduced subsequent methane utilization in mixed and pure cultures tested and reduced the number of viable cells in the pure culture Methylomonas sp. strain MM2 by an order of magnitude. Perchloroethylene, tested at the same concentration, had no effect on the cultures. Neither the TCE itself nor the aqueous intermediates were responsible for the toxic effect, and it is suggested that TCE oxidation toxicity may have resulted from reactive intermediates that attacked cellular macromolecules. During starvation, all methanotrophs tested exhibited a decline in TCE transformation rates, and this decline followed exponential decay. Formate, provided as an exogenous electron donor, increased TCE transformation rates in Methylomonas sp. strain MM2, but not in mixed culture MM1 or unidentified isolate, CSC-1. Mixed culture MM2 did not transform TCE after 15 h of starvation, but mixed cultures MM1 and MM3 did. The methanotrophs in mixed cultures MM1 and MM3, and the unidentified isolate CSC-1 that was isolated from mixed culture MM1 contained lipid inclusions, whereas the methanotrophs of mixed culture MM2 and Methylomonas sp. strain MM2 did not. It is proposed that lipid storage granules serve as an endogenous source of electrons for TCE oxidation during methane starvation.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs

The effect of growth in different mineral media on subsequent oxidation of trichloroethylene (TCE) by type I and type II aquifer methanotrophs was evaluated. Mixed culture MM1, containing a type II methanotroph, and a type I pure culture tentatively identified as aMethylomonas sp., were enriched and isolated from an uncontaminated groundwater aquifer. The second-order rate coefficients (k/K_s) for TCE oxidation by these cultures varied by more than an order of magnitude when the cultures were grown in different mineral media. The presence of a chelator (NaEDTA) in one of these media, termed Whittenbury, significantly enhanced rates of TCE oxidation by all the cultures tested. When pregrown in this mineral medium, the resting cells of the pure cultureMethylomonas sp. MM2 exhibited second-order TCE oxidation rates as great as 0.78 liter/mg·day, whereas when pregrown in Whittenbury lacking the chelator, the rates did not exceed 0.018 liter/mg·day. The rate of TCE oxidation byMethylomonas sp. MM2 pregrown in another mineral medium formulation, devoid of chelators (termed Fogel), was intermediate in value (0.26 liter/mg·day), and adding EDTA to this medium did not affect the rate. Adding 1.6 μM copper to both Whittenbury and Fogel mineral media reduced the TCE oxidation rates about an order of magnitude; subsequent addition of 84 μM EDTA partially alleviated this effect. The maximal rate coefficients (k) for TCE oxidation byMethylomonas sp. MM2 were significantly higher, and the half saturation coefficients (K_s) for TCE significantly lower, following growth in the presence of EDTA. Stationary phase TCE oxidation rates as great as 2.3 liter/mg·day were achieved whenMethylomonas sp. MM2, grown in Whittenbury medium was provided formate as a source of reducing power. Omitting EDTA from Whittenbury medium also significantly reduced the methane oxidation rate and the growth yield. Copper addition did not significantly affect the methane oxidation rate or growth yield. The internal membrane structures ofMethylomonas sp. MM2 evaluated by transmission electron microscopy showed the presence of internal membranes, the ultrastructure of which was the same regardless of growth medium or TCE oxidation rate. The methane monooxygenase responsible for TCE oxidation inMethylomonas sp. MM2 under the conditions of this study appears to be associated with the particulate fraction.     

丙烯经酶催化氧化制环氧丙烷——一株甲烷氧化细菌的分离筛选及生理特性的研究

环氧丙烷是聚氨酯、不饱和聚酯和优质洗涤剂的主要原料,还可用于油漆、化妆品等,是一种非常重要的精细化工原料。目前环氧丙烷主要用氯醇法和烷基过氧化氢法生产。1963年,Vender Lindent发现庚烷菌P.Seruginosa的休止细胞可使辛烯-1氧化成环氧辛烷,首次提出了烯烃经生物催化环氧化生成相应环氧化物的过程。1977年,Colby等报导了从Methylococcus capsu-latus(Bath)菌中提取了非专一性菌甲烷单加氧酶。1979年,C.T.Hou等分离出二十多种甲烷氧化细菌都能使C_2—C_4烯烃氧化成     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture.

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.     

Experimental evaluation of a model for cometabolism: Prediction of simultaneous degradation of trichloroethylene and methane by a methanotrophic mixed culture

A model for cometabolism is verified experimentally for a defined methanotrophic mixed culture. The model includes the effects of cell growth, endogenous cell decay, product toxicity, and competitive inhibition with the assumption that cometabolic transformation rates are enhanced by reducing power obtained from oxidation of growth substrates. A theoretical transformation yield is used to quantify the enhancement resulting from growth substrate oxidation. A systematic method for evaluating model parameters independently is described. The applicability of the model is evaluated by comparing experimental data for methanotrophic cometabolism of TCE with model predictions from independently measured model parameters. Propagation of errors is used to quantify errors in parameter estimates and in the final prediction. The model successfully predicts TCE transformation and methane utilization for a wide range of concentrations of TCE (0.5 to 9 mg/L) and methane (0.05 to 6 mg/L). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 492-501, 1997.     

甲烷利用细菌降解三氯乙烯的研究

GYJ3菌株细胞微细结构的电镜观察结果表明：它具有Ⅱ型甲烷利用细菌的特征,应归属于Ⅱ型菌。考察了Cu2+浓度、培养气相中甲烷浓度对菌株细胞中甲烷单加氧酶（EC1．14．13．25,简称MMO）活性的影响。结果表明,培养液中Cu2+浓度为1．5μmol／L,培养气相中甲烷：空气比为2∶1时,可溶性甲烷单加氧酶占细胞中MMO总量的95％。研究了GYJ3菌株细胞悬浮液降解三氯乙烯过程。实验结果表明,GYJ3菌株能够降解不同浓度的三氯乙烯,较高浓度的三氯乙烯对降解反应没有明最的抑制作用。加入甲酸盐作为电子给体能够提高三氯乙烯降解反应速率。实验中观察到GYJ3菌株降解三氯乙烯过程中反应速率随着反应的进行而下降,在三氯乙烯降解过程中三氯乙烯氧化产物是导致细胞失活的主要原因。实验室中测定了GYJ3菌株单位重量细胞降解三氯乙烯极限量,它可作为评价细菌降解三氯乙烯能力的重要指标。     

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.     

Methane oxidation and the competition for oxygen in the rice rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O(2) with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently < 5 microM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

Biodegradation of linear alkylbenzenesulphonates (LAS) by mixed methanotrophic-heterotrophic cultures

The biodegradation of undecylbenzenesulphonate (C₁₁LAS) was studied in shake flasks at 21°C using two mixed bacterial cultures. The first culture, MM1, contained a type II methanotroph and four heterotrophs, and was enriched from a groundwater aquifer. The second culture, MC, consisted of five heterotrophic strains, most of them belonging to the genus Pseudomonas , and was isolated from the wastewater of a detergent plant. Methane, carbon dioxide and oxygen concentrations were determined by gas chromatography. Concentrations of C₁₁LAS and the aromatic intermediates were determined by reversed-phase HPLC. In spite of faster transformation of the alkyl side-chain by the culture MC, the culture MM1 containing type II methanotroph was capable of further degradation of C₁₁LAS aromatic intermediates (sulphophenylalkanoates). The most probable mechanism for the degradation of the alkyl part of the C₁₁LAS molecule by both cultures was β-oxidation of the terminal methyl group followed by β-oxidation. Studies of methane utilization demonstrated an approximately three times higher second-order rate coefficient for methane consumption ( k _max/ K _s) in the absence of C₁₁LAS. This indicates a possible metabolic activity of methanotrophs in the transformation of the complex LAS molecule due to the methane monooxygenase enzyme system.     

Comparison of the microbial population dynamics and phylogenetic characterization of a CANOXIS reactor and a UASB reactor degrading trichloroethene

AIMS: To understand the microbial ecology underlying trichloethene (TCE) degradation in a coupled anaerobic/aerobic single stage (CANOXIS) reactor oxygenated with hydrogen peroxide (H2O2) and in an upflow anaerobic sludge bed (UASB) reactor. METHODS AND RESULTS: The molecular study of the microbial population dynamics and a phylogenetic characterization were conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). In both reactors, TCE had a toxic effect on two uncultured bacterial populations whereas oxygen favoured the growth of aerobic species belonging to Rhizobiaceae and Dechloromonas. No methanotrophic bacteria were detected when targeting 16S rRNA gene with universal primers. Alternatively, pmo gene encoding the particulate methane monooxygenase of Methylomonas sp. LW21 could be detected in the coupled reactor when H2O2 was supplied at 0.7 g O2 l day(-1). CONCLUSIONS: Methylomonas sp. LW21 that could be responsible for the aerobic degradation of the TCE by-products is not among the predominant bacterial populations in the coupled reactor. It seems to have been outcompeted by heterotrophic bacteria (Rhizobiaceae and Dechloromonas sp.) for oxygen. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained show the limitations of the coupled reactor examined in this study. Further investigations should focus on the operating conditions of this reactor in order to favour the growth of the methanotrophs.     

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.     

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells.

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.     

Transformation capacities of chlorinated organics by mixed cultures enriched on methane, propane, toluene, or phenol

The degradation of trichloroethylene (TCE), chloroform (CF), and 1,2-dichloroethane (1,2-DCA) by four aerobic mixed cultures (methane, propane, toluene, and phenol oxidizers) grown under similar chemostat conditions was measured. Methane and propane oxidizers were capable of degrading both saturated and unsaturated chlorinated organics (TCE, CF, and 1,2-DCA). Toluene and phenol oxidizers degraded TCE but were not able to degrade CF, 1,2-DCA, or other saturated organics. None of the cultures tested were able to degrade perchloroethylene (PCE) or carbon tetrachloride (CC(4)). For the four cultures tested, degradation of each of the chlorinated organics resulted in cell inactivation due to product toxicity. In all cases, the toxic products were rapidly depleted, leaving no toxic residues in solution. Among the four tested cultures, the resting cells of methane oxidizers exhibited the highest transformation capacities (T(c)) for TCE, CF, and 1,2-DCA. The T(c) for each chlorinated organic was observed to be inversely proportional to the chlorine carbon ratio (Cl/C). The addition of low concentrations of growth substrate or some catabolic intermediates enhanced TCE transformation capacities and degradation rates, presumably due to the regeneration of reducing energy (NADH); however, addition of higher concentrations of most amendments reduced TCE transformation capacities and degradation rates. Reducing energy limitations and amendment toxicity may significantly affect T(c) measurements, causing a masking of the toxicity associated with chlorinated organic degradation. (c) 1995 John Wiley & Sons, Inc.     

Anaerobic methane oxidation

To clarify the biological mechanism of anaerobic methane oxidation, experiments were performed with samples of the Black Sea anaerobic sediments and with the aerobic methane-oxidizing bacterium Methylomonas methanica strain 12. The inhibition-stimulation analysis did not allow an unambiguous conclusion to be made about direct and independent role of either methanogenic or sulfate-reducing microorganisms in the biogeochemical process of anaerobic methane oxidation. Enrichment cultures obtained from samples of water and reduced sediments oxidized methane under anaerobic conditions, primarily in the presence of acetate or formate or of a mixture of acetate, formate, and lactate. However, this ability was retained by the cultures for no more than two transfers on corresponding media. Experiments showed that the aerobic methanotroph Mm. methanica strain 12 is incapable of anaerobic methane oxidation at the expense of the reduction of amorphous FeOOH.