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The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification
Pergamon
0038-0717(94)00141-3
Soil Viol. Biochem. Vol. 27. No. I, pp. 17-22, 1995 Copyright C 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.04
THE USE OF 15N POOL DILUTION AND ENRICHMENT TO SEPARATE THE HETEROTROPHIC AND AUTOTROPHIC PATHWAYS OF NITRIFICATION D. BARRACLOUGH and G. PURI Department of Soil Science, University of Reading, London Road, Reading RGI 5AQ, England (Accepted I 7 June 1994)
Summary-The techniques of lSN pool dilution and enrichment were used to separate autotrophic and heterotrophic nitrification in an acid woodland soil. Results from laboratory incubations indicated that a maximum of 8% of the observed nitrification could be the result of heterotrophic nitrifiers oxidizing
organic nitrogen to nitrate without passing through the exchangeable soil ammonium pool. Autotrophic nitrification rates ranged from 0.96 to 2.64pg N g-l d-l, depending on the time of the year the soil was sampled. Heterotrophic nitrification rates ranged from 0.08 to 0.12 pegN g-’ d-’ and appeared unaffected by the nitrification inhibitor N-Serve.
INTRODUCTION
Nitrate can be produced in soil by two nitrification pathways. The first, and probably the most important in agricultural soils, is the oxidation of ammonia to nitrate via hydroxylamine and nitrite (Wood, 1986). The second is the oxidation of organic aminonitrogen to nitrate via organic nitroso compounds (Killham, 1990). The evidence to date suggests that chemo-autotrophic nitrifiers can only use the inorganic route for nit&cation while heterotrophic nitrifers can use both organic and inorganic routes. Because of their sensitivity to low pH, it is thought that autrotrophic nitrifiers are less important in acidic soils and that nitrate production in such soils is predominantly mediated by fungal heterotrophic nitrification (Killham, 1990). But, while the presence of heterotrophic nitrification has been demonstrated in pure culture (Schmidt, 1960), distinguishing heterotrophic from autotrophic nitrification in the more complex environment of soil is less straightforward. Most probable number (MPN) techniques often fail to detect sufficient autotrophic nitrifiers to account for observed nitrification rates but this cannot be interpreted as indicating heterotrophic nitrification. Pennington and Ellis (1993) found that, in some soils, insufficient autotrophic nitrifiers were detected by MPN to account for nitrifying activity even though acetylene (an inhibitor of ammonium oxidizers) reduced nit&cation by 96%. Schimel et al. (1984) determined autotrophic and heterotrophic nitrification in an acid Sierran forest soil by using acetylene and chlorate as selective physiological blocks for parts of the autotrophic oxidation pathway. Nitrate production was unaffected by either treatment suggesting that the majority of nitrate
production was via the heterotrophic pathway. “N labelled ammonium was also used to show that little of the nitrate produced originated from ammonium, although the details of this part of the work are brief, and as will become clear, the proportion of the nitrate pool derived from the added ammonium label is not a reliable way of determining the production of nitrate from the whole ammonium pool (i.e. soil ammonium + added labelled ammonium). De Boer et al. (1992) used the inhibition of nitrification by acetylene to conclude that nitrification in four acid forest soils was largely autotrophic. Killham (1986, 1987), again using biochemical blocks, concluded that nitrification in acid arable and grassland soil was largely autotrophic while in acid coniferous soils it was mediated by heterotrophic fungi. Thus, biochemical blocks have been a powerful way of separating autotrophic and heterotrophic nitrification, but their specificity is difficult to prove. For example, Landi et al. (1993) concluded that cycloheximide, used as a specific inhibitor of fungal activity, could have a non-target effect on autotrophic bacteria. A more direct way to study heterotrophic nitrification and to assess its environmental significance would be to determine rates of heterotrophic and autotrophic nitrification directly. If it is assumed that the organic (heterotrophic) nitrification pathway does not pass through the exchangeable ammonium pool in soil, the techniques of isotope dilution and isotope enrichment allow the organic and inorganic routes of nitrification to be separated. We report here results from such an approach in which the proportion of nitrate produced by heterotrophic nitrification is determined by using 15N pool dilution to measure the combined gross rate of autotrophic and heterotrophic nitrification while ‘*N
D.
18
BARRACLOUGH and G. PURI
pool enrichment is used to determine autotrophic and heterotrophic nitrification separately. Although heterotrophs can use both organic and inorganic pathways, we will use the term heterotrophic nitrification to indicate the oxidation of organic nitrogen to nitrate. Autotrophic nitrification will be used to describe ammonium oxidation, on the assumption that this is carried out predominantly by autotrophs. Theor) Isotope pool dilution techniques enable gross rates of nitrification (or mineralization) to be determined by monitoring the decline in the lSN abundance in a nitrate (or ammonium) pool, labelled at t = 0 and receiving unlabelled nitrogen via nitrification (or mineralization). The assumptions and restrictions in this approach were discussed by Barraclough (1990). Thus, if the size and 15N abundance of a nitrate pool are determined at at least two intervals following the addition of labelled nitrate, equation (I) can be solved to give n, the gross rate of nitrification. All notation is given in Table I. Gross rates of mineralization can be obtained from similar measurements on an
*N, =
*No
(1)
(I + 0t /N,)fl;”
MATERIALS
initially-labelled NH, pool. In the case of a labelled nitrate pool, isotope dilution strictly determines the gross rate at which unlabelled nitrate enters the pool. Thus, where the ammonium pool and the soil organic N pool are unlabelled, isotope dilution gives the rate of nitrate production by the autotrophic and heterotrophic routes combined. In contrast, pool enrichment involves labelling the ammonium pool and monitoring the subsequent appearance of “N nitrate. The rate at which 15N appears in the NO, pool is determined by the rate at which nitrate is produced by the autotrophic route. Thus, in principle, pool dilution gives the rates of nitrate production by the
Table I. List of symbols Symbol A ISA ‘A N “N ‘N m n
Meaning Ammonium N pool size Ammonium “N pool size “N atom excess in ammonium pool Nitrate N pool size Nitrate “N pool size “N atom excess in nitrate pool Gross rate of nitrogen mineralization Gross rate of nitrification (=n,+n,) Time Rate of ammonium consumption Rate of nitrate consumption Observed rate at which pool size changes
combined autotrophic and heterotrophic routes, while pool enrichment depends only on the autotrophic route. Interpreting pool enrichment in terms of gross rates is more complex than in pool dilution because the “N abundance of both the ammonium and nitrate pools changes over time and analytical solutions describing the changes in the “N abundance of the nitrate pool with time are only approximations (Wessel and Tietema. 1992). As a result, interpreting pool enrichment experiments in terms of rates of nitrogen transformation requires simulation modelling. The procedure in this work, then, was to perform an isotope dilution experiment using 14NHA5NOj to determine rates of nitrification by the combined autotrophic and heterotrophic paths (n, + nh). A parallel isotope dilution experiment with “NH:‘NOj, under the same conditions, yielded the gross rate of mineralization and the size and i5N abundance of the nitrate pool at intervals following label addition (pool enrichment). The pool enrichment experiment was analysed using simulation modelling to obtain the gross rate of autotrophic and heterotrophic nitrification separately.
Units figNg vgNg at.%
aNg pgNg
at.%
PcgNtz
d
/%Ng
d
days lrgNg pgNg FgNg
d d d
Subscripts0 and I refer to time 0 and I respectively.Subscripts h and a refer to hetero- and autotrophic nitrification, respectively.
AND METHODS
Isotope dilution and enrichment experiments were carried out on soil collected from the 0 to IOcm soil layer of a deciduous woodland in Berkshire, England, planted with oak in 1890 and little disturbed since. The soil is a Gleyic Brown Earth. Selected properties of the soil (O-IO cm) are: pH (in HzO) 3.8; total N 0.18%; organic C 2.41%; soil microbial biomass N 82 pg N gg’. Soil was collected in October 1992 and in September 1993, sieved gently through a coarse sieve and stored in sealed containers at 20’C. Three experiments were performed. The two on the soil collected in October 1992 are identified as E4 and El2, indicating the time in weeks after collection when the experiments were performed. One experiment was carried out on the soil collected in September 1993 within 1 week of collection. For experiments E4 and E12, 40g aliquots of soil were packed in plastic cylinders 1 cm deep and 7.5 cm dia at the bulk density in the field (0.85 g cm-‘) and either (‘5NH4)2 SO,+ KNO, or (NH4)2S04 + K15N0, added in 3 ml of deionized water. Tests had shown this gave a uniform distribution of label in the soil. The addition increased the soil moisture content by 7.5% (w/w) to 31 and 28% (w/w) in E4 and El2 respectively. The initial moisture content in the September 1993 experiment was 6% (w/w); after addition of the label and N-Serve (or water) it was 21% (w/w). In E4 2.5 pg Ng-’ as ammonium and nitrate was added in each injection, i.e. 5 fig N gg’ in total; in El2 the addition was Spg Ng-’ as ammonium and
19
Heterotrophic nitrification in a woodland soil Table2.Pool sizes and 'jN abundancesin thesoil collectedin September1993.Valuesaremeans of four replicates. Finuresin oarentheses are standarderrors
NO, pool
NH, pool NH, (OgNg-')
lSNH, (at.%)
NO, (PgNg-')
"NO, (at.%)
“NO, recovery (%)
Control
NH, label TO Tl T2 T3 T8
8.56(0.59) 6.61(0.26) 5.96(0.34) 5.16(0.74) 2.30(0.24)
7.590(0.563) 5.513(0.324) 4.407(0.087) 3.477(0.429) 0.814(0.138)
14.00(0.65) 17.68(0.30) 19.58(0.43) 21.94(0.32) 30.35(0.72)
0.379(0.020) 1.235(0.053) 1.673(0.093) 1.865(0.057) 1.835(0.041)
-
NO, label TO Tl T2 T3 T8
8.21(0.13) 6.63(0.28) 5.72(0.08) 4.45(0.26) 2.57(0.97)
0.374(0.010) 0.391(0.040) 0.401(0.020) 0.407(0.020) 0.408(0.005)
14.56(0.26) 17.64(0.26) 20.32(0.40) 23.31(0.56) 31.33(0.39)
2.599(0.100) 2.260(0.010) 1.968(0.050) 1.771(0.050) 1.353(0.010)
100 102 100 100 95
+N-Serve NH, label TO Tl T2 T3 T8
ll.22(0.53) 12.87(0.39) 13.30(0.42) 14.26(0.16) 15.03(0.36)
5.293(0.188) 4.275(0.100) 3.823(0.012) 3.261(0.050) 2.218(0.144)
13.22(0.16) 13.91(0.0) 13.93(0.22) 15.43(0.59) 16.99(0.41)
0.381(0.015) 0.633(0.052) 0.645(0.05) 0.751(0.013) 0.951(0.073)
NO, label TO Tl T2 T3 T8
12.13(0.37) 12.72(0.42) 13.04(0.52) 13.89(0.04) 14.88(0.28)
0.362(0.003) 0.378(0.012) 0.367(0.004) 0.362(0.001) 0.391(0.027)
13.43(0.26) 14.21(0.72) 14.73(1.16) 15.95(0.59) 16.79tO.32)
2.689(0.019) 2.612(0.156) 2.559(0.190) 2.491(0.050) 2.313tO.068)
nitrate. The cylinders were sealed in parafilm with four pin holes for gas exchange. Water loss throughout all experiments was negligible. In E4, Cfold replicate samples were taken 3 and 10 days after label addition. In E12, 3-fold replicate samples were taken at t = 0, 1, 2 and 3 days after label addition. In the September 1993 experiment a slightly different procedure was employed to aIIow the incorporation of the nitrification inhibitor N-Serve. 40g aliquots of fresh soil were incubated in Erlenmeyer flasks. Two days before label addition, 20 pg gg ’of N-Serve in an aqueous suspension (Bremner et nl., 1978) was added to half the flasks and well mixed; the corresponding amount of water was added to the remaining control flasks. Label addition was as for El2 with four replicate samples taken at t = 0, 1, 2, 3 and 8 days. Analyses
All analyses were performed on 1 M KC1 extracts (1: 5 soil solution ratio) of 40 g aliquots of fresh soil following filtration through glass-fibre filter paper. All the soil from each cylinder or flask was sampled. Soil ammonium and nitrate were determined by flow injection calorimetry. 15N:14N isotope ratios in the ammonium and nitrate fractions were determined on a VG Micromass 622 mass spectrometer linked to a Europa Scientific RoboPrep combustion analyser and referenced against IAEA “N quality control standard 305. No spiking was employed as reliable isotope ratios are possible on 20 pgN using this system.
100 102 103 103 I04
RESULTS
Tables 2 and 3 give the mean pool sizes and 15N abundances of the ammonium and nitrate pools, and the recovery of added 15N03 at each sampling in the three experiments. The results were processed to yield gross rates of nitrogen transformations as follows. Gross mineralization rates were obtained from the 15NH4 isotope dilution data by inserting the ammonium pool sizes and “N excess enrichments at two samplings into the ammonium version of equation (1) and solving for m, the gross rate of mineralization. The gross rate of nitrification (n = nh + n,) was obtained similarly by substituting nitrate pool sizes and abundances from the 15NOX isotope dilution experiments into equation (1) and solving for n, the gross rate of nitrification. The rates of hetero- and autotrophic nitrification were obtained by running a simulation model using the results from the pool enrichment experiment in which 15NH4was added. The model solves equations (2)-(S) describing the rate of change of the amounts of N and 15Nin the ammonium and nitrate pools. d”A = 0.3663m - a (“A/A) dt dA dr=m-a d”N = n,(“A/A) dt
+ 0.3663n, - p ( 15N/Nj
dN = n, + nh - 8. dt
(4)
D. BARRACLOUGH
20
and G. PURI
Table 3. Pool sizes and ‘sN abundances NH,
NH, (mNg
‘)
in soil collected NO,
pool ‘*NH, (at.%)
NO, (tizzNg
‘)
in October
1992
pool “NO, (at.%)
‘SNO, recovery (%)
E4 NH, label T3 TIO
7.90 (0.81) 7.64 (0.52)
I.383 (0.073) 0.540 (0.032)
8.33 (0.28) 15.07 (1.06)
0.751 (0.029) 0.778 (0.005)
NO, label T3 TIO
7.45 (0.42) IO. IS (2.67)
0.379 (0.013) 0.385 (0.015)
8.03 (0.46) 13.24 (0.47)
2. I I7 (0.053) I.356 (0.085)
90 91
El?
NH, label TO TI T2 T3
7.53 (0.16) 5.72 (0.09) 4.32(0.15) 2.66 (0. IO)
4.735 4.306 3.746 2.770
(0.133) (0.081) (0.098) (0.062)
47. I I 47.67 48.45 50. IO
(0.13) (0.79) (0.28) (0.27)
0.373 0.502 0.625 0.689
(0.007) (0.007) (0.016) (0.023)
NO, label TO Tl T2 T3
7.92 6.65 4.76 3. I4
0.356 0.351 0.357 0.331
(0.008) (0.003) (0.004) (0.009)
46.54 (I .47) 47.90 (0.41) 48.59(1.05) 49.93 (0.29)
I.206 I. 149 I. I I4 I .085
(0.046) (0.006) (0.024) (0.005)
(0.22) (0.15) (0.1 I) (0.07)
Equations (2) and (3) describe the rate at which the amounts of “N and N in the ammonium pool change. The rate of mineralization is that obtained from the pool dilution calculations described above. Equations (4) and (5) refer to the nitrate pool. Equation (4) is the key part of the model. It describes the rate at which the nitrate pool “N changes in terms of three processes. Autotrophic nitrification, n,, which introduces nitrate at the “N abundance of the ammonium pool at the time; heterotrophic nitrification, nhr converting organic nitrogen at natural abundance “N to nitrate; and nitrate consumption, /I (denitrification, immobilization etc.), which removes nitrate at the 15N abundance of the nitrate pool at the time. Nitrate consumption was calculated from the nitrate mass balance using the gross nitrification rate, n, determined from pool dilution. Thus: /j’= (N, + n . t - N,)/t.
100 94 91 90
nitrate pool in terms of the proportion produced from the ammonium pool must take into account the ammonium 15N pool dilution resulting from mineralization. The simulation model was used to find values of nh and n, that resulted in a simulated 15Nenrichment in the nitrate pool in the final sampling of the pool enrichment experiments equal to that measured. Initial pool sizes and abundances used in the model were those measured at the first sampling. The model was run with the constraint that the sum of the autoand heterotrophic nitrification rates in the pool enrichment experiment must equal the overall nitrification rate obtained from pool dilution. In E4, the increase in the nitrate pool is not the same in the
(6)
Equations (2)-(5) were solved using a variable timestep Runge-Kutta procedure from the Numerical Algorithms Library (NAG, 1983). Figure 1 illustrates a typical output from the simulation model. The points and line show the increase in “N abundance in an initially unlabelled nitrate pool as a function of the proportion of nitrification coming, via the autotrophic route, from a labelled ammonium pool. The gross nitrification rate was ISpg Ng-‘d-’ in all cases; only the proportion coming via the autotrophic route changes. The 15Nabundance in the nitrate pool is clearly very sensitive to the split between the two nitrification routes. The histogram shows the proportion of the increase in the nitrate derived from the added ammonium label. Even when all the nitrification is via the autotrophic route, only 47% of the increase in nitrate is derived from the ammonium label; the shortfall is unlabelled ammonium mineralized and then nitrified. Thus interpreting “N recoveries in the
1.6 t
0
/
Lh
20 40 60 80 100 Proportion of nifrification that is autotrophic
10 120
0
E 8
5
y
(%)
Fig. I. Illustration of simulation model output. The line shows the effect of increased proportion of autotrophic nitrification on “NO,. The histogram shows the proportion of the increase in nitrate derived from labelled ammonium.
Heterotrophic nitrification in a woodland soil
21
Table 4. Mean gross nitrogen transformation rates Mineralization rate (m)
Experiment
Nitrification rate (n)
Hcterotrophic rate (nh)
Autotrophic rate (n, )
Heterotrophic
0.88 1.59 1.52
8 36 (0)
Ph)
Ortoher1992 E4 El2 Corrected
1.99 0.92 0.92
0.96 2.50 1.18
0.08 0.91 -0.33
September 1993 Control TO-T1 TILT8
2.56 1.49
2.64 2.22
0.12 0.09
2.52 2.13
4 4
N-Serve T&T1 TILT8
2.78 1.59
0.72 0.44
-0.07 0.11
0.79 0.33
(0) 25
All rates are in pg N go ’d-‘.
dilution and enrichment experiments; a difference mirrored in the ammonium pools (see Table 3). “NO3 recoveries in the pool dilution experiment indicated no loss of nitrate. Accordingly, in this case only, the gross rate of nitrification was set to equal the increase in the nitrate pool size in the enrichment experiment. Table 4 gives the mean rate parameters obtained from the simulation modelling.
DISCUSSION The reliability of the approach clearly depends on the ability of the simulation model [equations (2)--(s)] to represent the decline in the r5N abundance of the ammonium pool and the rise in t5N abundance in the nitrate pool resulting from autotrophic nitrification. The more frequent sampling in the September 1993 experiment allows the reliability of the simulations to be checked. Figure 2(a) shows the lSN abundances measured in the ammonium pool at intervals between
t = 0 and t = 8 (points) and the simulated abundances obtained using the gross rate of mineralization obtained with the ammonium version of equation (1) (solid line). Figure 2(B) shows the average “N abundance in the nitrate pool (points); the solid line shows the results from the simulation model. Data are shown for both the control and the N-Serve treatments. The discontinuity in the simulated lines at t = 1 reflects the difference in rates of mineralization (and, to a lesser extent, nitrification) between the t = 0 to t = 1 period and the remaining period to t = 8. The higher rates in the t = 0 to t = 1 period, not apparent in the other two experiments, probably result from the extra soil disturbance caused by the incorporation of the inhibitor N-Serve (and the water in the control). The correspondence between the simulated and experimental results suggests that equations (2H5) afford an accurate description of nitrogen transformations in this soil. Interestingly, the gross rate of mineralization in the control and the N-Serve treatments are very similar, confirming that
W
(a)
IO r 9
0
2
Control 7 6 5 15N (at.%) 4 N-SeNe
3 N-Serve
2 1 0
Control ,“I”I”,
0123456789
0
J
11
11
”
“1
0123456789
Time after label addition (days)
Fig. 2. (a) Simulated (line) and observed (points) 15Nabundances in the ammonium pool in the September 1993 experiment. (b) Simulated (line) and observed (points) lSN abundances in the nitrate pool in the September 1993 experiment. Open symbols are control treatments; solid symbols are N-Serve.
22
D.
BARRACLOUGH and G. PURI
isotope dilution techniques can satisfactorily isolate individual processes in the soil nitrogen cycle. The results in Table 4 indicate that, with one exception, the rates of heterotrophic nitrification are very low. Only experiment El2 gives substantial rates of heterotrophic nitrification. It also differs from the others in that there was significant loss (10%) of r5N0, during the experiment. In E4 there was no loss of 15N0, between measurements at TJ and T,, and in the September 1993 experiment, 5% of the added nitrate label was lost, but over a longer period. Isotope dilution relies on the assumption that any loss processes exploit labelled and unlabelled N in proportion; i.e. in themselves the loss processes do not alter the “N abundance in the pool. If, for some reason, labelled nitrate were being preferentially lost in E12, the gross rate of nitrification would be overestimated since the loss of label would appear as dilution. To check this possibility, the results from El2 were reanalysed assuming the ISNO3 was preferentially lost. The results are shown in Table 4 as “corrected”. They suggest the significant heterotrophic nitrification may be an artefact arising from preferential loss of “NO,. The conclusion that heterotrophic nitrification is low in this soil is supported by the results from the N-Serve treatment in the September 1993 experiment. N-Serve reduced gross nitrification from around 2.4 to 0.44 to 0.72 pg N gg’ d-’ with most of the residual nitrification still being autotrophic in origin (Table 4). Overall these results suggest that heterotrophic nitrification is of only minor importance in these experiments, contributing, at most, 8% of nitrate production. The most reliable rates of heterotrophic nitrification were 0.08, 0.12 and 0.09 pg N gg’ d-‘, compared to rates of autotrophic nitrification between 0.88 and 2.64 pg N g- ’d-’ . Interestingly, following the addition of N-Serve, the estimated heterotrophic nitrification rate over a 7 day period was 0.11 pg N gg’ d-‘, compared to 0.09 /*g N g-’ d-’ without N-Serve. It is possible that the addition of ammonium stimulated autotrophic activity (Aarnio and Martikainen, 1992) indeed they may have caused heterogrophs to switch to ammonia oxidation. However, background concentrations of ammonium measured in situ in this soil are frequently in the range used in these experiments suggesting that any artificial stimulation of autotrophic nitrification is unlikely to be significant. In addition, the extent of heterotrophic nitrification was similar in E4, where 2.5 pg NH,-N gg’ was added, and in September 1993 where the addition was doubled. One final point concerns the possible disruption of fungal hyphae during the soil sampling. The integrity of fungal hyphae will almost certainly be affected by the soil sampling and sieving. This may reduce heterotrophic fungal activity in the laboratory exper-
iments compared to the field. Unfortunately, in situ spatial variability makes it very difficult to separate the two nitrification routes in the field. Field experiments carried out by the authors have proved very difficult to interpret for this reason. Thus while the combined use of pool dilution and pool enrichment appears to be a powerful method for separating autotrophic and heterotrophic nitrification in the laboratory, spatial variability might make it less useful in the field. Acknowledgements-This work was supported by The Natural Environment Research Council. The authors are grateful to Martin Heaps for help with ‘sN/i4N isotope ratio analyses.
REFERENCES Aarnio T. and Martikainen P. J. (1992) Nitritication in forest soil after refertilization with urea or urea and Soil Biology & Biochemistry 24, 951-954. dicyandiamide. Barraclough D. (1990) The use of mean pool abundances to interpret 15N tracer experiments I. Theory. Plant ond Soil 131, 89-96. Bremner J. M., Blackmer A. M. and Bundy L. G. (1978) Problems in use of nitrapyrin (N-Serve) to inhibit nitrification in soils. Soil Biology & Biochemistry 10, 441442. De Boer W., Tietema A., Klein Gunnewick P. J. A. and Laanbroek H. J. (1992) The chemolithotrophic ammonium-oxidising community in a nitrogen saturated acid forest in relation to pH-dependent nitrifying activity. Soil Biology & Biochemistry 24, 229-234. NAG (1983) The NAG PC50 Forfran Library. The Numerical Algorithms Group Ltd, Oxford. Killham K. (1986) Heterotroohic nitrification. In Nitrification (J. I: Presser, Ed.), \jol. 20, pp. 117-126. Special publications for Society for General Microbiology. IRL Press, Oxford. Killham K. (1987) A new perfusion technique for the measurement and characterization of potential rates of soil nitrification. Plant and Soil 97, 267-272. Killham K. (1990) Nitrification in coniferous forest soils. Processings of a workshop on nitrogen saturation in forest ecosystems. Plant and Soil 128, 3144. Landi L., Badalucco L., Pomare F. and Nannipieri P. (1993) Effectiveness of antibiotics to distinguish the contributions of fungi and bacteria to net nitrogen mineralization, nitrification and respiration. Soil Biolog), & Biochemistry 25, I77 1-I 778. Pennington P. I. and Ellis R. C. (1993) Autotrophic and heterotrophic nitrification in acid forest and native grassland soils. Soil Biolonv & Biochemis/rv 25, 1399-1408. Schimel J. P., Fireston; M. K. and Kihham K. S. (1984) Identification of heterotrophic nitrification in a Sierran forest soil. Applied and Environmental Microbiolog! 48, 802-806. Schmidt E. L. (1960) Nitrate formation by AspergillusJavus in pure and mixed culture environments. Transactions q/ rhe 7th International Congress of Soil Science 2, 600-605. Wessel W. W. and Tietema A. (1992) Calculating gross N transformations rates of “N pool dilution experiments with acid forest litter: analytical and numerical approaches. Soil Biology & Biochemistry 24, 931-942. Wood P. M. (1986) Nitrification as a bacterial energy source. In Nirrification (J. I. Prosser, Ed.), Vol. 20, pp. 39962. Special publications for Society for General Microbiology. IRL Press, Oxford.
0038-0717(94)00141-3
Soil Viol. Biochem. Vol. 27. No. I, pp. 17-22, 1995 Copyright C 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.04
THE USE OF 15N POOL DILUTION AND ENRICHMENT TO SEPARATE THE HETEROTROPHIC AND AUTOTROPHIC PATHWAYS OF NITRIFICATION D. BARRACLOUGH and G. PURI Department of Soil Science, University of Reading, London Road, Reading RGI 5AQ, England (Accepted I 7 June 1994)
Summary-The techniques of lSN pool dilution and enrichment were used to separate autotrophic and heterotrophic nitrification in an acid woodland soil. Results from laboratory incubations indicated that a maximum of 8% of the observed nitrification could be the result of heterotrophic nitrifiers oxidizing
organic nitrogen to nitrate without passing through the exchangeable soil ammonium pool. Autotrophic nitrification rates ranged from 0.96 to 2.64pg N g-l d-l, depending on the time of the year the soil was sampled. Heterotrophic nitrification rates ranged from 0.08 to 0.12 pegN g-’ d-’ and appeared unaffected by the nitrification inhibitor N-Serve.
INTRODUCTION
Nitrate can be produced in soil by two nitrification pathways. The first, and probably the most important in agricultural soils, is the oxidation of ammonia to nitrate via hydroxylamine and nitrite (Wood, 1986). The second is the oxidation of organic aminonitrogen to nitrate via organic nitroso compounds (Killham, 1990). The evidence to date suggests that chemo-autotrophic nitrifiers can only use the inorganic route for nit&cation while heterotrophic nitrifers can use both organic and inorganic routes. Because of their sensitivity to low pH, it is thought that autrotrophic nitrifiers are less important in acidic soils and that nitrate production in such soils is predominantly mediated by fungal heterotrophic nitrification (Killham, 1990). But, while the presence of heterotrophic nitrification has been demonstrated in pure culture (Schmidt, 1960), distinguishing heterotrophic from autotrophic nitrification in the more complex environment of soil is less straightforward. Most probable number (MPN) techniques often fail to detect sufficient autotrophic nitrifiers to account for observed nitrification rates but this cannot be interpreted as indicating heterotrophic nitrification. Pennington and Ellis (1993) found that, in some soils, insufficient autotrophic nitrifiers were detected by MPN to account for nitrifying activity even though acetylene (an inhibitor of ammonium oxidizers) reduced nit&cation by 96%. Schimel et al. (1984) determined autotrophic and heterotrophic nitrification in an acid Sierran forest soil by using acetylene and chlorate as selective physiological blocks for parts of the autotrophic oxidation pathway. Nitrate production was unaffected by either treatment suggesting that the majority of nitrate
production was via the heterotrophic pathway. “N labelled ammonium was also used to show that little of the nitrate produced originated from ammonium, although the details of this part of the work are brief, and as will become clear, the proportion of the nitrate pool derived from the added ammonium label is not a reliable way of determining the production of nitrate from the whole ammonium pool (i.e. soil ammonium + added labelled ammonium). De Boer et al. (1992) used the inhibition of nitrification by acetylene to conclude that nitrification in four acid forest soils was largely autotrophic. Killham (1986, 1987), again using biochemical blocks, concluded that nitrification in acid arable and grassland soil was largely autotrophic while in acid coniferous soils it was mediated by heterotrophic fungi. Thus, biochemical blocks have been a powerful way of separating autotrophic and heterotrophic nitrification, but their specificity is difficult to prove. For example, Landi et al. (1993) concluded that cycloheximide, used as a specific inhibitor of fungal activity, could have a non-target effect on autotrophic bacteria. A more direct way to study heterotrophic nitrification and to assess its environmental significance would be to determine rates of heterotrophic and autotrophic nitrification directly. If it is assumed that the organic (heterotrophic) nitrification pathway does not pass through the exchangeable ammonium pool in soil, the techniques of isotope dilution and isotope enrichment allow the organic and inorganic routes of nitrification to be separated. We report here results from such an approach in which the proportion of nitrate produced by heterotrophic nitrification is determined by using 15N pool dilution to measure the combined gross rate of autotrophic and heterotrophic nitrification while ‘*N
D.
18
BARRACLOUGH and G. PURI
pool enrichment is used to determine autotrophic and heterotrophic nitrification separately. Although heterotrophs can use both organic and inorganic pathways, we will use the term heterotrophic nitrification to indicate the oxidation of organic nitrogen to nitrate. Autotrophic nitrification will be used to describe ammonium oxidation, on the assumption that this is carried out predominantly by autotrophs. Theor) Isotope pool dilution techniques enable gross rates of nitrification (or mineralization) to be determined by monitoring the decline in the lSN abundance in a nitrate (or ammonium) pool, labelled at t = 0 and receiving unlabelled nitrogen via nitrification (or mineralization). The assumptions and restrictions in this approach were discussed by Barraclough (1990). Thus, if the size and 15N abundance of a nitrate pool are determined at at least two intervals following the addition of labelled nitrate, equation (I) can be solved to give n, the gross rate of nitrification. All notation is given in Table I. Gross rates of mineralization can be obtained from similar measurements on an
*N, =
*No
(1)
(I + 0t /N,)fl;”
MATERIALS
initially-labelled NH, pool. In the case of a labelled nitrate pool, isotope dilution strictly determines the gross rate at which unlabelled nitrate enters the pool. Thus, where the ammonium pool and the soil organic N pool are unlabelled, isotope dilution gives the rate of nitrate production by the autotrophic and heterotrophic routes combined. In contrast, pool enrichment involves labelling the ammonium pool and monitoring the subsequent appearance of “N nitrate. The rate at which 15N appears in the NO, pool is determined by the rate at which nitrate is produced by the autotrophic route. Thus, in principle, pool dilution gives the rates of nitrate production by the
Table I. List of symbols Symbol A ISA ‘A N “N ‘N m n
Meaning Ammonium N pool size Ammonium “N pool size “N atom excess in ammonium pool Nitrate N pool size Nitrate “N pool size “N atom excess in nitrate pool Gross rate of nitrogen mineralization Gross rate of nitrification (=n,+n,) Time Rate of ammonium consumption Rate of nitrate consumption Observed rate at which pool size changes
combined autotrophic and heterotrophic routes, while pool enrichment depends only on the autotrophic route. Interpreting pool enrichment in terms of gross rates is more complex than in pool dilution because the “N abundance of both the ammonium and nitrate pools changes over time and analytical solutions describing the changes in the “N abundance of the nitrate pool with time are only approximations (Wessel and Tietema. 1992). As a result, interpreting pool enrichment experiments in terms of rates of nitrogen transformation requires simulation modelling. The procedure in this work, then, was to perform an isotope dilution experiment using 14NHA5NOj to determine rates of nitrification by the combined autotrophic and heterotrophic paths (n, + nh). A parallel isotope dilution experiment with “NH:‘NOj, under the same conditions, yielded the gross rate of mineralization and the size and i5N abundance of the nitrate pool at intervals following label addition (pool enrichment). The pool enrichment experiment was analysed using simulation modelling to obtain the gross rate of autotrophic and heterotrophic nitrification separately.
Units figNg vgNg at.%
aNg pgNg
at.%
PcgNtz
d
/%Ng
d
days lrgNg pgNg FgNg
d d d
Subscripts0 and I refer to time 0 and I respectively.Subscripts h and a refer to hetero- and autotrophic nitrification, respectively.
AND METHODS
Isotope dilution and enrichment experiments were carried out on soil collected from the 0 to IOcm soil layer of a deciduous woodland in Berkshire, England, planted with oak in 1890 and little disturbed since. The soil is a Gleyic Brown Earth. Selected properties of the soil (O-IO cm) are: pH (in HzO) 3.8; total N 0.18%; organic C 2.41%; soil microbial biomass N 82 pg N gg’. Soil was collected in October 1992 and in September 1993, sieved gently through a coarse sieve and stored in sealed containers at 20’C. Three experiments were performed. The two on the soil collected in October 1992 are identified as E4 and El2, indicating the time in weeks after collection when the experiments were performed. One experiment was carried out on the soil collected in September 1993 within 1 week of collection. For experiments E4 and E12, 40g aliquots of soil were packed in plastic cylinders 1 cm deep and 7.5 cm dia at the bulk density in the field (0.85 g cm-‘) and either (‘5NH4)2 SO,+ KNO, or (NH4)2S04 + K15N0, added in 3 ml of deionized water. Tests had shown this gave a uniform distribution of label in the soil. The addition increased the soil moisture content by 7.5% (w/w) to 31 and 28% (w/w) in E4 and El2 respectively. The initial moisture content in the September 1993 experiment was 6% (w/w); after addition of the label and N-Serve (or water) it was 21% (w/w). In E4 2.5 pg Ng-’ as ammonium and nitrate was added in each injection, i.e. 5 fig N gg’ in total; in El2 the addition was Spg Ng-’ as ammonium and
19
Heterotrophic nitrification in a woodland soil Table2.Pool sizes and 'jN abundancesin thesoil collectedin September1993.Valuesaremeans of four replicates. Finuresin oarentheses are standarderrors
NO, pool
NH, pool NH, (OgNg-')
lSNH, (at.%)
NO, (PgNg-')
"NO, (at.%)
“NO, recovery (%)
Control
NH, label TO Tl T2 T3 T8
8.56(0.59) 6.61(0.26) 5.96(0.34) 5.16(0.74) 2.30(0.24)
7.590(0.563) 5.513(0.324) 4.407(0.087) 3.477(0.429) 0.814(0.138)
14.00(0.65) 17.68(0.30) 19.58(0.43) 21.94(0.32) 30.35(0.72)
0.379(0.020) 1.235(0.053) 1.673(0.093) 1.865(0.057) 1.835(0.041)
-
NO, label TO Tl T2 T3 T8
8.21(0.13) 6.63(0.28) 5.72(0.08) 4.45(0.26) 2.57(0.97)
0.374(0.010) 0.391(0.040) 0.401(0.020) 0.407(0.020) 0.408(0.005)
14.56(0.26) 17.64(0.26) 20.32(0.40) 23.31(0.56) 31.33(0.39)
2.599(0.100) 2.260(0.010) 1.968(0.050) 1.771(0.050) 1.353(0.010)
100 102 100 100 95
+N-Serve NH, label TO Tl T2 T3 T8
ll.22(0.53) 12.87(0.39) 13.30(0.42) 14.26(0.16) 15.03(0.36)
5.293(0.188) 4.275(0.100) 3.823(0.012) 3.261(0.050) 2.218(0.144)
13.22(0.16) 13.91(0.0) 13.93(0.22) 15.43(0.59) 16.99(0.41)
0.381(0.015) 0.633(0.052) 0.645(0.05) 0.751(0.013) 0.951(0.073)
NO, label TO Tl T2 T3 T8
12.13(0.37) 12.72(0.42) 13.04(0.52) 13.89(0.04) 14.88(0.28)
0.362(0.003) 0.378(0.012) 0.367(0.004) 0.362(0.001) 0.391(0.027)
13.43(0.26) 14.21(0.72) 14.73(1.16) 15.95(0.59) 16.79tO.32)
2.689(0.019) 2.612(0.156) 2.559(0.190) 2.491(0.050) 2.313tO.068)
nitrate. The cylinders were sealed in parafilm with four pin holes for gas exchange. Water loss throughout all experiments was negligible. In E4, Cfold replicate samples were taken 3 and 10 days after label addition. In E12, 3-fold replicate samples were taken at t = 0, 1, 2 and 3 days after label addition. In the September 1993 experiment a slightly different procedure was employed to aIIow the incorporation of the nitrification inhibitor N-Serve. 40g aliquots of fresh soil were incubated in Erlenmeyer flasks. Two days before label addition, 20 pg gg ’of N-Serve in an aqueous suspension (Bremner et nl., 1978) was added to half the flasks and well mixed; the corresponding amount of water was added to the remaining control flasks. Label addition was as for El2 with four replicate samples taken at t = 0, 1, 2, 3 and 8 days. Analyses
All analyses were performed on 1 M KC1 extracts (1: 5 soil solution ratio) of 40 g aliquots of fresh soil following filtration through glass-fibre filter paper. All the soil from each cylinder or flask was sampled. Soil ammonium and nitrate were determined by flow injection calorimetry. 15N:14N isotope ratios in the ammonium and nitrate fractions were determined on a VG Micromass 622 mass spectrometer linked to a Europa Scientific RoboPrep combustion analyser and referenced against IAEA “N quality control standard 305. No spiking was employed as reliable isotope ratios are possible on 20 pgN using this system.
100 102 103 103 I04
RESULTS
Tables 2 and 3 give the mean pool sizes and 15N abundances of the ammonium and nitrate pools, and the recovery of added 15N03 at each sampling in the three experiments. The results were processed to yield gross rates of nitrogen transformations as follows. Gross mineralization rates were obtained from the 15NH4 isotope dilution data by inserting the ammonium pool sizes and “N excess enrichments at two samplings into the ammonium version of equation (1) and solving for m, the gross rate of mineralization. The gross rate of nitrification (n = nh + n,) was obtained similarly by substituting nitrate pool sizes and abundances from the 15NOX isotope dilution experiments into equation (1) and solving for n, the gross rate of nitrification. The rates of hetero- and autotrophic nitrification were obtained by running a simulation model using the results from the pool enrichment experiment in which 15NH4was added. The model solves equations (2)-(S) describing the rate of change of the amounts of N and 15Nin the ammonium and nitrate pools. d”A = 0.3663m - a (“A/A) dt dA dr=m-a d”N = n,(“A/A) dt
+ 0.3663n, - p ( 15N/Nj
dN = n, + nh - 8. dt
(4)
D. BARRACLOUGH
20
and G. PURI
Table 3. Pool sizes and ‘sN abundances NH,
NH, (mNg
‘)
in soil collected NO,
pool ‘*NH, (at.%)
NO, (tizzNg
‘)
in October
1992
pool “NO, (at.%)
‘SNO, recovery (%)
E4 NH, label T3 TIO
7.90 (0.81) 7.64 (0.52)
I.383 (0.073) 0.540 (0.032)
8.33 (0.28) 15.07 (1.06)
0.751 (0.029) 0.778 (0.005)
NO, label T3 TIO
7.45 (0.42) IO. IS (2.67)
0.379 (0.013) 0.385 (0.015)
8.03 (0.46) 13.24 (0.47)
2. I I7 (0.053) I.356 (0.085)
90 91
El?
NH, label TO TI T2 T3
7.53 (0.16) 5.72 (0.09) 4.32(0.15) 2.66 (0. IO)
4.735 4.306 3.746 2.770
(0.133) (0.081) (0.098) (0.062)
47. I I 47.67 48.45 50. IO
(0.13) (0.79) (0.28) (0.27)
0.373 0.502 0.625 0.689
(0.007) (0.007) (0.016) (0.023)
NO, label TO Tl T2 T3
7.92 6.65 4.76 3. I4
0.356 0.351 0.357 0.331
(0.008) (0.003) (0.004) (0.009)
46.54 (I .47) 47.90 (0.41) 48.59(1.05) 49.93 (0.29)
I.206 I. 149 I. I I4 I .085
(0.046) (0.006) (0.024) (0.005)
(0.22) (0.15) (0.1 I) (0.07)
Equations (2) and (3) describe the rate at which the amounts of “N and N in the ammonium pool change. The rate of mineralization is that obtained from the pool dilution calculations described above. Equations (4) and (5) refer to the nitrate pool. Equation (4) is the key part of the model. It describes the rate at which the nitrate pool “N changes in terms of three processes. Autotrophic nitrification, n,, which introduces nitrate at the “N abundance of the ammonium pool at the time; heterotrophic nitrification, nhr converting organic nitrogen at natural abundance “N to nitrate; and nitrate consumption, /I (denitrification, immobilization etc.), which removes nitrate at the 15N abundance of the nitrate pool at the time. Nitrate consumption was calculated from the nitrate mass balance using the gross nitrification rate, n, determined from pool dilution. Thus: /j’= (N, + n . t - N,)/t.
100 94 91 90
nitrate pool in terms of the proportion produced from the ammonium pool must take into account the ammonium 15N pool dilution resulting from mineralization. The simulation model was used to find values of nh and n, that resulted in a simulated 15Nenrichment in the nitrate pool in the final sampling of the pool enrichment experiments equal to that measured. Initial pool sizes and abundances used in the model were those measured at the first sampling. The model was run with the constraint that the sum of the autoand heterotrophic nitrification rates in the pool enrichment experiment must equal the overall nitrification rate obtained from pool dilution. In E4, the increase in the nitrate pool is not the same in the
(6)
Equations (2)-(5) were solved using a variable timestep Runge-Kutta procedure from the Numerical Algorithms Library (NAG, 1983). Figure 1 illustrates a typical output from the simulation model. The points and line show the increase in “N abundance in an initially unlabelled nitrate pool as a function of the proportion of nitrification coming, via the autotrophic route, from a labelled ammonium pool. The gross nitrification rate was ISpg Ng-‘d-’ in all cases; only the proportion coming via the autotrophic route changes. The 15Nabundance in the nitrate pool is clearly very sensitive to the split between the two nitrification routes. The histogram shows the proportion of the increase in the nitrate derived from the added ammonium label. Even when all the nitrification is via the autotrophic route, only 47% of the increase in nitrate is derived from the ammonium label; the shortfall is unlabelled ammonium mineralized and then nitrified. Thus interpreting “N recoveries in the
1.6 t
0
/
Lh
20 40 60 80 100 Proportion of nifrification that is autotrophic
10 120
0
E 8
5
y
(%)
Fig. I. Illustration of simulation model output. The line shows the effect of increased proportion of autotrophic nitrification on “NO,. The histogram shows the proportion of the increase in nitrate derived from labelled ammonium.
Heterotrophic nitrification in a woodland soil
21
Table 4. Mean gross nitrogen transformation rates Mineralization rate (m)
Experiment
Nitrification rate (n)
Hcterotrophic rate (nh)
Autotrophic rate (n, )
Heterotrophic
0.88 1.59 1.52
8 36 (0)
Ph)
Ortoher1992 E4 El2 Corrected
1.99 0.92 0.92
0.96 2.50 1.18
0.08 0.91 -0.33
September 1993 Control TO-T1 TILT8
2.56 1.49
2.64 2.22
0.12 0.09
2.52 2.13
4 4
N-Serve T&T1 TILT8
2.78 1.59
0.72 0.44
-0.07 0.11
0.79 0.33
(0) 25
All rates are in pg N go ’d-‘.
dilution and enrichment experiments; a difference mirrored in the ammonium pools (see Table 3). “NO3 recoveries in the pool dilution experiment indicated no loss of nitrate. Accordingly, in this case only, the gross rate of nitrification was set to equal the increase in the nitrate pool size in the enrichment experiment. Table 4 gives the mean rate parameters obtained from the simulation modelling.
DISCUSSION The reliability of the approach clearly depends on the ability of the simulation model [equations (2)--(s)] to represent the decline in the r5N abundance of the ammonium pool and the rise in t5N abundance in the nitrate pool resulting from autotrophic nitrification. The more frequent sampling in the September 1993 experiment allows the reliability of the simulations to be checked. Figure 2(a) shows the lSN abundances measured in the ammonium pool at intervals between
t = 0 and t = 8 (points) and the simulated abundances obtained using the gross rate of mineralization obtained with the ammonium version of equation (1) (solid line). Figure 2(B) shows the average “N abundance in the nitrate pool (points); the solid line shows the results from the simulation model. Data are shown for both the control and the N-Serve treatments. The discontinuity in the simulated lines at t = 1 reflects the difference in rates of mineralization (and, to a lesser extent, nitrification) between the t = 0 to t = 1 period and the remaining period to t = 8. The higher rates in the t = 0 to t = 1 period, not apparent in the other two experiments, probably result from the extra soil disturbance caused by the incorporation of the inhibitor N-Serve (and the water in the control). The correspondence between the simulated and experimental results suggests that equations (2H5) afford an accurate description of nitrogen transformations in this soil. Interestingly, the gross rate of mineralization in the control and the N-Serve treatments are very similar, confirming that
W
(a)
IO r 9
0
2
Control 7 6 5 15N (at.%) 4 N-SeNe
3 N-Serve
2 1 0
Control ,“I”I”,
0123456789
0
J
11
11
”
“1
0123456789
Time after label addition (days)
Fig. 2. (a) Simulated (line) and observed (points) 15Nabundances in the ammonium pool in the September 1993 experiment. (b) Simulated (line) and observed (points) lSN abundances in the nitrate pool in the September 1993 experiment. Open symbols are control treatments; solid symbols are N-Serve.
22
D.
BARRACLOUGH and G. PURI
isotope dilution techniques can satisfactorily isolate individual processes in the soil nitrogen cycle. The results in Table 4 indicate that, with one exception, the rates of heterotrophic nitrification are very low. Only experiment El2 gives substantial rates of heterotrophic nitrification. It also differs from the others in that there was significant loss (10%) of r5N0, during the experiment. In E4 there was no loss of 15N0, between measurements at TJ and T,, and in the September 1993 experiment, 5% of the added nitrate label was lost, but over a longer period. Isotope dilution relies on the assumption that any loss processes exploit labelled and unlabelled N in proportion; i.e. in themselves the loss processes do not alter the “N abundance in the pool. If, for some reason, labelled nitrate were being preferentially lost in E12, the gross rate of nitrification would be overestimated since the loss of label would appear as dilution. To check this possibility, the results from El2 were reanalysed assuming the ISNO3 was preferentially lost. The results are shown in Table 4 as “corrected”. They suggest the significant heterotrophic nitrification may be an artefact arising from preferential loss of “NO,. The conclusion that heterotrophic nitrification is low in this soil is supported by the results from the N-Serve treatment in the September 1993 experiment. N-Serve reduced gross nitrification from around 2.4 to 0.44 to 0.72 pg N gg’ d-’ with most of the residual nitrification still being autotrophic in origin (Table 4). Overall these results suggest that heterotrophic nitrification is of only minor importance in these experiments, contributing, at most, 8% of nitrate production. The most reliable rates of heterotrophic nitrification were 0.08, 0.12 and 0.09 pg N gg’ d-‘, compared to rates of autotrophic nitrification between 0.88 and 2.64 pg N g- ’d-’ . Interestingly, following the addition of N-Serve, the estimated heterotrophic nitrification rate over a 7 day period was 0.11 pg N gg’ d-‘, compared to 0.09 /*g N g-’ d-’ without N-Serve. It is possible that the addition of ammonium stimulated autotrophic activity (Aarnio and Martikainen, 1992) indeed they may have caused heterogrophs to switch to ammonia oxidation. However, background concentrations of ammonium measured in situ in this soil are frequently in the range used in these experiments suggesting that any artificial stimulation of autotrophic nitrification is unlikely to be significant. In addition, the extent of heterotrophic nitrification was similar in E4, where 2.5 pg NH,-N gg’ was added, and in September 1993 where the addition was doubled. One final point concerns the possible disruption of fungal hyphae during the soil sampling. The integrity of fungal hyphae will almost certainly be affected by the soil sampling and sieving. This may reduce heterotrophic fungal activity in the laboratory exper-
iments compared to the field. Unfortunately, in situ spatial variability makes it very difficult to separate the two nitrification routes in the field. Field experiments carried out by the authors have proved very difficult to interpret for this reason. Thus while the combined use of pool dilution and pool enrichment appears to be a powerful method for separating autotrophic and heterotrophic nitrification in the laboratory, spatial variability might make it less useful in the field. Acknowledgements-This work was supported by The Natural Environment Research Council. The authors are grateful to Martin Heaps for help with ‘sN/i4N isotope ratio analyses.
REFERENCES Aarnio T. and Martikainen P. J. (1992) Nitritication in forest soil after refertilization with urea or urea and Soil Biology & Biochemistry 24, 951-954. dicyandiamide. Barraclough D. (1990) The use of mean pool abundances to interpret 15N tracer experiments I. Theory. Plant ond Soil 131, 89-96. Bremner J. M., Blackmer A. M. and Bundy L. G. (1978) Problems in use of nitrapyrin (N-Serve) to inhibit nitrification in soils. Soil Biology & Biochemistry 10, 441442. De Boer W., Tietema A., Klein Gunnewick P. J. A. and Laanbroek H. J. (1992) The chemolithotrophic ammonium-oxidising community in a nitrogen saturated acid forest in relation to pH-dependent nitrifying activity. Soil Biology & Biochemistry 24, 229-234. NAG (1983) The NAG PC50 Forfran Library. The Numerical Algorithms Group Ltd, Oxford. Killham K. (1986) Heterotroohic nitrification. In Nitrification (J. I: Presser, Ed.), \jol. 20, pp. 117-126. Special publications for Society for General Microbiology. IRL Press, Oxford. Killham K. (1987) A new perfusion technique for the measurement and characterization of potential rates of soil nitrification. Plant and Soil 97, 267-272. Killham K. (1990) Nitrification in coniferous forest soils. Processings of a workshop on nitrogen saturation in forest ecosystems. Plant and Soil 128, 3144. Landi L., Badalucco L., Pomare F. and Nannipieri P. (1993) Effectiveness of antibiotics to distinguish the contributions of fungi and bacteria to net nitrogen mineralization, nitrification and respiration. Soil Biolog), & Biochemistry 25, I77 1-I 778. Pennington P. I. and Ellis R. C. (1993) Autotrophic and heterotrophic nitrification in acid forest and native grassland soils. Soil Biolonv & Biochemis/rv 25, 1399-1408. Schimel J. P., Fireston; M. K. and Kihham K. S. (1984) Identification of heterotrophic nitrification in a Sierran forest soil. Applied and Environmental Microbiolog! 48, 802-806. Schmidt E. L. (1960) Nitrate formation by AspergillusJavus in pure and mixed culture environments. Transactions q/ rhe 7th International Congress of Soil Science 2, 600-605. Wessel W. W. and Tietema A. (1992) Calculating gross N transformations rates of “N pool dilution experiments with acid forest litter: analytical and numerical approaches. Soil Biology & Biochemistry 24, 931-942. Wood P. M. (1986) Nitrification as a bacterial energy source. In Nirrification (J. I. Prosser, Ed.), Vol. 20, pp. 39962. Special publications for Society for General Microbiology. IRL Press, Oxford.