Dynamics of reduction enzymes involved in the denitrification process in pasture soil

Soil Eiol. Biochem.Vol. 26, No. I I, pp. 1501-1506,1994 Copyright 0 I994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 003%0717/94 $7.00 + 0.00

0038-0717(94)00109-X

DYNAMICS OF REDUCTION ENZYMES INVOLVED IN THE DENITRIFICATION PROCESS IN PASTURE SOIL L. Department

of Biological

DENDOOVEN

and J. M.

ANDERSON

Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, U.K. (Accepted I May 1994)

Summary-When oxygen is depleted in soil, reduction enzymes involved in the denitrification process are activated and de nouo synthesis of enzymes starts within a few hours. The dynamics of these enzymes and the effect on the concentration of inorganic N formed were investigated for a soil from permanent pasture. Soil was incubated aerobically for 5 days and then amended with 100 mg NOT-N kg-‘. Treatments were with or without C,H, and with or without chloramphenicol (found to inhibit de noun synthesis of reduction enzymes), purged of all 02, shaken and anaerobically incubated for 48 h while CO, and N,O production and NO; and NO; concentrations were monitored. Chloramphenicol was found to have no inhibitory effects on nitrate reduction indicating that nitrate reductase activity persisted in the absence of de nouo synthesis. The persistence of nitrite reductase and nitrous oxide reductase was lower as the application of chloramphenicol increased NO; concentrations and reduced N, production. In the absence of chloramphenicol, de nwo synthesis of nitrite reductase started 5 h and that of nitrous oxide 16 h after anaerobiosis was imposed. It is concluded that the dynamics of nitrite reductase have only a small effect on the N,O production as NO; concentrations remained below 1 mg N kg-’ but the low persistence of N,O reductase in combination with its retarded de-repression results in a high N,O-to-N, ratio when anaerobic conditions are rapidly induced.

INTRODUCTION

The process of denitrification leading to the production of nitrous oxide (N,O) has been the subject of extensive research. The ratio of the nitrogen gases formed is largely controlled by pH, 0, and nitrate concentrations and the status of the reduction enzymes (Firestone et al., 1980). The different reduction enzymes involved in the denitrification process are nitrate reductase which catalyses the reduction of NO; to NO;, nitrite reductase which catalyses the reduction of NO; to N,O and nitrous oxide reductase which catalyses the reduction of N,O to N, (Knowles, 1982). When anaerobiosis is induced persistent reduction enzymes already present in the soil are rapidly activated while de nom synthesis of reduction enzymes starts within a few hours. The de-repression of nitrate and nitrite reductase generally starts between 3 and 6 h (Smith and Tiedje, 1979) but de nova synthesis of nitrous oxide reductase only starts 16-33 h after depletion of oxygen (Firestone and Tiedje, 1979). The different concentrations of reduction enzymes at the onset of anaerobiosis and differences in the dynamics of de now synthesis are therefore key proximate factors determining the ratio of N,O-to-N,. The difference in persistence and the timing of de nova synthesis of the reduction enzymes were examined when anaerobic conditions were imposed after a period of aerobic incubation. Soil was conditioned for 5 days and then anaerobically incubated for 48 h

with or without chloramphenicol, assumed to inhibit de nova synthesis of reduction enzymes (Smith and Tiedje, 1979), and with or without acetylene (Balderstone et al., 1976) whereby conditions for denitrification were optimized, i.e. strict anaerobiosis and unlimited nitrate availability. MATERIALS

AND METHODS

Experimental Site The experiments were carried out with soil from permanent pasture at the Agriculture and Food Research Council Institute for Grassland and Environmental Research at North Wyke, Devon, U.K. The soil, a clayey pelostagnogley is currently classified as belonging to the Hallsworth Series (Clayden and Hollis, 1984). The inorganic fraction contained 36.6% clay, 47.7% silt, 13.9% fine sand and 1.8% coarse sand (Armstrong and Garwood, 1991). The organic C content of the upper 10 cm was 5.3% and the organic N content 0.62%. At collection, the water content was 36.9% (w/w) and the pHcHIO) was 6.0. Experimental procedure Soil samples were collected on 18 November 1992 from the O-10 cm layer of an experimental plot that had been grazed by cattle but had received no N fertilizer for at least 10 years. The moist soil was sieved (5 mm) and sub-samples of 7.5 g soil were added to 100 ml Erlenmeyer flasks. The flasks were

1501

L.

1502

DENDOOVEN and .I.

air-tight sealed with a silicone rubber stopper and incubated aerobically for 5 days at 25-C. After 5 days, the headspace of each Erlenmeyer was sampled to ensure that aerobic conditions were maintained. Samples were analysed for 02, CO? and NzO using a Pye Unicam 4600 GC (U.K.) fitted with a thermal conductivity detector at 30-C. The CTR column from Alltech (a Porapak Q column of 2 m in line with a Molecular sieve + Porous Polymer of 1.82m) with the carrier gas He flowing at a rate of 35 ml min ‘, was maintained at 30-C. The Erlenmeyers were opened and 10ml of a degassed 3.4 mM KNO, solution was added to half of the sub-samples (CON treatment) while the other half received a solution containing 3.4 mM KNO, plus 3.7 mM chloramphenicol (CHL treatment). The samples were well mixed to create a soil slurry thereby minimizing the influence of diffusion of nitrate through the soil matrix. A concentration of cu 100 mg NO, -N kg ’ was thus obtained. The concentration of chloramphenicol was around 2500 mg kg ‘, sufficient to inhibit protein synthesis but not the activity of existing enzymes (Smith and Tiedje, 1979). After application of the solution, two sub-samples of each treatment were selected at random for assays of NO, and NO;. Each sub-sample was extracted for 30 min with 50 ml of distilled H,O. The extracts were filtered through a GFC Whatman filter paper and nitrate and nitrite were measured on an ion chromatograph (Dionex, U.K.). The Erlenmeyers were resealed, vacuum sucked for I5 min and flushed with He for 15 min. The production of N,O and N, was determined using the CzHz inhibition technique (Balderstone et al., 1976). After purging of all 02, IO ml of C2H? (10% v/v) was added to half of the sub-samples with KNO, alone (ACE treatment) and to half of those with KNO, + chloramphenicol (CHA treatment). All subsamples were then kept at 25-C for 48 h. After 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42 and 48 h, two sub-samples were selected at random from each treatment. The headspace of each Erlenmeyer was sampled and analysed for N,O and CO,. The concentrations of NzO and CO2 were corrected for gas

M.

ANDERSON

100 , 90 90

1

90 50 40 30 20 10 0

Time (hours) Fig. 1. C mineralization (mg CO,< kg-’ soil) of the Rowden soil anaerobically incubated for 48 h at 25°C following the application of 100mg NOT-N kg-’ soil. (&-fQ Control, (A--A) C,H, (10% v/v), (B-u) 2500 mg chloramphenicol kg-’ and (o-_O) 2500 mg chloramphenicol kg-’ plus C,H, (10% v/v). Bars indicate plus and minus SD.

dissolved in the water (Moraghan and Buresh, 1977). After measurement of N,O and CO,, samples were analysed for NO; and NO,. Data for NO; were corrected for the formation of NO, through the degradation of chloramphenicol (Dendooven et al., 1994). Statistical

analysis

Regression coefficients were calculated and co-variance analysis was carried out with the SAS statistical package (SAS Institute, 1988) using the general linear model procedure.

RESULTS Table I. CO, and N20 production rates of the Rowden sod amended with 100 mg NO<-N, 2500 mg chloramphenicol kg -‘, CZH, (10% v/v) or 2500 mg chloramphemcol kg ~’ plus C,H, (10% v/v). Anaerobic incubation for 4X h at 25 C Treatment Control C,H, chloramphenicol C>H,+ Chloramphenicol

CO, production (mg CO,-C kg-’ h ‘)

I .5I * At I .42 A

N,O production (mg N,O-N kg ’ h-‘)

(0.06)f (0.04) 2.06 B (0.07)

0.55 A (0.06) 0.64 B (0.05) 0.68 B (0.02)

2.00 B (0.05)

0.78 c (0.02)

‘Rates were determined by linear regression (Proc. GLM, SAS, 1988). W&es with the same character are not significantly different at the 0.05 level (co-variance analysis, Tukey’s Studentized Range (HSD) Test, Proc. GLM, SAS, 1988). fNumbers in parentheses are standard error of estimates.

AND DISCUSSION

C mineralization The rate of C mineralization in the CON treatment apparently followed zero-order kinetics. A small flush of CO* at the beginning of the incubation was presumably related to the decomposition of organic material set free after application of water and subsequent shaking of the samples (Fig. 1). C,H, had no effect on the CO, production but chloramphenicol significantly (at a 0.05 level) increased the CO, production rate in both the CHL and CHA treatment by 39% (Table I). These data are consistent with results reported in a study on the use of chloramphenicol (Dendooven et al., 1994). It was

1503

Denitrification reductases in pasture soils 75

z

1 1

measured from 16 h onwards and the N,O-to-N, ratio was around 1.9 after 48 h. The ratio between the CO2 and the N,O production rates was very low, i.e. 0.45, and was mainly the result of a flush in CO1 production coinciding with a lag in N,O production at the beginning of the incubation. The ratio between the CO, and the N,O production rates for the second day of the incubation was 0.82. These values were low if compared to the situation where glucose would be the only C substrate, nitrate the only electron acceptor and NO; completely reduced to N,O:

30

01

az

2(CH,O)

+ 2NO;

+ 2H+-+2CO,

+ N,O + 3H,O. (1)

15

0

Time (hours) Fig. 2. The N,O production (mg N,O-N kg-’ soil) of the Rowden soil anaerobically incubated for 48 h at 25°C following the application of 100mg NO, kg-’ soil. (A--A) Control, (A--A) C2H, (10% v/v), (0-U) 2500 mg chloramphenicol kg-’ and (m-m) 2500 mg chloramphenicol kg-’ plus C,H, (10% v/v). Bars indicate plus and minus SD.

found that chloramphenicol is an active reagent in the denitrification process and the increase in C mineralization is presumably related to a diversion of carbon supply from protein synthesis to respiratory processes (Smith and Tiedje, 1979) and a biological degradation of chloramphenicol (Smith and Worrell, 1950). Nitrous oxide The N,O production in the CON treatment followed a specific pattern. The production was very low for the first 3 h, increased sharply between 3 and 4 h and remained constant for the next 20 h (Fig. 2). Thereafter, the N,O production halted as most of the N,O produced was reduced to N,. The lag in N,O production was presumably not the result of an inhibition of the activity of the microbial biomass since there was a small but noticeable flush in CO, production. This may, however, be a consequence of the time required to de-repress the reduction enzyme system, partly because of the inhibitory effects of persisting 0, and because of the specific kinetics of the denitrification process, i.e. nitrite is a necessary intermediate in the reduction of nitrate to nitrous oxide retarding the release of N,O. In the ACE treatment, the N,O production followed the same time course but no inhibition of N,O production was observed after 24 h as C,H, blocked the N20 reduction. Production of N, could be

In this specific situation the ratio would be 1.17. Chloramphenicol, applied to inhibit de nova synthesis of reduction enzymes, increased the N,O production in both the CHL and the CHA treatment (Fig. 2). A possible explanation was that the persistence of the nitrate reductase and nitrite reductase was high, and that the aerobic conditioning for 5 days only slightly reduced the enzyme concentration. The increase in C mineralization, due to the application of chloramphenicol, increased the supply of electrons and thereby enhanced the reduction of nitrate and the production of N,O. This result was interpreted as indicating that the reduction potential of the soil was greater than electrons provided by oxidative processes, i.e. C substrate was limited. The production of N, in the chloramphenicol treatment started at the same time (after 16 h) but at half the rate of that in the CON treatment. Consequently, the NzO-to-N, ratio (7.1) was higher in the chloramphenicol treatment. This implies that the amount of nitrous oxide reductase was low at the onset of the anaerobic incubation and its synthesis or de-repression started after 16 h. The N,O production rate was not affected by the concentration of chloramphenicol and 150 mg chloramphenicol kg-’ soil seemed sufficient to inhibit the synthesis of reduction enzymes completely. The idea that C substrate was the limiting factor as compared to the reduction potential of the soil was supported in an experiment where a spike of glucose, equivalent to 100 mg C kg- ’ soil, was applied to treatments containing a range of concentrations of chloramphenicol (150, 300, 600, 1250 or 2500 mg kg-‘) and CzHz (10% v/v). The addition of glucose increased the N,O production by more than 2.3-fold in the control treatment while the increase was only 1.6-fold (ranging between 1.56 and 1.80-fold) when chloramphenicol was added (Table 2). Chloramphenicol reduced the NzO production indicating that reduction enzymes were synthesized in the control treatment. The de-repression of reduction enzymes appeared not to start within the first 5 h of the incubation as the rates of N,O production were the

L. DENDCOVEN and J. M. ANDERSON

1504

same over that period for the control and the chloramphenicol treatments. The N,O-to-CO, ratio in this experiment was 0.66 while the ratio increased to 0.83 after the application of glucose. The N,O produced as a direct result of glucose decomposition gave a N,O-to-CO, ratio of 1.03 and when we excluded the lag phase in N,O production (for this experiment we excluded data from the first 15 h) a value of 1.12 was obtained. This value corresponds well with a theoretical value of 1.17 obtained in a situation where glucose would be the only C substrate and all nitrate reduced to N,O (equation 1). These results show that the use of glucose to measure the denitrification potential of a soil must be carried out with care as the ratio between C mineralization and nitrate reduction can change giving higher losses of N,O than those that would be encountered in the field. Nitrite

The nitrite concentration in the CON treatment increased immediately and reached a maximum of 0.7mg NOT-N kg-’ after 3 h. C,H, slightly increased the maximum value measured, i.e. 0.9 mg NOT-N kg-‘, and retarded the timing of its peak value by 1 h. Thereafter, the concentrations decreased steadily in both treatments and were below 0.2 mg NOT-N kg-’ after 48 h (Fig. 3). Chloramphenicol sharply increased the nitrite

concentration from the 6 h to the 12 h of incubation. The increase in concentration continued but was much smaller until a maximum of 2 mg NOT-N kg-’ was obtained after 36 h. The increase in nitrite seemed to point at a difference in persistence and de-repression of nitrite and nitrate reductase. If the enzymes had the same dynamic properties then the kinetics of the nitrite would not be affected by chloramphenicol. This suggested that the persistence of nitrite reductase was lower than that of nitrate reductase. Its de-repression seemed to start after 34 h and was completed within 6 h while no nitrate reductase had to be formed. The IO-fold increase in nitrite concentration had no detectable effect on the N,O production as nitrite concentrations remained relatively low as compared to the N,O produced. It is possible, however, that the difference in persistence found between nitrate and nitrite reductase could be the result of an inhibition of nitrite reductase by chloramphenicol. Inhibition of existing denitrification enzyme activity by chloramphenicol was reported by Brooks et al. (1992). In the experiment where different concentrations of chloramphenicol were added, chloramphenicol increased the nitrite concentrations (Table 3). The changes in nitrite concentrations were slightly affected by the concentration of chloramphenicol applied but a possible inhibitory effect of chloram-

Table 2. CO, and N,O production of the Rowden soil amended with 100mg NO, -N kg-‘, amended with or without glucose and different concentrations of chloramphenicol. Treatments were incubated under anaerobic conditions for 48 hat 25°C without (A) and with(B) added glucose, equivalent to 100 mg C kg-’ soil Chloramphenicol Time (h)

0 0.0 3.8 7.1 18.3 31.9 49.6

(o.o)* (0.5) (0.5) (0.3) (0.3) (2.3)

0.0 8.2 19.5 44.8 59.0 65.9 90.8

(0.0) (0.9) (0.1) (4.0) (2.1) (5.2) (3.3)

5 14 29 48

0.0 0.7 2.6 10.8 21.0 33.7

(0.0) (0. I) (0.0) (0.4) (0.6) (5.7)

0 2 5 15 22 35 51

0.0 3.0 10.0 29.8 44.5 55.0 80.8

(0.0) (0.6) (0.1) (1.0) (4.0) (3.2) (0.2)

L

5 14 29 48

L

‘Numbers

in parentheses

150

300

concentration

(mg kg-’ soil)

600

I250

CO, production (mg CO,-C kg-‘) without glucose 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 4.0 (0. I) 4.2 (0.1) 3.8 (0.5) 4.5 (0.0) 9.6 (0.0) 10.5 (1.7) 9.3 (0.2) 8.7 (0.2) 26.5 (1.8) 24.8 (0.4) 23.5 (2.3) 22.1 (1.6) 48.4 (5.5) 50.9 (2.1) 46.3 (0.7) 43.7 (1.2) 68.2 (3.8) 65.1 (ND) 64.1 (5.9) 63.8 (1.6) CO, productjon (mg CO,-C kg~ ‘) with added glucose 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 7.7 (0.7) 1.7 (0.6) 6.9 (0.1) 8.3 (0.1) 19.4 (0.4) 20.2 (I .3) 22.1 (2.9) 22.5 (0.2) 48.9 (I .7) 54.0 (I .O) 53.9 (0.1) 50.2 (I .O) 68.7 (3.9) 68.8 (7.3) 68.9 (0.7) 65.6 (2.5) 72.9 (1.2) 77.0 (0. I) 81.8 (ND) 76.6 (3.4) I I2 (2.9) I IO (7.3) 107 (5.0) II6 (3.X) N,O production (mg N,O-N kg ‘) without glucose 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.8 (0.1) 0.9 (0. I) 0.7 (0.1) I .o (0.0) 4.1 (0.1) 3.9 (0.2) 3.4 (0.0) 3.2 (0.0) 14.5 (1.3) 13.3 (0.1) 13.1 (1.1) I I .9 (0.5) 27.8 (1.6) 31.7 (1.0) 29.9 (0.1) 29.1 (1.0) 46.3 (0.1) 42.7 (ND) 41.6 (2.9) 46.5 (0.7) N,O production (mg N,O-N kg ‘) with added glucose 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 2.5 (0.1) 2.8 (0.3) 2.7 (0.0) 2.8 (0.0) 9.1 (0.1) 9.4 (0.8) 10.5 (1.3) 10.4 (0.7) 27.9 (1.3) 29.8 (0.2) 30.6 (0.6) 29.3 (0.5) 41.0 (0.8) 39.0 (2.3) 42.6 (0.7) 40.4 (0.4) 50.5 (0.9) 50.2 (1.1) 55.7 (0.2) 51.1 (I 8) 78.1 (4.6) 76.6 (13) 73.7 (13) 73.3 (II) are SD

2500 0.0 4.4 10.7 26.5 49.1 72.7

(0.0) (0. I ) (0.4) (2.5) (2.4) (4.6)

0.0 8.2 22.4 57.0 69.9 91.9 121

(0.0) (0.2) (0.3) (2.5) (5.5) (2.4) (3.2)

0.0 (0.0)

I .o (0.0) 4.4 13.4 29.7 46.1

(0.1) (0.1) (I .8) (3.6)

0.0 2.4 9.5 28.7 37.6 45.8 67.9

(0.0) (0.1) (0.2) (1.2) (3.6) (4.8) (15)

Denitrification reductases

Time (hours)

Fig. 3. The NO, concentration (mg NOT-N kg-’ soil) of the Rowden soil anaerobically incubated for 48 h at 25°C following the application of 1OOmg NO;-N kg-’ soil. (A- -A) Control, (A--A) C,H, (10% v/v), (0-O) 2500 mg chloramphenicol kg-’ and (M--m) 2500 mg chloramphenicol kg-’ plus C,H, (10% v/v). Bars indicate plus

and minus SD. phenicol on nitrite reductase was considered to be negligible. In the soil with glucose added a maximum nitrite concentration was measured after 5 h and the concentration was higher than in the unamended soil (Table 3). These results again suggest a lower nitrite reductase activity as compared to nitrate reductase. The increased C mineralization due to the application

1505

in pasture soils

of a readily-decomposable C substrate increased the nitrate reduction but, as the nitrite reductase concentration was lower than the nitrate reductase concentration, nitrite accumulated in the soil. The nitrite concentration dropped quickly when nitrite reduction enzyme was synthesized: after about 5 h from the start of the event. In the soil where glucose was added with different concentrations of chloramphenicol, nitrite concentrations increased and concentrations were higher than in the unamended soil. Nitrite concentrations continued to increase and maxima were measured after 15-22 h. As more nitrate was reduced, a consequence of the increased C mineralization, maximum nitrite concentrations were higher than in the soil not amended with glucose. Further evidence that nitrite reductase was not in excess was found when nitrite was added as the determinant species of N oxide (against a background concentration of nitrate of less than 5 mg kg-‘). The N20 production rate decreased from 2.47 mg N,@N h-’ in the control soil to 2.26mg N,O-N h-t in the chloramphenicol amended soil although that the C mineralization increased from 1.55 to 1.65 mg CO& h-‘. The dynamics of nitrogen oxides reduction enzymes

This study showed differences in the resident activity of the reduction enzymes involved in the processes of denitrification. Nitrate reductase was found to be highly persistent in soil. An increase in carbon availability resulted in a 1.58-fold increase in N,O production without a de nouo synthesis of the enzyme. This result is consistent with the studies of Smith and Parsons (1985) who showed that even when soils were dried over 3 days only 29 and 16% of initial denitrification enzyme activity (DEA) was lost, even after 63 days, 27% of the initial activity remained.

Table 3. Nitrite concentrations in the Rowden soil amended with IOOmg NOT-N kg’, amended with or without glucose and different concentrations of chloramphenicol. Treatments were incubated under anaerobic conditions for 48 h at 25°C without (A) and with (B) added glucose, equivalent to 100 mg C kg-’ soil Chloramphenicol concentration (mg kg-’ soil) Time (h)

0

0 2 5 I4 29 48

0.33;:07)* 0.40 (0.20) 0.04 (0.08) ND ND

0 2 5 I5 22 35 51

ND I .78 (0.07) 3.44 (0.20) I .72 (0.08) 0.25 (0.00) 0.08 (0.12) ND

150

300

600

I250

Nitrite concentration (mg NO, -N kg ~’) without glucose ND ND ND 0.57N(:.02) 0.69 (0.07) 0.59 (0.14) 0.5 I (0.07) I .63 (0.06) I .74 (0.20) I .25 (0.01) I .34 (0.20) 2.3 I (0.00) 3.67 (0.08) 2.84 (0. IO) 2.28 (0.20) 3.38 (0.02) 3.43 (0.01) 3.76 (0.43) 3.43 (0.01) 3.05 (0.06) 2.51 (0.05) 3.12 (0.33) 3.24 (0.05) Nitrite concentration (mg NO, -N kg ‘) with added glucose ND ND ND 1.29 (0.15) 0.66N(:.05) I.10 (0.12) 1.59 (0.12) 3.07 (0.06) 1.48 (0.11) 2.61 (0.18) 3.45 (0.05) 6.51 (0.07) 4.95 (0.19) 5.34 (0.01) 6.13 (0.03) 7.10 (0.71) 5.31 (0.71) 5.44 (0.02) 6.78 (0.18) 6.16 (0.17) 4.04 (0.07) 5.08 (0.12) 6.07 (0.06) 5.44 (0.78) 4.06 (0.65) 5.50 (0.35) 4.54 (1.0)

*Numbers in parentheses are standard deviations. ND = Not detected: lower limit of detection was around 0.03 mg NO; -N kg ‘.

2500

0.9,“(:.06) 2.29 (0.08) 3.60 (0.63) 2.91 (0.13) 2.22 (0.03) ND 0.51 (0.22) 0.67 (0.48) 5.05 (0.49) 3.75 (0.03) 3.00 (0.03) 3.73 (0.96)

1506

L. DENDOOVENand J. M. ANDERSON

The persistence of nitrite and nitrous oxide reductase was much lower. They match results reported by J. Verbruggen (unpublished Ph.D. thesis Katholieke Universiteit Leuven, 1985) where a loamy soil, anaerobically conditioned for 3 days, lost 50% of nitrate reducing capacity within 24 h under aerobic conditions and 80% after 5 days, Extending the aerobic incubation did not further decrease the reduction capacity. Although the aerobic conditioning decreased the nitrite reductase concentration, its activity was still high. Nitrite concentrations remained below 4 mg NOT-N kg-’ when chloramphenicol was added while the additional application of glucose. equivalent to 1OOmg C kg-’ soil, kept nitrite concentrations below 7.2 mg NO;-N kgg Cho and Sakdinan (1978) reported a maximum concentration of 5.5 mg NOT-N kg-’ for a soil with a pH of 6.2. Differences in the dynamics of nitrite may be related to bacterial species involved in the denitrification process (Betlach and Tiedje, 1981). The effect of the lower persistence of nitrite reduc;ase on the NzO production was negligible as the nitrite concentrations remained so low. The dynamics of the reduction enzymes were different when anaerobic conditions were induced. Nitrate reductase was in excess while nitrite and nitrous reductase were synthesized at different times after oxygen depletion. The de no~‘o synthesis of nitrite reductase in the soil followed a pattern as described by Smith and Tiedje (1979). They reported that in a first phase (1-3 h) activity was not decreased by chloramphenicol, was increased slightly or not at all by organic carbon amendment. Phase I was attributed to the activity of pre-existing denitrifying enzymes. In a phase I1 (from 4-8 h), a full de-repression of the enzyme had occurred. The derepression of nitrous oxide reductase followed the pattern described by Firestone and Tiedje (1979). The de-repression of nitrous oxide (phase II) started after about 16 h. The low persistence of nitrous oxide under aerobic conditions in combination, with its retarded synthesis when anaerobic conditions were induced led to a high N>O-to-N, ratio as the reduction of nitrate to nitrous oxide was not inhibited. We concluded from our study that the persistence of the enzymes involved in the denitrification process were different and that their & noc’o synthesis foilowed different patterns. As the persistence of nitrous oxide was low and the de now synthesis under anaerobic conditions only started after 16 h, a high NzO-to-N? ratio was found. It is also shown that the use of chloramphenicol and glucose in denitrification

studies must be carried out with care and possible side-effects must be taken into account: chloramphenicol can enhance the NzO and CO2 production while the addition of glucose can change the relationship between nitrate reduction and C mineralization leading to higher losses of gaseous forms of N than encountered in the field. Acknowledgements-We are grateful to Dr D. Scholefield for access to the Rowden plots and for comments on an early draft. The research was funded by the Agricultural and

Food Research Council.

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Macalady D. L. (1992) Inhibition of existing denitrification enzyme activity by chloramphenicol. Applied and Enrironmental Microbiology 58, 1746-1753. Cho C. M. and Sakdinan L. (1978) Mass spectrometric investigation on denitrification. Canadian Journal of Soil Science 58, 443457.

Clayden B. and Hollis J. M. (1984) Criteria for differentiating soil series. Soil Suroey qf England and Wa1e.r. Technical Monograph, No. 17, Harpenden. Dendooven L., Splatt P. and Anderson J. M. (1994) The use of chloramphenicol in the study of the denitrification process: some side-effects. Soil Biology & Biochemisrry 26, 925%927.

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