Blotechnol. Prog. 1004, 10, 421-427
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Study of Nitrate Metabolism of Escherichia coli Using Fluorescence Hiren K. Trivedi and Lu-Kwang Ju* Department of Chemical Engineering, The University of Akron, Akron, Ohio 44325
Public concern about environmental nitrate contamination has increased significantly. To investigate the nitrate metabolism of Escherichia coli, profiles of NAD(P)H fluorescence responding to nitratehitrite additions to anaerobic E . coli cultures a t the resting or minimum-growth state were monitored by an on-line fluorometer. Two different types of fluorescence profiles were observed. Upon nitrate addition, the fluorescence signal dropped instantaneously, indicating the presence of NAD(P)Hdependent nitratehitrite reductases, without previous nitratehitrite induction. The signal remained a t that level until all of the nitrate was reduced. In some cases, the signal then directly rose back to the earlier anaerobic level. In others, it partiallyrecovered to an intermediate level, remained at that level for a period much longer than the first stage, and then returned to the anaerobic level. The single-stage response implied that the nitrite formed by nitrate reduction was totally converted to ammonium simultaneously. The two-stage response, however, resulted from intermediate nitrite accumulation: the rise in fluorescence a t the end of the first stage signified nitrate depletion, and the second stage corresponded to reduction of the nitrite accumulated in the first stage. This is supported by the specific rates of nitrate and nitrite reduction determined from the fluorescence profiles. While the former (0.0138 f 0.0026 g of NO,--N/(g of cells)*h)was about the same in all the runs conducted, the latter was found to be 1order of magnitude lower than the former in cultures showing the two-stage response. The presence of ammonium, a t 0-50 ppm NH4+-N, was found to have no appreciable effect on the nitrate reduction rate for the E . coli studied.
Introduction Providing crops with adequate fertilizers is vital to ensuring food supplies. The use of fertilizer nitrogen in modern agriculture, however, has led to increased nitrate levels in recipient subsoils, aquifers, inland waters, and coastal seas. When ingested in high amounts by humans and animals, nitrate may cause potentially adverse health effects, including methemoglobinemia, cancer, etc. (Council for Agricultural Science and Technology, 1985;Keeney, 1986; Panel on Nitrates, 1978). Public concern about nitrate contamination of groundwater and surface water due to agricultural activities therefore has increased significantly. The health standard for nitrate in public drinking water supplies in the U.S.has been set at 10 mg of NOs--N/L (Follett and Walker, 1989). However, nitrite is much more toxic than nitrate (Panel on Nitrates, 1978). In fact, the health hazards that are associated with nitrate chiefly result from the bacterial conversion of ingested nitrate to nitrite. Some fish specieswere found to be killed by acute exposure to less than 1mg/L N02--N (Russo and Thurston, 1976). To bacteria, nitrate and nitrite are both strong oxidizing agents and potential sources of nitrogen. Consequently, different groups of bacteria exploit them in different ways (Cole, 1988). In assimilative nitrate reduction, nitrate is reduced to ammonia for use as a nitrogen source for growth; in dissimilative nitrate reduction, nitrate is used as an alternative electron acceptor in energy generation. Assimilative nitrate reduction occurs in all plants and most fungi, as well as in many bacteria, whereas dissimilative nitrate reduction is restricted to bacteria, although a wide variety of bacteria can carry out this process. In both
* Author to whom correspondence should be addressed. 8756-7938/94/3010-0421$04.50/0
cases, nitrate is first reduced to nitrite. Nitrite can then be reduced to ammonia through either an assimilative or a dissimilative pathway, or it can be reduced to N2 (denitrification). In general, enzymes involved in an assimilative pathway are ammonia-repressed, while dissimilative reductases are repressed by 0 2 and are synthesized under anaerobic conditions (Brock and Madigan, 1988). In a previous study (Ju and Trivedi, 1992), denitrification by Pseudomonas aeruginosa was monitored using an on-line fluorometer (the BioGuide system, BioChem Technology Inc., King of Prussia, PA). The BioGuide system was developed to measure the level of intracellular NAD(P)H [i.e.,the reduced forms of coenzymes NAD(P), nicotinamide adenine dinucleotide (phosphate)], on the basis of the property that the reduced coenzymesfluoresce at 460 nm when irradiated with 340-nm light, while the oxidized forms, i.e., NAD(P)+,do not fluoresce (BioChem Technology Inc., 1983). As it is the only analyzer available that provides information on intracellular conditions without process interruption, the fluorometer has been applied to many microbial fermentations, animal cell cultures, and biological wastewater treatment processes to generate information on cellular metabolism and to provide better process control in large-scale industrial operations (Armiger et al., 1986; Armiger et al., 1990; BioChem Technology Inc., 1983; Siano and Mutharasan, 1991;Srinivas and Mutharasan, 1987; Winter et al., 1988). It has excellent stability for long-term operation, and yet ita response is rapid enough to follow metabolic transitions occurring on the order of seconds. It was demonstrated to be an effective tool for studying the NAD(P)Hdependent nitrate metabolism of microorganisms (Ju and Trivedi, 1992).
0 1994 American Chemical Society and American Institute of Chemical Engineers
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Escherichia coli is known to reduce NH4+ under anaerobic conditions through dissimilative pathways, termed nitrate ammonification (Herbert and Nedwell, 1990). When coupled with NADH oxidation, the two subsequent reactions involved in the process can be written as follows:
NO;
-
+ NADH + H+ + 3NADH + 5H'
NO;
NO;
+ NAD' + H 2 0
NH4++ 3NAD'
+ 2Hz0
(1) (2)
In the first step, nitrate serves as the terminal electron acceptor for the respiratory chain by virtue of a dissimilative nitrate reductase, which is generally believed to be induced when nitrate is provided in the medium (Cole, 1990). Nitrate reductase has been isolated from the cytoplasmicmembrane of E. coli by a number of detergentbased, alkaline heat treatment, or solvent extraction procedures (Clegg, 1976; Enoch and Lester, 1975;Forget, 1974; Kemp and Atkinson, 1966; MacGregor, 1975). Although formate and lactate are among the potential electron donors for nitrate respiration of glucose-grown cells, there is little doubt that NADH is a significant in vivo electron donor for the process. Many fermentation studies confirmed that ethanol production is greatly depressed during nitrate respiration, while the level of acetate is usually doubled (Ishimoto and Yamamoto, 1977; Keevil et al., 1979; Verhoeven, 1956). Anaerobic cultures of most E. coli strains reduce nitrite to NH4+, as shown in the second step. The NADHdependent nitrite reduction by a cytoplasmic nitrite reductase provides the host with a potential nitrogen source for growth, regenerates NAD+ from NADH, increases the pH of the environment, and removes the toxic product of nitrate reduction from the cytoplasm (Cole, 1990). It is the most active nitrite reductase for E. coli,and the activity is sufficient to account for at least 50%,and up to 95%, of the overall rate of nitrite reduction when glucose is the substrate (Cole, 1982). Two more enzymes are also capable of catalyzing the conversion of nitrite to ammonia: a NADPH-dependent sulfite reductase and a formatedependent nitrite reductase. The former has been reported to contribute at most 5% of the total nitrite reduction (Cole and Ward, 1973). The latter is linked to a membrane-bound, electron-transfer chain and probably serves to remove two potentially toxic compounds, extracellular nitrite and intracellular formate, with the additional advantage of generating a proton electrochemical gradient (Cole, 1990). However, Abou-Jaoude et al. (1977) reported that this pathway was undetectable in many strains and contributed only up to about 20% to the total rate of nitrite reduction by others. Furthermore, the presence of glucose in the growth medium was found to repress the formate-dependent nitrite reductase activity (Abou-Jaoude et al., 1979). It is apparent that the dissimilative ammonification of nitrate by E. coli is strongly NAD(P)H-dependent and can be monitored effectively by the BioGuide system. In this study, the fluorescence profiles responding to nitratelnitrite addition of anaerobic E. coli cultures at the resting or minimum-growth state, regulated by substrate availability, are investigated. It aims at examining the rates and mechanism of nitratelnitrite reduction by E. coli in the natural environment. Materials and Methods Escherichia coli (ATCC No. 11303)was maintained on agar slants containing 30 g/L tryptic soy broth (TSB).
After incubation in a liquid medium of 30 g/L TSB for 24 h at 37 "C, the culture was transferred to two 2-L Erlenmeyer flasks, each containing 300 mL of liquid medium with the following composition (per liter of tap water): 30 g of glucose, 1g of yeast extract, 4 g of (NH4)2HP04,2 g of M g S 0 ~ 7 H z 0 , 2g of KH2P04, and 0.5 g of FeCly6HzO. The cells were grown typically for 1618h under vigorous agitation by magnetic stir bars. To minimize the optical disturbances coming from sources other than intracellular NAD(P)H, the harvested cultures were centrifuged and washed to remove the spent medium. This procedure is necessary because E. coli is known to produce fluorophores interfering with the monitoring of NAD(P)H fluorescence during active growth. The fluorophores mainly responsible have been identified as folic acid and its derivatives (excitation maxima at 285 and 350 nm, fluorescence maximum a t 450 nm) (Maneshin et al., 1990). During anaerobic fermentation, tetrahydrofolate is released to the medium (Maneshin et al., 1990). Under aerobic conditions, the tetrahydrofolate released (excitation maximum at 360 nm) is oxidized by oxygen in the medium to folate derivatives and, subsequently, xanthopterin (excitation maximum at 395 nm, fluorescence maximum at 450 nm). Because the folate derivatives and xanthopterin have higher fluorescence efficiencies than tetrahydrofolate, the oxidation leads to an increase in the fluorescence intensity of an aerobic E. coli culture, even after cell growth has ceased. The washed cells were then resuspended in 800 mL of a lean medium contained in a double-side-arm Celstir bioreactor (Wheaton Scientific, Milliville, NJ). The lean medium contained 10 g/L NaC1, to prevent osmotic shock to cells, along with 0.1 g1L MgS0~7Hz0and 0.1 g/L KH2P04. The lean medium, partly reflecting substrate limitations commonly encountered in nature, was used to minimize the contribution to culture fluorescence from fluorophores produced with active cell growth. During the major part of the runs, nitrogen was sparged at a minimal rate into the system to ensure the complete absence of oxygen. Followingresuspension of centrifuged cells, the system was allowed to stabilize under anaerobic conditions. Air sparging was then switched on to create aerobic conditions. After the change in the culture fluorescence profile due to aeration was established, air sparging was stopped and the dissolved oxygen concentration (DO)was allowed to drop to zero. Once the culture had shifted back to anaerobic conditions, indicated by the return of fluorescenceto the previous anaerobic level, small volumes of concentrated potassium nitrate solutions were added to cause different nitrate concentrations in the reactor. The response of culture fluorescence to nitrate addition was followed. The fluorescence profile of the culture was monitored by the BioGuide system, which accessed the broth through an optical well mounted on the reactor wall. The reactor was equipped with a pH electrode, a DO probe, a gas sparger, and an exit gas line, all mounted on the reactor cap. The whole setup was covered with a black opaque plastic bag to avoid any interference from outside light. Samples were collected from one of the side-arms. The fluorescence signal and DO concentration were recorded by computer data acquisition and analysis software (BioGuide Software, BioChem Technology). The fluorescence intensity was expressed as a manufacturercalibrated normalized fluorescence unit (NFU). Cell concentration was determined by measuring the sample's absorbance at 600 nm with a spectrophotometer (Spectronic 20, Bausch & Lomb, Rochester, NY), on the
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180
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1
145
A
140 0
2
1
3
4
T i m IHd
Figure 1. Typical culture fluorescence profile for E. coli showing single-stage responses to nitrate additions: 0,culture fluorescence; A, NH,+-N.
-, dissolved oxygen; 0, NO,--N;
basis of a preestablished calibration curve of absorbance versus measured cell dry weight. An ammonia-selective electrode was used to determine the ammonium concentration and the combined nitrate and nitrite concentration; the latter was based on the titanous chloride reduction method (Clesceri et al., 1989). pH was maintained at 7.0 f 0.1 by acid-base addition. The agitation speed was kept at the minimum required to ensure complete mixing inside the reactor.
Results and Discussion Culture Fluorescence Profiles. As mentioned previously, the BioGuide system measures the level of intracellular NAD(P)H. In living cells, coenzymes, NAD(P), are the major intermediate electron carriers in the oxidation-reduction reactions of metabolism. Accompanying substrate catabolism, the oxidized forms of coenzymes, NAD(P)+,are reduced to NAD(P)H. NADPH primarily serves as the reducing power in biosynthetic reactions (anabolism), whereas NADH is directly involved in ATP generation via oxidative phosphorylation in aerobic metabolism. Through these processes, the reduced forms of coenzymes, NAD(P)H, are oxidized back to NAD(P)+. Similar reduction-oxidation cycles of coenzymes, NAD(P), exist for anaerobic respiration and fermentation. Nitrate and/or nitrite can replace the role of oxygen as the terminal electron acceptor in the reduction-oxidation cycle of NAD(P) for certain microorganisms, including E. coli. On the other hand, no externally supplied electron acceptor is required during fermentation, where the oxidation of NADH to NAD+ is coupled with the reduction of an oxidized organic compound (e.g., acetyl-coA or pyruvate in the case of E. coli) generated from the catabolism of fermentable substrates. The proportion of the reduced coenzymes,i.e., CNAD(P)H/ (CNAD(P)H + CNAD~)+), is determined by the balance between the rates of the reduction (generation) and oxidation (consumption) reactions. As the organic compounds involved in oxidizing NADH in fermentation have much weaker oxidizing power than oxygen, nitrate, and
nitrite, the level of NAD(P)H at the anaerobic state is higher than those at the aerobic and nitrate/nitritereducing states. Hence, a drop in the culture fluorescence from its anaerobic value 'is expected with nitrate/nitrite addition or the introduction of air into the system. Similarly, a rise in the fluorescence level is expected to accompanythereturn of the system to anaerobicconditions when nitratehitrite or DO is exhausted. Two different types of culture fluorescence profiies were observed during the study of the nitrate metabolism of E. coli, as shown in Figures 1 and 2. In both cases, cells responded similarly to aeration: the fluorescence signal immediately dropped to a lower value and later jumped back to its anaerobic level after air sparging was stopped and DO dropped below a critical concentration. However, the responses to nitrate addition were different. The fluorescence signal dropped upon the addition of nitrate in both cases. In Figure 1, the signal returned to ita anaerobic level in a single step once all of the nitrate was consumed, while in Figure 2, the fluorescence signal first rose to an intermediate level and remained relatively steady at that level for a much longer period of time, before making the second jump to ita anaerobic level. The observed fluorescence responses confirmed the NAD(P)H dependency of the nitratehitrite metabolism of E. coli. The single-stage response shown in Figure 1 implied that the nitrite formed by nitrate reduction was converted to ammonium immediately; no accumulation of intermediate nitrite occurred in the culture. This gained some support from the off-line analysis results of the combined concentrations of N0f-N and N02--N in the samples taken after nitrate addition: smooth, decreasing trends of NO,- content were observed, and both NO3- and NO2- were completely removed when the fluorescence signal began to climb back to the anaerobic value (Figure 1). On the other hand, the two-stage response shown in Figure 2 resulted from the difference in the reduction rates of nitrate and nitrite. The first jump of the fluorescence, following the drop caused by nitrate addition, cor-
Biotechnol, RoQ.., 1994, Vol. 10,
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N i t r a t e Addition (ppm NO3-- N )
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iz?
8 5
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-w
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i5i!
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Figure 2. Typical culture fluorescence profile for E. coli showing two-stage responses to nitrate additions: -, dissolved oxygen. responded to the exhaustion of nitrate in the medium; the second jump from the steady intermediate value corresponded to the depletion of nitrite. Nitrite accumulated in the first stage because nitrate reduction was faster than nitrite reduction, and it was reduced in the second stage. This phenomenon was confirmed in another run (Figure 31, where 1ppm of N02--N was added after the system had recovered from 1-ppm NO5-N addition. The signal dropped immediately upon the addition of nitrite, indicating that NADH-dependent nitrite reductase was active in this case. More importantly, when compared with the earlier two-stage response to the 1-ppm NO3--N addition, the drop was smaller than the drop observed in the firststage response, but was the same as the difference between the anaerobic value and the intermediate level observed in the second stage. This strongly supported the theory that the second stage observed in Figures 2 and 3 corresponded to nitrite reduction. To examine these phenomena more quantitatively, specific rates of nitrate and nitrite reduction were calculated for a number of repeated runs and are listed in Table 1. A detailed description of how the reduction rates were determined from the fluorescence profiles is given here. The effect of added nitrate concentration on the fluorescencedrop was found to follow saturation kinetics, similar to that observed in denitrification by P. aeruginosa (Ju and Trivedi, 1992). At low nitrate concentrations, not all of the enzymatic sites for nitrate/nitrite reduction were filled with the substrate. As the nitrate concentration increased, the fraction of occupied sites and the rate of NAD(P)H oxidation also increased, leading to lower NAD(P)H levels and larger drops in fluorescence. Beyond a certain nitrate concentration, the active sites were saturated; further increases in nitrate concentration did not increase the oxidation rate of NAD(P)H, and the fluorescence drop reached its maximum. Consequently, when a nitrate concentration higher than the critical level was added, the fluorescence signal dropped to the lowest level and remained there for some time before starting to rise.
0 , culture
fluorescence;
The sharp fluorescence drop indicated the instantaneous binding and reduction of nitrate when unfilled enzymatic sites were available. Therefore, the point at which the fluorescence started to rise signified the exhaustion of nitrate in the medium and the flat part corresponded to the complete coverage of active sites for steady-statenitrate reduction. Accordingly, the steady-state nitrate reduction rate could be calculated as *‘NOs-- ‘N08-,added
At
- ‘N08-,critical
Attlat part
(3)
where the duration of the steady state was taken as the flat part of the response curve between the nitrate addition and the starting point of the fluorescencerise. The nitrate concentration that was actually processed during the steady state was assessed by subtracting the critical nitrate concentration from the added concentration, because the former was removed from the medium during the transient stage immediately following nitrate addition. Subsequently,the specific rate of steady-state reduction of nitrate to nitrite could be calculated by dividing the nitrate reduction rate by the employed cell concentration. The calculations for the rates of nitrite reduction were done similarly. In the cases where two-stage responses were observed, specific steady-statenitrate reduction rates were calculatedfrom the flat part of the first stage following nitrate addition. If we assume that nitrate was reduced to nitrite in the first stage, and that the resultant nitrite was reduced to NH,+ simultaneously at a slower rate and continued to be reduced during the second stage, specific steady-state nitrite reduction rates were calculated by dividing the resultant nitrite concentration from nitrate reduction by the combined duration of both stages, Le., between the nitrate addition and the starting point of the fluorescence rise at the end of the second stage. In Table 1,the specific reduction rates of nitrate and nitrite were assumed to be the same for the runs with single-stage response. For the run shown in Figure 3, Le., run 6 in
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240 230 220 210 200 190 -
180 170
-
160
0
1
2
3
Time IHr)
Figure 3. Fluorescence profile of E. coli showing the two-stage response to nitrate addition and the response to external nitrite addition (legends are the same as for Figure 2). Table 1. Nitrate/Nitrite Reduction Rates. Ammonium Processing Rates, and Fluorescence Drop Ratios for Emherichia coli apparent nitrate nitrite ammonium ammonium run cell response to NFU nitrate addition disappearance ratea assimilation ratea drop ratio reduction ratea reduction rate" no. concentration (g/L) single-stage 0.013 0.013 0.0018 0.0155 0.61 1 1.3 single-stage 0.010 0.010 0.75 2 0.6 0.016 0.0018 0.0034d 0.74 0.0016 two-stage 3.7 3 0.014 4 0.W 0.0013 0.76 0.0013 two-stage 3.0 0.018 0.90 0.0010 two-stage 3.0 5 0.012 0.44 two-stage 0.0014,0.00085b 2.0 6 a All rates are reported in grams of NOa--, N O p , or NHd+-N per gram of cellsehour. Nitrite reduction rate calculated from the response to exogenous nitrite added to the system. Apparent disappearance rate for ammonium calculated from the difference between the assimilation rate and the nitrite reduction rate. d Ammonium assimilation rate calculated by addition of the nitrite reduction rate and the apparent disappearance rate.
Table 1, two nitrite reduction rates are given. One was calculated from the fluorescence response to the nitrite added externally, and the other was calculated from the two-stage response to nitrate addition. The two rates agree with each other very well. This lends more support to the explanation that nitrite reduction is responsible for the second-stagefluorescenceprofile. The rate data shown in Table 1 also confirm that the twostage responses to nitrate addition in some of the runs are due to the differences in the reduction rates of nitrate and nitrite. More significantly, the single- or two-stage response is shown to be determined by the markedly different nitrite reduction rates of the cultures, while the nitrate reduction rates are comparable in all of the runs. The cause of these different responses to nitrate addition deserves further investigation. Nevertheless, it appeared that the enzyme systems for nitrite reduction were more "fragile" (or less stable) than those for nitrate reduction in the E. coli strain studied, considering that this study was conducted under the substrate-limiting condition commonly occurring in nature. It should be noted that a similar two-stage phenomenon for anaerobic nitrate reduction by E.coli K-12 has been reported by Abou-Jaoude et al. (1979). They showed that nitrate was reduced and nitrite was accumulated during the first phase following nitrate addition. The nitrite
concentration reached the maximum when all of the nitrate was exhausted. The second phase started when the nitrate was used up and- corresponded to the utilization of the nitrite that had been accumulated in the first phase. They found that, in the presence of nitrate, the nitrite reduction rate was much lower (ca. 25%) than that observed in the absence of nitrate. It was therefore concluded that nitrate inhibited the reduction of nitrite; the level of inhibition depended on the concentration of nitrate. However, the different nitrite processing rates observed in this study were not caused by nitrate inhibition. In fact, the added nitrate concentrations were higher in the runs showing single-stage responses, where nitrite reduction rates were 1 order of magnitude larger than those found in the runs showing two-stage responses. Furthermore, in one case where the two-stage response was observed, the nitrite reduction rates were shown to be comparable, with or without the presence of nitrate in the system (run 6 in Table 1). Nitrate did not show a significant inhibitory effect on nitrite reductases in the current study. In all of the figures (1-31, the culture fluorescence was found to rise slightly throughout the study. Cell growth was not responsible, because cells were merely maintained in the lean medium employed in the study. This was confirmed by the cell concentrations measured a t the
420
beginning and the end of the runs. As described in Materials and Methods, the rise in fluorescence reflected the increased background fluorescence, resulting from the slow oxidation of the extracellular fluorophores that had been introduced to the reactor with the residual spent medium, despite the laborious centrifugation and washing during the preparation of the fluorescence study. Nitrate and Nitrite Reductase Syntheses. It is generally believed that the synthesis of nitrate and nitrite reductases is regulated by two control mechanisms: oxygen repression and substrate induction during anaerobic growth. As described in Materials and Methods, the synthetic medium used in this study for growing cells did not contain nitrate or nitrite, hence, cells were not subjected to substrate (nitrate or nitrite) induction. However, instantaneous response to nitrate addition was observed in all of the cases studied. Showe and DeMoss (1968) studied the kinetics of nitrate reductase synthesis of E. coli K-12. They reported that even in the absence of nitrate, there was significant nitrate reductase induction after the culture had shifted to an anaerobic state. In the current study, the cells were grown aerobically. This was confirmed in several runs by measuring the DO in the culture before centrifuging for the following fluorescence study. The NADH-dependent nitratehitrite reductases of E. coli observed in this study were therefore either constitutive (i.e.,free from oxygen repression and induction of substrate and anaerobiosis) or induced by the shift to the anaerobic state when the cultures were centrifuged and allowed to stabilize under anaerobic conditions at the beginning of the fluorescence study. The period available for anaerobic induction for the latter assumption was less than 1h, which is significantly shorter than the required 2-3 h of nitrate induction for denitrifying enzymes observed in our previous study of P. aeruginosa (Ju and Trivedi, 1992). Nitrate Reduction Rate. As shown in Table 1, the steady-state specific nitrate reduction rate of E. coli in this study was 0.0138 f 0.0026 g of NOs--N/(g of cells).h, which is only around 10% of the rates reported by others (Cole, 1988;Hasan and Hall, 1975)for derepressed cultures of E. coli. It can be explained as follows: In the current study, cells were not provided with nitrate for the induction of dissimilative reductases. It is therefore possible that the nitrate reductase system was only partially developed, supported by the results of Showe and DeMoss (19681, who have reported that the nitrate reductase activity was about 20-fold higher with nitrate induction compared to the induction by anaerobiosis in the absence of nitrate. Further, the cells in this study were kept at a maintenance state. Slower nitrate reduction rates were anticipated in the less active cells. Nevertheless, the nitrate reduction rate of E. coli observed in this study was also much lower than the denitrification rate (0.215 f 0.020 g of NOS-N/(g of cells).h) found in the culture of P. aeruginosa maintained under similar conditions (Ju and Trivedi, 1992). As shown in Figures 1-3, the fluorescence dropped instantaneously after each nitrate addition. The nitrate added externally had to diffuse through the cell membrane in order to be acted upon by nitrate reductase, located on the cytoplasmic side (Clegg, 1976;Enoch and Lester, 1975; Forget, 1974;Kemp and Atkinson, 1966;MacGregor, 1975). Hence, the instantaneous drop in culture fluorescence indicates that nitrate reduction for E. coli is not limited by mass transfer, similar to that for P. aeruginosa, suggesting the existence of facilitative transport systems for nitrate. Ingledew and Poole (1984)believed that nitrate
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was unlikely to enter the cells via a uniporter, as any membrane potential across the cytoplasmic membrane would tend to exclude nitrate for E. coli. They suggested an electroneutral nitrate/nitrite antiporter system. However, as mentioned earlier, the majority of the nitrite formed from nitrate reduction is reduced to ammonia by a NADH-dependent nitrite reductase in the cytoplasm (Cole, 1990). Only a small fraction of nitrite formed would be available for driving the antiporter. Together with the observation of instantaneous response to nitrate addition in the absence of nitrite, this suggests the existence of active transport systems other than the nitrate/nitrite antiporter. Effect of Ammonium. Two types of nitrate reduction have previously been recognized. Nitrate assimilation is a tightly regulated process, which normally proceeds slowly at the rate at which ammonia is required for growth, so that nitrite rarely accumulatesduring nitrate assimilation. In contrast, dissimilative nitrate reduction is a more rapid process that can lead to the rapid and massive accumulation of nitrite (Cole, 1988). Assimilative nitrate and nitrite reductases are soluble enzymes. Variations in oxygen tension have little effect on the assimilation process, and the enzyme synthesis is induced by nitrate or nitrite but repressed by ammonium (Hewitt, 1975). On the other hand, the enzymes involved in dissimilative nitrate reduction are normally anoxia-derepressed, i.e., they require the absence of oxygen and the presence of anoxic conditions (the presence of nitrate or nitrite) (Brock and Madigan, 1988). To check the effect of ammonium in the medium, the responses to nitrate addition were followed immediately after different concentrations of ammonium (Le., 0, 15, and 50 ppm of NH4+-N) had been added to the culture (Figure 1). The nitrate reduction rates determined from these responses had close values of 0.018,0.015, and 0.012 g of NO3--N/(g of cells)-h,respectively, corresponding to the ammonium concentrations of 0, 15, and 50 ppm of NHd+-N. The average nitrate reduction rate was around 0.015 g of NOs--N/(g of cells)*h,which is comparable to the rates found in other runs made in the absence of ammonium (Table 1).Hence, the presence of ammonium did not have a significant effect on nitrate reduction in the E. coli studied. Together with nitrite accumulation observed in the two-stage response, this supports the general finding of literature reports that the nitrate reduction pathway of E. coli is dissimilative in nature (Cole, 1990). Also shown in Table 1 are two different NH4+-N disappearance rates for some runs. The assimilation rate was determined by analyzing samples without any nitrate or nitrite being present; the apparent rate was found by analysis of samplestaken when nitratehitrite was present in the medium. In run 1, where the single-stage phenomenon was observed, the apparent rate of NH4+-N disappearance was found to be approximately the difference between ita assimilation rate and the nitrate reduction rate. This again reflects the fact that, in the single-stage case, the nitrate reduction rate is the same as the nitrite reduction rate, which in turn equals the generation rate of NH4+-N and accounts for the difference between the assimilation rate and the apparent disappearance rate of NH4+-N. Further, when a slow nitrite reduction rate was observed (two-stage response), the ammonium assimilation rate was also very slow. This probably indicates that the two-stage phenomenon occurred when the employed culture was less active, due to either previous culture history or more severe substrate limitation.
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Ratio of Fluorescence Drops. As seen from Figures 1-3, the fluorescence values at the aerobic states were smaller than the lowest value during the anoxic states in all of the cases. This can be explained by the difference in the reduction potential for the oxidation-reduction pair of N03-/NH4+, which is +0.695 V, and that for 1/2 02/ H20,which is +0.82 V (Brock and Madigan, 1988). Oxygen, with a stronger oxidation power, oxidizes the intracellular NADH to a lower level than does nitrate. The results with E. coli showed that the ratio of the two fluorescence drops, one corresponding to the shift from anaerobic to nitratehitrite-reducing (anoxic) states and the other corresponding to the shift from anaerobic to aerobic states, Le., (NFUmaerobjc- NFUmoxic)/ (NFUmerobic - NFU,,robic), had a value of 0.70 f 0.14 in different runs (Table 1). Large ratios such as these are expected for E. coli that can efficiently utilize nitrate as terminal electron acceptor in the absence of oxygen and are similar to those found in denitrifyingp. aeruginosa (Ju and Trivedi, 1992). Acknowledgment The work was supported by a Faculty Research Grant from The University of Akron. Literature Cited Abou-Jaoude, A,; Chippaux, M.; Pascal, M.; Casse, F. Formate: A New Electron Donor for Nitrite Reduction in Escherichia coli K12. Biochem. Biophys. Res. Commun. 1977, 78,579583. Abou-Jaoude, A.; Chippaux, M.; Pascal, M. Formate-Nitrate Reduction in Escherichia coli K12: 1. Physiological Study of the System. Eur. J. Biochem. 1979,95,309-314. Armiger, W. B.; Lee, J. F.; Roshong, W.; Doutrich, J. Keeping a Constant "Eye" on Plant Performance. Oper. Forum WPCF 1990,7 (91,30-32. BioChem Technology,Inc. Guide t o Culture Fluorescence, King of Prussia, PA, 1983. Brock, T. D.; Madigan, M. T. Biology of Microorganisms, 5th ed.; Prentice Halk Englewood Cliffs, NJ, 1988. Clegg, R. A,; Purification and Some Properties of Nitrate Reductase (EC 1.7.99.4)from Escherichia coli K12. Biochem. J . 1976,153,533-541. Clesceri, L. S.;Greenberg,A. E.; Trussell,R. R. Standard Methods for the Examination of Water and Wastewater, 17th ed.; American Public Health Assoc.: Washington, D.C., 1989. Cole, J. A. Independent Pathways for the Anaerobic Reduction of Nitrite to Ammonia in Escherichia coli. Biochem. SOC. Trans. (599th Meeting, Birmingham, England) 1982,10,476477. Cole, J. A. Assimilatory and Dissimilatory Reduction of Nitrate to Ammonia. In The Nitrogen and Sulfur Cycles; Cole, J. A., Ferguson,S.J., Eds.; CambridgeUniversity Press: Cambridge, U.K., 1988;p 281. Cole, J. A. In Denitrification in Soil and Sediment; Revsbech, N. P., Sorenson, J., Eds.; Plenum: New York, 1990;pp 57-76. Cole, J. A.; Ward, F. B. Nitrite Reductase-Deficient Mutants of Escherichia coli. J . Gen. Microbiol. 1973,76,21-29. Council for Agricultural Science and Technology, Agriculture and Groundwater Quality, Report 103, 1985. Enoch, H. G.; Lester, R. L. Purification and Propertiesof Formate Dehydrogenase from Escherichia coli. J. Biol. Chem. 1975, 250,6693-6705. Follett, R. F.; Walker, D. J. Groundwater Quality Concerns about Nitrogen. In Nitrogen Management and Groundwater Protection;Follett, R. F., Ed.; Elsevier: New York, 1989;pp 1-22.
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