On humus formation - Springer Link

Plant and Soil 7 7 , 3 0 5 - 3 1 3 (1984). Ms. 5492 9 1984 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands.

On humus formation CtfANDRIKA VARADACHARI and KUNAL GHOSH

Department of Agricultural Chemistry and Soil Science, Calcutta University, 35 Ballygunge Circular Road, Calcutta 700 019, India. Received 10 May 1983. Revised September 1983

Key words Fulvic acids Humic acids Humus degradation Humus synthesis Summary A scheme on humus formation has been proposed. This is as follows: Lignin/ carbohydrates, the chief sources of C for the microorganisms are first broken down by extracellular enzymes into smaller units. Soluble units are absorbed into the microbial cell where part of them are converted to phenols/quinones. These together with oxidising enzymes are discharged into the environment where they polymerise by a free radical mechanism. The formation of FA (fulvic acids) has also been explained and it has been concluded that the difference between FAs and HAs (humic acids) is merely in the degree of polymerisation and that FAs are not necessarily more aliphatic than HAs.

Introduction The synthesis of humus in the environment has been dealt with by many workers and consequently many different theories for humus formation have been proposed 8,~~ However, though each is successful from a particular angle, none can independently explain all aspects of humus synthesis. It is therefore thought necessary to unify the more redeeming features of the older theories into a new "concept" which will thus explain the phenomena more completely. The pioneer venture in this direction is perhaps the "polyphenol" (PP) theory, as elucidated recently by Stevenson is. However, Stevenson 18 himself was aware of the shortcomings of the scheme outlined in this theory, and pronounced it as "not completely satisfactory". A careful study of the scheme indicates that it cannot provide suitable answers to a number of questions regarding humus formation because it deals rather vaguely with some of the processes involved and is also not sufficiently comprehensive. Taking these points into account, a different scheme of humus formation has therefore been proposed in this work. The theory is similar to the PP theory, in that, the synthesis of polyphenols is regarded as the essential step of humus formation, however, it differs from the PP theory in the infrastructure of the processes. The individual steps have also been described in greater detail and each of these have been based on sound theoretical or experimental fact so that the entire framework rests on firm foundations. 305

306

VARADACHARI AND GHOSH

Lignin / Carbohydrates/Mocromoleculor C- compounds

I

Extrocellulor ottock by microorganisms

Smaller units

Small C-molecules

/

J

Unutilised

Soluble units ingested into the cell

/

Biochemical transformations

Residue

J

L

Phenols/Ouinones I ~

Discharged into

~

the surroundings

"-.

Oxidation to Cellular components COz, (GOOH)e, C2 Hs OH, Citric acid, "~ etc. Ring fission to oliphotic molecules

Enzymes ~

Nitrogenous compounds/Sugars

I

Free radical polymerisotion Humic polymers of various sizes Fig. 1. Proposed scheme on humus formation.

A schematic representation of the processes involved in the proposed scheme are depicted in Fig. 1. In the following section, the theoretical basis of this scheme has been discussed. This has been dealt with in three parts. The first, deals with the synthesis of the primary building units of humus; the second, explains the mechanism of formation of humic and fulvic acids from these primary units; the third, gives a short account of the probable mechanism of degradation of humus, this being necessary in order to clarify certain misconceptions regarding the synthesis of fulvic acids. Synthesis of humus-building units In this study, humus formation, only from lignin, carbohydrates and nitrogenous compounds are considered because the contribution of other plant materials like tannin, waxes, etc. are insignificant in this respect.

HUMUS FORMATION

307

Lignin The transformation of lignin in the soil is the result of lignin serving as an energy source for microorganisms. In order to serve this purpose, it is essential for the molecule to first penetrate into the organism. Since penetration into the organism is not possible owing to its impermeability to the microbial cell, the lignin must be first solubilised by depolymerisation to give smaller units that are capable of diffusing through the cell-wall 1. Isolation of various lignin building units during the decomposition of both natural and synthetic lignins 1~ has shown that splitting of the lignin molecule takes place along the aryl ether linkages or along the C-C bonds linking adjoining aromatic nuclei. According to Alexander 1, such splitting is effected by lignase, an extracellular enzyme(s). The aromatic products released by these enzymes are consumed by the organisms and are then utilised (a) partly as a C-source for mycelialsynthesis and (b) partly as a source of energy. Prior to serving as a source of C or energy, these substituted benzenoid structures may undergo a number of biochemical transformations within the celV, e.g. (i) aromatic methyl ethers are demethylated to the corresponding hydroxy derivative, (ii) methyl substituents may be oxidised by COOH but in some cases they may remain intact, (iii) COOH groups may be oxidatively decarboxylated, etc. That these transformations are essentially intracellular processes is well-known to the biochemists 7,11. It must be noted that this refutes the PP theory is according to which such transformations "occur within or on exposed edges of intact molecules by extracellular enzymes produced by the fungi". Therefore, grouping the sources of the polyphenols into that from lignin or from microorganisms as has been done in the PP theory is not right. Polyphenols have to be synthesised by the microorganisms and within the microbial cells and as such they are all "microbial" polyphenols differing only in their carbonaceous precursors which may be lignin or carbohydrates or any other Ccompound. The phenols produced by the above mentioned transformation follow one of two pathways: (A) Part of the phenols may be enzymatically oxidised to quinones with the liberation of energy which is utilised by the organism 6. Fig. 2a presents a typical oxidation of a polyphenol by enzymes like laccase, tryosinase and peroxidase. Further enzymic oxidation will lead to polymerisation (or more precisely, polycondensation). However, within the cell, the limited supply of oxygen limits the extent of reaction 2 (Fig. 2a) so that extensive polymerisation probably does not occur. On cell lysis, when excess oxygen is available due to rupture of the cell wall, extensive polymerisation takes place. Humic substances will not therefore, in general,

308

VARADACHARI AND GHOSH

@

OH

2 Cu (enzyme)+

1/0

4+ >

OH

2 Cu (enzyme) +

~O+2H

2-t-

I 2 Cul+(enzyme)+2H++ ~" 02

2 Cu (enzyme)+ H20

@ [~

O2 f"s'"GOOH ~"..-C~O COOH " ~)CH2CO0H

OH OH~H

(•

~OH OH OH OH OH OH

laccaSe[O]

COOH

'

f

Acetyl Co A +

H j H. . ~ COOH O~_.H~f~.CO0 I H

Succinote

Krebs Cycle

0 0 "

C~ O 0//

OH

(R)CH.NH I GOOH

\\o

I

I

OCH

[O]

;

§

C

"~l

GOOHOH ~ jO O=p= 0 ~ POLYMERS CHaO

-

OH

,

cleavage Fig. 2.

(2

NH.CH(R) I COOH

CH O OH GOOH 3~ laccaseC H 3 0 " ~ ~

OH

GH30..~

(1)

@

OH ~OH

OH

+

"~'OCHa 0

>-OH

oH

(utilised)

no cleavage

(a) Utilisation of phenols by enzymic oxidation. (b) Utilisation of phenols by oxidative ring fission. (c) Dimeric products of catechol oxidation. (d) Products of enzymic reaction of catechol with amino acids. (e) Oxidation of vanillic acid with loss of carboxyl groups. (f) Scheme on microbial degradation of humic substances. (g) Likely and unlikely modes of cleavage of humic acid during microbial degradation.

be formed within the living cells but can be formed within dead cells where membrane disintegration has set in. However, it must be noted that living cells m a y secrete excess phenolics, enzymes, etc. into their immediate environment where these may polymerise at the cell surface

HUMUS FORMATION

309

thus imparting a dark colour to the organism. (B) The remaining phenols undergo a whole sequence of reactions whereby they are cleaved to aliphatic acids and then incorporated into the Krebs cycle 7. Thus, the phenols which follow this path (Fig. 2b) cannot directly form humic substances.

Carbohydrates Like lignin, carbohydrates are also first cleaved into soluble units before being they are ingested in the cell. These may then be partly converted into aromatic compounds by some specific organisms. This aromatisation must precede the formation of humic substances from non-aromatic sources like the carbohydrates. The aromatic compounds which include various phenols and quinones are formed by (a) the shikimic acid pathway and (b) the acetate pathway 3. It must be noted in this connection that many organisms like Aspergillus niger which can form phenolic compounds from carbohydrates and thus synthesise humus from this source, cannot utilise lignin. However, the reverse is not true, i.e. all organisms that can utilise lignin also utilise carbohydrates 1.

Formation of humic substances Phenolic acids and quinones produced by the microorganisms from lignin and carbohydrates together with oxidising enzymes are discharged from the cell after its lysis. Under the influences of these enzymes, condensation occurs between the phenols themselves and also with amino acids 9. The condensing phenols include those synthesised by the organism as well as the phenolic units of lignin (both monomeric and polymeric) that are still present in the environment. Like all other phenol condensation reactions, the mechanism of polymerisation is a free radical one 4. Coupling occurs at the o- and ppositions to the hydroxyl group and also via the oxygen ~s as shown 4 in Fig. 2c. With amino acids, compounds like those shown in Fig. 2d may be formed ~~ These compounds can react further, in a similar manner, with other phenols and amino acids or polypeptides. Reactions with heterocyclic N-compounds and carbohydrates may also occur 1~ Polymerisation may cause loss of COOH groups, e.g. oxidation of vanillic acid by laccase yields a series of diphenyl compounds, as presented in Fig. 2e, some of which have been decarboxylated4. Since polymerisation causes loss of COOH groups, it follows that the more polymerised material will have less COOH groups per unit weight as compared to the less polymerised material. This can explain why

310

VARADACHARI AND GHOSH

Table 1. Percentage carbon data of some humic and fulvic acids on 'theoretical decarboxylation' Sample

Source

Reference

% ash

COOH (meq/g)

%C (analysed)

%C of theoretically decarboxylated samples*

HA

Beaverhills Boetica Brunizem Harpster La Plaine Leonardite Peat Podzol Pont Casse Rifle Peat

(a) (b) (c) (d) (b) (d) (d) (c) (b) (c)

0.9 1.8 1.8 0.9 1.53 0.6 0.5 0.8 0.69 0.0

4.5 4.05 3.89 3.30 4.48 4.56 3.84 3.94 3.78 3.84

56.4 54.40 53.6 55.8 54.92 57.1 54.1 53.7 54.52 54.1

63.6 60.3 59.0 60.6 61.7 64.6 59.6 59.2 60.0 59.6

FA

Armadale Farmville Halifax Millbrook Rawdon Thom

(e) (e) (e) (e) (e) (e)

0.4 1.4 0.0 1.5 1.6 0.0

49.0 45.2 44.5 45.2 49.0 45.5

63.8 57.2 58.5 57.6 63.5 59.3

9.2 9.1 10.2 7.8 9.1 9.8

(a) Ghosh K and Schnitzer M 1980 Soil Sci. 129,266-276. (b) Griffith S M and Schnitzer M 1975 Soil Sci. Soc. Am. Proc. 3 9 , 8 6 1 - 8 6 7 . (c) Ardakani M S and Stevenson F J 1972 Soil Sci. Soc. Am. Proc. 36,884-890. (d) Stevenson F J 1976 Soil Sci. Soc. Am. J. 4 0 , 6 6 5 - 6 7 2 . (e) Schnitzer M 1970 Can J. Soil Sci. 50, 199-204.

* Calculated as:

%C of sample - %C in CO0 groups 100 - % weight of CO0 groups

X 100

-

%C - 1.2x

• 100

100 - 4.4•

where x is COOH content in meq/g. Note: (i) The analysis data should be on moisture- and ash-free basis. (ii) COOH group content should be determined with purified samples of low (preferably < 2%) ash content.

fulvic acids (FAs) which are smaller polymers have more COOH groups per unit weight 17 than humic acids (HAs) which are larger polymers. If the above conclusion be true, there should not be much structural differences b e t w e e n FAs and HAs - since the major difference between these macromolecules (apart from their degree o f polymerisation) is in their COOH group content, it follows that %C content of these molecules, devoid o f their COOH groups, should be the same. To establish this, Table 1 has been constructed on the following basis: Per unit weight of sample, FAs contain more carboxylic oxygen as

HUMUS FORMATION

311

compared to HAs. Therefore in the calculation of %C of the samples, the %C of FAs will automatically be lower than that of HAs even if the basic structural framework of the two is the same. Hence the deduction, on the basis of %C data, that FAs are less aromatic than HAs, is erroneous. In Table 1, the %C of FAs and HAs has been calculated after 'theoretical decarboxylation' of the sampes. It can be seen that %C, of 'theoretically decarboxylated' samples of FAs and HAs, is nearly the same which means that the structural units of these molecules are also similar. Since HAs are larger molecules than FAs, one can conclude that FAs are only less polymerised forms of HAs and do not necessarily contain more aliphatic groups than HAs. In this connection, attention should be paid to the fact that computed aromaticity 17 of FA, viz. 7.1, is comparable to that of HA, viz. 6.9. Moreover the E4/E 6 ratio which has mainly been used as an evidence to establish the more aliphatic character of FA over HA, is mostly a measure of particle size and not of the aliphatic: aromatic ratio in the humic structure S. Support for the observation that FAs are less polymerised forms of humic substances also comes from the fact that they dominate under conditions where free radical polymerisation is suppressed, e.g. in anaerobic conditions, in presence of excess water, etc.

Degradation of humic substances Degradation of humic substances is likely to proceed as for lignin since organisms like Polystictus versicolor, Hypholoma fasciculare, Polyporus resinosus, Ustulina zonata, etc. that degrade lignin I can also degrade humic substances 12. This fact is further borne out by micromorphological evidence2. The conclusion appears to be justified, since phenolic units of both lignin and humic substances have been shown (earlier in this text) to be linked by similar bonds. Thus the phenolic units released from humic substances must be utilised in a manner similar to those of lignin, i.e. the phenols may either be converted to quinones and other phenols or they may be degraded to aliphatic acids. Thus schematically the degradation would proceed as described in Fig. 2f. This scheme of degradation has been borne out by experiments, on degradation of HA by Penicillium frequentans which leads to a large increase in total OH whereas COOH groups 16, %C and %H remains the same ~a. This indicates that the aromatic units are removed as a whole - side chain oxidation to COOH, without removal of the entire aromatic unit, does not occur (Fig. 2g). From this scheme of degradation of HAs, it follows that FAs cannot be

312

VARADACHARIAND GHOSH

produced by microbial degradation of HAs since such degradation does not cause an increase in COOH groups.

Conclusion A unique feature of the proposed scheme is that, it can explain the formation of humus under diverse situations and can also account for or predict the relative dominance of FAs and HAs in any kind of environment. In attempting to do so, the following points must be borne in mind: (1) any C - source that can be transformed by organisms into phenolic compounds can serve as the precursor of humus. (2) Only those organisms that are capable of utilising/transforming/ producing phenols are directly responsible for the synthesis of humus. (3) The humus-building units condense by a free radical mechanism to form humus. (4) The difference between FAs and HAs is merely in their degree of polymerisation and FAs are not necessarily more aliphatic than HAs.

References 1 2 3

4

5 6 7 8 9 10

11 12 13 14

Alexander M 1961 Introduction to Soil Microbiology. John Wiley, N.Y., 128, 2 0 5 209 p. Babel U 1975 Micromorphology of soil organic matter. In Soil Components. Vol. 1. Ed. J E Gieseking. Springer - Verlag, N.Y., 369-473 p. Birkinshaw J H 1965 Chemical constituents of the fungal cell. 2. Special chemical products. In The Fungi. Vol. 1. Eds. G C Ainsworth and A S Sussman. Academic, N.Y., 179-228 p. Brown B R 1967 Biochemical aspects of oxidative coupling of phenols. In Oxidative coupling of Phenols. Eds. W I Taylor and A R Battersby. Marcel Dekker, N.Y., 167201 p. Chen Y, Senesi N and Schnitzer M 1977 Information provided on humic substances by E,/E6 ratios. Soil Sci. Soc. Am. J. 4 1 , 3 5 2 - 3 5 8 . Doby G 1965 Plant Biochemistry. Interscience, London, 599-624. Evans W C 1963 The microbiological degradation of aromatic compounds. J. Gen. Microb. 32,177-184. Felbeck G T 1971 Chemical and biological characterisation of humic matter. In Soil Biochemistry. Vol. 2. Eds. A D McLaren and J Skujins. Marcel Dekker, N.Y., 36-59 p. Flaig W 1964 Chemische Untersuchungen an Humusstoffen. Z. Chemic 4 , 2 5 3 - 2 6 5 . Flaig W, Beutelspacher H and Rietz E 1975 Chemical composition and physical properties of humic substances. In Soil Components. Vol. 1. Ed. J E Gieseking. Springer Verlag, N.Y., 1-211 p. Hurst H M and Burges N A 1967 Lignin and humic acids. In Soil Biochemistry. Vol. 1. Eds. A D McLaren and G H Peterson. Marcel Dekker, N.Y., 260-283 p. Hurst H M, Burges N A and Latter P 1962 Some aspects of the biochemistry of humic acid decomposition by fungi. Phytochem. I, 227-231. Kononova M M 1966 Soil organic Matter. Pergamon, Oxford, 112, 125, 134,167, 261 p. Mathur S P and PauI E A 1966 Microbial utilization of soil humic acids. Can J. Microb. 13,573-580.

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Nonhebel D C and Walton J C 1974 Free radical chemistry. Cambridge University Press, Cambridge, 10-20, 328 p. Paul E A and Mathur S P 1967 Cleavage of humic acid by Penicillium frequentans. Plant and Soil 2 7 , 2 9 7 - 2 9 9 . Schnitzer M and Ghosh K 1979 Some recent advances in the chemistry and reactions of humic substances. J. Indian Chem. Soc. 56, 1090-1093. Stevenson F J 1982 ttumus Chemistry. John Wiley, N.Y., 195-220 p.