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Studies of nitrogen immobilization and mineralization in calcareous soils—I
sari mi.
B~O&W. vol. 9, pp. 309 to 318. Pergamon
STUDIES
Press 1977. Printed in
GreatBritain
OF NITROGEN
LIBERALIZATION
IMMOBILIZATION
IN CALCAREOUS
DISTRIBUTION OF IMMOBILIZED NITROGEN SOIL FRACTIONS OF DIFFERENT PARTICLE SIZE AND DENSITY
AND
SOILS-I. AMONGST
J. N. LADD, J. W. PARSONS* and M. AMATO Division of Soils, CSIRO, Glen Osmond, South Australia 5064 (Accepted 6 December 1976) Summary-’
$NO; was jmmobiliz~d in a calcareous clay and a calcareous sandy soif during incubation of each soil with glucose and wheat straw. Changes in the distribution of immobilized “N amongst soil extracts and soil fractions of different particle size and density were determined during periods of net N immobilization. The nature of the organic-C amendment, but not soil type, significantly influenced both the distribution of the immobilized 15N and the pattern of changes of the organic-r5N of soil fractions with time. In straw-amended soils, approx. 20% of the organic-‘“N became associated with a light fraction, sp. gr. < 1.59, the remainder becoming distributed mainly amongst the silt and clay fractions. In glucoseamended soils, very little (< 1.2%) of the “N was immobilized in the light fraction, sp. gr. <1.59, most being rapidly distributed amongst the silt and clay fractions. During a period of complete immobilization. organic-15N was transferred from the fine clay to the silt and coarse clay fractions. Silt, coarse clay and fine clay components from glucose-amended soils sampled at the end of the net immobilization phase were further fractionated densimetrically into light (sp. gr. <2.06) and heavy (sp. gr. >2.06) subfractions. The organic- 15N of respective light subfractions accounted for 4344% of the total organic-r5N of the silt, l-9% of that of the coarse clay and 19-21”/, of that of the fine clay fractions.
INTRODUCTION Studies of the relative availability of nitrogenous components of soils have mostly been chemically based. Of the chemically “defined” com~nents, acid-hydrolyzable amino acid-N appears to contribute most to inorganic-N if judged by the amounts of N lost from this organic-N pool during periods of net mineraliza-
tion. However, measurements of the percentage decrease of N from soil chemical fractions show that no component is consistently of greater biological availability (Keeney and Bremner, 1944, 1966; Broadbent, 1968). A clearer definition of “active” and “passive” nitrogenous pools formed in soils, in situ, may be achieved if fractionation procedures were to permit direct analysis of the nitrogen contents of recognizable biological entities (Jansson, 1967; Jansson and Persson, 1968; Persson, 1968). Oades and Ladd (1977) have suggested that suitable components may be (i) the soil biomass, separated according to organism size and its association with either larger plant fragments or with inorganic constituents of the soil or (ii) extracelluiar or lytic nitrogenous products, separated as soiubfe compounds of different mol. wts. or as insoluble constituents, either free or bonded to inorganic or organic soil colloids. In these respects a more advantageous approach may be to fractionate soils by *On leave from Department of Soil Science, University of Aberdeen. Aberdeen. Scotland. 309
physical methods before chemical analysis. Physical methods used have mainly involved fractionation of dispersed soils according to particle size (Chichester, 1970; Ladd and Paul, 1973; McGill et al., 1975) and density (Greenland and Ford, 1964; Ford and Greenland, 1968). Ford and Greenland (1968) concluded that the N of a light fraction, the partially humified material separated from soils by flotation in a solvent of sp. gr. 2.0, is a relatively labile component of soil organic-N. McGill et nl. (1975) have shown that transfer of N through fractions of different particle size and of pyrophosphate extracts took place during net N immobilization. Ladd and Paul (1973) using a modified and probably less destructive fractionation procedure, found that bicarbonate extracts of soil and also material of diameter ~0.2 pm, contained nitrogenous compounds (arid-hydrolyzable amino acid-N) which were readily formed and partly decomposed during the period of net N immobilization. This paper is the first of a series dealing with N immobilization and mineralization in calcareous soils. The aim is to describe eventually the transfer of organioN between components of biological significance, such as those mentioned above, and to measure the contribution of each component to the mineral-N pool. Although both immobilization and mineralization reactions take place concurrently, the initial approach has been to relate changes in the organic-N of several soil components to the net changes of mineral-N of soils during different incuba-
J. N. LAIX). J. W. PARSONS and M. AMATO
3 10
tion periods. In this study, a calcareous clay and a calcareous sandy soil were incubated with isNO; and glucose or wheat straw. The procedure of Ladd and Paul (1973) has been extended to permit measurements of the changes in the distribution of 15N amongst both soluble extracts and also particles of different size and density, during periods of net N immobilization. The changes of fraction organic-15N which accompany net N mineralization are described by Ladd et al. (1977).
MATERIALS
AND
METHODS
soils (50g dry soil equivalent) wcrc each incubated in duplicate with ’ 4C-glucose (3 &i) plus KN 03. Moist CO,-free air was drawn through the incubation flasks (250 ml) and evolved CO, was trapped in 20 ml 1 M LiOH. The traps were replaced every 0.552 days and all their contents transferred (50 ml. final volume). Total-CO, evolved was determined after titrating residual alkali of a 20 ml sample with standard HCl (Tinsley er ul.. 1951). 14C02 evolution was calculated from the radioactivity of a 0.5 ml subsample mixed with 20 ml scintillation solvent (Turner, 1968). Flasks without soil or substrate were included as controls. Soil fructionution
Soils A calcareous sand and a calcareous clay soil (Table 1) were sampled to 1Ocm depth from under mixed pastures at Two Wells and Northfield, South Australia, respectively. Bulked samples were dried at 35°C freed of coarse plant material, ground, sieved (2mm) and stored at 22°C. Soil incubations (I) Both soils were incubated in duplicate with K15N0,, with glucose or wheat straw as energy sources. 15NO; (0.1 mg N g-i dry wt. soil, 10.18 atom 0Aexcess ’ 5N) and glucose (2.5 mg C g-’ dry wt. soil) were supplied in solution, the straw as ground material (10 mg straw or 4.36 mg straw-C g- ’ dry wt. soil). The added 15N0; was assumed to have equilibrated immediately with the small amounts of unlabelled NO; of the soils (Table 1) and of the wheat straw (0.6 pg N g- ’ dry wt. soil equivalent). Incubations were carried out at 25°C in 31 Erlenmeyer flasks, each containing initially 1.25 kg airdried soil moistened with the appropriate nutrient solutions. The flasks were connected in parallel in aeration trains through which moist, NH,-free air was drawn for 160 days. Moisture contents of the sandy and clay soils were maintained at approximately 9.1% and 18.0’%, respectively, equivalent to holding capacities 22-24% of the soils’ water (Bremner and Shaw, 1958). When appropriate, the soils were briefly mixed before removing 50 g or 150 g subsamples, of which 5 g was immediately extracted for NHf-N and NO;-N determinations and 10 g was dried (90°C 2 h) for total-N measurements. The remainder of the sample was frozen in liquid N, then stored at - 15°C until fractionated or further analysed. (2) On a smaller scale, moistened sandy and clay
Soil samples from the larger scale experiments were fractionated into components of different particle size and density (Fig. I) by modified procedures of Ford et ul. (1969) Ladd and Paul (1973) and McGill et al. (1975).
Extractable carbohydrate was determined as glucose equivalents by shaking previously-frozen soil subsamples with 5 ~01s. 0.5 N KzS04 for IOmin, reacting samples of the extract with anthrone reagent (Oades, 1967) and relating absorbances at 630nm to those obtained with similarly-treated glucose standards. NHf-N and NOT-N of 2 M KC1 and water extracts were determined by distillation with MgO and Devarda’s alloy-MgO mixture (Bremner, 1965b). Total-N of dried. ground soils was determined by Kjeldahl digestion, after pretreatment to reduce NO; (Bremner and Shaw, 1958; Bremner, 1965a). After titration with standard 5 mN H2S04, to pH 5.0, duplicate distillates were pooled, “spiked” if necessary with 0.5--l mg unlabelled NH:-N, further acidified to pH 4.0. then dried (Ross and Martin. 1970). Atom ‘I{, i5N abundance was determined in an A.E.I. type MS3 mass spectrometer. Statistical analyses were based on data obtained with soil samples taken during both the net immobilization and mineralization phases. Calculations of the appropriate standard error of difference (S.E.D.) were based on four degrees of freedom. RESULTS
Changes in NOT-N
AND
DISCL’SSION
concentrations
In soils amended with glucose, added i5N0; was rapidly used after a brief lag period (0.5 days), and
Table 1. Properties
of soils Soil IccBtlOn Two
Factual key (Northcote. 1971) Orgamc c (““) CaCO, (” Extractable carbohydrate (rng glucose-C equl” go 1) Total N (“,,I NOT-N (fig g-0
n)
PH Total exchangeable (m-equiv IoOg-‘) Clay I”,,) Sand (“,,I Water-holdmp
Wells
GCl.2
Northfield IJg5.16
I 05 0 7 0.024 I). 4.4 x4
I I?
1I4
44 0.015 0. I ? 12.2 X?
I
cations
capacity
(“,,)
I5 I3 XI 40 8
41 47 35 75.3
311
Physical distribution of immobilized nitrogen in soils Moist soil (5091 Add 300 ml 0.2M NaHCOx pH 8.3 shake (1 h. 25'C) Centrifuge (12iiOO p, 30 min.) Filter supernatant Repeat extraction 1 Filtrate concentrate to 300 ml by freeze-drying Filter (Diaflo PM 10 membrane) _--L High mol. wt. a
(Whatman filter paper)
I
I
Floating material
Sediment Add 500 ml distilled water Disperse in macerator (max. speed, 4 min.) Wet sieve (200 urn) Wash with distilled water
LOW mol. wt. P w
Wash 3 times with distilled water Concentrate Freeze
Acidify to pH5.0 Concentrate Freeze
:a&
Freeze drv FRACTION
I
(dorm)
FRACTION , ~~~~~i_ 1 Sedimant ( 50um) ~~~
FlOlO material tight fractiog,.gr.<1.59) Dry 80 C 16h
(discard)
Heav~on:
sp.gr.>1.59 (sagd) Dry 80 C, 16h FRACTION 4
FRACTION 3
Centrifuge (18301, 15 min.1 Repeat twice on redispersed sediment
Redisperse in distilled water Centrifuge (669, 5 min.) Repeat twice on redispersed sediment
Freeze dry FRACTION
7
1 g subsample in 15 ml Nonagon containing surfactant
0.1% Aerosol
07-100
Disperse by sonication (2 min.) Stand 30 min. Centrifuge (ZOOOg, lh) Filter supernatant (Millipore Solvinert, membrane, 0.22 pm) I
Sediment
I
(>Zum,-$G$ Freeze-dry
(Coarse clay) Freeze-dry
FRACTION 5
FRACTlON 6
Floating material sp.gr. ~2.06 Dry BO'C, 16h FRACTION ?a
&dim&t spm06 Dry 80°C, 2d FRACTION
7b
I g subsample in 15 ml Nemagon contain-
I g subsample in I5 ml Nemason containing surfaclant 0.1% Aerosol OT-100 Disperse by sonication (2 min.) Stand 30 min. Centrifuge (2000s. lh) Filter supernatant (Millipore Solvinert, membrane, 0.22 urn)
0.1% Aerosol OT-100 Disperse by sonic&ion (2 min.) Stand 30 min. Centrifuge (20009, Ih) Filter supernatant
Floaiing Material sp.gr. ~2.06 Dry 80°C, 16h
* sp.gr.o>2.06 Dry 80 C. 2d
Floating material sp.gr.,<2.06 Dry 80 C, 16h
Sediment SP.gr.06 Dry 8&, 2d
FRACTION 5a
FRACTION 5b
FRACTION 6a
FRACTION 6b
Fig. 1. Fractionation
had disappeared within l-2 days (Fig. 2). “NO; disappearance was accompanied by a rapid decrease in the amounts of extractable carbohydrate, from an initial concentration of 2.5 mg to 0.12 mg glucose-C equivalents g - 1 soil after 2 days’ incubation. Both the rate and extent of glucose oxidation to COZ (Fig. 2) and the rates of utilization of r5NO; and soluble carbohydrate were greater in the sandy than in the clay soil. After 2.5 days’ incubation, when more than 96% of the added r4C-glucose had been metabolized in both soils, 38.5% and 29.2% of the glucose-C had been evolved as 14C02 from the sandy
of soils.
and clay soils respectively. Thereafter, in each soil 14C0, was evolved at a declining rate and formed a decreasing proportion of the total-CO, released. Nevertheless, after 64 days’ incubation, the percentage conversion (61.4%) of labelled substrates to 14C02 in the sandy soil continued to exceed that (54.4%) in the clay soil. Both soils retained some r4C02 but in amounts insufficient to account for the differences in 14C02 evolution rate; after 64 days’ incubation, 2.8% (clay soil) and 1.0% (sandy soil) of the 14C input could be released as r4C02 by acidification of the soils. These results are consistent with the general
TWO
WELLS-
STRAW
~bs~~~tion that organic matter stabilization is more The results suggest that a small amount of “N was favoured in soils of high cfay content (Colom and lost during the net ~mmobiiizat~on phase. DenitrificaWolcott, 1967; Jenkinson, 1971; Sorensen f972+ tion losses are unlikely since (a). there was no cfear $975). Added r’NO;-N ~~~~ed ~mi~obi~~zedduring in~~t~o~ that losses were associated with a part&rthe period 2-8 days, when glucose metabolic products far soil or C amendment and (b) soiis were incubated were oxidized rapidly, However, upon entering the at only 22-24% of their water-holding capacities. slower oxidative phase, same net mineralization of N became evident. Recoveries ofsoil weight and N fhm ~fkx-tionated soils Extractable carbohydrate concentrations in soils Reoveries of soii weight and of u~~abe~i~ native with added straw were low i~~t~~~~~(approx. 0.2mg so&N were essentiaially com@ete (Table 2). NowelTr, ghrcose-C e~u~~~ents g’- ’ soil) and ~o~~n~a~ons soil fractionation ted to a further loss of “N eabk? decreased within 0.5 days to about 0.05 mg glucose-t: 2); the proportion (1%13%) unaccounted for was equivalents gmi soil, this concentration being mainsimilar and unrelated to the net rates of r 5N immobitamed far at least 32 days. Most of the added ’ 5N0i lization in both soils; Some “N may have been lost had disappeared in both soils after 16 days’ incubaas ““NH, during ~r~~e-dr~jng of the soil fractions. tion (Fig. 2). NO; was formed from L~~~abe~~ed soil sources during the net N ~mrnob~l~zat~~~phase. NH:-N concentrations remained very low throughout, The distribution of unlabelled soil N amongst various soil fractions is based on mean values obtained with samples taken at selected times during the ~~~-da~ ~n~u~t~on (Table 3, Fig. 3). (Q.) ~ruci~~~~ 1 ~rrrd 2. 3~~ar~nat~ solutions extracted reiatively small amounts of organic-N from Table 2. Rec~~~~~esof soil weight and N from f~~~t~~n~t~dsoils
* Sums of the weights of the fractions and of the amounts of N in the fractions, expressed as percentages of tbe respective values obtained utith unfract~~~at~ soil. i_SE, standard error of Ihe mean.
Physical distribution of immobilized nitrogen in soils
313
Table 3. Distribution of soil weight and unlabelled soil N after fractionation
Fraction weight (mg g-’ soil) SOil Northfield Clay
Two Wells Sand
Fraction I(NaHC0; Z(NaHC0; 3(Sp. gr. 4(particle S(particle h(particle 7(particle I(NaHC0; Z(NaHCO;
Mean’
soluble, high mol. wt.) soluble, low mol. wt.) < 1.59) dia. > 50 pm) dia. > 2 urn, < 50 urn) dia. z0.i pm, <2’pm) dia, co.2 pm) soluble, high mol. wt.) soluble, low mol. wt.)
S.E.t
14N content of fraction (“/, by wt.) MfXUI
ND. N.D. 262 210 274 251 N.D. N.D.
3(Sp. gr. < 1.59) 4(particle di& > 50 pm) S(particle dia > 2 pm, < 50 pm) b(particle dia, >0.2 pm, ~2 am) 7(particle dia. co.2 pm)
SE
N.D. N.D. See Figure 3 3.1 3.4 7.8 6.0
See Figure 3 799 2.6 95.8 2.3 55.2 2.1 46.6 1.9
0.005 0.194 0.190 0.093 N D. N D.
0.001 0.004 0005 0.002
0.007 0.576 0.485 0.273
0.001 0.010 0.013 0.009
Distribution of soil “N (pg “N in fraction g-’ soil)
Percentage distribution of recovered 14N
MeaIl
SE.
MeaIl
8.7 10.3 3x.3 13 I 408 518 234 27.2 15.9
0.6 I.2 2.6 1.8 13.6 I5 5.7 2.1 29
0.7 0.X 3.1 1.0 32.9 41.7 18.8 2.4 1.4
78.0 46.9 553 267 I28
4.7 5.4 19.1 lU.5 7.8
6.9 4.2 48.9 23.6 II.4
I
S.E 0.05 00x 0.2 0.1 I.1 I.1 0.4 0.2 02 0.4 0.5 1.6 0.9 0.7
*Mean values obtained for fractions of a given soil, irrespective of C amendment, sampled 2, 8, 16, 64 and 160 days during incubation. As an exception, values for Fraction 2-N were obtained for soils sampled on days 2, 8 and 16. when net release of 14N-NO; from native soil sources was minimal. i S.E., standard error of the iean value.
each soil (Table 3). During the first 16 days of incubation, nitrogenous compounds of high mol. wt. (Fraction 1) predominated in the extracts of the sandy soil, but extracts of the clay soil contained approximately equal amounts of organic-N of high and low mol. wt. The amounts of N in Fraction 1 varied with sampling time, but except for an apparent maximal concentration in extracts of straw-amended soils sampled on day 16, no clear trend emerged. Irrespective of soil or C amendment, the N of Fraction 1 was highly correlated (P < 0.001) with the specific absorbance of the extracts at 260nm (r = 0.988) and at 400nm (1. = 0.975), suggesting that extractable nitrogenous compounds of high mol. wt. were present in the soils as loosely adsorbed humic complexes. r-
INORTHFIELD STRAW
T
Estimates of low mol. wt. organic-14N (Fraction 2), extractable from soils sampled after day 16, were unreliable because of the large amounts of unlabelled NOT-N present. Up to and including day 16, the amount of organic-14N of this fraction was related to the specific absorbances at 260 nm and at 400 nm of the extracts, but the relationship between the two soils was different for the two C amendments (P < 0.05). (b) Fraction 3. Fraction 3 consisted of material floated off from soils after aqueous extraction and after dispersion in Ccl,. It accounted for about 3% (clay soil) and 7% (sandy soil) of the recovered, unlabelled soil N (Table 3). The amounts of unlabelled N of Fraction 3. although significantly affected by
TWO WELLS STRAW
NORTHFIELD GLUCOSE
TWO WELLS GLUCOSE
l-
,
L
2 6 166J.160
2 6 16 SL 150 SAMPLING
2 8 16 6L 160
2 8 16 6L 160
DAY
Fig. 3. Changes in the weights and percentage 14N contents of Fraction 3 of glucose- and strawamended soils, with incubation period. S.E.D. (fraction weight), 0.72; S.E.D. (% N), 0.26.
3, N. LADD.J. W. PARSOM and M. AM.~TO
314
soil type (P < O.Ol), were independent of C amendment on sampling day. By contrast, the weight of Fraction 3 was dependent upon soil type (P < O.Ol), the nature of the initial C amendment (P < 0.001) and the period or incubation (P < 0.01) (Fig. 3). In straw-amended soils, weight loss from Fraction 3 was accompanied by a significant gain (P < 0.01) in percentage N content and by changes in physical appearance. Early in the incubation (days 2 and 8), the added straw predominated in the fraction, together with minor amounts of finely-divided, darkbrown material. Incubation led to loss of the straw component, which was obvious at day 16 and more so at day 160, when the fraction consisted mainly of the finely-divided material and small straw fragments. many of which were associated with brown products of decomposition. In glucose-amended soils, changes in the percentage N content of Fraction 3 were not statistically significant and changes in the appearance of the fraction were less obvious, with the fine, dark-brown material predominating throughout. Small amounts of light-brown and brown plant fragments, present at days 2, 8 and 16, diminished in size and quantity on continued incubation. (c) Fructions 4, 5, 6, 7. Approximately 75% of the unlabelled, native N of each soil was associated with the silt (Fraction 5) plus coarse clay (Fraction 6) components (Table 3). In the sandy soil, the silt fraction had the highest percentage N content and accounted for about half of the soil N. In the clay soil, silt and coarse clay fractions were of similar, high percentage N contents whereas sand fractions (Fraction 4) from both soils were of low and variable N content. Neither fraction weight nor amount of N of Fractions 4, 5,6 or 7, varied significantly with time of sampling. Fraction 6 alone for both soils showed a significant (P < 0.05) decline in percentage N content from a maximum at day 2. (d) Subfractions. Silt and clay fractions of each glucose-amended soil, sampled on day 16, were further separated into light and heavy subfractions, alter dispersing freeze-dried material in Nemagon, sp. gr. 2.06 (Table 4). Overall the mean percentage recoveries of fraction weight (101.1%, S.E., l.lV/;,) and fraction N (98.9%, SE.. 2.4%) were complete. Material in subfraction 5a, from silt size particles, was a black, finely-divided, amorphous powder mostly derived from material floating at the surface of the Nemagon after centrifugation. The silt light sub-
Table 4. Distribution
S&l North~eld flay
Distribution
Sub-fractmn
number
(part>& density. g ml-‘) %I( C2.06) 5q> 2.06)
72 920 6 1005 54 988 228 752 77 967
7a( < 2.06) 7b(>206)
74 923 so
7(
<50) <2)
according
to particle
’
6a( < 2.06) 6b(>206) 7a( ~2.06) 7b(>2.06) 5a( < 2.06) 5b( > 2.06) ha(<2.06) 6b( > 2.06)
5(:-Z,
separated
Sub-fraction weight ,mp gfraction)
5(> 2,
6(>0.2,
during the net imnobili-7ation
phase
During the net immobilization phase, 15N became distributed amongst all soil fractions. The nature of the C amendment in~uen~d both the extent to which N of soil fractions became labelled (Fig. 4) and the amounts of lSN distributed in the soil fractions with time (Fig. 5). However, the patterns of change of “N in soil fractions were, for a given C amendment, similar in both soils despite great differences in soil texture. (a) Fractions 1: 2. Rapid microbial growth during *5N0; immobilization resulted in the formation of small amounts of highly labelled, extractable, extracellular compounds of high and low mol. wts. Compounds of high mol. wt. extracted from the clay soil were, for a given C amendment, more highly labelled (Fig. 4) but in lower concentration (Fig. 5), than those from the sandy soil. This may reflect a greater but differential adsorption af both labelled and unlabelled products by the clay soil. However, differences may also occur in the nature of the microbial populations and in the proportions of the newly-formed. immobilized 15N present as extracellular and lytic products. In soils amended with straw, organic-15N of Fraction 1 increased si~ifi~ntIy (P < 0.05j between days 2 and 8. reflecting increasing (P < 0.001) ’ ‘N atom ‘;: enrichments of the N of the fraction during the early net immobilizatioil phase. Thereafter, the proportion of 15N compounds in Fraction 1 declined, commencing before immobilization was maximal at 16 days.
6(>0.2.
C2)
qf “N
of soil weight and unlabelled soil N in sub-fractions
Fraction numhtlr (particle dia El”&% pmi
7( C.O.2) Two Wells Sand
fractions had percentage N contents IO- I? times greater than those of the corresponding heavy subfractions and contained about 50.-75”;, of the total-N of the silt fractions. Material in subfraction 6a, which was derived almost entirely from suspended rather than surface8oating particles, had a percentage N content similar to that of the corresponding heavy subfraction and accounted for < IOU/(,of the N of the coarse clay fraction from which it originated. Subfraction 7a. prepared from the fine clay fractions, contained dark-brown. ~nely-divided material, most of which had floated at the surface of the Nemagon. The percentage N contents of the light subfractions were 3- 4 times greater than those of the corresponding heavy subfractions. Approximately 20:; of the fine clay N was contained in the light subfractions.
I 40 0.1: 0 I9 0. I H 0.25 0.08 I 90 0. I9 0 53 045 O.XI 0 2: 0.066
I Ofi6 !OXZ I3 I762 133 786 4310 1401 406 437s 59x 2149 160
48.2 51.x 0‘6 9Y4 i-t.? 85 ‘; 75.6 244 us 91.5 21.X 7x.2
density
Physical distribution of immobilized nitrogen in soils
L
9
NORTHFIELO
2
30
I 4 :
20
-GLUCOSE
315
TWO
WELLS
- STRAW
‘IWO
WELLS
-GLUCOSE
B 10 0 SAMPLINGDAY
2816
2616
2616
2616
1
2
3
L
FRACTION
2.916 2616 5
6
2616 7
2 6 16 2 676 2
I
2 8 16 2 616
2 6 16 2 616
L
3
5
2 6 16
6
7
Fig. 4. The proportion of N of sail fractions accountable as 15N in gkcose- and stra~“ameoded soils. For each fraction in tarn, values are shown far soils sampled on days 2, 8, and 16. S.E.D. (Fraction l), 1.52; (F2), 1.54: (F3), 3.88; (F4), 1.05; (F5), 0.33; (I%), 1.46; (F7), 1.01.
of the extractable, immobilized ‘% to actively-metabolizing microflora and its transfer to other organic residues before onset of net mineralization are in accord with the observations of Ladd and Paul-(1973) using bicarbonate extracts of a sandy loam and by McGill et al. (1975) using pyrophosphate extracts from a high clay soil. Extractable, ‘SN-labelled, organic compounds of low mol. wt. were rapidly formed in glucose-amended soils, to an extent which made Fraction 2 the most
In glucose-amended soils, nitrogenous compounds af high mol. wt. became labelkd more rapidly and changes in the r ‘N of Fraction I during a period (24 days) of complete immobilization were not statistically significant. However, in the sandy soil there was a significant (P < 0.05) decline in the relative ‘%I atom % enrichment of the N of Fraction 1 before net mineralization was observed, i.e. between days 2 and 8, indicating a preferential transfer of organic-“N to other fractions. The ready availability of at least part
77.0 TWO
TWO
0 SAMPLINGDAY FRACTION
2616
2616
1
2
2816 3
2616
2616
2816
2616
b
5
6
7
2616 1
WELLS
-
WELLS
2816
2616
2616
2
3
L
STRAW
-GLUCOSE
2616 5
28% 6
2&t? 7
Fig. 5. Distribution of recovered “?I in fractions of glucose- and straw-amended soiIs. For each fraction in turn, values are shown for soils sampled on days -2, 8 and 16. S.E.D. [Fraction I), 0.86: (F2), 2.14; fF3), 1.25; @?4),0.43; fF5f, 1.59; jF6), 3.73; QT), 3.73.
316
.I. !‘d. LAIN.
J. W.
PARSOhS
highly labclled of any from these soils. The ‘“N atom Y!{enrichments of this fraction were least at day 8 but did not change much during the entire incubation. In straw-amended soils. ’ 'N-labclled compounds formed a decreasing proportion of the N of Fraction 2 as incubation continued. The decreases corresponded with immobilization of the added “NO;. which was located in this fraction. (h) F~ctiort 3. The proportions and amounts of ‘“N-labelled compounds in the nitrogenous pool represented by soil material of sp. gr. < 1.59. were highly influenced by the nature of the C amendment (P < 0.001) (Figs. 4 and 5). In straw-amended soils. “N of the light fraction increased significantly (P < 0.001) to account at day 16 for about 20:; of all of the immobilized. labelled material in each soil (Fig. 5). The inclusion of decomposing straw residues influenced the percentage content of both unlabelled and labelled N of Fraction 3 (Figs. 3 and 4). Values for percentage “N + 14N content increased throughout. from 0.30”/;, (clay soil) and 0.561,, (sandy soil) on day 2, to 1.70:/, (clay soil) and 2.09”;, (sandy soil) by day 160 when the added straw was extensively decomposed. Nevertheless, the presence of relatively available C, as in straw, did not also increase significantly the amount of unlabelled N immobilized in the light fraction. (The decline in the atom”,, enrichment of i5N0; during the net immobilization phase demonstrated that some unlabelled NO; was being formed, but insufficient amounts were being immobilized in Fraction 3 to be detected as a net gain in N of the fraction.) In glucose-amended soils, the N of Fraction 3 was poorly labelled (Fig. 4) and changes in the small amounts of “N of this fraction were not statistically significant (Fig. 5). Obviously-recognizable root fragments, which otherwise would have been included in this fraction and which. like wheat straw, may have been a relatively available C source, had been removed soon after sampling the soils in the field. Thus the light fraction of glucose-amended soils, as judged by physical appearance. had already undergone extensive microbial decomposition before the experiment. This view is supported by the observation that the percentage unlabelled N content of Fraction 3 from glucose-amended soils remained constantly high (2.02”;. S.E., O.OS”,,) throughout the incubation (Fig. 3). (c) Fractions 4, 5, 6. 7. Most of the newly-immobilized ‘“N was associated with silt (Fraction 5) and clay (Fractions 6 and 7) (Fig. 5). Of the particle size fractions, the fine clay became the most highly labelled. irrespective of soil or C amendment (Fig. 4). In soils amended with glucose. all 15N0, was immobilized by day 2. At this stage. the atom 1, enrichment of the N of the fine clay fraction was maximal and this fraction contained 34’10(sandy soil) and 630, (clay soil) of the total immobilized r5N. During the “N was period when ‘“N remained immobilized, redistributed amongst the fractions, In particular. between days 2 and 8. statistically significant (P < 0.01) decreases in the amounts of 15N in the fme clay fraction were accompanied by significant increases in the amounts of “N of the silt (P < 0.05) and coarse clay (P < 0.01) fractions. In the silt frac-
and M. hf~ru
tion. further gains occurred between days Y and 16. Thus, during growth on microbial metabolites by successive microbial populations in glucose-amended soils. organic-‘“N was transferred from the fine clay fraction to the silt and coarse clay components (Fig. 5). Changes in 1‘N of the fractions were not significantly due to changes in fraction weight, resulting from differences in the extent of soil dispersion (or possibly reaggregation) of the soils. Rather changes in “N of the fractions directly reflected changes in the 15N atom “Clenrichments of the N of the fractions. The nature of the nitrogenous material transferred from the fine clay fraction is unknown. Possibly adsorbed extracellular and lytic products formed, like those of Fraction 1, during the initial flush of microbial growth, were further metabolized. Resynthesis of this extracellular ’ 5N into microbial cells may relocate it in fractions of larger particle size after dispersion of the soils. Alternatively. some of the 15N initially immobilized within the fine clay fraction may be part of a microbial biomass. The fine clay fraction could contain spherical organisms of diameter up to 0.8 /(rn. assuming a cell sp. gr. of 1.1. The viability of these newly-formed microbial cells may be low and the organic-“N associated with them may become incorporated into larger and more stable cells of a secondary population. Other alternatives arc possible. For example. rapidly growing microbial cells in soils sampled on day 2 may be more readily dispersed than cells growing more slowly in soils sampled on day 8. Further studies should involve measurements of the changes of biomass-“N and non biomass-“N of the particle size fractions during the net immobilization phase. Soil fractions obtained by McGill rr al. (1975) included material of particle diameter <0.04 Aim. This fraction contained a significant (approx. 25;.,) proportion of the recently-immobilized N. which they reasonably ascribed to extracellular and lytic products and released cytoplasmic compounds adsorbed to very fine clay particles. Their method involved extraction of dried soil with a pyrophosphate solution. followed by dispersion of the extracted soil by ultrasonic vibration. A difficulty introduced by their procedure lay in deciding the extent to which the distribution of the immobilized N was due to natural processes of cell growth and lysis or to cell rupture from drying. extraction and sonic&ion of the soil. If the latter were cxtensivc, then changes in N of fractions due to immobilization mineralization reactions ma\ have been obscured. In our study, soils were not dried before extraction, extraction with bicarbonate was brief, and dispersion was obtained by maceration. This procedure caused only a few cells to rupture, as judged by retention of dehydrogenasc activity (Ladd and Paul. 1973). Nevertheless the soils were well dispersed, based on a preliminary comparison of the perceptages of soil of particle diameter <2 /Irn, obtained by the above treatment and by that of Emerson (1971). However. the percentage of material of particle diameter <0.04pm was far less than that reported by McGill ct 01. (1975) and material in this size range was not analysed as a separate fraction. Thus strict comparison of our results with those of McGill rt al. (1975) are not possible because of differences in techniques
Physical distribution of immobilized
nitrosen
in soils
317
Table 5. Distribution of “N in subfractions separated according to particle density
Soil Northfield Clay
Fraction number (particle diameter range, pm) 5(>2,
Subfraction number (particle density. g ml-‘)
<50)
5a( <2.06) 5b( > 2.06)
<2)
6a( < 2.06) 6b( > 2.06) 7a( < 2.06) 7W> 2.06)
6(>0.2. 7( <0.2) Two Wells Sand
5(>2. 6(>0.2.
<50) <2)
7( <0.2) S.E.D.
Sa( < 5q> 6a( < 6bj > 7a( < 7bl>
2.06) 2.06) 2.06) 2.06) 2.06) 2.06)
“N (y” of subfraction-N) 2.95 3.62 3.x7 4.6X 15 55 1048 ?62 444 14.98 14.79 6.91 8.33 0 32
and in the particle sizes of fractions under consideration, but both studies support the view that during the net immobilization phase, newly-formed nitrogenous products are transferred from fine to coarser soil particles. In straw-amended soils, the organic-15N of all particle size fractions, except sand, increased significantly (Fraction 5, P < 0.001; Fractions 6 and 7, P < 0.05) during the first 16 days’ incubation (Fig. 5). The major gains occurred between days 2 and 8. Net immobilization of ’ 5NO; in straw-amended soils was complete only at day 16. Any prior transfer of organic-15N from the fine clay fraction may have been obscured by a net gain due to increased total amounts of 15N immobilized. However, from 8 to 16 days, when net immobilization was continuing, gains in the organic-15N of the silt fraction were significant (P < 0.01) but not in the clay fractions. (d) Subfractions. In the sandy soil, the N of the heavy subfraction of both the silt and fine clay components (Table 5) was more highly labelled than that of the respective light fraction (P < O.Ol), whereas labelling of the N of the coarse clay subfractions were very similar. By contrast, in the clay soil, the N of the light subfraction of the fine clay was more labelled than that of the complementary heavy subfraction (P < 0.001).
Thus in the silt and clay size fractions, a proportion of the labelled, organic nitrogenous products was apparently bound to inorganic components, but in the silt fraction, a large amount could be separated as “partially humified” components of sp. gr. ~2.06. The observed distribution of labelled and unlabelled N may have been influenced by the freeze-drying of the fractions before densimetric subfractionation. Work with undried preparations is needed to determine whether significant, differential gains and losses of N occur within these light and heavy subfractions during net immobilization or long-term net mineralization. Acknowledgements-We arc indebted to Mr J. Greenaway and Dr A. W. Moore, Division of Soils, St. Lucia, Queensland, for mass spectrometric data, to the late Mr A. R. P. Clarke for general soil analyses and Dr R. Correll, Division of Mathematics and Statistics, Glen Osmond, for statistical analyses. REFERENCES
J. M. (1965a) Total nitrogen. In Methods qf Soil Analysis (C. A. Black, Ed.), American Society of Agronomy Monograph. 9, (2) 1149-l 178.
BREMNER
Distrlbutlon of fraction “N (pg lSN in subfraction g- ’ fraction) 31 41 0.5 x7 24 93 117 66 72 75x 45 196 14
BREMNER
Percentage distribution of recovered
’ 'N
43.0 57.0 0.6 99.4 20 5 79.5 63.9 36. I 8.7 91.3 18.7 XI 3
J. M. (1965b) Inorganic forms of nitrogen. In Methods of Soil Analysis (C. A. Black, Ed.), American Society of Agronomy Monograph. 9, (2) 1179-1237. BREMNER J. M. and SHAW K. (1958) Denitrification in soil: I. Methods of investigation. J. agric. Sci. 51, 22-39. BROADBENT F. E. (1968) Turnover of nitrogen in soil organic matter. In Organic Matter and Soil Fertility. Pontif acad. Sci. Scripta I/aria 32. 61-82. CHICHESTER F. W. (1970) Transformations of fertilizer nitrogen in soil. II. Total and 15N-labelled nitrogen of soil organo-mineral sedimentation fractions. PI. Soil 33, 437456. COLOM J. and WOLCOTT A. R. (1967) Forms of organic nitrogen in clay-amended sandy soil. PI. Soil 26, 261-268. EMERSON W. W. (1971) Determination of the contents of clay-sized particles in soils. J. Soil Sci. 22, 50-59. FORD G. W. and GREENLAND D. J. (1968) The dynamics of partly humified organic matter in some arable soils. Trans. 9th int. Congr. Soil Sci. Adelaide 2, 403410. FORD G. W., GREENLAND D. J. and OADES J. M. (1969) Separation of the light fraction from soils by ultrasonic dispersion in halogenated hydrocarbons containing a surfactant. J. Soil Sci. 20, 291-296. GREENLAND D. J. and FORD G. W. (1964) Separation of partially humified organic materials from soils by ultrasonic dispersion. Trans. 8th int. Congr. Soil Sci., Bucharest 3. 137-148. JANSSON S. L. (1967) Soil organic matter and fertility. Trans. int. Sot. Soil Sci., Aberdeen, Commissions II and IV, l-10. JANS~ON S. L. and PERSSON J. (1968) Coordination of humus chemistry and soil organic matter biology by isotopic techniques. In Isotopes and Radiation in Soil Organic Mutter Studies. IAEA, Vienna 11 l-124. JENKIN~ON D. S. (1971) Studies on the decomposition of Cl4 labelled organic matter in soil. Soil Sci. 111. 64-70. KEENEY D. R. and BREMNER J. M. (1964) Effect of cultivation on the nitrogen distribution in soils. Proc. Soil Sci. Sot. Am. 28. 653-656. KEENEY D. R. and BREMNER J. M. (1966) Characterization of mineralizable nitrogen in soils. Proc. Soil Sci. Sot. Am. 30, 714-719. LADD J. N. and PAUL E. A. (1973) Changes in enzymic activity and distribution of acid-soluble, amino acidnitrogen in soil during nitrogen immobilization and mineralization. Soil Biol. Biochem. 5, 825-840. LADD J. N., PARSONS J. W. and AMATO M. (1977) Studies of nitrogen immobilization and mineralization in calcareous soils-II. Mineralization of immobilized nitrogen from soil fractions of different particle size and density. Soil Biol. Biochem. 9, 319-325. MCGILL W. B., SHIELDS J. A. and PAUL E. A. (1975) Relation between carbon and nitrogen turnover in soil organic fractions of microbial origin. Soil Biol. Biochem. 7. 57-63.
31x
J. N. LALII), J. W. PARSONS and M. AMTO
N~RTIKOTL K. H. (1971) A Factuul Keyfbr the Rrcogrzirion of Awstrulim Soils. CSIRO. Australia, Division of Soils.
Divisional Report. 3rd edition Pub. Rellim Technical Publications, Glenside, S.A. OArIFs J. M. (1967) Carbohydrates in some Australian soils. Aust. J. Soil Rrs. 5. 103-116. OADES J. M. and LADII J. N. (1977) Biochemical properties. Carbon and nitrogen metabolism. In Soil Factors in Crop Production in (I Srrni-arid Environmrnr (J. S. Russell and E. L. Greaten, Eds.), Australian Soil Sci. Sot. Monograph, Univ. of Queensland Press. in press. PERSSON J. (1968) Biological testing of chemical humus analysis. La&r-Hogs/c A/m/r 3. 81-217. Ross P. J. and MARTIN A. E. (1970) A rapid method for preparing gas samples for nitrogen-15 determination. ha/w. Land. 95. 817 822.
SOREMENL. H. (1972) Stabilization
of newly formed amino acid metabolites in soil by clay minerals. Soil Sci. 114. 5 ~1I. S~RIZNSENL. H. (1975) The influence of clay on the rate of decay of amino acid metabolites synthesised in soils during decomposition of cellulose. Soil Biol. Biochrm. I. 171-177. TINSLEY J., TAYLOR T. G. and MOORF. J. H. (1951) The determination of carbon dioxide derived from carbonates in agricultural and biological materials. Analyst, Lond. 76. 30&310. TURNER J. C. (1968) Triton X-100 scintillant for carbon-14 labelled materials. Int. J. appl. Radint. Isotopes 19. 557- 563.
B~O&W. vol. 9, pp. 309 to 318. Pergamon
STUDIES
Press 1977. Printed in
GreatBritain
OF NITROGEN
LIBERALIZATION
IMMOBILIZATION
IN CALCAREOUS
DISTRIBUTION OF IMMOBILIZED NITROGEN SOIL FRACTIONS OF DIFFERENT PARTICLE SIZE AND DENSITY
AND
SOILS-I. AMONGST
J. N. LADD, J. W. PARSONS* and M. AMATO Division of Soils, CSIRO, Glen Osmond, South Australia 5064 (Accepted 6 December 1976) Summary-’
$NO; was jmmobiliz~d in a calcareous clay and a calcareous sandy soif during incubation of each soil with glucose and wheat straw. Changes in the distribution of immobilized “N amongst soil extracts and soil fractions of different particle size and density were determined during periods of net N immobilization. The nature of the organic-C amendment, but not soil type, significantly influenced both the distribution of the immobilized 15N and the pattern of changes of the organic-r5N of soil fractions with time. In straw-amended soils, approx. 20% of the organic-‘“N became associated with a light fraction, sp. gr. < 1.59, the remainder becoming distributed mainly amongst the silt and clay fractions. In glucoseamended soils, very little (< 1.2%) of the “N was immobilized in the light fraction, sp. gr. <1.59, most being rapidly distributed amongst the silt and clay fractions. During a period of complete immobilization. organic-15N was transferred from the fine clay to the silt and coarse clay fractions. Silt, coarse clay and fine clay components from glucose-amended soils sampled at the end of the net immobilization phase were further fractionated densimetrically into light (sp. gr. <2.06) and heavy (sp. gr. >2.06) subfractions. The organic- 15N of respective light subfractions accounted for 4344% of the total organic-r5N of the silt, l-9% of that of the coarse clay and 19-21”/, of that of the fine clay fractions.
INTRODUCTION Studies of the relative availability of nitrogenous components of soils have mostly been chemically based. Of the chemically “defined” com~nents, acid-hydrolyzable amino acid-N appears to contribute most to inorganic-N if judged by the amounts of N lost from this organic-N pool during periods of net mineraliza-
tion. However, measurements of the percentage decrease of N from soil chemical fractions show that no component is consistently of greater biological availability (Keeney and Bremner, 1944, 1966; Broadbent, 1968). A clearer definition of “active” and “passive” nitrogenous pools formed in soils, in situ, may be achieved if fractionation procedures were to permit direct analysis of the nitrogen contents of recognizable biological entities (Jansson, 1967; Jansson and Persson, 1968; Persson, 1968). Oades and Ladd (1977) have suggested that suitable components may be (i) the soil biomass, separated according to organism size and its association with either larger plant fragments or with inorganic constituents of the soil or (ii) extracelluiar or lytic nitrogenous products, separated as soiubfe compounds of different mol. wts. or as insoluble constituents, either free or bonded to inorganic or organic soil colloids. In these respects a more advantageous approach may be to fractionate soils by *On leave from Department of Soil Science, University of Aberdeen. Aberdeen. Scotland. 309
physical methods before chemical analysis. Physical methods used have mainly involved fractionation of dispersed soils according to particle size (Chichester, 1970; Ladd and Paul, 1973; McGill et al., 1975) and density (Greenland and Ford, 1964; Ford and Greenland, 1968). Ford and Greenland (1968) concluded that the N of a light fraction, the partially humified material separated from soils by flotation in a solvent of sp. gr. 2.0, is a relatively labile component of soil organic-N. McGill et nl. (1975) have shown that transfer of N through fractions of different particle size and of pyrophosphate extracts took place during net N immobilization. Ladd and Paul (1973) using a modified and probably less destructive fractionation procedure, found that bicarbonate extracts of soil and also material of diameter ~0.2 pm, contained nitrogenous compounds (arid-hydrolyzable amino acid-N) which were readily formed and partly decomposed during the period of net N immobilization. This paper is the first of a series dealing with N immobilization and mineralization in calcareous soils. The aim is to describe eventually the transfer of organioN between components of biological significance, such as those mentioned above, and to measure the contribution of each component to the mineral-N pool. Although both immobilization and mineralization reactions take place concurrently, the initial approach has been to relate changes in the organic-N of several soil components to the net changes of mineral-N of soils during different incuba-
J. N. LAIX). J. W. PARSONS and M. AMATO
3 10
tion periods. In this study, a calcareous clay and a calcareous sandy soil were incubated with isNO; and glucose or wheat straw. The procedure of Ladd and Paul (1973) has been extended to permit measurements of the changes in the distribution of 15N amongst both soluble extracts and also particles of different size and density, during periods of net N immobilization. The changes of fraction organic-15N which accompany net N mineralization are described by Ladd et al. (1977).
MATERIALS
AND
METHODS
soils (50g dry soil equivalent) wcrc each incubated in duplicate with ’ 4C-glucose (3 &i) plus KN 03. Moist CO,-free air was drawn through the incubation flasks (250 ml) and evolved CO, was trapped in 20 ml 1 M LiOH. The traps were replaced every 0.552 days and all their contents transferred (50 ml. final volume). Total-CO, evolved was determined after titrating residual alkali of a 20 ml sample with standard HCl (Tinsley er ul.. 1951). 14C02 evolution was calculated from the radioactivity of a 0.5 ml subsample mixed with 20 ml scintillation solvent (Turner, 1968). Flasks without soil or substrate were included as controls. Soil fructionution
Soils A calcareous sand and a calcareous clay soil (Table 1) were sampled to 1Ocm depth from under mixed pastures at Two Wells and Northfield, South Australia, respectively. Bulked samples were dried at 35°C freed of coarse plant material, ground, sieved (2mm) and stored at 22°C. Soil incubations (I) Both soils were incubated in duplicate with K15N0,, with glucose or wheat straw as energy sources. 15NO; (0.1 mg N g-i dry wt. soil, 10.18 atom 0Aexcess ’ 5N) and glucose (2.5 mg C g-’ dry wt. soil) were supplied in solution, the straw as ground material (10 mg straw or 4.36 mg straw-C g- ’ dry wt. soil). The added 15N0; was assumed to have equilibrated immediately with the small amounts of unlabelled NO; of the soils (Table 1) and of the wheat straw (0.6 pg N g- ’ dry wt. soil equivalent). Incubations were carried out at 25°C in 31 Erlenmeyer flasks, each containing initially 1.25 kg airdried soil moistened with the appropriate nutrient solutions. The flasks were connected in parallel in aeration trains through which moist, NH,-free air was drawn for 160 days. Moisture contents of the sandy and clay soils were maintained at approximately 9.1% and 18.0’%, respectively, equivalent to holding capacities 22-24% of the soils’ water (Bremner and Shaw, 1958). When appropriate, the soils were briefly mixed before removing 50 g or 150 g subsamples, of which 5 g was immediately extracted for NHf-N and NO;-N determinations and 10 g was dried (90°C 2 h) for total-N measurements. The remainder of the sample was frozen in liquid N, then stored at - 15°C until fractionated or further analysed. (2) On a smaller scale, moistened sandy and clay
Soil samples from the larger scale experiments were fractionated into components of different particle size and density (Fig. I) by modified procedures of Ford et ul. (1969) Ladd and Paul (1973) and McGill et al. (1975).
Extractable carbohydrate was determined as glucose equivalents by shaking previously-frozen soil subsamples with 5 ~01s. 0.5 N KzS04 for IOmin, reacting samples of the extract with anthrone reagent (Oades, 1967) and relating absorbances at 630nm to those obtained with similarly-treated glucose standards. NHf-N and NOT-N of 2 M KC1 and water extracts were determined by distillation with MgO and Devarda’s alloy-MgO mixture (Bremner, 1965b). Total-N of dried. ground soils was determined by Kjeldahl digestion, after pretreatment to reduce NO; (Bremner and Shaw, 1958; Bremner, 1965a). After titration with standard 5 mN H2S04, to pH 5.0, duplicate distillates were pooled, “spiked” if necessary with 0.5--l mg unlabelled NH:-N, further acidified to pH 4.0. then dried (Ross and Martin. 1970). Atom ‘I{, i5N abundance was determined in an A.E.I. type MS3 mass spectrometer. Statistical analyses were based on data obtained with soil samples taken during both the net immobilization and mineralization phases. Calculations of the appropriate standard error of difference (S.E.D.) were based on four degrees of freedom. RESULTS
Changes in NOT-N
AND
DISCL’SSION
concentrations
In soils amended with glucose, added i5N0; was rapidly used after a brief lag period (0.5 days), and
Table 1. Properties
of soils Soil IccBtlOn Two
Factual key (Northcote. 1971) Orgamc c (““) CaCO, (” Extractable carbohydrate (rng glucose-C equl” go 1) Total N (“,,I NOT-N (fig g-0
n)
PH Total exchangeable (m-equiv IoOg-‘) Clay I”,,) Sand (“,,I Water-holdmp
Wells
GCl.2
Northfield IJg5.16
I 05 0 7 0.024 I). 4.4 x4
I I?
1I4
44 0.015 0. I ? 12.2 X?
I
cations
capacity
(“,,)
I5 I3 XI 40 8
41 47 35 75.3
311
Physical distribution of immobilized nitrogen in soils Moist soil (5091 Add 300 ml 0.2M NaHCOx pH 8.3 shake (1 h. 25'C) Centrifuge (12iiOO p, 30 min.) Filter supernatant Repeat extraction 1 Filtrate concentrate to 300 ml by freeze-drying Filter (Diaflo PM 10 membrane) _--L High mol. wt. a
(Whatman filter paper)
I
I
Floating material
Sediment Add 500 ml distilled water Disperse in macerator (max. speed, 4 min.) Wet sieve (200 urn) Wash with distilled water
LOW mol. wt. P w
Wash 3 times with distilled water Concentrate Freeze
Acidify to pH5.0 Concentrate Freeze
:a&
Freeze drv FRACTION
I
(dorm)
FRACTION , ~~~~~i_ 1 Sedimant ( 50um) ~~~
FlOlO material tight fractiog,.gr.<1.59) Dry 80 C 16h
(discard)
Heav~on:
sp.gr.>1.59 (sagd) Dry 80 C, 16h FRACTION 4
FRACTION 3
Centrifuge (18301, 15 min.1 Repeat twice on redispersed sediment
Redisperse in distilled water Centrifuge (669, 5 min.) Repeat twice on redispersed sediment
Freeze dry FRACTION
7
1 g subsample in 15 ml Nonagon containing surfactant
0.1% Aerosol
07-100
Disperse by sonication (2 min.) Stand 30 min. Centrifuge (ZOOOg, lh) Filter supernatant (Millipore Solvinert, membrane, 0.22 pm) I
Sediment
I
(>Zum,-$G$ Freeze-dry
(Coarse clay) Freeze-dry
FRACTION 5
FRACTlON 6
Floating material sp.gr. ~2.06 Dry BO'C, 16h FRACTION ?a
&dim&t spm06 Dry 80°C, 2d FRACTION
7b
I g subsample in 15 ml Nemagon contain-
I g subsample in I5 ml Nemason containing surfaclant 0.1% Aerosol OT-100 Disperse by sonication (2 min.) Stand 30 min. Centrifuge (2000s. lh) Filter supernatant (Millipore Solvinert, membrane, 0.22 urn)
0.1% Aerosol OT-100 Disperse by sonic&ion (2 min.) Stand 30 min. Centrifuge (20009, Ih) Filter supernatant
Floaiing Material sp.gr. ~2.06 Dry 80°C, 16h
* sp.gr.o>2.06 Dry 80 C. 2d
Floating material sp.gr.,<2.06 Dry 80 C, 16h
Sediment SP.gr.06 Dry 8&, 2d
FRACTION 5a
FRACTION 5b
FRACTION 6a
FRACTION 6b
Fig. 1. Fractionation
had disappeared within l-2 days (Fig. 2). “NO; disappearance was accompanied by a rapid decrease in the amounts of extractable carbohydrate, from an initial concentration of 2.5 mg to 0.12 mg glucose-C equivalents g - 1 soil after 2 days’ incubation. Both the rate and extent of glucose oxidation to COZ (Fig. 2) and the rates of utilization of r5NO; and soluble carbohydrate were greater in the sandy than in the clay soil. After 2.5 days’ incubation, when more than 96% of the added r4C-glucose had been metabolized in both soils, 38.5% and 29.2% of the glucose-C had been evolved as 14C02 from the sandy
of soils.
and clay soils respectively. Thereafter, in each soil 14C0, was evolved at a declining rate and formed a decreasing proportion of the total-CO, released. Nevertheless, after 64 days’ incubation, the percentage conversion (61.4%) of labelled substrates to 14C02 in the sandy soil continued to exceed that (54.4%) in the clay soil. Both soils retained some r4C02 but in amounts insufficient to account for the differences in 14C02 evolution rate; after 64 days’ incubation, 2.8% (clay soil) and 1.0% (sandy soil) of the 14C input could be released as r4C02 by acidification of the soils. These results are consistent with the general
TWO
WELLS-
STRAW
~bs~~~tion that organic matter stabilization is more The results suggest that a small amount of “N was favoured in soils of high cfay content (Colom and lost during the net ~mmobiiizat~on phase. DenitrificaWolcott, 1967; Jenkinson, 1971; Sorensen f972+ tion losses are unlikely since (a). there was no cfear $975). Added r’NO;-N ~~~~ed ~mi~obi~~zedduring in~~t~o~ that losses were associated with a part&rthe period 2-8 days, when glucose metabolic products far soil or C amendment and (b) soiis were incubated were oxidized rapidly, However, upon entering the at only 22-24% of their water-holding capacities. slower oxidative phase, same net mineralization of N became evident. Recoveries ofsoil weight and N fhm ~fkx-tionated soils Extractable carbohydrate concentrations in soils Reoveries of soii weight and of u~~abe~i~ native with added straw were low i~~t~~~~~(approx. 0.2mg so&N were essentiaially com@ete (Table 2). NowelTr, ghrcose-C e~u~~~ents g’- ’ soil) and ~o~~n~a~ons soil fractionation ted to a further loss of “N eabk? decreased within 0.5 days to about 0.05 mg glucose-t: 2); the proportion (1%13%) unaccounted for was equivalents gmi soil, this concentration being mainsimilar and unrelated to the net rates of r 5N immobitamed far at least 32 days. Most of the added ’ 5N0i lization in both soils; Some “N may have been lost had disappeared in both soils after 16 days’ incubaas ““NH, during ~r~~e-dr~jng of the soil fractions. tion (Fig. 2). NO; was formed from L~~~abe~~ed soil sources during the net N ~mrnob~l~zat~~~phase. NH:-N concentrations remained very low throughout, The distribution of unlabelled soil N amongst various soil fractions is based on mean values obtained with samples taken at selected times during the ~~~-da~ ~n~u~t~on (Table 3, Fig. 3). (Q.) ~ruci~~~~ 1 ~rrrd 2. 3~~ar~nat~ solutions extracted reiatively small amounts of organic-N from Table 2. Rec~~~~~esof soil weight and N from f~~~t~~n~t~dsoils
* Sums of the weights of the fractions and of the amounts of N in the fractions, expressed as percentages of tbe respective values obtained utith unfract~~~at~ soil. i_SE, standard error of Ihe mean.
Physical distribution of immobilized nitrogen in soils
313
Table 3. Distribution of soil weight and unlabelled soil N after fractionation
Fraction weight (mg g-’ soil) SOil Northfield Clay
Two Wells Sand
Fraction I(NaHC0; Z(NaHC0; 3(Sp. gr. 4(particle S(particle h(particle 7(particle I(NaHC0; Z(NaHCO;
Mean’
soluble, high mol. wt.) soluble, low mol. wt.) < 1.59) dia. > 50 pm) dia. > 2 urn, < 50 urn) dia. z0.i pm, <2’pm) dia, co.2 pm) soluble, high mol. wt.) soluble, low mol. wt.)
S.E.t
14N content of fraction (“/, by wt.) MfXUI
ND. N.D. 262 210 274 251 N.D. N.D.
3(Sp. gr. < 1.59) 4(particle di& > 50 pm) S(particle dia > 2 pm, < 50 pm) b(particle dia, >0.2 pm, ~2 am) 7(particle dia. co.2 pm)
SE
N.D. N.D. See Figure 3 3.1 3.4 7.8 6.0
See Figure 3 799 2.6 95.8 2.3 55.2 2.1 46.6 1.9
0.005 0.194 0.190 0.093 N D. N D.
0.001 0.004 0005 0.002
0.007 0.576 0.485 0.273
0.001 0.010 0.013 0.009
Distribution of soil “N (pg “N in fraction g-’ soil)
Percentage distribution of recovered 14N
MeaIl
SE.
MeaIl
8.7 10.3 3x.3 13 I 408 518 234 27.2 15.9
0.6 I.2 2.6 1.8 13.6 I5 5.7 2.1 29
0.7 0.X 3.1 1.0 32.9 41.7 18.8 2.4 1.4
78.0 46.9 553 267 I28
4.7 5.4 19.1 lU.5 7.8
6.9 4.2 48.9 23.6 II.4
I
S.E 0.05 00x 0.2 0.1 I.1 I.1 0.4 0.2 02 0.4 0.5 1.6 0.9 0.7
*Mean values obtained for fractions of a given soil, irrespective of C amendment, sampled 2, 8, 16, 64 and 160 days during incubation. As an exception, values for Fraction 2-N were obtained for soils sampled on days 2, 8 and 16. when net release of 14N-NO; from native soil sources was minimal. i S.E., standard error of the iean value.
each soil (Table 3). During the first 16 days of incubation, nitrogenous compounds of high mol. wt. (Fraction 1) predominated in the extracts of the sandy soil, but extracts of the clay soil contained approximately equal amounts of organic-N of high and low mol. wt. The amounts of N in Fraction 1 varied with sampling time, but except for an apparent maximal concentration in extracts of straw-amended soils sampled on day 16, no clear trend emerged. Irrespective of soil or C amendment, the N of Fraction 1 was highly correlated (P < 0.001) with the specific absorbance of the extracts at 260nm (r = 0.988) and at 400nm (1. = 0.975), suggesting that extractable nitrogenous compounds of high mol. wt. were present in the soils as loosely adsorbed humic complexes. r-
INORTHFIELD STRAW
T
Estimates of low mol. wt. organic-14N (Fraction 2), extractable from soils sampled after day 16, were unreliable because of the large amounts of unlabelled NOT-N present. Up to and including day 16, the amount of organic-14N of this fraction was related to the specific absorbances at 260 nm and at 400 nm of the extracts, but the relationship between the two soils was different for the two C amendments (P < 0.05). (b) Fraction 3. Fraction 3 consisted of material floated off from soils after aqueous extraction and after dispersion in Ccl,. It accounted for about 3% (clay soil) and 7% (sandy soil) of the recovered, unlabelled soil N (Table 3). The amounts of unlabelled N of Fraction 3. although significantly affected by
TWO WELLS STRAW
NORTHFIELD GLUCOSE
TWO WELLS GLUCOSE
l-
,
L
2 6 166J.160
2 6 16 SL 150 SAMPLING
2 8 16 6L 160
2 8 16 6L 160
DAY
Fig. 3. Changes in the weights and percentage 14N contents of Fraction 3 of glucose- and strawamended soils, with incubation period. S.E.D. (fraction weight), 0.72; S.E.D. (% N), 0.26.
3, N. LADD.J. W. PARSOM and M. AM.~TO
314
soil type (P < O.Ol), were independent of C amendment on sampling day. By contrast, the weight of Fraction 3 was dependent upon soil type (P < O.Ol), the nature of the initial C amendment (P < 0.001) and the period or incubation (P < 0.01) (Fig. 3). In straw-amended soils, weight loss from Fraction 3 was accompanied by a significant gain (P < 0.01) in percentage N content and by changes in physical appearance. Early in the incubation (days 2 and 8), the added straw predominated in the fraction, together with minor amounts of finely-divided, darkbrown material. Incubation led to loss of the straw component, which was obvious at day 16 and more so at day 160, when the fraction consisted mainly of the finely-divided material and small straw fragments. many of which were associated with brown products of decomposition. In glucose-amended soils, changes in the percentage N content of Fraction 3 were not statistically significant and changes in the appearance of the fraction were less obvious, with the fine, dark-brown material predominating throughout. Small amounts of light-brown and brown plant fragments, present at days 2, 8 and 16, diminished in size and quantity on continued incubation. (c) Fructions 4, 5, 6, 7. Approximately 75% of the unlabelled, native N of each soil was associated with the silt (Fraction 5) plus coarse clay (Fraction 6) components (Table 3). In the sandy soil, the silt fraction had the highest percentage N content and accounted for about half of the soil N. In the clay soil, silt and coarse clay fractions were of similar, high percentage N contents whereas sand fractions (Fraction 4) from both soils were of low and variable N content. Neither fraction weight nor amount of N of Fractions 4, 5,6 or 7, varied significantly with time of sampling. Fraction 6 alone for both soils showed a significant (P < 0.05) decline in percentage N content from a maximum at day 2. (d) Subfractions. Silt and clay fractions of each glucose-amended soil, sampled on day 16, were further separated into light and heavy subfractions, alter dispersing freeze-dried material in Nemagon, sp. gr. 2.06 (Table 4). Overall the mean percentage recoveries of fraction weight (101.1%, S.E., l.lV/;,) and fraction N (98.9%, SE.. 2.4%) were complete. Material in subfraction 5a, from silt size particles, was a black, finely-divided, amorphous powder mostly derived from material floating at the surface of the Nemagon after centrifugation. The silt light sub-
Table 4. Distribution
S&l North~eld flay
Distribution
Sub-fractmn
number
(part>& density. g ml-‘) %I( C2.06) 5q> 2.06)
72 920 6 1005 54 988 228 752 77 967
7a( < 2.06) 7b(>206)
74 923 so
7(
<50) <2)
according
to particle
’
6a( < 2.06) 6b(>206) 7a( ~2.06) 7b(>2.06) 5a( < 2.06) 5b( > 2.06) ha(<2.06) 6b( > 2.06)
5(:-Z,
separated
Sub-fraction weight ,mp gfraction)
5(> 2,
6(>0.2,
during the net imnobili-7ation
phase
During the net immobilization phase, 15N became distributed amongst all soil fractions. The nature of the C amendment in~uen~d both the extent to which N of soil fractions became labelled (Fig. 4) and the amounts of lSN distributed in the soil fractions with time (Fig. 5). However, the patterns of change of “N in soil fractions were, for a given C amendment, similar in both soils despite great differences in soil texture. (a) Fractions 1: 2. Rapid microbial growth during *5N0; immobilization resulted in the formation of small amounts of highly labelled, extractable, extracellular compounds of high and low mol. wts. Compounds of high mol. wt. extracted from the clay soil were, for a given C amendment, more highly labelled (Fig. 4) but in lower concentration (Fig. 5), than those from the sandy soil. This may reflect a greater but differential adsorption af both labelled and unlabelled products by the clay soil. However, differences may also occur in the nature of the microbial populations and in the proportions of the newly-formed. immobilized 15N present as extracellular and lytic products. In soils amended with straw, organic-15N of Fraction 1 increased si~ifi~ntIy (P < 0.05j between days 2 and 8. reflecting increasing (P < 0.001) ’ ‘N atom ‘;: enrichments of the N of the fraction during the early net immobilizatioil phase. Thereafter, the proportion of 15N compounds in Fraction 1 declined, commencing before immobilization was maximal at 16 days.
6(>0.2.
C2)
qf “N
of soil weight and unlabelled soil N in sub-fractions
Fraction numhtlr (particle dia El”&% pmi
7( C.O.2) Two Wells Sand
fractions had percentage N contents IO- I? times greater than those of the corresponding heavy subfractions and contained about 50.-75”;, of the total-N of the silt fractions. Material in subfraction 6a, which was derived almost entirely from suspended rather than surface8oating particles, had a percentage N content similar to that of the corresponding heavy subfraction and accounted for < IOU/(,of the N of the coarse clay fraction from which it originated. Subfraction 7a. prepared from the fine clay fractions, contained dark-brown. ~nely-divided material, most of which had floated at the surface of the Nemagon. The percentage N contents of the light subfractions were 3- 4 times greater than those of the corresponding heavy subfractions. Approximately 20:; of the fine clay N was contained in the light subfractions.
I 40 0.1: 0 I9 0. I H 0.25 0.08 I 90 0. I9 0 53 045 O.XI 0 2: 0.066
I Ofi6 !OXZ I3 I762 133 786 4310 1401 406 437s 59x 2149 160
48.2 51.x 0‘6 9Y4 i-t.? 85 ‘; 75.6 244 us 91.5 21.X 7x.2
density
Physical distribution of immobilized nitrogen in soils
L
9
NORTHFIELO
2
30
I 4 :
20
-GLUCOSE
315
TWO
WELLS
- STRAW
‘IWO
WELLS
-GLUCOSE
B 10 0 SAMPLINGDAY
2816
2616
2616
2616
1
2
3
L
FRACTION
2.916 2616 5
6
2616 7
2 6 16 2 676 2
I
2 8 16 2 616
2 6 16 2 616
L
3
5
2 6 16
6
7
Fig. 4. The proportion of N of sail fractions accountable as 15N in gkcose- and stra~“ameoded soils. For each fraction in tarn, values are shown far soils sampled on days 2, 8, and 16. S.E.D. (Fraction l), 1.52; (F2), 1.54: (F3), 3.88; (F4), 1.05; (F5), 0.33; (I%), 1.46; (F7), 1.01.
of the extractable, immobilized ‘% to actively-metabolizing microflora and its transfer to other organic residues before onset of net mineralization are in accord with the observations of Ladd and Paul-(1973) using bicarbonate extracts of a sandy loam and by McGill et al. (1975) using pyrophosphate extracts from a high clay soil. Extractable, ‘SN-labelled, organic compounds of low mol. wt. were rapidly formed in glucose-amended soils, to an extent which made Fraction 2 the most
In glucose-amended soils, nitrogenous compounds af high mol. wt. became labelkd more rapidly and changes in the r ‘N of Fraction I during a period (24 days) of complete immobilization were not statistically significant. However, in the sandy soil there was a significant (P < 0.05) decline in the relative ‘%I atom % enrichment of the N of Fraction 1 before net mineralization was observed, i.e. between days 2 and 8, indicating a preferential transfer of organic-“N to other fractions. The ready availability of at least part
77.0 TWO
TWO
0 SAMPLINGDAY FRACTION
2616
2616
1
2
2816 3
2616
2616
2816
2616
b
5
6
7
2616 1
WELLS
-
WELLS
2816
2616
2616
2
3
L
STRAW
-GLUCOSE
2616 5
28% 6
2&t? 7
Fig. 5. Distribution of recovered “?I in fractions of glucose- and straw-amended soiIs. For each fraction in turn, values are shown for soils sampled on days -2, 8 and 16. S.E.D. [Fraction I), 0.86: (F2), 2.14; fF3), 1.25; @?4),0.43; fF5f, 1.59; jF6), 3.73; QT), 3.73.
316
.I. !‘d. LAIN.
J. W.
PARSOhS
highly labclled of any from these soils. The ‘“N atom Y!{enrichments of this fraction were least at day 8 but did not change much during the entire incubation. In straw-amended soils. ’ 'N-labclled compounds formed a decreasing proportion of the N of Fraction 2 as incubation continued. The decreases corresponded with immobilization of the added “NO;. which was located in this fraction. (h) F~ctiort 3. The proportions and amounts of ‘“N-labelled compounds in the nitrogenous pool represented by soil material of sp. gr. < 1.59. were highly influenced by the nature of the C amendment (P < 0.001) (Figs. 4 and 5). In straw-amended soils. “N of the light fraction increased significantly (P < 0.001) to account at day 16 for about 20:; of all of the immobilized. labelled material in each soil (Fig. 5). The inclusion of decomposing straw residues influenced the percentage content of both unlabelled and labelled N of Fraction 3 (Figs. 3 and 4). Values for percentage “N + 14N content increased throughout. from 0.30”/;, (clay soil) and 0.561,, (sandy soil) on day 2, to 1.70:/, (clay soil) and 2.09”;, (sandy soil) by day 160 when the added straw was extensively decomposed. Nevertheless, the presence of relatively available C, as in straw, did not also increase significantly the amount of unlabelled N immobilized in the light fraction. (The decline in the atom”,, enrichment of i5N0; during the net immobilization phase demonstrated that some unlabelled NO; was being formed, but insufficient amounts were being immobilized in Fraction 3 to be detected as a net gain in N of the fraction.) In glucose-amended soils, the N of Fraction 3 was poorly labelled (Fig. 4) and changes in the small amounts of “N of this fraction were not statistically significant (Fig. 5). Obviously-recognizable root fragments, which otherwise would have been included in this fraction and which. like wheat straw, may have been a relatively available C source, had been removed soon after sampling the soils in the field. Thus the light fraction of glucose-amended soils, as judged by physical appearance. had already undergone extensive microbial decomposition before the experiment. This view is supported by the observation that the percentage unlabelled N content of Fraction 3 from glucose-amended soils remained constantly high (2.02”;. S.E., O.OS”,,) throughout the incubation (Fig. 3). (c) Fractions 4, 5, 6. 7. Most of the newly-immobilized ‘“N was associated with silt (Fraction 5) and clay (Fractions 6 and 7) (Fig. 5). Of the particle size fractions, the fine clay became the most highly labelled. irrespective of soil or C amendment (Fig. 4). In soils amended with glucose. all 15N0, was immobilized by day 2. At this stage. the atom 1, enrichment of the N of the fine clay fraction was maximal and this fraction contained 34’10(sandy soil) and 630, (clay soil) of the total immobilized r5N. During the “N was period when ‘“N remained immobilized, redistributed amongst the fractions, In particular. between days 2 and 8. statistically significant (P < 0.01) decreases in the amounts of 15N in the fme clay fraction were accompanied by significant increases in the amounts of “N of the silt (P < 0.05) and coarse clay (P < 0.01) fractions. In the silt frac-
and M. hf~ru
tion. further gains occurred between days Y and 16. Thus, during growth on microbial metabolites by successive microbial populations in glucose-amended soils. organic-‘“N was transferred from the fine clay fraction to the silt and coarse clay components (Fig. 5). Changes in 1‘N of the fractions were not significantly due to changes in fraction weight, resulting from differences in the extent of soil dispersion (or possibly reaggregation) of the soils. Rather changes in “N of the fractions directly reflected changes in the 15N atom “Clenrichments of the N of the fractions. The nature of the nitrogenous material transferred from the fine clay fraction is unknown. Possibly adsorbed extracellular and lytic products formed, like those of Fraction 1, during the initial flush of microbial growth, were further metabolized. Resynthesis of this extracellular ’ 5N into microbial cells may relocate it in fractions of larger particle size after dispersion of the soils. Alternatively. some of the 15N initially immobilized within the fine clay fraction may be part of a microbial biomass. The fine clay fraction could contain spherical organisms of diameter up to 0.8 /(rn. assuming a cell sp. gr. of 1.1. The viability of these newly-formed microbial cells may be low and the organic-“N associated with them may become incorporated into larger and more stable cells of a secondary population. Other alternatives arc possible. For example. rapidly growing microbial cells in soils sampled on day 2 may be more readily dispersed than cells growing more slowly in soils sampled on day 8. Further studies should involve measurements of the changes of biomass-“N and non biomass-“N of the particle size fractions during the net immobilization phase. Soil fractions obtained by McGill rr al. (1975) included material of particle diameter <0.04 Aim. This fraction contained a significant (approx. 25;.,) proportion of the recently-immobilized N. which they reasonably ascribed to extracellular and lytic products and released cytoplasmic compounds adsorbed to very fine clay particles. Their method involved extraction of dried soil with a pyrophosphate solution. followed by dispersion of the extracted soil by ultrasonic vibration. A difficulty introduced by their procedure lay in deciding the extent to which the distribution of the immobilized N was due to natural processes of cell growth and lysis or to cell rupture from drying. extraction and sonic&ion of the soil. If the latter were cxtensivc, then changes in N of fractions due to immobilization mineralization reactions ma\ have been obscured. In our study, soils were not dried before extraction, extraction with bicarbonate was brief, and dispersion was obtained by maceration. This procedure caused only a few cells to rupture, as judged by retention of dehydrogenasc activity (Ladd and Paul. 1973). Nevertheless the soils were well dispersed, based on a preliminary comparison of the perceptages of soil of particle diameter <2 /Irn, obtained by the above treatment and by that of Emerson (1971). However. the percentage of material of particle diameter <0.04pm was far less than that reported by McGill ct 01. (1975) and material in this size range was not analysed as a separate fraction. Thus strict comparison of our results with those of McGill rt al. (1975) are not possible because of differences in techniques
Physical distribution of immobilized
nitrosen
in soils
317
Table 5. Distribution of “N in subfractions separated according to particle density
Soil Northfield Clay
Fraction number (particle diameter range, pm) 5(>2,
Subfraction number (particle density. g ml-‘)
<50)
5a( <2.06) 5b( > 2.06)
<2)
6a( < 2.06) 6b( > 2.06) 7a( < 2.06) 7W> 2.06)
6(>0.2. 7( <0.2) Two Wells Sand
5(>2. 6(>0.2.
<50) <2)
7( <0.2) S.E.D.
Sa( < 5q> 6a( < 6bj > 7a( < 7bl>
2.06) 2.06) 2.06) 2.06) 2.06) 2.06)
“N (y” of subfraction-N) 2.95 3.62 3.x7 4.6X 15 55 1048 ?62 444 14.98 14.79 6.91 8.33 0 32
and in the particle sizes of fractions under consideration, but both studies support the view that during the net immobilization phase, newly-formed nitrogenous products are transferred from fine to coarser soil particles. In straw-amended soils, the organic-15N of all particle size fractions, except sand, increased significantly (Fraction 5, P < 0.001; Fractions 6 and 7, P < 0.05) during the first 16 days’ incubation (Fig. 5). The major gains occurred between days 2 and 8. Net immobilization of ’ 5NO; in straw-amended soils was complete only at day 16. Any prior transfer of organic-15N from the fine clay fraction may have been obscured by a net gain due to increased total amounts of 15N immobilized. However, from 8 to 16 days, when net immobilization was continuing, gains in the organic-15N of the silt fraction were significant (P < 0.01) but not in the clay fractions. (d) Subfractions. In the sandy soil, the N of the heavy subfraction of both the silt and fine clay components (Table 5) was more highly labelled than that of the respective light fraction (P < O.Ol), whereas labelling of the N of the coarse clay subfractions were very similar. By contrast, in the clay soil, the N of the light subfraction of the fine clay was more labelled than that of the complementary heavy subfraction (P < 0.001).
Thus in the silt and clay size fractions, a proportion of the labelled, organic nitrogenous products was apparently bound to inorganic components, but in the silt fraction, a large amount could be separated as “partially humified” components of sp. gr. ~2.06. The observed distribution of labelled and unlabelled N may have been influenced by the freeze-drying of the fractions before densimetric subfractionation. Work with undried preparations is needed to determine whether significant, differential gains and losses of N occur within these light and heavy subfractions during net immobilization or long-term net mineralization. Acknowledgements-We arc indebted to Mr J. Greenaway and Dr A. W. Moore, Division of Soils, St. Lucia, Queensland, for mass spectrometric data, to the late Mr A. R. P. Clarke for general soil analyses and Dr R. Correll, Division of Mathematics and Statistics, Glen Osmond, for statistical analyses. REFERENCES
J. M. (1965a) Total nitrogen. In Methods qf Soil Analysis (C. A. Black, Ed.), American Society of Agronomy Monograph. 9, (2) 1149-l 178.
BREMNER
Distrlbutlon of fraction “N (pg lSN in subfraction g- ’ fraction) 31 41 0.5 x7 24 93 117 66 72 75x 45 196 14
BREMNER
Percentage distribution of recovered
’ 'N
43.0 57.0 0.6 99.4 20 5 79.5 63.9 36. I 8.7 91.3 18.7 XI 3
J. M. (1965b) Inorganic forms of nitrogen. In Methods of Soil Analysis (C. A. Black, Ed.), American Society of Agronomy Monograph. 9, (2) 1179-1237. BREMNER J. M. and SHAW K. (1958) Denitrification in soil: I. Methods of investigation. J. agric. Sci. 51, 22-39. BROADBENT F. E. (1968) Turnover of nitrogen in soil organic matter. In Organic Matter and Soil Fertility. Pontif acad. Sci. Scripta I/aria 32. 61-82. CHICHESTER F. W. (1970) Transformations of fertilizer nitrogen in soil. II. Total and 15N-labelled nitrogen of soil organo-mineral sedimentation fractions. PI. Soil 33, 437456. COLOM J. and WOLCOTT A. R. (1967) Forms of organic nitrogen in clay-amended sandy soil. PI. Soil 26, 261-268. EMERSON W. W. (1971) Determination of the contents of clay-sized particles in soils. J. Soil Sci. 22, 50-59. FORD G. W. and GREENLAND D. J. (1968) The dynamics of partly humified organic matter in some arable soils. Trans. 9th int. Congr. Soil Sci. Adelaide 2, 403410. FORD G. W., GREENLAND D. J. and OADES J. M. (1969) Separation of the light fraction from soils by ultrasonic dispersion in halogenated hydrocarbons containing a surfactant. J. Soil Sci. 20, 291-296. GREENLAND D. J. and FORD G. W. (1964) Separation of partially humified organic materials from soils by ultrasonic dispersion. Trans. 8th int. Congr. Soil Sci., Bucharest 3. 137-148. JANSSON S. L. (1967) Soil organic matter and fertility. Trans. int. Sot. Soil Sci., Aberdeen, Commissions II and IV, l-10. JANS~ON S. L. and PERSSON J. (1968) Coordination of humus chemistry and soil organic matter biology by isotopic techniques. In Isotopes and Radiation in Soil Organic Mutter Studies. IAEA, Vienna 11 l-124. JENKIN~ON D. S. (1971) Studies on the decomposition of Cl4 labelled organic matter in soil. Soil Sci. 111. 64-70. KEENEY D. R. and BREMNER J. M. (1964) Effect of cultivation on the nitrogen distribution in soils. Proc. Soil Sci. Sot. Am. 28. 653-656. KEENEY D. R. and BREMNER J. M. (1966) Characterization of mineralizable nitrogen in soils. Proc. Soil Sci. Sot. Am. 30, 714-719. LADD J. N. and PAUL E. A. (1973) Changes in enzymic activity and distribution of acid-soluble, amino acidnitrogen in soil during nitrogen immobilization and mineralization. Soil Biol. Biochem. 5, 825-840. LADD J. N., PARSONS J. W. and AMATO M. (1977) Studies of nitrogen immobilization and mineralization in calcareous soils-II. Mineralization of immobilized nitrogen from soil fractions of different particle size and density. Soil Biol. Biochem. 9, 319-325. MCGILL W. B., SHIELDS J. A. and PAUL E. A. (1975) Relation between carbon and nitrogen turnover in soil organic fractions of microbial origin. Soil Biol. Biochem. 7. 57-63.
31x
J. N. LALII), J. W. PARSONS and M. AMTO
N~RTIKOTL K. H. (1971) A Factuul Keyfbr the Rrcogrzirion of Awstrulim Soils. CSIRO. Australia, Division of Soils.
Divisional Report. 3rd edition Pub. Rellim Technical Publications, Glenside, S.A. OArIFs J. M. (1967) Carbohydrates in some Australian soils. Aust. J. Soil Rrs. 5. 103-116. OADES J. M. and LADII J. N. (1977) Biochemical properties. Carbon and nitrogen metabolism. In Soil Factors in Crop Production in (I Srrni-arid Environmrnr (J. S. Russell and E. L. Greaten, Eds.), Australian Soil Sci. Sot. Monograph, Univ. of Queensland Press. in press. PERSSON J. (1968) Biological testing of chemical humus analysis. La&r-Hogs/c A/m/r 3. 81-217. Ross P. J. and MARTIN A. E. (1970) A rapid method for preparing gas samples for nitrogen-15 determination. ha/w. Land. 95. 817 822.
SOREMENL. H. (1972) Stabilization
of newly formed amino acid metabolites in soil by clay minerals. Soil Sci. 114. 5 ~1I. S~RIZNSENL. H. (1975) The influence of clay on the rate of decay of amino acid metabolites synthesised in soils during decomposition of cellulose. Soil Biol. Biochrm. I. 171-177. TINSLEY J., TAYLOR T. G. and MOORF. J. H. (1951) The determination of carbon dioxide derived from carbonates in agricultural and biological materials. Analyst, Lond. 76. 30&310. TURNER J. C. (1968) Triton X-100 scintillant for carbon-14 labelled materials. Int. J. appl. Radint. Isotopes 19. 557- 563.