different long-term treatments: soil I, prolonged arable cultivation; soil II, 17 years under grass after pro- longed arable cultivation; and soil III, old pasture.
Soii B:nlBuxkrax.Vol. 7.pp.65-71 Peigamon Press 1975. Pnnted
m Great Britam
COMPONENTS OF SOIL ORGANIC MATTER GRASS AND ARABLE CROPPING D.C. WHITEHEAD,HAZEL Grassland
Research
Institute,
Hurley,
(Acqred
UNDER
BUCHAN and R.D. HARTLEY Maidenhead, Berkshire, SL6 5LR, England 19 Auyusl 1974)
Summary-Components of the organic matter have been studied in three soils from adjacent sites with different long-term treatments: soil I, prolonged arable cultivation; soil II, 17 years under grass after prolonged arable cultivation; and soil III, old pasture. Contents of total organic C in the top 15cm were O+Q in soil 1. 1.7% in soil II and 4.8% in soil III. The light fraction, comprising partially decomposed materials with a specific gravity < 2.06, represented greater proportions of the organic C in soils II and III (2@-23 per cent) than in soil I(8.5 per cent). The light fraction of soil III had a relatively high N content. The proportions of the soil organic C released. by hydrolysis as neutral sugars, uranic acids, amino sugars, amino acids and phenolic acids were generally similar in the three soils, although uranic acids and phenolic acids constituted somewhat greater proportions in soils 11 and III than in I. The light fractions contained greater proportions of neutral sugars and phenolic acids, and smaller proportions of amino sugars and amino acids than the whole soils.
animal residues, and the organic matter in the latter being more fully decomposed and associated with inorganic soil colloids. The monomers released by hydrolysis of the two fractions were also determined.
After a period of years under grass a soil will usually have a higher content of organic matter and a higher degree of aggregate stability than a similar soil after a period of years under arable cropping. Aggregate stability is influenced by various factors but in many soils, particularly those containing large proportions of fine sand and silt, it is related to the content of organic matter (Cooke, 1967). Of the various components of soil organic matter, the polysaccharides synthesized by soil microorganisms are thought to be of particular importance in promoting aggregate stability (Harris, Chesters and Allen, 1966; Swincer, Oades and Greenland, 1969). However other components may be involved. Thus there is evidence tllat~henolic compounds may augment the effect of microbial polysaccharides (Griffiths and Burns, 1972) and that humic acid molecules may contribute to the stabilisation of aggregates (Swaby, 1949). While increases in total organic matter content in soils maintained under grass have been reported from several investigations (e.g. Richardson, 1938; Clement and Williams, 1964). there is little comparative information on the chemical composition of the organic matter in soils under grass and arable cropping. The aim of this investigation was to make such a comparison using three soils from sites, all within a small area and having similar parent material, that have been maintained either under grass or arable cultivation for prolonged periods. Analyses were made of the neutral sugar, uranic acid, amino sugar, amino acid and phenolic acid monomers released by hydrolysis of the three soils. In addition, samples of each soil were separated into light and heavy fractions. the former consisting mainly of partially decomposed plant and
MATERIALS AND METHODS
Soils The three soils used in the investigation were all from within an area of 150 x 250 m which overlies Upper Chalk, but the soil parent material is derived largely from Eocene deposits. Two of the soils were from an experiment at the Grassland Research Institute in which plots have been maintained since 1954 under continuous arable cropping (soil I) or under a ryegrass-white clover sward (soil II). Previously this area had been under continuous arable cultivation for about 100 years. Within the experiment there are four replicate and randomised plots of both the arable (I) and grass (II) treatments. The arable plots have grown barley continuously since 1962, the straw being baled and removed, and the stubble ploughed in. The grass plots have been defoliated at intervals by grazing. The third soil (III) was from an old pasture, thought to have been continuously under grass for several hundred years. Soils I and II were from the Frilsham series while soil III was obtained close to the boundary between this and the Windsor series. Thirty-two cores of each soil, 5 cm dia and 15 cm in depth were obtained in May 1971. With soils I and II, eight cores were taken from each of the four replicate plots. The cores of each soil were mixed and the soils passed through a 6-mm sieve to remove stones. roots and stubble. They were then freeze-dried and ground to pass a 2-mm sieve. 65
66
D. C.
WHITE.HI:AII. HAZIL BI:CHAN
Each soil was analysed for content of coarse sand, find sand. silt and clay; for total organic C by dichromate oxidation (Clement and Williams, 1964); for total N using a Coleman nitrogen analyser; and for pH in a 12.5 suspension in 0.01 M CaClz. In addition the structural stability of an air-dried, unground sample of each soil was assessed by the ratio dispersion method as described by Llermott (1967). Sep”‘“‘fior1 of’iighr U/U/f?ra7~~,fiwctioi7s The separation of samples of each soil into light and heavy fractions was carried out by the densimetric procedure of Greenland and Ford (1964) using Nemagon (1.2-dibromo-3-chloropropane. Shell ~iiternatio~al Ltd.)~ontainill~ 0.1”; w/v Aerosol OT (sodium dioctyl sulphosuccinate, Cyanamid Co. Ltd.) as the heavy liquid of specific g,ravity 206 (Oades and Swinccr, 196X). Batches of so11(6.0 g), dried over silica gel, were mixed with approximately 180 ml of heavy liquid and subjected to treatment for 3 min with an MSE ultrasonic disint~gr~~tor at a frequency of 20 kcis. A stream ofcold air was directed at the bottle contail~ing the soil undergoing disintegration to keep the temperature of the mixture below 35’C. The light fraction was obtained by centrifuging the mixture and filtering the liquid and suspended material. Both the light fraction and the residual heavy fraction were washed with acetone and dried over silica gel.
The neutral sugars, uranic acids, amino sugars, amino acids and phenolic acids were determined after acid or alkaline hydrolysis of the soils and their fractions. For the estimation of neutral sugars, liydroiysis was carried out by the procedure recommended by Oades, Kirkman and Wagner (1970). Individual neutral sugars were determined by the method of M. V. Cheshire (unpublished) which is similar to that of Albersheim, Nevins, English and Karr (1967) except that excess acetic anhydride is removed by evaporation with ethanol followed by extraction with ether to remove the last traces of the anhydride before gas chromatographv of the alditol acetate derivatives. A mixture of the de&atives prepared from the sugars listed in Table 3 was used as a reference solution. For the estimation of uranic acid content, the hydrolysis and purification procedure of McGrath (197 I) was employed, and uranic acid in the H,SO, eluatc was then determined in 0.5 ml samples by the method of Bitter and Muir (1962) using galacturonic acid standards. A factor of 1.25 was applied to the results as a correction for losses during hydrolysis (McGrath. 1971). Amino acids and amino sugars were determined after hydrolysis of 1.0 g of soil material or heavy fraction, or 0.2g of light fraction. with 25mI boiling 6~ HCI for 18 h under reflux in a stream of nitrogen. An aliquot of the hydrolysate was evaporated to dryness in a rotary evaporator and re-evaporated from water
and
R. D. HAKTL~Y
several times until the pH was about 3.0. The residue was dissolved in 5 ml of 209,, sucrose in a sodium citrate buffer of pH 2.875 (Technicon Instruments Corp.. 1970). Any solid material which precipitated at this stage was filtered of. Amino acids and amino sugars in 1 ml of the filtrate were then separated by gradient clution from an ion-exchange column and determined ~olorimetri~ally with ninliydrin. A Tcchnicon NC-1 Amino Acid Analvser was used with the ?Ih elution system described ‘in the Tcchnicon Handhook (Technicon Instruments Corp.. 1970) but with 70 ml of buffer in each cell of the ‘“Autograd”. Tho instrument was calibrated with a solution containing the amino acids. listed in Table 4. together with glucosamine and galactosamine, each at a concentr~~tion of 0.25 mM. Since amino sugars arc susceptible to losses during hydrolysis. a correction factor of 1.7 was applied to the results for glucosamine and galactosamine on the basis of data obtained by Sowdcn (1959). Amino acids arc relatively stable during the hydrolysis of soils with 6 N HCI (Bremner, 1965) and no corrcction factor was applied. Phenolic compounds were determined. after alkaline hydrolysis, by gas chromatography of their trimethylsilyl ether derivatives. The hydrolysis procedure of Morita (1965) was employed but after a preliminary 18 h extraction with cold 0.5 N NaOH in a NJ atmosphere. The two hydrolysis extracts wcrc combined. acidified with HCI to pH 2@, centrifuged. the solutions treated with a suspension of freshly precipitated rinc ferricyanide (15 ml!100 ml extract) to precipitate lipid material (Hamence. 1944) and recentrifuged. Tho solutions were then readjusted to pH 2.0 and extracted with ether. The ether extracts were dried over anhydrous Na,SO,, reduced to small volume. transfcrrcd to 3 ml glass tubes. evaporated to dryness and the residues dried over silica gel. Tri-sil (Pierce Chemical Co.) was added at the rate of 0.1 mljrng residue, the tubes stoppered, shaken and allowed to stand for 16 h at 3X’C. The trimethylsilyl ethers were determined using a Pye Series 104 gas chromdtograph with a l-5 m glass column containing 3”; SE30 on Gas Chrom P (80 -100 mesh). The temperature of the column was increased from 100 to 270 C at 2’ C min _ * and then maintained at this temperature. The rate of Row of Ar carrier gas was 80 ml min.- ’ and the flame ionization detector temperature was 250°C. A reference mixture of trimethylsilyl derivatives was prepared from p-hydroxyhenzoic. vanillic, syringic, ~)-collnlari~ and ferulic acids (0.25 mg of each) dissolved in 0.1 N NaOH and treated in the same manner as the soil hydrolysates. The retention times (min) of the derivatives of the acids were: phydroxybenzoic. 19.5; vanillic, 26.2: syringic, 33.2; pcoumaric. 35.0 and ferulic. 42.5. The identity of these acids in the ether extracts of hydrolysates of the soils was confirmed by TLC (Schleicher and Schiill cellulose plates, F 1440) using tolucne-formic acid-.water (40:45:15, upper layer) as solvent and diazotized I’nitroaniline (Swain, 1953) and diazotized sulphanilic acid (Krebs. Hcusser and Wimmer. 1969) as spray rea-
67
Components of soil organic matter gents. The R,r values of the acids were: p-hydroxybenzoic 0.06. vanillic 0.27. syringic 0.17, p-coumaric O3X. and ferulic 0.26.
RESULTS AND DISCUSSION
Since soils I and II were obtained from the randomiscd plots of a field experiment they were closely similar in particle size distribution and pH (Table I). The permanent pasture soil III, however, had a considerably higher content of clay. While both organic C and total N increased markedly from soil I to II to III. the C/N ratios were similar at approximately 10: I. The stability of the aggregates, as assessed by their dispersion ratios. increased in the order soil I. II, III.
A major difference between the grassland and arable soils was in the proportions of the soil organic C and N present in the light fractions: these proportions were considerably greater in the grassland soils (II and III) than in the arable soil (I). A similar difference between a grassland and cultivated soil was obtained in Australia by Oades (1967) who also reported a close correlation between the amount of light fraction and the stability of soil aggregates. The organic matter of the light fraction is important, firstly because it tends to prevent soil aggregates, once formed. from coalescing. and secondly, because it serves as a substrate for the synthesis of polysaccharides by soil microorganisms (Aliison, 1968). The light fractions had higher C/N ratios than the whole soils, the difference being more than twofold for soil I, rather less for II and much less for III (Table I).
Greenland and Ford (1964) also found that the C/N ratios of the light fractions were approximately double those of the whole soils when the soils were under continuous cultivation but that, under p~rmancnt pasture, the C/N ratios of the light fractions and whole soils were similar. The differences between the soils in the C/N ratios of the light fractions were due mainly to differences in N content, which when calculated as a percentage of the organic matter were 302. 3.15 and 4.50 for the light fractions of soils I, II and III, respectively. These results suggest that the light fraction of soil III had undergone much more microbial attack than those of the other soils, and that it would reiease, on further decomposition, more N in forms available for uptake by plants.
A major objective of the investigation was to compare the organic matter of the three soils in terms of the proportions of the various monomers released by hydrolysis. The contents of each class of monomer are therefore expressed, in Table 2, as percentages of the totai organic C in the three soils and in the light and heavy fractions. (The absoiute amounts of C in each class can be calculated from these values together with the C contents given in Table 1.) In addition, Table 2 includes the percentage of total N in amino sugars and amino acids. The atnounts of the individual neutral sugars are shown in Table 3, expressed on a percentage weight basis of the total neutral sugars. Results for individual amino acids and phenolic acids have been calculated similarly (Tables 4 and 5). The proportion of the total organic C that was hydrolysed to neutral sugars increased slightly in the order soil I, II, III (Table 2). With all three soils. considerably
Table I, Some physical and chemical properties of the three soils and their light fractions Soil I
Soil II
Soil III
31.7 42.8 9%
31.4 41.7 10.5
10.9 35.5 16.2
Clit y, y/C;
I36
14.3
17.0
Organic C. “;, Total N, ‘:, C/N ratio of whole soil pH (O-01M CaCl,) Dispersion ratio
0.949 0.102 9.3 6.70 24.4
I.729 0.163 IO6 6-58 13.0
0.36 8.5 4.1 22.68 I.178 19.3
20.2 Il.4 23.56 1.275
Whole soils Coarse sand, ‘I;, Fine sand, ‘I;, Silt. :‘c
Light fractions Light fraction, “~d.Rex. 5, 340-345. ALLISON F. E. (196X) Soil aggregation-some facts and fallacies as seen by a microbiologist. Soil Sci. 106, 136143. BtrrEK T. and M~:IK l-1. M. (1962) A modified uranic acid carbozole reaction. AIIN~~~.Biochrm. 4, 330-334. BKEMNER J. M. (1965) Organic forms of nitrogen. In /lil(~ri~~rtso{Soil Am~i!,sis (C. A. Black. Ed.) pp~l23& 1245. Am. Sot. Agron. Madison. C‘LEMIX~ c‘. R. and WILLIAMS T. E. (1964) Leys and soil organic matter-l: The accumulation of organic carbon in soils under difierent leys. J. ugric. Sci.. Cnfrlh. 63, 3773x3. COOKE G. W. (1967) The Cormol of Soil Ferri/it.r. Crosby Lockwood, London, p. 442.
GREENLANIJ D. J. and FOKI) G. W. (1964) Separation
of partially humiiied organic materials from soils by ultrasonic dispersion. Trans qf tlzr Eighth Gong. Int. Sot. Soil Sci., Bucharest, 1964. 3, 137-148. GRIFFI~HS E. and BURNS R. D. (1972) Interaction between phenolic substances and microbial polysaccharides in soil aggregation. PI. Soil 36, 599-6 12. HAMIZNCEJ. H. (1944) The detection and determination of auxins in organic manures-II. A,~lysr 69, 229-235. HAKRIS R. F., CHESTEKS G. and ALLEN 0. N. (1966) Dynamics of soil aggregation. -Ir/r~. Agrorl. 18, IO7- 169. HUNTJENS J. L. M. (1972) Amino acid composition of humic acid-like polymers produced by streptomycetes and of humic acids from pasture and arable land. Soil Bioi. BioChCJ117. 4, i3Y- 345.
JONES D. I. H. (1970) Cell-wall constituents of some grass species and varieties. J. Sci. Ftl Agric. 21, 559-562. KI(A& S. CT. (1969) Some carbohydrate fractions of a gray wooded soil as in~~ienced by cropping systems and fertilizers. Cuft. J. Soil Scj. 49, 2 1%224. KREBS K. G.. HEUSSEK D. and WIM%~~EK H. (1969) Spray reagents. In T&I-LUJU Chro,~wtol/r.np~~!! (E. Stahl, Ed.) pp. 855-9 I I. Springer, Berlin. MCGRA~H D. (197 I) The determination of uranic acid (and sugar) in soil. Geoder~ra 5, 261-269. MORIT~ H. (1965) The phenolic acids in organic soils. fin. J. Bi~}~b~/~l. 43, 127?- 1280. Onrt~s J. M. (1967) Carbohydrates in some Australian soils. ilust. J. Soii Rex 5, 103-l 15. 0~11~s J. M. (1972) Studies on soil polysaccharides~~lll: Composition of polysaccharides in some Australian soils. Aust. J. Soil Res. 10, I I3- 126. OAIXS J. M., KIRKMAN M. A. and WAC;NER G. H. (1970) The use of gas-liquid chromatography for the determination of sugars extracted from soils by sulphuric acid. Proc. Soil Sri. Sac. Am 34, 2X0-235. OAIJES J. M. and SWINGER G. D. (1968) Effect of time of ssmpling and cropping sequences on the carbohydrates in red brown earths. Tru~.s I$ t/w Ninth Cmg. IN. SW. Soil Ski., Adelaide, 1968, 3, 18%192. RICHARLJSONH. L. (lY38) The nitrogen cycle in grassland soils with especial reference to the Rothamsted Park grass experiment. J. ayric~. Scci., Cmfh. 28, 73.- 121. ROBERTS R. M., CETORTLLI J. J.. KIRBY E. C. and EIU~S~N M. (1972) Location ofglycoproteins that contain glucosamine in plant tissue. FI. Ph~~siol.. Ltr~~rstc~r, 50, 531 -535. SALAM~N M. (1963) Hcxosamines in soil aggregates. ,Ytrrurr. Lo,rrl. 200, 500. SOWDEN F. J. (1959) Investigations on the amounts of hexosamines found in various soils and methods for their determination. Soii Sci. 88, 138~143. SOWDIX F. J. (196X) Effect of long-term annual additions of various organic amendments on the nitrogenous components of a clay and a sand. CUU. J. Soil Sci. 48, 33 I-339. SWABS R. J. (1949) The intluencc of humus on soil aggregation. J. Soil Sci. 1, 182-194. SWAIN T. (1953) The identitication ofcoumarins and related compounds by filter-paper chromatography. Bi~~~~~~~~. J. 53,20&208. SWIK~ER G. D., OAIJ~~SJ. M. and GREENLANI) D. J. (1969) The extraction. characterization and significance of soil polysacchdrides. Adr. Apron. 21, 195. 235. TECHNI~ON IwrRuMfwrs CORPORATIOK (1970) Revised Operation Manual for the Technicon Amino Acid Analyser System Model No. NC-I. Technical Public~~tion No. TAO-O 155-00.
Components of soil organic matter WAGNERG. H. and MUTATKARV. K. (196X) Amino components of soil organic matter formed during humification of 14C glucose. Proc. Soii. Sci. Sot. Am. 32, 683-686.
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WEBLEYD. M. and JONESD. (1971) Biological transformation of microbial residues in soil. In Soil Bi~~~~~~?7isr~~. Vol. 2 (A. D. MCLARENand J. SKUJINS,Eds) pp. 4415485. Dekker. New York.
