CARBOHYDRATES IN THE SOIL

BY N. C. MEHTA, P. DUBACH AND H. DEUEL

Laboratory of Agricultural Chemistry, Swiss Feileral Znslitute o/ Technology, Zurich, Switzerland

I . Introduction. . . . . . . . . . . . . . . . . . . , . , , , . . . . . . . _ . _ . . . . , . . . . . , . . . . . . . . , . . 335 11. Isolation and Characterization , , . . , , . . . . , . , . , . , . , . , , . . , . . . . . . , . . _ , . . 337

1. Monosaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 2. Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 3. Other Carbohydrates.. . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . 343

111. Quantitative Determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 1. Hexoses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 2. Pentoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 3. Uronic Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 4 . Amino Sugars . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 346

347 6. Total Carbohydrates.. . . . . . , . . . . . , . . . . . . . . . . . , . . . . . . . . . , . . , . . . . , . . . . . 317

IV. Source arid Transformation. . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 1. Source. . . . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . , . . . . . . . . . 348 2. Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

V. State and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 1 . Interaction with Other Soil-constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 2. Function in the Soil.. . . . . . . . . . . . . . . . . . . 353

Surumitry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

. . . , . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . , . . . . .

VI.

I. INTRODUCTION

The soil is one station in the geochemistry of carbohydrates. Originating in higher plants, animals, and micro-organisms, carbohydrates occur in soils and peats, in lakes, rivers, and oceans, and in lignites, brown coals, fossils, and sediments in general.’ l2 Carbohydrates have even been isolakd from sedimentary rocks 180 to 300 million years

The soil is a complex mixture of numerous inorganic and organic con- stit,uent>s which vary in size, shape, chemical constitution, and reactivit,y, and it contains numerous organisms. The various constituents interact to form systems of higher order, thus contributing to the characteristic archi- tecture of various soils.4 The soil structure (that is, the arrangement of the

(1) P. H. Abelson, Fortschr. Chem. org. Naturstoge, 17, 379 (1959). (2) J . R. Vallentyne, in “Organic Geochemistry,” I. A. Breger, ed., Pergamon

(3) J. G . Palacas, F. M. Swain and F. Smith, Nature, 186, 234 (1960). (4) H. Deuel, Trans. Intern. Congr. Soil Sci., 7th Congr., Madison, Wisc., in press.

Press, London, in press.

335

336 MEHTA, DUBACH AND DEUEL

soil constituents in aggregates) determines to a large extent such properties as the air-water relationship, tillability, and stability toward erosion.

The kind of soil which is produced in a given location is controlled by the following factors: climate, parent material, organisms, topography, and age.6 The formation of soil is brought about by three groups of processes: the weathering of the inorganic parent material, the incorporation and transformation of organic material (humification), and the interaction and translocation of the inorganic and organic constituents. A soil “profile” with characteristic “horizons” is formed, the top soil being, in general, richest in organic matter. Various combinations of these processes lead to different, well recognized soil-groups, such as podzols, brown forest soils, chernozems, and laterites.e Normal soils are well drained and well aerated. They generally contain less than 10 % of organic matter. Under conditions of poor drainage and poor aeration, an abnormal accumulation of salts (as in saline soils) or of organic matter (as in peats) occurs. This review deals mainly with normal, well drained soils; only brief designations of the soils are given.

The organic constituents of the soil are collectively termed humus.’ The organic material originating from plants and animals (which is continually added to the soil) is rapidly decomposed. Under normal conditions, no continuous accumulation of any one substance is possible. While decomposi- tion is taking place, new substances are synthesized which, in turn, are decomposed. The decomposing and synthesizing processes usually reavh an approximate dynamic equilibrium.

Morphologically, two extreme humus forms can be distinguishede(”) : (a) mor, which is incompletely decomposed plant material, not incorporated into the lower, inorganic part of the soil, and (b) mull, namely, well de- romposed material, thoroughly mixed with the inorganic part of the soil.

Soil organic matter is a mixture of a great number of compounds of low to high molecular weight>#s Many compounds, most of which are known to occur in plant and animal tissues and in micro-organisms, have been iso-

(5) H. Jenny, “Factors of Soil Formation,” McGraw-Hill Book Co., Inc., New York, N . Y . , 1941.

(6) (a) J. S. Joffe, “Pedology,” Pedology Publications, New Brunswick, N . J., 2nd Edition, 1949. (b) W. L. Kubiena, “Bestimmungsbuch und Systematik der Boden Europas,” F. Enke, Stuttgart, 1963.

(7) Monographs on soil organic matter: (a) S. A. Waksman, “Humus,” Williams and Wilkins Co., Baltimore, Md., 1936. (b) M. M. Kononowa, “Die Humusstoffe des Bodens,” Deutscher Verlag der Wissenachaften, Berlin, 1958. (c) J. Pochon and H. de Barjac, “Trait6 de Microbiologie des Sols,” Dunod, Paris, 1958. (d) F . Scheffer and B. Ulrich, “Humus und Humusdhgung,” F. Enke, Stuttgart, 1980.

(8) F . E. Broadbent, Advance8 in Agron., 6 , 153 (1953). (9) J. M. Bremner, J . Soil Scsci., 2, 67 (1961); 6, 214 (1964).

CARBOHYDRATES I N THE SOIL 337

lated from soils.'O but these compounds constitute only an insignificant part of the total organic matter of soil. The major components of soil or- ganic matter, usually over 50%, are the so called humic substances; these are colored, organic acids of unknown (probably aromatic) constitution and of low (fulvic acids) to high (humic acids) molecular weight. Nitrogen accounts for 3 to 5 % of soil organic matter. About 4.5 to 55 of the total organic nitrogen is a-amino nitrogen, corresponding to a protein content of 5 to 1.5 % of soil organic matter. Total carbohydrates have been estimated to constitute between 5 and 20% (average 10%) of soil organic matter. Carbohydrates occur in the soil in living and in decaying organisms, as well as in extracellular form. As it is practically impossible to separate the living micro-organisms and tiny plant and animal tissues from the dead organic matter of soil, the whole system is usually treated as an entity. Carbohy- drates have been studied in the most diverse (and not always clearly de- fined) soils. In most cases, the top horizon of the soil profile has been ex- amined,

Although humus constitutes, generally, only a small part of the soil (below 10 %), it exerts a profound influence on the physical, chemical, and biological properties of the soil. Attempts have been made to attribute par- ticular influences to specific compounds in humus. The observation of the ameliorative effect12 of polysaccharides on soil structure has greatly stimu- lated research on the nature of soil carbohydrates.

The following questions may be asked. Which monomeric sugars are present in soil carbohydrates and in what quantities? What kinds of car- bohydrates occur in soil? What proportion of the organic matter of soil is carbohydrate? Are carbohydrates linked to other soil constituents? What is the source and what is the role of carbohydrates in soils? What are the differences between the carbohydrates of different soils?

11. ISOLATION AND CHARACTERIZATION

1 . Monosaccharides

Only trace amounts of monosaccharides have so far been detected in soils. The reducing sugars in cold-water extracts of Norwegian amounted to less than 1 % of the total soil organic matter (2 % for peats). Glucose, galactose, xylose, and rhamnose have been identified by paper chromatography in sodium hydroxide extracts of various Scottish soils.14

(10) E. C. Shorey, U . S D e p t . Agr., Bur. Soils, Bull., 88, 1 (1913). (11) See Ref. 7(d), pp. 132-139. (12) J. P. Martin, W. P. Martin, J . B. Page, W. A. Raney and J. D. De hlent,

(13) E. Alvsaker, Unio. Bergen Skrifter, 23, 1 (1948). (14) W. G. C. Forsyth, Chem. & Znd. (London), 515 (1948).

Advances i n Agron., 7 , 1 (1955).

338 MEH‘PA, DUBACH AND DEUEL

For these ext,racts, t,he Elson-Morgan test for amino sugars was also posi- tive. In a cold-water extract of a Norwegian forest soil, glucose (0.22 70 of the soil organic matter), galactose (0.02), fructose (0.035), xylose (0.03), arahinose (0.04), and ribose (<0.001%) have been det,ermined by quanti- tatdive paper-chromatography.l6 The fructose was identified by x-ray anal- ysis of it,s (2,4dinitrophenyl)hydrazone. This is, to date, the oirly refer- ence in the likrature to a definite identification of fructose in the soil. Sug- ars and uronic acids were detected in solutions pressed out of peat^.'^^ nlonosaccharides are strongly adsorbed by clay mineralslG; the proportioii of such carbohydrates could, therefore, be considerably greater than is in- dicat,ed by the amount, that has been extracted under mild conditions.

2 . Polusaccharides

Many methods have been used for the isolat,ion of polysaccharides from soil. Except for one ~ase , l7 (~ ) the maximum yield obtained awount,s for only about, 2 % of the soil organic matter. The average content of carbohydrat,e, however, is estimated to lie between 5 and 20% of soil organic matter.’ The nature arid state of the remaining carbohydrate material is not, known. So far, the primary aim of the various workers has actually been the iso- lation of an undegraded polysaccharide material free from inorganic and organic impurities, and the completeness of the extraction has only been of secondary interest.

a. Extraction.-Polysaccharides have been extracted from the soil (see Table I) wit#h buffers,l*Jg hot water,2°-2ss alkali,24.26 and acid;’ respectively.

(15) E. Alvsaker and K. Michelson, Acta Chem. Scand., 11, 1794, 1795 (1957). (15a) I. V. Aleksandrova, Pochuouedenie, 11, 85 (1960); Soils and Fertilizers, C o v -

monwealth Bur. Soil. S c i . , 24, 99 (1961). (16) D. J. Greenland, J . Soil Sci . , 7,319,329 (1956); R. A. Kohl and Y. A. Taylor,

Soil Sci . , 91. 223 (1961). (17)(a) J. W. Parson, “A Chemical Study of Polysaccharide Material Isolated from

Soil,” Ph.T). Thesis, University of Reading, Engl., 1958. (b) J . W. Purson and J . Tinsley, Soil Sci. Sac. A m . Proc. , 24, 198 (1960).

(18) W. N. Huworth, F. W. Pinkard and M. Stucey, Nature, 168, 836 (1943). (19) B. Bernier, Riochem. J., 70, 590 (1958); A. G . Oyston, ibid. , 70, 598 (1958). (20) R. B . Duff, J. Sci. Food Agr. , 3 , 140 (1952); Chem. & Znd. (London), 1104

(21) 0. Theander, Suensk Kem. Tidskr. , 64, 197 (1952); Acta Chem. Scand., 8 , 989

(22) R. L. Whistler and K. W. Kirby, J . A m . Chem. Sac., 78, 1755 (1958). (23) C. E. Clapp, “High Molecular Weight Water-soluble Muck: Isolation and

Determinution of Constituent Sugars of a Borate Complex-forming Polysaccharide, Employing Electrophoretic Techniques,” Ph.D. Thesis, Cornell University, N . Y. , 1957.

(23a) J. 1,. Mortensen, Trans. Intern. Congr. Soil Sc i . , 7th Congr., Madison, Wisc . , in press.

(1952); ib id . , 1513 (1954).

(1954).

CARBOHYDRATES IN THE SOIL 339

cir, s+ % of soil organac

malter

Extraction inetlzod Soils (Soil organic matter, yo)

---

Polysaccharides were extracted from various British soils with buffers, in yields of 0.05 to 0.15% of the soils.'* No further details of the isolation procedure were given. More recently, soils were extractcd with a phosphate buffer of pH 7, and the polysaccharides were recovered from the dialyzed and concentrated extract by precipitation with ethan01.l~ This procedure extracted less non-dialyzable, non-carbohydrate material than those em- ploying dilute alkali or sodium pyrophosphate. Yields were about the same as with hot-water extraction, but the polysaccharides isolated by means of phosphate buffer had a higher viscosity.

Ref- erences

TABLE I Extraction and Yield o j Polysaccharide Preparations jrom var ious soils

buffers (room temperature) phosphate buffer (room tem-

perature)

perature) alkali (0.5 N NaOH, room tem-

do. do.

water (4 hrs., 85") water (6 hrs., 85", N2) water (24 hrs., Soxhlet) acid (98% HCOzH, 30 min. re-

fluxing)

Yield of polysacclia- ride preparalions

0.05-0.15 - 18 0 . 0 1 4 . 0 4 - 19

- 1.32-1.94 24

0.02-0.13 0.10-1.00 25 0 . 0 2 4 . 2 5 - 20, 27

20 0 . 3 0.05 1.45 22 - 1.8 23

-

- 3.5-11.5 17(a)

British soils British forest soils

British and tropical

Swiss soils (3.0-32.5%) Wisconsin soils (0.8-

Scottish soil (5.6%) Indiana soil (3.38%) New York muck soil British mineral soils,

peats, and composts

soils

2.6%)

In a major investigation of their polysac~harides,~~ soils were extracted with 0.5 N sodium hydroxide. The humic substances of high molecular weight were precipitated by acidification of the extract to pH 2.5 to 3.0, and the centrifuged, light-colored solution was fractionated by chromato- graphy on a column of carbon. Four fractions were collected. Fraction A was eluted with 0.1 N hydrochloric acid, and contained amino acids, purine bases, and sugars; fraction B was eluted with 90 % aqueous acetone, and, on drying, gave a red powder with indications of the presence of phenolic glycosides; fraction C was eluted with water, and contained the polysac-

(24) W. G . C. Forsyth, Biochem. J . , 41, 176 (1947); 46, 1401 (1950). (25) P. Dubach, G. Zweifel, R. Bach and H. Deuel, 2. Pflanzenernahr. Dung. 'u..

Bodenk., 69, 97 (1955).

340 MEHTA, DUBACH AND DEUEL

charides; fraction D was eluted with 0.5 N sodium hydroxide, and contained colored humic material. The polysaccharide material was precipitated from fraction C by the addition of acetone; the yields were 1.32 to 1.94% of the soil organic matter. Polysaccharides have also been isolated from alkaline extracts by a slightly modified p r o c e d ~ r e ~ ~ * ~ 7 : the alkaline extract was acidified and centrifuged, and the polysaccharides were precipitated by pouring the neutralized, concentrated, supernatant solution into acetone.

Extraction of the polysaccharides with hot water20 dissolved only a small amount of humic substances, mainly of low molecular weight.22 The method has the disadvantage that polysaccharides may be degraded a t elevated temperatures. The soil was twice extracted a t 85" for 4 hours, and the polysaccharides were precipitated by pouring the dialyzed, concentrated extract into acetone.20 The original procedure has been modified to a 3-hour treatment21 a t 65" and to a 24-hour extraction in a Soxhlet apparatuv.23

A more complete e x t r a ~ t i o n ~ ~ ( ~ ) of polysaccharides was attempted by refluxing the soil for two 30-minute periods with 98 % formic acid contain- ing lithium bromide. The organic matter extracted was precipitated by the addition of ivopropyl ether and was redispersed in lithium chloride solu- tion. The colored humic substances were then precipitated with hexadecyltri- methylammonium bromide, while the acidic and neutral polysaccharides were kept in solution by the lithium chloride. The possible degradative effects of hot formic acid on soil polymccharides have not yet been investi- gated.

Except for the formic acid e x t r a c t i ~ n , l ~ ( ~ ) all the other methods yielded approximately the same amoun t of polysaccharide. However, even after more than 20 successive extractions of a Swiss brown-earth (Braunerde) with acid, water, and alkali, further extracts gave a positive anthrone re- action for sugars.28 The extraction of polysaccharides is probably made difficult by their interaction with inorganic surfaces and humic substanrcs.

b . Purification.-The raw polysaccharides have been purified by re-pre- ripitation, dialysis, formation of copper complexes, decolorization with carbon, and deproteinization with cadmium sulfate, and, also, by the Sevag method. These purification procedures always result, in appreciable loss of polysaccharide. None of the purified polysaccharide preparations were free from non-carbohydrates. Even after careful purification of the material, the carbohydrate ~ o n t e n t ~ 7 ( ~ ) ' ~ ~ was as low as 50 %. The non-rarbohydrate part contained humic substances and proteins.

(1954).

(1957).

(26) D. A. Rennie, E. Truog and 0. N. Allen, Soil Sci. SOC. Am. Proc., 18, 399

(27) G. Cheaters, 0. J. Attoe and 0. N. Allen, Soil Sci. SOC. A m . Proc., 21, 272

(28) Unpubliehed work of this laboratory.

CARBOHYDRATES IN THE SOIL 34 1

c. Fractionation.-Hydrolyzates of the polysaccharide preparations usu- ally contain more than ten sugars. Polysaccharides containing even 5 to 6 different kinds of sugar residues are rare, and none are yet known which have more than G different kinds of sugar residues.2g e30 The soil-polysac- charide preparations are, therefore, probably mixtures of different poly- saccharides.17Jg* 22, z 3 , 31 Consequently, many attempts have been made to fractionate the material, using the well established methods of polysac- charide chemistry.

The shape of the precipitation curve obtained on gradual addition of ethanol to an aqueous solution of the polysaccharide material indicated heterogeneity.22 However, the five fractions collected showed no significant quantitative differences in component sugars. Another attempt to obtain fractionation of the polysaccharides by precipitation with ethanol from a solution in water or fonnamide was unsuccessful.1g Precipitation with hexadecyltrimethylammonium bromide likewise produced no clear separa- tion.”(”) *l9 *32Electrophoresis1Q - 2 Z , 2 3 , 2 a a ~ f some preparations showed them to be heterogeneous without, however, giving an unequivocal separation. Acetyl- ation, followed by fractional precipitation in a series of solvents and by ultracentrifugation, produced no fractionation of the polysa~charides.’~ Some degree of fractionation was obtained by precipitation with Benedict copper s01ution.l~

Polysaccharides isolated from a Swiss river-soil by the carbon-adsorpt ion techniquez4 were fractionated by anion-exchange chromatography on a (2-diethylaminoethyl)cellulose column.31 Five fractions, having increasing uronic acid and decreasing (non-uronic acid) sugar content, were eluted with phosphate buffer and sodium hydroxide solutions of increasing concen- trations. The major portion of the polysaccharides was very low in uronic acid, and a small fraction was very high in uronic acids. After hydrolysis of the fractions, no differences in the sugar components could be detected by qualitative paper-chromatography. The approach is promising; it, may eventually lead to the isolation of individual polysaccharides whose constitution and origin can be studied.

d. Characterization.-The composition of polysaccharide preparations isolated from various soils is given in Table 11. Considering the differences in the soils and the methods used, the discrepancies in the findings are less

(29) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press Inc., New York, N . Y., 1953, p. 18.

(30) H. Deuel and H . Neukom, Kolloidchem. makromol. Naturslofe, 18, 91 (1958). (31) M. Miiller, N . C. Mehta and H. Deuel, 2. PJanzenerndhr. Dung. u. Rodenk.,

(32) H . Streuli, N. C. Mehta, M. Muller and H . Deuel, Mill. Gebiele Lebensm. u . 90, 139 (1960).

H y g . , 49, 396 (1958).

342 MEHTA, DUBACH AND DEUEL

ndiana soil22

surprising than the agreement in the main results. In the hydrolysates of extracted soil-polysaccharides, the following constituents have usually been found by paper chromatography and isolation of the pure sugars or their derivatives : glucose, galactose, mannose, xylose, arabinose, ribose, rham- nose, fucose, uroriic acids, amino sugars, and some unknown sugars.

mineral soils, 7s: peats, com-

New E'ork

posts'7'a'

TABLE I1

945 0.34 2 . 4

9 . 1 trace

-

Characterization of Polysaccharide P

- - - - - -

Equivalent weight N , % OCHa, % Reducing sugar, o/o Uronic anhydride, Yo Amino sugars, yo

Component sugars, % of total sugars in preparation

Glucose Galactose Mannose Arabinose Xylose Ribose Rhamnorre Fucose Unknown nugars

127 .8-36.4 22.2-22.6 28.6-29.8 - - -

7.7-13.6 -

3.5-6.6

iarations Isolaled frotn Various Soils

26.6-38.0 17.8-23.3 16.3-21 . 0 6.8-8.2 7.5-9.0 -

8.2-19 .O 0 0

,185 0 . 3 0 80 15.8 0

20.8 20.0 21.9 11.7 23.6

1 . 5 0 0 0 -

-

Scar lid

Soil'

- OOO

1 2

20 0

-

36

29

10 4

11 0 7

Soils

I I British

- - -

37.8-15.5 17.0-6.7 about 5

21.2 16.6 18.5 10.4 12.6

trace 14.2 0 6 . 5

Three unknown sugars of high Rl value have been found in traces.?" They have tentatively been identified as 0-methyl-hexoses and O-niethyl- heptoses. These 0-methyl sugars may be the same as the unknown sugars of high Rl values detected by other workers.22023,26J1 The presence of 0- methyl sugars has been demonstrated in nine out of ten soils and they seem to be of general occurrelice. One of them was found to be a constituent of a polysaccharide produced by Bacillus m~gatherium.~~ )34 The

(33) Macaulay Inst. Soil Research, Ann. Rept., 1964/66, 32. (34) W. G. C . Forsyth, Trans. Intern. Congr. Soil Sci., 6th Congr., Lhopoldville,

3, 119 (1954).

CARBOHYDRATES IN THE SOIL 343

reported presence of fructose in soil polysaccharides'8 has not been con- firmed, and no other ketoses have yet been found. Up to I 1 % of amino sugars has been determined (by the Elson-Morgan m e t h ~ d ' ~ ( ~ ) J ~ ) in the polysaccharide preparations. This extractable part of the amino sugars of soil is probably not chitin. Amino acids, also, have been found in the hy- drolyzates of the p r e p a r a t i o n ~ . ~ ~ ~ ~ ~ J ~

No structural work has been done on the polysaccharide preparations. In the hydrolyzates, the sugar components have been estimated quantita- tively by paper chromatography or other methods (see Table 11). It would be premature to try to interpret the ratios found between individual sugars or classes of sugars, or to discern a pattern in the variations between dif- feren t soils, because the preparations were heterogeneous and represented only a small part of the total polysaccharides of the soil.

Ultracentrif ugal studieslg have shown that the material isolated was poly- disperse and that the macromolecules were highly anisometric. Certain polysaccharide preparations contained from 60 to 90 % of dialyzable com- p o n e n t ~ . ~ ~

3 . Other Carbohydrates

The isolated monosaccharides and polysaccharides represent oiily a small part of the total carbohydrates of soil. The soil residue after extraction, and isolated fractions of soil organic matter (for example, humic substances), might contain sugars other than those which have been detected in the polysaccharidex isolated. However, hydrolysis of soils and of humic sub- stances isolated, followed by chromatography of the freed sugars, showed that this was not the case.22*36,36

Amino sugars have almost exclusively been investigated in hydrolyzates of the total soil. The presence of glucosamine and galactosamine has been definitely established by paper chromatography and ion-exchange chronia- tography37-42 arid by the isolation of both sugars in crystalline form.40 I t appears that 2-acetamido-2-deoxy-~-glucose (N-acetyl-~-glucosamiiie) is also present in the hydr~ lyza te s .~~ NO other amino sugars have as yet heeii detected .*2

Some other substances related to carbohydrates have been isolated from soils : glucaric acid,10 mannitol,1° inositol (present partly as esters of phos-

(35) D. L. Lynch, L. M. Wright and H . 0. Olney, Soil Sci., 84,405 (1957). (36) D. L. Lynch, H . 0. Olney and L. M. Wright, J . Sci. Food Agr., 9,56 (1958). (37) J. M. Bremner, J . Agr. Sci. , 39, 183 (1949). (38) J. M. Bremner, Biochem. J . , 47, 538 (1950). (39) J. M. Bremner, J . Agr. Sci., 46, 247 (1955). (40) J. M. Bremner, J . Sci. Food Agr. , 9, 528 (1958). (41) F. J. Stevenson, Soil Sci. SOC. Am. Proc., 18,373 (1954); 20,201 (1956). (42) F. J. Sowden, Soil Sci., 88, 138 (1959).

344 MEHTA, DUBACH AND DEUEL

phoric acid4a), ribonucleic acids, and deoxyribonucleic acids.44+46 All of these compounds are present in very small proportion. A rhamnoside has been isolated from an American soil.*O In the fractionat,ion of soil extracts on carbon,24 the presence of phenolic glycosides in fraction B (see Section II,2a) has been claimed. Four g. of fraction B per 100 g. of soil organic matter was obtained. The material was very unstable, darkening on stand- ing in the presence of air, until black tars were finally obtained. Ot,her worker^'^-^^ have obtained similar products. However, the evidence avail- able doe8 not unequivocally show the presence of phenolic glycosides.47

111. QUANTITATIVE DETERMINATION Investigations on the influence of the carbohydrates on soil properties,

and attempts to determine the composition of soil organic matter, have prompted many workers to determine, quantitatively, the proportion of carbohydrates in soils.

As pointed out in the preceding Section, complete extraction of carbohy- drates from the soil has not yet been accomplished. Moreover, the isolation of pure polysaccharides from the extracts is tedious and by no means quant,i- tative. It is, therefore, necessary to have quantitative methods for the determination of total carbohydrates or their individual components. The sugars have mostly been determined in hydrolyzates separated from the soil. It has not yet been found possible to determine either the completeness of hydrolysis or the losses occurring during hydrolysis. The diff ererit stabilities of the glycosidic linkages between the various sugars are not known. In addition, some of the sugars, particularly pentoses and uronic acids, may be partially destroyed during hydrolysis. It is difficult to determine ac- curately the substances or groups of substances present in such an extremely heterogeneous system as a soil. In no instance has a check, by isolation methods, of the results of the determinations yet been possible.

1. Hexoses

Attempts have been made to use colorimetric methods for t,he determina- tion of the hexoses in soils. The reaction with anthrone has been tried di- rectly on the soil, but was found to give irreproducible results.4* However, this method works with soil hydroly~ates.4~ In addition to what has been

(43) R. K. Yoehida, Soil Sci., 80.81 (1940). (44) A. P. Adams, W. V. Bartholomew and F. E. Clark, Soil Sci. SOC. Am. PTOC.,

(46) G . Anderson, Nature, 180, 287 (1967); Soil Sci., 86, 169 (1968). (40) T. V. Droadova, Pochuouedenie, 1, 83 (1966) ; Soils and Fertilizers, Common-

(47) E. Schlichting, 2. Pflanzenerndhr. DzZng. u. Boded . , 61, 97 (1953). (48) Macaulay Znst. Soil Research, Ann. Rept., 186S/M, 26. (49) R. H. Brink, P. Dubach and D. L. Lynch, Soil Sci., 88,167 (1960).

18.40 (1964).

wealth Bur. Soil Sci., 18, 27 (1966).

CARBOHYDRATES IN THE SOIL 345

said before concerning losses during hydrolysis, mention must be made of the fact that the various hexoses give different extinction coefficients in the anthrone method. The exact composition of the hexose mixture must, therefore, be known in order to permit of a calculation of the hexose con- tent. The hexose content of various American soils, determined by the anthrone method and expressed as glucose, was found to be between 4 and 13 % of the soil organic matter.@-498

Hexoees of Delaware soils have also been determined, by quantitative paper-chromatography of soil hydrolyzates, as amounting to 1 to 2 % of the soil organic matte1-.~6

2. Pentoses

Pentoses have frequently been determined in soils by the furfural-phloro- glucinol method.60 But phloroglucinol also gives precipitates with a variety of other aldehydes, such as 5-methyl-2-furaldehyde, 5-(hydroxymethyl)-2- furaldehyde, and formaldehyde. Orcino161 and aniline acetate62 are much more specific reagents, and no aldehyde present in the hydrochloric acid distillate from soils has been found to interfere in the furfural determin- ation by the orcinol method.63 The orcinol and aniline acetate methods give, for various Swiss and Norwegian soils, a “pentose anhydride” con- tent of 0.5 to 8.5 % of the soil organic matter (see Table 111) ; no corrections were made for the furfural derived from uronic acids.

Pentoses have also been determined in the hydrolyzates of soils (1 N sulfuric acid, 1 hour, 120°), after the removal of the uronic acids with anion exchanger, by Bial’s orcinol meth0d.6~8 They constituted 3 to 5 % of soil organic matter.

3. Urmic Acids

The Lefbvre-Tollens decarboxylation method for the determination of uronic acids has been applied to s o i l ~ . ~ ~ J ~ This method gives good results with plant material adequately prepared. With soils, however, unbelievably high values for uroiiir acid, up to 40% of the soil organic matter, are oh- tained. The decarboxylation method has been shown to be unsuited for the

(49a) D. N. Graveland and D. L. Lynch, Soil Sci. , 91, 162 (1961). (50) See Ref. 7(a), pp. 138-141, 160-163. (51) A. Johansson, Svensk Papperstidn., 66,820 (1952). (52) G. A. Adams and A. E. Castagne, Can. J . Research, BI. 314 (1948). (53) N. C. Mehta and H. Deuel, 2. PJlaneenernUhr. Dung. u. Bodenk., 90,209 (1960). (53a) R. L. Thomas and D. L. Lynch, Soil Sci. , 91,312 (1961). (54) E. C. Shorey and J. B. Martin, J . A m . Chem. SOC., 63,4907 (1930). (55) For a review see: (a) H. Deuel, P. Dubach and R. Bach, 2. PJlanzenerndlhr.

Dung. u. Bodenk., 81, 189 (1958). (b) H. Deuel and P. Dubach, ib id . , 82, 97 (1958). (c) H . Deuel and P. Dubach, Helu. Chim. A d a , 41, 1310 (1958). (d) H. Deuel, P. Dubach and N. C. Mehta, Sci. Proc. Roy. Dublin Soc., 1, 115 (1960).

346 MEHTA, DUBACH AND DEUEL

Reducing sugars as glucose

anhydride, Yo of soil organic

mallera

determination of uroriic acids in soils (as well as in decomposed arid ccrtain fresh plant materials). Only a small part of the evolved carbon dioxide originates from uronic acids. Ready decarboxylation has been found to be a general property of the colored humic substances.66 Preparations of humic substances that are free from uronic acids and sugars evolve carbon dioxide, even in a neutral medium a t 70".

TJroiiic acids in soil extracts have been determined by the curt)azole method in proportions of 0.07 to 0.16 % of the total soil.6fi In another study, the uronic acids constituted 1 to 4 % of the organic matter of the The glycosiduroriic acids arc not completely extracted from the soil, and

Ref- erences

TABLE I11 Fudural-yieldinu Substances and Ked?tcing Sugars of Various Soils

range average ~

G.Ck18.1 12 5.2-21.6 12 1.8-45.2 24

6.2-18.0 10

- - - -

Organic carbon of soils, of

sod

~

13

7(~),84, 68 53 52

<5 5-10 > 10

<5

<6 > 10

Soils

15 Norwegian soils 13 38

' I I 1

' I I 1

39 American soih

4 Swiss soils 2 Canadian soil8

Furfural- yielding substances as penlose anhydride, % of soil

organic mallera

range

0.5-3.6 0.74.9 2.4-8.5

-

1.5-3.5 6.6 and 10.

zverage

1.8 2.6 5.4

-

2.6 -

a Soil organic matter = organic carbon of soil X 1.72.

they are partially coprecipit,utJed with the humic acids3S which have to t)c removed heforr the determinat#ion. The carbazolc method, t hercforc, givcs only a minimum value when applied to extracts. The prohlem of the csti- mation of the total uroiiic acids of soils remains to bc solved.

4. Amino Sugars

Although amino sugars in the soil were not detected unambiguously until a decade ag0,~7 they are now the best investigated of the carbohydrates of the soil. Various independent, methods have been used for the determina- tion of amino sugars in soil hydrolyzates. The alkaline deamination of amino sugars followed by the determinat,ion of the ammonia liberated,37J!J

(56) D. L. Lynch, E. E. Hearns and L. J . Cotnoir, Soil Sci . SOC. Am. Proc., 21, 160 (1957); P. Dubach and D. L. Lynch, Soil Sci. , 87,273 (1959).

CARBOHYDRATES IN THE SOIL 347

42,67 the Elson-Morgan colorimetric rnethodl40~67~~ and adaptations of the Moorestein method69 for the fractionation and determination of amiiio compouiids on ion-exchange resins41 t41 have heen applied and found to give concordant results. There is also agreement on t,he optimal hydrolysis roil-

ditions, namely, 6 N hydrocshloric acid at 100" for Ci to 9 hours. The values are multiplied by a factor (for example, 1.25) to compensate for losses oc- curring during hydrolysis.

The results reported in the above-mentioned papers show consistently that between 5 and 10% of the organic nitrogen in surface soils consists of amino sugars. In many soils, the percentage increases with the depth of the soil sample removed, approaching 25 % in mature, clay-rich subsoils. In somc tropical soils, there is no accumulation with d e ~ t h . 6 ~ " The ratio of glucosamine to galactosamine varies with the kind of soi140~42*60 from 1.2 to 4.6, the highest ratio having been found in forest soils with acidic litter. If it is assumed that, in surface soils, the ratio of organic matter to nitro- gen is 20:1, the content of amino sugar reported above corresponds to roughly 5 % of the organic matter. As the total carbohydrates account for about 5 to 20% of the organic matter, the amino sugars constitute a sub- s tantial proportion of the carbohydrates.

5. Other Sugars

Among the other sugars detected in the soil, only the 6-deoxyhexoses have been quantitatively determined. In Delaware soils, rhamnose and fucose, determined by quantitative paper-chromatography, amounted to 20 % of the Under the conditions of furfural formation from pentoses, t,he 6-deoxyhexoses yield 5-met,hyl-2-furaldehyde; this has been determined by the differential solubilities of the phloroglucides in alcohol.61*6* The pro- portion of 6-deoxyhexoses in some cases exceeded that of pen toses.

6. Total Carbohydrates

The total carbohydrates of various soils13,63*64 have been determined, after hydrolysis, by measuring the reducing substances by the Bertrand and Hagedorn-Jensen methods. Soil hydrolysates always contain humic substances which reduce Fehling solutions6(") ,66; they do not,, however,

(57) J. M. Brernner and K. Shaw, J . Agr . Sci. , 44, 152 (1954). (58) F . J. Stevenson, Soil Sci. , 89, 113 (1957); 84,99 (1957). (59) S. Moore and W. H. Stein, J. B i d . Chem., 192, 663 (1951). (59a) 8. Singh and P. K. Singh, J . Indian SOC. Soil Sci. , 8 , 125 (1960). (60) F. J. Sowden and K. C. Ivarson, Plant and Soil, 11,249 (1959). (61) E. Michelet and J. Sebelien, Chemiker-Ztg., 30, 356 (1906). (62) R. Balks, Landwirtsch. Vers.-Sta., 103, 221 (1925). (63) For a review, see Ref. 7(a). (64) S. A. Wakeman and I. J. Hutchings, Soil Sc i . , 40,347 (1935). (65) T. B6res and I. Kiraly, AgrokCmia I s Talajtan, 6,245 (1956).

348 MEHTA, DUBACH AND DEUEL

interfere in the Somogyi method for the determination of reducing sugars.6 Soils with up to 10 % of organic carbon have, according to these methods, a carbohydrate content of about 12% of the soil organic matter (see Table 111). The proportion of carbohydrates seems to increase with the content of organic matter; soils high in organic matter usually contain a high propor- tion of poorly decomposed plant-material.

By analogy with plant analysis, the reducing sugars in soil hydrolyzates obtained with dilute acid were presumed to originate from “hemicelluloses,” and those liberated from the residue (by digestion with 72% sulfuric acid and subsequent hydrolysis with dilute sulfuric acid) were believed to origi- nate67 from “cellulose.” Since most of the carbohydrates in aerated soils are probably not plant polysaccharides, these terms are inappropriate. More- over, part of the so-called “cellulose” might actually be polymers of amino sugarsBB This terminology is, therefore, more applicable to soils containing humus of the more@ type (poorly decomposed plant material) than of the mull type (well decomposed organic matter).

The carbohydrates in soil hydrolyzates have been measured by quantita- tive paper-chromatography.96 The summation of individual sugars gives, for two Delaware soils examined, a carbohydrate content of 2.3 and 5.6%, respectively, of the soil organic matter. Considering the complexity of the material, accurate determination of the individual sugars is probably the only way in which to estimate the total carbohydrate content of soils. Quantitative column-chromatography has been successfully used for the determination of individual sugars in the hydrolyzate of decomposing fore~t-litter.~~8

IV. SOURCE AND TRANSFORMATION In the soil, a continuous addition, degradation, and synthesis of carbo-

hydrates takes place. A particular sample of soil gives a momentary glimpse into a dynamic (partly cyclic) system which might, except for seasonal vari- ations, be in equilibrium. The relatively constant level of soil organic matter and of carbohydrates therein over a long period of time does not, therefore, reflect a long “life” of the individual carbohydrate molecules.

1. Source

a. Plants.-The main primary source of carbohydrates is the added plant material; of this, carbohydrates comprise more than 50% of the dry matter. There is a wide variety of carbohydrates and carbohydrate-containing

(66) Unpublished results of this laboratory. (67) See Ref. 7(a), p. 408. (68) S. A. Waksman and K. R. Stevens, Soil Sci., 26, 113 (1928); SO, 97 (1930). (69) See Ref. 6(b), pp. 40-60. (69a) F. J. Sowden, personal communication.

CARBOHYDRATES IN THE SOIL 340

compounds in plants : mono-, oligo-, and poly-saccharides, glycosides, gal- lotannins, iiucleic acids, phytin, and so on; among these, polysaccharides predominate. Cellulose makes up the main part of the plant carbohydrates; other polysaccharides are starch, pectic substances, fructans, mannans, and xylans.70 The carbohydrates are incorporated into soils either as dead tissues or as exudates of living roots.71

b. Animals.-Animals are a minor source of carbohydrates for the soil. They may contribute glycogen, mucoids, chitin, nucleic acids, and so on.

c. Microorganisms.-It is believed that micro-organisms (bacteria, ac- tinomycetes, fungi, and algae) which decompose the primary plant, and ani- mal material synthesize the major part of soil c a r h o h y d r a t e ~ ~ ~ ~ ~ ~ ~ ~ ~ in aerated soils (see Section IV, 2b).

2. Transformation The carbohydrates in the soil are transformed mainly by endo- and exo-

enzymes.72 Most of the enzymes found in the soil are believed to be of mi- crobial origin, the contribution of plant enzymes being small. Considering the presence of a multitude of micro-organisms, it is not surprising that numerous enzymes have been identified in soils-for example, amylase, cellulase, hemicellulase, polygalacturonase, and invertase. In addition to the above-mentioned carbohydrases, the soil must contain other enzymes involved in the transformation and synthesis of carbohydrates. Only phy- t a ~ e 7 ~ and glucose oxidaseT4 have been detected up to now. During the deter- mination of soil-enzyme activity, the formation of additional enzymes has to be avoided. The effectiveness of toluene is a subject of contr0versy.~6

The enzymes in the soil may be adsorbed on other soil constituents; their activity is, thereby, decreased or increased, as shown by the results of model reactions.76

a. Decomposition of Carbohydrates.-Many studies have been made on the decomposition of total plant-materials and individual carbohydrates in

(70) See Ref. 29, p. 1. (71) H. Borner, Botan. Rev., 26, 383 (1980). (72) F. Richard, Mitt. Schweiz. Anstalt Forstl. Versuchswesen, 24, 297 (1945); H.

Sqjrensen, Nature, 176, 74 (1955) ; G. 5. Davtyan, Pochvovedenie, 5.83 (1958) ; I. Kiss, Nature, 182.203 (1968); for citations of papers by E. Hofmann and coworkers, see G . Hoffmann, 2. Pjlanzenerndhr. Dung. u. Bodenk., 86.97 (1959) ; J. Drobnlk, Plant and Soil, 12, 199 (1960); J. Augier and R. Moreau, Ann. inst. Pasteur, 99. 130 (1960); V. Turkovh and M. bogl , Rdstlinnct Vyroba, 6, 1431 (1960).

(73) R. H. Jackman and C. A. Black, Soil Sci., 73, 117 (1952). (74) A. S. Galstyan, Izvest. Akad. Nauk Armyan. S . S . R . , Biol. i Sels’skokhoz.

Nauki, 12 (No. 4), 75 (1959); Chem. Abstracts, 64, 11354 (1980). (75) D. Claus and K. Mechsner, Plant and Soil, 12, 195 (1960); J. Drobnlk, ibid.,

14.94 (1961); E. Hofmann and G. Hoffmann, ibid., 14.96 (1961). (76) A. D. McLaren, Soil Sci. SOC. Am. PTOC., 18, 170 (1954).

350 MEHTA, DUBACH AND DEUEL

soils and in compost^.^ Many of the results have been obtained by the acidic hydrolysis (sce Section 111, 6) and decarboxylation methods,77 and are thus subject to reservations. The results may be summarized as follows. All plant carbohydrates are more or less rapidly decomposed in the soil, a rough order of increasing stability of some of them being: monosaccharides, oligo- saccharides, starch, pectin, mannan, xylan, and cellulose.7(c) -7(d),77s Plant carbohydrates persist in different soils for various periods of time. Relatively less cellulose is found in the lower than in the upper soil horizons. In high- moors, there is an accumulation of cellulose and hemicelluloses. Chitin of fuiigal or animal origin seems to be rather stable. The microbial polysac- charides are not resistant,, although the rate of decomposision is lower than that of some plant polysaccharides.22.78 The carbohydrates are decomposed primarily by microbes. Purely chemical degradation is probably only im- portant under special soil conditions, as in acid peats. The carbohydrat)es may be protected against decomposition by lignin,7(*) protein-phenol com- plexes,79 humic substances, and clays.80 The soil aggregates contain many micro-pores (of diameter less than 1 p ) which are inaccessible to micro- organisme8* ; polysaccharides situated in such pores would be comparatively immune to degradhon. Extracellular polysaccharides are more likely to be protected by the above mechanism than those polysaccharides which are part of plant and animal tissues. It has been shown that drying of the soil increases the amount of water-soluble mono- and oligo-saccharides.sl*

The degradat,ion of carbohydrates leads, directly or indirectly, to various products, including carbon dioxide, organic acids, microbial polysaccharides, and humic substances. It! has often been maintained that carbohydrates are transformed into the dark-colored humic substances by chemical and microbial processes.’

The chemical degradation of carbohydrates, particularly under acidic conditions, produces reductones, furan derivatives, pyruvaldehyde, and so on, which can condense, either among themselves or with amino compounds (Maillard reactions), to produce dark-colored, amorphous products, similar to humic subst,ances.s2 Pyruvaldehyde, which has been held to be an inter- mediate in Maillard reactions, has been identified in many soils.8s Such con-

(77) A. G. Norman and W. V. Bartholomew, Soil Sci. Soc. Am. Proc., 6 , 848 (1940). (77a) H . K. Juin and A. K. Bhutt,acharya, 2. Pflanzenerndhr. Dung. u . Bodenk., 91,

(78) J . P. Martin, J . Bacteriol., 60, 349 (1945); Soil Sci., 61, 157 (1946). (79) W. R . C. Handley, “Mull und Mor Formation in Relation to Forest Soils,”

(80) D. L. Lynch and L. J . Cotnoir, Soil Sci. Soc. Am. Proc., 20, 367 (1956). (81) A. D. Itovira and E. L. Graecen, Australian J . Agr . Besearch, 8,659 (1958). (81a) B. Bernier, Lava1 Uniu., Foresl Research Foundation, Contrib. NO. 6, (1960). (82) See Ref. 7(d), pp. 120-125. (83) C. Enders and S. Sipirdsson, Biochem. Z., 313,174 (1942).

233 (1980).

Her Majesty’s Stationery Ofice, London, 1954.

CARBOHYDRATES IN THE SOIL 35 1

densation reactions may occur in very acid soils, but are certainly of minor importance in the formation of soil humic substances in general. The sug- gestion that phenolic glycosides are intermediates in humic-substance for- mationZ4 has not been substantiated.

According to one theory, the synthesis of humic substances is supposed to be brought about, primarily, by the condensation of the autolysis products from micro-organisms growing on carbohydrates (mostly cellulose) .7 The biosyrithesis of aromatic compounds from ~arbohydrstes8~” may be of im- portance in the soil.

It has been shown by tracer techniques that, when labeled glucose, hemi- cellulose, or cellulose is allowed to decompose in the soil, the activity is rapidly distributed in all soil organic fractions examined.84 If the experiments were to be repeated on better defined fractions, the role of carbohydrates iii humic-substance formation could be considerably clarified.

b. Synthesis of Carbohydrates.-Micro-organisms are capable of synthesiz- ing polysaccharides and other carbohydrates, frequently as LZ major meta- bolic product.86 The polysaccharide formation may be endo- or exo-cellular. Polysaccharides produced by a few soil-bacterial species, in pure cultures, have been intensively investigated.8R Not much information is available about polysaccharides produced by the bulk of soil bacteria,34J8,86a*87 and even less is known about the fungal polysaccharides of soil.88 Furthermore, the behavior of micro-organisms in pure cultures under optimum conditions does not indirate how they might behave under natural competitive condi- tions.

It has been found that 5 to 16% of the bacterial species isolated from various British and tropical soils are capable of producing exocellular poly- saccharides on synthetic rnedia.T8 A large majority of these produce either levans or glucose-uronic acid p o l y m e r ~ . ~ ~ ~ ~ 7 Since t8he levan-producing bac- teria require a sucrose or raffinose substrate, and since these sugars have not been found in the soil, the contribution of levans to soil polysaccharides is probably negligible.I4 The levan-producing bacteria produce non-levan polysaccharides when grown on monomeric sugars. Two other groups of soil bacteria, present in lower numbers, produce polymers of the glucose-man-

(83a) See D. B. Sprinson Advances i n Carbohydrate Chem., 16,235 (1960). (84) See J. Mayaudon and P. Simonart, Plant and Soil, 11. 181 (1959) and earlier

papers by these authors. (85) (a) T. H. Evans and H. Hibbert, Advances i n Carbohydrate Chem., 2,203 (1946).

(b) M. Stacey and S. A. Barker, “Polysaccharides of Microorganisms,” Clarendon .Press, Oxford, 1900.

(86) E. A. Cooper, W. D. Daker and M. Stacey, Biochem. J . , S2, 1752 (1938). (86a) D. L. Lynch, Can. J . Microbiol., 6 , 673 (1960). (87) (a) W. G . C. Forsyth and D. M. Webley, J . Gen. Microbiol., 3, 395 (1949).

(88) B. Bernier, Can. J . Microbiol., 4, 195 (1958). ( b ) Biochem. J . , 44,455 (1949).

352 MEHTA, DUBACH AND DEUEL

nose-uronic acid and glucose-mannose-rhamnose-uronic acid type, re- spectively. Some strains of Bacillus megathetiurn produce polysaccharides containing glucose, fructose, mannose, rhamnose, xylose, uronic acids, and, probably, a methylated sugar. Paper chromatography of hydrolyzed, bac- terial cultures invariably showed spots corresponding to ribose, probably from ribonucleic acid, and fucose.“ Nucleic acid might also be the source of ribose found in isolated soil polysaccharide preparations. Fucose, which is present in considerable amounts in the soil, is not known to be a common constituent of the higher plants, but it does occur in algal and bacterial polysaccharides in appreciable proportions.

Soil bacteria are capable of producing polysaccharides containing all the sugars found in soils, except arabinose and galactose. An intensive searchs4 to isolate, from soil, micro-organisms capable of incorporating these two sugars into polysaccharides failed. Arabinose and galactose do occur, how- ever, in polysaccharides produced by pathogenic bacteria.86(*)

The glucosamine found in soil could originate partially from chitin, which is a constitutent of the cell wall of fungi and of the exoskeleton of various invertebrate spe~ies.8~ Part of the glucosamine may be a component of microbial polysaccharides. The origin of galactosamine, which occurs in large amounts in some forest soils, is not clear; however, t,here is an indica- tion that it has a mainly bacterial rigi in.'^-^^

and other sugars are proportionally higher. It may be concluded that the major part of the polysaccharides in most aerated soils is, in fact, of mi- crobial origin.20 ,22 e P 4 This explains also their extreme heterogeneity.

V. STATE AND FUNCTION

The previous Sections have dealt with the chemistry, abundance, and transformation of soil carbohydrates, with little reference to the larger system, soil, in which they occur. The carbohydrates are in intimate con- tact with other organic and inorganic soil constituents and enter into inter- actions with them. Such interactions have an influence on the behavior of carbohydrates on the one hand and on soil properties and plant nutrition on the other.

1. Interactions with Other Soil-constitutents a. Organic Constituents.-A part of the carbohydrates is associated with,

and difficult to separate from, other organic substances, such as proteins and humic substances. This circumstance has led to the assumption that there is a covalent bond between the carbohydrate and the other material. Polysaccharide preparations isolated from soils always contain appreciable

(89) P. W. Kent and M. W. Whitehouse, “Biochemistry of the Aminosugare,” Butterworths Scientific Publications, London, 1968, p. 92.

The proportion of glucose and xylose is lower in soils than in

CARBOHYDRATES IN THE SOIL 353

amounts of non-carbohydrates. Carbohydrates have also been determined in humic acid preparation^,^^,^^'^^,^^ of which they were found to constitute 2 to 20%. However, by careful purification, sugar-free, humic fractions have been obtained from some podeol soils.48 ,56(a) It seems that carbo- hydrates are linked to other organic constituents by van der Waals, hydro- gen, or ionic bonds.

b. Inorganic Constituents.-Sugars, nucleic acids, and polysaccharides are adsorbed on mineral s ~ r f a c e s ~ ~ ~ ~ ~ J ' 2 J ' ~ and are partially protected from microbial degradation by this a d s ~ r p t i o n , ~ ~ s e a Sugars have been detected upon hydrolysis of a naturally occurring, clay-organic complex extracted from an Ohio The adsorption of neutral sugars and polysaccharides is, supposedly, mainly due to hydrogen bonding.le Acidic polysaccharides can form ionic and coordinate bonds with metal cations, either free or in mineral surfaces. Neutral soil-polysaccharides may form complexes with borate.23

Microscopic, electron-microscopic, and histochemical techniques would help in studying the actual state of carbohydrates in soils.

2. Function in the Soil

a. ZnfEuence on Physical Properties.-Perhaps the most important role attributed to polysaccharides is their influence on soil structure. Polysac- charides may flocculate2s or deflocculate clay minerals,96 affecting their mobility and distribution in the soil profile. It has repeatedly been shown that long-chain polysaccharides are capable of binding inorganic soil-par- ticles into stable aggregates.12 A statistical correlation has also been estab- lished between t,he amount of polysaccharides extracted26 from, or deter- minedge in, soils and their degree of aggregation; such a correlation does not, however, necessarily prove that polysaccharides are the main aggregat- ing agents.g7 The specific destruction of carbohydrates of natural soil-ag-

(89a) D. E. Coffin, W. A. Delong and B. P. Warkentin, Trans. Intern. Cvnyr. Soil

(90) F. Jacquin, Compl. rend., 960, 1892 (1960). (91) F. J. Sowden and H. Deuel, Soil Sci., 91, 44 (1961). (92) D. L. Lynch, L. M. Wright and L. J. Cotnoir, Soil Sci. Soc. Am. Proc., 20,

6 (1966); D. L. Lynch, L. M. Wright, E. E. Hearns and L. J. Cotnoir, Soil Sci., 84, 113 (1967).

Sci., 7th Congr., Madison, Wise. , in press.

(93) C. A. I. Goring and W. V. Bartholomew, Soil Sci. , 74, 149 (1952). (94) F. J. Stevenson, J. D. Marks, J. E. Varner and W. P. Martin, Soil Sci. Soc.

(95) C . Bloomfield, Tram. Intern. Congr. Soil Sci., 6th Congr., Paris, B, 27 (1956). (96) J. A. Toogood and D. L. Lynch, Can. J . Soil Sci., 39,151 (1959); A. Kullmann

(97) N. C. Mehta, H. Streuli, M. Milller and H. Deuel, J . Sci. Food Agr., 11, 40

Am. PTOC., 16, 69 (1962).

and K. Koepke, Z. Pjlanzenerndhr. Dilng. u. Bodenk., 93.97 (1961).

(1960).

354 MEHTA, DUBACH AND DEUEL

gregates by treatment with periodate, hot acid, and so on, failed to affect the stability of aggregates, whereas artificial aggregates, prepared with various polysaccharides, were destroyed by these treatment^.^' It was con- cluded that polysaccharides do not contribute essentially to the aggregation of the Swiss soils studied.

b. Influence on Chemical Processes.-Carbohydrates may inhibit the pre- cipitation of iron and aluminum by p h o ~ p h a t e , ~ ~ and favor the leaching of sesquioxides from the upper to lower horizons in some soils.QQ Bacteria iso- lated from soils can produce ~-arabino-2-hexulosonic acid (“2-ketogluconir arid”) , a chelatirig agent.loO Chelating carbohydrates can accelerate the weathering of minerals.

c. Influence on Microbial Activity.-Carbohydrates, particularly those from fresh plant-material, are a readily available source of carbon and energy for micro-organisms, and, consequently, they control to a great extent the microbial activity in soils. The decomposing carbohydrates are helieved by many workers to be the ultimate source of humic ~ubstances.~ On the other hand, the microbial decomposition of humic substances is ac- celerated in the presence of carbohydrates.101

d. Influence on Plant Nutrition.-Monomeric sugars can be absorbed and utilized by plants. Sugars niay stimulate seed germination and root elonga- tion.1n2 Soil rarbohydrates may have many different, indirect effects on plant nutrition. For instance, mineralization of phytin and nucleic acids supplies phosphorus to the plant. The carbohydrates may keep phosphate in u readily convertible form and prevent it from forming irisoluhle precipi- tates with calrium, iron, or aluminum.

VI. SUMMARY

A part of the carbohydrates of various soils has been isolated, purified, and shown to consist of polysaccharides composed of many sugars. Fruc- tionation and characterization of these preparations showed the extreme heterogeneity of the polysuccharides, confirming their predomiiiant ly nii- crobial origin.

The quantitative determinations, although not always satisfactory, have shown the carbohydrates to constitute about 10% of soil organic matter. The major immediate problem is development of better methods for the determination of t,he total carbohydrates and their individual monosac- (98) D. B. Bradley and D. H. Sieling, Soil Sci. , 76, 175 (1953). (99) M. Schnitzer and W. A. DeLong, Soil Sci. SOC. Am. Proc., 19, 363 (1955). (100) R. B . Duff and D. M. Webley, C‘hem. & Ind. (London), 1376 (1959). (101) H. Thiele and G . Andersen, Zentr. Bakleriol. Parasilenk., Abt. 11, 107, 247

(102) R. Brown, A . W. Johnson, E. Robinson and A . R. Todd, Proc. Roy. SOC. (1963).

(London), B136, 1 (1949); R . Brown and E. Robinson, ibid. , B136, 577 (1950).

CARBOHYDRATES IN THE SOIL 355

charide components. This is a prerequisite to (a) a more intensive study of the role and transformation of carbohydrates in the soil and (b) the elucida- tion of the nature and abundance of the remaining, possibly non-polysac- charide, part of soil carbohydrates. The study of the amount and kind of carbohydrates in different soils is of pedological interest.

Acknowledgment

We wish to thank the Schweizerischer Nationalfonds zur Forderung wissen- schajtlicher Forschung for financial support.