Control of denitrification enzyme activity in a streamside soil

FEMS MicrobiologyEcology102 (1993)225-234 © 1993 Federation of European MicrobiologicalSocieties0168-6496/93/$06.00 Published by Elsevier

225

FEMSEC 00439

Control of denitrification enzyme activity in a streamside soil Per Ambu8 Institute of Population Biology~ Copenhagen University, Copenhagen, Denmerk

Received 25 September 1992 Revision received 25 November 1992 Accepted 25 November 1992 Key words: NO 3 and NO~- reduction; Chloramphenicol; Kinotic parameter ( K m and Vm~x); Temperature control; Substrate availability; Denitrification 1. SUMMARY Progress curve analysis of NO 3 and NO~ reduction in surface soil samples from a streamside soil gave K m values of 4.24 and 6.33 tzM, and Vma~ values of 2.16 and 1.83 /zmol 1- I min -~, respectively. Recoveries of reduced NO 3 and NO~- as gaseous N averaged 82 and 108%. The unrecovered N O 3 - N was presumably dissimilated to NH~-N. The denitrification enzyme activity (DEA) was examined throughout a year and showed seasonal and spatial variabilities of only 10% to 26%, suggesting a high persistency of denitrifying enzymes. Soil moisture and D E A correlated significantly (r = 0,7671; P < 0.01), The D E A in saturated subsoil also showed a relatively little variation, with spatial variabilities of between 28 and 38%, Amendment with NO 3 rarely enhanced the activity more than two-fold at either depth. Addition of glucose increased the activity 2.3 and 2.5 times in the surface soil and Correspondence to: Per Ambus, Institute of Population Biology, Copenhagen University,Universitetsparken 15, DK-2100, Denmark.

subsoil respectively, indicating a moderate carbon limitation of denitrification. The activation energy of D E A was found to be 64.9 kJ t o o l - t and Qt0 values for the 2-12°C and 12-22~C temperature ranges were 2.71 and 2,53, respectivel~/, Extrapolation suggested tll0jFe would be a 4.4-fold increase in D E A i( ~ ¢ tenaper~,t~ro was changed from 0 to 15~C. Sttb,~t~a~ diffusion limited the denitrifioa.tion 10 to 25 fold. Thus, under anaerobic moist conditions it appears that changes in denitrification might primarily be due to varying diffusion of substrates into the anaerobic soil centers. Over a year, fluctuations in DEA, temperature changes and fluctuations of electron-acceptor and -donor supply will only have a minor effect on natural denitrification activity. 2. I N T R O D U C T I O N R e c e n t studies have d e m o n s t r a t e d that streamside soils have the capacity to support high rates of biological denitrifieation [1-3] which may be an important sink for NO 3 from agricultural discharge, Yet, knowledge about the dynamics

226 and the control of denitrification in ecotones between terrestrial and aquatic systems is incomplete. Soil denitrification is primarily regulated by (a) oxygen, (b) N O 3, and (c) available carbon [4]. The process is highly variable and the ability to predict the extent of denitrification is limited. The level of active denitrifiers is a combined product of a variety of soil processes [5], and is usually expressed as the potential denitrification enzyme activity ( D E A ) given by Smith and Tiedje [6]. D E A should therefore provide valuable information about the combined effect of interaction between the various biological, chemical and physical factors known to influence denitrification. D E A has successfully been used as a parameter for modelling denitrification spatial variability [7] and annual denitrification N-loss at the landscape level [8], but it may [9] or may not [8,9] be a reliable predictor of daily denitrification activity. The aim of this study was to identify key factors in the regulation of denitrification activity in a streamside soil. Investigations were made into (a) N O 3 and N O 2 reduction kinetics ( K m and Vma~); (b) the seasonal variation of D E A and substrate availability; (c) the temperature effect on DEA; and (d) the magnitude of diffusion limited denitrification.

3. M A T E R I A L S A N D M E T H O D S

3.1. Study site The study was carried out in a narrow riparian strip bordered by a small stream on one side and by agricultural fields on the other. The site was located in a quaternary morainic landscape 50 km south-west of Copenhagen. The vegetation was Table 1 Characteristics of riparian soil 0-5 cm 5-0 cm 15-20 cm

PHH20 7.3 7.4 7.0

a Loss on ignition.

% tot-N 2.74 2.58 2.37

% tot-C 33.76 33.02 29.69

% LOI 65.3 56.7 57.5

a

dominated by Baldfngera arundindcea and Carex sp. Soil characteristics are listed in Table 1. In the period from October to May, water often formed stagnant pools in inter-tussock depressions. Soil samples were collected at 4 locations, 1, 12, 23 and 34 metres from the stream.

Kinetics of NO 7 and NO 7 reduction Surface 0 - 1 0 cm samples were collected at the 4 locations in August 1990, composited and stored under field-moist conditions at 4°C until analysed. Analysis took place within two weeks. Breitenbeck and Bremner [10] showed that storage of field-moist soil samples at 4°C for up to 30 days did not alter the denitrification capacity. The soil was thoroughly mixed and bulky residual plant material was removed. Soil slurries were prepared by adding 15 g field-moist soil to 120 ml serum bottles containing 70 ml of phosphate buffer (40 mM; p H 7.3) with 1 g 1-1 of glucose-C. The serum bottles were sealed, evacuated and flushed with N 2 three times, and 12 ml of the headspace was replaced with pure C2H 2. After 15 min, 5 ml degassed solutions of K N O 3 or N a N O 2 were added to triplicate flasks to give final concentrations of 150 or 100 /zM, respectively. Soil slurry portions of 3.0 ml were removed at 10-min intervals using a 13G x 4 needle permanently positioned through the stopper and closed off with a 3 way stopcock. The 3.0 ml soil slurry samples were immediately added to centrifuge tubes containing 3.0 ml of 100% C H 3 O H to stop denitrification activity. The samples were then centrifuged (30 min; 3800 x g) and the supernatant was passed through a 0.45 /zm filter before being analyzed for N O 3 and N O ~ . Preliminary investigations showed that N O 3 reduction and denitrification in anaerobic soil slurries with 50% C H 3 O H was repressed by 96% for at least 2 h. In addition, 1-ml headspace samples were taken from the serum bottles to measure the accumulation of N20. The gas samples were stored in 3.5-ml Venojects® preflushed with pure N 2• After each sampling, 4.0 ml of 10% C 2 H 2 in N 2 was added to the headspace to replenish the volume sampled. The incubation was performed

227 at room temperature for 90-120 min and all flasks were shaken by hand every 5 min. The kinetic parameters K m and Vm~x were estimated by fitting the NO 3 and NO 2 depletion data to the integrate form of the MichaelisMenten equation [11] by using P R O C NLIN of SAS [12]. Chloramphenicol interfered strongly with the N O 3 / N O 2 analysis and could not be included in this experiment. For that reason, the extent of denitrifier growth was estimated from patterns of N 2 0 evolution in the absence and presence of growth inhibitor. Three flasks received 15 g field-moist soil and 30 ml of a solution containing chloramphenicol (1 g 1-1), KNO3-N (0.2 g 1-1) and glucose-C (2 g L -1) (see (iv) below); 3 flasks received the same but without chloramphenicol. The flasks were flushed with N 2 and amended with CEH 2 as described above, and the headspace was analyzed for N 2 0 every 15 rain for 2 h.

slurries were incubated at room temperature and shaken thoroughly by hand every 15 min; 3 1-ml gas samples were taken at regular intervals over 30 to 60 min. Subsamples (20 g each of field-moist soil) were extracted with 40 ml of deionized water (rotary shaker; 1 h). Portions (20 ml) of the water extracts were centrifuged and filtered as described above and analyzed for N O 3 , NO~- and watersoluble carbon.

3.3. Temperature response Subsamples (15 g each) of field-moist soil were mixed with 30 ml of solution (iv) (see above), buffered with 40 mM of phosphate (pH 7.3) and preadjusted to 2, 6, 8, 15 or 23°C. The soil slurries were flushed with N 2 and amended with C2H2; triplicate flasks were incubated at each of the above temperatures. The flasks were shaken and sampled for N 2 0 analysis at 20-min intervals over a 160-min incubation period.

3.2. Denitrification enzyme activity (DEA) Soil samples for examination of D E A and substrate control of the denitrification activity were collected on 13 October in 1988 and on 16 January, 8 May and 4 September in 1989. The samples were taken with an auger from the surface 5 cm and from the top 5 cm of the saturated soil layer. The water table was 5 cm below the surface in October and 15 cm below in May and September. In January the whole area was flooded and soil was consequently collected only from the surface 5 cm. Samples from each location and depth were treated separately and were stored as described above, usually for less than 3 days. After removing residual plant material, soil slurries were made by mixing 10-20 g field-moist samples with 20 ml of one of four solutions: (i) chloramphenicol (1 g l-1); (ii) chloramphenicol (1 g 1-1) and KNOa-N (0.2 g 1-1); (iii) chloramphenicol (1 g L - I ) and glucose-C (2 g 1-1); and (iv) chloramphenicol (1 g 1-1), KNO3-N (0.2 g 1-1) and glucose-C (2 g L - l ) . Solution (iv) is a modification of the denitrification enzyme activity assay solution used by Smith and Tiedje [6]. Triplicate incubations were performed in 120-ml serum bottles which were flushed with N 2 and amended with C2H 2 as described above. The

3.4. Suspension experiment Sixteen soil cores 15 cm in length were collected in PVC tubes 30 cm long by 4.5 cm inner diameter. The tubes were sealed at the bottom, and brought to the laboratory for injection with 30-ml portions of deionized water containing either (i) chloramphenicol (3 g I-1); (ii) chloramphenicol (3 g 1-1) and KNO3-N (0.17 g 1-1); (iii) chloramphenicol (3 g 1-1) and glucose-C (1.8 g 1-1); or (iv) chloramphenicol (3 g I-1), KNO3-N (0.17 g 1-1) and glucose-C (1.8 g 1-1). The solutions were injected along the length of each of 4 replicate cores, and they were followed by 30 ml of C2H2, which was also injectedalong the length of each core. By the addition of the 30-ml portions the soil moisture content ended up at 104% water filled porespace. The samples were preincubated under anaerobiosis for 16 h. The preincubation took place in a 60-L jar containing 10 kPa CEH 2 in pure N 2 and was performed to allow dispersion of CEH 2 into the soil pores. Following preincubation the PVC tubes were immediately capped and 1-ml headspace samples were removed at hourly intervals over 2 h to determine the N 2 0 accumulation at room temperature. After gas sampling, the cores were

228 Table 2 H 2 0 , N O 3 - N , and water soluble C concentrations at two depths in a riparian soil on four occasions H20 (kg k g - ')

NOa-N (mg k g - l)

COD-C (mg k g - 1)

Pet 13, 1988 0 - 5 cm 5 - 1 0 cm

3.40 2.45

ND ND

348.3 229.2

J a n 16, 1989 0 - 5 cm

4.44

8.5

May 8, 1989 0 - 5 cm 15-20 cm

3.94 2.19

13.1 6.8

392,7 34~.6

Step 4, 1989 0-5 m 15-20 cm

3.34 2.39

12.7 5.8

517.3 323.9

flasks were sealed and 50 ml of the headspace gas was reptit~,d with pure C 2 H 2. The denitrification l~tes were determined from the amount of N20 evolved at 0, 1 and 2 h by the stirred slurries. 3.5. Analysis

N20 was analyzed on a gas chromatograph a Porapak-Q column and 63Ni EC-d~tector (Shimadzu GC-8A). F | ~ j ~l Samples of the filtrates were analyzed for NO 3 and NO 2 on a HPLC operated with an anion column (Waters, IC-PAK TM) and 5 mM phosphate buffer at pH 9 as solvent. NO~- and NO2 was detected by UV-absorption at 210 nm. The detection limit by this method was about 0.5 ~mol 1-1 of N. The chemical oxygen demand (COD) of the soil extracts was determined from wet-oxidation with dichromate [13]. The COD are expressed as glucose-C equivalents. Inorganic N and COD are given on a dry-weight basis (105°C, 24 h). The soil variables are listed in Table 2.

operated with

ND

ND, not determined. transferred to 500-ml Erlenmeyer flasks and suspended in 200 ml deionized water. The slurries were flushed for 1 min with pure N 2 (1 1 s-i), the 2.5

4. RESULTS AND DISCUSSION

• no chloramphenicol 0 with chloramphenicol

i

4.1. K m and I/ma x o f N O 3 and N O 2 reduction Z

1.5

//'

/6

z%`

0.5

o.o

/~

0

20

40

610

810

' 100

' 120

140

minutes

Fig. 1. N 2 0 evolution in soil slurries with and without chloramphenicol. Vertical bars indicate two standard errors, n = 3. W h e r e no error bars are shown the S.E. was less than size of symbol. The dashed lines shows the position of the best fit to a sequential linear-exponential expression: Y = a - ( t ~ T ) + b, t
N20 evolution from soil slurries without chloramphenicol suggested a biphasic pattern as com. pared with the chloramphenicol amended soil (Fig, 1). The N20 evolution pattern in non-chloramphenieol flasks is in accordance with the observations of Smith and Tiedje [6] and reflects derepression of de novo enzyme synthesis, in the present study, denitrification enzyme activity in non-chloramphenicol amended soil was constant during about one hour. The Michaelis-Menten expression is only valid under steady-state conditions, so efforts to retard microbial growth must be taken. Here, the soil/solution ratio in the samples was adjusted to minimize the duration of the experiment; the initial steady phase indicates that the N O f - and NO 2 reduction will not have been accelerated by an increase in biomass during the course of the experiment. The experiment also indicated that the denitriflcation enzyme activity was not affected by the presence of chip-

229 200 Aceumulotion I of N20 from NO3 . and NO2 V Reduction of NO:~ • and NO2 0 150' ~\ \

"6

\

o

t

0

35

~

"-'~J=c

70

c'

105

c

c

140

minutes

Fig. 2. Progress curves of N O 2- and NO 3 reduction and accumulation of N 2 0 from N O 2 and NO~- reduction in slurries of surface soil. Vertical bars indicate one standard error, n = 3. Where no error bars arc shown the S.E. was less than size of symbol.

ramphenicol which is in accordance with the finding of Smith and Tiedje [6] but opposed to the results in a recent study by Brooks et al. [14]. Analysis of the progress curves (Fig. 2) gave K_.m values of 4.24 + 4.96 and 6.33 __+1.03 /xM (X _+S.E.), and Vm~x values of 2.16 + 0.22 and 1.83 + 0.08 /zmol 1-1 min -1 for NO~ and NO2 reduction, respectively. Recoveries of NO 3 and NO~- as N20 averaged 82 and 108; %, respectively (Fig. 2), indicating that whilst NO2 reduction occurred entirely through denitrification, other processes contributed to the reduction of NO~-. In the NO3-amended flasks, the appearance of NO~- was transient (data not shown), as expected from the differences in VmaxThe high affinities for both N O f and NO2 utilization found here are in accordance with K m values ranging between 1.7 and 50 ~M reported for soils [15], sediments [15-17] and pure cultures [18-20], although a K m of 450 lzM has been reported from a study with soil slurries [21]. A high affinity for NO 3 utilization enables the bacteria to grow on the low concentrations of NO~usually found in habitats like mires [22] and riparian forests [23]. Even in systems receiving high NO~- inputs, anaerobic zones may exhibit low

NO~- concentrations when the NO 3 consumption rate exceeds the supply by diffusion [17,21]. In the present soil, bulk NO 3 concentrations rarely exceeded 200/zM (data not shown) which makes it likely that the NO 3-reducing community was exposed to NO 3 concentrations in the firstorder range [21]. Such conditions make it difficult to relate the results obtained from stirred soil slurries with homogeneous and relatively high NO~- concentrations to the field situation. The outcome of competition for NO~- between denitriflers and other NO 3 reducers is strongly affected by changing the NO 3 concentration, especially in the first- and mixed-order ranges which favour denitrifiers [24]. Thus, it may well be that denitrification in this community is responsible for appreciably more than the apparent 80% of the total NO3 reduction observed in the slurry study (Fig. 2). The low recovery of NO 3 in N20 could be due to the presence of other NO 3 reducing processes or an incomplete blockage of the nitrous oxide reduction [25,26], or both. But since N20 accumulated in stochiometric amounts from NO 2, it seems most likely that the missing N from NO 3 might be accounted for in the form of intracellular and biomass-incorporated N or as N dissimilated to NH~-. The chloramphenicol experiment discussed above indicated that no growth occurred during the relatively brief incubation, and a separate tracer study showed that NO 3 was not assimilated during anaerobiosis [27]. The tracer study also showed that net production of NH~- was responsible for about 6% of the dissimilatory NO 3 reduction, suggesting that the NO 3 not recovered in N20 in this experiment was reduced to NH~-. This does not explain why NO 2 amendment did not result in N-loss since non-denitrifying soil bacteria are also capable of reducing NO 2 to NH~- by dissimilation. However, dissimilatory NO~- reduction to NH~- takes place apparently only following prolonged growth under NO3-free conditions when NO~- is in excess [28]. Under natural conditions, NO£ concentrations are usually in the nM range at the present study site and the indigenous community may not be adapted to carrying out dissimilatory reduction of NO 2 to NH~-.

230 20

A

7

20

0 oct • jan • may

I

sep

/" / t~ /

~T-- ~ ..~

15

//// 10

oct jan may aep

16

7 Z I

..

0 • • A

\

5/

7

\

t

Z [

12 / /

Oe~ Z ca

Z

E

A~

8 /

0

1,

ll2 metres from

2'3

A

4-

3'~

25

310 315 ,i0

stream

Fig. 3. Denitrification enzyme activity at 4 dates in surface soil samples collected at different distances from the stream. Vertical bars indicate two standard errors, n = 3. Where no error bars are shown, the S.E. was less than size of symbol.

4.2. Variability of DEA and substrate control T h e d e n i t r i f i c a t i o n e n z y m e activity in the surface soil varied b e t w e e n 5.2 a n d 17.9 mg N 2 0 - N k g - t h - 1 , showing the highest a n d lowest rates in J a n u a r y a n d S e p t e m b e r respectively (Fig. 3). T h e seasonal variability of D E A in the surface soil c o r r e s p o n d e d to a coefficient of variation (CV) of 26% ( n = 4) a n d the spatial variability corres p o n d e d to a C V of b e t w e e n 10 a n d 23% ( n = 4 o n the 4 occasions). O f the variables e x a m i n e d (Table 2), it a p p e a r e d that D E A in the surface soil was significantly r e l a t e d to the soil water c o n t e n t (Fig. 4). D E A in the subsurface soil varied b e t w e e n 1.3 a n d 6.1 mg N 2 0 - N kg -1 h -1 ( T a b l e 3). Since s a m p l i n g d e p t h varied according to season a n d D E A d e c r e a s e d m a r k e d l y with increasing d e p t h (data not shown) it is n o t possible to c o m m e n t o n seasonal variability in subsurface soil activity. However, w h e n samples were collected at the same depth, i.e., in May a n d S e p t e m b e r , the D E A differed significantly only at o n e location (Table 3). T h e spatial variability c o r r e s p o n d e d to a CV of b e t w e e n 28 a n d 38% which is slightly higher c o m p a r e d with the surface variability. A d d i t i o n of 0.2 g I-1 NO~--N to surface samples caused a two-fold increase in activity u n d e r b o t h a m b i e n t a n d h i g h - c a r b o n c o n d i t i o n s (N

,15

510 55

kg H20 kg-1 Fig. 4. Relationship between surface denitrification enzyme activity and soil moisture (r = 0.767; P < 0.01). Horizontal and vertical bars indicate two standard errors (n = 3). Where no error bars are shown the S.E. was less than size of symbol. The dashed line show the position of the geometric mean regression line.

a d d e d / n o a d d i t i o n ratio, a n d N a n d C a d d e d / C a d d e d ratio; T a b l e 4). It was only in J a n u a r y , w h e n a m b i e n t N O 3 was relatively low in the surface soil (Table 2), that the m e a n effect of N O 3 a d d i t i o n exceeded a t w o - f o l d increase in activity (data n o t shown). I n the subsurface, N O 3

Table 3 Denitrification enzyme activity in saturated riparian subsurface soil at different distances from stream on three occasions Depth and date

Meters from stream 1 12

23

34

mg N20-N kg- 1 h - l Oct 13, 1988

5-10 cm

4.4 b

4.6 b

3.1 c

6.1 a

3.3 aA b

3.5 aA

1.7 hA

1.8 bA

2.3 abA

1.3 cB

1.7 bcA

2.4 aA

a

May 8, 1989

15-20 cm Sep 4, 1989

15-20 cm

Within same sampling date, means followed- by the same lower-case letter are not significantly different (Duncan's multiple-range test; P < 0.05). b Within same depth and distance from stream, means followed by the same capital letter are not significantlydifferent (t-test; P < 0.05).

a

231 Table 4 Effects on denitrification in anaerobic slurries followingtreatment with different combinations of substrates Treatments compared Surface

Ratio of rates between treatments

a

N added/no addition b N and C added/C added N and C added/N added N and C added/no addition

1.7 + 0.4 c 2.0 + 0.6 2.3 + 0.l 4.0 + 1.0

Subsurface d

N added/no addition N and C added/C added N and C added/N added N and C added/no addition

1.1 + 0.1 1.4 + 0.2 2.5 + 0.3 2.5 + 0.3

a n = 16. Four locations on 4 dates. b N added = 0.2 g 1-1 KNO3-N;C added = 2.0 g 1-I glucoseC; N and C added=0.2 g 1-1 KNO3-N and 2.0 g 1-1 glucose-C. e Mean + standard error of the mean. d n = 12. Four locations on 3 dates.

addition caused increases in activity below a factor of 1.4 at both ambient and high carbon levels (Table 4). Nitrate amended samples, from surface and subsurface, gave an average 2.4-fold increase in activity when 2 g I-1 of glucose-C was added (N and C a d d e d / N added ratio; Table 4). The carbon effect in September was the highest observed: a 2.8-fold and 3.9-fold activity increase in surface and subsurface samples, respectively. Adding both N O 3 and glucose increased the activity about two-fold more than either substrate alone in samples from the surface (N and C a d d e d / n o addition ratio; Table 4). In subsurface samples the effect of adding N and C simultaneously was similar to the effect of adding glucose alone, but two-fold higher than the N O 3 effect. The seasonal and spatial variability of the D E A was remarkably low compared with the variability of natural denitrification found in this habitat [41] and other soil ecosystems [7,29-31]. The low variability of D E A demonstrates that bacteria maintain their denitrifying enzymes under non-denitrifying conditions [5,9,32], which may make it impossible to use D E A to predict the short-term dynamics of naturally-occurring denitrification in such a system. Smith and Parsons [32] suggested

that the rate of enzyme synthesis is less important than the activation-inactivation of existing denitrification enzymes by fluctuating soil aeration (moisture). On a long-term scale, soil moisture is seemingly a good indicator of DEA, as also observed by Parsons et al. [33]. In January, D E A increased with increasing distance from the stream (Fig. 3). The reason for this is not obvious. However, effluent soil water from the adjacent agricultural area may expose the microbial community at the field margin to higher concentrations of substrates compared with the microbial community at the stream margin, although this pattern could not be derived from any of the variables analysed. Generally, the limitation of the activity imposed by carbon availability was greater than that imposed by N O ; availability. At both depths the bulk N O 3 concentration appeared to be in the zero to mixed-order range and no profound N O 3 limitation was observed. The consistent two- to three-fold increase in activity upon glucose addition indicated that easily-degradable carbon might be the limiting factor in this organic soil, yet, the carbon limitation was never very intense. Carbon limitation has been found in other organic [34] and mineral soils [6,23,35]. The highest level of water-soluble carbon occurred in September (Table 2), presumably because of decaying plants. Since this coincided with the highest glucose response, water-extractable carbon seems to be a poor measure of available carbon. However, the data does not rule out other possibilities. Raised levels of water-extractable carbon might have induced an increase in the microbioal population size which would give a higher activity upon glucose addition. The results given here provide strong evidence that denitrification in this type of riparian system is a potential NO 3 sink since a high DEA, in both surface water and shallow groundwater, is maintained throughout the year. Yet, available carbon is a moderate limiting factor.

4.3. Temperature response of DEA A plot of Log e (DEA) obtained at different temperatures versus the reciprocal absolute temperature (Fig. 5) suggests that the temperature

232 6

O

8''r-~

v

O

4-

"-

0

"-

O -x

O "-.O

3:

2 3,35

I 3.40

I 3,45

I 3,50

I 3.55

I 3.60

3.65

1000 * T -1 Fig. 5. Arrhenius plot o f denitrification enzyme activity. The dashed line show the position of the regression line (r = 0.929; P < 0.001).

response can be described by the Arrhenius equation over the temperature range examined. The study focused only on temperatures likely to occur in the field and consequently no attempts were made to determine the temperature for maximum activity. An activation energy of 64.9 kJ mol-~ for the denitrification enzyme activity was calculated from the slope of the regression line, equal to Ea/R, where E a is the activation energy and R is the universal gas constant. By interpolation, Q10 values of 2.71 and 2.53 were obtained for the 2 to 12°C and 12 to 22°C temperature ranges respectively. It can also be calculated that the activity decreases about 4.4 times when the temperature is lowered from 15°C to 0°C. The activation energy and the corresponding Q10 values are within the range of temperature coefficients reported by other workers. McKenney et al. [36] found E a values of between 50 and 55 kJ mo1-1 and a mean Q10 of 2.1(15_25) for NO~- reduction in 2 mineral soils, and Jacobson and Alexander [37] reported a Q10 of 1.9(15_30) for NO 3 loss in a sandy loam. In anaerobic slurries from an alder swamp E a and Q10 for N 2 0 evolution varied between 70 and 76 kJ mo1-1, and 2.8 and 3.0(0_25), respectively [38]. Hence, it seems as if denitrifiers from different communities respond differently to temperature

changes and that the highest activation energy may apply to denitrifiers in organic soils. Different responses to temperature may also be due to adaption of denitrifying populations to different local temperatures [39]. Yet, the alder swamp [38] was situated less than 70 km from the field site used here. According to Focht and Verstraete [40] it is most likely that disparate temperature effects can be explained by differences in the amount and quality of organic substrates. The present data show, that the temperature effect is more important than the effects caused by NO 3 and carbon additions (Table 4), at least when a change from 0 to 15°C (representing the transition from prevalent winter to prevalent summer temperatures) is considered. Under in situ conditions, however, it might often be impossible to differentiate between the direct effect of temperature changes on denitrification enzyme activity and indirect effects caused by the impact of temperature changes on for example respiration, nitrification and 0 2 solubility, all of which are important factors in the regulation of denitrification. Therefore, the relative importance of temperature compared with substrate supply might change depending on the conditions.

4.4. Effect of suspending soil cores under suboptimum and optimum substrate conditions Denitrification increased markedly when both untreated and substrate-amended cores were made into slurries (Table 5), indicating an intense limitation of denitrification in intact soil due to substrate diffusion. The suspension effect in untreated samples and in samples where only one substrate has been added was not significantly

Table 5 Ratio between denitrification in anaerObic slurries and cores unde r suboptimum and optimum substrate conditions Substrate treatment

Slurry/core ratio

ambient + 5.1 mg N O 3 - N s a m p l e - 1 + 54 mg glucose-C sample - 1 + NO~-, glucose

24 + 8 a 21 + 3 215:2 10 + 0.2

a Mean 5: standard error, n = 4.

233 differetit. However, t h e r e was a t e n d e n c y towards a lower degree of diffusional l i m i t a t i o n w h e n o n e substrate was a d d e d c o m p a r e d with u n t r e a t e d samples. W h e n N O 3 a n d glucose were a d d e d s i m u l t a n e o u s l y the s u s p e n s i o n effect was two-fold lower t h a n with u n t r e a t e d samples or with a single additive. Overall, these results d e m o n s t r a t e that the detlitrifiers in this c o m m u n i t y possess a high substrate affinity a n d are limited slightly by the a m o u n t of available substrate. T h e d e n i t r i f i c a t i o n p o t e n t i a l is r e l a t e d to soil m o i s t u r e a n d varies rolatively little t h r o u g h o u t the year, It is importarif tO n o t e that d e n i t r i f i c a t i o n c a n p r o c e e d even at t e m p e r a t u r e s close to 0°C, which is the prevalent t e m p e r a t u r e at times w h e n r u n o f f from adjacent agricultural areas c o m m o n l y takes place. Thus, w h e n N O f e n t e r s the soil pores d u r i n g the winter r u n o f f period it is likely to b e r e d u c e d , at least 80% t h r o u g h denitrification. However, the efficiency of the r i p a r i a n z o n e as a N O 3 filter is severely c o n s t r a i n e d by the limiting effect of substrate diffusion o n the rate of denitrification.

ACKNOWLEDGEMENTS T h e technical assistance of L o n e Schou is greatly appreciated, a n d the v a l u a b l e criticism of the m a n u s c r i p t by S ~ r e n C h r i s t e n s e n a n d H e l e n Clayton is gratefully acknowledged. T h e work was s u p p o r t e d by the D a n i s h N a t u r a l Science Research Council a n d by the N a t i o n a l A g e n c y of E n v i r o n m e n t a l P r o t e c t i o n in D e n m a r k .

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