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Transformations of added and indigenous nitrogen in gnotobiotic soil: A comment on the priming effect
00384717/87 $3.00 + 0.00 Pergamon Joumds Ltd
Soil Bid. Biochcm. Vol. 19. No. 6. pp. 673-618. 1987 Printed in Great Britain
TRANSFORMATIONS OF ADDED AND INDIGENOUS NITROGEN IN GNOTOBIOTIC SOIL: A COMMENT ON THE PRIMING EFFECT L. E.
b%ODS
USDA Agricultural Research Service, 8408 Hildreth Road, Cheyenne, WY 82009, U.S.A.
C. v.
COLE
USDA Agricultural Research Service and Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523, U.S.A. L. K.
PORTER
USDA Agricultural Research Service, P.O. Box E, Fort Collins, CO 80522, U.S.A. and D. C. COLEMAN Entomology Department and Institute of Ecology, University of Georgia, Athens, GA 30602, U.S.A. (Accepted
20 February
1987)
Summary-When ‘5N-labehed fertilizer is added to soil. it frequently appears that more indigenous soilN is mineralized-a phenomenon called the “priming effect”. We have made an experimental study of the interaction of added and indigenous N during laboratory incubations with welldefined N concentrations. microbial populations and incubation conditions. Populations of a single species each of bacteria and amoebae were inoculated into propylene-oxide sterilized soil and their growth, respiration and Nmineralization were monitored for 34 days. In one experiment, bacterial numbers doubled (from 17.2 to 36.7 x 10 g-‘) and respired C nearly doubled (from 966 to 1892 pg g-‘) as added N (ammonium sulfate) increased from 0 to 7Opgg-‘. In a second experiment, mineral-N following incubation increased from 2.6 to 55.9flgg- and mineral-N of soil origin increased from 2.6 to 16.3pgg-’ as added N increased from 0 to 100 pg g-‘. However, in the same experiment. the amount of unlabelled soil-N required to dilute Mineralthe ‘sN-Iabelled ammonium to the observed atom% 15N decreased from 72.1 to 41.2uaa-‘. . _ization of soil-N was enhanced (an apparent priming effect) in spite of decreased interaction with unlabelled-N. This “priming effect” resulted from increased net N-mineralization that accompanied increased N-fertilization so long as mineral-N concentrations remained low enough to limit soil microbial activity.
INTRODtXt’tON When fertilizer-N is added to soil, it interacts with indigenous soil-N sometimes increasing the mineralization of the soil-N--the “priming effect” (Barrow, 1960; Broadbent, 1965; Westerman and Kurtz, 1974; Dormaar, 1975). Many mechanisms have been proposed that could produce this result (Parnas, 1976; Jansson and Persson, 1982; Jenkinson et al., 1985). Westerman and Kurtz (1973) and Westerman and Tucker (1974) reported increased uptake by plants of native soil-N with increasing additions of “N-1abeJled urea or oxamide and suggested that increased microbial activity was the likely cause. Fried and Broeshart (1974) contended that root growth increased with added N and that the plants consequently assimilated N from a larger soil volume, thereby diluting the ‘rN. They held that soil-N availability was not affected by added N since the calculated A-values did not increase. Westerman and Kurtz (1974) replied that “None of us have counts of microbes or measures of nitrate and ammonium ions and their sources in the soil at appropriate time periods before and after addition of fertilizer. Lacking some such direct evidence, neither of s.xB.‘PbB
us can offer proof that our speculation was correct and that the other one was wrong.” The purpose of this paper is to provide some direct evidence concerning this phenomenon. We introduced populations consisting of a single species each of a bacterium (Pseudomonas paucimobilis) and an amoeba (Acanthamoeba polyphaga) into sterilized soil. Amoeba were included because they enhance both respiration and N-mineralization (Coleman et al., 1977; Woods et al., 1982). This procedure allowed for accurate measurements of population, respiration and mineral-N. Counts of microbes and measures of respired C and mineral-N at increasing concentrations of N-fertilizer and their relationship to N “priming” were simplified to the greatest possible extent. Because we first sterilized the soil, biological. activity at the time fertilizer-N was added was zero in all flasks. Consequently, all subsequent N transformations were performed by the population whose numbers and activity were measured. Although it may be argued that this approach is not in many senses representative of field conditions, it does effectively isolate the phenomena of interest and allows for sufficient control of experi673
L. E.
674
WOODS ef al.
mental conditions that we were able to directly address the relationship between biological activity and soil N transformations. MATERIALS AND METHODS
Shingle-Renohill sandy clay loam soil was used in the two experiments. This soil is an association of two soils (Shingle, a mesic shallow Ustic Torriotthent and Renohill, a mesic Ustollic Hapiargid), from the USDA-ARS Central Plains Experimental Range (CPER), 50 km northeast of Fort Collins, Colorado. The soil was sieved through 6 mm (quarter-in) hardware cloth in the field, then through a I.0 mm sieve to remove root fragments and stones. In the first experiment, 20g of air-dry soil was weighed into 50 ml Erlenmeyer flasks, wetted to field capacity (15% moisture by wt), incubated for 2 days at 25°C in the dark to establish microbial activity and sterilized by exposure to a saturated atmosphere of propylene oxide for 48 h. For the second experiment, the soil was treated similarly, except that IOOg soil was added to 250 ml Erlenmeyer flasks. Sterilization by this method was found to add about 1600 pg g-’ of glycol-C to a similar soil (Anderson et al., 1978). After sterilization, the flasks were allowed to air-dry at room temperature. Nutrients were added in solution. Inocula and sterile nutrient solutions were mixed and added to the soil to create field capacity moisture (15%). When organisms were not added to a flask, they were replaced by a like quantity of sterile media. Gnotobiotic populations of bacteria or of bacteria and amoebae together were introduced into the sterilized soil. This procedure assured that N entered the bacteria as it was immobilized and provided better estimates of bacterial numbers in soil than those normally obtained. The bacterium chosen for these studies (P. pauc~mobilis) was easily counted on dilution plates and was readily consumed by the amoeba1 species (A. polyphaga). Both organisms were isolated from soil obtained from the CPER (Anderson er al., 1978). Bacteria were grown alone or in combination with amoebae. Before inoculation, bacteria and amoebae were maintained in solution culture (Anderson er a!., 1978). The bacterial inoculum was from a log-log culture (the second 24 h incubation). The amoeba1 inoculum was also from log phase (7 day-old) culture. Inocula provided I x lo6 bacteria and 1.7 x 10’ amoebae g-’ soil. These bacteria (P. paucimobilis) did not excrete ammonium when incubated alone. They metabolize both mineral and organic-N. Amoebae reduce the bacterial biomass and increase the concentration of mineral-N (Woods et al., 1982). In the first experiment, respiration was measured by collecting evolved CO2 in 1 M NaOH and titrating the excess base to the phenol-phthalein end point with standard acid (1 M HCI), after precipitating the carbonate with I M BaCl. In the second experiment respiration was not measured. The contents of each flask were destructively sampled and aliquots of soil were removed for analy ses on each sample date. One g of soil was diluted in 1.0% saline solution and plated in serial dilutions onto nutrient agar plates for bacterial counts. Soil
from each uninoculated flask was placed directly onto nutrient agar to check for recolonization. In the first experiment, there was no growth on any of the samples. In the second experiment, most of the samples had fungi growing on the agar after t wk. This test is much more sensitive than dilution plating and 15N dilution was increased in only two cases. Therefore we believe that there was little microbial contamination of uninoculated soil in either of the experiments. In the first experiment, 5g of wet soil was extracted with 50 ml of 1 M KC1 and in the second, lOOg soil was extracted with 500 ml of 1 M KC1 (Bremner, 1965) for “N and ammonium-N anlayses. Ammonium-N was determined using modified Conway microdiffusion dishes (Stanford et al., 1973). Atom-% lSN was determined with an AEI MS-20 isotope ratio mass spectrometer using the conversion procedures of Porter and O’Deen (1977). Initial nitrate-N values were low (less than 1 pgg-‘) and, since nitrifiers were absent, nitrate concentrations did not change. In the first experiment, we added IOOpggglucose-C and 0,20,70 or 200 pegg-i of ammoniumN in a complete block design with bacteria alone (B) or bacteria plus amoebae (AB). Glucose was added as a C source in addition to propylene oxide products and native soil C in the first experiment. Ammonium sulfate was added as the N source in both experiments. Ammonium-N and bacterial numbers were sampled on days 4, 20 and 34. Amended but uninoculated flasks were sampled on the first sample date in order to evaluate the initial conditions. In the second experiment, we added no glucose because in the first experiment results for 0 and IOO~gg- glucose-C treatments did not differ. We also added 0, 20, 70 or 100 pg g-’ of ammonium-N, enriched to approximately 3.9 atom-% “N (exact values reported below). Two biological treatments were established: uninsulated controls (UC) and amoebae and bacteria together (AB). Both sets of flasks were sampled after 34 days of incubation. The sterile media of UC and the inocula of AB treatments contained equal amounts of labelled ammonium sulfate solution, bacterial media and amoeba1 media. A small amount of unlabelled-N from the media is included in the “N analysis, causing the atom-% ‘IN to increase as the solution becomes more concentrated in ammonium (Table 1). If no N, other than indigenous ammonium-N, diluted the isN-labelled ammonium sulfate, the final atom-% “N can be predicted from the sum of concentration of fertilizer (ammonium sulfate) N multipli~ by its atom-% iSN and the concentration of soil ammonium-N multiplied by its atom-% “N, divided by the sum of the concentrations of soil and fertilizer ammonium-N. The concentration of unlabelled soil-N that contributed to the isotopic dilution of the added labelted-N can be calculated by the formula: X2 = [(CljC2) - IJ(Xl), where XI is the amount of labelled tracer added, X2 is the amount of unlabelled compound, C2 is the atom-% excess of the compound recovered from the mixture and Cl is the atom-% excess of the added label (Hauck and Bremner, 1976). Three replicates were sampled in both experiments and analyses of variance performed. Means separ-
675
N transformations fable I. The effect of added ammonium on the amount and atom-% “N of KCkxtractable ammonium after 34 days. without oraanivw
N added
Fertilizer solution
(JIRR-‘1
Extracted ~rno~~-N (atom-% “M
Predicted
0.374r 0.3650 0.3742
0
Diluting the Extracted lab&d N ammonium-N firr g-v
1I.P
Il.8 11.7
40
3.7572
3.0590 3.0907 2.6045
3.0011
47.5 48.6 53.4
10.8 10.1 21.5
70
3.9109
2.7633 3.4484 3.4120
3.4114
75.5 78.2 7s. I
35.9 10.8 II.9
3.9528
3.6567 3.6704 3.6427
3.5834
97.3
9.3 8.8 9.8
too
‘Mean atom-% ‘%I soi13 0.3711. “Mean i~i~~~ ammonium-N pg 5-l = 11.5.
ations were calculated by Tukey’s Honestly Significant Differences (Kirk, 1968). All reported differences were significant at least at P = 0.05. RESULTS
AND DISCUSSION
Two experiments are described in this paper. The first establishes the growth, N uptake and mineralization patterns for bacteria and amoebae during incubation. The second examines the interaction of indigenous N with added fertilizer-N during similar incubations and in sterile unin~uIat~ soil. Observations on the trophic interactions of the bacteria and amoebae and their relationship to glucose and ammonium sulfate additions have been described by Bryant ef al. (1982), Woods et al. (1982) and Fairbanks Edal. (1984). This paper is limited to the N interactions accompanying the biological processes. Nitrogen flows through these ~pulations can be. visualized as in Pig. I. Ammonium, either from fertilizer or from indigenous sources, is immobilized by the bacteria after inoculation. During growth, bacteria assimilate both organic-N and ammoniumN. Amoebae consume the bacteria and excrete ammonium, which is then either assimilated by the bacteria again, or remains in the soil as ammonium. In this figure there is no Bow from bacteria to mineral-N because this species does not produce
Fig. 1. Nitrogen
transformations in the gnotobiotic populations of bacteria and amoebae.
mineral N (Woods er al., 1982) and no flow from mineral-N to amoebae because these protozoans grow by consuming bacteria and do not consume mineral-N directly (Bryant er al., 1982). Bacteria grow faster than amoebae and therefore incorporate N into their biomass before the amoebae increase enough to affect them when both are introduced simultaneously. As the amoeba1 population increases, bacteria are consumed and N is mineralized (Woods et al., 1982). Because bacteria and amoebae were inoculated into sterilized soil, immobilized N could only enter the ~pulations that were subsequently measured and mineralized N could only come from those same populations. Two factors affect the amount of indigenous soil-N that is ultimately mineralized-the quantity of it immobilized by the bacteria, and the total amount of mineral-N that is produced (Jansson and Persson, 1982). When a readily available source of N, such as ammonium, is added to a soil in which low mineral-N concentrations limit microbial activity, microorganisms will use more of it and less indigenous soil N, as long as N remains limiting. As the N supply increases, more N will be mineralized when C is lost to microbial respiration. If net N minerali~tion increases more than the immobili~tion of indigenous N decreases, then mineralization of indigenous N will increase. Both numbers and activity of P. paucimobilis and A. polyphaga increased with increasing N additions up to 7Opg g-i (Table 2). The soil was low in available N (11.5 pg g-i) and propylene oxide sterilization probably added some C residues (Anderson et al., 1978). Bacteria in the presence of amoebae immobilized up to 82 pg g-l of ammonium-N after 4 days (Table 3). Bacteria alone immobilized up to 96 yg g-‘. but they mineralized N only at the highest N addition (2OOggg-‘) and only between days 20 and 34. Amoebae caused bacterial numbers to decrease, respiration to increase and N mineralization to increase when 20 pig g-i or more of N were added. After 34 days, both 70 and 200 pg g-r N treatments contained measurable mineral N (Table 3). In the uninoculated (UC) treatments of the second experiment, the only soil N that diluted the labelledN was indigenous ammonium, Assuming that only
L. E.
676
WOODS
et al.
Table 2. The effect of N amendments from 0 to 200 pg g-’ on bacteria and amoeba1 numbers and cumulative respiration with bacteria alone (8) or with a mixed population of bacteria and amoebae (AB) on days 4 and 34 of incubation
N Biologicat treatment
(ccgg-‘1
added
B
0
i
ZO
34 Number of amoebati wg-‘)
B B
2:
AB AB AB AB
2. I 4.1 4.4 3.3
2:
4
34
4 34 Cumulatives moiration’
Number of bacteriab w g-l)
&II-‘)
19.8 22.6 53.5 42. I
15.0 17.5 24.9 33. I
E 525 510
1354 1600
17.4 17.5 36.1 33.6
6.1 6.4 9.4 13.0
224 365 536 490
I;: I 892 1795
610
a99
‘HSD (0.05) for number of amoebae = 1.8. ‘For number of bacteria between N additions within a date and biological treatment = 10.2. CFor cumulative respiration between N additions within a date and biological treatment = 84.
Table 3. Tbc e&ct of N amendment on extractable mineral-N, with bacteria alone (B) or with a mixed popufation of bacteria and amoebae CAB), during 34 days of incubation (experiment one) h,‘S
Biological treatment
N added
0*
4 keg-‘)
20
34
B
0
1.9
4.8
4.1
5.4
:: B
20 2:
75.1 25.9 197.1
4.1 4.4 103.6
4.4 3.9 63.0
6.7 5.2 84.9
1.9 25.9 7s. 1 197.1
4.4 6.3 10.2 115.2
5.9 5.9 14.7 121.2
6.1 6.3 32.2 127.5
AB AB AB AB
2:
‘Measured in unin~uiated Raskson day 4. not included in statistical analyses. HSD (0.05) = 5.9 between N additions for each date and biological treatment, 5.4 between dates for each N addition and biological treatment, 4.5 between biological treatments for each date and N addition.
the 1 I .5 pg g-l of soil ammonium-N contributed to the dilution of tagged ammonium, the final atom-% r5N of ammonium-N would be as shown in Table 1. These values were very close to the measured “N dilutions in all but two cases (Table 1 and Fig. 2). Jansson (1958) also found that the “N abundance of
mineral N was not diluted in sterile soiI and concluded that ammonium did not interact with other forms of soil N in the absence of organisms. In the inoculated (AB) treatments of this experiment, organic soil N also diluted the labelled-N. When populations were N-limited (less than that of 70 pg g-l), the amount of organic-N that contributed to the dilution of the label decreased as added N increased (Fig. 2). That is, microbes used less indigenous organic N. However, their use of organic-N decreased less than net mineralization increased (Fig. 4). The microbial use of soil N decreased by 3 I pg g-‘; net mineralization increased by 53 pg g-‘. Mineralization of soil-N increased by 14pgg-’ because of the increase in net mineralization which accompanied the increase in microbial activity (see Tables 2 and 3 and Fig. 3). Thus there was a “priming effect” even though microbial assimilation of indigenous N actually decreased (Table 4 and Fig. 4). When 70 pg g-’ of ammonium-N or more were added to this soil, microbial populations and respiration were no longer N-limited (Table 2). When the
r SO
0 o a
Tom1 From httilimr From soit
/ ./
z i
2oNo WQWliunS
0
.-*-*I t
I
I
40
70
ioo
Added NH:-N
trg
Q-’
1
Soil)
of soif N that diluted ‘sN-labelled added N after incubation (34 days, ZS’C) with bacteria and amoebae or with no inoculum. Bars at ends of lines represent HSDs (0.05). Fig. 2. Concentrations
Added NH;-N
+g
Q-’
So&f
Fig. 3. Ammonium-N concentrations after jR~bation (34 days, 25°C) with bacteria and amoebae and its sources. Bars at ends of lines represent HSDs (0.05).
N transformations
.-; 0
40
Added NH:-N
I 70
I 100
(
(pg g-’ soioil)
Fig. 4. Soil-N appearing as ammonium-N after incubation contrasted with soil-N diluting the 15N-labelled added N. Bars at ends of lines represent HSDs (0.0s).
populations were not N-limited (70 and 100 pg g-’ of N), an average of 43 pg g-t of soil N contributed to the dilution of the added label. Of this, it appears that 1I.5 pg g-i were ammonium (the amount of ammonium present in the soil before amendment) and the remaining 31.5 pg g-i were soil organic-N. Fried and Broeshart (1974) Jenkinson et al. (1985) and others have pointed out that increased dilution of labelled-N in plants results when roots explore a larger soil volume containing equally available N. While this no doubt can occur in field experiments, we found increased dilution of labelled mineral-N without plants and this dilution increased while microbial uptake of soil-N decreased. Stimulated mineralization of soil-N did not mean that more indigenous soil-N was utilized by the microorganisms. Jenkinson et al. (1985) list several sets of circumstances that might give rise to both “apparent” and “real” added nitrogen interactions (ANI) or priming. They point out that “apparent” priming can result from “displacement reactions with the soil microbial biomass”. This would come about by rapid incorporation of labelled ammonium into microbial cells and into their organic molecules via enzyme-mediated reactions with subsequent release of unlabelled-N into the inorganic pool. Without any change in the biomass, these processes would cause dilution of the tagged N in the soil inorganic-N pool. This did not occur in our experiments because we began with sterile soil, which had no microbial biomass when the fertilizer was added. Microbial biomass increased for the first few days, immobilizing a large portion of the inorganic-N. A second potential source of apparent AN1 is via
677
pool substitution caused by immobilization. Considerable immobilization occurs even in soils experiencing net mineralization. Substitution results in an AN1 directly proportional to the amount of immobilization, regardless of the amount of mineralization. Anything that increases immobilization will increase the ANI. In our experiments, there was substantial immobilization which differed between N-amendment levels. Immobilization and mineralization rates did not remain constant throughout the incubation because we introduced fresh inoculum into soils containing rich sources of both C and N. Even in the presence of amoebae, bacteria assimilated up to 94 pg N g-i after 4 days (Table 3). In other experiments with the same soil and microorganisms, 75pgg-’ of mineral-N were assimilated into the bacterial biomass in 64 h (Elliott et al., 1983). Immobilized tagged ammonium was remineralized during the incubation. Thus, while pool substitution can cause an “apparent” AN1 because of immobilization, even if mineralization of untagged organic-N remains constant, the conditions assumed by Jenkinson er al. (1985) in deriving this relationship did not apply to our experiments. A “real” ANI in the absence of plants can occur when soils “contain fresh organic residues whose C:N ratios are wide enough that the N requirements of the population cannot be met from inorganic reserves”. In our experiments glycol residues and killed microorganisms provided a source of fresh organic matter whose C:N ratio was no doubt wide. In the first experiment, respiration and bacterial numbers increased with increased N amendments indicating that the populations were limited by low mineral-N concentrations when less than 70 /Ig g-i were added, thus allowing for a “real” priming effect. Above this amount, populations were no longer N-limited. There was a priming effect in response to N-amendment as long as soil mineral-N limited microbial activity, as suggested by Jenkinson et al. (1985). However, this priming effect increased while the amount of organic-N diluting the tagged N decreased (Fig. 4). Calculations of the amount of unlabelled N required to dilute the added tagged N are analogous to the “A-value” calculation of Fried and Dean (1952) except that the measurements were made in KCIextractable mineral-N instead of in plant-N. This value decreased with increasing N additions as long as N concentrations limited microbial activity (Tables 2, 4 and Fig. 4). This decrease would violate the assumption that A-values do not change when fertilizer additions increase. The assumption is valid only when mineral-N concentrations are sufficiently large that they do not limit soil microbial activity. This
Table 4. The c&t of added ammonium on net mineralization, atom-% ‘?J and the amount of soil N needed to produce the observed “N, dilution, following 34 days of incubation, with bacteria (P. prucimobifis) and amoebae (A. polyphago) Added N Mineral N (JC3i3-‘) 0 40 70 100
2.6 II.0 41.3 55.9
Added N Mineral N (atom-% “N) 3.6297 3.8048 3.8867
1.5339 2.4820 2.8601
Soil N Bacterial uptake 01BB“) 72. I 43.9 41.2
Mineralized 2.6 7.1 IS.9 16.3
L. E.
678
WOODS et al.
situation is likely in agricultural soils, which are disturbed by tillage operations and therefore do not have constant C inputs from root exudation and turnover. “N dilution did not increase further between the two highest N additions (70 and lOO~gg_‘) and there was no further increase in soil-N mineralization (Fig. 4). In incubations with a single species of soil bacterium and protozoan, there was an apparent “priming effect” of added ammonium, which was not, however, accompanied by increased microbial uptake of indigenous soil-N. As N additions increased from 0 to IOOfigg-’ the population used 31 pgg-’ less soil-N, but mineralized 52 pgg-’ more N and I4 pg g-’ more soil N. This increased mineralization of soil-N was accompanied by increased microbial activity and increased net N mineralization. Priming resulted as long as N concentrations remained low enough to limit microbial activity, even though dilution by soil organic-N decreased. In the exchange between Fried and Broeshart (1974) and Westerman and Kurtz (1974) both sides are correct depending on the N status of the microbial population. When microbial needs for mineral-N were satisfied, further increases in N-amendment did not alter the mineralization of soil organic-N or the contribution of soil organic-N to the dilution of tagged mineral-N. When soil microbial populations were limited by low concentrations of mineral-N, a “real” priming effect accompanied the increased microbial activity that resulted from mineral-N additions. This priming effect, however, was not accompanied by an increase in the use of soil organicN by the population. It occurred because net mineralization increased more than the use of soil organic N decreased. Ac/cnowledgemen~s-Support for this research was provided by a National Science Foundation to Colorado State University. isN and analytical assistance were provided by USDA-Agricultural Research Service. We thank Ric Morgan, Jan Gumsey and Bill O’Decn for valuable laboratory assistance.
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Barrow N. J. (1960) The effects of varying the nitrogen, sulphur and phosphorus content of organic matter on its decomposition. Australian Journal Agricultural Research 11.317-330. Bremner J. M. (1965) Inorganic forms of nitrogen. In Merho& of Soil Analysis, Part 2, Chemical and Microbiological Properties (C. A. Black, Ed.). pp. 1179-1237. American Society of Agronomy, Madison. Broadbent F. E. (1965) Effect of fertilizer nitrogen on the release of soil nitrogen. Soil Science Sociery of America Proceedings 29, 692652.
Bryant R. J., Woods L. E., Coleman D. C., Fairbanks B. C., McClellan J. F. and Cole C. V. (1982) Interactions of bacterial and amoeba1 populations in soil microcosms with fluctuating moisture content. Applied and Enoironmenral Microbiology 43, 747-752.
Coleman D. C.. Cole C. V., Anderson R. V., Blaha M., Camnion M. K.. Clarholm M.. Elliott E. T.. Hunt H. W.. Schaefer B. and Sinclair J: (1977) An analysis of rhirosphere-saprophage interactions in terrestrial ecosystems. Ecological Bulletin, Stockholm 25, 299-309. Dormaar J. F. (1975) Susceptibility of organic matter of chemozemic Ah horizons to biological decomposition. Canadian Journal of Soil Science 55, 473-480.
Elliott E. T., Cole C. V., Fairbanks B. C., Woods L. E., Bryant R. J. and Coleman, D. C. (1983) Short-term bacterial growth, nutrient uptake and ATP turnover in sterilized, inoculated and C-amended soil: the influence of N availability. Soil Biology & Biochemistry _lS, 35-91. Fairbanks B. C., Woods L. E., Bryant R. J.. Elliott E. T.. Cole C. V. and Coleman D. C. (1984) Limitations of ATP estimates of microbial biomass. Soil Biology & Biochemistry 16, W-558.
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Fried M. and Dean L. A. (1952) A concept concerning the measurement of available soil nutrients. Soil Science 73, 263-271. Hauck R. D. and Bremner J. M. (1976) Use of tracers for soil and fertilizor nitrogen research. Advances in Agronomy 2%. 219-266.
Jansson S. L. (1958) Tracer studies on nitrogen transformations in soil with mineralization-immobilization
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Kirk R. E. (1968) Experimental Design: Procedures for the Behavioral Sciences. Brooks/Cole Publications Company, Belmont. McGill W. B., Shields J. A. and Paul E. A. (1975) Relation between carbon and nitrogen turnover in soil organic fractions of microbial origin. Soil Biology & Biochemisfry 7, 57-63.
Pamas H. (1976) A theoretical explanation of the priming effect based on microbial growth with two limiting substrates. Soil Biology & Biochemistry 8, 139-144. Porter L. K. and O’Deen W. A. (1977) Apparatus for preparing nitrogen from ammonium chloride for nitrogen-15 determinations. Analytical Chemistry 49, 514-516.
Stanford G.. Carter J. N., Simpson E. and Schwaninger D. E. (1973) Nitrate determination by a modified Conway microdXusion method. Journal of Oficial Agricultural Chemists 56, 1365-1368.
_
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Westerman R. L. and Kurtz L. T. (1973) Priming effect of “N-labelled fertilizers on soil nitrogen in fieid experiments. Soil Science Society of America Proceedings 37, 72%727.
Westerman R. L. and Kurtz L. T. (1974) Reply to Fried and Broeshart. Soil Science Society of America Proceedings 38, 858-859.
Westerman R. L. and Tucker T. C. (1974) Effect of salts and salts plus nitrogen-l5-labelled ammonium chloride on mineralization of soil nitrogen nitritication and immobilization. Soil Science Sociery of America Proceedings 38, 602-605.
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Soil Bid. Biochcm. Vol. 19. No. 6. pp. 673-618. 1987 Printed in Great Britain
TRANSFORMATIONS OF ADDED AND INDIGENOUS NITROGEN IN GNOTOBIOTIC SOIL: A COMMENT ON THE PRIMING EFFECT L. E.
b%ODS
USDA Agricultural Research Service, 8408 Hildreth Road, Cheyenne, WY 82009, U.S.A.
C. v.
COLE
USDA Agricultural Research Service and Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523, U.S.A. L. K.
PORTER
USDA Agricultural Research Service, P.O. Box E, Fort Collins, CO 80522, U.S.A. and D. C. COLEMAN Entomology Department and Institute of Ecology, University of Georgia, Athens, GA 30602, U.S.A. (Accepted
20 February
1987)
Summary-When ‘5N-labehed fertilizer is added to soil. it frequently appears that more indigenous soilN is mineralized-a phenomenon called the “priming effect”. We have made an experimental study of the interaction of added and indigenous N during laboratory incubations with welldefined N concentrations. microbial populations and incubation conditions. Populations of a single species each of bacteria and amoebae were inoculated into propylene-oxide sterilized soil and their growth, respiration and Nmineralization were monitored for 34 days. In one experiment, bacterial numbers doubled (from 17.2 to 36.7 x 10 g-‘) and respired C nearly doubled (from 966 to 1892 pg g-‘) as added N (ammonium sulfate) increased from 0 to 7Opgg-‘. In a second experiment, mineral-N following incubation increased from 2.6 to 55.9flgg- and mineral-N of soil origin increased from 2.6 to 16.3pgg-’ as added N increased from 0 to 100 pg g-‘. However, in the same experiment. the amount of unlabelled soil-N required to dilute Mineralthe ‘sN-Iabelled ammonium to the observed atom% 15N decreased from 72.1 to 41.2uaa-‘. . _ization of soil-N was enhanced (an apparent priming effect) in spite of decreased interaction with unlabelled-N. This “priming effect” resulted from increased net N-mineralization that accompanied increased N-fertilization so long as mineral-N concentrations remained low enough to limit soil microbial activity.
INTRODtXt’tON When fertilizer-N is added to soil, it interacts with indigenous soil-N sometimes increasing the mineralization of the soil-N--the “priming effect” (Barrow, 1960; Broadbent, 1965; Westerman and Kurtz, 1974; Dormaar, 1975). Many mechanisms have been proposed that could produce this result (Parnas, 1976; Jansson and Persson, 1982; Jenkinson et al., 1985). Westerman and Kurtz (1973) and Westerman and Tucker (1974) reported increased uptake by plants of native soil-N with increasing additions of “N-1abeJled urea or oxamide and suggested that increased microbial activity was the likely cause. Fried and Broeshart (1974) contended that root growth increased with added N and that the plants consequently assimilated N from a larger soil volume, thereby diluting the ‘rN. They held that soil-N availability was not affected by added N since the calculated A-values did not increase. Westerman and Kurtz (1974) replied that “None of us have counts of microbes or measures of nitrate and ammonium ions and their sources in the soil at appropriate time periods before and after addition of fertilizer. Lacking some such direct evidence, neither of s.xB.‘PbB
us can offer proof that our speculation was correct and that the other one was wrong.” The purpose of this paper is to provide some direct evidence concerning this phenomenon. We introduced populations consisting of a single species each of a bacterium (Pseudomonas paucimobilis) and an amoeba (Acanthamoeba polyphaga) into sterilized soil. Amoeba were included because they enhance both respiration and N-mineralization (Coleman et al., 1977; Woods et al., 1982). This procedure allowed for accurate measurements of population, respiration and mineral-N. Counts of microbes and measures of respired C and mineral-N at increasing concentrations of N-fertilizer and their relationship to N “priming” were simplified to the greatest possible extent. Because we first sterilized the soil, biological. activity at the time fertilizer-N was added was zero in all flasks. Consequently, all subsequent N transformations were performed by the population whose numbers and activity were measured. Although it may be argued that this approach is not in many senses representative of field conditions, it does effectively isolate the phenomena of interest and allows for sufficient control of experi673
L. E.
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WOODS ef al.
mental conditions that we were able to directly address the relationship between biological activity and soil N transformations. MATERIALS AND METHODS
Shingle-Renohill sandy clay loam soil was used in the two experiments. This soil is an association of two soils (Shingle, a mesic shallow Ustic Torriotthent and Renohill, a mesic Ustollic Hapiargid), from the USDA-ARS Central Plains Experimental Range (CPER), 50 km northeast of Fort Collins, Colorado. The soil was sieved through 6 mm (quarter-in) hardware cloth in the field, then through a I.0 mm sieve to remove root fragments and stones. In the first experiment, 20g of air-dry soil was weighed into 50 ml Erlenmeyer flasks, wetted to field capacity (15% moisture by wt), incubated for 2 days at 25°C in the dark to establish microbial activity and sterilized by exposure to a saturated atmosphere of propylene oxide for 48 h. For the second experiment, the soil was treated similarly, except that IOOg soil was added to 250 ml Erlenmeyer flasks. Sterilization by this method was found to add about 1600 pg g-’ of glycol-C to a similar soil (Anderson et al., 1978). After sterilization, the flasks were allowed to air-dry at room temperature. Nutrients were added in solution. Inocula and sterile nutrient solutions were mixed and added to the soil to create field capacity moisture (15%). When organisms were not added to a flask, they were replaced by a like quantity of sterile media. Gnotobiotic populations of bacteria or of bacteria and amoebae together were introduced into the sterilized soil. This procedure assured that N entered the bacteria as it was immobilized and provided better estimates of bacterial numbers in soil than those normally obtained. The bacterium chosen for these studies (P. pauc~mobilis) was easily counted on dilution plates and was readily consumed by the amoeba1 species (A. polyphaga). Both organisms were isolated from soil obtained from the CPER (Anderson er al., 1978). Bacteria were grown alone or in combination with amoebae. Before inoculation, bacteria and amoebae were maintained in solution culture (Anderson er a!., 1978). The bacterial inoculum was from a log-log culture (the second 24 h incubation). The amoeba1 inoculum was also from log phase (7 day-old) culture. Inocula provided I x lo6 bacteria and 1.7 x 10’ amoebae g-’ soil. These bacteria (P. paucimobilis) did not excrete ammonium when incubated alone. They metabolize both mineral and organic-N. Amoebae reduce the bacterial biomass and increase the concentration of mineral-N (Woods et al., 1982). In the first experiment, respiration was measured by collecting evolved CO2 in 1 M NaOH and titrating the excess base to the phenol-phthalein end point with standard acid (1 M HCI), after precipitating the carbonate with I M BaCl. In the second experiment respiration was not measured. The contents of each flask were destructively sampled and aliquots of soil were removed for analy ses on each sample date. One g of soil was diluted in 1.0% saline solution and plated in serial dilutions onto nutrient agar plates for bacterial counts. Soil
from each uninoculated flask was placed directly onto nutrient agar to check for recolonization. In the first experiment, there was no growth on any of the samples. In the second experiment, most of the samples had fungi growing on the agar after t wk. This test is much more sensitive than dilution plating and 15N dilution was increased in only two cases. Therefore we believe that there was little microbial contamination of uninoculated soil in either of the experiments. In the first experiment, 5g of wet soil was extracted with 50 ml of 1 M KC1 and in the second, lOOg soil was extracted with 500 ml of 1 M KC1 (Bremner, 1965) for “N and ammonium-N anlayses. Ammonium-N was determined using modified Conway microdiffusion dishes (Stanford et al., 1973). Atom-% lSN was determined with an AEI MS-20 isotope ratio mass spectrometer using the conversion procedures of Porter and O’Deen (1977). Initial nitrate-N values were low (less than 1 pgg-‘) and, since nitrifiers were absent, nitrate concentrations did not change. In the first experiment, we added IOOpggglucose-C and 0,20,70 or 200 pegg-i of ammoniumN in a complete block design with bacteria alone (B) or bacteria plus amoebae (AB). Glucose was added as a C source in addition to propylene oxide products and native soil C in the first experiment. Ammonium sulfate was added as the N source in both experiments. Ammonium-N and bacterial numbers were sampled on days 4, 20 and 34. Amended but uninoculated flasks were sampled on the first sample date in order to evaluate the initial conditions. In the second experiment, we added no glucose because in the first experiment results for 0 and IOO~gg- glucose-C treatments did not differ. We also added 0, 20, 70 or 100 pg g-’ of ammonium-N, enriched to approximately 3.9 atom-% “N (exact values reported below). Two biological treatments were established: uninsulated controls (UC) and amoebae and bacteria together (AB). Both sets of flasks were sampled after 34 days of incubation. The sterile media of UC and the inocula of AB treatments contained equal amounts of labelled ammonium sulfate solution, bacterial media and amoeba1 media. A small amount of unlabelled-N from the media is included in the “N analysis, causing the atom-% ‘IN to increase as the solution becomes more concentrated in ammonium (Table 1). If no N, other than indigenous ammonium-N, diluted the isN-labelled ammonium sulfate, the final atom-% “N can be predicted from the sum of concentration of fertilizer (ammonium sulfate) N multipli~ by its atom-% iSN and the concentration of soil ammonium-N multiplied by its atom-% “N, divided by the sum of the concentrations of soil and fertilizer ammonium-N. The concentration of unlabelled soil-N that contributed to the isotopic dilution of the added labelted-N can be calculated by the formula: X2 = [(CljC2) - IJ(Xl), where XI is the amount of labelled tracer added, X2 is the amount of unlabelled compound, C2 is the atom-% excess of the compound recovered from the mixture and Cl is the atom-% excess of the added label (Hauck and Bremner, 1976). Three replicates were sampled in both experiments and analyses of variance performed. Means separ-
675
N transformations fable I. The effect of added ammonium on the amount and atom-% “N of KCkxtractable ammonium after 34 days. without oraanivw
N added
Fertilizer solution
(JIRR-‘1
Extracted ~rno~~-N (atom-% “M
Predicted
0.374r 0.3650 0.3742
0
Diluting the Extracted lab&d N ammonium-N firr g-v
1I.P
Il.8 11.7
40
3.7572
3.0590 3.0907 2.6045
3.0011
47.5 48.6 53.4
10.8 10.1 21.5
70
3.9109
2.7633 3.4484 3.4120
3.4114
75.5 78.2 7s. I
35.9 10.8 II.9
3.9528
3.6567 3.6704 3.6427
3.5834
97.3
9.3 8.8 9.8
too
‘Mean atom-% ‘%I soi13 0.3711. “Mean i~i~~~ ammonium-N pg 5-l = 11.5.
ations were calculated by Tukey’s Honestly Significant Differences (Kirk, 1968). All reported differences were significant at least at P = 0.05. RESULTS
AND DISCUSSION
Two experiments are described in this paper. The first establishes the growth, N uptake and mineralization patterns for bacteria and amoebae during incubation. The second examines the interaction of indigenous N with added fertilizer-N during similar incubations and in sterile unin~uIat~ soil. Observations on the trophic interactions of the bacteria and amoebae and their relationship to glucose and ammonium sulfate additions have been described by Bryant ef al. (1982), Woods et al. (1982) and Fairbanks Edal. (1984). This paper is limited to the N interactions accompanying the biological processes. Nitrogen flows through these ~pulations can be. visualized as in Pig. I. Ammonium, either from fertilizer or from indigenous sources, is immobilized by the bacteria after inoculation. During growth, bacteria assimilate both organic-N and ammoniumN. Amoebae consume the bacteria and excrete ammonium, which is then either assimilated by the bacteria again, or remains in the soil as ammonium. In this figure there is no Bow from bacteria to mineral-N because this species does not produce
Fig. 1. Nitrogen
transformations in the gnotobiotic populations of bacteria and amoebae.
mineral N (Woods er al., 1982) and no flow from mineral-N to amoebae because these protozoans grow by consuming bacteria and do not consume mineral-N directly (Bryant er al., 1982). Bacteria grow faster than amoebae and therefore incorporate N into their biomass before the amoebae increase enough to affect them when both are introduced simultaneously. As the amoeba1 population increases, bacteria are consumed and N is mineralized (Woods et al., 1982). Because bacteria and amoebae were inoculated into sterilized soil, immobilized N could only enter the ~pulations that were subsequently measured and mineralized N could only come from those same populations. Two factors affect the amount of indigenous soil-N that is ultimately mineralized-the quantity of it immobilized by the bacteria, and the total amount of mineral-N that is produced (Jansson and Persson, 1982). When a readily available source of N, such as ammonium, is added to a soil in which low mineral-N concentrations limit microbial activity, microorganisms will use more of it and less indigenous soil N, as long as N remains limiting. As the N supply increases, more N will be mineralized when C is lost to microbial respiration. If net N minerali~tion increases more than the immobili~tion of indigenous N decreases, then mineralization of indigenous N will increase. Both numbers and activity of P. paucimobilis and A. polyphaga increased with increasing N additions up to 7Opg g-i (Table 2). The soil was low in available N (11.5 pg g-i) and propylene oxide sterilization probably added some C residues (Anderson et al., 1978). Bacteria in the presence of amoebae immobilized up to 82 pg g-l of ammonium-N after 4 days (Table 3). Bacteria alone immobilized up to 96 yg g-‘. but they mineralized N only at the highest N addition (2OOggg-‘) and only between days 20 and 34. Amoebae caused bacterial numbers to decrease, respiration to increase and N mineralization to increase when 20 pig g-i or more of N were added. After 34 days, both 70 and 200 pg g-r N treatments contained measurable mineral N (Table 3). In the uninoculated (UC) treatments of the second experiment, the only soil N that diluted the labelledN was indigenous ammonium, Assuming that only
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676
WOODS
et al.
Table 2. The effect of N amendments from 0 to 200 pg g-’ on bacteria and amoeba1 numbers and cumulative respiration with bacteria alone (8) or with a mixed population of bacteria and amoebae (AB) on days 4 and 34 of incubation
N Biologicat treatment
(ccgg-‘1
added
B
0
i
ZO
34 Number of amoebati wg-‘)
B B
2:
AB AB AB AB
2. I 4.1 4.4 3.3
2:
4
34
4 34 Cumulatives moiration’
Number of bacteriab w g-l)
&II-‘)
19.8 22.6 53.5 42. I
15.0 17.5 24.9 33. I
E 525 510
1354 1600
17.4 17.5 36.1 33.6
6.1 6.4 9.4 13.0
224 365 536 490
I;: I 892 1795
610
a99
‘HSD (0.05) for number of amoebae = 1.8. ‘For number of bacteria between N additions within a date and biological treatment = 10.2. CFor cumulative respiration between N additions within a date and biological treatment = 84.
Table 3. Tbc e&ct of N amendment on extractable mineral-N, with bacteria alone (B) or with a mixed popufation of bacteria and amoebae CAB), during 34 days of incubation (experiment one) h,‘S
Biological treatment
N added
0*
4 keg-‘)
20
34
B
0
1.9
4.8
4.1
5.4
:: B
20 2:
75.1 25.9 197.1
4.1 4.4 103.6
4.4 3.9 63.0
6.7 5.2 84.9
1.9 25.9 7s. 1 197.1
4.4 6.3 10.2 115.2
5.9 5.9 14.7 121.2
6.1 6.3 32.2 127.5
AB AB AB AB
2:
‘Measured in unin~uiated Raskson day 4. not included in statistical analyses. HSD (0.05) = 5.9 between N additions for each date and biological treatment, 5.4 between dates for each N addition and biological treatment, 4.5 between biological treatments for each date and N addition.
the 1 I .5 pg g-l of soil ammonium-N contributed to the dilution of tagged ammonium, the final atom-% r5N of ammonium-N would be as shown in Table 1. These values were very close to the measured “N dilutions in all but two cases (Table 1 and Fig. 2). Jansson (1958) also found that the “N abundance of
mineral N was not diluted in sterile soiI and concluded that ammonium did not interact with other forms of soil N in the absence of organisms. In the inoculated (AB) treatments of this experiment, organic soil N also diluted the labelled-N. When populations were N-limited (less than that of 70 pg g-l), the amount of organic-N that contributed to the dilution of the label decreased as added N increased (Fig. 2). That is, microbes used less indigenous organic N. However, their use of organic-N decreased less than net mineralization increased (Fig. 4). The microbial use of soil N decreased by 3 I pg g-‘; net mineralization increased by 53 pg g-‘. Mineralization of soil-N increased by 14pgg-’ because of the increase in net mineralization which accompanied the increase in microbial activity (see Tables 2 and 3 and Fig. 3). Thus there was a “priming effect” even though microbial assimilation of indigenous N actually decreased (Table 4 and Fig. 4). When 70 pg g-’ of ammonium-N or more were added to this soil, microbial populations and respiration were no longer N-limited (Table 2). When the
r SO
0 o a
Tom1 From httilimr From soit
/ ./
z i
2oNo WQWliunS
0
.-*-*I t
I
I
40
70
ioo
Added NH:-N
trg
Q-’
1
Soil)
of soif N that diluted ‘sN-labelled added N after incubation (34 days, ZS’C) with bacteria and amoebae or with no inoculum. Bars at ends of lines represent HSDs (0.05). Fig. 2. Concentrations
Added NH;-N
+g
Q-’
So&f
Fig. 3. Ammonium-N concentrations after jR~bation (34 days, 25°C) with bacteria and amoebae and its sources. Bars at ends of lines represent HSDs (0.05).
N transformations
.-; 0
40
Added NH:-N
I 70
I 100
(
(pg g-’ soioil)
Fig. 4. Soil-N appearing as ammonium-N after incubation contrasted with soil-N diluting the 15N-labelled added N. Bars at ends of lines represent HSDs (0.0s).
populations were not N-limited (70 and 100 pg g-’ of N), an average of 43 pg g-t of soil N contributed to the dilution of the added label. Of this, it appears that 1I.5 pg g-i were ammonium (the amount of ammonium present in the soil before amendment) and the remaining 31.5 pg g-i were soil organic-N. Fried and Broeshart (1974) Jenkinson et al. (1985) and others have pointed out that increased dilution of labelled-N in plants results when roots explore a larger soil volume containing equally available N. While this no doubt can occur in field experiments, we found increased dilution of labelled mineral-N without plants and this dilution increased while microbial uptake of soil-N decreased. Stimulated mineralization of soil-N did not mean that more indigenous soil-N was utilized by the microorganisms. Jenkinson et al. (1985) list several sets of circumstances that might give rise to both “apparent” and “real” added nitrogen interactions (ANI) or priming. They point out that “apparent” priming can result from “displacement reactions with the soil microbial biomass”. This would come about by rapid incorporation of labelled ammonium into microbial cells and into their organic molecules via enzyme-mediated reactions with subsequent release of unlabelled-N into the inorganic pool. Without any change in the biomass, these processes would cause dilution of the tagged N in the soil inorganic-N pool. This did not occur in our experiments because we began with sterile soil, which had no microbial biomass when the fertilizer was added. Microbial biomass increased for the first few days, immobilizing a large portion of the inorganic-N. A second potential source of apparent AN1 is via
677
pool substitution caused by immobilization. Considerable immobilization occurs even in soils experiencing net mineralization. Substitution results in an AN1 directly proportional to the amount of immobilization, regardless of the amount of mineralization. Anything that increases immobilization will increase the ANI. In our experiments, there was substantial immobilization which differed between N-amendment levels. Immobilization and mineralization rates did not remain constant throughout the incubation because we introduced fresh inoculum into soils containing rich sources of both C and N. Even in the presence of amoebae, bacteria assimilated up to 94 pg N g-i after 4 days (Table 3). In other experiments with the same soil and microorganisms, 75pgg-’ of mineral-N were assimilated into the bacterial biomass in 64 h (Elliott et al., 1983). Immobilized tagged ammonium was remineralized during the incubation. Thus, while pool substitution can cause an “apparent” AN1 because of immobilization, even if mineralization of untagged organic-N remains constant, the conditions assumed by Jenkinson er al. (1985) in deriving this relationship did not apply to our experiments. A “real” ANI in the absence of plants can occur when soils “contain fresh organic residues whose C:N ratios are wide enough that the N requirements of the population cannot be met from inorganic reserves”. In our experiments glycol residues and killed microorganisms provided a source of fresh organic matter whose C:N ratio was no doubt wide. In the first experiment, respiration and bacterial numbers increased with increased N amendments indicating that the populations were limited by low mineral-N concentrations when less than 70 /Ig g-i were added, thus allowing for a “real” priming effect. Above this amount, populations were no longer N-limited. There was a priming effect in response to N-amendment as long as soil mineral-N limited microbial activity, as suggested by Jenkinson et al. (1985). However, this priming effect increased while the amount of organic-N diluting the tagged N decreased (Fig. 4). Calculations of the amount of unlabelled N required to dilute the added tagged N are analogous to the “A-value” calculation of Fried and Dean (1952) except that the measurements were made in KCIextractable mineral-N instead of in plant-N. This value decreased with increasing N additions as long as N concentrations limited microbial activity (Tables 2, 4 and Fig. 4). This decrease would violate the assumption that A-values do not change when fertilizer additions increase. The assumption is valid only when mineral-N concentrations are sufficiently large that they do not limit soil microbial activity. This
Table 4. The c&t of added ammonium on net mineralization, atom-% ‘?J and the amount of soil N needed to produce the observed “N, dilution, following 34 days of incubation, with bacteria (P. prucimobifis) and amoebae (A. polyphago) Added N Mineral N (JC3i3-‘) 0 40 70 100
2.6 II.0 41.3 55.9
Added N Mineral N (atom-% “N) 3.6297 3.8048 3.8867
1.5339 2.4820 2.8601
Soil N Bacterial uptake 01BB“) 72. I 43.9 41.2
Mineralized 2.6 7.1 IS.9 16.3
L. E.
678
WOODS et al.
situation is likely in agricultural soils, which are disturbed by tillage operations and therefore do not have constant C inputs from root exudation and turnover. “N dilution did not increase further between the two highest N additions (70 and lOO~gg_‘) and there was no further increase in soil-N mineralization (Fig. 4). In incubations with a single species of soil bacterium and protozoan, there was an apparent “priming effect” of added ammonium, which was not, however, accompanied by increased microbial uptake of indigenous soil-N. As N additions increased from 0 to IOOfigg-’ the population used 31 pgg-’ less soil-N, but mineralized 52 pgg-’ more N and I4 pg g-’ more soil N. This increased mineralization of soil-N was accompanied by increased microbial activity and increased net N mineralization. Priming resulted as long as N concentrations remained low enough to limit microbial activity, even though dilution by soil organic-N decreased. In the exchange between Fried and Broeshart (1974) and Westerman and Kurtz (1974) both sides are correct depending on the N status of the microbial population. When microbial needs for mineral-N were satisfied, further increases in N-amendment did not alter the mineralization of soil organic-N or the contribution of soil organic-N to the dilution of tagged mineral-N. When soil microbial populations were limited by low concentrations of mineral-N, a “real” priming effect accompanied the increased microbial activity that resulted from mineral-N additions. This priming effect, however, was not accompanied by an increase in the use of soil organicN by the population. It occurred because net mineralization increased more than the use of soil organic N decreased. Ac/cnowledgemen~s-Support for this research was provided by a National Science Foundation to Colorado State University. isN and analytical assistance were provided by USDA-Agricultural Research Service. We thank Ric Morgan, Jan Gumsey and Bill O’Decn for valuable laboratory assistance.
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_
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