Bicarbonate Uptake by Nitrifiers: Effects of Growth Rate, ph,

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1984, p /84/1211-5$2./ Copyright ) 1984, American Society for Microbiology Vol. 48, No. 6 Bicarbonate Uptake by Nitrifiers: Effects of Growth Rate, ph, Substrate Concentration, and Metabolic Inhibitors L. W. BELSER Cawthron Institlte, Nelson, New Zealand Received 6 April 1984/Accepted 6 August 1984 The ratios of bicarbonate uptake to substrate oxidation were measured for three genera of nitrifying bacteria. The ratios for the two ammonium oxidizers tested were essentially the same;.863 ±.55 and.868 ±.91,umol of bicarbonate were taken up per umol ammonium oxidized for Nitrosomonas europaea and a Nitrosospira strain, respectively. For, a ratio of.236 ±.13,umol of bicarbonate taken up per umol of nitrite oxidized was obtained. Cells were grown in substrate-limited continuous culture and in batch culture, with generation times ranging between 16 and 189 h for the ammonium oxidizers and 18 and 69 h for All ratios appeared to be independent of growth rates and ph. However, shortterm changes in substrate concentration and certain metabolic inhibitors significantly changed the efficiency of bicarbonate uptake. The significance of these results to the application of the nitrapyrin-sensitive bicarbonate uptake method for measuring nitrification rates in natural samples is discussed. Estimating nitrification rates in samples from natural environments by measuring absolute changes in ammonium, nitrite, or nitrate is often very difficult for the following reasons: other organisms can be simultaneously producing and utilizing these ions; the rates of change in the concentration of these ions can be small in relation to the absolute concentrations; and the absolute concentrations of the compound may be near the detection limits of standard analytical methods. To overcome this sensitivity problem, it has been proposed by Billen (3) and Somville (9) that nitrification can be estimated indirectly by measuring bicarbonate uptake by nitrifying bacteria, the underlying assumption being that there is a constant stoichiometric ratio between the rate of substrate oxidation and the rate of bicarbonate uptake for a given species of nitrifier. It must also be assumed in any application of these methods that the uptake ratio is independent of the growth rate and environmental conditions. Recently, Hall (7) questioned whether these ratios are constant. He observed that the bicarbonate uptake method consistently underestimated observed rates of nitrification in the hypolimnion of a mesotrophic lake. There is only a limited amount of information in the literature dealing with the stoichiometry of bicarbonate uptake to substrate oxidation rate. Billen (3), who has summarized the work of others and supplemented this with batch culture studies of his own, estimated the ratio for ammonium oxidizers to be.1 FLmol of bicarbonate taken up per v.mol of ammonium oxidized; the ratio for nitrite oxidizers was estimated to be.2,umol of bicarbonate per Fmol of nitrite oxidized. The purpose of this study was to measure the ratios of bicarbonate uptake to substrate oxidation for representative members of the nitrifiers and to determine whether these ratios were independent of the growth rate and ph. Ratios determined in this study were independent of the growth rate and in good agreement with the estimates of Billen (3). However, it was also found that these ratios could be changed significantly by certain metabolic uncouplers or by rapid changes in substrate concentrations. 11 MATERIALS AND METHODS Cultures. The ammonium oxidizers (Nitrosomonas europaea and ) used in this study have been described previously (2). The nitrite oxidizer Nitrobacter winogradsky was provided by E. L. Schmidt (University of Minnesota, Minneapolis). The base medium was the same for both the ammonium and nitrite oxidizers, except for substrate. This base medium consisted of 1. mg of CaC12, 2.5 mg of MgSO4 * 7H,O, 2 mg of KH2PO4, 1 ml of chelated iron (8), and 1 ml of trace elements (8) per liter of distilled water. The ph of the medium was adjusted with 2.5% Na2CO3. Continuous cultures. Continuous culture vessels were constructed of glass, with Teflon magnetic stirrers and silicone rubber stoppers and tubing. Media flow in and effluent flow out of the vessels were controlled with peristaltic pumps. The vessels were water jacketed, and the temperature was maintained at 25. ±.2 C. The volumes of the vessels ranged from 35 to 1, ml. A ph stat was used in conjunction with one vessel: medium of ph 5.8 was added, which adjusted automatically with 2.5% Na2CO3 to the desired ph. The other culture vessels were run with media previously adjusted to the desired ph. Ammonium oxidizer cultures were run with medium added at ammonium concentrations of.5 and 2.5 mm, and the nitrite oxidizer cultures had medium added with a substrate concentration of 1. mm. Vessels were normally inoculated with 1 ml of exponentially growing cells (between 16 to 17 cells in total); cells were allowed to grow exponentially in the vessels as a batch culture until 5 to 9% of the substrate was oxidized before the pumps were turned on. Flow rate and nitrite concentrations were measured daily, and ph and ammonium concentrations were measured periodically. The steady state was assumed to be obtained when the nitrite concentration remained constant with time. Purity of the cultures was checked weekly (8). Nitrite and ammonium were analyzed as described previously (1). Bicarbonate uptake experiments. Periodically during the

2 VOL TABLE 1. Growth conditions for nitrifying bacteria and ammonium oxidizers Expt Generation time (h) ph Substrate concn (mm) Continuous culture of nitrifying bacteria ± ± ± ± ± ± ± ± ± ± ±.5 Nitroso,nonas sp ± ± ±.5 3"' 189. ± ±.55 4"1 5.2 ± ± ± ± ±.1 2) ± Batch cultures of ammonium oxidizers Nitrosoinonas sp ± ±.2 > ± ±.1 > ± > ± >.3 "Contaminated cultures. study, 6- to 15-ml samples were taken from the vessels. On most occasions, the ammonium oxidizers were washed on.45-p.m (pore size) Millipore filters and suspended in nitrogen-free medium, although unwashed cells were used in some studies. The cell suspension was then divided into 3- ml samples; substrate and, when required, inhibitors were added. The samples were then divided into 1-ml aliquots; two were amended with [14C]bicarbonate and one was unamended. The unamended sample was used to determine the substrate oxidation rate. Uptake studies were done in tubes sealed with silicone rubber septa. These tubes were flushed with CO,-free air before cell suspensions were added. The [t4c]bicarbonate (7,uCi in.1 ml of 1 mm NaOH) was injected through the septum, and the tube was preincubated for 15 min. Incubations lasted between 1 and 6 h, depending on the activity, with measurements being made periodically. In initial studies with each organism, time courses on bicarbonate uptake were done. Since reproducibility was very good, later studies only measured initial (after 15 min of preincubation) and final fixed bicarbonate. Three or four measurements of nitrite were made during the same incubation period. Bicarbonate incorporated into cells was measured by collecting 1. or 2. ml of cell suspension on a Nucleopore filter (.2 p.m [pore size]), washing with buffer, fuming with HCl to remove unincorporated label, and then drying the filters. The filters were then dissolved in.5 ml of Protosol and counted with 1 ml of a fluor made of.1 g of dimethylpopop (1,4-bis-[2(4-methyl-5-phenyloxazolyl)]benzene),.67 g of PPO (2,5-diphenyloxazole) per liter of 1% methanol in toluene. BICARBONATE UPTAKE BY NITRIFIERS 111 Specific activities of the bicarbonate were determined for each incubation tube by acidifying the sealed tube at the end of the experiment and injecting replicate samples into a gas chromatograph and into stoppered scintillation vials containing 1. ml of 3-phenylethylamine in the fluor used for counting cellular uptake. The gas chromatograph was equipped with a 2-m Porapak Q column and a thermal conductivity detector. Inhibitors. Metabolic inhibitors carbonyl cyanide-rnchlorophenylhydrazone, N,N'-dicyclohexylcarbonimide, and 2,4-dinitrophenol were dissolved in dimethyl sulfoxide to final concentrations of 1, 4, and 2 mm, respectively. Between 5 and 1 [I of the inhibitors was added to 3 ml of cell suspension to give the desired concentration. RESULTS Continuous and batch cultures. Continuous cultures were done successfully with Nitrob(ater sp., Nitrosolionas sp., and Nitrosospir-a sp.. was grown at four different generation times between 18 and 69 h and at three different phs from 6.2 to 8.. Nitrosoinonatis sp. was grown at four generation times from 35 to 189 h at three different phs from 6.7 to 7.7. was grown in continuous culture with one generation time (53.3 h) and two different phs (7.3 and 6.7). Generation times, phs, and limiting substrate concentrations for these continuous cultures are summarized in Table 1. To obtain data on more rapidly growing ammonium oxidizer cells than could be attained in continuous culture, several batch cultures were run; culture conditions for these studies are also summarized in Table 1. Effect of growth rate on bicarbonate uptake/substrate oxidation ratio. Table 2 shows the ratios between bicarbonate uptake and substrate oxidation for the nitrifying bacteria grown under conditions described in Table 1. The substrate concentrations given in Table 2 are those present in the cell suspension at the beginning of the uptake experiment. The only ratio that seems to deviate from the norm for was that determined in study SA (Table 2). Due to low ph (6.2), there was very little carbon dioxide present; the concentration was approaching the detection limit by gas chromatography. To determine whether the high ratio could be an artifact caused by errors in the carbon dioxide measurement, an experiment was done with cells grown at ph 6.2 but with enough added bicarbonate in half of the tubes to double the carbon dioxide concentration. The remaining tubes were unamended. The unamended incubations gave the same high ratios which were obtained in the initial two experiments of study SA, whereas the bicarbonate-amended samples gave ratios compatible with those measured in studies 1 to 4. The value determined is designated as SB in Table 2. Of the ammonium oxidizers tested, all had similar efficiencies in taking up bicarbonate in relation to the substrate oxidized. However, when the substrate concentration was increased, thus increasing the rate of substrate oxidation, more efficient uptake was observed. Therefore, a series of studies was performed to investigate the effect of changes in the rate of substrate oxidation on the ratio of the two activities. Effect of substrate oxidation rate on bicarbonate uptake/ substrate oxidation ratio. Substrate concentrations were varied in such a way that the relative oxidation rate varied between Vmax and 1% of Vmax for ammonium oxidizers and between Vmax and 4% of Vmax for All three nitrifiers showed significant changes in efficiency with which

3 112 BELSER APPL. ENVIRON. MICROBIOL. TABLE 2. Ratios between bicarbonate uptake and substrate oxidation for nitrifying bacteria: effects of growth rate and ph Culture and study no. Generation time (h) ph Uptake ratio' Substrate concn (mm)b No. of replicates" ± ±.3 3, ± ±.9 5, ±.39.8 ±.3 3, ± ±.16 5, 2 SA ±.5.47 ±.8 3, 2 SB ±.3.47 ±.8 1, 2 Nitrosomonas sp. id ± , 2 2d ± , 2 3d ± ±.5 3, 2 4d ± , ±.7 >.3 1, ±.27 >.3 1, 3 id ± , 2 2d ± , ±.45 >.5 1, 3 4d ± , 3 a The average uptake ratios for Nitrobac ter cultures (excludes data from study 5A [see text for explanation]), Nitrosomnonas cultures, and Nitrosospira cultures were.236 ±.13,.863 ±.55, and.869 ±.91, respectively. bwith the exception of the values for studies 2 and 4 of the Nitrosomonas cultures, substrate concentrations were similar in magnitude to the in situ continuous culture substrate concentrations. The concentrations represent the substrate concentrations at the beginning of the uptake study. ' The first number in the column represents the number of experiments done on different days at a given continuous culture condition. The second number represents the number of replicates of bicarbonate uptake on a given day. In study 5B of the Nitrobac ter cultures, bicarbonate was added to the tubes, thereby doubling the concentration; otherwise, the experiment was the same as in study 5A. d Washed cells. bicarbonate was taken up in relation to the relative oxidation rate. For, increasing the relative oxidation rate decreased the efficiency by which bicarbonate was taken up per micromole of substrate oxidized (Fig. 1). The opposite was seen for the ammonium oxidizers, with the maximum efficiencies (uptake ratios) occuring near Vmax (Fig. 2). In one study ( ; culture 1), uptake measurements were made on four occasions during a 14-day interval. These uptake incubations were run in the presence of.5 and.5 mm ammonium. Both oxidation rates relative to Vmax remained constant during the 14-day period and were % for the.5 mm incubation and 14.5 ± 1.3% for the.5 mm incubation. However, the o'e.5 L QL U n o -I Oi o I A U A *A 4 2 Oxidation Rate (% Vmax) FIG. 1. Bicarbonate uptake efficiency of as a function of relative oxidation rate. Oxidation rate was varied by adding nitrite to unwashed cells. In situ oxidation rate was between 3 and 4% of Vmax. Symbols: U, culture 4; *, culture 5. Oxidation Rate (% Vmax) FIG. 2. Bicarbonate uptake efficiency of ammonium oxidizers as a function of the relative oxidation rate. Oxidation rate was varied by adding various concentrations of substrate to washed cells. In situ oxidation rates (continuous culture) were between 4 and 8% of Vmax. Symbols: A, Nitrosomonas culture 3; *, Nitrosomonas culture 4; *, Nitrosospira culture 1; and *, Nitrosospira culture 2.

4 VOL. 48, c -W QL 1i 8^ 61 f 2 44t Time (days) FIG. 3. Bicarbonate uptake efficiency as a function of time for growing with a generation time of 53 h (ph 7.3) for two concentrations of ammonium. Uptake efficiency is expressed relative to the uptake ratio in the presence of 2 mm ammonium. Symbols: *,.5 mm; and,.5 mm. ratios decreased progressively with the.5 mm incubations, although they remained relatively constant in the presence of.5 mm ammonium (see Fig. 3). Effects of inhibitors. Inhibitors appeared to cause a greater decrease in uptake efficiency with than with the ammonium oxidizers. These results are summarized in Table 3. The uptake efficiency for a given inhibitor concentration is expressed as the percentage of the uptake ratio determined for that inhibitor concentration to the uptake ratio without inhibitor added. The percentage decrease in BICARBONATE UPTAKE BY NITRIFIERS 113 TABLE 4. Effect of changing ph on the efficiency of bicarbonate uptake by Nitrosospira and Nitrosomonas cultures Culture Clue Uptake ph Relative efficiency (%)a rate () 1 1. ± ± , ± ± ± 7.3 2, ± ± ± 4.5 2, 2 p oxidation ReplicateSb Nitrosomonas sp. 3A 1. ± ± , 2 3A 14. ± ± , 2 3A 15.7 ± ± , 2 a Uptake efficiency and oxidation rates are expressed relative to rates and ratios at ph 7.4 to 7.5. All incubations were with 2. mm ammonium. bthe first number in the column represents the experiments done on different days at a given continuous culture condition. The second number represents the number of replicates of bicarbonate uptake on a given day. substrate oxidation rates are also given. N,N'-Dicyclohexylcarbonimide appeared to have the greatest effect on uptake efficiency Inhibitors that might occur in the environment, such as ammonium at high ph and chlorate, did not appear to inhibit bicarbonate uptake selectively with respect to nitrite oxidation. Decreasing ph will also inhibit ammonium oxidation rates. Two ammonium oxidizers were used to determine whether rapidly changing ph would affect the efficiency of uptake. Our results showed that changing ph does not have a short-term effect on the uptake efficiency (Table 4). DISCUSSION The results presented in Table 2 seem to indicate that the ratio between the rate of bicarbonate uptake and the rate of TABLE 3. Effect of inhibitors on the efficiency of bicarbonate taken up by Nitrobacter, Nitrosomonas, and Nitrosospira cultures Inhibitor and Substrate Inhibition of Uptake Culture MIcncnanM concn concn (n oxidation efficiency Replicatesc ~(MM) rate (%)b () 4 NH4+ (4) ± 9.1 1, 2 4 C13- (2) ± 7.4 1, 2 4 DCCD (7) ±.5 1, 2 4 DNP (4) ± 2.5 1, 2 5 DCCD (7) ± ± 2.1 2, 2 5 DNP (1) ± 2.1 1, 2 Nitrosomonas sp. 2 Ni (2) ± 3. 1, 2 2 CCCP (2) ± 3. 1, 2 2 DNP (24) ± 2.1 1, 2 4 CCCP (17) ± ± , 2 4 CCCP (34) ± ± 1.1 2, 2 4 DNP (34) ± 3.3 2, 2 4 DNP (68) ± ± , 2 2 CCCP (17) ± ± 1.2 2, 2 2 DNP (2) ± 3.2 1, 2 2 DNP (333) , 2 a DCCD, N,N'-Dicyclohexylcarbonimide; DNP, 2,4-dinitrophenol; and CCCP, carbonyl cyanide-m-chlorophenyl hydrazone. buptake efficiencies and oxidation ratios are expressed in terms of controls to which no inhibitors were added. c The first number in the column represents the experiments done on different days at a given continuous culture condition. The second number represents the number of replicates of bicarbonate uptake on a given day.

5 114 BELSER substrate oxidation was independent of the growth rate and ph for each organism tested. There did not appear to be any difference in the ratios for the two ammonium oxidizers Nitrosomonas sp. and. If the standard deviations for the average uptake rates are presented as percentages of the averages, the precision of the measurements for, Nitrosomonas sp., and was 5.6, 6.3, and 1.5%, respectively. All these variations would be within the experimental error of the technique. The main source of error during measurements was in measuring the CO2 for calculation of the specific activity. This was mainly the result of standardizing the gas chromatograph from day to day. Thus, the reproducibility on a given day was much greater than on different days. During the course of the study, the standardization technique improved, which resulted in better day-to-day reproducibility. Fewer day-to-day replicates were needed in later experiments, due to this improved reproducibility. The average values determined here agree reasonably well with the values of.1 and.2 for ammonium and nitrite oxidizers initially used by Billen (3) and now widely used in in situ nitrification measurements. One purpose of this work was to determine whether the ratio between bicarbonate uptake and suibstrate oxidation was indeed a constant. To do this, various environmental parameters were altered to determine whether the ratio would change. Variations in substrate concentrations and the addition of certain metabolic inhibitors were found to significantly alter the uptake ratios (Fig. 1, 2, and 3; Table 3). However, not all inhibitory compounds affected the ratio (Table 3). Changing the ph also did not affect the ratio (Table 4). One possible explanation of the data in Table 2 and Fig. 1, 2, and 3 would be that the cells adapt their enzyme levels to maintain a constant uptake ratio which is independent of the growth rate. For example, Nitrobacter cells adapted to grow at 18 and 61 h of generation time had similar uptake ratios of.254 ±.64 and.214 ±.38, respectively (Table 2). The former uptake ratio occurred in the presence of.38 mm nitrite, whereas the latter ratio occurred in the presence of.35 mm nitrite. When the substrate concentration of the latter was increased to.5 mm, the uptake ratio decreased to.13 ±.2 (Fig. 1; 6% of Vmax). Thus, when the substrate concentrations were similar, the uptake ratio was significantly lower for the slower growing cells. The data presented for (Fig. 3), showing a decrease in the ratio with time and low concentrations (concentrations below in situ level), also indicate an adaption of the enzyme system. The effect of the concentration of oxygen (the electron acceptor for nitrification) was not tested in this study, since the continuous culture vessels were not designed to control the oxygen tension or to be sampled without disturbing oxygen tension. However, there are several reports in the literature (4-6) indicating that reduced oxygen levels stimulate growth (bicarbonate uptake [4]) while decreasing nitrification rates. Thus, until further work is done, it is not clear whether the ratio remains constant at low oxygen tensions. It was not the purpose of this paper to survey the effect of inhibitory compounds on the uptake ratio (i.e., to determine whether the inhibitors could selectively inhibit bicarbonate uptake with respect to substrate oxidation); our purpose was only to establish whether the ratio could be affected or not. Initial tests with three inhibitory compounds that might be found in natural environments had no effect on the ratio APPL. ENVIRON. MICROBIOL. (Table 3; ammonium and chlorate for, nickel for Nitrosomonas sp.). To improve the chance of finding inhibitory compounds that could affect the ratios, metabolic inhibitors that affect the production of ATP were used (N,N'-dicyclohexylcarbonimide, 2,4-dinitrophenol, and carbonyl cyanide-m-chlorophenyl hydrazone). The results presented in Table 3 indicate that such inhibitors can greatly affect the ratio. For the nitrapyrin-sensitive bicarbonate uptake method to be effective in estimating in situ nitrification rates, two criteria must be met. The first of these is that the ratio of bicarbonate uptake to the rate of substrate oxidation remain constant and independent of the growth rate and environmental factors such as ph. This criterion is supported by the work presented here. The second of these criteria is that nitrapyrin selectively inhibit the bicarbonate uptake of autotrophic nitrifiers and leave all other processes that involve bicarbonate uptake unaffected. This has not been investigated here. This work supports the former assumption only and thus cannot be considered a demonstration of the effectiveness of the technique with natural samples. This work also puts restraints on the utilization of the bicarbonate uptake technique. Clearly, it cannot be used to measure kinetic parameters (i.e., Km and Vmax) of natural populations of nitrifiers, since the data presented in Fig. 1 and 2 show that there is not a stoichiometric relationship between bicarbonate uptake and substrate oxidation rate when the substrate concentration is varied. This might also limit the usefulness of the technique in environments in which substrate concentrations are changing rapidly, such as in rivers and estuaries. There is also a possibility that inhibitory compounds could affect the ratio. A survey of the effects of inhibitory compounds on coupling efficiency would be necessary to elucidate this possibility. ACKNOWLEDGMENTS I thank E. L. Mays for technical assistance and D.. Mountfort and D. W. Grant for helpful discussions. LITERATURE CITED 1. Belser, L. W., and E. L. Mays Specific inhibition of nitrite oxidation by chlorate and its use in assessing nitrification in soils and sediments. Appl. Environ. Microbiol. 39: Belser, L. W., and E. L. Schmidt Growth and oxidation kinetics of three genera of ammonia oxidisers. FEMS Microbiol. Lett. 7: Billen, G A method for evaluating nitrifying activity in sediments by dark ["4C]-bicarbonate incorporation. Water Res. 1: Carlucci, A. F., and P. M. McNally Nitrification by marine bacteria in low concentrations of substrate and oxygen. Limnol. Oceanogr. 19: Goreau, T. J., W. A. Kaplan, S. C. Wofsy, M. B. McElroy, F. W. Valois, and S. W. Watson Production of NO- and N,O by nitrifying bacteria at reduced concentrations of oxygen. Appl. Environ. Microbiol. 4: Gundersen, K The growth and respiration of Nitrosocystis oceanus at different partial pressures of oxygen. J. Gen. Microbiol. 2: Hall, G. H Apparent and measured rates of nitrification in the hypolimnion of a mesotrophic lake. Appl. Environ. Microbiol. 43: Schmidt, E. L., and L. W. Belser Nitrifying bacteria, p In A. L. Page, R. H. Miller, and D. R. Keeney (ed.), Methods of soil analysis, 2nd ed. American Society for Agronomy, Madison, Wis. 9. Somville, M A method for measurement of nitrification rates in water. Water Res. 12: