New Insights into Methyl Bromide Cooxidation byNitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions

ABSTRACT We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (∼6 × 107 cells ml−1) of the NH3-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH4+ and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH4+, 18% of the NH4+, and 35% of the NH4+, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH4+-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells−1). Approximately 90% of the NH3-dependent O2uptake activity and the NO2−-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO2−production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO2− accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO2−-producing and MeBr-oxidizing activities could not be attributed directly to NH4+ or NH3 limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO2− and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH3-oxidizing activity.

Nitrosomonas europaea, a chemolithoautotrophic NH 3 oxidizer, oxidizes a variety of compounds, including alkanes, alkenes, alkynes (6,10), halogenated hydrocarbons (12,18,27), and aromatic compounds (9,13), with ammonia monooxygenase (AMO). AMO is the broad-substrate-range oxygenase that is responsible for oxidation of NH 3 to hydroxylamine (NH 2 OH), the first step in oxidation of NH 3 to NO 2 Ϫ (30). Previously described studies of cooxidation of halogenated hydrocarbons by NH 3 -oxidizing bacteria have focused primarily on determining the range of compounds cooxidized by N. europaea (6,7,13,(16)(17)(18) and, to a lesser degree, on kinetic parameters (12). The majority of these studies were conducted by using short incubation periods (Յ1 h), high-density cell suspensions (10 9 to 10 11 cells ml Ϫ1 ) exhibiting high rates of NO 2 Ϫ production (ϳ3 mol ml Ϫ1 h Ϫ1 ), and pH values considered to be optimal for NH 3 oxidation (pH 7.8 to 8.0). Comprehensive studies have not been performed yet with lowerdensity cell suspensions (Ͻ10 8 cells ml Ϫ1 ) that exhibit NH 3 oxidation rates more typical of environments like nitrifying bioreactors (ϳ0.1 mol of NH 4 ϩ ml Ϫ1 h Ϫ1 ) (1,2), in which cooxidation may occur (15,23). Furthermore, the sustainability of cooxidation and the relationship of cooxidation to NO 2 Ϫ production could not be examined adequately in our previous studies because total ammonium (NH 4 ϩ plus NH 3 ) became limiting very quickly because of high rates of consumption and because a decrease in pH reduced NH 3 availability. The issue of sustainability and the factors that affect it need to be studied in order to understand the long-term effects of cooxidation of halogenated hydrocarbons on NH 3 oxidizers and to better assess the potential use of these organisms in bioremediation of contaminants.
Methyl bromide (MeBr) is a soil fumigant that is used to control weeds, soilborne plant pathogens, and nematodes (25,29,31). MeBr has been categorized as a class 1 ozone-depleting chemical by the U.S. Environmental Protection Agency and is scheduled for complete phase-out within a few years (25). Thus, the fate of MeBr has some applied significance, and this compound also is an excellent model compound for examining cooxidation by N. europaea because the end products of MeBr cooxidation (formaldehyde and HBr) have been identified (11,12,17). The objective of this study was to examine cooxidation of MeBr by a moderately low-density suspension of N. europaea cells (ϳ6 ϫ 10 7 cells ml Ϫ1 ) that oxidized NH 3 at a rate similar to the rates measured in nitrifying bioreactors (1,2). with orbital shaking (150 rpm). The growth medium consisted of 25 mM (NH 4 ) 2 SO 4 and other constituents as described elsewhere (4). Cells were harvested by centrifugation (11,000 ϫ g, 15 min) after the late exponential phase was reached (3 days), washed twice in buffer (50 mM potassium phosphate, pH 7.2), and resuspended in buffer to an optical density at 660 nm of approximately 1.0. All assays were initiated with aliquots of this cell suspension within 1 h of preparation. Epifluorescence microscopic counting of 4Ј,6Ј-diamidino-2-phenylindole (DAPI)-stained cells confirmed that the cell density was 1.2 ϫ 10 9 Ϯ 0.3 ϫ 10 9 cells ml Ϫ1 when the optical density at 660 nm was 1.0. The average dry weight of the cell suspension was 0.32 Ϯ 0.08 mg ml Ϫ1 .
Preparation of assay vials. Portions (5 ml) of sterile assay buffer were added to sterile glass vials (capacity, 74 ml), which were sealed with gray butyl stoppers (Kimble, Owens, Ill.) and aluminum crimp top seals (Wheaton, Millville, N.J.). The assay buffer contained 5 mM (NH 4 ) 2 SO 4 and 50 mM KH 2 PO 4 -K 2 HPO 4 (pH 7.2) unless otherwise noted. An MeBr stock vial was prepared by flushing a sealed vial containing 5 to 10 glass beads for 1 min (with periodic shaking to disrupt air pockets) with MeBr (99.5% pure; Matheson Gas Products, Inc., Newark, Calif.) from a lecture bottle. After an overpressure of gas was established in the stock vial, appropriate amounts of MeBr were added to assay vials by using gas-tight Hamilton microsyringes equipped with sidebore needles. Experiments were initiated by adding an aliquot of the cell suspension to each vial so that the final cell density was 6 ϫ 10 7 Ϯ 1.5 ϫ 10 7 cells ml Ϫ1 (16 Ϯ 4 g [dry weight] of cells ml Ϫ1 , 80 Ϯ 20 g vial Ϫ1 ) unless otherwise noted. The vials were inverted and incubated in the dark at 27°C.
Analytical procedures. MeBr oxidation was measured by monitoring the disappearance of MeBr from the gas phase in the assay vials by using a Shimadzu model GC-14 gas chromatograph. The gas chromatograph was equipped with a stainless steel column (outside diameter, 0.32 cm; length, 91 cm) packed with Porapak-Q (80-100 mesh; Waters Associates Inc., Framingham, Mass.) (column temperature, 120°C) and a flame ionization detector (detector and injector temperature, 200°C) interfaced with a Shimadzu model CR501 integrator. At time intervals, 60-to 200-l aliquots of headspace gas were removed from the assay vials with a gas-tight Hamilton microsyringe equipped with a sidebore needle. To check for AMO-independent oxidation of MeBr, we included control vials that contained 1% (vol/vol) acetylene, a specific mechanism-based inactivator of AMO (4). By using these controls we determined that about 10 and 20% of the MeBr that disappeared from vials in 24-h assays and 48-to 72-h assays, respectively, could be considered AMO independent. These quantities were routinely subtracted from the MeBr depletion values that were obtained with vials that did not contain acetylene. The amounts of MeBr in the vials were determined by comparison with standards containing known amounts of MeBr in 74-ml vials containing 5 ml of sterile assay buffer. A dimensionless Henry's Law constant for MeBr of 0.25 was utilized (24), and approximately 22% of the total amount of MeBr added to the vials partitioned into the liquid phase. For all assays the amount of MeBr added was expressed as the concentration in the liquid phase, while the amount transformed was expressed in micromoles. For purposes of comparison, when 10 mol of MeBr was added to a vial, the aqueous phase concentration of MeBr was 0.44 mM. NO 2 Ϫ production was determined by removing aliquots (20 to 100 l) of cell suspensions from the sealed vials with a gas-tight syringe and determining the NO 2 Ϫ contents colorimetrically (3). Response of the initial rate of MeBr oxidation to cell density, pH, and NH 4 ؉ and MeBr concentrations. (i) Cell density. Aliquots of diluted cell suspensions of N. europaea were added to triplicate assay vials containing buffer, 10 mM NH 4 ϩ , and 0.5 to 3 mol of MeBr vial Ϫ1 (aqueous concentration, 0.02 to 0.13 mM) in order to obtain cell densities of 6 ϫ 10 6 , 6 ϫ 10 7 , and 6 ϫ 10 8 cells ml Ϫ1 . Samples of headspace gas were recovered at 3-to 12-h intervals for up to 3 days and examined to determine if MeBr depletion could be measured.
(ii) pH. N. europaea cell suspensions were prepared in phosphate buffer preadjusted to pH 6.2, 7.2, or 8.2. One-milliliter aliquots (6 ϫ 10 7 cells ml Ϫ1 ) were injected into vials that already contained either 0.11, 0.22, or 0.44 mM MeBr and 10 mM NH 4 ϩ in the appropriate buffer. Samples of the headspace gas and liquid contents of the vials were obtained and used to determine the MeBr and NO 2 Ϫ contents at 3-to 6-h intervals over a 24-h period.
(iii) NH 4 ؉ and MeBr concentrations. N. europaea cells (6 ϫ 10 7 cells ml Ϫ1 ) were incubated in a factorialized design experiment with combinations consisting of 0.11, 0.22, or 0.44 mM MeBr and 2.5, 5, or 10 mM NH 4 ϩ . MeBr oxidation and NO 2 Ϫ production were monitored at 2-to 6-h intervals. The rates of MeBr oxidation and NO 2 Ϫ production were calculated by using the linear regression feature in SigmaPlot 3.0 (Jandel Scientific, San Rafael, Calif.). The correlation coefficients for all regressions were Ն0.96. The initial rates were expressed in micromoles of MeBr oxidized or NO 2 Ϫ produced per milligram (dry weight) per hour.
Examination of the factors that might influence the sustainability of MeBr oxidation. (i) Influence of buffer, growth medium, and Na 2 CO 3 on the sustainability of MeBr oxidation. N. europaea cells (6 ϫ 10 7 cells ml Ϫ1 ) were incubated in either phosphate buffer or complete growth medium (pH 7.2) that was supplemented or not supplemented with Na 2 CO 3 (4 mM). The pH of each solution was adjusted to 7.2, and 4 mM Na 2 SO 4 was added to vials that did not receive Na 2 CO 3 . Both the cell suspensions and the NH 4 ϩ stock solution were prepared by using the appropriate buffer or growth medium. MeBr disappearance was monitored over a 36-h period.
(ii) Influence of NH 4 ؉ limitation or cell inactivation on the sustainability of MeBr oxidation. To determine if either NH 4 ϩ limitation or cell inactivation was the primary reason for the loss of MeBr-oxidizing activity, a preparation containing 6 ϫ 10 7 cells ml Ϫ1 was incubated with 10 mM NH 4 ϩ and 0.22 mM MeBr until the initial rate of MeBr oxidation declined (after approximately 9 h of incubation). Then replicate vials received either (i) additional cells (3 ϫ 10 8 cells), (ii) additional cells plus NH 4 ϩ (equivalent to an additional 10 mM), or (iii) NH 4 ϩ alone. MeBr oxidation and NO 2 Ϫ production were monitored for another 15 h.
(iii) Effects of the end products of NH 3 and MeBr oxidation, NH 4 ؉ depletion, and pH decline on sustainability of MeBr oxidation. The effects of accumulation of the end products of MeBr and NH 3 oxidation on the sustainability of MeBr oxidation were examined by monitoring the oxidation of 0.22 mM MeBr in 50 mM phosphate buffer (pH 7.0) supplemented with 7.5 mM NH 4 ϩ . Factorialized combinations consisting of 2.5 mM NO 2 Ϫ and 0.4 mM formaldehyde were added to the assay vials; these concentrations were chosen because they represented the approximate conditions in the assay vials after 24 h of oxidation of 0.22 mM MeBr and 10 mM NH 4 ϩ . The reactions were started by adding 1-ml aliquots of cells suspended in phosphate buffer (pH 7.0), and MeBr disappearance and NO 2 Ϫ production were monitored as described above. (iv) Influence of inhibition of protein synthesis on NO 2 ؊ production during MeBr cooxidation. Either chloramphenicol (final concentration, 200 or 400 g/ ml) or kanamycin (10 to 50 g/ml) was dissolved in 50 mM phosphate buffer (pH, 7.2). Aliquots of the buffer were then supplemented with 10 mM NH 4 ϩ and injected into sealed vials containing enough MeBr so that the MeBr concentration in the aqueous phase was 0.22 or 0.44 mM. The reactions were started by adding N. europaea (6 ϫ 10 7 cells ml Ϫ1 ), and NO 2 Ϫ production was monitored during 7 h of incubation.
Residual NH 4 ؉ -and NH 2 OH-dependent O 2 uptake activity after oxidation of MeBr. To further examine the effect of MeBr oxidation on the residual activity of N. europaea, cells (6 ϫ 10 7 cells ml Ϫ1 ) were incubated with 10 mM NH 4 ϩ and 0.11, 0.22, or 0.44 mM MeBr for 24 h. Following incubation, the vials were opened and vented for 5 min. The cell suspensions were filtered through 25-mm diameter 0.4-m-pore-size polycarbonate filters and washed by filtering 9 ml of sterile buffer over the cells. Control vials containing cells, 10 mM NH 4 ϩ , and no MeBr were treated exactly like the vials that contained MeBr were treated. To determine residual O 2 uptake activity, the filters were placed in 2-ml portions of buffer, and the cells were washed off with gentle shaking. An aliquot of the cell suspension (1.6 ml, 64 g [dry weight] of cells) was added to an O 2 electrode chamber. After 3 to 5 min of stirring, NH 4 ϩ (final concentration, 10 mM) was added to the chamber, and the NH 4 ϩ -dependent O 2 uptake rate was measured over a 2-to 5-min interval. NH 4 ϩ -dependent O 2 uptake was stopped by adding 1-allyl-2-thiourea (final concentration, 0.1 mM), a reversible inhibitor of AMO (14). Subsequently, NH 2 OH (final concentration, 0.6 mM) was added to the chamber to measure NH 2 OH-dependent O 2 uptake.
Recovery of NO 2 ؊ -producing ability by N. europaea after oxidation of 0.44 mM MeBr for 24 h. N. europaea (6 ϫ 10 7 cells ml Ϫ1 ) was exposed to 0.44 mM MeBr in phosphate buffer (pH 7.2) for 24 h as described above. Vials containing cells that were not exposed to MeBr were included as controls. Cells were harvested from the buffer, washed, and resuspended in complete growth medium at either pH 7.2 or pH 8. Unexposed cells were diluted another 10-fold in growth medium so that the initial rates of NO 2 Ϫ production were similar for both exposed and unexposed cells. At 6-h intervals, samples of cells were recovered, and NO 2 Ϫ production was determined as described above.

RESULTS
In preliminary experiments (data not shown) we established that oxidation of MeBr at concentrations up to 0.44 mM could be measured accurately with a cell density of 6 ϫ 10 7 cells ml Ϫ1 (16 g [dry weight] of cells ml Ϫ1 ). Oxidation of MeBr at concentrations of Ͼ0.66 and Յ0.88 mM could be measured, but the rates decreased rapidly after only 1 to 4 h of incubation and thus were not studied in detail. At cell densities of Ͻ10 7 cells ml Ϫ1 , the incubation time required to accurately measure MeBr disappearance with a gas chromatograph equipped with a flame ionization detector was 24 h or more, and such determinations could be made only at low MeBr concentrations (Յ0.04 mM). The MeBr-oxidizing ability of N. europaea was examined at three pH values (pH 6.2, 7.2, and 8.2) representing the range of pH values likely to be encountered in many natural environments. At each of the three MeBr concentrations evaluated (0.11, 0.22, and 0.44 mM) MeBr was oxidized significantly faster at pH 7.2 than at either pH 6.2 or pH 8.2 (data not shown). After 24 h of incubation the pH had changed very little in the pH 7.2 preparation (final pH, 7.0 to 7.1), which implied that the buffering capacity of 50 mM phosphate was Ϫ production decreased threefold as the NH 4 ϩ concentration decreased from 10 to 1 mM. In the presence of 0.22 mM MeBr, the initial rates of MeBr oxidation were relatively insensitive to changes in the NH 4 ϩ concentration at concentrations between 1 and 10 mM, despite the fact that the level of NO 2 Ϫ production changed fivefold over this concentration range. In the presence of 0.44 mM MeBr, the initial rate of MeBr oxidation responded to most incremental changes in the NH 4 ϩ concentration and decreased at NH 4 ϩ concentrations between 5 and 2.5 mM and between 2.5 and 1 mM. When we examined the ratio of amount of MeBr oxidized to amount of NO 2 Ϫ produced (M/N ratio), we found that the maximum initial rates of MeBr oxidation occurred at almost identical M/N ratios (the M/N ratios were 0.24, 0.21, and 0.21 for MeBr concentrations of 0.44, 0.22, and 0.11 mM, respectively) regardless of the MeBr and NH 4 ϩ concentrations. We also observed another trend: M/N ratios of Ն0.30 and Ͻ0.1 were associated with suboptimal initial rates of MeBr oxidation.
The rates of MeBr oxidation invariably declined when the cells were incubated for more than 12 h, and they declined to zero within 12 to 24, 36 to 48, and 48 to 72 h in the presence of 0.44, 0.22, and 0.11 mM MeBr, respectively (Fig. 1a). Although the rate of NO 2 Ϫ production was constant for at least 12 h in the control lacking MeBr, in the presence of 0.11, 0.22, and 0.44 mM MeBr the rates of NO 2 Ϫ production were constant for approximately 12, 6, and 3 h, respectively, and then gradually declined to zero over ranges of time similar to the ranges of time as described above for MeBr oxidation (Fig.  1b). Whereas the rates of oxidation of 0.11, 0.22, and 0.44 mM MeBr definitely decreased at different times (36 to 48, 24 to 36, and 12 to 24 h, respectively), the corresponding rates of NO 2 Ϫ production were very similar (2 to 3 mol of NO 2 Ϫ produced mg [dry weight] of cells Ϫ1 h Ϫ1 ). Despite relatively large differences in the rate and duration of active MeBr oxidation for the different NH 4 ϩ -MeBr combinations, similar amounts of MeBr were oxidized with a large number of the NH 4 ϩ -MeBr combinations (1 to 2 mol per vial, 10 to 20 mol mg [dry weight] of cells Ϫ1 ) ( Table 2). In general, this was attributed to the fact that while the rates of MeBr oxidation were about two-to threefold lower when 0.11 MeBr was used than when 0.22 and 0.44 mM MeBr were used, the length of the period of active oxidation was inversely proportional to the MeBr concentration (e.g., 72, 48, and 24 h) ( Table 2).
We obtained no evidence which supported the possibility that either (i) NO 2 Ϫ and/or formaldehyde accumulation, (ii) a decrease in pH (pH 7.2 to 7.0), (iii) NH 4 ϩ limitation (10 to 7.5 mM), or (iv) inadequate medium composition (buffer versus growth medium) was directly responsible for the decreases in NO 2 Ϫ -producing ability and MeBr-oxidizing ability. For example, when assays were initiated at pH 7 in the presence of 7.5  a Because the durations of the initial rates of NO 2 Ϫ production and MeBr oxidation varied considerably for the different combinations of NH 4 ϩ and MeBr, the linear regression feature of SigmaPlot 3.0 was used to determine the time interval over which rates were constant. The rates are expressed in micromoles per milligram (dry weight) per hour.
b The values in parentheses are the standard deviations of the means based on the results obtained for three replicates per treatment. mM NH 4 ϩ and different combinations of NO 2 Ϫ (2.5 mM) and formaldehyde (0.3 mM), the characteristics and amounts of MeBr oxidized were similar to the characteristics and amounts obtained under typical assay conditions (Fig. 2). Furthermore, the initial rates of MeBr oxidation and NO 2 Ϫ production could be sustained in the same assay vials for several additional hours if a second aliquot of cells (30 ϫ 10 7 cells) was added to the assay mixture after 9 h of incubation (Fig. 3). Although 10 mM NH 4 ϩ added along with the cells increased the rate of NO 2 Ϫ production, it did not increase the rate of MeBr oxidation to a value greater than the value obtained when only cells were added. The increase in NO 2 Ϫ production without a concomitant increase in MeBr oxidation is consistent with other data which showed that the same rate of oxidation of 0. 22 (Table 3) activities had been lost. Much less of the whole-cell hydroxylamine (NH 2 OH)-dependent O 2 uptake activity was lost (20 to 30%) after exposure to MeBr. Recovery of NO 2 Ϫ production by MeBr-exposed cells was monitored after the cells were resuspended in fresh growth medium (pH 7.2 or 8) containing 20 mM NH 4 ϩ (Fig. 4). Cells that were not exposed to MeBr but otherwise treated identically were diluted 10-fold to obtain a similar initial rate of NO 2 Ϫ production, and these cells were used as a control. We found that the NO 2    Ϫ produced during the first 6 h of incubation by the cells exposed to MeBr was about the same as the amount produced by the unexposed cells, the rate of NO 2 Ϫ accumulation was lower in the former preparation. A 48-to 60-h recovery period was required before the cells exposed to MeBr exhibited the same rate of NO 2 Ϫ production that they exhibited before they oxidized MeBr.

DISCUSSION
By experimenting with moderately low-density cell suspensions we gained insight into characteristics of MeBr oxidation by N. europaea that were not detected in previous studies performed in our laboratory. For example, Rasche et al. (17) and Keener and Arp (12) used 0.5 to 4 mg (dry weight) of cells ml Ϫ1 in their analyses of MeBr oxidation by N. europaea. Because the capacity of N. europaea to transform MeBr is between 10 to 20 mol of MeBr mg (dry weight) of cells Ϫ1 , we know that the quantities of cells used by our colleagues could transform approximately 10-fold more MeBr than the amounts used routinely in these types of studies (2 to 10 mol per assay mixture). It is not surprising, therefore, that they did not determine the finite capacity of N. europaea to oxidize MeBr and that NO 2 Ϫ production and NH 4 ϩ -dependent O 2 uptake activities declined considerably as a consequence of prolonged MeBr oxidation.
At first we were confused by our finding that both NO 2 Ϫproducing and MeBr-oxidizing activities were lost during transformation of MeBr because Rasche et al. (16) had concluded from short-term studies that monohalogenated aliphatic compounds could be degraded by N. europaea without AMO inactivation by the end products of cooxidation. Nonetheless, it has been reported that formaldehyde (28) and NO 2 Ϫ in the absence of NH 4 ϩ (20) inhibit NH 3 oxidation in N. europaea, yet we obtained no evidence that these end products were inhibitory to MeBr oxidation at the concentrations generated in our assays and under our experimental conditions. At this time, however, we cannot rule out the possibility that formaldehyde generated intracellularly might be more toxic to N. europaea than externally applied material is or that some oxidatively generated brominated chemical species might be the cause of toxicity. Although NH 4 ϩ -dependent O 2 uptake was reduced more severely by exposure to MeBr than NH 2 OH-dependent O 2 uptake was reduced, our data indicate that prolonged MeBr oxidation resulted in a more general toxic effect on the cells than inactivation of AMO per se. For example, previous studies in our laboratory showed that when approximately 90% of the NH 4 ϩ -dependent O 2 uptake activity in N. europaea was eliminated by specifically inactivating AMO with strong light, NO 2 Ϫ -producing activity could be restored completely within 4 h of the time when cells were resuspended in fresh growth medium (8). In contrast, our studies showed that cultures exposed to MeBr for 24 h, in which approximately 90% of the NH 4 ϩ -dependent O 2 uptake activity was debilitated, required about 48 to 60 h of incubation to exhibit a rate of NO 2 Ϫ production comparable to the initial rate detected in unexposed cells. Indeed, the effect of long-term oxidation of MeBr on NO 2 Ϫ -producing activity is more similar to what occurred when N. europaea lost approximately 90% of its NH 4 ϩ -dependent O 2 uptake activity during short-term cooxidation of trichloroethylene. In that case, very little NO 2 Ϫ -producing activity was observed after 8 h of incubation, presumably because the cells had suffered too much nonspecific damage during trichloroethylene oxidation (8). Because cells exposed to MeBr exhibit about one-tenth the rate of NO 2 Ϫ production that unexposed cells exhibit, it seems reasonable to conclude that approximately 10% of the cells survived the 24-h MeBr oxidation period and that the exponential recovery of NO 2 Ϫ production probably reflected proliferation of the surviving cells. It is not clear, however, why development of NO 2 Ϫ production by the cells exposed to MeBr lagged behind development of NO 2 Ϫ production by the diluted, unexposed control cells when the two inocula were adjusted so that the initial activities were similar. It is possible that some of the residual NO 2 Ϫ production by the cells exposed to MeBr originated from cells that were no longer capable of cell division. A recent study has shown that when methane-grown Methylocystis trichosporium OB3b oxidizes some chlorinated ethylenes, cell viability decreases more rapidly than the activity of methane monooxygenase decreases (26). Other studies performed in our laboratory have shown that AMO activity can be either upregulated or downregulated in response to NH 4 ϩ availability (21,22), that de novo protein synthesis is extremely limited in cells exposed to 10 mM NH 4 ϩ at pH 7 (5), and that production of the mRNA transcript for AMO is limited when rates of NO 2 Ϫ production are supported by Յ2 mM NH 4 ϩ at pH 7.5 (19). The faster development of NO 2 Ϫ production by the unexposed cells might have been due to upregulation of NH 3 -oxidizing activity (21), and the cells exposed to MeBr might have lacked this ability.
Finally, during the initial optimum phase of cooxidation of 0.44 mM MeBr, the rate of NO 2 Ϫ production declined to approximately 30% of the initial rate before any effect on MeBr oxidation was observed (Fig. 1), and the M/N ratio increased from 0.13 to 0.47. Although it is not known how MeBr oxidation could cause NH 3 oxidation to decrease while it allows MeBr oxidation to continue unabated, cell viability might decrease if reductant generation became insufficient to meet the combined needs of NH 3 oxidation, MeBr cooxidation, and the essential maintenance requirements of the cell.
By carrying out cooxidation experiments with moderately low cell densities before we conducted ecologically based studies, we identified a number of additional physiological and molecular biological questions worth pursuing with N. europaea. Additional studies will be required (i) to determine the mechanism responsible for the MeBr-induced decreases in NH 3 -oxidizing activity and cell viability in N. europaea; (ii) to examine in more detail the sequence of events that occur Ϫ production by N. europaea cells resuspended in fresh growth medium after they were exposed (solid symbols) or not exposed (open symbols) to 0.44 mM MeBr in phosphate buffer (pH 7.2). The growth medium contained 20 mM NH 4 ϩ . Symbols: OE, growth medium, pH 7.2; s, growth medium, pH 8; ‚, growth medium, pH 7.2; ᮀ, growth medium, pH 8. For clarity error bars are not shown. The standard deviations were Յ10% of the mean values regardless of the treatment.
during recovery of cells that have reached their cooxidative transformation capacity; and (iii) to determine if cooxidative activity interferes with regulation of AMO activity and gene regulation in response to NH 4 ϩ availability.