metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at...

FEMS Microbiology Ecology 101 (1992) 133-143 0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00 Published by Elsevier

133

FEMSEC 00397

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Michael Schuster ’ and Ralf Conrad b

a Fakrtltiit fiir Biologic, Uttioersitiit Kotrstanz, Kottstanz, ad ” Ma.u-Plnttck-lnstitlcl fiir Terrestrische Mikrobiologie, Marbrtrg, FRG

Received 10 January 1992 Revision received 14 April 1992

Accepted 14 April 1992

Key words: Nitric oxide production; Nitric oxide consumption; Nitrification; Denitrification; Acetylene inhibition; Soil moisture content; Nitrogen fertilizer

1. SUMMARY

NO production and consumption rates as well as N,O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed. us to distinguish between nitrification and denitrification as sources of NO and N,O. Under aerobic conditions untreated soil showed very low release of NO and N,O but high consumption of NO. Fertilization with NH: or urea stimulated both NO and N,O production by nitrification. Addition of glucose at high soil moisture contents led to increased N, and N,O

Correspondence ro: R. Conrad, Max-Planck-Institut fiir Terrestrische Mikrobiologie, Karl-von-Frischstrasse, D-3550 Marburg, FRG.

production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N,O production by denitrification. The production of NO and N,O was further stimulated at low moisture contents and after addition of glucose or NO;. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO;. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N,O release.

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2. INTRODUCTION

Nitric oxide (NO) and nitrous oxide (N,O) are trace gases which play a central role in the chem- istry of the atmosphere [l]. Although the global emission is still uncertain, it is clear that soils are a significant source of these gases. Field experiments [2,3] as well as laboratory experiments [4,5l have shown that production and consumption of NO occur simultaneously in soils. At a certain NO-mixing ratio, the compensation point, the gas flow between soil and atmosphere, is zero. De- pending on the ambient NO-mixing ratio a soil may therefore act as a net source or a net sink for NO. By contrast, the compensation mixing ratio of N,O is usually higher than the ambient mixing ratio, so that soils seem to be only sources for N,O [6,7]. (NO mixing ratio = volume NO per volume gas phase; ppmv = ~1 I-‘)

Nitrification and denitrification are considered to be the most important processes involved in metabolizing NO and N,O in soils. The production of NO and N,O depends on the overall activity of nitrification and/or denitrification and in addition on the percentage of the nitrogen flow that is partitioned into NO and N,O as end products [8]. Nitrification is an aerobic process which is predominantly regulated by the availability of NH: 181. Fertilization with NH: has been shown to enhance emissions of NO and N,O [2,9]. By contrast, denitrification is basically an anaerobic process. Thus the most important fac- tor regulating denitrification activity is the oxygen partial pressure [lo]. Despite contact with atmospheric oxygen, anaerobic microsites exist in soils [ll]. Under in situ conditions a high soil-moisture content in combination with a high rate of oxygen consumption may thus stimulate denitrification. In laboratory experiments, high soil-moisture contents increased N,O formation by denitrification [12].

The dependence of NO emissions on soil- moisture content has so far not been investigated in laboratory experiments. The soil-moisture content affects not only the availability of O,, but also the diffusion of NO and N,O in the soil and thus the flux between soil and atmosphere [13]. Diffusion limitation may exist between the atmo-

sphere and the bulk-soil phase, between different soil crumbs, and within an individual soil crumb. For example, diffusion limitation may decrease the flux of NO or N,O from the site of production out into the atmosphere, thus increasing the residence time and the possibility of consumption.

In this study, NO and N,O metabolism were measured at different soil-moisture contents and under various conditions that stimulated and/or inhibited nitrification and/or denitrification. The examined soil contained both nitrifiers and denitrifiers and showed an increasing production of NO and N,O with decreasing oxygen partial pressures [14].

3. MATERIALS AND METHODS

Soil samples were taken from the A,, horizon of a maize field in the Donau valley at Ottmaring in the vicinity of Straubing (Bavaria). The content of nitrogen compounds was 1.3 pg NH:-N g-‘d.w. and 21.4 pg NO:-N g-‘d.w. respec- tively. The maximum water holding capacity (WHC) was reached at a water content of 41% (41 g Hz0 per 100 g d.w. soil). The other charac- teristics have already been described [15]. The soil was passed through a sieve (2 mm mesh) and stored at ambient moisture content (47% WHC) in a cold room (4°C). At the beginning of each experiment the soil was sprayed with water or solutions of NH&l, urea, KNO, or glucose, in order to reach the required water content and to fertilize the soil. The pH value was not influenced by this procedure. In some experiments the soil was partially dried in an air stream before- hand. The exact soil-moisture content was determined gravimetrically for each treatment. Nitro- gen compounds were analyzed calorimetrically after extraction with 1 M KC1 solution [16]. All experiments were carried out at 25°C.

N,O release was measured using 10 g of moist soil that was placed in serum bottles (120 ml) which were closed with black rubber stoppers and incubated under a N, (anaerobic conditions) or an air atmosphere. Different amounts of acetylene (C,H,) were injected into the headspaces to

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give partial pressures of 1 Pa or 20 kPa C,H,. This treatment was applied to determine N,O release by nitrification, denitrification and the release of N, + N,O by denitrification [17,18]. Gas samples were taken at different times with a gastight syringe (Glenco, Housten, TX) and analyzed for N,O in a gas chromatograph [19]. The detection limit was 5 ppbv N,O. Values of mean and standard deviation were obtained from 3 parallel experiments.

A partial pressure of 1 Pa C,H, was used to inhibit nitrification. The residual N,O production was considered to be due to denitrification (N,O,), and the difference in N,O production between the uninhibited control and the flasks containing 1Pa C,H, was considered to be due to N,O production by nitrification (N,O,). Tests showed that 1 Pa C,H, inhibited ammonium consumption as much as 10 kPa C,H,, but less than 0.1 Pa C,H, suggesting that ammonium oxidation was completely inhibited by 1 Pa C,H,. Partial pressures of C,H, higher than 1 Pa were not suitable for inhibiting nitrification, as they resulted in N,O accumulation higher than the uninhibited control, probably by partially inhibiting the N,O reductase of the denitrifiers. A partial pressure of 20 kPa C,H, showed the highest rates of N,O formation and was thus considered to be completely inhibitory for the N,O reductase. The N,O production measured under these conditions was considered to represent the production of N,O + N, by denitrification (N,O, + N,,). The production of N,, was thus calculated from (N,O, + N,,) - N,O,. The statistical sig- nificance of differences in rates between different incubation conditions was tested by paired t-tests at P < 5%.

The system for measuring release and uptake rates of NO and NO, has already been described in detail [5,20]. Briefly, 50 g of moist soil were placed in a glass flask (1-l) and continuously flushed (0.1-2.0 I min-‘) with humidified NO-free synthetic air (20% O,, 80% N,) or N,. NO production was maintained proportional to the amount of soil used, thus excluding the possibility that the transfer of NO between bulk-soil phase and gas phase became rate-limiting. In order to determine the uptake of NO at different mixing

ratios, small amounts (l-20 ml min-‘) of a cali- bration gas (83.5 ppmv NO) were added to the gas stream at the inlet of the flask. The gas streams were regulated with flow controllers (HiTech, Wagner, Offenbach, FRG). AI1 gases were obtained from Messer-Griesheim (Diissel- dorf, FRG). At the outlet of the flask the gas stream was dried in a cooling trap (- 30°C). NO was continuously measured at the inlet and outlet of the flask using a chemiluminescent NO- analyzer (Tecan CLD 770 AL ppt, Hom- brechtikon, Switzerland). The detection limit was 0.2 ppbv NO. At mixing ratios > 0.5 ppmv NO the gas stream was diluted with NO-free air or N, .before analysis. The rate of NO production (P) and the pseudo-first-order rate constant of NO consumption (k) were determined by linear regression of the NO fluxes at different NO mixing ratios [5]. Alternatively, the measured NO fluxes were correlated to the corresponding NO mixing ratios according to the Michaelis-Menten kinetics using the approach described by Remde and Conrad [20]. Nitrification was inhibited by appli- cation of C,H,. Since C,H, interfered in the chemoluminescent detector with the NO signal, C,H, could not be fed continuously into the gas stream. Therefore, the gas stream was turned off and C,H, was injected into the flask to establish a partial pressure of 1000 Pa. After 3 h the gas stream was switched on again thus removing most of the C,H, from the gas phase. Some C,H, apparently remained dissolved in the soil phase which was sufficient to inhibit NO production. Then, the parameters of NO metabolism (P and k) were determined. NO, was also measured at the outlet of the flask, but could never be detected.

4. RESULTS

4.1. Production of NO and N,O The production of N,O in absence and in

presence of low and high concentrations of acetylene was measured in closed serum bottles (Fig. 1). In unfertilized soil (Fig. 1A) N,O production was partially inhibited by 1 Pa C,H, indicating that the N,O originated from both nitrification

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N20 II-IQ N/Q d.w.1 10 N20 LIJQ N/Q d.w.1

50 -- 0 Pa 02H2 ‘.“” 1 PB C2H2 - 20 kPs C2H2 ---- OOnlrd ““” I P0 C2H2 - 20 kP0 C2H2

8 - untreated soil

J

0 100 200 300 400 500 600 700 0 200 400 800 800 1000 1200 Time [h] Time [h]

Fig. 1. N,O accumulation under aerobic conditions at 3 different partial pressures of C,H, in closed serum bottles using unamended soil (A), or soil amended with 25 pg NH:-N g-‘d.w. soil (B) both at a soil moisture content of 47% WHC. Bars

indicate SD of triplicate measurements.

and denitrification. N,O production in presence measured in the following experiments after 100 of 20 kPa C,H, was higher than in the presence h and 300 h of incubation. The amounts of N,O of 1 Pa C,H, indicating that denitrification re- accumulated at 300 h are summarized in Tables sulted in production of both N,O and N,. In soil l-4 for the different experiments. The data at fertilized with NH: (Fig. 1B) N,O production 100 h gave equivalent results (not shown). The was greatly stimulated. The inhibition of N,O tables also show the rates of NO production accumulation in presence of 1 Pa C,H, indicates which were measured three times over a period that the fertilizer-induced N,O production was of about 5-10 h. Because of the different time then mainly due to nitrification. To compare dif- intervals used, we only express NO production in ferent soil conditions, accumulated N,O was rates of NO-N produced per hour, but N,O

NO flux [,,Q N/h Q d.w.1 3

emmonlum-ferlillzed

I 30 40 60 60 70 80

Time [hl _.

NO flux [nQ N/h Q d.w.1 7

30 Time [hl

Fig. 2. Flux of NO under aerobic conditions after addition of 142 pg NH:-N g-‘d.w. (A) or 100 /.q urea-N g-‘d.w. (B) both at a soil moisture content of 47% WHC. The flow rates of air through the incubation flasks were 1175 and 123 ml min-‘, respectivly.

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Table 1

Influence of soil moisture content on the accumulation of N, by denitrification (N,,), of N,O by denitrification (NaOo) or nitrification O$ON), and on the production rate (P) of NO under aerobic conditions U

WHC NZD (%I

NZOD NO production rate (ng N g-‘d.w.)

&ON (ng N h-‘g-‘d.w.)

4 0 o.s*o.3 b 1.2* 1.4 0.1 f 1.0 26 2.0* 0.5 2.3kO.7 b 0.2kO.7 0.5 * 0.3 47 0.9* 1.3 2.5*2.1 0 0.3$-0.6 55 3.1* 4.9 b lSf0.9 0.2f0.9 68 117.3*53.0 b 1.1 rto.1 0.6f0.5 70 0.3 f 0.5

’ Average* SD of triplicate measurements; N,O by denitrification (N,O,) was obtained from the N,O accumulation in presence of 1 Pa C,H,; N,O by nitrification (N,O,) from the difference in N,O accumulation without C2H2 minus with 1 Pa C2H2; N, by denitrification (N,,) from the difference in N,O accumulation with 20 kPa C,H, minus with 1 Pa C2H2. The data show the amount of N,O accumulated in closed serum bottles after 300 h incubation at 25°C (cf. Fig. 1). The NO production rate (P) was determined from linear regression of NO fluxes against increasing NO mixing ratios and extrapolation to zero NO. The NO fluxes were measured in continuously flushed flasks three times over a period of about 5-10 h.

b Marked values are significantly (P < 5%) different from each other.

production in total N,O-N accumulated per 300 h.

The release of NO (positive flux between soil and gas phase) was measured in flasks which were continuously flushed with a gas stream. It should be noted that in contrast to the experiments with closed bottles, NO is largely removed from the soil and thus can no longer be reduced to N,O or N, under the flushed conditions. Addi- tion of NH: or urea stimulated NO release (Fig. 2). NO release was enhanced but rapidly decreased again in treatments with NH: (Fig. 2A). The reason for the relatively rapid decrease of NO release after fertilization with NH: was probably due to immobilisation of NH:, since

part of the added NH: was also consumed when nitrification was inhibited by acetylene (experiment not shown). The permanently decreasing NO release rate made it impossible to measure the NO flux as function of different NO mixing ratios and thus did not allow determination of the kinetic parameters of NO turnover (P, k) in NH:-fertilized soil. However, the maximum rate of NO production (P) was probably greater than the maximum NO release rate (Fig. 2A), i.e. P > 2.9 ng NO-N h-‘g-‘d.w. After addition of urea, on the other hand, NO release increased slowly to a maximum, stayed constant for about 5 h and then decreased again (Fig. ‘2B). The time period with maximum NO release was sufficiently

Table 2

Influence of soil moisture content on the accumulation of Ns by denitrification (N,,), of N,O by denitrification (N,O,) or nitrification (N20N), and on the production rate (P) of NO under aerobic conditions after addition of 0.5 mg glucose g-‘d.w. soil

WHC NZD &OD &ON - NO production rate (o/o) (ng N g-‘d.w.) (ng N h-‘g-‘d.w.)

47 2.9* 2.2 4.7f 1.2 1.5*1.3 0.2* 1.0 50 33.8* 20.7 b 3.9* 1.9 3.Ok2.0 60 173.4k111.6 4.4& 2.6 b 4.6f4.5 0 68 143.2* 41.7 44.1 f 20.8 b 0 72 a 757.3 f 468.3 b 156.7 f 12.4 b 0 0.3*0.5

n Addition of 1.0 mg glucose g-‘d.w. soil. b Marked values are significantly (P < 5%) different from each other.

13s

Table 3

Influence of addition of ammonium (25 /.~g N g-‘d.w.), urea (25 /.~g N g-‘d.w.), nitrate (100 /.~g N g-‘d.w.1, or glucose (0.5 mg g-‘d.w.) on the accumulation of Nz by denitrificntion (N,,), of N,O by denitrification (N,Oo) or nitrification (N,O,), and on the production rate (P) of NO under aerobic conditions at soil moisture contents of 45-50% WHC

Addition N ZD %OD &ON NO production rate (ng N g-‘d.w.1 (ng N h-‘g-‘d.w.1

Control 0.9* 1.3 2.5f2.1 0.0 f 2.0 0.3 f 0.6 NH: 4.Sf 6.8 1.9kO.6 31.4f5.1” > 2.9 ’ Urea 19.5fl9.1 4.4 f 2.6 41.5k9.2 ” 10.0*0.5” N0.i 2.2* 0.2 0.9 f 0.2 0.4f0.2 0.3 f 0.6 Glucose 2.9* 2.2 4.7* 1.2 1.5rtl.3 0.2* 1.0

n marked values are significantly (P < 5%) different from the control.

long to measure the NO flux at different NO mixing ratios so that the kinetic parameters of NO turnover could be measured in presence of urea.

Untreated soil released very low amounts of NO and N,O under aerobic conditions. The production rates (PI of NO were independent of the soil moisture content (Table 1). The same was true for the amounts of N,O accumulated by nitrification (N,O,) and denitrification (N,O,). Only the amounts of N, accumulated (N,,) were significantly stimulated at 6S% WHC. All the other values were not significantly different from each other. Moisture contents higher than 70% WHC further stimulated accumulation of N,, however, gas diffusion became already limiting at this high soil-moisture content so that N,O accumulation was no longer proportional to the amount of soil used (experiment not shown).

Glucose was added in order to stimulate respi- ratory 0, consumption and/or denitrification. With glucose, denitrification was increasingly stimulated at soil-moisture contents > 68% WHC and resulted in an increased accumulation of both N,O and N, (Table 2). Production of N,O due to nitrification (N,O,) remained low under these conditions. The rates of NO production also remained low. Even after addition of 5 mg glucose g-‘d.w. at 100% WHC the NO production rate (no longer proportional to amount of soil) was only 0.8 ng NO-N h-‘g-‘d.w.

Experiments with addition of nitrogen compounds under aerobic conditions are summarized in Table 3. Addition of either ammonium or urea resulted in significantly increased accumulation of N,O by nitrification (N,O,) and in increased production rates of NO (Table 3). Addition of C,H, almost completely inhibited NO produc-

Table 4

Influence of soil moisture content on the accumulation of N, by denitrification (N,,), of N,O by denitrification (NsOo) or nitrification (N,ON), and on the production rate (P) of NO under anaerobic conditions

WHC (%I

26 30 3s 47 60 4s n

N 2D

25300*2900

26 700 f 1300 30900*3500 32900&5000

%OD (ng N g-‘d.w.1

1630*389 b

276 f 455 b 28-f 13b

0

NZON

0

0 0 0

NO production rate (ng N h-‘g-‘d.w.1

131.0f7.1

120.5 f 1.5 b 64.5 f 0.0 b 39.7 f 1.6 b

423.1 f 1.0 b

’ Addition of 2 mg glucose g-td.w. soil. b Marked values are significantly (P < 5%) different from each other.

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NO llux [ng N/h g U.W.]

-5t \ I

0 1

1 2 3 4 NO,mixing ralm lppmvl

Fig. 3. Influence of C,H, on the aerobic NO flux in soil amended with 100 pg urea-N g-‘d.w. at 47% WHC. 0, control measured after 24 h of pre-incubation in presence of urea; A, as control, but with a further 3 h of incubation

with C,H,.

tion in urea-treated soil (Fig. 3) indicating that NO was exclusively produced by nitrification. Ad- dition of nitrate, on the other hand, had neither a significant effect on N,O accumulation (N,O, and N,O,), nor on N, accumulation (N,,), nor on the NO production rates indicating that denitrification was not stimulated by nitrate. The same was true for the effect of glucose, a potential electron donor for denitrifiers.

To investigate NO production by denitrification, anaerobic experiments were carried out. NO fluxes were measured after 24, h of anaerobic pre-incubation when NO production rates were highest. NO production rates were much higher under anaerobic than under aerobic conditions (Table 4). Similarly, N, and N,O accumulation (both by denitrification) were much higher under anaerobic than under aerobic conditions. Mois- ture contents > 30% WHC increasingly inhibited the accumulation of N,O in the closed incubation bottles (Table 4). Under these conditions N,O was re-utilized after NO; was completley reduced (not shown). The reutilization of N,O, presumably by reduction to N,, was possible since N,O was not flushed out of the incubation bottles. In the closed serum bottles, NO accumulated only to amounts > 30 ng NO-N g-‘d.w. after 300 h of incubation, indicating ,that it was

probably further reduced to N,O. However, moisture contents > 38% WHC inhibited the NO production rate also under flushed anaerobic conditions (Table 4), indicating increasing partial reduction of the NO to N,O (e.g., within soil crumbs) before it could be flushed out of the flask. Under flushed conditions (47% WHC), the apparent N,O production rate was indeed higher (about 40 ng N,O-N h-‘g-‘d.w.) than in the closed bottles (about 1 ng N,O-N h-‘g-‘d.w.), but still was lower than the apparent NO production rate (about 60 ng NO-N h-‘g-‘d.w.). Addi- tion of glucose stimulated NO release (flushed flasks), but N,O release (closed serum bottles) was then no longer detectable (Table 4). N,O release was probably also stimulated, but the accumulated N,O was re-utilized in the closed bottles before the first sampling and analysis of N,O. In contrast to anaerobic bottles, the N,O which accumulated in aerobic bottles was not consumed again (Fig. 1B).

4.2. Consumption of NO NO consumption under anaerobic conditions

was studied in the flow-through system measuring the flux of NO as a function of increasing NO mixing ratios (Fig. 4). The NO release decreased linearly with increasing NO mixing ratios up to about 1 ppmv NO indicating that NO consumption followed a first-order reaction. At higher NO mixing ratios the NO flux slowly reached a con-

NO llux Imy N/h g d.w.1 1401

snseroblc

0 0 1 2 3 4 5 8 7

NO mlxlng rello lppmvl

Fig. 4. Influence of soil moisture content (26-60% WHQ on the NO flux in soil incubated under anaerobic conditions.

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Table 5

Influence of the addition of nitrate (100 pg N g-‘d.w.1 and gluose (2 mg g-‘d.w.1 on production and consumption of NO under anaerobic conditions at soil moisture contents of 45- 50% WHC

Addition NO production rate (ng N h-‘g-‘d.w.1

NO consumption rate constant (cm3h-‘g-‘d.w.)

Control 64.5 f 0.0 26.71bO.O NO, 122.5 f 0.3 47.0 f 1.0 Glucose 423.1 f 1.0 65.6* 2.4

The values of the columns were significantly (P < 5%) different from each other.

stant value. The overall kinetics of NO consumption can be explained by Michaelis-Menten kinetics as reported earlier [20]. K, and V,, values were determined from the data in Fig. 4 using Eadie-Hofstee plots. The Vmax of NO consumption ranged between 43 and 74 ng NO-N h-‘g-‘d.w. and the K, ranged between 0.9 and 4.6 ppmv NO, showing no significant trend at soil-moisture contents between 26 and 60% WHC. The first order rate constants (k = 27-49 cm3h-‘g-‘d.w.) of NO consumption at low-NO mixing ratios also did not show a significant trend with increasing soil moisture content (Fig. 4).

NO flux [ng N/h g d.w.1

0 1 2 3 rl NO ml+g .!atJ ipam~l \

Fig. 5. Influence of soil moisture content (4-70% WHC) on the aerobic NO flux in soil incubated under aerobic conditions. Negative NO flux indicates net uptake of NO by the soil. The numbers in the figure show the NO consumption rate constants (cm3h-‘g-‘d.w.1 calculated by linear regres-

sion of the fluxes versus the NO mixing ratios [5].

However, addition of glucose or of NO; apparently stimulated both the NO production rate (P) and the NO consumption-rate constant (Table 5).

Aerobic NO consumption experiments showed completely different kinetics than the anaerobic ones. The NO consumption followed apparent first-order kinetics and was not saturated up to a mixing ratio of 14 ppmv NO (Fig. 5). The analyti- cal system did not allow measurements at higher NO mixing ratios. NO consumption was due to microbiological processes, as it was nearly abolished by autoclaving (80-95% inhibition). NO, as a possible product of NO consumption was not detected. Under aerobic conditions NO consumption was significantly inhibited by increasing soil moisture content (Fig. 5). Air-dried soil (4% WHC) showed the highest rate constants of NO consumption (67 cm3h.-‘g-‘d.w.), humidified soil (70% WHC) the lowest (5 cm3h-‘g-‘d.w.). Inhi- bition of nitrification by acetylene abolished NO production, but had no effect on NO consumption (Fig. 3).

5. DISCUSSION

Our results show that both nitrification and denitrification contributed to the production of N,O and NO, however, to a different extent for each gas. Production of either NO or N,O was obviously regulated differently by the prevailing soil conditions (content of water, nitrogen, organic carbon). NO production was not influenced by the soil-moisture content or by addition of glucose, and was predominantly due to nitrification as long as the soil was incubated under air. N,O production increasingly originated from denitrification when soil respiration was stimulated by addition of glucose especially at high soil- moisture contents. Increased soil-moisture contents alone stimulated N,O production by denitrification in other investigations [181, but were not effective in this soil, at least at soil moisture contents < 68% WHC. The reason was possibly slow oxygen consumption because of the soil’s low organic carbon content.

The investigated soil produced only low amounts of N,O and NO under aerobic condi-

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tions. The low rates were most likely due to the limited nitrification activity because of the low availability of NH: and to the limited denitrification activity because of inhibition by 0,. While addition of NO; stimulated neither N,O production nor NO production, fertilization with NH: or urea strongly increased the production of NO and N,O by nitrification. Hence, under aerobic conditions nitrification was more important for production of NO and N,O than denitrification. Under anaerobic incubation conditions, on the other hand, both NO and N,O were produced by denitrification. Production of NO was then stimulated by addition of NO; or glucose.

In unamended aerobic soil, rates of NO production (approx. 100-500 pg N h-‘g-‘d.w.1 were generally much higher than the rates of N,O production (approx. O-8 pg N h-‘g-‘d.w.) or N, production (approx. O-400 pg N h-‘g-‘d.w.; being calculated from the N,O accumulated in serum bottles during 100 h of incubation). The high rates of NO production versus N,O/N, production are consistent with the conclusion that NO production was due to nitrification rather than to denitrification. In unamended anaerobic soil, on the other hand, the rates of N, production were similar to those of NO production (e.g., 90 ng N,-N h-‘g-‘d.w. vs 64 ng NO-N h-‘g-‘d.w. at 47% WHC, Table 4) being consistent with NO production by denitrification. How- ever, the comparison of the production rates of NO and N,O has to be interpreted with care, since the former were measured during much shorter periods than the latter. NO production rates may not have been constant during the long periods used to measure N,O accumulation. At least in soils amended with nitrogen, NO production rates changed with time and thus were not comparable with the rates of N,O accumulation.

Most of the NO produced in anaerobically incubated soil was removed from the continuously flushed incubation flask before it could be reduced to N,O and N, by the denitrifiers. In the closed bottles, however, where NO and N,O could be further reduced, the major product of denitrification was N,. This conclusion is consistent with earlier observations [14]. The anaerobic NO-production rate in flushed flasks decreased with in-

creasing soil-moisture content. Possibly, the NO- production process itself was unaffected by the moisture content, but the diffusion of NO into the gas phase may have been limited. Although the diffusion between the bulk soil and the gas phase was apparently not limiting up to 70% WI-K (because of proportionality between NO release rate and amount of soil), diffusion still may have been limiting within the soil crumbs. If the NO production was mainly taking place inside of soil crumbs, diffusion limitation by the increased soil moisture content may have allowed the consumption of part of the produced NO before it could escape into the bulk-soil gas phase. In this case, the measured NO-production rate (P) would actually be an apparent NO-production rate by the soil crumbs, and this rate would be lower than the actual NO-production rate by the denitrifiers inside of a soil crumb because of unaccounted NO consumption within the soil crumbs.

The rates of anaerobic consumption of NO from the gas phase, however, were not significantly influenced by increasing soil-moisture content indicating that diffusion of NO into the soil crumbs was not limiting for the consumption process. A conceivable interpretation would be that most of the NO diffusing into the soil crumbs was consumed at the outside rather than in the interior of the soil crumbs by denitrifiers with high affinity for NO. Indeed, anaerobic NO consumption exhibited K, values which were in an ex- tremely low range of l-5 ppmv NO equivalent to 2-10 nM NO in the soil water phase. Similarly low K, values were also observed in other soils and in pure cultures of denitrifying bacteria [20,211. Anaerobic NO consumption was stimulated by nitrate and glucose, as observed similarly in other soils [9,211.

Aerobic consumption of NO, on the other hand, showed the kinetics of a first-order reaction up to 14 ppmv NO. This behaviour is different from that described earlier for other aerobic soils [201. It is unlikely that this reaction was caused by denitrifiers which typically show Michaelis-Menten kinetics with K, values in the lower ppmv range [20]. Possibly, NO was consumed by an oxidative rather than reductive pro-

142

cess. NO consumption has been demonstrated in aerobically growing methanotrophic bacteria [22]. Oxidative consumption of NO was suggested for Nitrobacter [23]. Oxidation of NO to NO; was also observed in a Pseudomonas species [211. NO may also be consumed by microbially mediated nitrosation of soil organic material [24]. NO consumption by ammonium oxidizers, on the other hand, has so far not been observed (unpublished results), although Nitrosomonas is able to reduce nitrite to NO and N,O [25]. More research is necessary to characterize the aerobic NO-con- suming process.

In contrast to anaerobic conditions, the rate constants for aerobic consumption of NO from the gas phase decreased with increasing soil- moisture content. This result suggests that processes in the interior of soil crumbs may con- tribute to the NO consumption, and that diffusion of NO into the soil crumbs may have been limiting. It should be noted that at 26% WHC (favourable for diffusion) the rate constant of NO consumption was similar for aerobic (30 cm3h- ’ g-‘d.w.1 and for anaerobic (49 cm3h-‘g-‘d.w.) conditions, whereas at 60% WHC (unfavourable for diffusion) it was significantly smaller for aerobic (7 cm3h-lg-‘d.w.) than for anaerobic (36 cm3h-‘g- ‘d.w.1 conditions. This result is consistent with our observation that denitrification op- erated under anaerobic but not under aerobic conditions at moisture contents lower than 68% WHC (Table 1). However, a dire& inhibitory effect of water on the unknown aerobic NO-consumption activity also cannot be excluded. In contrast to NO, N,O was not consumed under aerobic conditions. We conclude that the regulation of the NO consumption activity by soil moisture and the thus resulting diffusional constraints are as important for NO release as the regulation of the NO production processes.

ACKNOWLEDGEMENTS

This work is a contribution to the EURO- TRAC-BIATEX project and was financically sup- ported by the Bundesministerium fiir Forschung

und Technologie (07 EU 719) and the Fonds der Chemischen Industrie.

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metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at...

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