Retrospective on microbial transformations of halogenated organics.

Prior to the 1960s, knowledge of biological transformations of highly halogenated aliphatic compounds was limited, except in mammalian organisms where enzymatic transformations occurred to rid the body of ingested harmful chemicals. Limited abiotic transformation of such compounds had also been observed, with half-lives varying from days to centuries. Commonly believed was that aerobic transformation might occur by cometabolism rather than to conserve energy for respiration, while anaerobic transformations were in general thought not to occur. However, in the late 1960s anaerobic transformation of chlorinated pesticides was noted, and then in the early 1980s, partial microbial dehalogenation of chlorinated solvents such as tetrachlorethene, trichloroethene, trichlorethane, and carbon tetrachloride was also found to occur. With only partial dechlorination, complete detoxification was not achieved. And at the time, dehalogenation reactions were not believed to yield energy for growth to the degrading microorganisms. However, in the 1990s bacteria began to be found that obtain energy from anaerobic transformations, often enabling complete dechlorination and detoxification. Since then such ability has been found among several bacterial species, many of which use molecular hydrogen as a donor substrate and halogenated organics as electron acceptors, thus conserving energy through reductive dehalogenation. Growth of knowledge in this field has grown rapidly since the 1960s. Broad usages of such microorganisms are now underway to rid contaminated groundwater of hazardous halogenated chemicals.

Estimating reaction rate coefficients within a travel-time modeling framework.

A generalized, efficient, and practical approach based on the travel-time modeling framework is developed to estimate in situ reaction rate coefficients for groundwater remediation in heterogeneous aquifers. The required information for this approach can be obtained by conducting tracer tests with injection of a mixture of conservative and reactive tracers and measurements of both breakthrough curves (BTCs). The conservative BTC is used to infer the travel-time distribution from the injection point to the observation point. For advection-dominant reactive transport with well-mixed reactive species and a constant travel-time distribution, the reactive BTC is obtained by integrating the solutions to advective-reactive transport over the entire travel-time distribution, and then is used in optimization to determine the in situ reaction rate coefficients. By directly working on the conservative and reactive BTCs, this approach avoids costly aquifer characterization and improves the estimation for transport in heterogeneous aquifers which may not be sufficiently described by traditional mechanistic transport models with constant transport parameters. Simplified schemes are proposed for reactive transport with zero-, first-, nth-order, and Michaelis-Menten reactions. The proposed approach is validated by a reactive transport case in a two-dimensional synthetic heterogeneous aquifer and a field-scale bioremediation experiment conducted at Oak Ridge, Tennessee. The field application indicates that ethanol degradation for U(VI)-bioremediation is better approximated by zero-order reaction kinetics than first-order reaction kinetics.

Estimating kinetic mass transfer by resting-period measurements in flow-interruption tracer tests.

Flow-interruption tracer test is an effective approach to identify kinetic mass transfer processes for solute transport in subsurface media. By switching well pumping and resting, one may alter the dominant transport mechanism and generate special concentration patterns for identifying kinetic mass transfer processes. In the present research, we conducted three-phase (i.e., pumping, resting, and pumping) field-scale flow-interruption tracer tests using a conservative tracer bromide in a multiple-well system installed at the US Department of Energy Site, Oak Ridge, TN. A novel modeling approach based on the resting-period measurements was developed to estimate the mass transfer parameters. This approach completely relied on the measured breakthrough curves without requiring detailed aquifer characterization and solving transport equations in nonuniform, transient flow fields. Additional measurements, including hydraulic heads and tracer concentrations in large pumping wells, were taken to justify the assumption that mass transfer processes dominated concentration change during resting periods. The developed approach can be conveniently applied to any linear mass transfer model. Both first-order and multirate mass transfer models were applied to analyze the breakthrough curves at various monitoring wells. The multirate mass transfer model was capable of jointly fitting breakthrough curve behavior, showing the effectiveness and flexibility for incorporating aquifer heterogeneity and scale effects in upscaling effective mass transfer models.

Estimating first-order reaction rate coefficient for transport with nonequilibrium linear mass transfer in heterogeneous media.

A travel-time based approach is developed for estimating first-order reaction rate coefficients for transport with nonequilibrium linear mass transfer in heterogeneous media. Tracer transport in the mobile domain is characterized by a travel-time distribution, and mass transfer rates are described by a convolution product of concentrations in the mobile domain and a memory function rather than predefining the mass transfer model. A constant first-order reaction is assumed to occur only in the mobile domain. Analytical solutions in Laplace domain can be derived for both conservative and reactive breakthrough curves (BTCs). Temporal-moment analyses are presented by using the first and second moments of conservative and reactive BTCs and the mass consumption of the reactant for an inverse Gaussian travel-time distribution. In terms of moment matching, there is no need for one to specify the mass transfer model. With the same capacity ratio and the mean retention time, all mass transfer models will lead to the same moment-derived reaction rate coefficients. In addition, the consideration of mass transfer generally yields larger estimations of the reaction rate coefficient than models ignoring mass transfer. Furthermore, the capacity ratio and the mean retention time have opposite influences on the estimation of the reaction rate coefficient: the first-order reaction rate coefficient is positively linearly proportional to the capacity ratio, but negatively linearly proportional to the mean retention time.

Correlation of functional instability and community dynamics in denitrifying dispersed-growth reactors.

Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing Delftia- and Achromobacter-like T-RFs. The isolate representing the Acidovorax-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The Acidovorax-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.

Stability in a denitrifying fluidized bed reactor.

This study evaluates changes in the microbial community structure and function of a pilot-scale denitrifying fluidized bed reactor during periods of constant operating conditions and periods of perturbation. The perturbations consisted of a shutdown period without feed, two disturbances in which biofilms were mechanically sheared from carrier particles, and a twofold step increase in feed nitrate concentration. In the absence of perturbations, nitrate removal was stable and consistently greater than 99%. The structure and dynamics of the microbial community were studied using cloning and sequencing techniques and terminal restriction fragment length polymorphism (T-RFLP) of the SSU rRNA gene. Under unperturbed operating conditions, stable function was accompanied by high constancy and low variability of community structure with the majority of terminal restriction fragments (T-RFs) appearing throughout operation at consistent relative abundances. Several of the consistently present T-RFs correlated with clone sequences closely related to Acidovorax (98% similarity), Dechloromonas (99% similarity), and Zoogloea (98% similarity), genera recently identified by molecular analyses of similar systems. Significant changes in community structure and function were not observed after the shutdown period. In contrast, following the increase in loading rate and the mechanical disturbances, new T-RFs appeared. After both mechanical disturbances, function and community structure recovered. However, function was much more resilient than community structure. The similarity of response to the mechanical disturbances despite differences in community structure and operating conditions suggests that flexible community structure and potentially the activity of minor members under nonperturbation conditions promotes system recovery.

Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination.

Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetrachloroethene, 99Tc, No3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (deltaC(xy) values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites.

Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor.

High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n = 500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.

A derivative of the menaquinone precursor 1,4-dihydroxy-2-naphthoate is involved in the reductive transformation of carbon tetrachloride by aerobically grown Shewanella oneidensis MR-1.

Transformation of carbon tetrachloride (CT) by Shewanella oneidensis MR-1 has been proposed to involve the anaerobic respiratory-chain component menaquinone. To investigate this hypothesis a series of menaquinone mutants were constructed. The menF mutant is blocked at the start of the menaquinone biosynthetic pathway. The menB, menA and menG mutants are all blocked towards the end of the pathway, being unable to produce 1,4-dihydroxy-2-naphthoic acid (DHNA), demethyl-menaquinone and menaquinone, respectively. Aerobically grown mutants unable to produce the menaquinone precursor DHNA (menF and menB mutants) showed a distinctly different CT transformation profile than mutants able to produce DHNA but unable to produce menaquinone (menA and menG mutants). While DHNA did not reduce CT in an abiotic assay, the addition of DHNA to the menF and menB mutants restored normal CT transformation activity. We conclude that a derivative of DHNA, that is distinct from menaquinone, is involved in the reduction of CT by aerobically grown S. oneidensis MR-1. When cells were grown anaerobically with trimethylamine-N-oxide as the terminal electron acceptor, all the menaquinone mutants showed wild-type levels of CT reduction. We conclude that S. oneidensis MR-1 produces two different factors capable of dehalogenating CT. The factor produced under anaerobic growth conditions is not a product of the menaquinone biosynthetic pathway.

The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions.

We compared carbon flow under constant low-substrate conditions (below 20 microM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring 14C-labeled metabolites derived from a feeding of [U-14C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.

Pseudomonas sp. strain KC represents a new genomovar within Pseudomonas stutzeri.

Pseudomonas sp. strain KC (= ATCC 55595 = DSM 7136) is a denitrifying aquifer isolate that produces and secretes pyridine-2,6-bis(thiocarboxylate) (PDTC), a chelating agent that fortuitously transforms carbon tetrachloride without producing chloroform. Although KC has been used successfully for full-scale bioremediation of carbon tetrachloride, its taxonomy has proven difficult to resolve, as it retains properties of both Pseudomonas stutzeri and Pseudomonas putida. In the present work, a polyphasic approach was used to conclude that strain KC represents a new genomovar (genomovar 9) within the species P. stutzeri.

Parallel processing of substrate correlates with greater functional stability in methanogenic bioreactor communities perturbed by glucose.

Parallel processing is more stable than serial processing in many areas that employ interconnected activities. This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations. The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors. To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration. The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product. A new stability parameter, "moment of amplification envelope," was used to compare the soluble compound stability. These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing. The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.

Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose.

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).

Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride.

Under iron-limiting conditions, Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CT) to CO2 and nonvolatile products when activated by reduction at cell membranes. Pseudomonas fluorescens and other cell types activate the factor. Triparental mating was used to generate kanamycin-resistant lux::Tn5 recombinants of strain KC. Recombinants were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous 14C-carbon tetrachloride. CT+ recombinants generated nonvolatile 14C-labeled products, but four CT- recombinants did not generate significant nonvolatile 14C-labeled products and had lost the ability to degrade carbon tetrachloride. When colonies of P. fluorescens were grown next to colonies of CT+ recombinants and were exposed to gaseous 14C-carbon tetrachloride, 14C-labeled products accumulated around the P. fluorescens colonies, indicating that the factor secreted by CT+ colonies had diffused through the agar and become activated. When P. fluorescens was grown next to CT- colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor. Expression of lux reporter genes in three of the CT- mutants was regulated by added iron and was induced under the same iron-limiting conditions that induce carbon tetrachloride transformation in the wild-type.

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.

Effects of phenol feeding pattern on microbial community structure and cometabolism of trichloroethylene.

Cometabolism of trichloroethylene (TCE) by phenol-fed enrichments was evaluated in four reactors with distinct phenol feeding patterns. The reactors were inoculated from the same source, operated at the same average dilution rate, and received the same mass of phenol over time. Only the timing of phenol addition differed. Reactor C received phenol continuously; reactor SC5 received phenol semicontinuously--alternating between 5 h of feed and 3 h without feed; reactor SC2 alternated between 2 h of feed and 6 h without feed; and reactor P received a single pulse every 24 h. The structure of the enrichments and their capacity for TCE transformation were analyzed. In long-term operation, reactors C and SC5 were dominated by fungi, had higher levels of predators, were more susceptible to biomass fluctuations, and exhibited reduced capacity for TCE transformation. Reactors P and SC2 were characterized by lower levels of fungi, higher bacterial biomass, higher concentrations of TCE-degrading organisms, and higher rates of TCE transformation. After 200 days of operation, rates of TCE transformation increased 10-fold in reactor P, resulting in TCE transformation rates that were 20 to 100 times higher than the rates of the other reactor communities. The cause of this shift is unknown. Isolates capable of the highest rates of TCE transformation were obtained from reactor P. We conclude that cometabolic activity depends upon microbial community structure and that the community structure can be manipulated by altering the growth substrate feeding pattern.

Mass transfer and temperature effects on substrate utilization in brewery granules.

Liquid film and diffusional resistances of brewery granules during acetate, propionate, and ethanol utilization were investigated. Substrate utilization rate increased with decreased granule size. Effectiveness factors for acetate, propionate, and ethanol were calculated by comparing the maximum rates of substrate utilization of whole granules (1.8 to 3.0 mm) and fine flocs (20 to 75 mum) derived by disrupting whole granules. For acetate, propionate, and ethanol, maximum specific substrate utilization rates (k(m') g/g VS . d) for the flocs, were 5.11, 6.25, and 5.49, respectively, and half-velocity coefficients (K(g') mM) were 0.45, 0.40, and 3.37, respectively. Calculated effectiveness factors were 0.32, 0.41, and 0.75 for acetate, propionate, and ethanol, respectively. The effect of temperature on substrate utilization was examined at 26 degrees C, 31 degrees C, and 37 degrees C using acetate as sole carbon source. Utilization rates increased with temperature. Flocs were most sensitive to temperature, and whole granules were least affected. The behavior of flocs was well described by the Van't Hoff-Arrhenius equation. Effectiveness factors for acetate utilization by the granules were 0.36, 0.35, and 0.32 at 26 degrees C, 31 degrees C, and 37 degrees C, respectively, indicating little effect of temperature. Based on these results, we conclude that both liquid film and diffusional resistances influenced the rate of substrate utilization in a UASB reactor with granular sludge. Temperature effects were much less important than diffusional limitations within the granules. (c) 1995 John Wiley & Sons, Inc.

Localization and Characterization of the Carbon Tetrachloride Transformation Activity of Pseudomonas sp. Strain KC.

Previous research has established that Pseudomonas sp. strain KC rapidly transforms carbon tetrachloride (CT) to carbon dioxide (45 to 55%), a nonvolatile fraction (45 to 55%), and a cell-associated fraction ((equiv)5%) under denitrifying, iron-limited conditions. The present study provides additional characterization of the nonvolatile fraction, demonstrates that electron transfer plays a role in the transformation, and establishes the importance of both extracellular and intracellular factors. Experiments with (sup14)C-labeled CT indicate that more than one nonvolatile product is produced during CT transformation by strain KC. One of these products, accounting for about 20% of the [(sup14)C]CT transformed, was identified as formate on the basis of its elution time from an ion-exchange column, its boiling point, and its conversion to (sup14)CO(inf2) when incubated with formate dehydrogenase. Production of formate requires transfer of two electrons to the CT molecule. The role of electron transfer was also supported by experiments demonstrating that stationary-phase cells that do not transform CT can be stimulated to transform CT when supplemented with acetate (electron donor), nitrate (electron acceptor), or a protonophore (carbonyl cyanide m-chlorophenylhydrazone). The location of transformation activity was also evaluated. By themselves, washed cells did not transform CT to a significant degree. Occasionally, CT transformation was observed by cell-free culture supernatant, but this activity was not reliable. Rapid and reliable CT transformation was only obtained when washed whole cells were reconstituted with culture supernatant, indicating that both extracellular and intracellular factors are normally required for CT transformation. Fractionation of culture supernatant by ultrafiltration established that the extracellular factor or factors are small, with an apparent molecular mass of less than 500 Da. The extracellular factor or factors were stable after lyophilization to powder and were extractable with acetone. Addition of micromolar levels of iron inhibited CT transformation in whole cultures, but the level of iron needed to inhibit CT transformation was over 100-fold higher for washed cells reconstituted with a 10,000-Da supernatant filtrate. Thus, the inhibitory effects of iron are exacerbated by a supernatant factor or factors with a molecular mass greater than 10,000 Da.

Effects of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.

Under denitrifying conditions, Pseudomonas sp. strain KC transforms carbon tetrachloride (CT) to carbon dioxide via a complex but as yet undetermined mechanism. Transformation rates were first order with respect to CT concentration over the CT concentration range examined (0 to 100 micrograms/liter) and proportional to protein concentration, giving pseudo-second-order kinetics overall. Addition of ferric iron (1 to 20 microM) to an actively transforming culture inhibited CT transformation, and the degree of inhibition increased with increasing iron concentration. By removing iron from the trace metals solution or by removing iron-containing precipitate from the growth medium, higher second-order rate coefficients were obtained. Copper also plays a role in CT transformation. Copper was toxic at neutral pH. By adjusting the medium pH to 8.2, soluble iron and copper levels decreased as a precipitate formed, and CT transformation rates increased. However, cultures grown at high pH without any added trace copper (1 microM) exhibited slower growth rates and greatly reduced rates of CT transformation, indicating that copper is required for CT transformation. The use of pH adjustment to decrease iron solubility, to avoid copper toxicity, and to provide a selective advantage for strain KC was evaluated by using soil slurries and groundwater containing high levels of iron. In samples adjusted to pH 8.2 and inoculated with strain KC, CT disappeared rapidly in the absence or presence of acetate or nitrate supplements. CT did not disappear in pH-adjusted controls that were not inoculated with strain KC.

The kinetics of cometabolism.

Experimental observations indicate that the rates of cometabolic transformation are linked to the consumption of growth substrate during growth and to the consumption of cell mass and/or energy substrate in the absence of growth substrate. Three previously proposed models (models 1 through 3) describing the kinetics of cometabolism by resting cells are compared, and the interrelationships and underlying assumptions for these models are explored. Models 1 to 3 are shown to converge at high concentrations of the nongrowth substrate. An expression describing nongrowth substrate transformation in the presence of growth substrate is proposed, and this expression is integrated with an expression for cell growth to give a single unstructured model (model 4) that encompasses models 1 to 3 and describes cometabolism by both resting and growing cells. Model 4 couples transformation of nongrowth substrate to consumption of growth substrate and biomass, and predicts that cometabolism will result, and decreased specific growth rates for a cometabolizing population. Competitive inhibition can also be incorporated in the model. Experimental aspects of model calibration and verification are discussed. The need for models that distinguish between the exhaustion of cell activity and cell death is emphasized.