P l a n t a n d Soil 36, 295-299 (1972) Ms. 1676

O R G A N I C M A T T E R T R A N S F O R M A T I O N S

I N S O I L

II. NATURE OF CARBOHYDRATES IN SOILS INCUBATED WITH Cla-LABELLED OAT ROOTS UNDER AEROBIC AND

ANAEROBIC CONDITIONS

by M. K. S INHA*

Timiryazev Academy of Agricultural Sciences, Moscow

SUMMARY

I n a n i n v e s t i g a t i o n on t h e b iochemica l changes of a d d e d C14-tagged oa t roo ts i n c u b a t e d w i t h soils unt ie r aerobic a n d anae rob i c condi t ions , b iosynt t les - is of po lysacchar ides of v a r y i n g compos i t i on was n o t e d d u r i n g t h e humif i - ca t ion process. Poss ib le causes of pe r s i s t ence of c a r b o h y d r a t e s in soils are ind ica t ed .

INTRODUCTION

Plant residues when added to soils bring about a flux of micro- biologieal activity during which part of the added organic material is mineralized while another part may be transformed into substances having varying degrees of stability, uniformity of composition and extractibility with the conventional humus extractants. It has been emphasized recently that the soil organic matter represents an ecosystem representing a chain of biochemical and colloid chemical reactions with attached pools and side links. The main fractions of the soll organic matter consist of plant and animal debris, hetero- trophic biomass and stabilized residues including recently formed humic substances 5. These fractions are subject to continuous trans- formations including formation or renewal and decay or decompo-

* Present address: Department of Soils, Ranchi Agricultural College, Kanke, Ranchi, Bihar, INDIA.

296 M.K. SlNHA

sition. A more or less steady state with regard to the size and func- tions of the fractions is reached in soils commensurate with the pre- vailing environmental conditions. The use of C14-tagged plant resi- dues has greatly amplified the possibilities of registering biochemical changes during their decomposition and humification. The determi- nation of carbohydrates, amino acids and phenolic compounds in different organic matter fractions may aid more effectively in tracing the biological events during humus formation.

In a previous publication on the distribution of C la activity of added plant residue into different organic matter fractions, a differ- ential C14-enrichment of the individual fractions was noted. The data suggested of the biosynthesis of polysaccharides during the decomposition of Cl4-tagged oat roots. Persistence and presence of carbohydrates in soils has been demonstrated by several workers 3 10 Gaponenkov and Schatzman 4 detected glucuronic and galacturonic acids in the acid hydrolyzates of humic acids.

The present investigation relates to the determination of the qualitative composition of sugars and uronic acids in different organic matter fractions of soils incubated with C14-1abelled oat roots under aerobic and anaerobic conditions. It also aimed at explaining the possible significance of carbohydrates in humus formation.

METHODS NAI) MATERIALS

The soils used were o b t a i n e d f rom a c u l t i v a t e d podzol (Az/A2 hor.) a n d 0-15 cm layer of a Krasnozem. T he phys icochemica l cha rac te r i s t i c s of t h e solls h a v e been r e p o r t e d eartier. De ta i l s of l abe l l ing of t h e p l a n t ma te r i a l s a n d in- c u b a t i o n m e t h o d s were t h e same as r e p o r t e d in t he p rev ious pub l i ca t ion .

The soils a f t e r a pe r iod of t h r ee m o n t h s were e x t r a c t e d a n d organic m a t t e r f r a e t i o n a t e d accord ing to t h e scheme p re sen t ed earlier.

I n i t i a l l y t h e soils were e x t r a c t e d w i t h acidifiecl w a t e t (pH - 2.0) to s e p a r a t e low-molecula r we igh t s u b s t a n c e s f rom t he speciIic h u m i c subs tances . A l igh t yel lowish coloured e x t r a c t was o b t a i n e d ind i ca t i ng a p a r t i a l e x t r a c t i o n of free fu lv ic aeid. The e x t r a c t was s u b s e q u e n t l y e x t r a c t e d w i t h e ther . The aqueous e x t r a e t was adso rbed on a e t i v a t e d charcoa l a n d e x t r a c t e d w i t h e t h y l a leohol (15 pe r cent) for t h e s e p a r a t i o n of sugars a n d po!ysacchar ides . The res idue on t h e co lumn was e lu ted wi th 0 .1N NaOH. This e x t r a c t r ep resen ted t h e free fu lv ic acid f rac t ion .

The res idua l soil was r e p e a t e d l y e x t r a c t e d w i t h 0 .1N N a O H solut ion. I-Iumic a n d fulvic acids were s e p a r a t e d b y ac id i f iea t ion of t h e e x t r a c t w i t h H2SO4 to p H 1.0. The h u m i c acid f r ac t ion was f u r t h e r pur i f ied b y r e p e a t e 4

CARBOHYDRATES IN SOIL ORGANIC MATTER 297

prec ip i ta t ion and solut ion with acid and alkali. Final ly, the humic acid f rac t ion was dialysed. The fulvic acid f rac t ion was f rac t iona ted by elution ch roma tog raphy on ac t iva ted charcoal according to Forsy th ' s me thod wi th cer ta in modificat ions. Free sugars were de te rmined by paper ch roma tog raphy in the e thyl alcohol f rac t ion alld f ract ion A of fulvic acid. In o ther fulvic acid fract ions (ethyl alcohol, aqueous ext rac t , f ract ions C and D), and in humic acid fract ion, sugars were de te rmined in the 0.5N H2SO4 hydrolysa te . The excess acid was neutra l ized wi th BaCO3 wi th eongo-red paloer.

The composi t ion of sugars in t he hydrolys tes was de te rmined by ascending loaper ch roma tog raphy in a solvent sys tem: n-butanol-acet ic ac id-water (4:t :5). The chromatograms were sprayed wi th alcoholic sola t ion of p-ana- zidin (3% solut ion acidified wi th HC1). The chromatograms were dried for 10 minutes at 12õ°C when sugar sports were visible on them.

RESULTS AND DISCUSSION

The results of chromatographie analysis of sugars in different fractions and their acid hydrolysates are presented in Table 1. Free sugars in the alcoholic Iraction of the aqueous extract were detected only in podzol soil. In soils incubated under aerobic conditions glucose and xylose were detected, whereas in anaerobically incu- bated soil galacturonic acid, galactose and xylose were noted. In the polysaccaride of the alcoholic extract only xylose was detected in aerobically incubated soil. In anaerobically incubated soil galactu- ronic acid, xylose, glucose, and one unidentified sugar were detected. This sugests the possible biosynthesis of a more complex poly- saccharide under anaerobic eonditions. The fulvic acid fraction of the aqueous extract of all soils upon acid hydrolysis yielded glucuro- nic and galacturonic acids.

Xylose only was detected in Iraction C of the fulvic acid Iraction (0.1N NaOH extract) in the anaerobically incubated Krasnozem soil. The polysaccharide fraction (fraction D) of the fulvic acid upon hydrolysis yielded galactose, mannose, xylose, one unidenti- Iied sugar and traees of glucuronic acid in all soils. In acid hydroly- sate of fraction D of fulvic acid, glucuronic and galacturonic acids were detected. The humic acid fraetion upon acid hydrolysis yielded galacturonic acid and galactose.

Polysaccharides and their derivatives form one of the essential components of the soll organic marter and their persistence in soil is attributed to their biosynthesis by soil micro-organisms and their resistance to further decomposition is due to their interaction and

298 M.K. SINHA

TABLE 1

Nature of carbohydrates in organic marter fractions of solls incubated with C x4-tagged oat roots under aerobic and anaerobic conditions.

Soils and conditions

of incubation.

Anqueous extract (pH,2.0) Alkaline extract (0.1 N NaOH)

Alcoholie Alkalilie Humic acid Fulvic acid fractions fraction fraction

(in tlydrolysate) C D (in hydrolysate)

1. Podzol

Aerobie

Anaerobic

2. Krasnozem

Aerobic

Anaerobic

F~88 SUgörS :

Glueose, xylose, Glucuronic and Glucuronic I n hydrolysate: galacturonic acid, galactose Xylose acids Free sugars:

Galacturonic Glucuronic and Glucuronic acid, galactose galacturonic acid, galactose xylose acids

Not detected

Not detected

Galactose, Galacturonic mannose, xylose, acid, unknown unknown

Galactose, Galacturonic mannose, xylose, acid, unknown llnknown

Glucuronic and galacturollic -- -- -- acids Glucuronic and Glucuronic Galactose, Glucuronic and galacturonic acid, galactose mannose, xylose, galacturonic acids unknown acids

association with polyvalent metallic ions and with the stable humic and fulvic acid fraction s • s. The biosynthesis of polysaccharides during the decomposition of plant residues has been reported by several workers 1 9. Complexing with metals may be an important factor in the persistence of polysaccharides in the soll. Additional possible factors include natural resistance to decomposition of cer- tain polysaccharides, and combination with other soil constituents including clays, humic acids, and other polysaccharides. Polysaccha- ride fractions should thus be regarded as a natural product of soll environment as are humic substances. In view of the present con- cept of humus formation which visualizes that the products of the resynthesis of microbial plasma participate in the formation of humic acid molecule, and as polyuronides a r e a common component of bacterial slime, the presence of carbohydrate residues in the humic acid molecule is not surprising. The possible participation of carbohydrates during the condensation of aromatic compounds

CARBOHYDRATES IN SOIL ORGANIC MATTER 299

with products of protein decomposition in the formation of humic acid, particularly as peripheral grouping may not be excluded 2 a s

I t may be noted that anaerobic conditions of decomposition of plant materials lead to the biosynthesis of polysaccharides to a rela- tively greater extent and of greater complexity. In the krasnozem soils, there is a relatively lower degree of persistence and accumula- tion of these substances than in podzol soils.

It may be emphasized on the basis of the present investigation that in studying humus formation the entire sequence of biochemical changes of added organic marter may provide an integrated know- ledge of the differences so offen observed between different soils. Refinements of the conventional humus analysis methods and study of the biochemical changes during organic matter transformations may~greatly help in elucidating the complicated chain of reactions involved in humus formation.

ReGeived February 9, 1971

REFERENCES

1 Chaha l , K. S., Biosynthesis and eharacterization of soiI polysaecharides. Isotopes and Radiatiorl in Soff Organie Marter Studies. IAEA, Vienna (1968).

2 D r a g u n o v , S. S., A Gomparative study of humiG acids from soils and peats. PoGhvo- vedenie, No. 7 (1948).

3 D r a g u n o v , S. S. and P a p o v a , L. N., Fraetional Gomposition of humie aeids. PoGhvovedenie, No. 5, 33-40 (1969).

4 G a p o n e n k o v , T. K. and S G h a t z m a n , I. M., Content of uroniG aeids in the main humus fraGtions of some soils and their role in soil proGesses. Doklady BASKHNIL. No. 11, 1-3 (1965).

5 J a n s s o n , S. L., Soll organie marter and fertility. Trans. Intern. SOG. Soll Sei. Comm., I I and IV, 1-10 (1967).

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7 M a r t i n , J. P., E r v i n , J. O. and S h e p e r d , R. A., DeGomposition and binding aetion of polysacGharides from Aztobacter indicus (Beijerinckia) and other baGteria in soil. Soll Sei. Soe. Am. Proe. 29, 297-400 (1965).

8 M a r t i n , J. P., E r v i n , J. O. and S h e p e r d , R. A., Decomposition of the Iron, Aluminum, Zinc, and Copper salts or complexes of some miGrobial and plant poly- saeGharides in soll Soil Sei. SoG. Am. Proc. 30, 196-200 (1966).

9 Meh ta , N. C., D u b a e h , P., and Deuel , H., Carbohydrates in the soll. Advances Carbohydrate Chem. 16, 335-355 (1961).

10 P a r s o n , J, W. and T i n s l e y , J., ChemiGal studies of polysaGeharide material in soils and eomposts based on extraGtion with anhydrous formiG aGid. Soil SGi. 92, 46-53 (1961).