carbon and nitrogen dynamics of soil organic matter fractions from cultivated grassland soils

Carbon and Nitrogen Dynamics of Soil Organic Matter Fractionsfrom Cultivated Grassland Soils

C. A. Cambardella* and E. T. Elliott

ABSTRACTThe amount of organic matter present in soil and the rate of soil

organic matter (SOM) turnover are influenced by agricultural man-agement practices. Because SOM is composed of a series of fractions,management practices will also influence the distribution of organicC and N among SOM pools. Our study examined SOM fractions thatare occluded within the aggregate structure. Aggregates were dis-rupted by sonication and the disrupted soil suspensions were passedthrough a series of sieves to isolate size fractions. Densiometric sep-arations were carried out on the size fractions, creating size-densityfractions. Fine-silt-size particles having a density of 2.07 to2.22 g/cm3 isolated from inside macroaggregates contained the highestpercentage of total soil C and N for all cultivation treatments and,because of its properties, will be referred to as the enriched labilefraction (ELF). As cultivation intensity was reduced, the amount ofN in the ELF increased from 110 mg N/kg in the bare fallow treatmentto 405 mg N/kg in the no-till treatment. About 5% of the N in theELF was mineralized during a 28-d laboratory incubation, averagedacross treatments. The proportion of N mineralized from the ELF(4.7%) was significantly higher than from intact macroaggregates(2.1%), which suggests this fraction may be protected from decom-position within the aggregate structure. We postulate that the ELF isa byproduct of microbial activity and that it contributes to bindingmicroaggregates into macroaggregates in cultivated grassland soils.

AGRICULTURAL MANAGEMENT PRACTICES influencethe amount of organic matter present in soil and

cause changes in the rate of SOM turnover. Soil or-ganic matter is composed of a series of fractions fromvery active to passive (Schimel et al., 1985). Thesefractions have been conceptualized in mathematicalmodels as kinetically defined pools with differentturnover rates (Jansson, 1958; Parnas, 1975; Jenkin-son and Raynor, 1977; Paul and van Veen, 1978; vanVeen and Paul, 1981; van Veen et al., 1984; Partonet al., 1987). The distribution of organic C and Namong labile and stable pools is affected by manyfactors including crop rotation (Janzen, 1987), typeand length of tillage (Tiessen and Stewart, 1983; Dalaiand Mayer, 1987; Balesdent et al., 1988; Cambardellaand Elliott, 1992a), and fertilizer applications (Stan-ford and Smith, 1972; Christensen, 1988). The mostlabile fractions'decline with cultivation.

Parton et al. (1987) and Metherell (1992) predictthat changes in the size of the slow (i.e., intermediateturnover) organic matter pool account for most of thedifferences in SOM content between managementtreatments after 20 yr of cultivation. These predicteddifferences in soil organic C and N between treatmentscan be partially accounted for by organic C and N inthe particulate organic matter (POM) fraction (Cam-bardella and Elliott, 1992a). The remainder of the slow

C.A. Cambardella, USDA-ARS, National Soil Tilth Lab., 2150Pammel Drive, Ames, IA 50011; and E.T. Elliott, Natural Re-source Ecology Lab., Colorado State Univ., Fort Collins, CO80523. Received 31 Aug. 1992. * Corresponding author.

Published in Soil Sci. Soc. Am. J. 58:123-130 (1994).

pool not accounted for by POM was hypothesized tobe microbially produced decomposition products thatare physically protected within the structure of the soilaggregates (Cambardella and Elliott, 1992a; Mether-ell, 1992).

The greatest effects of cultivation on the nutrientand microbial characteristics of soil are observed inthe C- and N-enriched small macroaggregate fraction(250-2000 urn) (Tisdall and Oades, 1980a,b; Dor-maar, 1983; Elliott, 1986; Gupta and Germida, 1988;Cambardella and Elliott, 1993). Soil microorganisms,especially fungi, may play an important role in theformation of these macroaggregates (Tisdall and Oades,1982; Molope et al., 1987; Gupta and Germida, 1988).Microbial mucilage and polysaccharides are thoughtto be involved in the binding together of microaggre-gates into macroaggregates (Harris et al., 1963; Tis-dall and Oades, 1979; Foster, 1981; Molope et al.,1987). Transient organic matter (Tisdall and Oades,1982) has been suggested to exist between and on thesurfaces of microaggregates that are protected insidemacroaggregates (Elliott, 1986; Gupta and Germida,1988) and hypothesized to be the major organic matterpool depleted as a result of cultivation (Elliott, 1986).The purpose of the research described here was toisolate this fraction.

The movement of material into and out of SOMfractions can be mathematically described with sim-ulation models but, to determine how well these modelsdescribe nature, one must be able to selectively isolatemeaningful organic matter fractions from soil andcharacterize them. Historically, most scientists study-ing the nature of organic matter have utilized chemicalextractants to fractionate SOM (Stevenson and Elliott,1989). However, chemical extracts of the soil are notclearly related to the dynamics of organic matter innatural and cultivated systems (Jenkinson, 1971; Oadesand Ladd, 1977; Duxbury et al., 1989) because theyextract SOM that may be physically protected frommicroorganisms and not readily available for decom-position (Elliott and Cambardella, 1991; Elliott et al.,1992).

Physical fractionation techniques can be less de-structive and more selective. Results obtained fromphysically separated soil fractions may relate moredirectly to the structure and function of SOM in situ(Christensen, 1992) than chemical extracts. Sonica-tion has been used extensively to disperse soil priorto physical fractionation (McKeague, 1971; Turche-nek and Oades, 1979; Anderson et al., 1981; Tiessenand Stewart, 1983; Christensen, 1985; Catroux andSchnitzer, 1987; Gregorich et al., 1988, 1989; Angersand Mehuys, 1990). Sequential fractionation proce-dures that further separate ultrasonically dispersed sizefractions using density flotation have been success-

Abbreviations: SOM, soil organic matter; ELF, enriched labilefraction; POM particulate organic matter; BSD, effective sphericaldiameter; ANOVA, analysis of variance.

123

124 SOIL SCI. SOC. AM. J., VOL. 58, JANUARY-FEBRUARY 1994

Table 1. Selected chemical and physical properties of cultivatedsoils (0-20-cm depth) from Sidney, NE, July 1990.t

Cultivationpractice

Bare fallowStubble mulchNo-till

C

15.7a17.3a

N. g/kg& "&

: 1.29b1.46a1.51a

C/N

10.4clO.Sbc11. 4a

Sando.

40a38a36a

Clay

30a30a31a

f Data adapted from Cambardella and Elliott (1992a) from same site.t Values in the same column followed by the same letter are not

significantly different at P > 0.05 according to Fisher's LSD meanseparation test.

fully used to isolate SOM fractions (Turchenek andOades, 1979; Tiessen and Stewart, 1983).

The greatest potential problem associated with theuse of sonication for soil fractionation is the redistri-bution of organic matter among size fractions, whichcan complicate efforts to selectively isolate organicmatter fractions from specific sources in the soil andconfuse interpretation of results. The recovered sizefractions, usually obtained by sedimentation, mayconsist of an unknown mixture of free organic matter,primary particles, and organo-mineral particles. Sep-aration of size fractions using sedimentation is basedon Stokes' law that allows the calculation of the ESDof a settling particle. Exceptions to the assumptionsof Stokes' law are well known; deviations from aspherical shape and particle densities less than thoseused for the calculations of settling times (usually 2.65g/cm3) result in particles larger than desired remainingin the supernatant. Violation of these assumptions ismore likely for organic matter or organo-mineral par-ticles than for primary particles. Errors derived fromviolation of the density assumptions of Stokes' lawcan result in silt-size particles residing in the clay frac-tion or sand-size particles residing in the silt fraction(Elliott and Cambardella, 1991). Clay-associated orsilt-associated organic matter, defined by sedimenta-tion, can be a heterogeneous pool of organic matter,as observed by Balesdent et al. (1988) and Tiessenand Stewart (1983). To conceptually resolve the in-formation obtained from physical fractionation meth-ods, it is important to know the actual sizes of particlesresiding in the size fractions and not just their ESD(Elliott and Cambardella, 1991).

The objectives of this study were to isolate andcharacterize biologically meaningful fractions that areprotected within the aggregate structure of cultivatedgrassland soil and to use this information to enhanceour understanding of how aggregate structure controlsthe turnover of SOM in managed systems. We haveused the specific combination of (i) wet sieving toobtain macroaggregate size fractions, (ii) gentle son-ication of the macroaggregates to produce the con-stituent parts, (iii) sieving of the sonicated soil slurryto isolate size fractions, and (iv) density flotation ofthe size fractions to yield discrete size-density frac-tions, all of which were originally contained withinthe aggregate structure of the soil (Cambardella andElliott, 1992b). We have avoided the problems asso-ciated with sedimentation described above by sepa-rating size fractions with a series of sieves. Thisfractionation sequence is unique in that the sonication

energies used to break apart the aggregates are severalorders of magnitude lower than those commonly usedin SOM studies (Christensen, 1985). The sonicationenergy employed was empirically determined to be theminimum energy required to reduce the macroaggre-gates into the next hierarchical level (Tisdall and Oades,1982) of constituent parts. Our intent was to mini-mally disrupt macroaggregates in order to obtain frac-tions that were meaningful at the spatial scale of thesoil microbial community.

MATERIALS AND METHODSField Sampling

Soil was collected from an experimental site located at theHigh Plains Agricultural Research Laboratory near Sidney, NE.The soil type is a Duroc loam (a fine-silty, mixed, mesic PachicHaplustoll) developed on mixed loess and alluvium. Selectedcharacteristics of the Duroc loam are given in Table 1 (Cam-bardella and Elliott, 1992a). The site had never been cultivatedprior to 1969, when it was plowed and sown to winter wheat(Triticum aestivum L.). Plow depth at sodbreaking was 20 cm.Three management treatments were established: bare fallow(plowing), stubble mulch (subtill), and no-till. One wheat cropis removed every other year and the alternate year the groundis fallow. Fertilizer is not applied to any of the plots. The barefallow treatment is tilled to a depth of 10 to 15 cm using amoldboard plow in the spring of the fallow year followed bytwo to three cultivator or rotary rod-weeder operations. Stubblemulch fallow is cultivated with 0.9- and 1.5-m sweeps two tofour times a year at a depth of 10 cm, followed by a rotaryrod weeder. Weed control during fallow in the no-till treatmentis accomplished with herbicides (Fenster and Peterson, 1979).

The experimental design is a randomized complete blockwith three field replicates. The three cultivation treatments arerandomly assigned plots within each of the three field repli-cates. The entire design is repeated in an adjacent area of thefield so that the crop and fallow portion of the rotation isrepresented every year instead of every other year. Treatmentplots within field replicates are 8.5 by 46.0 m (Fenster andPeterson, 1979).

We sampled the west rotation of the experiment in late Julyof 1990, which had been in fallow since the summer of 1989.Cores were taken with an 8-cm-diam. steel coring bit to a depthof 20 cm without removing surface residues. One core wasremoved every 4.5 m along the length of each plot, for a totalof 10 cores per plot. The exact horizontal location along thewidth of the plot for each core was randomly located. Tominimize edge effects, a 0.50-m buffer zone was establishedalong the edges of each plot. The soil was gently broken apartby hand in the laboratory and passed through a 2.8-mm sievewhile still moist. The large pieces of stubble and root that hadpassed through the sieve were removed by hand. The sievedsoil was dried overnight at 50 °C, after which the soil takenfrom each plot was composited and stored at 4 °C.

Aggregate SeparationsA 100-g subsample of soil from each plot was wet sieved

through a series of three sieves to obtain four aggregate frac-tions: (i) >2000 /xm (large macroaggregates), (ii) 250 to 2000fjan (small macroaggregates), (iii) 53 to 250 /j,m (microaggre-gates), and (iv) <53 /im (silt- plus clay-size particles) (Fig.1). Soil was capillary-wetted overnight at 4 °C prior to sieving.The capillary-wetted soils were consistently brought up to fieldcapacity plus 5% (w/w), the water content at which maximumaggregate stability is attained for these soils (Hofman and deLeenheer, 1975). The prewetted soil samples were suspendedin room temperature water on the largest sieve for 5 min beforesieving. Aggregate disruption was accomplished by moving

CAMBARDELLA & ELLIOTT: C AND N DYNAMICS OF ORGANIC MATTER FRACTIONS 125

100 g oven-dry soil

T

I2000pm sieve

250 \un sieve

I 53 (am sieve

SMALL MACROAGGREGATE FRACTION(250-2000 pm)

MICROAGGREGATE FRACTION(53-250 pn)

SONICATION10 g/55 ml H O

MACROAGGREGATE

MICROAGGREGATE

COARSE SILT-SIZE

1

I

250 |im sieve

53 urn sieve

20 \ua sieve

SONICATION10 g/55 ml H O

MICROAGGREGATE

COARSE SILT-SIZE

CENTRIFUGATION

9min/100xg

tPellet

FINE SILT-SIZE

tPellet

tSupernatent

| 38 min/2500 x g

tSupernatent

Fig. 1. Size fractionation sequence.COARSE CLAY-SIZE FINE CLAY-SIZE

the sieve 3 cm vertically 50 times during a period of 2 min,being careful to break the surface of the water with each stroke.Material remaining on the sieve was backwashed into a roundaluminum pan (23-cm diam.) and dried at 50 °C overnight ina forced-air oven. Soil plus water that passed through the sievewas poured onto the next finer sieve size and the process re-peated (Elliott, 1986). The smallest fraction (silt plus clay)

was centrifuged 10 min at 2500 x g and the pellet backwashedto a round aluminum pan and dried overnight at 50 °C. Thedried aggregate size fractions were weighed and stored in wide-mouth vials at room temperature.

Prior to chemical analysis, plant residue and roots that werelarger than =1 mm in length were removed by hand with aforceps from subsamples of the aggregate-size fractions. The


aggregates were ground in a mortar and pestle and subsampledto determine total organic C (Snyder and Trofymow, 1984)and total Kjeldahl N (Nelson and Sommers, 1980) by wetoxidation.

Intraaggregate Soil Organic Matter FractionsWe disrupted small macroaggregates (250-2000 /xm) and

microaggregates (53-250 jum) into their constituent parts usinga probe-type ultrasonic unit (Ultrasonics Heat Systems ModelW-375). The probe output power was calibrated by measuringthe heating and cooling temperature changes produced by son-icating a known mass of water for a specific time (North,1976). The energy imparted to the known mass of water wasdetermined to be 6.87 ± 0.09 J/s with the sonicator set at15% of maximum output power on the meter. The sonicationtime was adjusted to produce 1236 J (22.5 J/mL) of sonicationenergy. This energy level was empirically determined to bethe minimum energy level required for complete breakdownof small macroaggregates (250-2000 jum) into sand- and silt-size microaggregates, nonassociated organic matter, and pri-mary particles.

Aggregate disruption was accomplished by suspending 10 gof aggregates in 55 mL of room-temperature water and im-mersing the ultrasonic probe to a depth of 3 to 5 mm into thesoil suspension. The disrupted soil suspensions were passedthrough a series of sieves in order to separate size fractionsthat were contained within the structure of the original aggre-gates. Four size fractions were isolated from the sonicatedsmall macroaggregates: (i) 250 to 2000 /nm, (ii) 53 to 250 ju,m,(iii) 20 to 53 fj,m, and (iv) <20 fan. The three smaller sizefractions were also isolated from sonicated microaggregates.Fine-silt- (100 x g for 9 min) and clay-size material (2500 xg for 38 min) were isolated by sequential centrifugation (mod-ified from Ladd et al., 1977) (Fig. 1). Material <20 ju,m insize was determined to be 96% fine-silt size (2-20jam) and4% coarse-clay size (0.2-2 fim) on a mass basis. Less than0.5% of the total soil C and N was associated with the coarse-clay-size material. There was no fine-clay-size material pro-duced by the low sonication energies used in this study. Allsize fractions were dried overnight at 50 °C in a forced airoven .

Density separations were carried out by suspending 0.75-gsubsamples of the aggregate size fraction in 15 mL of sodiummetatungstate (Oades, 1989) adjusted to a density of 1.85,2.07, or 2.22 gVcm3 (Fig. 2). Minor deviations in the densityof the heavy liquid significantly affect the amount of organicC and N in organic matter fractions (Christensen, 1992). Wetested a range of densities, in the laboratory in order to deter-mine which densities yielded size-density fractions with thehighest percentage of total soil C and N for the sonicated sizefractions. The suspended soil was evacuated for 10 min undera 186 kPa vacuum to remove entrapped air from the soil porespace. For simplicity, we will refer to the isolated organo-mineral complexes in the size-density fractions as organic mat-ter, but acknowledge that the organic matter in the fractions isassociated with the soil mineral fraction.

Organic matter that was equal to or lighter than the liquidin which it was suspended floated to the top after sitting over-night at room temperature. This organic matter was removedby aspiration, washed several times with deionized water, trappedon a preashed (400 °C) glass fiber filter (GF/A, 5.5-cm diam.,Whatman) and dried overnight at 50 °C. The organic matterthat sank in the 2.22 g/cm3 density liquid was also isolated inthe same fashion. Total organic C (Snyder and Trofymow,1984) and N (Nelson and Sommers, 1980) were estimated bywet oxidation of the filter plus adhering organic matter. So-dium metatungstate does not interfere with the digestion pro-cedures.

Total organic C or N was expressed as the total amount ofC or N in the size-density fraction per gram of original soil.

fp.75g Oven-Dry Size FractioTT)l Na-metatungstate

Floaters

Sinkers

LIQUID DENSITY1.85 g/cm3 2.07g/cm3 2.22g/cm3

IRT OVERNIGHTASPIRATE

| FLOATERS] Filter[SINKERS]

JRinse 3x HaO

•JOven-Dry /<*'«» Fiber Filterl Overnight (50=C)

I Organic Material J

TOTAL ORGANIC CARBONor

TOTAL ORGANIC NITROGEN

Fig. 2. Densiometric fractionation sequence.

Data could not be expressed per unit weight of size-densityfraction because the amount of contaminating metatungstatecould not be accurately determined. The organic C or N con-tent of the two middle density (1.85-2.07 and 2.07-2.22 g/cm3) fractions was calculated by subtracting the amount oforganic C or N in the density fraction from that in the nextlighter density fraction (Cambardella and Elliott, 1992b).

Potentially mineralizable N was estimated on selected ag-gregate-derived size-density fractions and for intact aggregatefractions. Comparison of specific rates of mineralization forintact aggregates with those determined for size-density frac-tions provides information on the degree of physical protectionfrom decomposition afforded to the organic matter by the soilstructure. The flotation and separation procedures for isolatingmaterial for laboratory incubation were the same as describedin the preceding paragraph except size-density fractions werenot dried prior to incubation. The moist size-density fractionadhering to the glass fiber filter was placed in 30 mL of an N-free mineral salts solution adjusted to pH 7.0 (Catroux andSchnitzer, 1987) in a wide-mouth 250-mL Erlenmeyer flask.The incubation chambers were inoculated with 1 mL of soilsuspension (0.001 g preincubated moist soil/mL deionized H2O),closed with neoprene rubber stoppers, and incubated at 26 °Cfor 28 d on an orbital shaker rotating at 56 rpm (Catroux andSchnitzer, 1987). Rotating the aggregates in liquid culture at56 rpm for 28 d did not result in a significant loss of structuralintegrity for this soil. We calculated the amount of O2 availablein the head space of the incubation flasks to be =2500 ^imol.The metabolic demand of the size-density fractions did notexceed 50 ^imol of O2 during the course of a 28-d incubation.Therefore, estimates of the mineralization potential were as-


Table 2. A comparison of tillage effects on the amount oforganic C and N in aggregate-size fractions in surface soil(0-20 cm).t

Table 4. The amount of organic N in small-macroaggregate-derived size-density fractions.

Cultivationpractice



>2000/tm

0.70b:f1.09ab1.38a

0.07bO.lOab0.13a

i 250-2000 /tm 53-250 /tm——— g/kg original soil ——

Carbon6.77b 3.72a8.60ab 3.60a

10.30a 3.80aNitrogen

0.66b 0.37a0.84ab 0.37aI.Ola 0.37a

<53 fan

2.37a1.68a1.29a

0.21a0.16a0.13a

t Data adapted from Cambardella and Elliott (1993) from same site,t Values in the same column followed by the same letter are not

significantly different at P > 0.05 according to Tukey's HSD meanseparation test.

Table 3. The amount of organic C in small-macroaggregate-derived size-density fractions.

Densityg/cm3

<1.851.85-2.072.07-2.22

>2.22

<1.851.85-2.072.07-2.22

>2.22

<1.851.85-2.072.07-2.22

>2.22

250-2000 fan 53-250 tim 20-53 /tm 2-20 /tin—————————— % of total soil C ———————————

Bare fallow

1.50ct

0.38c0.3 Id

1.38ct

0.28de0.35d

3.11eft

2.02ef0.23i

4.64b1.67cO.lSd1.09c

Stubble mulch4.38b1.69c1.07c1.35cNo-till4.66cd3.45de1.43g1.52fg

0.73cd1.94c4.90b7.13b

1.52c1.51C5.44b8.45ab

0.94gh0.84g8.10b9.52bc

O.lSd0.25d

20.4a1.91c

O.lOe0.92c

17.3a9.75a

0.072i0.70h

18.2a6.13bc

t Values within each tillage matrix followed by the same letter are notsignificantly different at P > 0.05 according to Tukey's HSD meanseparation test.

$ There was no detectable organic C in this fraction.

sumed to be aerobic. Baseline mineral N values were deter-mined by extracting duplicate moist size-density fractions with15 mL of 2 M KC1. After 28 d, the incubated organic matterfractions were extracted in 2 M KC1 and inorganic N (NH^-and NOj-N) in the filtrate was determined using a Lachatflow-injection system (Lachat Instruments, Milwaukee, WI).

Statistical analysis of the data was conducted using the SASstatistical package (SAS Institute, 1990) for analysis of vari-ance (ANOVA/GLM). The data were log-transformed andgrouped by aggregate size (Tables 2 and 3) or by tillage andaggregate size (Tables 4, 5, 6, 7, and 8). Main and interactiveeffects were compared using Tukey's procedure for pairwisecomparison of means at a 0.05 level of significance (Steel andTorrie, 1980).

RESULTS AND DISCUSSIONBare fallow soil had significantly less total organic C

and N than stubble mulch and no-till soils, which werenot statistically different in their total organic C and Ncontents (Table 1). Six percent of the soil, averaged acrosstreatments, was found in the large macroaggregate sizeclass (>2000 jam). Small macroaggregates (250-2000

Densityg/cm3

<1.851.85-2.072.07-2.22

>2.22

<1.851.85-2.072.07-2.22

>2.22

<1.851.85-2.072.07-2.22

>2.22

250-2000 /i

1.40det

l.llc1.17d

2.00ef*

0.57ij1.25gh

1.81det

0.65fgl.OSfg

m 53-250 /tm——— % of total

Bare fallow2.98cdl.SOf1.97g2.50cd

Stubble mulch3.85cd2.62e0.5 Ij2.69de

No-till4.21c2.90cd0.70g2.70cd

20-53 /tmsoilN

1.23de1.32e5.64bc5.57b

2.05ef0.83W5.12c9.32b

1.92d1.14ef7.04b9.59b

2-20 /tm

l.OSe0.44h8.47a6.90ab

t1.70fg

15.9a10.6b

1.79de24.8a9.44b

t Values within each tillage matrix followed by the same letter are notsignificantly different at P > 0.05 according to Tukey's HSD meanseparation test.

$ There was no detectable organic N in this fraction.

Table 5. The effect of tillage on the amount of organic C andN in the enriched labile fraction (ELF) and in microaggregate-derived fine-silt-size particles (density = 2.07-2.22 g/cm3).

Cultivationpractice


Small-macroaggregatederivedt

C Nmg/kg

- original soil -2670at llOc2780a 233b3020a 405a

C/N

24.311.97.5

Microaggregate derivedC N

mg/kg- original soil -

1420a 92a973b 99a787c 79b

C/N

15.49.8

10.0t ELF fraction.j Values in the same column followed by the same letter are not


fj.m) contained 49, 55, and 60% of the soil on a dry-weight basis for the bare fallow, stubble mulch, and no-tillage treatments, respectively. The amount of organicC and N associated with small macroaggregates washighest in the no-till soil and lowest in the bare fallowsoil (Table 2). This pattern was also observed in largemacroaggregates, although the amount of C and N wasan order of magnitude lower than for small macroaggre-gates (Table 2). The organic C and N contents of mi-croaggregates (53-250 pirn) and silt-plus-clay-size particles(<53 /jon) were not significantly different among thethree cultivation treatments (Table 2). There were nosignificant differences in the sand-free organic C and Nconcentrations of the aggregates within each cultivationtreatment (Cambardella and Elliott, 1993). The distri-bution of organic C and N in the aggregate fractionsappears to be controlled primarily by the amount of soilpresent in the aggregates and not by the organic C andN concentration of the aggregates.

Twenty percent of the total soil organic C and 8.5%of the total soil organic N in bare fallow soil was asso-ciated with fine-silt-size (2-20 /un) particles having adensity of 2.07 to 2.22 g/cm3 (Tables 3 and 4) isolatedfrom small macroaggregates. This fraction consistently


Table 6. A comparison of tillage effects on the concentrationof organic C and N in the enriched labile fraction (ELF)and in microaggregate-derived fine-silt-size particles (density= 2.07-2.22 g/cm3).

Cultivationpractice

Small-macroaggregatederivedt

C NMicroaggregate derived

C Nw.~/lrn nn~..Ann*« «•_«_*:»...


4564a5032a

224b 4306a382b 3892a672a 3626a

279a396a305a

t ELF fraction.i Values in the same column followed by the same letter are not


Table 7. The distribution of organic C and N within organicmatter fractions for cultivated surface soil (0-20 cm).

Cultivationpractice

POMt ELF (POM + ELF)N N


18.419.524.7

16.316.312.2

20.417.318.2

8.515.924.8

38.8 24.836.8 32.242.9 37.0

t Data from Cambardella and Elliott (1992a) from same site. POM,particulate organic matter; ELF, enriched labile fraction.

had the highest percentage of the total soil organic C (F= 52.31, P < 0.0001) and N (F = 17.96, P < 0.0001)associated with it for all the cultivation treatments (Ta-bles 3 and 4) and, because of its properties, will sub-sequently be referred to as the ELF. With reduced intensityof cultivation (bare fallow > stubble mulch > no-till),the percentage of the total soil organic N associated withthe ELF increased from 8.5 to 24.8%. The amount oforganic N associated with no-till ELF was significantlyhigher than that for stubble mulch and bare fallow (Table5), suggesting that no-till soil is particularly efficient atsequestering organic N within the structure of small ma-croaggregates.

Fine-silt-size particles having a density of 2.07 to 2.22g/cm3 were also isolated by sonicating intact microag-gregates. This size-density fraction had significantly higheramounts of organic C (F = 87.90, P < 0.0001) and N(F = 78.71, P < 0.0001) associated with it than anyother microaggregate-derived size-density fraction (datanot shown). However, it had considerably lower amountsof organic C and N than the ELF for all cultivation treat-ments (Table 5).

The amount of organic C or N in the ELF (microgramsof C or N in ELF per gram of original soil) is a functionof the amount of soil in the small macroaggregates andthe concentration of organic C or N (micrograms of Cor N in ELF per gram of small macroaggregates). Anincrease in the amount of organic C or N in the ELF isindicative of an increase in aggregate soil mass, an en-richment of the fraction in C or N, or some combinationof these two processes. An increase in the concentrationof organic C or N in the ELF, however, can only indicatean enrichment of that fraction in C or N, since the con-founding effects of differences in aggregate soil masshave been removed.

The concentration of organic C in the ELF was similaracross treatments (Table 6). Similarly, the concentration

Table 8. Percentage of total N mineralized for the enrichedlabile fraction (ELF), microaggregate-derived fine-silt-sizeparticles (FS; density = 2.07-2.22 g/cm3), and intactaggregates for the three treatments.

N mineralization within fractionsSmall macroaggregates Microaggregates

Cultivationpractice


Intactaggregates

2.8a,Bf1.2a,B2.3a,A

IntactELF aggregates

6.4a,A 0.9a,B5.2a,A 0.4a,B2.5a,A 0.2a,B

FS

9.5a,A3.9a,A5.9a,A

t Values in the same column followed by the same lowercase letterand values in the same row within a tillage treatment and within anaggregate size followed by the same uppercase letter are notsignificantly different at P > 0.05 according to Student's f-test.

of organic C and N in fine-silt-size particles (density =2.07-2.22 g/cm3) isolated from inside microaggregatesdid not vary with cultivation treatment. No-till ELF,however, had a higher concentration of organic N thanbare fallow or stubble mulch ELF (Table 6).

No-till ELF is enriched in organic N when comparedwith the other cultivation treatments. One explanationfor this observation is the accumulation of fungal hyphaldebris in treatments where residues are left on the soilsurface. It has been reported that fungi dominate micro-bial communities in no-till systems (Holland and Cole-man, 1987; Doran, 1980; Hendrix et al., 1986). Microbialbiomass, however, can account for only 2 to 6% of totalsoil N under most conditions (Brookes et al., 1985). Thecell walls of many fungal hyphae contain chitin, a pol-ymer of N-acetyl glucosamine, which has a C/N ratio of=7 (Muzzarelli, 1977). Fungal cell wall debris may haveaccumulated in no-till ELF resulting in the observed highpercentage of total soil N in this fraction. We suggestthat chitin, encrusted with mineral particles, along withother microbial byproducts, may be accumulating be-tween microaggregates but within small macroaggre-gates in the no-till treatment. This hypothesis is supportedby the data of Gupta and Germida (1988), who reportedfinding the highest amounts of fungal biomass associatedwith macroaggregates isolated from a Brown Chernoz-emic soil.

The partitioning of organic C and N between the ELFand the POM fraction (Cambardella and Elliott, 1992a)differs for the three cultivation treatments (Table 7). Barefallow soil had the highest proportion of organic C (20.4%)and the lowest proportion of organic N (8.47%) associ-ated with the ELF. The opposite is true for no-till soil,where 18.2% of the organic C and 24.8% of the organicN is associated with the ELF. Balesdent et al. (1990)reported that the accumulation of maize (Zea mays L.)C in topsoil (0-30 cm) was significantly lower for no-till soil than for conventionally tilled soil. Together withthe POM fraction, the ELF accounts for =40% of thetotal soil C in all the treatments. No-till soil had thehighest proportion of the total soil N in the POM andELF fractions, and bare fallow soil had the lowest pro-portion of the total soil N in these two fractions (Table7)-

The percentage of the total N mineralized (microgramsof net mineralized N in the fraction per microgram oftotal N in the fraction) for the ELF was not significantlydifferent for the three cultivation treatments (Table 8).


This suggests that the quality of the organic matter as-sociated with the ELF is similar for the three cultivationtreatments. Averaging across treatments, a larger per-centage of the total soil N was mineralized for the ELF(4.7%) than for intact small macroaggregates (2.1%).There was no difference, however, in the percentage ofthe total N mineralized for no-till ELF compared withintact small macroaggregates isolated from no-till soil(Table 8). The fungal hyphal fragments that we hypoth-esized to be accumulating in no-till ELF may have alower potential for mineralization than other byproductsof microbial metabolism. The accumulation of more la-bile products of microbial metabolism, such as muci-lages, could account for the relatively higher potentialfor mineralization observed for stubble mulch and barefallow ELF compared with intact aggregates (Table 8).

The slower mineralization rate assessed for intact ag-gregates compared with the ELF may indicate that son-ication lysed microbial cells and increased the netmineralization of microbial biomass N. However, lysedmicrobial cell contents would probably be soluble in thewater used during sieving to separate size fractions. An-other explanation for the observed results is that soni-cation may segregate substrates with a high immobilizationpotential and result in decreased N immobilization in theELF. Alternatively, the ELF may be protected from de-composition within the aggregate structure of cultivatedgrassland soils. Intact microaggregates exhibited a lowerpotential for mineralization than fine-silt-size particleshaving a density of 2.07 to 2.22 g/cm3 isolated frommicroaggregates (Table 8). This difference in mineral-ization potential was more pronounced than for the ELF(Table 8), indicating the microaggregate-derived fractionmay be protected to a greater extent than the macroag-gregate-derived ELF. Since pore neck diameters asso-ciated with microaggregates (0.2-2.5-/im-diam. pores)are an order of magnitude smaller than those associatedwith macroaggregates (25-100-/im-diam. pores) (Oades,1984), this observation is consistent with the hypothesisthat the soil pore space is controlling the access of soilmicroorganisms to the organic material protected withinthe aggregate structure (Elliott and Coleman, 1988; El-liott et al., 1980; Gregorich et al., 1989).

Although the fine-silt-size fraction (density 2.07-2.22g/cm3) isolated from microaggregates is enriched in la-bile C and N, we suspect it is essentially inaccessible tothe soil microorganisms and therefore should not be in-cluded in the same pool of organic matter as the ELF.The characteristics of the ELF (i.e., relatively low den-sity, association with soil minerals, relative high min-eralization potential, position within the soil structure)indicate it may be part of the transient organic matterpool suggested to exist between and on the surfaces ofmicroaggregates that are protected inside macroaggre-gates (Tisdall and Oades, 1982). The soil aggregatestructure may be spatially constraining decomposition ofthe ELF and effectively lowering its potential for min-eralization in situ. Therefore, we suggest the ELF, whichis physically occluded inside macroaggregates, is an im-portant part of the slow organic matter pool.

CONCLUSIONSThe fractionation methods used in this research were

developed to specifically isolate organic matter fractionsinvolved in the binding of microaggregates into ma-

croaggregates. We suggest this organic matter is part ofthe transient organic matter pool proposed by Tisdall andOades (1982) and hypothesized to be the major organicmatter pool depleted as a result of cultivation of grass-land soils (Elliott, 1986).

No-till and stubble mulch soil had more organic C andN associated with macroaggregates than bare fallow soil,suggesting that reduced-tillage management can improveor maintain the macroaggregate structure of soil and in-crease the ability of the soil to sequester organic matterrelative to conventional tillage. Gentle sonication of smallmacroaggregates (250-2000 ju.m) isolated from culti-vated grassland soils followed by density flotation re-vealed an organic matter fraction that contained 17 to20% of the total soil C and 9 to 25% of the total soil N.Because of its properties, this fraction is referred to asthe ELF. The amount of organic N associated with theELF increased as the intensity of cultivation decreased,with no-till ELF having the greatest amount of organicN. Averaged across treatments, a larger percentage ofthe total soil N was mineralized from the ELF than fromintact small macroaggregates. Therefore, the ELF is la-bile, but may be physically protected from decomposi-tion within the aggregate structure of the soil.

The ELF is clearly an important pool of soil organicmatter in the grassland soils we studied based on its highconcentration of C and N and its lability. We suspectthat it is the product of microbial activity because it ismineral-associated but has a relatively low density, andbecause of its position within the soil structure. The ELFmay be functioning to bind microaggregates into ma-croaggregates and, as such, may be part of the transientorganic matter pool. Together with the POM fraction,the ELF is probably responsible for the intermediate-term nutrient-supplying capacity of cultivated grassland-derived soils.

ACKNOWLEDGMENTS

Funding for this research was provided by National ScienceFoundation Grant no. BSR-8605191. The authors wish to thankMike Beare, Indy Burke, Vern Cole, and John Doran for theircritical review of this manuscript.

carbon and nitrogen dynamics of soil organic matter fractions from cultivated grassland soils

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