effects of soil structure disturbance on mineralization of organic soil nitrogen

Effects of Soil Structure Disturbance on Mineralization of Organic Soil NitrogenH. L. Kristensen, G. W. McCarty,* and J. J. Meisinger

ABSTRACTDisturbance of soil structure by tillage operations is thought to

make soil organic N accessible for mineralization which was otherwiseprotected from degradation. The origin of N released by disturbanceof soil structure is, however, poorly understood and needs to be relatedto microbial activity. This study was performed to investigate theeffect of soil structure disturbance on the release of active or protectedorganic N pools in surface soils (0-2 cm) under plow- (FT) or no-tillage (NT) management. Active soil N was defined as the poolparticipating in mineralization-immobilization turnover during short-term incubation (6 d) while protected pools were considered inactiveduring this period. The active pool of soil N was labelled with 1SN inintact samples of PT and NT soils. The samples were either keptintact or sieved and repacked, and then leached weekly during a35-d incubation period. The disturbance of soil structure increasedmineral N release from 6 to 15 mg kg"1 in the NT soil within the firstweek after disturbance. This release was found to originate from bothactive and physically protected N pools as could be assessed by therelative differences in I5N content of mineralized N by intact anddisturbed soil samples. In contrast, the release from the PT soil was7 to 9 mg N kg ' after disturbance, with only a minor contribution fromprotected N pools. These results support the theory that disturbance ofsoil structure by tillage may destabilize and release protected poolsof soil N. Over the entire period of incubation, protected N accountedfor 27% of total N release in the NT soil and 12% in that of PT. Thecalculation of availability ratios, defined as the ratio between the 15Nenrichment of mineralized N and that of total soil N, showed thatrecently added 15N was less available for mineralization in the NTsoil as compared to that of PT. The probable cause for this differencewas the higher C/N ratio of organic matter in NT surface soil indicatingmore nonhumified organic matter when compared to PT organicmatter.

SOIL MANAGEMENT PRACTICES THAT MINIMIZE SOIL DIS-TURBANCE have been introduced and gained accep-

tance in crop production with the purpose of reducingsoil erosion and conservation of soil organic matter.There is substantial evidence that tillage managementinfluences the dynamics of C and N in agricultural soils.For example, NT management generally increases theamount of active soil N and organic and microbial bio-mass C and N, and results in substantial stratificationof these pools within the profile surface soil (Doran,1987; Follett and Schimel, 1989; Beare et al., 1994a;McCarty and Meisinger, 1997).

Greater retention of organic C and N in soils underNT management has been associated with increased soilaggregation (Beare et al., 1994a, 1994b). It is thoughtthat disruption of soil aggregates by tillage operationscauses physically protected pools of organic C and N

G.W. McCarty and J. J. Meisinger, USD A-ARS Environmental Chem-istry Lab., Beltsville, MD 20705 USA; and H. L. Kristensen, Dep. ofTerrestrial Ecology, National Environmental Research Inst., Vejls0-vej 25, DK-8600 Silkeborg, Denmark. Received 22 Mar. 1999. *Corre-sponding author ([email protected]).

Published in Soil Sci. Soc. Am. J. 64:371-378 (2000).

to become available for microbial degradation (Watersand Oades, 1991; Ladd et al., 1993). The physically pro-tected organic matter is thought to be less available formicrobial degradation than unprotected matter becauseof location inside micro- and macro-aggregates andsmall pores, or because of encrustation of organic matterby clay minerals (Tisdall and Oades, 1982; Gupta andGermida, 1988; Hassink, 1992). Another source of pro-tected organic N, however, may consist of N pools thatare immobilized within living but inactive microorgan-isms or within components of the active microbial com-munity with low rates of turnover. For example, organicN may be immobilized in fungal hyphae which havebeen proposed to act as binding agents of microaggreg-ates into macroaggregates (Tisdall and Oades, 1982).With disturbance of the hyphal network, the fungal tis-sue could be subjected to microbial degradation. Thiskind of protection would be dependent on the abilityof the living cell to maintain the integrity of the cellularenvironment and, with death of the organism, the pro-tecting mechanism would be lost. This could be consid-ered to be a form of biological protection.

Investigations of the influence of tillage on soil Nmineralization have often involved the use of soil sam-ples prepared for investigation by either fractionationor sieving (Craswell and Waring, 1972; Gupta and Ger-mida, 1988; Follett and Schimel, 1989; Beare et al.,1994a; McCarty and Meisinger, 1997). Bundy and Mei-singer (1994), however, stressed the importance of usingexperimental techniques with minimal disturbance ofthe soil in studies where the management systems underevaluation are themselves characterized by differencesin the degree of soil disturbance. Most studies of distur-bance effects of soil structure on mineral N release fromintact samples of agricultural soil have made no attemptto differentiate the origin of released N (Rice et al.,1987; Cabrera and Kissel, 1988; Stenger et al., 1995).By use of 15N techniques, Grace et al. (1993) foundsimulated cultivation increased the release of recentlysynthesized microbial metabolites, while Hassink (1992)reported indirect evidence for pools of physically pro-tected N in soils by relating N release to pore size distri-bution. Disturbance of soil structure may influence min-eralization-immobilization turnover by increasingavailability of protected N to microbes or by stimulatingprotozoa-soil fauna grazing on microbes (Ladd et al.,1993). Thus it is likely that released N may originateboth from formerly inactive (protected) and active soilN pools. There is a need to relate N release caused bysoil structure disturbance to microbial activity to enableassessment of protection mechanisms.

The purpose of this study was to investigate the effectof soil structure disturbance on mineralization and im-

Abbreviations: PT, plow-tillage; NT, no-tillage; MIT, mineralization-immobilization turnover; AR, availability ratio.

371

372 SOIL SCI. SOC. AM. J., VOL. 64, JANUARY-FEBRUARY 2000

Fig. 1. One subunit of the system for leaching intact soil samples withunsaturated flow. Components of assembled subunit: (A) funnelbottom (Nalgene part no. 4280-0550,45 nun i.d.); (B) No. 9 rubberstopper; (C) three hypodermic needles (0.8 mm o.d., 2.5 cm length);(D) air hole (3 mm); (E) polycarbonate tube (41 nun i.d., 1.6 mmwall); (F) soil core; (G) Biichner funnel (Nalgene part no. 4280-0550, 45 mm i.d.); (H) 2.5 (xm glass fiber filter (Fisher Scientific);(I) 0.45 (un cellulose nitrate membrane filter (Whatman) prerinsedwith water; (J) vacuum manifold (-80 kPa); (K) 120 mL bottle.The leaching solution (0.01 M CaCI:) was supplied to each subunitof the system by use of a peristaltic proportioning pump (TechniconModel III, pump tubing 0.51 mm i.d.).

mobilization of N in the surface layer of soils subjectedto long-term FT or NT management, with particularfocus on the release of protected N.

For this study, we posited that the protected pool ofsoil N does not participate significantly in short-termbiological cycling such as occurs in mineralization-immobilization turnover (MIT) (Jansson, 1958). Thus,the protected N pool was operationally defined as thatwhich was not isotopically enriched by immobilizationof 15NH4

+ during an incubation designed to label thepool of active soil N (active phase, Follett et al., 1989).The contribution from protected soil N pools to mineral-ization after disturbance of soil structure could then bedetected as a dilution of the 15N enrichment of N re-leased from the active N pool, the dilution being causedby release of 14N from the protected pools. By use ofthis dilution of mineralized N in disturbed relative tointact samples, different pools of protected soil N couldbe characterized according to the mechanism of protec-tion. For example, we also considered biological protec-tion to be a potentially significant mechanism. In theassessment of types of protected soil N, no assumptionwas made regarding the extent by which they were mu-tually exclusive or additive.

MATERIALS AND METHODS

Study Site and SamplingIntact soil samples were collected from experimental field

plots established in 1976 to study the influence of annualmoldboard PT and NT management on fertilizer uptake bya continuous monoculture of maize [Zea mays (L.)]. The fieldwas located in the Piedmont region in central Maryland on amoderately well drained Delanco silt loam (fine-loamy, mixedmesic Aquic Hapludults). The experimental plots were estab-lished in a split-plot design with different N fertilizer ratesnested within tillage treatments. Soil samples used in the pres-ent study were from two field replicates of PT and NT plotsthat received annual surface applications of 135 kg ha"1 fertil-izer N. Previous work showed that this rate represents thesufficiency rate of N fertilization for maize production (Mei-singer et al., 1985). In March 1996, intact soil samples werecollected from each field plot by pressing polycarbonate plastictubes (41 mm diam.) against the soil surface that gave minimalcompaction of the soil. Tillage had not been performed sincethe previous harvest and fresh litter remained on the surfaceof both PT and NT plots. Prior to sampling, pieces of freshlitter were therefore removed from the soil surface. The sam-ples were brought to the laboratory and remained in the sam-pling tubes throughout the experiment to ensure minimal dis-turbance of soil structure. The excessively moist samples(25-35% H2O of soil dry weight) were partially dried for 3 dat 22°C. Each of these intact soil samples were then cut to anapproximate dry weight of 40 g (2.0-2.5 cm length) by gentlypushing the sample partly out of the tube and cutting it fromthe lower end of the sample. The dry weight obtained wasbased on bulk density measurements of three extra samplescollected from each field site. Each tube containing soil wasthen placed in the top part of a Biichner-type filtering funnelwith a two component filter. All details on the materials usedin the setup are provided in Fig. 1. The average pH in the PTand NT soil samples was 5.8 and 5.3, respectively (soil/waterratio 1:2).

Labeling the Active Nitrogen PoolThe active microbial N pool in the intact soil samples was

labeled with 15N by injection of a mixture of 15(NH4)2SO4 (5mg N kg"1 soil, 99% enriched) and glucose (150 jjig C g"1

soil) followed by an incubation period of 6 d. This permittedincorporation of the label into the portion of active N involvedin mineralization-immobilization turnover (MIT) within theperiod of incubation. A 1-mL hypodermic syringe fitted witha needle (0.51 mm o.d., 16 mm length) was used to make aseries of 5 injections of the labeling solution to a depth of 1.5cm. With each injection, the needle was pulled out evenly asthe solution was dispensed. A total of 0.35 mL of solution wasinjected into each sample giving a final soil water content ofapproximately 19 and 24% of soil dry weight for PT and NTsoil, respectively (70% of field capacity). The treated soilsamples were placed together with a wet paper towel into0.9 L bottles and incubated for 6 d at 25°C.

The Leaching SystemThe soil samples in the tubes were leached by an unsatu-

rated flow of solution; a leaching method that prevents pond-ing at the soil surface and thus reduces the risk of preferentialflow through soil pores as well as of NOj" loss by denitrifica-tion. The leaching system was composed of multiple subunitsof which details are given in Fig. 1. A multichannel propor-tioning pump supplied leaching solution (60 mL 0.01 M CaCl2,flow rate 8.9 ± 0.14 mL h"1) to the surface of each sample

KRISTENSEN ET AL.: SOIL STRUCTURE DISTURBANCE ON SOIL NITROGEN 373

and a vacuum manifold system acting on membrane filterssupplied tension to soil water (-80 kPa) which enabled collec-tion of leachate in a bottle. After each leaching event, thevacuum was maintained for approximately 2 h until the soilwater content in each sample reached the content previousto leaching (19-24% of dry weight). The collected leachateswere diluted with 4 M KC1 to a final concentration of 1 MKC1, shaken, and stored at 3°C for a maximum of 14 d untilanalysis for amount and isotopic content of inorganic N.

The efficiency of the leaching system for removal ofNO3"~ from intact and disturbed samples was tested and theuse of 60 mL of leaching solution removed an average of 96to 98% of the NO3~" in the soil cores during incubation. Thisefficiency was acceptable for use of the system in this study.The amount of NH4

+ in leachates or soil extracts was belowdetection limits.

Structral Disturbance ExperimentTo study the effect of soil structure disturbance on mineral-

ization of active and protected soil N pools, intact samples ofPT and NT soil were sieved after their active soil N pool hadbeen labeled. With this study, three replicate samples weretaken from each of two replicate field plots, sieved (mesh size2 mm), and repacked to the original bulk density, while threeother replicate samples from each plot were left intact. Bothdisturbed and intact samples were leached on a weekly basisduring a 35-d incubation with the first leaching occurring theday before the disturbance of soil structure by sieving to re-move any initial soil NOf.

Between leaching events, the soil samples in the subunitcomponents of the leaching system were removed from thevacuum manifold and placed in 0.9-L bottles with a wet papertowel and incubated at 25°C. The bottles were opened foraeration every third day of the incubation.

Biomass Perturbation ExperimentTo study the possible origin of protected N from biologically

protected pools in the soil, the microbial biomass was per-turbed in samples without disturbing the soil structure. Thiswas done by fumigation with chloroform to perturb the bio-mass in intact samples of PT and NT soil. Six replicate soilsamples were taken from one of each field plot and labeledwith 15N in the active microbial N pool as previously described.Before start of incubations, the samples were placed on theleaching system to remove any initial soil NO3~. Half of theseintact replicate samples were fumigated with ethanol-freeCHC13 for 20 h and evacuated repeatedly to remove residualchloroform. All samples were then placed in 220-mL bottles,sealed with rubber stoppers, and treated with 2 mL acetyleneto inhibit nitrification. The samples were incubated at 25°Cfor 14 d to allow equilibration of I5N enrichment between theperturbed biomass and the released NH|. Then the sampleswere extracted in 1 M KC1 (soil/solution ratio 1:10), and soilsand extracts analyzed for amount and isotopic content of N.The acetylene treatment was included to eliminate the influ-ence of nitrification on MIT processes because the fumigatedsoils would have reduced or no populations of nitrifying bacte-ria in contrast to control soils without fumigation. This proce-dure was judged effective as the amount of NO-T in the samplesat the end of the experiment was below detection limits.

Sample AnalysisConcentrations of NOj" and NH/ in leachates and soil ex-

tracts were determined colorimetrically by flow-injection anal-ysis (Lachat Instruments, 1989,1990). The samples were alsoprepared for analysis of isotopic content of NO-f-N and

NH4+-N by use of the diffusion method by Brooks et al. (1989)

prior to analysis by an isotope mass spectrometer interfacedwith an automated N-C analyzer (Europa Scientific Ltd.,Cheshire, UK). The soil water content was obtained by dryingsamples for 24 h at 105°C.

After termination of the structural disturbance experiment,each sample was sieved (mesh size 2 mm), mixed, and subsam-pled for analysis of pH, total soil C, and microbial C contentas well as the amount and isotopic content of total soil N andmicrobial N pools. The total C and N content was measuredby dry combustion on a C and N analyzer (Leco CNS - 2000).The isotopic content of total N in soil samples was determinedby dry combustion of soil using the same N-C analyzer inter-faced with the mass spectrometer for N isotope analysis(Hauck et al., 1994). Microbial biomass C and N pool sizeswere estimated by use of the chloroform fumigation-incuba-tion technique (Voroney and Paul, 1984) and with fcc = 0.41and kK = 0.3. Soil (14 g) from each sample was fumigated withethanol-free CHC13 for 20 h followed by incubation for 10 dat 25°C in 250-mL bottles. Headspace gases were sampledwith a gas syringe and analyzed for CO2 content by gas chroma-tography (McCarty and Blicher-Mathiesen, 1996). The fumi-gated soil samples were extracted in 1 M KC1 (soil/solutionratio 1:10) and analyzed for amount and isotopic content ofNOr and NH4

+.

Data AnalysisThe contributions of active and protected pools of soil or-

ganic N to the mineral N pool formed during incubation ofthe disturbed soil samples were calculated using amounts and15N enrichments of the NO3~ pools obtained from both intactand disturbed samples. The data were analyzed using the prin-ciple of isotope dilution (Jansson, 1958) which can be ex-pressed in the following equation:

Amin = (AJC + Ay Y)/(X + Y)which can be solved for both X and Y when

[1]

Z = X+Ywhere

Z = amount of N mineralized to NOf in each disturbedsoil sample

X = amount of active N mineralized to NO3~ in each dis-turbed soil sample

A^ = atom percent 15N in the active N pool assumed to bethe atom percent 15N measured in the mineralizedNOr pool in each intact soil sample

Y = amount of protected N mineralized to NO3~ in eachdisturbed soil sample

Ay = atom percent 15N in the protected N pool (assumedto be natural abundance 0.366%)

^4min = atom percent 15N in the mineralized NO3~ pool ineach disturbed soil sample.

The mineralization rate of recently 15N-labeled relative toindigenous organic soil N was determined by calculating theavailability ratio (AR) as originally defined by Broadbent andNakashima (1967):

_ atom percent 15N in the NO3~ pool (leachate)atom percent 15N in total N (soil)

[3]

Potential ErrorsAn assumption made with use of the above equations is

that the 15N label was evenly distributed in the soil sample.

AR


This may have been violated to some degree since the distribu-tion was a result of injection of solution and subsequent diffu-sion of the label into the soil matrix during the preincubation.Sensitivity analysis performed by Davidson et al. (1991)showed, however, that this assumption may not be critical inthis type of labeling procedure as long as the distribution isnot biased concurrently to a nonrandom occurrence of theprocesses under study.

The fact that the 15N-labeling procedure was performedprior to imposition of the structural disturbance treatmentson the soil ensured that the label was incorporated undersimilar conditions for all samples resulting in similar distribu-tions of label in the active N pool. The treatment, imposedby sieving and repacking half of the samples involved in thestructural disturbance experiment, probably redistributed thelabeled pool more uniformly than that originally in the intactsamples. Because mineralization of soil organic N can be de-scribed by a decay function with first-order kinetics, the simpleimposition of a more uniform distribution of the 15N labeledsoil organic matter should have little or no influence on thekinetics of subsequent N mineralization. Hence, little or nobias in 15NO3~ production should have resulted from the redis-tribution of 15N labeled organic matter in these experiments.

The average recovery of added 15N at the end of the soil

0.5

0.4-

oat

5" 0.3 -\zO)

0.2-

0.1 -

en

0

14-

12-

10-

8-

6-

4-

2-

• NT intactNT disturbed

• PT intactPT disturbed

NT intactNT disturbed

• PT intactPT disturbed

114 21

Incubation time (day)28 35

Fig. 2. The amount of 15NOf-N and NO3 -N collected during leachingof incubated soil samples obtained from fields under plow- (PT)or no-tillage (NT) management. The soil structure of the sampleshad been disturbed or was kept intact, and the arrow indicatesthe time of the disturbance. Different letters indicate significantdifferences between collected amounts within day of leaching (n =6; t test, P < 0.05).

structural experiment was high and of equal size irrespectiveof soil type and treatment (90 and 89% for disturbed PTand NT samples, 90 and 92% for intact PT and NT samples,respectively). This indicates that losses of 15N due to denitrifi-cation and leaching of dissolved organic N compounds, wereof minor importance during the experiment.

Statistical AnalysisStatistical significance of differences in soil properties and

distribution of 15N between PT and NT soils were tested byanalysis of variance (F test). Differences in mineralizationrates and availability ratios between disturbance treatmentsand PT and NT soils were tested by analysis of variance fol-lowed by pairwise comparisons by Tukey's studentized rangetest (t test). Relationships between soil properties were investi-gated by simple linear regression modeling and homogeneityof slopes were tested using general linear models procedures(SAS Institute, 1988). In the assessment of statistical signifi-cant differences between results, tests with P < 0.05 wereconsidered statistically significant and tests with 0.05 < P <0.15 were considered as trends.

RESULTS AND DISCUSSIONMineralization of Nitrogen

Comparison of amounts of 15NO3~ and total NO-T col-lected in leachates from the intact and disturbed NT soilsamples showed that soil structure disturbance causedincreased release of 15NO3~ within the first week aftertreatment but had little influence on release in subsequentweeks (Fig. 2). The results obtained with the PT soilsamples showed that structure disturbance had no effecton the amount of 15NOf released with any of the leachingevents. Comparison of data from corresponding PT andNT soil samples showed that the amount of 15NO3' re-leased from the PT soil was generally higher than thatfrom the NT soil for all periods of incubation. However,the total amounts of NO3~ mineralized from the intactsamples of PT and NT soil did not differ significantly(Ftest P < 0.22). But, disruption of the NT soil structurecaused a two- to threefold increase in the total amountof NO3~ mineralized within the first week after the dis-ruption. This influence decreased markedly in the subse-quent weeks of incubation. Thus soil structure distur-bance seemed to increase availability of organic N formineralization in the NT soil with little correspondinginfluence in PT soil. The increased release of 15N in theNT soil did indicate, however, that at least part of thereleased N came from a pool of recently synthesizedorganic N.

Mineralization of Active and Protected Nitrogen

The involvement of different soil N pools in mineral-ization was investigated further by calculation of theportions of NOf mineralized from active and protectedN pools with disruption of soil structure (Fig. 3). In themathematical treatment of data (Eq. [1] and [2]), weassigned production of NOf in the intact soil samplesas being from the active N pool with the assumptionthat the 15N enrichment of the mineralized NO3~ re-flected the 15N enrichment of the active N pool for both


20

01

15-

I D -

5-

Plow-tillage ^j |ntact core active N

1 . 1 Disturbed core, protected NDisturbed core, active N

14 21i

28 35 28 35

Incubation time (day) I Incubation time (day)Fig. 3. The amount of NOf-N released from active or protected soil N pools during incubation of intact and disturbed plow- (PT) or no-tillage

(NT) soil samples. The arrows indicate the time of soil structure disturbance. The bars indicate standard errors of the total amount releasedat each leaching event (n = 6).

intact and disturbed soil samples. With this it can becalculated that soil structure disturbance significantlyincreased the amount of active N mineralized in the NTsamples by 3 mg kg"1 soil within the first week (F testP < 0.02) while no corresponding increase was detectedin the PT soil samples. The amount of physically pro-tected soil N released from the NT soil only tended tobe higher than from the PT soil due to variability amongreplicate soil samples (F test P < 0.12). This tendencyis consistent with the findings of Beare et al. (1994b),who found that soil under long-term NT managementhad a better soil structure with a higher number andincreased stability of macroaggregates than under PT.Macroaggregates are believed to be closely associatedwith the pool of protected organic matter in the soil(Tisdall and Oades, 1982; Gupta and Germida, 1988).When calculated for the entire period of incubationafter soil disturbance (Days 7-35), the protected N ac-counted for 27% of total N release in the surface layerof the NT soil and for 12% of total N release in the PTsoil. These results are comparable to those reported byBeare et al. (1994a) (29% and 6.5%, respectively), whoused a nonlabeling technique with measurements of Nrelease from crushed macroaggregates of the 0 to 5 cmsurface layer of soil.

Origin of Protected NitrogenThe rate for release of N in the disturbed NT soils

increased rapidly with a subsequent corresponding de-crease to approximately that of intact samples withinthe first two weeks after the treatment (Fig. 2). Thisindicated that readily decomposable organic matter wasmade accessible for degradation by soil microorganismsby structure disturbance. To determine if this may berelated to perturbation of the soil microbial biomassresulting in release of biologically protected N, we stud-ied the flush of soil N mineralization and the 15N isotope

dilution of this pool resulting from chloroform fumiga-tion in intact samples of NT and PT soils. It is wellestablished that fumigation of soil causes release of Nfrom the microbial biomass which is then subject tomineralization (Voroney and Paul, 1984). The fumiga-tion of soil samples caused a 3.2 to 3.6 factor increasein amount of soil N mineralized when compared tountreated samples (Table 1). The 15N enrichment of theN released from the fumigated PT samples tended tobe lower than that from the untreated samples (F testP < 0.06) indicating that the PT soil contained a poolof biologically protected N. This contrasts the resultsfor the NT soil where no significant difference was foundin the 15N enrichment between the fumigation treat-ments (Ftest P < 0.22). This is surprising given the factthat the NT soil had a much higher pool of microbialbiomass-C and N (Table 2; F test P < 0.001) and may,for example, be expected to have greater fungal biomassthan the PT soil due to a lack of soil disruption and theinvolvement of hyphae in macroaggregate formation(Gupta and Germida, 1988; Beare et al., 1992).

Distribution and Availability of Nitrogen-15Figure 4 shows the distributions of added 15N label

and indigenous N in the PT and NT soils at terminationTable 1. Effects of perturbation of microbial biomass by chloro-

form fumigation on amount and T5N content of NHJ releasedduring incubation of plow-tillage (PT) and no-tillage (NT) soilsamples. The P value of F tests (n = 3) of differences betweenfumigated and intact samples are indicated.

Total NH4+ release 15N enrichment

PT, nonperturbed

PT, perturbedNT, nonperturbed

NT, perturbed

mg N kg 1

11.6P < 0.01

36.918.3

P < 0.0166.0

%4.88

P < 0.063.571.66

P < 0.221.92


Table 2. The properties of the soil organic C and N pools of the plow-tillage (PT) and no-tillage (NT) soil samples and the range andcoefficient of variation (CV) of these properties. The P values give significance of the differences between tillage means (Ftest, n = 12).

SoilN SoilC Soil C/N Biomass N Biomass C Biomass C/N

PTRangeCVNTRangeCV

g N kg '1.06

0.97-1.113.712.05

1.66-2.7214.5

P < 0.001

g C kg '11.2

10.0-11.95.17

24.418.5-36.9

21.2P < 0.001

10.510.3-10.8

1.8111.8

11.1-13.66.1

P < 0.001

mg N kg '120

79-16621.4

283147-459

36.5P < 0.001

mg C kg >424

357-45512.01

1181597-1821

30.2P < 0.001

3.613.14-4.51

14.854.29

3.06-5.3114.0

P < 0.01

of the 35-d incubation in the soil structural disturbanceexperiment. As can be expected, a single event of struc-tural disturbance had no measurable influence on thedistribution of N in the different pools of soil N (compar-ison not shown). By contrast, the distribution of the 15Nlabel was substantially different in the PT and NT soils.The proportion of label that was mineralized in PT soilwas significantly greater than that of the NT soil (F testP < 0.001). Correspondingly, the 15N content of the NTsoil (residual + biomass) remained significantly higher(F test P < 0.001) as well as, with slight approximation,that portion in the biomass fraction alone (F test P <0.06).

The distribution of total soil N also differed substan-tially between the PT and NT soils when viewed onabsolute and relative bases (Fig. 4). For example, theaverage size of total N and biomass N pools in theNT soil was approximately twice those of the PT withgreater absolute amounts of soil N being mineralizedin the NT soil (42 mg kg"1) when compared to that ofPT (30 mg kg"1) (F test P < 0.04). When viewed on a

Plow-tillagetotal 1SN

0.27

No-tillagetotal 15N

0.95

0.38

0.50

2.96 3.25

Plow-tillagetotalN

No-tillagetotal N

120 283

941 1768

• Leachate • Biomass Jill Residual soilFig. 4. The distribution of I5N and N after the 35 day incubation of

plow- (PT) or no-tillage (NT) soil samples (« = 12). The valuesindicate size of the specified N pool on a mg N kg'1 soil basis.

basis relative to total soil N, however, mineralizationwas greater in the PT soil (2.8%) as compared to NTsoil (2.0%) (Ftest P < 0.001). Follett and Schimel (1989)suggested that it is likely that the differences in distribu-tion of 15N label are at least partly due to the differencesin the total amounts of N in the surface layer of PT andNT soils.

To further investigate this hypothesis, the 15N mineral-ization data were normalized on a basis of amounts oftotal N in the incubated samples of PT and NT soils. Thiswas done by calculation of the AR of 15N enrichment ofthe NOf produced relative to the 15N enrichment in theoverall soil N pool (see Eq. [3]). Figure 5 shows the ARcalculated for N mineralized at various times during theincubation. The resulting values of AR for all soils weresubstantially greater than unity which is expected as therecently added 15N has been immobilized in a pool ofactive soil N which is more available for mineralizationrelative to other indigenous soil N. It can be seen thatstructural disturbance of soil caused a decrease in theAR relative to that of the intact samples at the firstleaching event after disturbance for the PT soil (Fig. 5).This decrease is indicative of the release of physicallyprotected N from an unlabeled pool of soil N as alsoshown in Fig. 3. Interestingly, the adjustment of 15Nmineralization data on the basis of total soil N did not

8

o2Il-ia.23

1

7-

6-

5 -

4-

3-

2-

1 -

NT intactNT disturbedPT intactPT disturbed

7 14 21Incubation time (day)

28 35

Fig. 5. The availability ratio for N in leachates collected during theincubation of plow- (PT) or no-tillage (NT) soil samples. The soilstructure of the samples had been disturbed or was kept intact. Thearrow indicate the time of disturbance. Different letters indicatesignificant differences between availability ratios within day ofleaching (n = 6; t test, P < 0.05).


bring equivalence between PT and NT soils with respectto availability of 15N label for mineralization. This find-ing indicates that factors other than soil N pool sizeinfluence the availability of recently added N for miner-alization by soil microorganisms.

Relationship Between Availability Ratioand Soil Properties

The characterization of the individual soil samples,collected as intact cores from the field, showed not onlywide divergence in properties of the soil surface layerbetween the tillage treatments but also within the repli-cate samples from each treatment (Table 2). Both thesize and coefficient of variation (CV) for the total andmicrobial soil N pools were two to four times higher inthe NT soil as compared to that of PT, and similarpattern of divergence was found for total and microbialC in these samples.

Linear regression analysis between the total N con-tent of all soil samples and the AR of added 15N, ascalculated for the entire experimental period after dis-turbance, (Fig. 6A) shows a strong negative correlationbetween AR and total N (r2 = 0.79). In this analysis,both disturbed and intact samples were pooled eventhough other analysis showed that there was good prob-ability that this treatment caused variation in the slope(P < 0.06). An initial conclusion from these results maybe that the use of availability ratios was not effectivefor adjusting the 15N mineralization data to the differ-ences in size of N pools within soils. Further analysis ofdata, however, provides another explanation.

For this study, soil samples were collected within thetop 2 cm of surface soil. McCarty and Meisinger (1997)found substantial stratification with depth of the compo-sition of soil organic matter with considerable increasein the C/N ratio at the surface layer of NT soil in contrastto that of PT. It was concluded that this increase wasdue to annual deposition of fresh plant residue withhigh initial C/N ratio, which is left on the soil surfaceof the NT soil, while being mixed into deeper soil layersof the PT soil. Characterization of the soil samples usedin this study showed this same pattern with higher C/Nratios in the surface soils under NT than PT manage-ment but also with substantial variation in this ratiowithin replicate soil samples (Table 2). Regression anal-ysis shows a strong positive relationship between totalN content and C/N ratio in the samples (r2 = 0.89)which indicates that nonhumified plant residue is animportant component of soil organic matter in the sur-face layer (Fig. 6B). We surmise that the strong covari-ance between total N content and C/N ratio accountsfor the apparent relationship between total N and AR.This indicates that the variation in the C/N ratio is theprimary factor inducing variation in AR for these soils.Regression analysis provides evidence for a negativecorrelation (r2 = 0.67) between AR and the C/N ratioof the samples (Fig. 6C). It is thus suggested that theC/N ratio and the degree of decomposition of the or-ganic matter in the soils were important factors de-termining the availability of recently added N to micro-

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Fig. 6. Linear regressions between properties of plow- (PT) or no-tillage (NT) soil samples that were incubated and leached weeklyduring a period of 35 d. The soil structure of the samples had beendisturbed or was kept intact. The linear regressions shown are (A)the availability ratio against the soil N content; (B) the soil Ncontent against the soil C/N ratio; and (C) the availability ratioagainst the soil C/N ratio.

organisms. This conclusion gains support from work byBlackmer and Green (1995), who found that 15N addedto fresh corn stover was quickly immobilized and notreadily released with the residue decomposition. An-other factor, which may contribute to the observed dif-ferences in AR, is that the microbial community of theNT surface soil may be relatively more dominated byfungi when compared to the PT soil (Beare et al., 1992).A microbial community with a higher proportion offungi relative to bacteria may be expected to have aslower turnover of biomass and a higher immobilizationof added N in fungal components with a low potentialfor mineralization (Holland and Coleman, 1987; He etal., 1988). It is also possible that differences in soil struc-ture between PT and NT soils may have influenced15N availability for mineralization, for example, due to


protection of microbes from grazing by protozoa andsoil fauna (Ladd et al., 1993)

Relevance of Experimental ApproachSieving was used here as a treatment for disturbing

the soil structure and the results obtained have twoimplications for the experimental approach in studiesof soil N dynamics. The sieving treatment was found torelease mineral N from protected N pools in the soils,however, this release was not well reflected by the flushin total mineral N release after sieving. This flush in netN mineralization has been recommended by Hassink(1992) as a measure of protected N release from soils.But, the current experimental approach with use of siev-ing and 15N-labeling techniques showed that the flushin mineral N release may originate from both active andprotected N pools as in the case of the NT soil, or thatprotected N may be released despite the lack of a flushin mineral N release as found in the PT soil (Fig. 3).Thus, the use of 15N labeling of the active soil N poolcan be a useful approach for studying the involvementof protected organic N pools in MIT activities. Thesieving treatment was found, however, to influence netN mineralization rates to a different degree in the soilsunder study, with the influence declining within 7 to14 d after the treatment. These findings add support tothe conclusion of other studies (Craswell and Waring,1972; Hassink, 1992); that use of freshly sieved soil sam-ples (through 2-mm mesh size or less) should be avoidedwhen preparing samples for studies of N metabolism insoils which are characterized by different degrees ofdisturbance (Bundy and Meisinger, 1994).

ACKNOWLEDGMENTSThis work was supported in part by a fellowship from the

National Environmental Research Institute of Denmark, theDanish Research Academy, and Aalborg University.

effects of soil structure disturbance on mineralization of organic soil nitrogen

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