Kinetics of denitrification by Pseudomonas fluorescens: Oxygen effects

Soil Bid. Biochem. Vol. 26, No. 7. pp. 90-908, I994 Copyright ci; 1994EkevierScienceLtd 0038-0717(93)EOO38-N Printed in Great Britain.All rightsreserved 003%0717/9457.00+ 0.00

Pergamon

KINETICS PSEUDOMONAS

OF DENITRIFICATION BY FLUORESCENS: OXYGEN EFFECTS

D. J. MCKENNEY,‘* C. F. DRURY,’ W. I. FINDLAY,~B. MUTUS,’ T. MCDONNELL’ and C. GAJDA’ ‘Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4 and ‘Research Station, Agriculture Canada, Harrow, Ontario, Canada NOR IGO (Accepted 24 November

1993)

Summary-To determine the inhibitory effect of O2 on denitrification in pure cultures of Pseudomonas Juorescens, measurements of net NO and N,O production rates, fNo and fNXo,under anaerobic conditions were compared to rates under partially aerobic conditions. Nitrate or NO, solutions were injected into buffered suspensions and gaseous NO and N,O were sparged by constant flows of N, carrier gas, or mixtures of N2 with measured small quantities of 0, (cu &60 Pa). Apparent Michaelis-Menten parameters were calculated from saturation kinetic plots obtained by monitoring net NO or N,O production following periodic addition of NO;, NO; or NO to suspensions of cells cultured aerobically in the absence of NO, With NO, substrate added under anaerobic conditions the apparent KM= 3.74 + I .80 mM, and under partially aerobic conditions KM= 2.10 f 0.54 mM; with NO; substrate (anaerobic conditions) KM= 5.28 + 0.76 pM (partially aerobic conditions), KM= 6.05 k 1.30 PM, and with NO substrate, anaerobic conditions, KM= 7.3 f 7.4 nM. The addition of 0, resulted in short-term transient effects indicating rapid and reversible inhibition of NO and N,O production. When 0, was present, NO,, accumulated in greater amounts than observed under completely anaerobic conditions. The transient effects suggest that the reduction of NO;, NO and NzO are each inhibited to increasingly greater extents by 0,.

INTRODUCTION The enzymatic reductive steps in the denitrification pathway (Payne, 1981) viz.

appear to operate most efficiently in the absence of oxygen. Oxygen inhibits the process very effectively and suppresses the synthesis of denitrifying enzymes (Payne, 1981; Tiedje et al., 1982). Although the inhibitory effect of 0, addition to anaerobic soils, cultured whole cells or enzyme suspensions undergoing denitrification has been known for many years, very little quantitative information of the effect of 0, on the specific steps or even on the overall process is available (Remde and Conrad, 1991). Davies et aI. (1989), using mass spectrometry to monitor dissolved gases, found that in Paracoccus denitrificans N,O production increased and N, production decreased with O2 present whereas with Pseudomonas aeruginosa both N20 and N2 increased. In a study of O2 addition to denitrifying Pseudomonas aeruginosa, Hernandez and Rowe (1987) found that NO, reduction to NO; in cell extracts was apparently not affected by 0, although inhibition was observed in whole cell suspensions. They proposed that OX inhibited NO; transport across the cell membrane. Nitrite uptake was not affected by low, *Author for correspondence.

-0.3% of 0, saturation, concentrations although NO; reduction was partially inhibited. Hochstein et al. (1984), in a study using Paracoccus halodenitrrjicans observed that sensitivity to 0, inhibition increased inversely with N oxidation state, i.e. NO; reduction was less sensitive than NO; reduction which in turn was less sensitive than N,O reduction. Samuelsson et al. (1988) reported significant NO; accumulation when Pseudomonas juorescens had access to both NO; and 02, but only minor accumulation of NO; in the absence of 02. Similar effects have been reported with partially aerobic soil systems (Kroeckel and Stolp, 1985; Drury et al., 1991). Whether similar inhibition mechanisms operate in these and other systems cannot be answered at the present time. Gamble et al. (1977) showed that Pseudomonas fluorescens are among the most common denitrifying microbes in world soils. It is therefore important to determine the response of these bacteria to changing environmental factors such as the concentration of O,, NO,, NO;, etc. Therefore our objectives were to examine the response of Pseudomonas fluorescens to added NO< or NO; under partially aerobic and fully anaerobic conditions. MATERIALSAND METHODS

Cultures of Pseudomonas JIuorescens (ATCC No. 17822) were grown at 30°C on tryptone, yeast extract, agar slants (5, 1.5 and Sg in I litre distilled Hz0 901

902

D. J. MCKENNEY et al.

respectively). All media were autoclaved at 127°C for 20 min. For harvesting, suspensions were prepared by inoculating 100 ml tryptone and yeast growth media, and shaking 12-16 h at 30°C. Bacteria were centrifuged at 5000 rev min’ for 15-20min (or 10,000 rev mm’ for 5 min), washed with phosphate buffer (pH = 7) or Tris-HCI buffer (20m~, pH = 7), recentrifuged, and resuspended in buffer. Optical densities (O.D.) of suspensions were typically ca 1.5 at 620 nm corresponding to ca IO9 cells ml-‘. Calibration of O.D. vs number of cells was determined by serial dilution. Colonies were counted with a Quebec colony counter (Darkfield, model 3327, American Optical, Buffalo, New York) and optical densities were determined using a Shimadzu U.V. 240 spectrophotometer (Shimadzu Corp., Kyoto, Japan). The flow system was a slightly modified version of that described by McKenney et al. (1982, 1984). A constant flow, ca 380 ml min’, of carrier gas, Nz (99.995%), sparged product gases from the cell culture suspensions for analysis of gaseous NO and N>O. The cell suspensions were contained in a Pyrex column (2.5 cm dia x 20 cm) that had a mediumporosity fritted disc sealed into the bottom. A sidearm (8 mm dia), ca 15 cm above the disc at an angle of about 60” and closed with a septum allowed direct injection of substrate solution into the suspension. The relatively rapid flow of carrier gas and its passage through the fritted disc produced vigorous mixing during runs, which minimized diffusion or mixing rate limitations on product formation. Net NO and N,O production rates, fNo and fNlo, respectively were calculated as fNo = f,[NO] and fN20 = f,[N,O], where f, is the measured total gaseous flow rate through the reactor column and [NO] and [N,O] are concentrations in the effluent gas stream. Further details were given by McKenney et al. (1982, 1984). Oxygen from a 1% 0, in N, mixture (Liquid Carbonic, Toronto) was added to the carrier gas as required, before passage through the suspension. Flow rates were measured with calibrated rotameter flow meters. Nitric oxide was measured using a NO/N02/N0, analyzer (Model 14B/E, Therm0 Electron Corp., Hopkinton, Mass). Nitrous oxide was analyzed by gas chromatography (Hewlett-Packard, Model 588OA) with a 63Ni electron capture detector and Porapak Q column. Nitrite was analyzed periodically following the Cox method (Cox, 1980) as modified by Nagashima et al. (1985) by withdrawing w 1 ml of suspension, immediately filtering through a 0.2 pm cellulose acetate membrane filter then injecting a _ 200 fll aliquot into 15 ml of an aqueous solution of NaI (130 mM) and Hj PO, (13 M). Nitrite was rapidly reduced to NO which was then swept by carrier gas from the solution to the NO/NO*/NO, analyzer. Dissolved O2 was measured using a dissolved 0, electrode (Model 57, Yellow Springs Instrument Co., Yellow Springs, Ohio).

Michaelis-Menten K, and V,,, values were calculated using ENZFITTER (Elsevier Biosoft, Cambridge, England), a non-linear regression data analysis program. RESULTS

Addition of NO; to suspensions of Pseudomonas Jlworescens under anaerobic conditions resulted in net production of NO. Nitrate conversion of NO, was obviously slow relative to formation of NO from NO; because NO; accumulated to concentrations that were very low relative to the concentration of added NO;. For example, a number (6-1 I) of 0.5 ml aliquots were taken for NO, analysis in each of six kinetics trials with added NO; concentrations ca 60 pM. The average accumulated NO; concentration was only 0.11 + 0.09 ,u~ (n = 60). The dependence of fNo on [NO,] was determined by the addition of various quantities of NO, to the cell suspensions and measuring the maximum rates produced following each addition. Figure I shows data obtained from successive injections of 20 pil.Oml vol of 39.6m~ KNO, aqueous solution. The NO; concentrations obtained ranged from 0.056 to 9.78 ITIM. Each injection resulted in a stepwise increase in fNo, and was marked by a sudden decrease followed by a rise in NO production. This was probably due to the introduction and rapid consumption of small quantities of 0, dissolved in the NO, solution thereby temporarily interrupting production of NO. Following a final addition at -60 min the run was terminated. Typical plots of fNo vs [NO<] are shown in Fig. 2. Because rates tended towards saturation at high [NO,], it was convenient to express the data in terms of apparent Michaelis-Menten

4o I

0

I

I

,

,

0

10

20

30 Time

5

40

1

I

50

60

(min)

Fig. I. Net NO production with successive additions of NO, to a 14ml suspension of 5 1.5 x IO’ cells at T = 298 K. An aliquot of 20~1 to I.0 ml (0.79-39.6 pmol NOj) was added after each maximum NO production was obtained.

Denitrification by P. juorescens: oxygen ellkcts 40

Table

2. Apparent

under partially

903

MichaelisMenten

aerobic

conditions

values with

- I.5 x IO’ cells ml Substrate

PO,

NO;

for

NO

or NO,

14.5

1OJ’

Khl

V,,X

0%7

2.lO;:68)+

2

4

6

8

Nitrate concentration

0.210

3.23 (0.67)

22.1 (1.0)

18.2

0.235

2.35 (0.061)

10.2(1.0)

20.3

0.262

I .36

14.1 (1.9)

26.2

0.338

2.02 (0.67)

26.6

0.343

1.78 (0.55)

6.02 (0.67)

31.1

0.401

I .83 (0.80)

4. I6 (0.67)

I.

anaerobic

Apparent

Michaelis-Menten conditions -1.5

NO,

7.Ol(Y.73)

72.6 (7.2)

4.15(1.45)

44.5 (6.8)

13.0

0.168

7.58 (2.37)

47.8 (6.1)

16.2

0.209

5.57 (1.61)

51.6(6.0)

4

18.0

0.232

7.03 (I .94)

54.1 (6.1)

10

21.0

0.271

6.68 (I .45)

46.6 (4.0)

25. I

0.324

4.26 (0.75)

22.6(1.4)

Mea”

for NO;

numbers means

production

substrate

nmol min-’ 8

3.74ny.86)*

43.5 (37.5)

301

2

3.72 (1.52)

9.11 (1.49)

3.74 (I .80)

36.6 (36.7)

(I I .6)

283

6

2&.70)

20.0

290

2

7.26 (0.80)

58.1 (20.9)

295

I

8.81

69.3

303

6

6.86 (2.72)

29.3 (19.5)

in parentheses

where

standard

and

Vma.

KU

K 298

Mean = 5.28(0.76) *The

NO

of

trials

Mean = NO;

or

are standard

deviations

errors,

are given.

32.1(5.8) except

(5.5)

0.0743

x 109 cellsml-’

Number

T

Substrate

NO;

II.1

0.127

(mM)

values

with

IO (0.54)

ll.9(1.5)

5.76

parameters, Table 1. The average KM from 10 such plots with added [NO;] between 0.021 and 12.7 mM was 3.74 + 1.80 mM and the average If,,,., value was 36.6 f 36.7 nmol min-’ under anaerobic conditions. These values, based on the rate of NO production, closely approximate the apparent Michaelis-Menten parameters for NO; reduction to NO; because the NO production rate from NO; was several hundred times faster (below). In addition NO was rapidly removed from the suspension by carrier gas thereby minimizing subsequent reactions (McKenney et al., 1982). With added constant flows of O,N, carrier gas mixtures net NO production rates were reduced compared to rates in the absence of O2 (Fig. 2). Table 2 shows results of experiments over a range of low partial pressures of O2 (14.5-3 1.1 Pa). Although NO is oxidized by 0, via a termolecular reaction to form NO,, the rate would be negligibly slow under our experimental conditions (Galbally and Roy, 1978).

under

= 2.

(0.54)

9.81

Fig. 2. Net NO production rate as a function of NO; concentration under anaerobic conditions (T = 298 K) and in the presence of a constant flow of 0, (P, = 18 Pa. T = 297 K). The solid lines are Michaelis-Menten curves computed to fit the data.

Table

8.97(1.10)

16.3

NO;

0

for

the

*Calculated

’

nmol min

Mean

0

and

T = 297 K

‘,

Pa

NO,

production

substrate

assuming

Henry’s

= 6.05 (I .30)

Law

applies.

48.5 (13.7)

KH = 4.3 x IO9 Pa at

297 K. tThe

numbers errors

in parentheses

estimated

of points

by the

except

for each curve ranged

parentheses

for

ENZFITTER

for the means

from

are standard

the means program. 6 to 14. The

are standard The

number

numbers

in

deviations.

No significant amount of NO, was detected using the NO/NO,/NO,, analyzer. On average the estimated V,,,,, and KM values were similar in the presence or absence of 02. No obvious trend of either apparent V,,, or KM vs [0,] was evident. The concentrations of OX dissolved in the suspensions were estimated assuming that Henry’s Law applied. This assumption was checked by a few measurements of O2 dissolved in distilled water in experiments carried out under similar conditions, with 0,-N, carrier gas mixtures ranging between 0.1 and 5% O2 (_ 100 Pa-5 kPa) and with flow rates normally employed. The results showed that only about l-l .5 min were required to reach 0, saturation with these partial pressures of 0,. The final measured concentrations were in good agreement with those predicted by Henry’s Law. With the lower partial pressures of 0, we used (6-60Pa), which were below the detection limit of the O2 electrode, longer times would be required to saturate the suspensions. In order to estimate whether the experiments were of long enough duration for the low partial pressures of O2 to reach Henry’s Law equilibrium values in solution we calculated the mass-transfer coefficient and rate of 0, transfer to the suspension following Geankoplis (1983). Assuming a reasonable N,-O, mixture bubble diameter of 1 mm and 1 x lo4 bubbles SC’, only ca 3 s would be required to reach saturation at 298 K and O2 partial pressures in the range 6-60 Pa, provided that the O2 consumption rate by the microbes was ~6 nmol min-‘. Oxygen uptake rates of 109 and 128 nmol min-’ mg-’ protein for Pseudomonasjuorescens have been reported by Robertson and Kuenen (1991). If we assume a mass of 5 x lo-l3 g cell-’ (Powell, 1974) with _ 10% protein, an O2 uptake rate of about 0.1 nmol min-’ is obtained. Therefore we are reasonably

D. J. MCKENNEYet al.

904

11”4

7

.9

c

&

z

Anaerobic

E E 0’ 5

0 1

0

2 3 Time (min)

4

5

Fig. 3. Net NO production rate following NO; addition under anaerobic conditions and with added 0, (P,, = 22 Pa) in the carrier gas. [NO;] = 0.718 PM, T = 297 K.

that Henry’s Law equilibrium values of O2 were achieved in our experiments and that the observed effects were not limited by mass Or transfer. In similar experiments with NO; as substrate, production of NO was usually much more rapid than was the case with NO; substrate. Figure 3 shows an example, fNo vs time following NO; addition under anaerobic or partially-aerobic conditions (22 Pa). In the absence of 0, the maximum net rate was obtained in _ 0.4 min and by _ 3.5 min the reaction was complete. When a small quantity of O2 was present the rates were substantially reduced although the total amount of accumulated NO was approximately equal to that obtained in the absence of 0,. The ratio of total NO produced without O2 to total NO produced confident

702 60

0

I

0

5

I

10

1

15

I

20

Nitrite concentration

25

30

35

(PM)

Fig. 4. Net NO production rate as a function of NO; concentration under anaerobic conditions (T = 295 K) and with added 0, (P, = 21 Pa) in the carrier gas. T = 297 K. The solid lines are Michaelis-Menten curves computed to fit the data.

with Or (PO*= 8-60 Pa, [NO;] = 0.719 p(M) was 0.99 kO.11 (n = 13). A typtcal plot of fNo vs [NO,] is shown in Fig. 4, and the calculated apparent Michaelis-Menten constants are shown in Table 1. In this case data were obtained over the temperature range 283-303 K, however, we observed no significant trend with temperature. The average KM was 5.28 f 0.76 PM and I’,,,,, was 32. I + 5.8 nmol min-’ under anaerobic conditions. With added constant flows of 0,-N, carrier gas mixtures the average values were KM = 6.05 f 1.30~~ and V,,,,,= 48.5 + 13.7 nmol min-’ (Table 2). These KM values are ca 700-400 times smaller than the respective apparent KM values with NO; substrate. Some NO was reduced in situ to form a small quantity of N,O. For example, N,O production rates were usually < 10% of NO production in the absence of OX and ~25% when 0, was present following NO, addition. Rates of net N,O production always tended to parallel net NO production rates (data not shown). When NO, was the substrate added, similar behavior was observed. No effort was made to measure N, since removal of its precursors NO and N,O by carrier gas would significantly reduce the N, production rate (McKenney et al., 1982). A few attempts were made to determine N,O production with NO as substrate by introducing NO contained in the carrier gas at constant flow rates through the cell suspensions and measuring subsequent production of N,O. Unfortunately the low solubility of NO in water, Henry’s law constant, KH = 1.72 + lo6 kPa (Battino and Clever, 1966) imposes a limit on the amount of NO that can be supplied to the cells and hence on the amount of N,O that can be produced by utilizing exogenous NO as substrate. The concentrations of N,O produced were only about 0.8-2 pmol ml-’ (gas phase at STP). Concentrations of NO in solution calculated using Henry’s Law were only about 2-25 nM. The estimated Michaelis-Menten parameters (three trials, data not shown) were KM = 7.3 f 7.4 nM and V,,,,, = 0.7 + 0.5 nmol min-‘. In order to observe the initial response to 0, addition, we added small flows of O2 to the N2 carrier gas and the mixture was bubbled through the suspensions during a number of experiments initially started under anaerobic conditions. In these cases transient short-term changes in the rates of production and removal of NO;, NO and NzO were observed prior to the establishment of ‘steady-state’ rates as obtained with constant flows of 02-Nz carrier gas mixtures. The initial response to addition of Or depended on several factors: the concentration of NO;, the number of viable cells, the time following NO; injection that 0, was added, and the quantity of O2 added. With initial [NO;] = 707 pM and 3 1 Pa O2 added at 6 min [Fig. 5(a)], fNo rapidly decreased then increased through a maximum at 9 min eventually leveling off at 13 min. When the O2 flow was discontinued fNo again increased then decreased to the approximate

Denitrification by P. fluorescens: oxygen effects

905

(b)

(a)

I

0, off

0

10

5

15 20 Time (min)

25

30

0

: 0 4

I I

0,

on

0, off

:

I

5

10

15

Time (min)

Fig. 5. Transient effects of Oz on net NO production with NO; substrate (707 PM). The 0, was added and turned off at the time indicated. 5(a) PO, = 31 Pa. T = 294 K. (b) PO1= 33 Pa. T = 299 K.

rate expected without 0, addition. Figure 5(b) shows other results with the same concentration of added NO;. The maximum fNo obtained in this case was ca

off

25

(a)

0 NO

A NO;

02 off

iO200

:O2

off

(b)

. NO I

0

10 20

ni

30 40 50 Time (min)

n

60 70

N20

80

Fig. 6. Transient effectson, 6(a) net NO production rate and accumulation of NO;, [NO; ] = I .36 mM, P, = 23 Pa, 1.4 x IO9cells ml-’ and (b) net NO and N,O production rates, [NO;] = 87.5 PM, P,,, = 9.7 Pa, 1.1 x lo9 cells ml-‘.

45% higher suggesting that more viable cells were in the suspension or more enzyme activity. With addition of 33 Pa O2 at 4 min NO production decreased rapidly then more slowly with complete inhibition at 6.5 min. When 0, flow was turned off, fNo rapidly increased to a value greater than the value obtained before 0, was added. With 1.36 mM NO; initially [Fig. 6(a)] and with 23 Pa O2 added, the fNo rapidly decreased by - 50% eventually leveling off at - 1.1 x lo-’ mol min-’ while NO; continued to accumulate, apparently not altered much by 0, addition. When anaerobic conditions were restored at 45 min, fNo increased sharply similar to the by -58% to - 1.8 x 10-8molmin-‘, rate expected in the absence of OZ. A relatively small increase in NO; concentration (- 11%) was also observed indicating that NO; formation had been partially inhibited. Figure 6(b) shows results where only 9.7 Pa O2 was added to a suspension containing only 87.5~~ NO;. With this small concentration of NO;, fNo was relatively slow, but increased in the presence of O,, the increase in NO and N,O occurring some 10min following O2 addition. The data [Fig. 6(b)] show that production of N,O responds to O2 addition in a similar fashion to NO, and the progress curves were more or less parallel. Both show an increase after O2 was added by cu 15-fold, followed by a sharp decrease to apparently level off at rates still greater by about 5-fold than rates measured before admission of OZ. When O2 was turned off, the rates again increased through a maximum. By - 70 min the rates returned to levels approximately equal to those existing before O2 addition. The short-term transient effect on NO production when O2 was added using a relatively large quantity

D. J. MCKENNEY ef al.

906

120 h 100 ; Z *I) B E a 60 0’ 5 40 20 0

1

0

10

20 30 Time (min)

40

50

Fig. 7. Transient effects of 0, on net NO production rate with NO, substrate. The 0, was added and turned off at the

times indicated. [NO;] = 205 PM. (205 PM) of NO; as substrate is illustrated in Fig. 7. Nitric oxide was very rapidly produced when NO; was injected into the suspension at zero time. The rapid increase in NO was followed by a slower decrease indicating consumption of NO;. Addition of only 9 Pa O2 resulted in a considerable decrease followed by a markedly slower increase in NO production. When O2 was turned off at 19 min, NO production increased to almost the same rate as obtained just prior to O2 addition. Repetition of the experiment with a slightly greater amount of O2 (PO, = 27 Pa) added to the same suspension resulted in similar changes in NO production. Nitric oxide production was inhibited totally using only slightly larger quantities of 0, (- 100 Pa). In similar experiments with NO; substrate, the effect of addition of small quantities of O2 on both fNo and fN20was determined., Both rates decreased rapidly, apparently in parallel, following O2 addition. For example in three cases using 210 FM NO;, fNSowas ca 2% of fNo (fNo = 9.43 + 0.18 x lo-’ and fNjo = 1.59 +_0.45 x lo9 mol min-‘) before 0, addition. Within about l-5 min after addition of 3, 14 or 16 Pa of 02, fN*o ranged between -4-22% of fNo. The immediate effect on fNo was to decrease by -93, 99 and 9 1% respectively before slowly rising, similarly to the rates shown in Fig. 7. The fNlo decreased by 70,91 and 76% respectively. Because rates change so quickly in response to 0,) large variations in NO and particularly NzO analyses were not unexpected in these short-term experiments. DISCUSSION

Under both anaerobic and plots of rate vs added substrate cated typical saturation kinetics, ent Michaelis-Menten constants, obtained. The maximum rates,

aerobic conditions, concentration indifrom which apparKM and V,,,, were V,,,,,, are not very

informative as they depend on the number of viable cells. The apparent KM values, on the other hand, are independent of enzyme concentration and can therefore be compared to previous related studies. For example, the average KM = 5.28 + 0.76 PM obtained in our work using NO; substrate agrees well with the value of 5.5 + 1.1 PM obtained by Betlach and Tiedje (1981) based on NO; loss in pure cultures of Pseudomonasjluorescens grown under denitrifying conditions. This result suggests that nitrate reductase is constitutive in this organism because it was apparently not repressed by the aerobic conditions and lack of NO; in our growth media. In contrast, Betlach and Tiedje (1981) obtained a KM < 15 PM for NO; reduction in Pseudomonas jhorescens grown under denitrifying conditions whereas in our study with cells grown aerobically in the absence of NO;, KM was much larger, 3.74m~, similar to values of N 0.1-3.5 mM reported for various soils (Betlach and Tiedje, 1981). This discrepancy was probably due to repression of nitrate reductase synthesis in our culture growth media. Betlach and Tiedje (1981) suggested that the higher KM values for denitrification in soils may be explained by carbon limitation and diffusion effects. Our rough estimate of KM = 7.3 nM for N,O production with NO substrate agrees with the values obtained by Remde and Conrad (1991) of 0.6-6.0 nM, based on NO consumption in Pseudomonasjuorestens under various growth conditions. LineweaverBurk, Hanes, and other graphical representations were non-linear, as expected for coupled enzyme systems (Cornish-Bowden, 1979). Although the detailed mechanism remains a matter of considerable conjecture (Heiss et al., 1989), NO is considered to be produced from NO;, the primary product of NO; reduction, and NO undergoes subsequent reaction. Determination of net NO production, fNo, would have provided KM and V,,, values more closely approximating ‘true’ values if constant steady-state concentrations of NO; were maintained. This was clearly not the case here. Because the apparent KM values presented in Table 2 show no clearcut trend with added 02, standard plots (Cornish-Bowden, 1979) of KM/V,,,,, or l/V,,,,, vs O2 could not be expected to yield reliable estimates of apparent inhibition constants for 0,. The apparent KM values for the three substrates range over about six orders of magnitude with 3.74 f 1.80 mM for NO;, 5.28 k 0.76 PM for NO;, and 7.3 f 7.4 nM for NO reflecting their increasing reactivity. With the flow-through system used in these experiments, rapid removal of NO from the suspension decreased the rate of N,O production, which therefore was usually considerably less than NO production. Anderson and Levine (1986) observed a significantly higher production rate of NzO (10.09 nmol cell-‘d-l) than NO (0.09mmol cell-’ d-l) using Pseudomonas Jitlorescens in a batch culture sparged

907

Denitrification by P. Jluorescens: oxygen effects with 0.5% 0,.

The lower flow rate (150 ml min-‘) used in their experiments resulted in a relatively long flushing time (3.7 min) and consequently more opportunity for NO to undergo reaction. The residence time in our reactor was only -0.3min. Betlach and Tiedje (1981) presented a MichaelisMenten kinetic model for the successive steps in the denitrification process using data obtained from experiments with pure cultures, including Pseudomonas Jluorescens. They found that O2 inhibited denitrification and increased production of N,O relative to N,. Their results could be reasonably explained by their mode1 in which they assumed that O2 inhibited each step equally in the pathway, and the patterns of accumulation of intermediates could be attributed to different rates of reduction. They did not rule out the possibility that N,O reductase may have been more sensitive to 0, than the reductase leading to N,O formation. Our experiments show that O2 inhibited all steps in the denitrification process. The inhibition was rapid and reversible. Furthermore, the observed patterns of fall and rise can be readily explained if the sensitivity to inhibition increases in the order NO; -+NO;
fiers by Hochstein et al. (1984), Anderson and Levine (1986), Hernandez and Rowe (1987), Samueisson et al. (1988), Davies et nl. (1989) and Remde and Conrad (1990). Completely anaerobic conditions are clearly not essential for denitrification to proceed. In fact Davies et al. (1989) found an increase in N,O and N, production from Pseudomonas aeruginosa when 0, was present. The different sensitivity of the various steps towards 02, together with the reversible nature of the inhibition are probably partly responsible for the variable ratios of NO:N,O, and N,O:N, emissions observed from soils under natural conditions where 0, availability may be restricted by water, porosity, etc. The relatively high sensitivity to O2 of N,O and NO in contrast to NO; reduction and particularly the relative insensitivity of NO; reduction may perhaps be a general phenomenon. However, quantitative differences in production rates in different species are evident (Anderson and Levine, 1986; Davies et al., 1989; Remde and Conrad, 1990). Remde and Conrad (1990) suggested that NO and N,O production by the nitrifier Nitrosomonas may proceed by the same sequential pathway as in denitrifiers. The inhibition of NO; reduction by 0, which could profoundly influence the concentration of NO; in soils may play a pivotal role in favoring either nitrification, or denitrification or dissimilatory nitrate reduction. Acknowledgements-We

thank Dr K. E. Taylor, Department of Chemistry and Biochemistry, University of Windsor for helpful discussions. This work was supported by a grant from the National Sciences and Engineering Research Council of Canada. REFERENCES

Anderson I. C. and Levine J. S. (1986) Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers. Applied and Enoironmenfal Microbiology 51, 938-945.

Battino R. and Clever H. L. (1966) The solubility of gases in liquids. Chemical Reviews 66,.395-463. _ Betlach M. R. and Tiedie J. M. (1981) Kinetic explanation for accumulation of nitrite, nitric oxide and nitrous oxide during bacterial denitrification. Applied and Environmental Microbiology 42, 1074-1084.

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