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Soil Science Society of America Journal 65:1430-1436 (2001)
© 2001 Soil Science Society of America

DIVISION S-4 - SOIL FERTILITY & PLANT NUTRITION

Nitrogen-15 Recovery in Soil Incubated with Potassium Nitrate and Clover Residues

Anne W. Muriuki*,a, Larry D. Kingb and Richard J. Volkb

a Kenya Agricultural Research Inst., P.O. Box 57811, Nairobi, Kenya
b Dep. of Soil Science, North Carolina State University, Raleigh, NC 27695-7619

* Corresponding author (AWMuriuki{at}kari.org)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
In the southeastern USA, legumes are used as green manure to meet crop N requirements of a following crop, but recovery is usually lower than from conventional fertilizers. We conducted a laboratory study for 26 wk under aerobic conditions to monitor recovery of 15N-labeled KNO3 (fertilizer) and crimson clover (Trifolium incarnatum L.) residues (clover) in organic, inorganic, and microbial biomass N pools. Volatilization of NH3 from decomposing clover residues was monitored for 12 wk. Three hundred–gram samples of a Typic Kanhapludult soil were amended with N (0.051 mg N kg-1 dry soil in fertilizer and 0.049 mg N kg-1 dry soil in clover). A control with no N was also included. Although inorganic N (NH4, NO2, and NO3) accumulated throughout (fertilizer > clover > control), the rate of accumulation did not differ among treatments. Organic and microbial biomass N concentration did not differ among treatments, but applied N recovery in microbial biomass was greater in clover than fertilizer (P < 0.05) throughout. Ammonia volatilized was negligible. After 26 wk, applied N recovered in soil inorganic N was 66% for fertilizer and 40% for clover; in soil organic N, 18% for fertilizer and 50% for clover; and in microbial biomass N, 0.75% for fertilizer and 1.5% for clover. Applied N presumed denitrified was 16% in fertilizer and 10% in clover. We concluded that clover green manure can meet the N requirements of a following crop from the time of emergence in the southeastern USA.

Abbreviations: ANI, added N interaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
THE ADVERSE EFFECTS of excess N in the environment and associated environmental concerns (Newbould, 1989; Shaviv and Mikkelsen, 1993; Bumb, 1995) have resulted in renewed interest in the use of green manure as fertilizers (Harris and Hesterman, 1990; Peoples and Craswell, 1992; Crozier et al., 1998). In green manuring, the whole plant is incorporated into the soil while the N content is still high, assuming that a significant amount will be readily available to plants as the green manure decomposes. Various studies have indicated that biologically fixed N2 is only partially available to plants during the first growing season (Azam et al., 1986; Harris and Hesterman, 1990; Crozier et al., 1998). As most legume N resides in the soil organic pool (Ladd et al., 1981; Ladd and Amato, 1986; Harris et al., 1994), the main contribution of legumes to cropping systems presumably is to increase soil fertility in the long term by building up soil organic N reserves. Legume N becomes available for crop uptake only after tissue decomposition and subsequent mineralization of organic N. These processes are mediated by microbial populations; therefore, inorganic N usually increases following legume N incorporation (Patra et al., 1992; Fauci and Dick, 1994; Harris et al., 1994). That is why soil microbial biomass combined with the C/N ratio of organic matter is considered a good indicator of soil fertility because of its role as both a source and a sink for soil N (McGill et al., 1986; Jenkinson and Parry, 1988).

Uptake of legume N by crops or microorganisms can be studied using 15N-labeled plant residues. Recovery of fertilizer N by a first-year crop has been found to vary from <20 to >50%. Crozier et al. (1998) reported that a mature corn crop took up 11% of fertilizer N applied as 15NH4 and 20% of fertilizer N applied as 15NO3 in aboveground corn (Zea mays L.) biomass. Timmons and Cruse (1990) found that corn took up more spring banded (53%) than fall surface-applied fertilizer N (17%). Varvel and Peterson (1990) found N recovery to be greater for corn grown in rotations (59%) than as a monocrop (52%).

Recoveries of 15N from legume residues by a first-year crop vary from <5% to more than 30% (Azam et al., 1986; Ladd and Amato, 1986; Varco et al., 1989; Harris and Hesterman, 1990; Harris et al., 1994; Jensen, 1994a). When legumes with a low C/N ratio are incorporated into soil, NO3 leaching can be enhanced (Drury et al., 1991; Azam et al., 1993; Campbell et al., 1994; Jensen, 1994b) because rapid mineralization increases the ability of soil to supply mineral N (Sarrantonio and Scott, 1988; Patra et al., 1992; Francis et al., 1994). Other losses of mineral N can occur through NH3 volatilization and denitrification of NO3. Microbial immobilization can temporarily immobilize NO3 or NH4 (Aulakh et al., 1991; Janzen and McGinn, 1991; Myrold, 1998).

A laboratory study was conducted to measure recovery of 15N-labeled KNO3 and clover residue N in various soil N pools. The objectives of the study were to (i) monitor the levels of N and recovery of 15N in inorganic, organic, and microbial biomass N pools and (ii) monitor the loss of N via volatilization from decomposing clover residues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
Production of Nitrogen-15-Labeled Clover
Nitrogen-15 labeled Tibee crimson clover was grown in a laboratory growth chamber in two bins (42 by 28 by 20 cm deep). Light in the growth chamber (108 µmol m-2 s-1) was provided by a combination of flourescent and incandescent lights for a day length of 18 h. Seeds were planted on 30 Aug. 1996 in sand culture and watered with distilled water until germination was complete. Thereafter, plants were watered with a Hoagland solution (Hoagland and Arnon, 1950) containing 5 mmol K15NO3 (40.388 atom% 15N). Average temperatures above the clover canopy were 29°C at day and 23°C at night. Unlabeled clover also was grown in two bins, but it was watered with a Hoagland solution containing unlabeled KNO3. Clover plants were thinned at 10 and 24 d after sowing. The remaining plants were harvested whole (shoots and roots) on 22 Oct. 1996 (53 d), just before full bloom. Roots were washed thoroughly with running water to remove sand particles. Whole plants were cut into pieces 0.5 to 1.0 cm long and dried at 65°C. A subsample of labeled and unlabeled clover residues was ground and analyzed for total N and C on a Perkin Elmer 2400 CHN Elemental Analyzer (The Perkin-Elmer Corp., Norwalk, CT).

Soil Collection and Preparation
On 25 Nov. 1996, bulk soil sample was collected from the upper 15 cm of a Cecil sandy clay loam (clayey, kaolinitic thermic Typic Kanhapludult) located at the North Carolina State University Lake Wheeler Road Field Laboratory. The soil was mixed well, passed through a 7-mm sieve to remove gravel, put into a bucket, and sealed immediately to minimize moisture loss. Two nonsieved subsamples were collected and placed into polyethylene bags and the gravimetric moisture content determined. Soil comprised of 470 g kg-1 sand, 210 g kg-1 silt, and 320 g kg-1 clay (Gee and Bauder, 1986). The pH was 6.3 (Thomas, 1996). Total N (860 mg kg-1) and total C (14.4 g kg-1) were analyzed using a Perkin Elmer 2400 CHN Elemental Analyzer (The Perkin-Elmer Corp.).

Treatments
Field-moist soil (~180 g kg-1) equivalent to 300 g of oven-dry soil was weighed into 30 by 27 cm polyethylene bags and used as the laboratory media for each treatment. The bags were left open for 4 d on a laboratory bench before imposing treatments on 2 Dec. 1996. The fertilizer N treatment supplied 0.051 6 g N kg-1 soil (5 mL of 0.2 mol L-1 K15NO3, 60.73 atom% 15N). The clover treatment supplied 0.049 6 g N kg-1 soil (0.32 g labeled clover, 34.55 atom% 15N, 46 g N kg-1, 418 g C kg-1). A control treatment received no N addition. Nitrogen sources were mixed thoroughly with soil and the moisture content of samples adjusted to 180 g kg-1 with distilled water. Thirty samples were prepared for each treatment. A completely randomized design was used to assign three sample replicates to one of the 10 sampling dates (i.e., treatments were replicated three times). Samples were incubated in darkness at 22 to 24°C. To maintain aerobic soil conditions, samples were removed from the incubator every 2 wk, aired for 1 h on a laboratory bench, and the moisture content adjusted to 180 g kg-1 with distilled water. The contents of the bags were mixed well before returning to the incubator. Destructive sampling was performed as soon as possible after setting up the experiment (18 h), and subsequently after 1, 2, 4, 6, 8, 10, 12, 17, and 26 wk of incubation. Total N and recovery of 15N in inorganic, organic, and microbial biomass N was measured and also the loss of N via volatilization from decomposing clover residues. Losses through denitrification were calculated by difference from the measured N pools.

Laboratory and Statistical Analyses
Microbial biomass N was estimated by the chloroform fumigation–extraction method (Brookes et al., 1985) after applying a pretreatment procedure to volatilize NO3-N (Wyland et al., 1994) prior to Kjeldahl digestion (Bremner and Mulvaney, 1982). Fifty grams of air-dry soil (<2 mm) was extracted with a 0.5 mol L-1 K2SO4 solution (1:3 soil/solution ratio) by shaking and subsequent centrifugation. The soil was removed from the centrifuge tubes and air dried for 2 to 3 wk. Total inorganic N (NH4, NO3, and NO2) was determined in a subsample of the supernatant by steam distillation methods (Keeney and Nelson, 1982). Organic N was determined in a 2.0-g subsample by the micro-Kjeldahl procedure (Bremner and Mulvaney, 1982).

The 15N enrichment of total N in clover, microbial biomass, and organic N samples was determined in samples containing ~0.4 mg N following a modification of the diffusion procedure described by MacKown et al. (1987). A borosilicate test tube (12 by 75 mm) containing the trapping acid solution (H2SO4) was placed into a 120-mL container with the sample. The sample was made alkaline (pH > 12) with 12.5 mol of NaOH. Containers were sealed immediately, swirled briefly (5 s) to mix contents, placed in an oven at 65°C, and retrieved after 10 d. In samples containing inorganic N, we followed a modification of the diffusion procedure described by Brooks et al. (1989). A borosilicate test tube (12 by 75 mm) containing the trapping acid solution (H2SO4) was placed in a specimen container with the sample (~0.35 mg N). To start diffusion, 0.2 g of MgO and ball-milled 0.4 g of Devarda's alloy were added to the sample and the containers sealed immediately. Samples were placed in an oven set at 65°C and retrieved after 18 d. After retrieval, contents in the borosilicate test tubes were transferred quantitatively into clean ones and the solutions evaporated to dryness.

Isotope ratio analyses were performed using a CEC 21-620 mass spectrometer (Consolidated Electrodynamics Corp., Pasadena, CA), after converting NH4-N to N2 gas by the NaOBr freeze layer technique (Volk and Jackson, 1979). We calculated the recovery of applied N as the difference between applied N in fertilizer or clover and N found in control for inorganic N samples and by the 15N dilution tracer method for samples containing inorganic and organic N (Hauck and Bremner, 1976). The amount and percentage of 15N in microbial biomass samples was calculated as described by Ehaliotis et al. (1998).

Ammonia volatilized from decomposing clover residues was measured over a period of 12 wk and calculated as the difference between NH3 volatilized from soil plus clover residue (clover) and soil alone (control). Fresh soil equivalent to 100 g (oven dry weight) was weighed into seventy 980-mL mason jars. To the clover treatments, approximately 0.11 g of unlabelled clover residue (0.045 g N kg-1) was added to 35 jars and mixed well with soil. The control treatment received no N addition. Soil moisture was adjusted to 180 g kg-1 soil with distilled water. A 50-mL beaker containing 10 mL of 0.3 mol L-1 H3BO3 was placed into each jar before sealing tightly. The jars were kept in a dark locker in the laboratory at 22 to 24°C. Jars were opened weekly for 30 min and the soil moisture content adjusted to 180 g kg-1 with distilled water. The amount of water required for moisture adjustment was determined from the weight lost by soil in five jars not assigned any treatment, but included in the experiment for that purpose. Destructive sampling was performed at 0 1, 2, 4, 6, 9, and 12 wk, and the amount of NH3 trapped in H3BO3 was determined as described by Keeney and Nelson (1982).

Analyses of variance were performed using the general linear model procedure (SAS Institute, 1989), and comparisons of treatment means were made by protected LSD tests.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUMMARY AND CONCLUSIONS
 REFERENCES
 
Inorganic Nitrogen and Nitrogen-15
At any given time, the amount of inorganic N in soil increased in the order: fertilizer > clover > control, but the rate of N accumulation did not differ statistically among treatments (Fig. 1A) . Net immobilization occurred during the first week in all treatments, indicating that microbial biomass immobilized N initially, but net N mineralization occurred from Week 2 onwards. Fertilizer N not immobilized or denitrified during the first week of incubation remained constant at 65% of applied N throughout the study (Fig. 1B). No difference was found between recoveries calculated by the difference method and 15N dilution methods in fertilizer, so recovery of applied N by the difference method data is not shown.



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  		Fig. 1. Soil inorganic N accumulation following addition of 50 mg kg-1 fertilizer and clover residue. Recovery of applied N in clover was determined by the difference ({blacktriangledown}) and by the 15N dilution methods ({blacktriangleup}).

 
Addition of clover residues resulted in net N immobilization initially (<1 wk), followed by a net N mineralization (Fig. 1C). A regression model applied to clover data from Weeks 1 through 10 showed significant accumulation of inorganic N, indicating that net N mineralization was rapid initially (Fig. 2) . After 2 wk of incubation, 29% of clover N was present in the inorganic N pool (Fig. 1C). Thereafter, the rate of increase was low but statistically significant. At the end of the study, 40% of the clover N was recovered as inorganic N. Thus, decomposition of clover residues (4.6% N) followed a two-phase pattern, a rapid initial phase (<14 d), followed by a slower phase. These results are consistent with previous research (Jensen, 1994c; Mary et al., 1996; Watkins and Barraclough, 1996).



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  		Fig. 2. Soil plus clover inorganic N accumulation from addition of clover during the first 10 wk of the study.

 
To determine the effect of fertilizer and clover on mineralization of native soil organic N, inorganic N from the applied N sources was subtracted from total inorganic N (Fig. 3) . No differences were noted between clover and the control at any date. However, native inorganic N was significantly greater in fertilizer than in control at 1, 2, and 8 wk. Therefore fertilizer addition resulted in an added-N interaction (ANI) or priming effect (Broadbent, 1965; Jenkinson et al., 1985; Azam et al., 1993). An ANI can be apparent or real. Jenkinson et al. (1985) suggested that apparent ANIs occur when microorganisms immobilize fertilizer N instead of native soil N—microbial activities are presumably unaffected, but native inorganic N is increased from the pool substitution. In real ANIs, mineralization of native soil organic N exceeds immobilization of added N (Azam et al., 1994), as in our study. By the end of the study, mineralization of native soil N had contributed much more inorganic N than had mineralization of clover N: 50 vs. 20 mg N kg-1 (Fig. 1C). However, because of the difference in size of the two organic N pools, the fraction of each pool mineralized was reversed, i.e., native N: 50/900 = 0.055, clover N 20/50 = 0.40.



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  		Fig. 3. Effect of treatments on native soil inorganic N. Significant differences indicated with LSD0.05 bar.

 
Organic Nitrogen and Nitrogen-15
Total (applied plus native soil) organic N did not differ between treatments, and was not influenced by time (Fig. 4) . Organic N from applied sources was statistically greater in clover than in fertilizer on each sampling date and was not influenced by time. At the end of the incubation period, 50% of the applied clover N and 18% of the applied fertilizer N was in the organic N form (Fig. 4). As in other studies, most fertilizer N (12%) that entered the organic pool did so within the first 2 wk of study (Broadbent and Tyler, 1962; Azam et al., 1994).



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  		Fig. 4. Soil organic N recovery of applied N after addition of 50 mg kg-1 clover residue and fertilizer.

 
Microbial Biomass Nitrogen and Nitrogen-15
Initial microbial biomass N accounted for 1.3% of total N and was within the range (1–7%) reported for agricultural soils (Olfs and Scherer, 1996). The amount of applied N recovered in microbial biomass was statistically higher in clover than in fertilizer on all sampling dates (Fig. 5) . The influence of time was not significant in fertilizer, but in clover, a statistically significant decline was observed after the first week. The first analysis for microbial biomass N was conducted 18 h after applying treatments. The concentration was 29 mg N kg-1 in clover and 12 mg N kg-1 in fertilizer and control. Such a rapid conversion of clover N into microbial biomass N within an 18-h period (29 - 12 = 17 mg N kg-1, ~35% of clover N) was unexpected. We surmise that fumigation with clover may have facilitated a breakdown of clover cells, and hence, some release of N from clover residue (Sparling et al., 1985; Olfs and Scherer, 1996). This N would then be incorrectly included in the measurement of microbial biomass N. Consequently, no data are reported for clover for the first sampling date. As in other studies (Jensen, 1994b; Sparling et al., 1995), after 1 wk of incubation, total biomass N was elevated in clover. It was unchanged in fertilizer and control. Then in clover, microbial biomass N declined rapidly between 1 and 4 wk, after which the general pattern was similar among treatments (i.e., peaked between Weeks 6 and 10, then generally declined). Decline of microbial biomass between Weeks 1 and 4 showed that microbial biomass responded to disappearance of N from a very labile pool. This suggests that the labile N pool resided in clover itself and consisted mainly of salt extractable inorganic N, which in this study comprised 21% of total N. During decomposition, one source of N for microbial biomass can be the plant residue itself (Mary et al., 1996). The second peak of microbial biomass N was observed between Weeks 6 and 10, perhaps in response to remineralized N from decomposing microbial biomass (Marumoto et al., 1982).



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  		Fig. 5. Changes in microbial biomass N after additions of clover residue and fertilizer.

 
Losses through Volatilization and Denitrification
The total quantity of NH3 recovered during the 12-wk volatilization study was <1 mg kg-1 (data not shown). We therefore assumed that N not accounted for in our measurements was lost due to denitrification. At the end of the study, 84% of fertilizer N and 90% of clover N was recovered. Possible losses of applied N may have occurred from discarding soil from the measured fractions. Initially, soil was sieved (<7 mm) to remove gravel, and then ground (<2 mm) to facilitate analyses of organic and inorganic N. Discarded soil N in these fractions was not quantified.

Denitrification losses occurred early in the incubation period: after 2 wk of incubation, 81% of fertilizer N and 90% of clover applied N was recovered. Cooper and Smith (1963) reported that 60 mg of N was lost via denitrification 60 to 90 h after applying NO3 in two western U.S. soils with a pH of 6.1. Moreover, losses of NO3 from fertilizer via denitrification have been reported even when the soil is well drained (aerobic), presumably because microsites of anaerobic soil can exist in a largely aerobic soil (Guthrie and Duxbury, 1978). Denitrifying microbial populations require a readily available C source, NO3, and anaerobic soil conditions. In both fertilizer and clover, these conditions were fulfilled early in the incubation period when the soil moisture content was still high (180 g kg-1). We surmise that in fertilizer, fine roots not removed through sieving provided a C source to denitrifying microbial populations soon after applying NO3. In clover, rapid decomposition of clover residues in the first 2 wk provided denitrifiers with a readily available C and NO3 source. Moreover in clover, the elevated microbiological activity may have rapidly depleted O2 in microsites, thus producing anaerobic zones where denitrification occurred. With longer aerobic incubation, we presume that denitrification decreased because of declining availability of C (Drury et al., 1991).


   SUMMARY AND CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
REFERENCES
 
Inorganic N (NH4, NO3, and NO2) accumulated with time at the same rate in each treatment. At a given sampling date, the concentration of inorganic N was in the order: fertilizer > clover > control. Organic N concentration did not differ among treatments. At the end of 26 wk, applied N recovered in soil inorganic N was 66% for fertilizer and 40% for clover, in soil organic N was 18% for fertilizer and 50% for clover, and in microbial biomass N was 0.75% for fertilizer and 1.5% for clover. Clover decomposition was rapid initially and slow afterwards: 30% was mineralized in <2 wk and 40% after 26 wk. Losses of inorganic N through denitrification (estimated by difference) were substantial (fertilizer = 16%; clover = 10%). Because soil pH was <7, ammonia volatilized from decomposing clover residues was negligible. We observed an ANI with addition of fertilizer N, but not with incorporation of clover residues. Microbial biomass N did not differ among treatments, but applied N recovered in microbial biomass was significantly greater in clover than in fertilizer throughout incubation. Therefore, when incorporating clover residues as green manure to meet N requirements of a following crop in the southeastern USA, the following should be taken into consideration: 1. Clover decomposition was rapid initially and slow afterwards, indicating that clover green manure can meet the N requirements of a following crop from the time of emergence. 2. Losses of inorganic N from NO3 can be quite substantial, even under aerobic soil conditions.

3. Volatilization of NH3 from decomposing clover residues is negligible for this soil.

Received for publication July 3, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 





This Article
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Right arrow Articles by Muriuki, A. W.
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Right arrow Articles by Volk, R. J.
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Related Collections
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