- Email: [email protected]
Contributions to gross N mineralization from 15N-labelled soil macroorganic matter fractions during laboratory incubation
Pergamon
0038-0717(%)ooo77-1
Soil Eio!. Biochem. Vol. 27, No. 12, pp. 1623-1628, 1995 Elsevier Science Ltd. Printed in Great Britain
CONTRIBUTIONS TO GROSS N MINERALIZATION FROM “N-LABELLED SOIL MACROORGANIC MATTER FRACTIONS DURING LABORATORY INCUBATION R. MONAGHAN*
and D. BARRACLOUGH
Department of Soil Science, The University of Reading, Whiteknights, P.O. Box 233, Reading RG6 2DW, England (Accepted 30 April 1995)
Summary-We examined the role of macroorganic matter contributions to rates of gross nitrogen mineralization measured during a laboratory incubation at 15°C. Two r5N-labelling techniques were employed. The first, referred to as the direct method, involved a mirror-image approach whereby labelled or unlabelled macroorganic matter from two different soils was incorporated into soil, and the contribution of this material to gross mineralization was measured using a combination of isotope dilution and pool enrichment procedures. Macroorganic matter was extracted from a sandy loam (Sonning soil) and an organic loam (Black soil). The second technique, a difference method, involved comparing measured gross rates of mineralization in soil with or without the incorporated unlabelled macroorganic matter. During the 66 day incubation, approx. 2.4 and 10.8% of the incorporated macroorganic matter was released from the Sonning and Black soil macroorganic matter fractions, respectively. Following correction to the amounts of macroorganic matter normally recovered in the soils, it was estimated that the Black macroorganic matter fraction contributed from 3 to 21.9% of the gross N mineralization, with a mean of 12%. The contribution of the Sonning macroorganic matter ranged from 0% initially to approx. 14% at the end of the incubation, with a mean of 4.5%. Agreement between the two techniques was reasonable, although the variation encountered in the difference method was considerably less than that observed in the direct method
INTRODUCIION
The mineralization of soil nitrogen is the sum of a number of proce:sses involving the decomposition of various organic substrates. For simplicity, we can divide the source of mineralized soil N into at least four distinct and -measurable pools: current plant residues; the soil microbial biomass (undergoing cryptic growth and including microbial products); light-fraction organic matter; and, by difference, the more stabilized humus. To advance our understanding of the dynamics of N mineralization in soil it is necessary to understand the role of each of these pools in contributing to the gross rate of N mineralization (or acting as a sink for inorganic N) under a range of soil, climate and management conditions. Macroorganic matter is a significant component of light-fraction organic matter in grassland soils. This fraction is generally thought to consist of dead fibrous material in a state of partial decomposition, largely derived from roots, but in principle, not including living root material (Whitehead et al., 1990). Methods for separating this fraction from soil involve dispersion of the soil followed by densimetric separation of the light organic matter from the *Author for correspondence, presently at: Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel, New Zealand.
heavier organic matter which has become more intimately associated with the colloidal material. The specific gravity of the dispersing agent generally ranges from 1.0 to 2.0 gem-’ (Sollins et al., 1984; Warren and Whitehead, 1988; Christensen, 1992). Although it is probable that a continuum exists in properties and composition of fractions between density extremes (Spycher and Young, 1979), the use of densimetric techniques to isolate light-fraction organic matter provides a conceptual framework within which studies of C and N movement through this light-fraction (‘free’ or non-complexed soil organic matter) can be made. We followed the approach of Warren and Whitehead (1988) who used a density fraction of 1.0 g crne3 to isolate light-fraction organic matter (or ‘macroorganic matter’) from 27 soils from England and Wales. They suggested that the macroorganic matter fraction in grassland soils may contribute substantially to the available N. This conclusion was derived from the observation that plant uptake of N was appreciably increased in soils where macroorganic matter was returned, compared with plant uptake in soil where macroorganic matter had been removed. The amounts of macroorganic matter recovered in soils in the South East of England were shown to increase with the number of years under grass, and represented a pool of organic N similar in size to that of living plant root material
1623
1624
R. Monaghan and D. Barraclough Table
1. Chemical
properties
series
Black series
of the 2 grassland Particle size distribution (%)
Soil texture
Soil type Sonning
and physical
matter extraction
PH
Land use
Sandy loam
2 mm-200 /Inl 200-50 /Hn 50-2 pm <2/Hn
36.4 46.3 7.9 10.6
6.2
0.38
5.8
Old grass (> 30 years)
Organic
2 mm-200 pm 200-50 pm 50-2 pm <2pm CaCO,
IO.8 22. I 5.0 30.6 10.2
7.3
0.84
10.3
Old grass ( > 30 years)
loam
(Whitehead et al., 1990). Concentrations of N in this fraction were found to be higher than in the corresponding root material. Our objective was to quantify N mineralization rates from this fraction in the laboratory using two ‘5N-labelling techniques. The first technique uses a mirror-image approach whereby (i) unlabelled (NH,)*S04 is added to soil which contains “Nlabelled macroorganic matter, and (ii) “N-labelled (NH& SO, is added to soil which contains unlabelled macroorganic matter. From the measured gross rates of N mineralization calculated in (ii) and the increase in ammonium pool enrichment measured in (i), the 15N atom% of the N being mineralized can be calculated, and thus the N mineralization solely from the macroorganic matter determined. This technique is referred to subsequently as the “direct method”. The second technique involves measuring gross rates of N mineralization in a set of control soils to which no macroorganic matter has been added. The difference in gross rates between this treatment and (ii) above is also an estimate of the N mineralized from the incorporated macroorganic matter. This technique is referred to subsequently as the “difference method”. MATERIALS
soils used for macroorganic
AND METHODS
Macroorganic matter was obtained from established pasture on two soils of contrasting texture in the spring of 1993. The pastures had received either 15N-labelled (50 atom% 15N) or unlabelled ammonium sulphate fertilizer applied in the spring of 1992 at a rate of 10 g m-*. Both pastures had been fertilized annually with ammonium nitrate at a rate of 120-170 kg N ha-‘. Some chemical and physical properties of the two soils are given in Table 1. The macroorganic matter fraction (Warren and Whitehead, 1988; Whitehead et al., 1990) was separated by dispersing approximately 2 kg portions
of soil in a solution of sodium hexametaphosphate (20 g 1-l). The suspension was then filtered through 0.2 mm Nybolt mesh sieve, the process being repeated three times. The second and third mixing of soil was done in water instead of the sodium hexametaphosphate solution. Material retained on the sieve was then re-suspended in water a final time to allow heavier colloidal material to settle before this snspension was again filtered through the mesh. At this stage visible root fragments were carefully removed from the material using a pair of tweezers. Roots were distinguished from macroorganic matter according to shape and colour. The roots removed were cylindrical in form and coloured milky-white, whereas the macroorganic matter remaining was an amorphous dark brown-coloured material. The organic material retained on the 0.2 mm sieve was then rinsed three times in distilled-deionized water, air-dried at 40°C and ground (0.5 mm). Analysis for ash content was determined by heating a sample of the material at 550°C for 4 h in a muffle furnace; C and N content and 15Nenrichments were determined on a Roboprep C and N Analyzer (Europa Scientific, Crewe, U.K.) linked to a VG622 mass spectrometer. Some chemical characteristics of the two macroorganic matter fractions are given in Table 2. Lignin determination was based on the acid-detergent fibre method (Van Soest and Wine, 1968). Cellulose and hemicellulose was determined by ashing the material remaining following the removal of lignin by oxidation with buffered permanganate solution. Re-incorporation of macroorganic Sowing series soil
The mineralization of macroorganic matter N under laboratory conditions was measured in the sandy textured Sonning soil. Moist soil was taken from the University Farm, Sonning, and transported back to the laboratory where it was sieved (5 mm) and then uniformly mixed. The experiment was set up
Table 2. Chemical characteristics of macroorganic matter extracted from the Sonning and Black series soil. Concentrations are calculated assuming that soil-free macroorganic matter contains 10% ash Soil Sonning Black
matter into the
Nitrogen (%)
Carbon (%)
Cellulose (%)
Lignin (%.)
C-to-N
2.64 2.54
43.1 38.4
36.9 34.9
14.7 II.1
16.3 15.1
Amino N (Irg N g-‘) 6.6 33.5
N mineralization from macroorganic matter using small pots made of PVC drainpipe, sealed at the
base with 0.2 mm Nybolt mesh. The size of these pots was 75 mm dia x 40 mm depth. The macroorganic matter from either the Sonning or Black grassland soil (labelled or unlabelled) was uniformly mixed with 176 g portions (o.d. equivalent) of the moist soil, by mixing soil and macroorganic matter in a plastic bag before being packed into the small pots to the field bulk density. The macroorganic matter was incorporated into the soil at a rate of 200 mg N kg-‘, a rate approx. 4-S-fold greater than was measured in the unamended field soils. There were five treatments in total, two treatments receiving the Sonning macroorganic matter (labelled or unlabelled); two treatments receiving the Black macroorganic matter (labelled or unlabelled); and a set of pots to which no macroorganic matier had been added, referred to as the control. The soil pots were then kept in a constant temperature room at 15°C and maintained at a soil moisture content of 0.33 g gg ‘. Measurement
of glposs rates of mineralization
Gross rates of N mineralization in the control soils and the soils receiving unlabelled macroorganic matter were measured 7 times over the 9 weeks following macroorganic matter incorporation using an isotopic dilution technique (Barraclough, 1991). Briefly, 10mg NH:-N kg-’ soil of labelled ammonium sulphate at 10 atom% 15N was injected into the soil using a syringe and hypodermic needles in a volume of 10 5 ml of solution. There were seven injection points per PVC pot, each point receiving 1.5 ml of solution spread over the 40 mm soil depth. The dilution in the enrichment of the soil ammonium pool was then measured over 4 days following label application, and l;he gross rate of N mineralization calculated from equation 1 (from Barraclough, 1991): A: = A,*/(1 +&/A,)““’
(1)
where A,* and Aic are the “N atom% excess values 3 h and 4 days following label injection, respectively; 0 is the rate of change in the size of the ammonium pool; A0 is the size of the ammonium pool measured 3 h after label injection, and m is the rate of mineralization. These rate estimates were made using 5-fold replication. Estimates of t’he rate of N mineralization solely from the incorporated macroorganic matter were made for the two treatments which received labelled macroorganic matter. These estimates were made for 3 intervals soon after macroorganic matter incorporation, and coincided with the measurements of gross mineralization rates in the treatments receiving unlabelled macroorganic matter (O-4, 6-10 and 14-18 days following incorporation). The theory behind these estimates of direct N mineralization rates has been described in detail by Watkins and Barraclough (1995). Unlabelled ammonium sulphate was injected into these treatments at the same rate as labelled ammonium sulphate was applied to the
1625
unlabelled macroorganic matter treatments, again sampled 3 h (A $) and 4 days (A :) injection.
and after
Analyses
Soils were crumbled and thoroughly mixed in a plastic bag immediately following sampling. Moist soil (50 g) was sub-sampled from each bag and shaken with 200 ml of 1 M KC1 for 1 h before being filtered through glass-fibre filter papers. Extracts were stored at 4°C until determinations of mineral N were made. Ammonium concentrations in the filtered extracts were determined by Flow Injection procedures (Tecator Ltd.) and samples for measurement of “N enrichment were prepared by diffusion of the alkaline extract over 5 days in 250 ml conical flasks sealed with large rubber bungs. Ammonia yielded by this procedure was trapped on small glass microfibre discs (Whatman GF/D) acidified with 10 ~1 of 2.5 M potassium hydrogen sulphate (Brooks et al., 1989). 15N/14Nisotope ratios of these diffusion discs were determined on a VG Micromass 622 mass spectrometer linked to a Roboprep elemental analyzer. Soil moisture was determined gravimetrically by drying sub-samples at 105°C for 24 h. Gross rates of N mineralization were calculated within each treatment by pairing replicates for samplings at A,* and A: according to label recovery. Statistical comparisons between treatments at each sampling date were made using Student’s t-test. RESULTS
The cumulative gross N mineralization in the unlabelled macroorganic matter and control treatments over the 66 day incubation is shown in Fig. 1. Gross rates in the soil receiving incorporated macroorganic matter extracted from the Sonning soil did not differ significantly from those measured in the control soils, with the exception of the rate measured between days 48 and 52 post-incorporation
??
+ Sonning
A + Black 0
s
0
IO
MOM MOM
Control
20
30
40
50
60
v
Days after incorporation Fig. I. Cumulative gross N mineralization in soils with and without the incorporated unlabelled Black and Sonning macroorganic matter fractions.
R. Monaghan and D. Barraclough
1626 Table 3. Soil ammonium
pool sizes and atom/o0 “N values for treatments where “N-labelled was incorporated (SD in brackets: n = 5 unless otherwise stated) mg N kg-’
Macroorganic matter
macroorganic
matter
“N atom%
Rate interval (davs after incoro.)
A”
A.
‘SA,
‘iA.
Sonning
o-4 6-10 14-18
10.6 (0.3) 8.5 (1.5) 9.0 (0.9)
4.0 (0.4) 2.3 (0.6) 3.2 (0.6)
0.399 (0.004) 0.366 (0.004) 0.416 (0.055)
0.456* (0.082) 0.393** (0.020) 0.392” (0.008)
Black
o-4 6-10 14-18
llX(1.1) 6.8 (0.7) 9.5 (0.8)
3.8 (0.3) I.1 (0.2) 2.5 (0.3)
0.534 (0.014) 0.401 (0.050) 0.414 (0.057)
0.590 (0.045) 0.523* (0.177) 0.623’ (0.245)
*, = 4; **n = 3.
(P < 0.05). In contrast, in soil which had the Black macroorganic matter incorporated, consistently higher rates of gross mineralization were observed relative to the control soils, with the exception of the sampling between days 62 and 66 when no significant differences were found between the three treatments. There is some indication of a secondary decomposition phase around day 50, when gross rates increased in all treatments, particularly in the Black macroorganic matter treatment. By the end of the incubation approx. 49.7, 30.8 and 27.4mg N kg-’ soil had been mineralized in the unlabelled Black, Sonning and control treatments, respectively. The sizes and enrichments of the ammonium pool in the treatments which had labelled macroorganic matter incorporated are presented in Table 3. Considerable variability was observed in the enrichments in the ammonium pool measured at the samplings made 4 days after injection of the unlabelled ammonium sulphate (‘5A,). In addition, some replicates contained insufficient N for analysis by mass spectrometry, thus adding to the problem of variability in these treatments. Estimates of the rate of N mineralization directly from the added macroorganic matter are shown in Table 4. As a comparison, rates of N mineralization from the unlabelled macroorganic matter are also shown, as calculated by subtracting rate estimates measured in the control soils from that measured in the unlabelled macroorganic matter treatments (difference method). Expressed as a proportion of gross rates, the rates of N mineralization from the labelled Black macroorganic matter treatment as calculated by the direct method, agree reasonably well with those calculated
Table 4. The proportion of gross nitrogen mineralization derived from the incorporated macroorganic matter for the O-4, 6-10 and 14-18 day intervals Sonning Days after incorporation 2 8 I6
macroorganic matter
Black macroorganic matter
Direct method*
Difference methodt
Direct method*
Difference method?
0.0 0.13 0.0
0 0 0.05
0.38 0.21 0.69
0.49 0.33 0.54
*Calculated from treatments receiving labelled macroorganic matter. Walculated as the difference in gross mineralization rates between soils receiving unlabelled macroorganic matter and the control soils.
as the difference between rates measured in the control soils and in the unlabelled Black macroorganic matter soils (difference method). Agreement between the direct and difference methods where Sonning macroorganic matter was incorporated was poor, however. The reason for this is not clear, but may partly be due to the fact that very little of the added Sonning macroorganic matter was decomposed over the experimental period. The high variability in ‘jN enrichments in the labelled macroorganic matter treatments will also account for much of the discrepancy between the two techniques. DISCUSSION
The relatively high rates of N mineralization observed at the first two sampling dates in Fig. 1 reflect the decomposition of readily-soluble N contained in the incorporated macroorganic matter and the N made available due to the disruption of soil aggregates during sieving and packing of soil into the pots. The secondary phase of decomposition noted between days 48 and 52 following incorporation of the Black macroorganic matter may reflect either the death and re-mineralization of the original biomass population involved in the early stages of decomposition or the growth and activity of a second group of microorganisms capable of acting upon macroorganic matter-N that was protected from the original biomass. Temperature and soil moisture content were constant throughout the incubation and cannot account for the large increase in gross mineralization rates between days 48 and 52. Gross rates of N mineralization also increased slightly in the control soils at this time. The reason why the two sources of macroorganic matter displayed different patterns of N release is unclear. The C-to-N ratios of the two macroorganic matter fractions were approximately similar (16.3 and 15.1 for the Sonning and Black fractions, respectively), as were the N concentrations in each. The Black macroorganic matter fraction had a lower lignin concentration which may account for the much greater release from this fraction. This fraction also contained a higher concentration of soluble amino acid N, as shown in Table 2, but this difference cannot account for the variation in gross N mineralization. Using the differences in gross mineralization
N mineralization from macroorganic matter rates between the unlabelled macroorganic matter treatments and the control soils, an approximate estimate of N mineralization over the 66 day incubation can be made. Using this approach, approx. 2.4 and 10.8% of the incorporated macroorganic matterN was released frclm the Sonning and Black macroorganic matter, respectively. Based on this difference, it is clear that C-to-N ratio alone is not a useful indicator of N release from this fraction in the short term (several months). Fauci and Dick (1994) also observed that net N mineralization over a growing season from animal and green manures did not relate well to the C-to-N ratio of the material alone. Reports in the literature on the role of light-fraction organic matter in the release of mineral N show contrasting results. Janzen (1987) noted that the net amount of N mineralized from surface soils under various rotations of arable crops was significantly related to the content of light fraction (sp. gr < 1S9) organic matter. Conversely, Sollins et al. (1994) found that net mineralization during anaerobic incubation was greater from the heavy fraction than from the light fraction (sp. gr < 1.59) organic matter extracted from seven forest soils. Correlation between net N mineralization and C-to-N ratio was negative for the light fraction but positive for the heavy fraction. Soil type may account for the observed difference in the pattern of N release from the two macroorganic matter fractions. The Black series soil is a poorly-drained calcareous soil high in organic matter and subjegct to periodic waterlogging during the winter. In contrast, the Sonning soil is a freelydrained sandy 1oa.m formed over alluvial gravels and is subject to drought during late spring and summer. It is possible that the macroorganic matter extracted from the Black soil in the spring of 1993 had undergone relatively little decomposition due to the cool, wet soil conditions that were present throughout the winter and early spring, but released significant amounts of N when incubated under the favourable conditions of temperature and moisture used in this study. Working on the same soil type, but under an arable cropping system. Watkins and Barraclough (1995) reported that winter wheat with a C-to-N ratio of 80-to-1 and oilseed rape residues contributed 8-9 and 13-15% of the gross N mineralization rate over a 54 day period, respectively. The pattern of N release for these crop residues was similar to that we found for the Black ma.croorganic matter fraction; initially there was a rapid release of N from the incorporated residue (presumably the water-soluble component) followed by a slower but more consistent release. Estimates of 1V mineralization from the labelled macroorganic matter using the direct method varied considerably, particularly for the Sonning macroorganic matter treatment where relatively little of the incorporated macroorganic matter was decomposed over the 66 day incubation. Ammonium pool enrich-
1627
ments also varied widely in the Black treatment, particularly for the iSA, sampling. Estimates of N mineralization from the unlabelled macroorganic matter using differences in rates of gross mineralization from the control soils (difference method) were, however, more uniform, and statistically significant differences could be measured between gross rates measured in the control and Black treatments. Estimates of net N mineralization rates were misleading due to losses of mineral N (estimated as the difference between gross and net mineralization during the incubation), presumably via denitrification, during the early and later stages of the incubation. The proportions of gross mineralization derived solely from the incorporated macroorganic matter, as calculated using the difference method, are shown in Fig. 2. These values are corrected for the amount of macroorganic matter N recovered in each soil type originally (approx. one-quarter to one-fifth of that added). The proportion of gross mineralization derived from the incorporated Black macroorganic matter ranged from 3 to 21.9%, with a mean of 12%. In contrast, the proportion of gross mineralization derived from the incorporated Sonning macroorganic matter ranged from 0 to 14%, with a mean of 4.5%. During the first 4 weeks of the incubation, the incorporated Sonning macroorganic matter contributed relatively little to the measured gross mineralization rate, but increased gradually thereafter to account for 14% of the gross mineralization rate between days 62-66. Due to the large amounts of macroorganic matter present in grassland soils, it was expected that this fraction would be a major contributor to gross mineralization in the two established grassland soils we examined. As this was not the case, however, it appears that a heavier (or higher mol. wt) fraction is acting as a major contributor of mineralized N in these soils. The absence of growing plants during this incubation would tend to lead to an underestimate of macroorganic matter contributions
10
20
.
Black
??
Sonning
30
40
50
60
Days after incorporation Fig. 2. The proportion of gross N mineralization derived from the macroorganic matter fraction (corrected to the amounts normally recovered in the soil).
1628
R. Monaghan and D. Barraclough
to gross mineralization as dead root material is the origin of this fraction. An alternative concept to the fractionation approach to studies of soil organic matter proposes that N is present in a continuum of an almost infinite number of pools or fractions which release mineral N according to a complex interaction of chemical and physical factors which protects it from breakdown by soil micro-organisms. This latter hypothesis implies, however, that N is mineralized at a rate proportional to the total amount of organic N present. In our study, the macroorganic matter-N incorporated into this soil accounted for 5.4 and 5.2% of the total soil N for the Black and Sonning fractions, respectively; the N released from this incorporated macroorganic matter, however, accounted for 45 and 15% of the total N mineralized during the 9 week incubation, respectively, suggesting that the organic N in these soils was not present as a continuum in terms of decomposability. While it has been demonstrated that the macroorganic matter extracted from the Black soil is a significant source of mineralized N, this fraction may also act as a significant sink of mineral N. Whitehead et al. (1990) noted that concentrations of N in the macroorganic matter fraction were higher than in corresponding root material, suggesting that in the early stages of decomposition N is immobilized by this fraction. Further investigation is required to define the role of this organic fraction as a source and sink of mineral N in soil, particularly following the ploughing of established grass swards. Acknowledgements-We would like to thank Martin Heaps for assistance in i5N analysis. This work was funded by the Natural Environment Research Council.
REFERENCES
Barraclough D. (199 1) The use of mean pool abundances to interpret lSN tracer experiments. I. Theory. Plant and Soil 131, 89-96.
Brooks P. D., Stark J. M., McInteer B. B. and Preston T. (1989) Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society ofAmerica, Journal 53, 1707-I 7 1I. Christensen B. T. (1992) Physical fractionation of soil and organic matter in primary particle size and density separates. Advances in Soil Science 20, l-90. Fauci M. F. and Dick R. P. (1994) Plant response to organic amendments and decreasing inorganic nitrogen rates in soils from a long-term experiment. Soil Science Society of America, Journal 58, 134-l 38.
Janzen H. H. (1987) Soil organic matter characteristics after long-term cropping to various spring wheat rotations. Canadian Journal of Soil Science 67, 845-856.
van Soest P. J. and Wine R. H. (1968) Determination of hgnin and cellulose in Acid-Detergent Fibre with permanganate. Journal of the Association of Oficial . Agrict&ural
Chemists 51, 780-785.
Sollins P.. Snvcher G. and Glassman C. A. (1984) Net nitrogen mineralization from light- and heavy-fraction forest soil organic matter. Soil Biology & Biochemistry 16, 31-37.
Spycher G. and Young J. L. (1979) Water-dispersible soil organic-mineral particles. II. Inorganic amorphous and crystalline phases in density fractions of clay-size particles. Soil Science Society of America, Journal 43, 328-332.
Warren G. P. and Whitehead D. C. (1988) Available soil nitrogen in relation to fractions of soil nitrogen and other soil properties. P/am and SoiP 112, 155-165. Watkins N. and Barraclough D. (1995) Gross rates of nitrogen mineralization associated with the decomposition of plant residues. Submitted. Whitehead D. C., Bristow A. W. and Lockyer D. R. (1990) Organic matter and nitrogen in the unharvested fractions of grass swards in relation to the potential for nitrate leaching after ploughing. Plant and Soil 123, 39949.
0038-0717(%)ooo77-1
Soil Eio!. Biochem. Vol. 27, No. 12, pp. 1623-1628, 1995 Elsevier Science Ltd. Printed in Great Britain
CONTRIBUTIONS TO GROSS N MINERALIZATION FROM “N-LABELLED SOIL MACROORGANIC MATTER FRACTIONS DURING LABORATORY INCUBATION R. MONAGHAN*
and D. BARRACLOUGH
Department of Soil Science, The University of Reading, Whiteknights, P.O. Box 233, Reading RG6 2DW, England (Accepted 30 April 1995)
Summary-We examined the role of macroorganic matter contributions to rates of gross nitrogen mineralization measured during a laboratory incubation at 15°C. Two r5N-labelling techniques were employed. The first, referred to as the direct method, involved a mirror-image approach whereby labelled or unlabelled macroorganic matter from two different soils was incorporated into soil, and the contribution of this material to gross mineralization was measured using a combination of isotope dilution and pool enrichment procedures. Macroorganic matter was extracted from a sandy loam (Sonning soil) and an organic loam (Black soil). The second technique, a difference method, involved comparing measured gross rates of mineralization in soil with or without the incorporated unlabelled macroorganic matter. During the 66 day incubation, approx. 2.4 and 10.8% of the incorporated macroorganic matter was released from the Sonning and Black soil macroorganic matter fractions, respectively. Following correction to the amounts of macroorganic matter normally recovered in the soils, it was estimated that the Black macroorganic matter fraction contributed from 3 to 21.9% of the gross N mineralization, with a mean of 12%. The contribution of the Sonning macroorganic matter ranged from 0% initially to approx. 14% at the end of the incubation, with a mean of 4.5%. Agreement between the two techniques was reasonable, although the variation encountered in the difference method was considerably less than that observed in the direct method
INTRODUCIION
The mineralization of soil nitrogen is the sum of a number of proce:sses involving the decomposition of various organic substrates. For simplicity, we can divide the source of mineralized soil N into at least four distinct and -measurable pools: current plant residues; the soil microbial biomass (undergoing cryptic growth and including microbial products); light-fraction organic matter; and, by difference, the more stabilized humus. To advance our understanding of the dynamics of N mineralization in soil it is necessary to understand the role of each of these pools in contributing to the gross rate of N mineralization (or acting as a sink for inorganic N) under a range of soil, climate and management conditions. Macroorganic matter is a significant component of light-fraction organic matter in grassland soils. This fraction is generally thought to consist of dead fibrous material in a state of partial decomposition, largely derived from roots, but in principle, not including living root material (Whitehead et al., 1990). Methods for separating this fraction from soil involve dispersion of the soil followed by densimetric separation of the light organic matter from the *Author for correspondence, presently at: Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel, New Zealand.
heavier organic matter which has become more intimately associated with the colloidal material. The specific gravity of the dispersing agent generally ranges from 1.0 to 2.0 gem-’ (Sollins et al., 1984; Warren and Whitehead, 1988; Christensen, 1992). Although it is probable that a continuum exists in properties and composition of fractions between density extremes (Spycher and Young, 1979), the use of densimetric techniques to isolate light-fraction organic matter provides a conceptual framework within which studies of C and N movement through this light-fraction (‘free’ or non-complexed soil organic matter) can be made. We followed the approach of Warren and Whitehead (1988) who used a density fraction of 1.0 g crne3 to isolate light-fraction organic matter (or ‘macroorganic matter’) from 27 soils from England and Wales. They suggested that the macroorganic matter fraction in grassland soils may contribute substantially to the available N. This conclusion was derived from the observation that plant uptake of N was appreciably increased in soils where macroorganic matter was returned, compared with plant uptake in soil where macroorganic matter had been removed. The amounts of macroorganic matter recovered in soils in the South East of England were shown to increase with the number of years under grass, and represented a pool of organic N similar in size to that of living plant root material
1623
1624
R. Monaghan and D. Barraclough Table
1. Chemical
properties
series
Black series
of the 2 grassland Particle size distribution (%)
Soil texture
Soil type Sonning
and physical
matter extraction
PH
Land use
Sandy loam
2 mm-200 /Inl 200-50 /Hn 50-2 pm <2/Hn
36.4 46.3 7.9 10.6
6.2
0.38
5.8
Old grass (> 30 years)
Organic
2 mm-200 pm 200-50 pm 50-2 pm <2pm CaCO,
IO.8 22. I 5.0 30.6 10.2
7.3
0.84
10.3
Old grass ( > 30 years)
loam
(Whitehead et al., 1990). Concentrations of N in this fraction were found to be higher than in the corresponding root material. Our objective was to quantify N mineralization rates from this fraction in the laboratory using two ‘5N-labelling techniques. The first technique uses a mirror-image approach whereby (i) unlabelled (NH,)*S04 is added to soil which contains “Nlabelled macroorganic matter, and (ii) “N-labelled (NH& SO, is added to soil which contains unlabelled macroorganic matter. From the measured gross rates of N mineralization calculated in (ii) and the increase in ammonium pool enrichment measured in (i), the 15N atom% of the N being mineralized can be calculated, and thus the N mineralization solely from the macroorganic matter determined. This technique is referred to subsequently as the “direct method”. The second technique involves measuring gross rates of N mineralization in a set of control soils to which no macroorganic matter has been added. The difference in gross rates between this treatment and (ii) above is also an estimate of the N mineralized from the incorporated macroorganic matter. This technique is referred to subsequently as the “difference method”. MATERIALS
soils used for macroorganic
AND METHODS
Macroorganic matter was obtained from established pasture on two soils of contrasting texture in the spring of 1993. The pastures had received either 15N-labelled (50 atom% 15N) or unlabelled ammonium sulphate fertilizer applied in the spring of 1992 at a rate of 10 g m-*. Both pastures had been fertilized annually with ammonium nitrate at a rate of 120-170 kg N ha-‘. Some chemical and physical properties of the two soils are given in Table 1. The macroorganic matter fraction (Warren and Whitehead, 1988; Whitehead et al., 1990) was separated by dispersing approximately 2 kg portions
of soil in a solution of sodium hexametaphosphate (20 g 1-l). The suspension was then filtered through 0.2 mm Nybolt mesh sieve, the process being repeated three times. The second and third mixing of soil was done in water instead of the sodium hexametaphosphate solution. Material retained on the sieve was then re-suspended in water a final time to allow heavier colloidal material to settle before this snspension was again filtered through the mesh. At this stage visible root fragments were carefully removed from the material using a pair of tweezers. Roots were distinguished from macroorganic matter according to shape and colour. The roots removed were cylindrical in form and coloured milky-white, whereas the macroorganic matter remaining was an amorphous dark brown-coloured material. The organic material retained on the 0.2 mm sieve was then rinsed three times in distilled-deionized water, air-dried at 40°C and ground (0.5 mm). Analysis for ash content was determined by heating a sample of the material at 550°C for 4 h in a muffle furnace; C and N content and 15Nenrichments were determined on a Roboprep C and N Analyzer (Europa Scientific, Crewe, U.K.) linked to a VG622 mass spectrometer. Some chemical characteristics of the two macroorganic matter fractions are given in Table 2. Lignin determination was based on the acid-detergent fibre method (Van Soest and Wine, 1968). Cellulose and hemicellulose was determined by ashing the material remaining following the removal of lignin by oxidation with buffered permanganate solution. Re-incorporation of macroorganic Sowing series soil
The mineralization of macroorganic matter N under laboratory conditions was measured in the sandy textured Sonning soil. Moist soil was taken from the University Farm, Sonning, and transported back to the laboratory where it was sieved (5 mm) and then uniformly mixed. The experiment was set up
Table 2. Chemical characteristics of macroorganic matter extracted from the Sonning and Black series soil. Concentrations are calculated assuming that soil-free macroorganic matter contains 10% ash Soil Sonning Black
matter into the
Nitrogen (%)
Carbon (%)
Cellulose (%)
Lignin (%.)
C-to-N
2.64 2.54
43.1 38.4
36.9 34.9
14.7 II.1
16.3 15.1
Amino N (Irg N g-‘) 6.6 33.5
N mineralization from macroorganic matter using small pots made of PVC drainpipe, sealed at the
base with 0.2 mm Nybolt mesh. The size of these pots was 75 mm dia x 40 mm depth. The macroorganic matter from either the Sonning or Black grassland soil (labelled or unlabelled) was uniformly mixed with 176 g portions (o.d. equivalent) of the moist soil, by mixing soil and macroorganic matter in a plastic bag before being packed into the small pots to the field bulk density. The macroorganic matter was incorporated into the soil at a rate of 200 mg N kg-‘, a rate approx. 4-S-fold greater than was measured in the unamended field soils. There were five treatments in total, two treatments receiving the Sonning macroorganic matter (labelled or unlabelled); two treatments receiving the Black macroorganic matter (labelled or unlabelled); and a set of pots to which no macroorganic matier had been added, referred to as the control. The soil pots were then kept in a constant temperature room at 15°C and maintained at a soil moisture content of 0.33 g gg ‘. Measurement
of glposs rates of mineralization
Gross rates of N mineralization in the control soils and the soils receiving unlabelled macroorganic matter were measured 7 times over the 9 weeks following macroorganic matter incorporation using an isotopic dilution technique (Barraclough, 1991). Briefly, 10mg NH:-N kg-’ soil of labelled ammonium sulphate at 10 atom% 15N was injected into the soil using a syringe and hypodermic needles in a volume of 10 5 ml of solution. There were seven injection points per PVC pot, each point receiving 1.5 ml of solution spread over the 40 mm soil depth. The dilution in the enrichment of the soil ammonium pool was then measured over 4 days following label application, and l;he gross rate of N mineralization calculated from equation 1 (from Barraclough, 1991): A: = A,*/(1 +&/A,)““’
(1)
where A,* and Aic are the “N atom% excess values 3 h and 4 days following label injection, respectively; 0 is the rate of change in the size of the ammonium pool; A0 is the size of the ammonium pool measured 3 h after label injection, and m is the rate of mineralization. These rate estimates were made using 5-fold replication. Estimates of t’he rate of N mineralization solely from the incorporated macroorganic matter were made for the two treatments which received labelled macroorganic matter. These estimates were made for 3 intervals soon after macroorganic matter incorporation, and coincided with the measurements of gross mineralization rates in the treatments receiving unlabelled macroorganic matter (O-4, 6-10 and 14-18 days following incorporation). The theory behind these estimates of direct N mineralization rates has been described in detail by Watkins and Barraclough (1995). Unlabelled ammonium sulphate was injected into these treatments at the same rate as labelled ammonium sulphate was applied to the
1625
unlabelled macroorganic matter treatments, again sampled 3 h (A $) and 4 days (A :) injection.
and after
Analyses
Soils were crumbled and thoroughly mixed in a plastic bag immediately following sampling. Moist soil (50 g) was sub-sampled from each bag and shaken with 200 ml of 1 M KC1 for 1 h before being filtered through glass-fibre filter papers. Extracts were stored at 4°C until determinations of mineral N were made. Ammonium concentrations in the filtered extracts were determined by Flow Injection procedures (Tecator Ltd.) and samples for measurement of “N enrichment were prepared by diffusion of the alkaline extract over 5 days in 250 ml conical flasks sealed with large rubber bungs. Ammonia yielded by this procedure was trapped on small glass microfibre discs (Whatman GF/D) acidified with 10 ~1 of 2.5 M potassium hydrogen sulphate (Brooks et al., 1989). 15N/14Nisotope ratios of these diffusion discs were determined on a VG Micromass 622 mass spectrometer linked to a Roboprep elemental analyzer. Soil moisture was determined gravimetrically by drying sub-samples at 105°C for 24 h. Gross rates of N mineralization were calculated within each treatment by pairing replicates for samplings at A,* and A: according to label recovery. Statistical comparisons between treatments at each sampling date were made using Student’s t-test. RESULTS
The cumulative gross N mineralization in the unlabelled macroorganic matter and control treatments over the 66 day incubation is shown in Fig. 1. Gross rates in the soil receiving incorporated macroorganic matter extracted from the Sonning soil did not differ significantly from those measured in the control soils, with the exception of the rate measured between days 48 and 52 post-incorporation
??
+ Sonning
A + Black 0
s
0
IO
MOM MOM
Control
20
30
40
50
60
v
Days after incorporation Fig. I. Cumulative gross N mineralization in soils with and without the incorporated unlabelled Black and Sonning macroorganic matter fractions.
R. Monaghan and D. Barraclough
1626 Table 3. Soil ammonium
pool sizes and atom/o0 “N values for treatments where “N-labelled was incorporated (SD in brackets: n = 5 unless otherwise stated) mg N kg-’
Macroorganic matter
macroorganic
matter
“N atom%
Rate interval (davs after incoro.)
A”
A.
‘SA,
‘iA.
Sonning
o-4 6-10 14-18
10.6 (0.3) 8.5 (1.5) 9.0 (0.9)
4.0 (0.4) 2.3 (0.6) 3.2 (0.6)
0.399 (0.004) 0.366 (0.004) 0.416 (0.055)
0.456* (0.082) 0.393** (0.020) 0.392” (0.008)
Black
o-4 6-10 14-18
llX(1.1) 6.8 (0.7) 9.5 (0.8)
3.8 (0.3) I.1 (0.2) 2.5 (0.3)
0.534 (0.014) 0.401 (0.050) 0.414 (0.057)
0.590 (0.045) 0.523* (0.177) 0.623’ (0.245)
*, = 4; **n = 3.
(P < 0.05). In contrast, in soil which had the Black macroorganic matter incorporated, consistently higher rates of gross mineralization were observed relative to the control soils, with the exception of the sampling between days 62 and 66 when no significant differences were found between the three treatments. There is some indication of a secondary decomposition phase around day 50, when gross rates increased in all treatments, particularly in the Black macroorganic matter treatment. By the end of the incubation approx. 49.7, 30.8 and 27.4mg N kg-’ soil had been mineralized in the unlabelled Black, Sonning and control treatments, respectively. The sizes and enrichments of the ammonium pool in the treatments which had labelled macroorganic matter incorporated are presented in Table 3. Considerable variability was observed in the enrichments in the ammonium pool measured at the samplings made 4 days after injection of the unlabelled ammonium sulphate (‘5A,). In addition, some replicates contained insufficient N for analysis by mass spectrometry, thus adding to the problem of variability in these treatments. Estimates of the rate of N mineralization directly from the added macroorganic matter are shown in Table 4. As a comparison, rates of N mineralization from the unlabelled macroorganic matter are also shown, as calculated by subtracting rate estimates measured in the control soils from that measured in the unlabelled macroorganic matter treatments (difference method). Expressed as a proportion of gross rates, the rates of N mineralization from the labelled Black macroorganic matter treatment as calculated by the direct method, agree reasonably well with those calculated
Table 4. The proportion of gross nitrogen mineralization derived from the incorporated macroorganic matter for the O-4, 6-10 and 14-18 day intervals Sonning Days after incorporation 2 8 I6
macroorganic matter
Black macroorganic matter
Direct method*
Difference methodt
Direct method*
Difference method?
0.0 0.13 0.0
0 0 0.05
0.38 0.21 0.69
0.49 0.33 0.54
*Calculated from treatments receiving labelled macroorganic matter. Walculated as the difference in gross mineralization rates between soils receiving unlabelled macroorganic matter and the control soils.
as the difference between rates measured in the control soils and in the unlabelled Black macroorganic matter soils (difference method). Agreement between the direct and difference methods where Sonning macroorganic matter was incorporated was poor, however. The reason for this is not clear, but may partly be due to the fact that very little of the added Sonning macroorganic matter was decomposed over the experimental period. The high variability in ‘jN enrichments in the labelled macroorganic matter treatments will also account for much of the discrepancy between the two techniques. DISCUSSION
The relatively high rates of N mineralization observed at the first two sampling dates in Fig. 1 reflect the decomposition of readily-soluble N contained in the incorporated macroorganic matter and the N made available due to the disruption of soil aggregates during sieving and packing of soil into the pots. The secondary phase of decomposition noted between days 48 and 52 following incorporation of the Black macroorganic matter may reflect either the death and re-mineralization of the original biomass population involved in the early stages of decomposition or the growth and activity of a second group of microorganisms capable of acting upon macroorganic matter-N that was protected from the original biomass. Temperature and soil moisture content were constant throughout the incubation and cannot account for the large increase in gross mineralization rates between days 48 and 52. Gross rates of N mineralization also increased slightly in the control soils at this time. The reason why the two sources of macroorganic matter displayed different patterns of N release is unclear. The C-to-N ratios of the two macroorganic matter fractions were approximately similar (16.3 and 15.1 for the Sonning and Black fractions, respectively), as were the N concentrations in each. The Black macroorganic matter fraction had a lower lignin concentration which may account for the much greater release from this fraction. This fraction also contained a higher concentration of soluble amino acid N, as shown in Table 2, but this difference cannot account for the variation in gross N mineralization. Using the differences in gross mineralization
N mineralization from macroorganic matter rates between the unlabelled macroorganic matter treatments and the control soils, an approximate estimate of N mineralization over the 66 day incubation can be made. Using this approach, approx. 2.4 and 10.8% of the incorporated macroorganic matterN was released frclm the Sonning and Black macroorganic matter, respectively. Based on this difference, it is clear that C-to-N ratio alone is not a useful indicator of N release from this fraction in the short term (several months). Fauci and Dick (1994) also observed that net N mineralization over a growing season from animal and green manures did not relate well to the C-to-N ratio of the material alone. Reports in the literature on the role of light-fraction organic matter in the release of mineral N show contrasting results. Janzen (1987) noted that the net amount of N mineralized from surface soils under various rotations of arable crops was significantly related to the content of light fraction (sp. gr < 1S9) organic matter. Conversely, Sollins et al. (1994) found that net mineralization during anaerobic incubation was greater from the heavy fraction than from the light fraction (sp. gr < 1.59) organic matter extracted from seven forest soils. Correlation between net N mineralization and C-to-N ratio was negative for the light fraction but positive for the heavy fraction. Soil type may account for the observed difference in the pattern of N release from the two macroorganic matter fractions. The Black series soil is a poorly-drained calcareous soil high in organic matter and subjegct to periodic waterlogging during the winter. In contrast, the Sonning soil is a freelydrained sandy 1oa.m formed over alluvial gravels and is subject to drought during late spring and summer. It is possible that the macroorganic matter extracted from the Black soil in the spring of 1993 had undergone relatively little decomposition due to the cool, wet soil conditions that were present throughout the winter and early spring, but released significant amounts of N when incubated under the favourable conditions of temperature and moisture used in this study. Working on the same soil type, but under an arable cropping system. Watkins and Barraclough (1995) reported that winter wheat with a C-to-N ratio of 80-to-1 and oilseed rape residues contributed 8-9 and 13-15% of the gross N mineralization rate over a 54 day period, respectively. The pattern of N release for these crop residues was similar to that we found for the Black ma.croorganic matter fraction; initially there was a rapid release of N from the incorporated residue (presumably the water-soluble component) followed by a slower but more consistent release. Estimates of 1V mineralization from the labelled macroorganic matter using the direct method varied considerably, particularly for the Sonning macroorganic matter treatment where relatively little of the incorporated macroorganic matter was decomposed over the 66 day incubation. Ammonium pool enrich-
1627
ments also varied widely in the Black treatment, particularly for the iSA, sampling. Estimates of N mineralization from the unlabelled macroorganic matter using differences in rates of gross mineralization from the control soils (difference method) were, however, more uniform, and statistically significant differences could be measured between gross rates measured in the control and Black treatments. Estimates of net N mineralization rates were misleading due to losses of mineral N (estimated as the difference between gross and net mineralization during the incubation), presumably via denitrification, during the early and later stages of the incubation. The proportions of gross mineralization derived solely from the incorporated macroorganic matter, as calculated using the difference method, are shown in Fig. 2. These values are corrected for the amount of macroorganic matter N recovered in each soil type originally (approx. one-quarter to one-fifth of that added). The proportion of gross mineralization derived from the incorporated Black macroorganic matter ranged from 3 to 21.9%, with a mean of 12%. In contrast, the proportion of gross mineralization derived from the incorporated Sonning macroorganic matter ranged from 0 to 14%, with a mean of 4.5%. During the first 4 weeks of the incubation, the incorporated Sonning macroorganic matter contributed relatively little to the measured gross mineralization rate, but increased gradually thereafter to account for 14% of the gross mineralization rate between days 62-66. Due to the large amounts of macroorganic matter present in grassland soils, it was expected that this fraction would be a major contributor to gross mineralization in the two established grassland soils we examined. As this was not the case, however, it appears that a heavier (or higher mol. wt) fraction is acting as a major contributor of mineralized N in these soils. The absence of growing plants during this incubation would tend to lead to an underestimate of macroorganic matter contributions
10
20
.
Black
??
Sonning
30
40
50
60
Days after incorporation Fig. 2. The proportion of gross N mineralization derived from the macroorganic matter fraction (corrected to the amounts normally recovered in the soil).
1628
R. Monaghan and D. Barraclough
to gross mineralization as dead root material is the origin of this fraction. An alternative concept to the fractionation approach to studies of soil organic matter proposes that N is present in a continuum of an almost infinite number of pools or fractions which release mineral N according to a complex interaction of chemical and physical factors which protects it from breakdown by soil micro-organisms. This latter hypothesis implies, however, that N is mineralized at a rate proportional to the total amount of organic N present. In our study, the macroorganic matter-N incorporated into this soil accounted for 5.4 and 5.2% of the total soil N for the Black and Sonning fractions, respectively; the N released from this incorporated macroorganic matter, however, accounted for 45 and 15% of the total N mineralized during the 9 week incubation, respectively, suggesting that the organic N in these soils was not present as a continuum in terms of decomposability. While it has been demonstrated that the macroorganic matter extracted from the Black soil is a significant source of mineralized N, this fraction may also act as a significant sink of mineral N. Whitehead et al. (1990) noted that concentrations of N in the macroorganic matter fraction were higher than in corresponding root material, suggesting that in the early stages of decomposition N is immobilized by this fraction. Further investigation is required to define the role of this organic fraction as a source and sink of mineral N in soil, particularly following the ploughing of established grass swards. Acknowledgements-We would like to thank Martin Heaps for assistance in i5N analysis. This work was funded by the Natural Environment Research Council.
REFERENCES
Barraclough D. (199 1) The use of mean pool abundances to interpret lSN tracer experiments. I. Theory. Plant and Soil 131, 89-96.
Brooks P. D., Stark J. M., McInteer B. B. and Preston T. (1989) Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society ofAmerica, Journal 53, 1707-I 7 1I. Christensen B. T. (1992) Physical fractionation of soil and organic matter in primary particle size and density separates. Advances in Soil Science 20, l-90. Fauci M. F. and Dick R. P. (1994) Plant response to organic amendments and decreasing inorganic nitrogen rates in soils from a long-term experiment. Soil Science Society of America, Journal 58, 134-l 38.
Janzen H. H. (1987) Soil organic matter characteristics after long-term cropping to various spring wheat rotations. Canadian Journal of Soil Science 67, 845-856.
van Soest P. J. and Wine R. H. (1968) Determination of hgnin and cellulose in Acid-Detergent Fibre with permanganate. Journal of the Association of Oficial . Agrict&ural
Chemists 51, 780-785.
Sollins P.. Snvcher G. and Glassman C. A. (1984) Net nitrogen mineralization from light- and heavy-fraction forest soil organic matter. Soil Biology & Biochemistry 16, 31-37.
Spycher G. and Young J. L. (1979) Water-dispersible soil organic-mineral particles. II. Inorganic amorphous and crystalline phases in density fractions of clay-size particles. Soil Science Society of America, Journal 43, 328-332.
Warren G. P. and Whitehead D. C. (1988) Available soil nitrogen in relation to fractions of soil nitrogen and other soil properties. P/am and SoiP 112, 155-165. Watkins N. and Barraclough D. (1995) Gross rates of nitrogen mineralization associated with the decomposition of plant residues. Submitted. Whitehead D. C., Bristow A. W. and Lockyer D. R. (1990) Organic matter and nitrogen in the unharvested fractions of grass swards in relation to the potential for nitrate leaching after ploughing. Plant and Soil 123, 39949.