Enhancement by acetylene of the decomposition of nitric oxide in soil

Soil Biol. Biochem. Vol. 29, No. 7, pp. 1057-1066, 1997 0 1997 Elsevier Science Ltd. All rinhts reserved Printed in &eat Britain

PII: s0038-0717(!27)oooo6-0

ENHANCEMENT DECOMPOSITION ANNETTE

0038-0717/97$17.00+ 0.00

BY ACETYLENE OF THE OF NITRIC OXIDE IN SOIL

BOLLMANN

and RALF CONRAD*

Max-Planlck-Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Str.,

D-35043 Marburg, Germany

(Accepted IO November 1996)

Summary--The acetylene inhibition technique is a widely used method to measure denitrification rates in soil. This technique is based on the inhibition of the NsO reductase with high concentrations of acetylene (about 10%). We tested possible artifacts created by using this technique under oxic conditions. Parts per billion concentrations of NO, an intermediate of the denitrification, were converted to NOs within seconds when both O2 and acetylene were present. There was no conversion of NO under anoxic conditions or with acetylene concentrations < 0.1%. Propyne and I-butyne also caused the conversion of NO to NOs, although to a lesser extent. In the absence of soil, the reaction stopped when an equilibrium between NO and NO2 was reached. The NO2 concentration at equilibrium increased with increasing temperature and with increasing acetylene concentrations up to 1%. Addition of small glass beads, qua.rtz sand or sea sand also increased the NO2 concentration. We assume that acetylene ( > O.l%), especially in the presence of surfaces, stimulated the chemical reaction 2NO + 02 --t 2NOs. In the presence of soil, NOs accumulated only transiently and was then taken up by the soil. Use of “NO resulted in 50% recovery of the label in the nitrate and nitrite fractions of the soil, indicating that NO* was probably chemically converted to nitrate, nitrite and other N compounds. The NO decomposition in the presence of acetylene was observed in all 14 soils tested and occurred in autoclaved and non-sterile soil with the same rate. The rate of NO decomposition increased with increasing acetylene concentration and with an increasing amount of soil, and decreased with increasing soil water content. In general, acetylene ( > 0.1%) enhanced the decomposition of NO in soil by factors of 5-557. 0 1997 Ehevier Science Ltd.

MTRODUCTION

Nitric oxide (No11 is a reactive trace gas that plays an important rob: in the chemistry of the troposphere (Crutzen, 1979; Singh, 1987). Soils are an important source of NO and contribute in the order of 20% to the total atmospheric NO budget (Conrad, 1995). Nitric oxide is turned over in soil by simultaneously operating production and consumption processes that determine, together with the soil diffusional characteristics, the magnitude of the net flux of NO between soil and atmosphere (Galbally and Johansson, 1989; Remde et al., 1993; Rudolph et al., 1996b; Rudolph and Conrad, 1996). Experiments have shown that gross rates of NO production and NO consumption change independently when soil conditions change (Krlmer and Conrad, 1991; BaumgPrtner and Conrad, 1992; Saad and Conrad, 1993). Different microbial and chemical processes contribute to the production and consumption of NO in soil (Conrad, 1996). Nitric oxide can be chemically produced from nitrite, by dismutation at low pH, or by other reactions (VanCleemput and Baert, 1976; Chalk and. Smith, 1983; Blackmer and *Author for correspondence.

Cerrato, 1986; McKenney et al., 1990). Nitrite, however, would be produced by microbial nitrification and denitrification. Nitric oxide is also directly produced by these two processes, which are both most important for NO production in soil (Conrad, 1990, 1996; Davidson, 1991; Williams et al., 1992). Microbial consumption of NO seems to be due either to reduction to N20 by denitrification (Remde and Conrad, 1991; Schafer and Conrad, 1993), to oxidation to nitrate by heterotrophic bacteria (Baumglrtner et al., 1996; Koschorreck et al., 1996; Rudolph et al., 1996a), or to a consumption reaction (not further characterized) by methanotrophic bacteria (Kramer et al., 1990; Bender and Conrad, 1994). Chemical NO consumption, however, plays no role, since autoclaved soil does not consume NO (Johansson and Galbally, 1984; Remde and Conrad, 1991). Whereas low partial pressures (I-10 Pa) of acetylene selectively inhibit the ammonium monooxygenase of the nitrification pathway, high partial pressures (10 kPa) inhibit the nitrous oxide reductase of the denitrification pathway (Davidson et al., 1986; Robertson and Tiedje, 1987; Klemedtsson et al., 1988). The inhibitory effect of high acetylene concentrations was exploited to determine denitrification rates in soil samples, intact cores and under

1057

A. Bollmann and R. Conrad

1058

et al., 1977; field conditions (Klemedtsson Yoshinari et al., 1977; Parkin et al., 1984; Aulakh et al., 1992). The inhibitory effect of low acetylene concentrations was used to determine the relative contribution of nitrification to production or net release of NO from soil (Davidson, 1992; Schuster and Conrad, 1992; Davidson et al., 1993; Parsons and Keller, 1995) Recently, Paul et al. (1993) noted a possible reaction of NO with acetylene but did not characterize it any further. In the course of experiments in which we attempted to measure the percentage of NO production relative to that of N20 and N2 production during denitrification by using the acetylene inhibition technique, we observed a bias that was possibly caused by a reaction of NO with acetylene. Therefore, we investigated the chemical reactions of NO in the absence or presence of acetylene and soil in more detail.

MATERIALS

AND METHODS

Soil samples

Soil samples listed in Table 1 were taken from the A, (agricultural soils) or Ah (forest and meadow soils) horizons. The soil samples were air-dried, sieved (< 2 mm in diameter), and stored in polyethylene bottles at 4°C. For the experiments, soil samples were sprayed with water to obtain a defined soil moisture content and conditioned at 25°C for 2 days. Soil moisture was determined gravimetrically and expressed as a percentage of the maximum water holding capacity (% whc) (Schlichting and Blume, 1966). The pH was measured in soil suspensions in 0.01 M CaClz solution using a glass electrode. Soil organic carbon was determined by wet combustion (Schlichting and Blume, 1966). Gas analysis NO was analysed in distinct gas samples (1 ml) using two different NO, analysers: a Therm0 electron chemoluminescent NO, analyser (series 14; Hopkinton, MA) (Remde and Conrad, 1991) and a

Scintrex Luminox NO, analyser (Scintrex Ltd.; Concord, Ontario, Canada) (Rudolph et al., 1996b). The Luminox analyser operated with a chemical converter (containing CrOs) that oxidized NO to NOz. Without the converter, it was used to analyse NO*. Calibration was done with standard gases (101.7ppmv NO in N2; 97.1 ppmv NO1 in NZ; 1 ppmv = ~11~’ gas phase) which were diluted with synthetic air (80% Nz, 20% 02). The detection limits were 10 ppbv NO or NO2 for both analysers. Gas samples with acetylene concentrations > 0.2% created a background signal in the Therm0 electron analyser, which then allowed only the detection of >20 ppbv NO (‘20 nl NO 1-l gas phase). The Luminox analyser, on the other hand, showed no interferences with acetylene. Experimental set-up

All experiments (with or without soil) were carried out in serum bottles (120 ml) with Teflon stoppers. We used Teflon stoppers since black rubber stoppers showed uptake of NOz. The serum bottles were evacuated and flushed with synthetic air five times. The NO concentration was adjusted to about 1000 ppbv (1000 nl NO 1-l gas phase). A gas sample was immediately taken to analyse the initial NO concentration. Then, a defined amount of acetylene (Messer-Griesheim, Siegen) was injected. Acetylene that was obtained from Linde (Mainz) or Air Liquide (Dusseldorf) was also tested, but gave the same results. The acetylene was purified by passage through 5N H$04, 5N NaOH, and granular CaClz (Hyman and Arp, 1987). Alternatively, propyne or I-butyne (a gift from Dr R. Koppmann, KFA, Jiilich) was used. Immediately after injection of acetylene, gas samples were taken repeatedly and analysed for NO and NOz until the concentrations stabilized. Unless specified otherwise, soil V3 at 60% whc was used for the experiments. In some experiments, glass beads, quartz sand or sea sand (5 g dw each) were poured into the serum bottles to increase the surface area. The glass beads (3 mm in diameter) were obtained from the local laboratory supplier.

Table I. Characteristics Soil BL BW Gl G2 G3 NB PBE RA Tl T2 Vl ;: WB

Soil twe

Cambisol Cambisol Cambisol Luvisol Luvisol Luvisol Cambisol Cambisol Cambisol

of the soils

Use

oH value

OTP c (%I

Forest Forest Barley field Barley field Barley field Pine forest Beech forest Rape field Wheat field Wheat field Barley field Barley field Barley field Brleadow, ploughed

3.9 2.8 6.8 6.1 6.4 3.6 3.9 5.7 1.5 7.4 6.5 6.2 5.8 4.7

4.3 18.7 I.1 1.4 1.0 7.5 7.5 1.3 1.3 1.3 1.1 I.1 1.1

1.6

Decomposition of NO by acetylene The quartz sand! (0.2-0.8 mm in diameter) and sea sand (0.1-0.3 mm in diameter) were obtained from Merck (Darmstadt). The gas analyses were done with the Luminox analyser if both NO and NO* had to be determined in the same experiment. The concentrations of NO and NOz were rneasured in alternate samples. The gas analyses were done with the Therm0 electron analyser if only NO was measured. NO consumption rate constants were routinely determined by placing 5 g (freslh weight) soil into the serum bottles. The rate constants were determined from the decrease of NO with time, assuming either a firstorder reaction (regression of the logarithm of NO concentrations against time) or a second-order reaction (regression of reciprocal NO concentrations against time). The rate constants were expressed in units of cm3 h-’ g-’ dw soil and cm3 molec-’ s-l, respectively. In the absence of acetylene, consumption by soil of 1000 ppbv NO was followed with time until a stable final NO concentration was reached, i.e. the compensation concentration between simultaneous NO production and consumption. The first-order rate constant of NO consumption was calculated from the decrease of NO with time and the compensation concentration, as described by Seiler et al. (1977) and Bollmann et al. (1995). The NO production rate was determined by multiplying the NO consumption rate constant with the compensation concentration. The experiments were usually conducted in three or four replicates. Statistical differences between treatments were tested according to Lord (1947) at P < 0.01, a statistical test that is suitable for a low number of replicates. Labelling experim
1059

1

1200 AAA

1000

]t

O.,,

6

A

~‘0

‘A

time [sl

D

Fig. 1. Conversion of NO to NO2 in synthetic air after application of 0.2% acetylene. NO* (0) and NO plus NOz (A) were measured with the Luminox NO, analyser, and NO (0) was calculated by difference. (University of Marburg). The percentage recovery of “N was determined as described by Rudolph et al. (1996a).

RESULTS

NO decomposition in the absence of soil The addition of 0.2% acetylene to 1000 ppbv NO (1000 nl NO I-’ gas phase) in synthetic air caused the conversion of part of the NO to NOz within seconds (Fig. 1). After 30 s, the reaction reached a stable equilibrium. Because of the rapidity, we were unable to determine the kinetic order of the reaction. However, we assumed a second-order reaction and determined a rate constant of 7.8 x lo-l6 cm3 molec-’ s-l (ra = 0.72) for the first 20 s of the reaction. When NO plus acetylene were mixed into a Nz atmosphere instead of air, the NO concentration did not decrease, and NOz was not formed. We tested acetylene, which had been obtained from three different sources (Messer-Griesheim, Linde, Air Liquide), and found no difference in the conversion of NO to NOz (results not shown). We also tested propyne and I-butyne, which, analogously to acetylene, are unsaturated hydrocarbons with a triple carbon bond. Again, conversion of NO was observed, but with decreasing efficiency when using propyne and 1-butyne instead of acetylene (Table 2). Different NO concentrations were incubated in the presence or absence of acetylene. The NO concentrations chosen (80-800 ppbv) were well within of NO concentrations (20 to the range > 1000 ppbv) that were observed in the pore space of soils (Rudolph and Conrad, 1996). The results show that a nearly constant percentage (52~64%) of the NO was lost in the presence of 0.39% acetylene (Fig. 2). Hence, the extent of conversion of

A. Bollmann and R. Conrad

1060

Table 2. Percentage conversion of loo0 ppbv NO to NO;! after the addition of different unsaturated hydrocarbons to the gas mixture in synthetic air (n = 4) Conversion (%)

Used hvdrocarbon

60-70 30-40 5-15

Acetylene HC = CH Propyne HC = C-CH, I-butyne HC = C-CHz-CH,

NO to NOz was apparently not a function of the NO concentration. However, it was a function of the acetylene concentration: the final NO concentrations that were reached at equilibrium decreased, and the NO2 concentrations increased, when 1000 ppbv NO was incubated with increasing acetylene concentrations (Fig. 3). At acetylene concentrations higher than l%, the increase in reaction rate apparently slowed down. At acetylene concentrations lower than O.l%, there was no conversion of NO to NO* at all. The recovery of NO plus NO2 was 100% at low ( < 0.5%) acetylene concentrations and decreased to about 60% at higher acetylene concentrations. The reasons for this incomplete recovery are not clear. We assume that there are absorption effects between NO or NO2 and acetylene, or between NOz and traces of water vapour contained in the injected acetylene. It should be noted, however, that most of the NO was recovered as N02, so we are confident that the disappearance of NO from the gas phase in presence of acetylene was due to its conversion to NOz. The incubation experiments were routinely carried out at room temperature (23-26°C). Incubation of 1000 ppbv NO together with 0.2% acetylene at temperatures increasing from 0 to 45°C resulted in an increasing depletion of NO (Fig. 4). Whereas no conversion of NO was observed at O”C, 95% of the NO was converted at 45°C. The conversion of NO in presence of acetylene (0.77%) was also enhanced when dry glass beads < quartz sand < sea sand were placed into the serum bottles (Fig. 5). We assume that this stimulating effect was due to enlargement of the surfaces in the incubation system.

without acetylene

OC

5 acetylene [%I

Fig. 3. Final concentrations of NO (0) and NO2 (0) after addition of different acetylene concentrations to a mixture of 1000ppbv NO in synthetic air. NO decomposition in the presence of soil The acetylene-catalysed conversion of NO to NO2 was also observed when soil was placed into the serum bottle (Fig. 6). In the presence of soil, however, NO* accumulated only transiently and was then decomposed together with NO within 20 min. Repetition of the experiment with “Nlabelled NO resulted in 50% recovery of the label within the nitrate + nitrite fraction of the soil. In the absence of acetylene, NO was consumed at a much slower rate (see below), and a transient accumulation of NO* was never detected. The NO decomposition rate constant was proportional to the amount of soil, at least up to a total of 5 g (fresh weight) soil, indicating that the overall reaction was dependent not only on the gas phase conversion of NO to NO*, but also on the uptake of NO2 by soil (Fig. 7). Nevertheless, the kinetics of NO decomposition in the presence of acetylene usually could be slightly better described by a second-order reaction (r* = 0.96-0.99) than by a first-order reaction (r* = 0.84-0.99). A secondorder reaction would be typical for the gas phase conversion of NO (Olbregts, 1985), whereas a firstorder reaction would be typical for the uptake of

1

800-

with acetylene

00 0

200 of NO

400 NO

600

800

1000

[ppbvl

before (0) and after (0) addition of 0.39% acetylene to different mixing ratios of NO in synthetic air. Fig. 2. Recovery

Fig. 4. Effect of incubation temperature mixing ratio after application of 0.3%

on the final NO

acetylene to a mixture of IOOOppbv NO in synthetic air. NO was not depleted in the absence of acetylene. Mean k SD of n = 3.

Decomposition of NO by acetylene b

b

b

&ss beads

quartzsand

sea sand

1061

7

m-

x &

B

400: 3CO-

zooIOO-

ocontml

Fig. 5. Effect of additional silicate surfaces (5 g) on the final NO concentration after application of 0.77% acetylene to a mixture of 1000 ppbv NO in synthetic air. NO was not depleted in the absence of acetylene. Mean k SD of n = 3; significant differences are labelled with different letters.

et al., 1973a,b; NO or NOz by soil (Prather Johansson and Galbally, 1984; Remde et al., 1989). The second-order rate constants of NO decomposition in the presence of acetylene were not different were done with autoclaved when the experiments soil instead of non-sterile soil (Table 3). However, NO decomposition was much slower when non-sterile soil was incubated with NO in the absence of acetylene (Fig. 8). In this case, NO was apparently decomposed by soil microorganisms, since this activity could be drastically decreased by autoclaving (Table 3). The residual NO decomposition in autoclaved soil, which. was practically zero, was in the order of the gas phase reaction of 2 NO with Oz described by Olbregts (1985). Decomposition rate constants of NO were measured in different soils that were incubated with 1000 ppbv NO in the presence and absence of 0.77% acetylene. ‘The NO decomposition rate constants were determined, assuming either a firstorder or a second-order process (Table 4). In the absence of acetylene, the NO decomposition rate constants (l.l-64.:5 cm3 h-r g-’ dw) were within the range that has been observed for microbial NO

0

4

2

6 soil Igl

8

10

12

Fig. 7. Effect of increasing amounts of soil (60% whc) on the NO decomposition rate constant determined in the presence of 0.77% acetylene. Mean + SD of n = 4. consumption

in

soil

(Baumgktner

and

Conrad,

1992; Saad and Conrad, 1993). Addition of acetylene, however, stimulated the NO decomposition by factors of S-557, irrespective of the reaction order that was assumed for the determination of the rate constants (Table 4). This result indicates that NO uptake by soil was enhanced by the acetylene-catalysed conversion of NO to NOz. The stimulation of NO decomposition was dependent on the acetylene concentration. Acetylene concentrations > 0.1% increasingly enhanced NO decomposition until the stimulation levelled off at > 1% acetylene (Fig. 9). On the other hand, acetylene concentrations of < 0.1% did not stimulate NO decomposition and resulted in relatively low rate constants (< lo-” cm3 molec-’ s-‘) that were typical for the microbial NO consumption in soil (Table 4). We also tested the effect of acetylene at very low concentrations (O.l-10Pa) that are typically used to inhibit nitrification specifically (Berg et al., 1982; Klemedtsson et al., 1988) and observed no significant effect on NO decomposition (Table 5). However, the rate of NO production was decreased to about 20% of the control, indicating that 80% of the NO production in this particular soil (V3) was due to nitrification (Table 5). The NO decomposition rate constants were influenced by the moisture content of the soil (Fig. 10). In the presence of acetylene, the rate constants were largest at 10% whc and gradually decreased with increasing soil moisture. Even air-dry soil (4% whc) still showed a relatively high NO decomposition (4.5 x lo-l6 cm3 molec-’ s-l) when acetylene was Table 3. NO decomposition rate constants (cm3 molcc-’ s-‘) determined for non-sterile and autoclaved soil (V3) in the presence of acetylene (n = 3; mean f SD) Acetylene (%)

Fig. 6. Decomposition of NO (a) and intermediate accumulation of NO2 (0) by 5 g soil V3 (60% whc) incubated under an atmosphere of synthetic air containing 8% acetylene.

0 (control) 0.58 0.77

Non-sterile

soil

Autoclaved

soil

7.57 x IO-” * 4.24 x lo-l9 5.65 x 10wt9 f 4.14 x 10e2c 4.05 x lo-l6 * 2.41 x IO-” 4.84 x lo-l6 f 1.07 x IO-” 5.53 x 10-‘6f 1.72 x lo-” 4.54 x lo-l6 f 2.22 x IO-”

A. Bollmann and R. Conrad

1062

5 10-16

E -g 4 lo-‘6 3

3 IO_‘6

i

2 lo16

._ .-; 8, 1 lo-‘6 E, B 8

Fig. 8. Decomposition of NO by 5 g soil V3 (60% whc) in the presence (0) or absence (0) of 0.77% acetylene.

0 0

0.2

0.4

0.6 0.8 acetylene [%I

1

Fig. 9. Effect of increasing acetylene concentrations on the NO decomposition rate constant measured using 10 g soil (60% whc). Mean f SD of n = 4.

present. Smaller rate constants ( c 7 x lo-l7 cm3 molec-’ s-l) were observed when soil was replaced by pure water, glass beads, quartz sand or sea sand (Fig. 11). However, the NO decomposition rate constants were higher if the glass beads, quartz sand or sea sand were moist. NO* was not measured in this experiment. In analogy to soil samples, however, we assume that NO;, will have accumulated only transiently and will then have been absorbed in the water phase.

ing autoclaved soil without any addition of acetylene. However, the reaction was strongly enhanced when acetylene was added. Although the reaction was so fast that exact kinetics could not be determined, the rate constant was at least 3 orders of magnitude higher in the presence than in the absence of acetylene. Because of the high background concentration of acetylene and the low concentrations of NO that were converted, we could not check for possible reaction products of acetylene with NO. However, a stoichiometric reaction of NO with acetylene is unlikely, since the rate constant of this reaction is extremely low. Frank-Kamenetskii (1944) measured the rate constant of the reaction at 600-900°C to be only k = 0.0093 exp(-32004 K/T), or, at 25°C: k = 2.12 x lOA cm3 molec-’ s-l. Acetylene is a gaseous hydrocarbon with a triple carbon bond. Propyne and 1-butyne which also contain a triple carbon bond also caused a stimulation of the oxidation of NO to NOl, although the amounts of NO* produced at the end of the reaction were smaller than with acetylene. It appears that propyne and l-butyne were involved in the oxidation of NO to NO2 in a similar way as acetylene.

DISCUSSION The oxidation of NO to NO* in the gas phase is a second-order reaction with respect to NO (Olbregts, 1985): 2N0 + O2 ----) 2N02. The rate constant decreases with increasing temperature up to 600 K, after which it increases. Under atmospheric conditions at 298 K, the rate constant is about 611 mol-’ s-l 1.0 x lo-l9 cm3 molec-’ s-’ (Olbregts, 1985). Gz found NO decomposition rate constants in the same range when NO decomposition was measured in empty serum bottles or in serum bottles contain-

Table 4. NO decompositionrate constants(first-orderand second-order)of various soils determined in the presence and absence of 0.77% acetylene Soil

NO decomposition rate constant (first-order) (cm’ h-’ g-’ d.w.) Without acetylene With acetylene Ratio with/without

NO decomposition rate constant (second-order) (cm’ molec-’ SC’) Without acetylene With acetylene Ratio with/without

BL :Y

9.1 64.8 3.2

182.5 380.2 150.6

20 48 6

1.5 x 10-1’ 6.2 1.8 x lo-l6 lo-‘*

6.4 x IO@ 5.1 x IO-l6 8.9 10-16

42 82 5

G2 G3 NB PBE RA TI T2 Vl v2 v3 WB

3.2 3.5 12.5 1.1 10.5 9.3 7.7 8.2 6.4 5.3 9.2

147.9 137.8 245.9 172.9 156.6 160.1 180.6 144.5 184 142 167.4

46 39 20 163 15 17 23 18 29 27 18

7.6 x 5.4 x 1.4 x 5.2 x 4.5 x 2.1 x 1.8 x 1.7 x 1.2 x 7.5 x 1.1 x

5.9 x 4.9 x 4.1 x 2.9 x 6.5 x 6.2 x 6.2 x 5.6 x 6.1 x 5.4 x 6.5 x

77 90 29 557 14 30 35 32 50 12 62

lo-‘s lo-‘8 1o-17 lo-l9 10-1’ 10-I’ 10-l’ 10-1’ 10-1’ lo-‘* lo-”

lo@6 1ov lo-l6 lo-l6 IO-lb lo-l6 lo-l6 IO-” IO-” lo-‘6 lo-”

Decomposition of NO by acetylene

1063

Table 5. NO consumptionrate constant and NO production rate in the presence and absence of 10 Pa acetylene using 10 g soil V3 (60% whc) (n = 4; mean f SD)

2 .¶

---

1a

a

bc

a

b

NO consumption rate NO production rate constant (ng NO(cm3 h-’ g-’ d.w.) N h-’ g-’ d.w.) Without acetylene With 10 Pa acetylene

12.1 * 0.2 12.2 + 0.4

0.55 * 0.04 0.11 f0.02

However, the mechanism of the stimulation remains unclear. The adsorption of NO and its following oxidation to NO2 by soil particles was described by Mortland (1965) and Miyamoto et al. (1974). Burdick (1922) investigated the catalysis of the NO oxidation by testing crushed glass and charcoal. The charcoal enhanced the NO oxidation by a factor of > 500. This, enhancement was very high compared to the enhancement by crushed glass. The author proposed that carbon compounds in the charcoal exerted a catalytic activity with respect to the oxidation of NO. We hypothesize that the stimulation of NO oxidation to NO2 by acetylene that was observed by us was due to the action of acetylene that was adsorbed to the surface of the serum bottles or to other surfaces (e.g. glass beads, etc.) present in the serum bottle. This hypothesis is supported by the following observations: (1) the amounts of converted NO increased with the concentration of acetylene up to about 1% when the glass surface of t.he serum bottle was presumably saturated with acetylene; (2) the amounts of converted NO increased when an additional surface as in the form of glass beads, etc. was added which could have adsorbed additional acetylene: however, adsorption isotherms were not determined, and the conversion of NO to NO2 was not analysed with respect to the theory of heterogenous catalysis; and (3) the acetylene-catalysed NO decomposition rate constants in the presence of soil decreased with

dE “g

8 lo-l6 T

8

6

IO-

8 B

I? 4 lo-l6 6 ‘; ._ B 2 10.16 0

4

B

O!--Trn

% moisture[%whc]

120

Fig. 10. Effect of increasing soil moisture content on the NO decomposition rate constants determined with 10 g soil V3 in the preseno: of 0.77% acetylene. Mean f SD of n = 4.

z

water

glass beads wet dry

L quartz sand dry wet

a

C

1

sea sand dry wet

Fig. 11. Effect of dry and wet (with 1ml water) silicate surfaces (5 g each) on the NO decomposition rate constant in the presence of 0.77% acetylene. NO was not depleted in the absence of acetylene. Mean + SD of n = 3; significant differences are labelled with different letters. increasing soil moisture (> 20% whc), suggesting decreased access of NO to soil particle surface. The amount of NO that was converted after the application of acetylene increased with increasing temperature. This temperature effect is opposite to that which would be expected from the termolecular gas phase reaction of 2 NO with O2 as described by Olbregts (1985), and is also opposite to that expected for the adsorption of NO to soil particles as described by Prather and Miyamoto (1974). Burdick (1922), however, also observed an increase of the charcoal-catalysed NO oxidation rate with increasing temperature, but only in the presence of water vapour. In the absence of water vapour, the effect of temperature was the opposite. In our experiments, we used synthetic air which contained only traces (10 ppmv = 10 ~1 Hz0 1-l gas phase) of water vapour. However, since we also used much lower NO concentrations (< 1 ppmv NO) than Burdick (3% NO), we cannot exclude the fact that water vapour was also involved in the stimulation of NO conversion by acetylene. In the presence of soil and acetylene, NO was decomposed to near completeness, since the NO2 that was produced was obviously taken up by the soil and thus allowed the further conversion of NO to NO*. In the presence of soil, NO* accumulated only as an intermediate and was subsequently taken up by the soil. The NO decomposition rate constants in the presence of acetylene were the same for autoclaved and for non-sterile soils demonstrating that not only the conversion of NO to NOz, but also the uptake of NO2 by soil was non-biological, and thus consistent with previous observations (Baumggrtner et al., 1992). The NO* was obviously further converted in the soil, forming nitrate and nitrite, since 50% of 15N-labelled NO was recovered in this nitrogen fraction. This observation is consistent with earlier reports (Ghiorse and Alexander, 1978) and may be ascribed to dismutation of NO1

1064

A. Bollmann and R. Conrad

in aqueous solution to nitrite plus nitrate (Huie, 1994). The remainder of the labelled NO2 probably reacted with soil organic matter (Cooney and Ross, 1987). With acetylene, the NO decomposition rate constants of 14 different soils were higher by factors of 5-557 than without acetylene. The enhanced NO decomposition occurred in all soils that were tested. Similarly, as observed for the conversion of NO to NO2 in the gas phase, the NO decomposition in the presence of soil increased with increasing acetylene concentration up to about 1% and then levelled off. Likewise, acetylene concentrations < 0.1% did not result in stimulated NO decomposition. Based on our results, we suggest the following reaction sequence for the acetylene-stimulated NO decomposition by soil: acetylene concentrations > 0.1% stimulate the oxidation of NO to NO* in a reaction that is possibly heterogenously catalysed after adsorption of acetylene to surfaces (e.g. the glass wall of the incubation flask). This fast reaction reaches an equilibrium that is characterized by stable NO and NO2 concentrations. The acetylenestimulated decomposition of NO continues if the produced NO2 is taken up by soil through adsorption, dissolution in soil water, dismutation to nitrite and nitrate, or reaction with soil constitutents. All these reactions are non-biological processes. This chemical decomposition of NO in the presence of acetylene creates a potential bias in all assays of soil nitrogen turnover that apply acetylene at concentrations > 0.1%. The most relevant assay is the acetylene blockage technique that is used to determine denitrification rates in soil (see Introduction). The presence of the l-10% acetylene that is typically applied in this assay may result in scavenging of NO that is an obligate intermediate in the microbial denitrification pathway (Zumft, 1993; Ye et al., 1994). Indeed, measurements of rates of NO production and of denitrification in various soil samples indicated that acetylene may create a bias in the acetylene blockage assay when applied to oxic soil samples, and may result in substantial underestimation of the true denitrification rate (Bollmann and Conrad, 1997). Another assay is the specific inhibition of the ammonium monooxygenase of nitrifiers by low acetylene partial pressures (l-10 Pa). Our results show that these low acetylene concentrations should not create an artifact when the relative contribution of nitrification and denitrification to NO production in soil is determined by the acetylene inhibition technique (Davidson, 1992; Schuster and Conrad, 1992; Davidson et al., 1993). However, application of higher acetylene partial pressures (10 kPa) may result in NO release rates that are too low, since not only the NO release by nitrifiers, but possibly also the NO release by denitrifiers and other processes are inhibited (Bollmann and Conrad, 1997;

McKenney et al., 1996). This artifact may lead to the erroneous interpretation that nitrification is the dominant NO-producing process in soil, although the eventual NO production by denitrification was obscured by the acetylene-enhanced decomposition of the produced NO (Parsons and Keller, 1995). We just learned that McKenney et al. (1997) have also studied the reaction of NO with O2 and acetylene. Similar to us, they observed that acetylene catalysed the depletion of NO in the gas phase as soon as O2 was present at concentrations higher than 200-300 ppmv. The authors suggest a reaction mechanism in which NO and 02 react subsequently with acetylene to form an organic radical which then reacts with a second NO to produce NOz and regenerate acetylene. Acknowledgements-We thank Katja Zinkan for technical assistance, Ulrike Bokisch from the laboratory of Dr D. Werner (Marburg) for the lSN analyses, Dr M. Koschorreck (Marburg) and Dr P. Warneck (Mainz) for helpful discussion, and Dr E. A. Kaiser (Braunschweig) for providing soil samples, and Dr R. Koppmann (Jiilich) for a gift of propyne and butyne. This study is a contribution to the BMBF program German ‘Klimaschwerpunkt Spurenstoff-Kreisliufe’, and was financially supported by the Fonds der Chemischen Industrie.

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