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Applied and Environmental Microbiology, December 2005, p. 7980-7986, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7980-7986.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cometabolism of Trihalomethanes by Nitrosomonas europaea

David G. Wahman, Lynn E. Katz, and Gerald E. Speitel Jr.*

The University of Texas at Austin, Department of Civil, Architectural and Environmental Engineering, 1 University Station, Austin, Texas 78712

Received 28 June 2005/ Accepted 4 August 2005


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ABSTRACT
 
The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 µg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants () increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH3-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (Tc), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.


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INTRODUCTION
 
Balancing the competing goals of disinfection and disinfection by-product (DBP) regulations is a challenge for many drinking water utilities. The proposed stage 2 disinfection and DBP regulations (15, 16) will only make this task more difficult. Although chlorine disinfection remains quite popular in the United States (6, 7), many utilities now use combinations of chlorine and chloramines to avoid excessive trihalomethane (THM) and haloacetic acid formation. A typical treatment scheme consists of an initial period of chlorination to help achieve disinfection goals followed by quenching with ammonia at some point in the treatment train to meet DBP goals through the lower DBP formation rates associated with chloramines. Nevertheless, significant formation of THMs and haloacetic acids can occur within treatment plants even during relatively short periods of chlorination (24, 28). Therefore, approaches for minimizing the formation of these DBPs or for removing the DBPs within treatment plants are potentially of much practical value.

Much effort over the past two decades has gone into approaches for minimizing DBP formation through modification of disinfection practices and removal of precursor materials (23), while comparatively little effort has been expended on approaches for removing DBPs formed in treatment plants before finished water is sent into distribution systems. Very early on, both technological and philosophical problems with the removal of THMs through activated carbon adsorption and air stripping were identified (28), and approaches for removing DBPs after formation have been largely ignored since that time. Recent developments in biological treatment, however, strongly suggest that revisiting treatment processes for THM removal is worthwhile.

No evidence indicates that THMs can support microbial growth. Considerable evidence is available, however, for cometabolism of chloroform, or trichloromethane (TCM), by bacteria growing on other chemicals (2, 5, 9). Of particular interest is the observation that nitrifying bacteria can cometabolize chloroform at a reasonable rate (0.03 to 0.1 liter/mg/day). The premise of this research is that THM removal should be possible within drinking water treatment plants by introducing a biological treatment step based on THM cometabolism by nitrifying bacteria. In this way, cometabolism would be sustained by adding a chemical commonly used in drinking water treatment while avoiding the addition of simple organic chemicals (i.e., methanol), which is highly undesirable in drinking water treatment.

Most of the cometabolism research with nitrifiers has been done with the soil bacterium Nitrosomonas europaea, which has been used as an example of the ubiquitous soil- and water-dwelling nitrifying bacteria. Vannelli et al. (30) showed that this organism could cometabolize various halogenated methanes, ethanes, and ethenes including chloroform, dichloromethane, and dibromomethane. THMs other than chloroform were not studied. Chloroform cometabolism by N. europaea was subsequently confirmed by Rasche et al. (17) and Ely (8), who also conducted detailed kinetic experiments. Melin et al. (14) and Ginestet et al. (10) showed that a mixed culture of nitrifiers from a marine sediment and activated sludge, respectively, could cometabolize chloroform, thereby providing some evidence that nonspecific ammonia monooxygenase (AMO) enzymes may be widely distributed in the environment, which would be advantageous for easy implementation of the proposed THM cometabolism process.

This research extended the previous work on TCM cometabolism kinetics to the other three regulated THMs to provide a basis for assessing the feasibility of the proposed treatment process. The key question was whether or not nitrifying bacteria can reliably cometabolize all four THMs at a sufficient rate to make the process attractive to utilities that practice (or want to practice) prechlorination, in particular, utilities practicing a combination of chlorination and chloramination. Information was developed on TCM, bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM) cometabolism kinetics as well as the toxicity of their intermediate by-products. N. europaea was chosen as a starting point for this evaluation to build on the large body of literature on this organism and to provide a pure-culture baseline for evaluating the performance of the mixed cultures that are likely to dominate in practice.


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MATERIALS AND METHODS
 
Bacterial strain and culture conditions.
N. europaea was obtained from the American Type Culture Collection (ATCC 19718) and grown in a series of autoclaved 1-liter brown glass bottles with foam plugs on 500 ml of the medium used by Ely (8). An inoculum of approximately 10% (by volume) was used when the cultures were transferred between bottles. After inoculation, the bottles were placed on a rotary shaker for approximately 1 week and then placed in a 4°C refrigerator and subsequently transferred to fresh medium within 6 weeks. To check for contamination during each bottle transfer, a portion of each bottle transferred was used to make plates on R2A agar. These plates were incubated for 3 weeks at 30°C and inspected for any visible cultures. If cultures were detected, the bottle was discarded.

To culture a larger mass for kinetic experiments, N. europaea was grown axenically in a Bioflow III fermentor with a 2.5-liter working volume (New Brunswick Scientific, NJ) operating in batch mode using the same nutrient solution and growth conditions as described previously by Ely (8), except that the temperature was maintained at 30°C. The fermentor was inoculated from bottle cultures and covered in aluminum foil to prevent inactivation of the culture by light (12, 22). The pH was controlled by the automatic addition of 5% (by weight) sodium carbonate (Na2CO3) to maintain the pH at 7.8, and the dissolved oxygen was automatically maintained in the range of 2.0 to 4.0 mg/liter by adjusting the agitation and/or by adding air into the fermentor.

Batch kinetic experiments.
Organisms were harvested from the batch reactor by centrifugation 3 days after inoculation, washed, centrifuged again, and resuspended in fresh buffer medium (8 mM phosphate and 10 mM carbonate, pH 8) for kinetic studies. The fresh buffer medium was aerated with pure oxygen to increase the dissolved-oxygen concentration to levels that would not be fully consumed by ammonia degradation during the experiment. To verify that no adverse effects occurred from the elevated dissolved-oxygen concentrations, baseline experiments (data not shown) for ammonia degradation were conducted before commencing with the batch experiments at the higher oxygen levels. When these experiments were compared with those conducted at the higher dissolved-oxygen concentration, kinetic parameters were similar. In addition, previous work by Uemoto et al. (29) suggested that exposure to high dissolved-oxygen concentrations of a longer duration (>12 h at greater than 30% O2 for suspended cultures) than that in these experiments (60 to 80 min) would be needed to see any adverse effects.

The approach described previously by Aziz et al. (5) was used in this research. Briefly, batch kinetic assays were carried out headspace free in 250-ml or 500-ml glass gas-tight syringes. Each syringe contained a small Teflon-coated stir bar so that the contents were well mixed using a magnetic stirring plate and wrapped in aluminum foil to prevent inactivation by light. The chemicals to be studied were injected through the nose of the syringe to start an experiment. Samples for measuring chemical concentrations, cell concentrations, pH, and dissolved oxygen were collected over time by depressing the syringe plunger and ejecting the samples into a smaller gas-tight syringe. Thus, headspace-free conditions were maintained throughout the experiment, thereby virtually eliminating the loss of chemicals through volatilization. Also, control experiments under abiotic conditions showed no loss of THMs from the syringe (data not shown).

The kinetic experiments were run rapidly (60 to 80 min) to avoid significant changes in the metabolic state of the organisms during the experiments. Five batch kinetic experiments were performed at room temperature (22 to 23°C). Nominally, the starting individual THM concentrations were 100 µg/liter each with either 4 mg/liter (experiment 3) or 8 mg/liter (experiments 1, 2, 4, and 5) of total reduced inorganic nitrogen (TRIN) present initially. TRIN represents the sum of ammonia-nitrogen (NH3-N) and ammonium-nitrogen (NH4+-N). From these experiments, ammonia kinetic and THM cometabolism kinetic parameters were determined. Bicinchoninic acid protein analysis was performed with bovine serum albumin standards to determine the protein content of the N. europaea culture in order to compare our data with those of Ely (8).

Low-temperature experiment.
A low-temperature experiment was conducted to determine the effect of temperature on kinetics. This experiment was undertaken by running two batch kinetic experiments simultaneously, one at room temperature (22°C) and one in a temperature-controlled, 14°C room. Samples were taken from the syringe in 10-ml volumes and measured with an alcohol thermometer before the experiment was started to ensure that the temperature of the medium was equal to the temperature in the room. The bacteria were kept at 14°C for approximately 30 min before the start of the experiment. The nitrifiers used in the simultaneous experiments were taken from the same batch reactor, and biomass, ammonia, and THM initial concentrations for each experiment were equivalent so that temperature was the only variable in the experiment.

Transformation capacity experiments.
Transformation capacity (Tc) represents the maximum mass of cometabolite than can be degraded per unit cell mass or, in other words, the mass of cometabolite degradation required to completely inactivate the cells (1). Product toxicity was established and transformation capacities were calculated in experiments run in a manner similar to that for the batch kinetic experiments. In these experiments, however, the organisms were exposed to a higher concentration of THMs for a longer period of time. The experiments were also conducted with increased initial ammonia and dissolved-oxygen concentrations to compensate for the increased experiment duration. The completion of the experiment was defined as the point when the nitrifiers ceased to degrade ammonia with excess ammonia and dissolved oxygen present. This criterion ensured that the cells were inactivated because of product toxicity and not because of some other limiting reactant.

Determination of kinetic parameters.
For ammonia kinetics, Monod kinetic coefficients were estimated by nonlinear regression analysis using the Solver routine in Excel. The model was formulated (Table 1) to account for the fact that TRIN was measured experimentally, while NH3 is the only form that attaches to the active site of the AMO (27); therefore, pH was inherently included in the model to convert from measured TRIN to NH3 through the use of a common acid/base parameter, {alpha}1, using measured pH values. A fourth-order Runge-Kutta numerical approximation of the Monod equation was fitted to the data by minimizing the normalized residual sum of squares (NRSS) between the predicted and the experimental values. The normalization was achieved by dividing the residual sum of squares by the experimental value squared, resulting in a dimensionless sum-of-squares error (5). For THMs, four different kinetic models were initially evaluated. For each of these four models, the same fitting method was performed as for the ammonia kinetics, and if required, the ammonia kinetic parameters determined in this research were used in the THM parameter determination without adjustment. As a result, the only adjustable parameters for the THM kinetic models were the THM rate constant and the initial concentration of each THM. A summary of the kinetic models and their major assumptions is provided in Table 1. The nonlinear regression analysis yielded estimates of THM rate constant (k1THM), ammonia maximum specific rate of degradation (kTRIN), and ammonia half-saturation constant (KSNH3-N) as well as the initial concentrations for ammonia and each THM. Further statistical analyses permitted estimates of the approximate 95% joint confidence limit (CL) for each parameter (19, 25, 26). For the batch kinetic experiments, transformation capacity was ignored in estimating the kinetic parameters because of the small amount of transformation capacity utilized in these experiments (12 to 26%, with an average of 19%). The validity of this assumption was confirmed by analyzing all the data from experiment 2, which started at 8 mg/liter TRIN, and a subset of the data at concentrations of 4 mg/liter TRIN and below, which corresponded to a point where significant THM transformation had occurred. In addition, 4 mg/liter matched the starting concentration of experiment 3, which was conducted with the same batch of organisms as in experiment 2. The ammonia kinetic parameters were essentially the same for all three analyses (data not shown), indicating that by-product toxicity can be ignored in kinetic experiments that consume a small fraction of the transformation capacity.


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  		TABLE 1. Summary of ammonia and THM kinetic models evaluated in kinetic experiments

Analytical methods.
The concentrations of individual THM species were analyzed on a Hewlett Packard 5890A gas chromatograph equipped with an autosampler. A J&W DB-5 column was used with constant pressure and splitless injection. The initial oven temperature was 32°C and was held for the first 3.5 min. The temperature was then increased at a rate of 20°C/minute to a temperature of 72°C, and the oven remained at this temperature for another 3.5 min. The total time for an analysis was 9 min. THM concentrations for transformation capacity calculations were determined using the same gas chromatograph but with a slightly modified method due to the higher concentrations of THMs required for this type of experiment. The initial temperature of 32°C was held for 9 min. The oven temperature was increased to 40°C at a rate of 20°C/minute and remained at this temperature for 3 min. The oven temperature was increased again at the same rate to 72°C and stayed at this temperature for 4 min, resulting in a total injection time of 20 min.

An Agilent 8453 UV-visible spectrophotometer was used to measure ammonia according to the HACH colorimetric method 10023 (salicylate method) using HACH Low Range Test 'N Tube Nitrogen-Ammonia AmVer reagent sets for 0.02 to 2.5 mg/liter NH3-N. The concentration of biomass was measured on an Agilent 8453 UV-visible spectrophotometer at a wavelength of 600 nm and by analysis of total suspended solids (TSS) (11). The two techniques provided comparable results. Dissolved oxygen was measured with a YSI 5905 probe on a YSI model 54ARC oxygen meter. pH was measured with an Orion model 920A pH/ISE electrode.


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RESULTS AND DISCUSSION
 
Model selection and kinetic parameter determination.
Four models were evaluated for describing the kinetics of THM cometabolism; they differed based on the assumptions made (Table 1) about the presence or absence of competition between ammonia and THMs and whether or not the reductant (NH3) is viewed as a second limiting reactant. Based on the analysis of the four THM degradation models, the reductant model, as described previously by Arcangeli and Arvin (4), was chosen, as it best represented the experimental data by visual inspection and NRSS analysis. As an example of this comparison, Table 2 provides the NRSS comparison for experiment 3. The absence of competition among the THMs is in accordance with previous work of Aziz et al. (5), where no competition was observed between cometabolites at low concentrations (<~5 mg/liter). The absence of competition between THMs is not a direct conclusion of this research but rather is implied from the modeling results. No competition was observed between TRIN and the THMs at the TRIN concentrations tested (<8 mg/liter as N), as evidenced by the better fit of the reductant model in comparison to the competition model.


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  		TABLE 2. Experiment 3 NRSS comparison between THM kinetic models evaluated for batch kinetic experiments

Figure 1 details experimental and model results for experiment 1 for all four THMs and TRIN using the reductant model for THM kinetic parameter determination. The fit of the data shown in Fig. 1 is typical of all experiments. A summary of the ammonia and THM kinetic parameters determined from the experiments is presented in Tables 3 and 4, respectively. The Monod half-saturation coefficient for ammonia ranged from 0.088 to 0.27 mg/liter NH3-N, and the maximum specific substrate utilization rate ranged from 1.9 to 4.3 mg TRIN/mg TSS/day, which are typical kinetic coefficients for nitrifiers (8, 18).



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  		FIG. 1. Typical batch kinetic experiment showing a reductant model kinetic model fit to experimental data for experiment 1.


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  		TABLE 3. 95% joint CL summary for ammonia kinetic parameters ( and kTRIN) determined during batch kinetic experiments


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  		TABLE 4. THM kinetic parameter summary and THM:TRIN kinetic coefficient ratios from batch kinetic experiments

The THM rate constants ranged from 0.050 to 0.16 liters/mg TSS/day for TCM, 0.081 to 0.22 liters/mg TSS/day for BDCM, 0.11 to 0.28 liters/mg TSS/day for DBCM, and 0.14 to 0.34 liters/mg TSS/day for TBM. Based on previous work of Segar et al. (21), treatment process feasibility has been demonstrated for cometabolic degradation reactors operating with rate constants of 0.03 to 0.1 liters/mg/day. The values determined for the THMs meet this requirement. In addition, these rate constants fall in the middle of the range observed in the literature for the breadth of halogenated aliphatic chemicals and bacteria that have been tested (3). Taking this in total, the THM rate constants seen in these experiments indicate that cometabolism occurred at rates that are typical and at a level that suggests practical feasibility for process implementation.

A protein content of 0.4 mg albumin protein per mg TSS was determined and was used to convert Ely's kinetic coefficients to the units shown in Tables 3 and 4. Although the TCM rate constants were lower than those reported previously by Ely, the THM degradation rates () varied in proportion to the ammonia degradation rate (kTRIN), as shown in Table 4, and the ratios for TCM compare well with that determined previously by Ely (8). The similar ratios among experiments for each THM suggest that the differences in observed rate constants shown in Table 4 may have resulted from differing enzyme levels or activity among the different batches of organisms.

Table 4 also indicates that as THM species became more bromine substituted, the magnitude of the rate constants increased. Keener and Arp (13) previously found that chlorinated compounds were generally less effective inhibitors of ammonia oxidation than brominated compounds, with the iodinated compounds being the most inhibitory of the monohalocarbons studied. They proposed that this trend may be related to the increasing size of halogen atoms or nucleophilicity (both orders are Cl < Br < I). Their proposed active-site model for AMO predicts that THMs would bind at a hydrophobic site, where NH3 also binds. Because bromine is more nucleophilic (less electrophilic) than chlorine, as bromine substitution increases, the hydrogen on the THM might reduce its partial charge and become relatively more hydrophobic, making it more attractive to the AMO active site. Therefore, the relative nucleophilicity of bromine compared to chlorine is the probable cause of the difference in the rate constants observed among the THM species.

Estimation of confidence limits for the kinetic coefficients.
Table 3 summarizes the analysis of the ammonia kinetic parameters for all the experiments with the corresponding 95% joint CLs. This analysis was also completed for the THM kinetic parameters (a summary of these results is shown in Fig. 2). The 95% joint CL analysis shows that the rate constants for both ammonia and THM degradation were similar across experiments. Looking at the individual THM rate constants, a statistically significant difference existed between TCM and TBM, verifying the general trend of increasing degradation rates with an increase in bromine substitution on the THM.



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  		FIG. 2. THM 95% joint CL summary for batch kinetic experiments.

Temperature effects.
The THM rate constants at 14°C ranged from 35% to 50% of their values at 22°C, and the impact of decreased temperature increased with an increasing degree of THM bromine substitution. Correspondingly, the calculated activation energies varied from 59 kJ/mol for TCM to 92 kJ/mol for TBM (Table 5). The reason for the apparent trend in activation energies, increasing with THM bromine substitution, is not obvious. The Monod maximum specific substrate utilization rate was affected to an even greater extent than the THM kinetic coefficients and was only 24% of its value at 22°C. Because the 14°C experiment did not progress to an adequately low TRIN concentration, the could not be determined from the experimental data; therefore, the was fixed to the value determined from the 22°C experiment in the data analysis. Overall, the temperature effect on the THM cometabolism rate was less than that on the nitrification rate but was nevertheless significant.


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  		TABLE 5. Comparison of temperature effects on kinetic parameters between room temperature kinetic experiment (22°C) and reduced-temperature kinetic experiment (14°C)

The activation energy for the TRIN maximum specific substrate utilization rate was calculated as 130 kJ/mol. This value is greater than, but similar to, values reported previously by Wong-Chong and Loehr (31) for ammonia oxidation (67 to 90 kJ/mol). When THM and TRIN activation energies are compared, it seems reasonable that those for the THMs should be less than that for the TRIN maximum specific substrate utilization rate. Relative to its primary substrate, an enzyme should not be as efficient in catalyzing the reaction of a cometabolite; therefore, only chemicals with activation energies less than that of the primary substrate should be amenable to cometabolism. Using the calculated activation energies, the THM rate constants maintain the trend of increasing with increasing bromine substitution over the range of expected applicable temperatures in practice (approximately 10 to 30°C).

Transformation capacity.
Product toxicity associated with the cometabolism of chlorinated aliphatics is most often described by a transformation capacity (Tc) term (1). Mathematically, Tc is defined as follows:

(1)
where Tc is the transformation capacity (micrograms THM per milligram TSS or nanomoles THM per milligram TSS), is the initial THM concentration (micrograms per liter THM or nanomolar concentration of THM), is final THM concentration (micrograms per liter THM or nanomolar concentration of THM), and X is the initial biomass concentration (milligrams per liter TSS).

For a Tc experiment to reach completion, ammonia degradation must stop with excess TRIN and oxygen present. This assures that the cells have been inactivated from the degradation of the THMs and not from some other limiting reactant. Experiments with high concentrations of individual THMs and several experiments with all four THMs present were conducted with various initial THM and cell concentrations to arrive at an experimental design that would meet these requirements. Figure 3 shows an experiment run with all four THMs simultaneously to examine the combined toxicity of the four THMs and to determine if this combined toxicity could be predicted from the individual Tc values. Ammonia, DBCM, and TBM degradation ceased at approximately 200 min, but TCM and possibly BDCM degradation continued beyond this point. Nevertheless, the organisms were considered to be inactivated at the point where ammonia degradation ceased, as the bacteria were no longer able to degrade their energy source. Keener and Arp (13) have proposed a two-site model for AMO that may account for the continued degradation of TCM and BDCM once ammonia degradation ceased.



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  		FIG. 3. Transformation capacity experiment with a THM mixture.

Table 6 details each THM's Tc on both a mass and a molar basis and compares the experiments with individual THMs to that with all four THMs present simultaneously. TCM had by far the lowest by-product toxicity, with a transformation capacity of 77 nmol TCM/mg TSS. The presence of any bromine substitution caused a substantial increase in product toxicity; transformation capacities ranged from 45 nmol/mg for BDCM to 22 nmol/mg for TBM. The increase in product toxicity associated with increasing THM bromine substitution may be a result of the expected half-lives of the intermediates formed during cometabolism for each THM. It would be expected that the proposed intermediate half-lives decrease with increasing bromine substitution (20). As THM bromine substitution increases, the shorter half-life would translate into the intermediates not diffusing away from the enzyme before reacting with it, possibly leading to a greater product toxicity as THM bromine substitution increases.


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  		TABLE 6. Transformation capacities for single solutes and a mixture of all THMs

In considering the mixture, it was assumed that each THM contributed to the overall toxicity in proportion to the mass of the THM degraded and its relative toxicity. Thus, the mass of each THM degraded was normalized by the total mass of each THM that would have been required to inactivate the cells had that THM been present individually. In this way, the percentage of Tc realized in the mixture for each THM was calculated. These percentages where then summed for all four THMs to arrive at the overall percentage of the Tc that was utilized, with a value of 100% indicating a perfect match between the assumed contributions to toxicity and the results observed. The calculated percentage was 103%, which verified the approach for estimating the combined effect of product toxicity in mixtures.

The results of the Tc experiments in this research compared to values measured previously by others are shown in Table 7. The Tc determined for the TCM compared well with the literature value for TCM, which had a relatively low toxicity compared to the other chemicals. BDCM, DBCM, and TBM, however, showed toxicity comparable with 1,1-dichloroethene, a chemical with high product toxicity. The relative differences in Tc values among the THMs indicate that both the relative speciation of THMs and their individual concentrations will be important in determining the probable toxicity associated with their degradation. Also, the trends of increasing product toxicity and increasing THM rate constants as bromine substitution increases illustrate an inherent tradeoff between reaction kinetics and product toxicity. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance. Thus, both kinetics and product toxicity must be evaluated for process implementation, especially when significant concentrations of bromine-substituted THMs are present.


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  		TABLE 7. Transformation capacity comparison for N. europaea with different chemicals


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ACKNOWLEDGMENTS
 
This research was funded by the American Water Works Association Research Foundation and the Texas Advanced Technology Research Program, which we thank for their financial, technical, and administrative assistance with the project.

The comments and views detailed herein may not necessarily reflect the views of the American Water Works Association Research Foundation or its officers, directors, affiliates, or agents.


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FOOTNOTES
 
* Corresponding author. Mailing address: The University of Texas at Austin, 1 University Station, Austin, TX 78712. Phone: (512) 471-4996. Fax: (512) 471-0592. E-mail: speitel{at}mail.utexas.edu. Back


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Applied and Environmental Microbiology, December 2005, p. 7980-7986, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7980-7986.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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