SOIL ORGANIC MATTER FRACTIONATION AND ITS EFFECT ON AGGREGATE

STABILITY

ROLL NO: 021325

SESSION: 2001-2002

SOIL SCIENCE DISCIPLINE

KHULNA UNIVERSITY
KHULNA
JUNE, 2007
SOIL ORGANIC MATTER FRACTIONATION AND ITS EFFECT ON AGGREGATE
STABILITY

COURSE TITLE: PROJECT THESIS

COURSE NO: SS- 4106

This project thesis paper has been prepared and submitted to Soil Science Discipline,
Khulna University, Khulna, for the partial fulfillment of the four years professional B. Sc.
(Hon’s) degree in Soil Science.

SUBMITTED BY

ROLL NO: 021325

SESSION: 2001-2002
SOIL SCIENCE DISCIPLINE
KHULNA UNIVERSITY
KHULNA.
SOIL ORGANIC MATTER FRACTIONATION AND ITS EFFECT ON AGGREGATE
STABILITY

APPROVED AS TO STYLE AND CONTENT BY

MD. SADIQUL AMIN

LECTURER
SOIL SCIENCE DISCIPLINE
KHULNA UNIVERSITY
KHULNA.
 DEDICATED
TO
MY BELOVED PARENTS AND SISTER
The Almighty God, the most merciful, most benevolent to man and His action,
make me enable to get the light of education. So honestly my all honors go to Him
for His kindness at first.

Then I owe an undying debt of gratitude to the person, my supervisor, Md. Sadiqul
Amin, Lecturer, Soil Science Discipline, Khulna University who made me
possible to bring the successful completion of this research work. Without his
endeavor, encouragement, continuous support and appreciation, it would not be
in its current shape. I would like to express my heartfelt gratitude to him. He has
provided me with many insights and various papers, journals and other materials
which made me courageous for walking in this way.

Special thanks are extended to Khandoker Qudrata Kibria, Sheikh Mohammad

Fazle Rabbi, and Md. Zaber Hossain, Soil Science Discipline, Khulna University
for their constructive guidance, valuable advice and affectionate feelings
throughout the period of research work and also in the preparation of the
manuscript.

I would like to express my special gratitude to all the teachers of Soil Science
Discipline of Khulna University for their continuous guidance and friendly attitude
during my varsity life. It is also a pleasure for me to thank all staffs of Soil Science
Discipline of Khulna University especially the lab assistant, Mr. Abdullah and Mr.
Mahfuz.

This acknowledgment would be incomplete without acknowledging my mates,

especially Mukut, Neela and Setu for their always support to do the best. I find
myself very blessed to have such friends.

I am out of word to articulate my expression about my parents and sister. It’s

better, not to ornament anything to their love, support, encouragement, and
patience. I’m really grateful to the almighty Allah as He awards me such parents
and sister.

Finally, I wish the friendly environment of Soil Science Discipline as well as

Khulna University as I saw in the first year of university life to live long that made
me aware of many thoughts and ideas of life.

Page
Table No: Caption
No:
List of Organic compounds in terms of ease of
Table-2.1
decomposition
Environmental issues involving soil organic
Table-2.2
matter
Table-2.3 Fractions of soil organic matter

Table-2.4 Elemental composition of humic substances

The contribution of individual sugar types to
Table-2.4
soil organic matter
Mean weight diameter, % total soil OC, % total
Table-4.1 extractable C, % humic acid and % fulvic acid
of the studied soil
Chapter Figure No: Caption Page
No: No:
2 Figure-2.1 Organic compounds in soil
Figure-2.2 Distribution of soil organic matter.
Figure-2.3 The process of decomposition of organic residues
is central to biogeochemical cycles atmosphere,
biosphere, pedosphere and the hydrosphere
Figure-2.4 Processes of organic matter decomposition
Figure-2.5 Flowsheet for the separation of humic
compounds into the different humic fractions.
Figure-2.6 Mechanism for the formations of soil humic
substances
Figure-2.7 Structure of humic acid
Figure-2.8 Structure of fulvic acid.
Introduction

All organic substances, by definition, contain carbon. Organic matter in the

world’s soils contains about three times as much carbon as is found in the world’s
vegetation. Soil organic matter, therefore, plays a critical role in the global carbon
balance that is thought to be the major factor affecting global warming, or the
greenhouse effect. Although organic matter comprises only a small fraction of the
total mass of most soils, this dynamic soil component exerts a dominant influence
on many soil physical, chemical, & biological properties (Brady & Weil, 2002).
Soil organic matter (SOM), and in particular its humic fraction, is an important
factor that controls aggregate stability by promoting the binding of primary soil
particles into stable aggregates. Although each fraction expresses several distinct
and important properties, humic acids are of immense interest rather than the rest
of the fractions, (Schnitzer, 1989). Several studies have found a close relationship
between the level of aggregation and SOM (Tisdal and Oades, 1982), and
numerous others have shown that incorporation of organic residues usually results
in the formation of water-stable aggregates (Lynch and Bragg, 1985). Organic
matter and its various fractions can contribute to both the formation and the
stabilization of soil aggregates but, under some circumstances, specific fractions of
organic matter can also destabilize aggregates and increase the dispersibility of
clay and silt sized materials. Increasing total organic C content normally results in
an increase in the size and stability of aggregates (Joerg et al., Institute of Soil
Science, University of Hannover, Herrenhaeuser). But the amount of organic
matter changes continuously in the soil and It depends on rainfall, temperature,
aeration, and many other factors (Skene et al., 1996).

A lot of experimental works have been performed on soil humic substances in

different countries of the world and the vast volume of literature on humus study
presents much information about the composition and nature of humus originated
from different sources in different soils under different climatic and environmental
condition. However, in Bangladesh, only a few investigations have been made on
the composition and characterization of soil organic matter and humic substances
(Khan, 1973; Gerzaback and Ullah, 1989) though it is essentially related with the
plant growth and overall fertility status of soil. Besides, there is no systematic
study has been conducted for soils. In this literature, investigation was carried out
to fulfill the following objectives:

(1) To determine the amount of total organic carbon in the soil.

(2) To determine the different fractions of the organic mater in the soil.

(3) To determine the effect of different fractions of the organic mater on aggregate
stability of the soil under study.
 LITERATURE REVIEW

2.1 SOIL ORGANIC MATTER:

The term ‘soil organic matter’ defines the totality of organic matter in the soil
(Jenkinson, 1988) to consist of a whole series of products which range from
undecayed plant and animal tissues through ephemeral products of decomposition
to fairly stable brown to black material bearing no trace of the anatomical structure
from which it was derived (Russel, 1988) resulting from microbial and chemical
transformations of organic debris (Googlee.com.).

The end product of organic matter decomposition is humus (Gupta, 1999). Humic
compounds are defined as amorphous, colloidal polydispersed substances with
yellow to brown-black color and relatively high molecular weights (Tan, 1993).

2.2 SOURCES OF SOIL ORGANIC MATTER:

Organic matter in the soil comes from remains of plants and animals (Sahai,
1999). The plants and animals (both macro and micro) grow in weathered
materials and the organic residues left behind decay with time and become an
integral part of the soil (Rai, 1995). As new organic matter is formed in the soil, a
part of the old becomes mineralized (mineral form such as sulphur, phosphorus
etc.) (Sahai, 1999).

Plant tissue is the main original source of soil organic matter. Under natural
condition, trees, shrubs, grasses and other native plants supply large quantities of
organic residues annually by way of their roots and tops (Rai, 1995). Even with
harvested crops, one tenth to one third of the plant tops commonly fall to the soil
surface and remain there or are incorporated into soil. Naturally, all the roots
remain in the soil (Brady, 1999). These residues serve as a source of nutrients and
energy for the life processes of the microorganisms. As these organic materials are
decomposed and digested by soil organisms the become part of the underlying soil
by infiltration. Accordingly, the residues of higher plants provide food for soil
organism, which in turn create stable compounds that help to maintain the organic
levels (Brady, 1999).

Animals are the subsidiary or secondary source of soil organic matter. When they
consume plants as their fees, they excrete their waste products as excreta, besides
contributing their own bodies after completing their life cycle (Rai, 1995). Certain
forms of animal life, especially the earthworms, termites and ants, also play an
important role in the translocation of soil and plant residues (Brady, 1999).

Another sources of organic matter are livestock manure, composted manure,

composted urban green waste, shredded paper or cardboard, peat and peat moss,
wood residues (bark, chips sawdust) etc. (Googlee. Com.).

2.3 TYPES OF HUMIC MATTER:

Currently it is known that humic matter exists not only in soils, but also in
streams, rivers, lakes, oceans, and their sediments. It can also he found as geologic
deposits, e.g., lignite or leonardite, coal, and oil shale. These deposits are the
sources for the production of commercial humates, which are used as soil
amendments (Lobartini et al., 1992).
According to Tan (2000), on the basis of present knowledge, humic matter can be
classified into three broad groups:

1. Terrestrial humic matter: humic matter in soils

2. Aquatic humic matter: humic matter in streams, lakes oceans, and their
sediments.
3. Geologic humic matter: humic matter in lignite or leonardite, coal, and other
geologic deposits.

Terrestrial humic matter can be subdivided again into three categories: softwood,
hardwood, and grass humic matter (Tan, 1993). It is composed of humic and fulvic
acids, although their contents may differ considerably from soil to soil. Aquatic
humic matter is composed mainly of fulvic acid and can be subdivided into
allochthonous and autochthonous humic matter. The allochthonous humic matter
is believed to originate from soil humic matter, but after leaching into streams it
has been subjected to further distinct changes (Jackson, 1975). The autochthonous
humic matter originates from indigenous aquatic plant material, which has a
different composition than terrestrial plants. Especially in the oceans, where
plankton, kelp, and other seaweed are major forms of organisms, the nature of
humic matter formed is quite different from soil humic matter. Aquatic humic
matter consists of carbohydrate-protein complexes as opposed to terrestrial or soil
humic matter, which is mainly a ligno-protein complex (Tan, 2000).

2.4 AGRICULTURAL IMPORTANCE OF ORGANIC MATTER:

Organic matter is the vast array of carbon compounds which is very important soil
constituents. Originally created by plants, microbes, and other organisms, these
compounds play a variety of roles in nutrient, water, and biological cycles
(Googlee. Com.). Depending on climatic conditions and cultural practices, the
humus content of soils often stabilizes at a fairly definite amount. For many plant
forms, the humus reserves in a meter-deep layer of soil reach 282-426 ton/ha
(Kononova, 1972, 1968), which far exceeds the biomass. In the southern region of
the United States, with a prevailing subtropical climate, soil humus content seldom
exceeds 3.5% (Tan, 1975), and the carbon/nitrogen (C/N) ratio usually narrows
down in a humification process from a value in excess of 20 for fresh material, to
a value of 8-20 for humus. In the semihumid regions of the Mollisols, the humus
content stabilizes at values between 6 and 10%. The organic matter content of a
soil is intimately related to its productivity (Tan, 1993).

Some of the functions of organic matter are as follows:

1. Soil organic matter is one of the major sources if nutrient elements for
plants. In this respect, nitrogen is probably the most important. It is the
source of 90-95% of the nitrogen in unfertilized soils.
2. Organic matter can be the major source of both available phosphorus and
available sulfur when soil humus is present in appreciable amounts (about
2% or more). The nitrogen, phosphorus and sulfur cycles have emphasized
the manner in which organic matter becomes a source of nutrients and a key
substance in nutrient transformations in soil.
3. Organic matter supplies directly or indirectly through microbial action the
major soil aggregate-forming cements, particularly the long sugar chains
called polysaccharides.
4. Organic matter is a carbon supply for many microbes that perform other
beneficial functions in soils (e.g. free dinitrogen fixers, denitrifiers).
5. When left on top of soil as a mulch, organic matter reduces erosion and
surface runoff, shades the soil (which prevents rapid moisture loss) and
keeps the soil color in very hot weather and wormer in winter (Donahue et
al., 1983).
 6. Aids growth of crops by improving the soil’s ability to store and transmit
air and water, as measured by improved porosity; water holding capacity,
and drought resistance. Higher water retention by soils owing to high
organic matter content is beneficial for water conservation and water use
efficiency for crops.
7. Provides a source of carbon and energy for soil microbes which cycle
nutrients and fight plant diseases.
8. Reduces the negative environmental effects of pesticides, heavy metals and
other pollutants by binding contaminants (Googlee. Com.).
9. Organic matter binds soil particles into structural units called aggregates.
These aggregates help to maintain a loose, open, granular condition, water
infiltrate and percolate readily. The granular condition of soil maintains
favorable condition of aeration and permeability.
10. Organic acids released from decomposing organic matter help to reduce
alkalinity in soils.
11. Fresh organic matter supplies food for such soil life as earthworms, ant and
rodents. This microorganism improves drainage and aeration (Sahai, 1999).
12. The humus portion of soil organic matter is, by virtue of its very high
cation exchange capacity, capable of acting as a reservoir of cationic
nutrients.
13. Another important property of humus is its ‘chelating’ power for some of
the essential trace elements which could have otherwise been lost (Biswas
and Mukherjee, 1997).

2.5 ORGANIC MATTER DECOMPOSITION PROCESS:

Organic matter does not add any new plant nutrients but releases nutrients in a
plant available form through the process of decomposition. In order to maintain
this nutrient cycling system, the rate of addition from crop residues and manure
must equal the rate of decomposition. If the rate of addition is less than the rate of
decomposition, soil organic matter will decline and, conversely if the rate of
addition is greater than the rate of decomposition, soil organic matter will increase.
The term steady state is a condition where the rate of addition is equal to the rate
of decomposition (Googlee. Com.).
The general term soil organic matter encompasses all the organic components of
a soil:
1. Living biomass ( intact plant animal tissues and microorganisms),
2. Dead roots and other recognizable plant residues,
3. A largely amorphous and colloidal mixture of complex organic substances
no longer identifiable as tissues is referred to as soil humus (Brady and
Weil, 2002).

For simplicity, organic matter can be divided into two major categories: stabilized
organic matter which is highly decomposed and stable, and the active fraction
which is being actively used and transformed by living plants, animals, and
microbes (Googlee. Com.).

When the plants and animals die, their dead remains are acted upon by a number
of microorganisms and are finally degraded or decomposed into simple organic
compounds. A product of this microbial decomposition is humus which is a dark
colored, jelly-like amorphous substance, composed of residual organic matters not
readily decomposed by soil microorganisms. The process of humus formation is
called humification (Shukla and Chandel, 2000).

Duchaufour (1998) stated that the “humic compounds” are involved in more or
less close bonding with mineral compounds (clay minerals and oxides); they are in
turn mineralized, but more slowly than fresh organic matter (Secondary
mineralization, Fig 2.1).

Fresh organic primary

matter (litter) CO2, SO42-, PO43-, NH4+, NO3-, etc.
Mineralization

Secondary
Humus CO2, SO42-, PO43-, NH4+,
Mineralization NO3-, etc.

Fig 2.1: Decomposition of fresh organic matter, humification and mineralization

(Duchaufour, 1998).

The humic and non-humic compounds collectively make up humus. The humic
substances comprise about 60-80% of the soil organic matter. Two more complex
substances polyphenols and polyquinones are formed by decomposition, synthesis
and polymerization. The non-humic group make up about 20-30% of the organic
matter in soils. The non-humic substances consist of polymers, polysaccharides,
and polyuronides. Non-humic group also have some simpler compounds such as
organic acids and some protein-like materials. Non-humic substances are less
complex and resistant to microbial attack than humic substances (Sahai, 1999).
2.6 MODERN VIEW OF HUMUS FORMATION:

It is now generally excepted that humic acid and fulvic acids are formed by a
multiple stage processes that includes- (1) Decomposition of all plant components
including lignin, into simpler monomers; (2) Metabolism of the monomers with an
accompanying increase in the soil biomass; (3) Repeated recycling of the biomass
C (and N) with synthesis of new cells; and (4) Concurrent polymerization of
reactive monomers into high-molecular-weight polymers (Flaig, 1966 and
Kononova, 1966).

Although many different types of reactions can lead to the production of dark-
colored pigments (e.g. humic acids), the main pathway appears to be through
condensation reactions involving polyphenols and quinines (Haider et al.,1974;
Flaig, 1966; Kononova, 1966; Haider and Martin, 1975; Haider and Martin, 1970;
Martin and Haider, 1969; Martin and Haider, 1971; Martin et al.,1972; Stevenson,
1982).

According to present day concepts, polyphenols derived from plants (e.g. lignin)
or synthesized by microorganisms, are enzymatically converted to quinones,
which undergo self-condensation or combine with amino compounds to form N-
containing polymers. An overall scheme for the formation of humic acid from
polyphenols is shown in Fig. 2.2 (Stevenson, 1986).

Flaig’s concept of humus formation is as follows:

1. Lignin, freed of its linkage with cellulose during decomposition of plant

residues, is subjected to oxidative splitting with the formation of primary
structural units (derivatives of phenylpropane).
 2. The side-chains of the lignin-building units are oxidized, demethylation
occurs, and the resulting polyphenols are converted to quinones by
polyphenoloxidase enzymes.
3. Quinones arising from lignin (as well as from other sources) react with N-
containing compounds to form dark-colored polymers.

Lignin and other plant Organic residues in

Polyphenolic compounds general

Microbial degradation Microbial degradation

Small polyphenolic Sugars, organic acids

units, methoxyphenols and other simple
and intermediate phenols organic compounds

Microbial synthesis Degradation Microbial

Oxidation of side and transformation synthesis
chains and methyl CO2, H2O, NH3, and Microbial
groups, hydroxylation other simple inorganic cells and
of rings, and compounds products
decarboxylation
Ring cleavage,
oxidation Release and alteration of
microbial phenolic polymers
Numerous mono-, di-,
and trihydroxyphenols
and benzoic acids Peptides, amino
acids, amino sugars,
Enzymatic and and polysaccharides
autooxidation containing amino
sugars from decom-
Phenolic radicals and posing organic
hydroxybenzoquinone residues and micro-
bial cell autolysis
 Polymerization

Humic acid

Fig. 2.2: Overall scheme for the formation of humic acid from polyphenols
derived from lignin and synthesized by microorganism (Martin and
Focht, 1977).

Stages leading to the formation of humic substances were postulated by Stevenson

(1986) to be as follows:
Stage 1: Fungi attack simple carbohydrates and parts of the protein and cellulose
in the medullary rays, cambrium, and cortex of plant residues.
Stage 2: Cellulose of the xylem is decomposed by aerobic myxobacteria.
Polyphenols synthesized by the myxobacteria are oxidized to quinones by
polyphenoloxidase enzymes, and the quinones subsequently react with N
compounds to form brown humic substances.
Stage 3: Lignin is decomposed. Phenols released during decay also serve as
source materials for humus synthesis.

2.7 FRACTIONATION OF HUMIC MATTER:

The classical method for fractionating the humic substances that disperses in the
alkali to acidify the suspension with H2SO4 or HCl, which causes a part of the
dispersed organic matter to precipitate. The part that stays in solution is known as
fulvic acid, that which precipitated out as humic acid, and that part of the organic
matter which dose not disperse in the alkali but remain in the soil as humin.
Although these fractions are given definite names, they are not homogeneous and
each contain particles with a wide range of molecular weight and constitution, nor
is there any clear cut division between the fractions (Russell, 1973).

Based on solubility in acids and alkalis, the humic compounds can be separated
into several humic fractions (Flaig et al., 1975):

Fractions Alkali Acid Alcohol

Fulvic acid Soluble Soluble
Humic acid Soluble Insoluble Insoluble
Hymatomelanic acid Soluble Insoluble Soluble
Humin Insoluble Insoluble Insoluble
According to the German workers, humic acid can be further separated with
neutral salt solutions into:
a. Brown humic acid (Soluble in NaCl)
b. Grey humic acid (insoluble in NaCl)
In addition to several authors have been reported the isolation of a
c. Green humic acid fraction (Kumada and Hurst, 1967; Tan, 1993).

Duchaufour (1973) studied the nature of humins and grouped the humins into
following four types:
a. Inherited or residual humins: These are found somewhat similar to the
partially decomposed litters and they are loosely bonded to mineral parts of
soils.
b. Insolubilized humins: These are the products of precipitation and
insolubilization of phenolic compounds bonded to peptides. These are
found to be partially extractable.
 c. Microbial humins: These are found to be the products of neoformation of
the compounds, like polysaccharides, poly uronides and amino- sugars in
the active microenvironment of interaction. These constitute about 10% of
humin fractions.
d. Developed humins: These are found to be the products of slow process of
humification or it is humus maturation. Structurally these are strongly
condensed aromatic ring, poor in functional groups and found to be contain
high content of carboxylic (-COOH) groups with uniform structure
(Kononova, 1961, 1968; Bhattacharjee,1997).

According to Tan (1993), the classical method of fractionation of humic

substances is based on difference in solubility of the constituents in aqueous acidic
and alkaline solutions and ethanol. The steps in the separation are shown
schematically in Fig 2.3.

Soil Organic Matter

With Alkali

Humic substances Humin + Non-humic matter

(soluble) (insoluble)

With Acid

Fulvic acid Humic acid

(soluble) (insoluble)

Adjust to pH 4.8 With alcohol

Fulvic acid β Humus Humic acid Hymatomelanic acid

(soluble) (insoluble) (insoluble) (soluble)

With neutral salt

 Brown humic matter Grey humic
matter
(soluble) (insoluble)

Fig 2.3: Flow sheet for the separation of humic compounds into the different
humic
fractions (Tan, 1993).

2.8 METHOD OF EXTRACTION OF HUMIC SUBSTANCES FROM

SOIL:

Chemical investigations on humic substances go back to more than 200 years. The
capacity of humic substances to adsorb water and plant nutrients was one of the
first observations. Humic substances were thought to arise from the prolonged
rotting of animal and plant bodies. From the beginning several thousand scientific
papers have been written on humic materials, yet much remains to be learned
about their origin synthesis, chemical structure and reactions and their functions in
terrestrial and aquatic environments (Kononova, 1966; Schnitzer and Khan, 1972).

The great diversity of soil organic substances present often in negligible amounts,
their dynamic state and the difficulty of isolating them from the soil in unchanged
form have so far been serious obstacles to the study of soil organic matter. There
difficulties can, however, be overcome (Kononova, 1966). Tan (1993) stated that
several methods are available for the extraction and isolation of humic substances
from soils. The selection of a suitable extractant is based on two conditions:

(1) The reagent should have no effect on changing the physical and chemical
nature of the substances extracted, and
 (2) The reagent should be able to quantitatively remove the humic substances
from soils.

Whitehead and Tinsley (1964) have proposed four criteria for solvents for the
extraction of humic substances. In their view, effective solvents should have:

(1) A high polarity and a high dielectric (or permittivity) constant to assist the
dispersion of the charged molecules.
(2) A small molecular size to penetrate into the humic substances.
(3) The ability to disrupt the existing hydrogen bonds and to provide
alternative groups to form humic solvent hydrogen bonds.
(4) The ability to immobilize metallic cations.

Over the years many inorganic and organic solvents have been evaluated for their
effectiveness in extracting humic compounds (Stevenson, 1965; Schnitzer et al.,
1959).

According to Tan (1993) some of the reported inorganic reagents used in

extraction are:
Acids Bases and salts
0.1 N HCl 0.1 N NaOH
0.25 N HF 0.5 N NaOH
1% H3BO3 0.1 M Na2CO3
0.1 M NaF
0.1 M Na4P2O7, PH 7.0
0.1 M Na4P2O7, PH 9-10
0.2 M Na2-EDTA
0.1 M Na2B4O7

Among these reagents, NaOH and Na4P2O7 are most widely employed in
extraction (Tan, 1993). The most efficient and commonly used extractant are
dilute aqueous alkaline solutions, principally NaOH (either 0.1N or 0.5 N)
solutions (Posner, 1966; Schnitzer, 1969). The use of dilute alkali, however, has
been criticized from time to time because it could alter organic matter through
hydrolysis and auto-oxidation (Stevenson, 1982). Although not as effective as
NaOH, Na4P2O7 is used frequently for extraction of humic fractions from soils
high in sesquioxides content (Kononova, 1961). To increase effectiveness of
extraction, a solution with pH 9-10 is recommended by Tan (1993). A comparative
study on the effectiveness of NaOH and Na4P2O7 extraction of humic acids by
Orioli and Curvetto (1980) yielded indications that with the pyrophosphate method
three high-molecular-weight fractions of humic acids were not extracted.

Extraction with acids as proposed by Schnitzer et al. (1959) technically yields

only fulvic acids. The organic solvents used for extraction of humic substances
were oxalic acid, formic acid, phenol, benzene, chloroform, or mixtures of these,
acetylaacetone, hexamethylenetetramine, dodecylsulfate, and urea (Schnitzer and
Khan, 1972).

2.8.1 Extraction of humic substances with sodium pyrophosphate and

sodium hydroxide mixture:

Kononova and Bel’Chikova (1961) recommended the use of a combination of 0.1

M Na4P2O7 + 0.1 N NaOH (pH 13). Amount of humus substances were extracted
by a single treatment of the soil with this mixture gives a yield of humus
substances similar to the quantity of humic acids and fulvic acids obtained by
Tyurin’s method (repeated extraction of a previously decalcified soil with 0.1 N
NaOH), but significantly smaller quantities of humus were extracted from the soil
with 0.1 M Na4P2O7 solution alone (Kononova, 1966). Single extraction with 0.1
M NaOH or 0.1 M NaOH + 0.1 M Na4P2O7 seemed to be enough for estimating
soluble humus from acid soils, but successive extraction with NaOH and Na4P2O7
at 100oC for 30 minutes was more desirable (Kumada and Ota, 1964).

The pH of the sodium pyrophosphate solution is very important; raising the pH

from 7.0 to 9.5 increases the yield of humus substances. When the mixture
Na4P2O7 and NaOH is used, calcium, iron and aluminium are replaced by sodium,
so that soluble sodium humates and fulvates and insoluble pyrophosphates of the
corresponding cations are formed (Kononova, 1966).

Sodium pyrophosphate is convenient because it extracts organic matter quickly (in

10-13 hours). In contrast to when NaOH is used, neither the preliminary soil
decalcification with dilute HCl or H2SO4, nor repeated extraction of the soil with
the solution, substantially influence the exchangeable calcium or carbonates are
present (Kononova ,1966). Use of this mixture avoids the lengthy decalcification
of soils (Kononova and Bel’Chikova, 1961) with high prior to extraction low in N,
(Dormaar, 1972; Vila et al., 1974) and shows lower molecular weights, E4/E6
ratios (Tomer et al., 1987), different electrophoratic patterns and behaviour on gel
filtration than do humic materials extracted from similar soils with 0.1 N NaOH
(Vila et al., 1974). Without decalcification of the soil, this mixture was the most
efficient for extracting humus from soil and NaOH was the least effective (Tomer
et al., 1987).

Yoshida et al. (1979) stated that hexose and neutral sugars in hydrolyses of fulvic
acid fraction extracted successively with 0.1 N NaOH and 0.1 N Na4P2O7 were
determined. The proportion of mannose in hydrolyses of fulvic acid extracted with
NaOH showed a tendency to increase with age, but the proportion of galactose
decreased. The proportion of arabinose and xylose in hydrolysates of the fulvic
acid extracted with Na4P2O7 decreased with increasing age.
2.9 CHEMICAL COMPOSITION OF HUMIC MATTER:

2.9.1 Elemental composition:

The first information on the elemental composition of humic acids dates back 150
years. Humic acids (HA) contain (w/w) 46 to 62% C, 3 to 6% H and 32 to 38% O
(Orlov, 1992). In the 19th century it was established that humic acids contained not
only C, N, H and O but also sulfur and phosphorus. According to Eggertz (1988),
humic acids contained 0.6-1.1% S and 0.2-3.7% P. He also observed 5.6%
aluminum and iron oxides, 0.05-0.15% sodium, up to 0.6% potassium and traces
of magnesium and manganese in these products (Orlov, 1985).

For fulvic acids (FA) lower carbon content, 36 to 44%, is typical, along with 3 to
4.5% N, 3 to 5% H and 45 to 50% O. That is to say these acids differ from humic
acids by a lower content of carbon and higher content of oxygen (Orlov, 1992).

In general, humic acid is higher in C and N, but lower in O and S content than
fulvic acid (Table 2.1). Comparison between terrestrial, aquatic, and geologic
humic acids indicates that geologic humic acid tends to be higher in C, but lower
in H, and especially N content (Tan, 2000).

Table 2.1: Elemental composition of humic matter

Origin C (%) H (%) N (%) O+S (%)

Terrestrial 50.6 5.6 4.3 38.5

Humic
acid

Aquatic 51.7 4.8 2.3 41.1

Geologic 57.1 3.8 1.6 37.5
Terrestrial 48.1 4.5 1.7 45.7
Fulvic
acid

Aquatic 47.4 5.0 1.5 46.1

Geologic 45.0 4.2 1.4 49.4
The variability of the elemental composition of humus acids is due to three groups
of factors: 1) variability of soils and humus substances in time and space; 2)
different techniques used to isolate humus substances from the soil; and 3) errors
in sample preparation and in analytical procedure (Orlov, 1985).

2.9.2 Total acidity and functional groups:

The total acidity also shows some variations because of origin (table 2.2). These
values are attributed to the sum of the carboxyl and phenolic-OH group content,
and indicate the cation exchange and complexing capacities of humic matter. A
high total acidity value is indicative of a high CEC and complexing power. The
data show that the lowest total acidity is exhibited by geologic humic acid and the
highest by geologic fulvic acid. The data in the literature often show that fulvic
acids exhibit higher total acidity values than humic acids. Geologic humic acid is
also more aromatic, whereas aquatic humic acid tends to be more aliphatic in
nature than terrestrial humic acids. In the literature, the tendency can be noticed
for fulvic acids to contain more aliphatic compounds than humic acids (Tan,
2000).
Table 2.2: Aliphatic C, aromatic C, COOH C contents, total acidity, COOH and
phenolic-OH group contents

Origin Aliphatic C Aromatic COOH Total Phenolic Phenolic

(%) C (%) C (%) acidity COOH OH
(mol/kg) (mol/kg ) (mol/kg)

Terrestrial 48.7 36.4 14.9 7.0 4.3 2.7

Humic

Aquatic 50.3 37.2 12.5 7.0 4.4 2.6

acid

Geologic 45.1 43.0 11.9 5.8 3.8 2.0

Terrestrial 61.0 25.3 13.7 6.8 4.7 2.1
F
u
 Aquatic 62.6 21.7 15.7 7.0 4.4 2.6
Geologic - - - 9.4 8.2 0.8

2.10 CHARACTERIZATION OF HUMIC SUBSTANCES:

Humic substances are composed of humic acid, fulvic acid and humin (Felbeck,
1965, 1971). One useful concept that has evolved over the years, and has been
popularized by Kononova (1966), is that the various humic fractions represented a
system of polymers which vary in a systematic way in elemental content, acidity,
degree of polymerization, and molecular weight. The proposed interrelationships
are shown in Fig. 2.4.

For characterization of humic substances various methods are employed.

Elemental composition is an important chemical property, which can be used to
establish the nature and source of humic substances. The elemental composition is
neither unique to humic substance nor does it define humic substances. However,
it does set boundaries for probably chemical structures, which are confirmed by
other properties, such as: 1) molecular weight, 2) functional groups, 3) absorption
spectra, 4) magnetic resonance spectra, 5) comhplexation ability, 6) wetting
properties, 7) absorbing properties, 8) solubilities, 9) exchange capacity, 10)
paramagnetism, 11) fluorescence and 12) titration curves (Steelink, 1985; Perdue,
1985).

Humus
(Decomposition products of organic residue)

Non humic substances Humic substances

 (Known classes of organic compound) (Pigmented polymer)

Fulvic acid Humic acid

(Oden) (Berzelius)
Crenic acid Apocernic acid Brown humic acid Grey humic acid
(Berzelius) (Springer)
Light yellow Yellow-brown Dark brown Grey-black

increase in degree of polymerization

2000 increase in molecular weight
300,000
45% increase in carbon content
62%
48% decrease in oxygen content
30%
1400 decrease in exchange capacity
500

Fig. 2.4: Classification and chemical properties of humic substances, Stevenson

and
Butler (1969).

According to Schnirzer (1978), aside from elementary and functional group

analyses, the methods are most frequently used for the characterization of humic
substances and can be divided into (i) non-degradative and (ii) degradative ones.
(i) Non-degradative methods: Non- degradative methods used for the
characterization of humic substances are: 1) Spectrophotometry in the UV
and visible spectrophotoflu-orometry, 2) Infrared spectrophotometry, 3)
electron spin resonance spectrometry, X-ray analysis, 4) Nuclear magnetic
 resonance spectrometry, 5) Electron microscopy, electron diffraction
analysis, 6) Visco-sity measurements, 7) Surface tension measurements, 8)
Molecular weight measurements, 9) Electrometric titrations.
(ii) Degradative methods: Degradative methods used for the characterization
of humic substances include: 1) Oxidation in alkaline and acidic media, 2)
Re-duction, 3) Hydrolysis, 4) Thermal degradation, 5) Radio chemical
degradation, 6) Biological degradation (Schnirzer, 1978).

2.10.1 Characterization of humic acids:

In Ultraviolet-visible light spectrophotometry, humic solutions are scanned and

the absorbance recorded at various wavelengths between 300 and 800 nm (Tan,
1993) whereas 465 and 665 nm is widely used by soil scientists (Schnizer, 1978).

A low color ratio, 3-5 is usually observed for humic acids and other related
compounds with high molecular weights. It can be noticed that humic acids with
high molecular weights (MW >30,000) have lower E4/E6 values (4.32-4.45) than
humic acids with lower molecular weights (MW = 15,000). The lower-molecular-
weight fractions exhibit E4/E6 values of 5.47-5.49 (Tan, 1993). Chen et al. (1977)
reported that a low E4/E6 ratio is associated with a relatively large molecular size
or high molecular weight that has high carbon content, but is relatively low in
oxygen, COOH groups and total acidity. A high E4/E6 ratio by contrast, indicated a
smaller molecule which contained less C but more O, -COOH groups and total
acidity than the larger molecule.
Kononova (1966) believes that the magnitude of the E4/E6 is related to the degree
of condensation of the aromatic C network, with a low ratio indicative of a
relatively high degree of condensation of aromatic humic constituents.
Conversely, a high E4/E6 ratio reflects a low degree of aromatic condensation and
infers the presence of relatively large proportions of aliphatic structures. Tsutsuki
and Kuwatsuka (1979b) again stated that the absorbance of humic acids decreased
stepwise with decreasing pH value. Tomar, et al. (1987) observed that the E4/E6
ratio for humic acid decreased due to decalcification. The optical density of humic
acids from the lower horizons is higher than that of humic acids from the upper
horizons in dark grey forest soils (Kononova, 1966). The color quotient of humic
acids generally decreased with depth in case of the Alfisol and Mollisol soils
(Sharma and Gupta, 1987).

Udupa et al. (1990) showed that the extinction ratio (E4/E6) of HA’s is largely
governed by totat carbon, exchangeable acidity, phenolic OH and total iron
contents; it increases abruptly with depth unto subsurface layer and then gradually
in subsequent layers, showing relatively slight increase in average particle size due
to molecular association through chemical bonding at surface layer than at the
subsurface layers. The molecular associations are more pronounced in humic acids
at lower depth as observed from the chemical composition of humic acids (Ghosh
and Schnitzer, 1979). Singhal and Kumar (1992) reported that the humic acids
from surface layers are different from those of the subsurface layers which are
more chain-like and is thus more sable due possibly to advanced microbial
activity. Similar observation had been made by Singhal and Soni (1991). During
comparative studies of the chemical structure and the optical properties of humic
acids of different origin, it was found that “younger” humic acids, in a chemical
cense, have a lower optical density, than move “mature” humic acids. The general
observation is due to the high condition of the aromatic carbon net in the “mature”
humus and to the predominance of side chains in the “young” humic acids
Kononova (1966).

The behavior of humic acids towards electrolytes (Ca, Fe, Al, etc.) is an important
characteristic. It is a complex phenomena which includes the precipitation of salts
of humic acids and is complicated by the occurrence of coagulation, depending on
the degree of dispersion of the substances. The degree of dispersion of humic acids
is inversely related to the ratio between aromatic (with hydrophobic properties)
and aliphatic (with hydrophilic properties) structures of the molecules.
Consequently, the greater the predominance of the aromatic ring in molecules of
humic acids the less resistant they are towards electrolysis (Kononova, 1966).
Joshi (1981) reported that lower threshold values of humic acids of Entisols,
Mollisols, Alfisols and Vertisols of Banswara profile indicated their well
condensed structure and higher coagulation threshold values of humic acid of Kota
soil suggested their less condensed aliphatic structure. It was also found from the
experimental data that humic acids of the upper horizons are distinguished from
those of the lower horizons by their higher degree of dispersion. In general the
humic acids of dark grey forest soils are sensitive to electrolytes for the presence
of a fairly well-expressed aromatic ring due evidently to the moisture regime and
the neutral reaction, reported by Kononova (1966).

Higher reducing power of the humic acids suggests that they might have been
originated from mono- and oligosaccharides (Rashid and Schaefer, 1992). Udupa
et al. (1988) reported that the exchange acidity and phenolic content of HA
increased with depth in khanapur forest soils and the trend was reversed in
semiarid soils of Badami. Tsutsuki and Kuwatsuka (1984) emphasized that humic
acid molecule was fully expanded a low ionic strength at pH 7.5, but it shrank
considerably with increasing ionic strength (Chakraborly et al., 1982).
.

2.10.2 Characterization of fulvic acids:

Odén (1919) introduced the new term ‘fulvic acids’ for the group of humic
substances occurring in peat water. He described these substances as compounds
of high molecular weight characterized by a reduced (less than 55 %) carbon
content and wishes solubility in water, alcohol and alkali; their salts are also
readily soluble in water. At low concentration these substances are slightly yellow
in color, in concentrate solution, they are orange-yellow resembling potassium
dichromate solution.

Fulvic acids have an extremely low optical density (Kononova, 1966). Joshi
(1982) showed that the FA’s increased in optical density with decreased in wave
length. A high color ratio, 7-8 or higher, corresponds with curves with steep slopes
and is usually observed for fulvic acids or humic fractions with relatively low
molecular weights (Tan, 1993). The E4/E6 ratios of FA’s in arctic region is 11.5,
cool temperate (acid soil) region is 9.0, subtropical region is 8.4-9.5 and tropical
region is 7.6-11.2 (Schnitzer, 1977). Chen et al. (1977) reported that a high E4/E6
ratio by contrast, indicated a smaller molecule which contained less C but more O,
-COOH groups and total acidity than the larger molecule. Baes and Bloom (1990)
showed that the E4/E6 ratios were independent of concentration of FA and the
E4/E6 ratio is due to absorbance of photons by chromatographic groups. Prasad and
Kumar (1989) examine that E4/E6 ratios and values varied with pH, passing
through a maximum at about pH 6. Prasad and Kumar (1988) indicated that the
contents of carboxyl and phenolic-OH groups, total acidity and values of E4/E6
were higher in the FA of soils as compared with FA of waste materials. The E4/E6
ratios of FA isolated from biogas slurry, poultry manure, compost, sewage sludge
and pressmud decreased with increasing molecular weight (Prasad and Kumar,
1989). Gupta and Bhardwaj (1989) proposed that the E4/E6 ratio of FAs for conifer
forest soils were higher than the FA’s of deciduous forest soils.
Joshi (1981) reported that fulvic acids of Entisols and Vertisols of Banswara
profiles studied had wider E4/E6 ratio and higher coagulation threshold values.
Kononova (1966) reported that fulvic acids are the most stable towards
electrolytes (CaCl2).

Sarker (1987) found that the FA fractions are mainly composed of molecules
having molecular weight ranges from 700-1500, 1500-5000, 5000-10,000. Joshi
(1982) examined that the negative relationship between organic carbon content of
the soils and FA implies the soils low in organic carbon are higher in FA contents.
Watanabe and Kuwatsuka (1992) suggested that abundant plant components
remain in the FA of the low land paddy soil while may aliphatic components were
incorporated into the FA of the brown forest soil.

2.10.3 Characterization of humin:

Humin is commonly defined as the class of sedimentary humic matter that remains
insoluble when sediments are treated with dilute alkali to extract the soluble humic
acids and fulvic acids. Because of its insolubility and macromolecular nature,
humin has been the least studied of all humic fractions. The classification of
humin as a separate class of humic substances was initially proposed at the turn of
the century by Odén (1919), and this classification has been in use since then
(Stevenson, 1982).

Najmr (1960) stated that in soils, the humin group may be partly represented by
carbonized plant residue whose presence is not always the result of forest burning;
small pieces of carbon are present in certain amounts in all soils, particularly in
moist meadow soils with restricted aeration. Shmuk (1930) stated that soil humins
were components of plant residues occurring in various stages of humification.
Consequently, Kononova (1966) reported that with regard to the other groups of
humus substances, humins of the soil medium do not appear to be a separate group
of humus substances but are apparently humic acids occurring in a stable link with
the mineral part of the soil; the humins group is in part represented by plant and
animal residues which have become carbonized.

Humin has been shown to have nearly identical functional group and elemental
compositions (Schnitzer and Khan, 1978; Hatcher, 1980) as humic acids. Humin
is slightly less aromatic than humic acids (Schnitzer and Khan, 1972). Saiz-
Jimenez et al. (1979) indicated that complex polysaccharides account for a
significant fraction of humin. Thus, soil humins are presently considered similar to
humic acids but having greater polysaccharide content (Hatcher et al., 1985).
Hatcher et al. (1980) showed that humin in peat is composed of aromatic
structures derived from lignin but also contains large amounts of aliphatic
structures that are paraffinic in nature. Humin differ in many respects from
associated humic acids, suggestive of the fact that humin is not a clay-humic acid
complex, and that humin is composed of a significant fractions of paraffinic
carbons derived most likely from algal or microbial sources (Hatcher et al., 1985).

2.11 EFFECT OF HUMIC MATTER ON SOILS, PLANT GROWTH

AND THE ENVIRONMENT:

Humic matter is an essential soil component and is known to have a beneficial

effect on soil physical, chemical, and biological properties (Table 2.3). It may
affect plant growth directly and indirectly. Directly, humic acid is capable of
improving seed germination, root initiation and elongation, respiration, uptake of
plant nutrients, and development of green mass. Indirectly, humic acid can modify
the properties of the medium in which plants grow. Humic acid is reported to
enhance soil structure formation and increase soil water-holding capacity, aeration
and permeability (Tan, 2000).
Table 2.3: General properties of soil organic matter and associated effects on soil
properties (Tisdale et al., 2002).

Effect on soil
Property Remarks

The typical dark color of many soils is

Color May facilitate warming.
caused by organic matter.
Helps prevent drying and
Organic matter can hold up to 20 shrinking. May significantly
Water retention
times its weight in water. improve the moisture-retaining
properties of sandy soils.
Permits exchange of gases
Combination with
Cements soil particles into aggregates. stabilizes structure, increases
clay minerals
permeability.
Forms stable complexes with Cu2+,
May enhance the availability of
Chelation Mn2+, Zn2+ and other polyvalent
micronutrients to higher plants.
cations.
Insolubility of organic matter is Little organic matter is lost by
Solubility in water
because of its association with clay. leaching.
Helps to maintain a uniform
Buffer action Organic matter exhibits pH buffering.
reaction in the soil.
May increase the soil CEC.
Total acidities of isolated fractions of
From 20 to 70% of CEC of
Cation exchange humus range from 300 to 1400
many soils(e.g. Mollisols) is
meq/100g.
due to organic matter
Decomposition of organic matter
A source of nutrients elements
Mineralization yields CO2, NH4+, NO3-, H2PO4, and
for plant growth.
SO42-.
Combines with Affects bioactivity, persistence and Modifies application rate of
organic molecules biodegradability of pesticides. pesticides for effective control.

Chemically humic acids increase the cation exchange capacity, thereby providing
to the soil a stronger buffer capacity for resisting sudden drastic chemical changes,
which is very important from an environmental standpoint (Tan, 2000). Humic
acids can also reduce micronutrient toxicity in acid soils, especially Al toxicity
(Tan and Binger, 1986; Ahmad and Tan, 1986; Brogon, 1964). This is attributed to
its capacity for complexing or chelating the metals (Tan, 1993). Organic matter
equally influences the oxidation-reduction potential of soils (Orlov, 1985). It is a
regulator of the conditions of mineral nutrition of plants. In particular, humus
acids change the conditions of fixation of phosphorus in the soil (Hasimoto, 1966),
and bind the nitrogen of ammonia and urea non-exchangeably (Stepanov, 1969).
Organic matter is capable of reducing the effectiveness of some fertilizers (Tuev,
1967). On the other hand, it decreases the fixation of ammonia by the soil (Porter
and Stewart, 1970) and the adverse effects of high doses of mineral fertilizers, etc.
(Kononova, 1972).

Unconfirmed reports state that humic acids or at least the degradation products of
humic acids can be taken up as food by plant roots (Flaig, 1975). Tan (1993)
stated that humic acid compounds and the like can improve plant growth directly
by acceleration of respiratory processes, by increasing cell permeability, or by
hormone growth action.

The dependence of the color of the soil organic matter content is well known
(Orlov, 1985). Considering the differential coefficients of extinction, G.I. Glebova
(1966) was able to estimate the role of individual components of humus in the
formation of the color of brown soils. The heat balance of soil, its volume weight,
consistency, specific gravity of the solid phase (Reppo, 1968) and other physical
properties (Rod and Guillet, 1970) are also associated with the content and
composition of humus. A direct relationship has even been discovered between the
content of available moisture in the soil, the content of moder-humus and fulvic
acids (Diaz-Fierros, 1970).

In industry, humic acid finds application as drilling muds for oil wells and as
emulsifiers. Its emulsification properties and other attributes of humic acids are
attracting the interest of medical science and the pharmaceutical industry (Tan,
2000). The use of humic acid as an anticoagulant to treat thrombosis has been
investigated with success (Kloecking, 1994). It is also a potential antiviral agent,
since application of ammonium humate at concentrations of 0.5 mg/L is reported
to prevent infection of herpes simplex viruses on surfaces of host cells (Thiel et
al., 1977). Clinical tests in Hungary show possibilities for humic acid to be used in
cancer therapy and in healing crush, cut, and especially burn wounds (Jurcsik,
1994). Peat baths were taken in the old days for therapy of gynecological and
rheumatic diseases, even today mud baths are offered in many European health
clinics (Tan, 2000). In Russia, a medicine prepared from peat, called torfot, is used
at the Ukranian Institute of Eye Diseases at Odessa as a topical treatment for
myopia, opacification of the cornea, and early retinal degeneration (Fuchsman,
1980). Because of its importance in industry and agriculture, humic acid is
produced today commercially on a large scale (Tan, 2000).

The presence of humic and fulvic acids in lakes and streams may stimulate the
growth of phytoplankton. Humic acid is capable of detoxifying lakes that are
affected by metal pollution (Tan, 2000). Some adverse phenomena are also
associated with humic acids. In particular, this refers to pollution of industrial and
potable water (Orlov, 1985). Orlov (1985) showed that fulvic acids have been
contained alkanes, fatty acids and alkylphthalates. Some of these are toxic. On
interaction with fulvic acids they become soluble in water, promoting their
migration and contamination of the environment (Ogner and Schnitzer, 1970;
Schnitzer and Ogner, 1970).
Soil sample was collected from specific locations and was processed to carry out
analytical measurement.

3.1 Collection and preparation of soil samples:

The soil sample of Pirojpur series was collected from specific locations and was
processed for the subsequent analysis. The sample was collected in bulk to depth
of 0-15 cm of surface layer on the basis of composite sampling method as
suggested by the soil survey staff of the USDA (1951).

The collected soil sample was air dried. Roots & other undecomposed & partially
decomposed organic residues were removed carefully. Then the sample was
grounded and a portion of the large & massive aggregates were sieved through a
number of sieves. The sample was then preserved for determining texture,
microaggregate, aggregate stability, pH, EC& fraction of soil organic matter.

3.2 Description of Pirojpur soil series:

According to Reconnaissance Soil Survey report (SRDI, 1973), the Pirojpur series
comprises seasonally shallowly to deeply flooded, poorly drained soils developed
in tidal clay deposits. They have dark non-calcareous silty clay to clay subsoil with
strong prismatic and blocky structure with dark grey cutans along ped faces
overlying buried peat or muck within a depth of 4 feet.

3.3 Methods for analysis of physical and chemical properties of soil:

Measurements of selected physical and chemical properties of the soils were

carried out according to conventional methods.

3.3.1 Physical properties:

Among different physical properties of soil, particle size classes and wet sieving
of aggregates were carried out for this measurement.

3.3.1.1 Particle size analysis:

Particle size analysis of the soils was done by hydrometer method as described by Day
(1965). Textural classes were determined by Marshall’s Triangular co-ordinate system.

3.3.1. 2 Wet sieving of the aggregates:

Aggregate separations and soil stability assessments were done by wet sieving.
Two pretreatments were applied before wet sieving: (i) air-dried soil was rapidly
immersed in water (slaked treatment) and (ii) air-dried soil was capillary-rewetted
before immersion in water (rewetted treatment). For capillary rewetting, dried soil
was placed on filter paper that was slowly moistened until a water content of 1.05
times field capacity was reached. The volumetric water content of the soil at field
capacity was determined for all individual samples. In aggregate sieving analysis
the aggregates, passed through 8mm sieve but retained on 2mm sieve (rewetted
and not-rewetted) and passed through 0.1mm sieve but retained on 0.05mm sieve
(grinded) were used. Two pretreatments were applied before wet sieving for
aggregates which were retained on 2mm sieve but passed through 8mm sieve: (i)
air-dried soil was rapidly immersed in water (slaked treatment) and (ii) air-dried
soil was capillary-rewetted before immersion in water (rewetted treatment). For
capillary rewetting, dried soil was placed on tissue paper for 10 minutes that was
slowly moistened. Wet sieving analyses were performed according to the methods
as described by Jalota et al. (1998). For wet sieving, 2mm, 1mm, 0.5mm and
0.25mm sieves were used for pre-treated samples (rewetted and not-rewetted) but
only0.1mm and 0.05mm sieveswere used for grinded sample. In ‘grinded’ sample
density separation, with benzene, was carried out to remove plant root and other
extra organic substances from soil. The mean weight diameter (MWD) of the
aggregates was calculated by the following formula (Van Bavel, 1949):
n
MWD = x w
i 1
i i
Where xi is the mean diameter of any particular size range of aggregates separated
by sieving and wi is the weight of aggregates in that size range as a fraction of the
total dry weight of the sample analyzed.

3.3. 2 Chemical properties:

Among different chemical properties of soil, reaction of the soil, electrical

conductivity, soil organic carbon, fractions of organic carbon were carried out for
this measurement.

3.3.2.1 pH:

Reaction of the soil or pH was measured electrochemically by using a bench pH

meter (Hanna instruments, model pH-211). The ratio of soil to water was 1: 2.5 as
suggested by Jackson (1962).

3.3.2.2 Electrical conductivity (EC):

The EC of the soil was measured at a soil: water ratio of 1: 5 by EC meter (Hanna
instruments, model EC-214) as described by USSL Staff (1954).

3.3.2.3 Soil organic carbon:

Soil organic carbon was determined by Tyrin’s (1936) method. Tyrin’s method
does not function well under diluted condition. But it is more suitable to
determine organic C at lower concentration than the method described by Walkley
& Black. In this work, half of a gram air-dried soil was taken into a 100 ml conical
flask. Ten milliliter 0.4 N K2Cr2O7 was added followed by the addition of some
quartz sand for the reduction of over frothing. A Funnel was placed into the neck
of the conical flask. The mixture was heated at boiling point for exactly five
minutes. The funnel and the neck of the flask were washed with minimum amount
of distilled water (less than 10 ml). Three drops of phenylanthranylic acid
indicator was added. It was titrated against 0.1 N Mohr’s salt solution. A blank
titration was run to standardize Mohr’s salt solution.
3.3.2.4 Extraction of humic matter from soil

The humic matters were extracted with 0.1M sodium pyrophosphate (Na4P2O7.10
H2O) and 0.1N NaOH mixtures, which had pH of 13. Thirty grams of air-dried
soils were taken from each sample into 500 ml conical flasks. 150 ml freshly
prepared extractants was added into each of the conical flask and mixed
thoroughly by shaking. The contents were left overnight (for 16-18 hours) after
sealing the conical flasks with rubber bung. After then the contents were
centrifuged and filtered by using Whatman no. 42 filter papers. Soil residues on
the filter papers were discarded.

3.3.2.4.1 Total organic carbon in the extract:

Ten milliliter extracts were taken into 100 ml conical flasks and neutralized by
adding H2SO4 (concentrated) drop by drop until a slight cloudiness appeared in the
solution. The flasks were then placed on a boiling water bath until the contents
were evaporated to dryness. The contents of the conical flasks were diluted with 5-
10 ml distilled water. After then Tyrin’s method was followed to determine total
organic C in the extract.

3.3.2.4.2 Humic acid carbon in the extract:

Fifty milliliter extracts were pipetted into beakers. Concentrated sulfuric acids
(H2SO4) were added drop by drop for coagulating humic acid gels. The solutions
were stirred with glass rods, until cloudiness appeared in the solutions (at about
pH 2-3). Then the beakers were heated (<80°) for 30 minutes with continuous
stirring with glass rods and left the contents at room temperature for overnight to
complete the precipitation of humic gel. After then the extracts were filtered. The
filter papers were first moistened with 0.05 N H2SO4 and then the acid solutions
were filtered followed by the filtration of humic gel. The precipitates, (brown) on
the filter papers were washed several times with cold 0.05 N H2SO4 until the
filtrates were colorless. The filtrates were discarded as they didn’t contain humic
acids. The funnels with the filter papers which carried humic acids were placed
into the necks of 50 ml volumetric flasks. The precipitates were dissolved in warm
0.05 N NaOH solutions. The Na-humate solutions were cooled to room
temperature and made up the volume with distilled water. 20 ml of the solutions
were taken to determine the humic acids C by Tyrin’s method.

3.3.2.4.3 Fulvic acid carbon in the extract:

The amounts of fulvic acids carbon were found by subtracting the humic acids
carbon from the total organic carbon in the extracts.
The knowledge about the composition of humic substances and their effect on
aggregate is essential because a stable soil structure is important to maintain
agricultural productivity and reduce environmental pollution and organic matter
and its different fractions lead to a stabilizing effect on aggregates as well as soil
structural. Results of the fractions of humic matter & the effect of those fractions
on aggregate stability have been briefly discussed in this section.

4.1 Important physical and chemical properties of soil:

Soils have different physical and chemical properties associated with it. Among
them, only texture, mean weight diameter, pH, EC organic carbon and its fraction
were carried out for this analytical purpose. Table- 4.1 represents the results that
have been found from analysis.

Table- 4.1: Mean weight diameter, % total soil OC, % total extractable C, %
humic acid and % fulvic acid of the studied soil.

% of total soil OC
MWD % Total Soil Total
Pretreatment
(mm) OC Extractable Humic Acid Fulvic Acid
Carbon
Not rewetted
3.66 2.674 6.53 3.21 3.35
(8-2)
Rewetted (8-2) 4.40 2.625 13.91 3.12 10.79
Grinded (0.05) 0.0142 1.06

4.1.1 Mean weight diameter (MWD):

The values of MWD of different pretreatment soil samples are presented in table-
4.1. MWD of ‘not-rewetted’ sample (slaked treatment) was found 3.66 mm where
as for capillary ‘rewetted’ sample it was 4.40 mm. MWD of the ‘grinded’ soil
sample was 0.0142 mm. The size of water-stable aggregates is greatly affected by
the method of wetting of soil sample. Direct immersion of dry soil in water at
atmospheric pressure causes disintegration of large aggregates into smaller ones.
This disintegration can be considerably reduced by wetting soil sample under
vacuum or by spraying with fine mist of water. This is because air plays a role in
aggregates disintegration which is known as air slaking. A higher amount of
disruptive energy occurs upon slaking because rapid wetting of dry soil leads to an
entrapment of air and a buildup of air pressure within the aggregates (Kemper et
al., 1985). Aggregates of lower stability disrupt because they cannot withstand this
pressure. In contrast, rewetted soil shows maximum stability because they
potentially conquer the pressure. He also observed that the amount of soil eroded
was related to how quickly the soil was wetted. Quick wetting allows the water to
encompass more of the aggregates before the entrapped air have escaped from
their interior.

4.1.2 Percent of total organic carbon (% TOC):

The values of % TOC of different pretreatment soil samples are presented in table-
4.1. Here ‘not-rewetted’ soil sample contains 2.674 %, ‘rewetted’ sample contains
2.625% and ‘grinded’ sample contains 1.06% total organic carbons. Sample of
not-rewetted contains more % TOC than sample of rewetted. But when the size of
aggregates is too small like ‘grinded’ sample, % TOC decreases nearly half. This
is because organic matter can disperse as well as it flocculates clay. On the other
hand, the stability of macroaggregates varies with the changes in organic matter
content or in management practices. Macroaggregates are stabilized by transient or
temporary binding agents such as roots, hyphae, and microbial- and plant-derived
polysaccharides. Here, in sieving process, different pre-treatments were applied on
rewetted, not-rewetted and grinded samples. In case of ‘grinded’ sample, it can be
concluded that organic matter decreases with decreasing aggregate size
(Cambardella & Elliott, 1993; Elliott, 1986). Microaggregates are bound by
persistent aromatic humic material associated with amorphous Fe and Al
compounds. The organic matter in the smaller aggregate size fractions are older,
less liable and more highly processed than the organic matter in larger aggregates
(Gupta & Germida, 1988; Paul, 1984). Also, Monreal et al. (1995) found that the
macroaggregate stability is correlated to many types of organic matter structures
such as lignin dimers, alkylaromatics, lipids, sterols, organic carbon and nitrogen.
The microaggregate stability, however, failed to correlate to any of the organic
matter structures. He concluded that soil organic matter and its entities may be less
important than the inorganic components in stabilizing microaggregates. Cohesion
between clay particles, mediated by humic substances linked to polyvalent cations,
may play a stronger role in microaggregate stability. Microaggregate stability of
different pedogenesis was related mainly to soil mineralogy; on the other hand,
organic carbon had little effect (Nwadialo & Mbagwu, 1991).

4.1.3 Percent of total extractable carbon (% TEC):

In this analysis, sodium hydroxide & sodium pyro-phosphate mixture was used as
an extractant for extracting carbon from differently managed soil sample i.e., not-
rewetted, rewetted and grinded. The values of % TEC of different pretreatment
soil samples are presented in table-4.1. Here ‘not-rewetted’ soil sample contains
6.53%, ‘rewetted’ sample contains 13.91% and ‘grinded’ sample contains 31.78 %
total organic carbons. Here ‘grinded’ sample contains more extractable carbon
than rewetted and not-rewetted samples. As explained earlier, microaggregates are
bound by persistent aromatic humic materials which are older, less liable and more
highly processed. When a mild extractant was used to extract carbon, all form of
extractable carbons was come in reaction. On the other hand macroaggregates are
stabilized by transient or temporary binding agents such as roots, hyphae etc.
which was removed before from carbon extraction as it is possible.

4.1.4 Humic Acid:

The results of humic carbon fraction of differently pretreated soil samples are
presented in table-4.1. ‘Not-rewetted’ soil sample contains 3.21%, ‘rewetted’
sample contains 3.12 % and ‘grinded’ sample contains 6.35% humic
carbons. Approximately no differences were found between ‘not-rewetted’&
‘rewetted’ samples in humic acid content. But there is a difference between
‘grinded’ & other two samples. Generally humic acids increase the clay dispersion
(Mbagwu et al., 1993). Theng (1976) explained that at high concentrations, humic
acids may penetrate into the clay domains and form complex chelates with the
polyvalent cations in the intercrystalline domains. This action breaks weak
electrostatic linkage between clay and organic matter; hence, displacing and
dispersing the clay particles. Another possible but vague explanation given by
Mbagwu et al. (1993) is that the interaction among humic acids, clay minerals and
iron oxides is in such a way that causes dispersion instead of flocculation.

4.1.5 Fulvic Acid:

Investigation results about fulvic carbon fractions are presented in table-4.1. The
amount of fulvic acids in ‘not-rewetted’ sample is 3.35%, in ‘rewetted’ is 10.79%
and in ‘grinded’ sample is 25.43%.

Shanmuganathan and Oades (1983) concluded that organic anions like fulvic acid
increases the negative charge on the clay particles. With increasing amounts of
absorbed anions, progressively more clay was dispersed.On the other hand, Soong
(1980) found that fulvic acid was more superior to humic acid in promoting
aggregation. But at higher rates, while the aggregating effect of humic acid surged
upward, the effect of fulvic acid, on the other hand, saturated. Theory suggests, for
two broad reasons, fulvic acids ought to be better than humic acids. For one, fulvic
acids are smaller, more highly charged and more polar than humic acids (Hayes,
1991). Secondly, fulvic acids are more acidic than humic acids due to their higher
concentrations of carboxylic functional group. But Theory appears to conflict with
experimental results. Previous experimental results, however, have shown that
humic acids are the better one.

Several lines of evidence suggest that the observation that higher organic matter
led to lower aggregate stability is not an anomaly.

Dell'agnola and Ferrari (1971) discovered that the humic substances in stable
aggregates have higher molecular weights (> 100, 000) than the humic substances
in the unstable aggregates. These higher molecular weights are dominated by
humic acids, and that phenolic-OH groups here are more effective than carboxyls
to promote aggregate stability. Larger contribution of higher weight humic
fractions was also found by Piccolo and Mbagwu (1990). Moreover, Chaney and
Swift (1984) found some evidence to indicate that less oxidized and higher
molecular weight humic substances were better in aggregation than the highly
oxidized and lower molecular weight substances. And there is always Soong's
(1980) experiment as mentioned earlier. Although a slower start, humic acid,
ultimately, had a longer and stronger effect on aggregation than fulvic acid.

Cations

Studies on clay and organic matter frequently overshadow the importance of cations.
From the C-P-OM model (Edwards and Bremner, 1967), polyvalent cations are crucial to
bridge clay and organic matter. Otherwise, clay and organic matter ought to repel each
other because both carry negative surface charge (Hsu, 1989). This is probably why
cations are related more to soil dispersibility than to slaking. Without this bridging, clay
and organic matter complexes would repel each other, causing dispersion. Giovannini
and Sequi (1978) hypothesize that these cations serve as junctions in a net or mesh
composed of polymeric chains of organic matter. When these cations are removed, this
mesh is weakened at the junctions; thus, stability decreases.
Conclusion

Aggregates are soil particles that are associated in quasi-stable small clods. Both
biological & physical–chemical processes are involved in the formation of soil
aggregates & its stability. They are a potent means of influencing ecosystem
function. This type of structure is generally the most desirable condition for plant
growth, especially in the critical early stages of germination & seedling
establishment. The formation & maintenance of stable aggregates is the essential
feature of soil tilth. It has pronounced influenced on crop rotation, fertilization
practices, erosion, or other soil management problems. Among other things,
organic matter is the major agent stimulating the formation & stabilization of
aggregates like granular, crumb-type etc. These compounds orient the clays into
packets (domains), which form bridges between individual soil particles, thereby
binding them together in water-stable aggregates. Changing in soil C content
changes the distribution and stability of soil aggregates. In laboratory
investigation, conducted in Soil Science Discipline, Khulna University, to study
the fractionation of soil organic matter and their effect on aggregate stability, it
was found that organic matter not only stabilizes aggregates but also disperses it.
This dual role of organic matter further confirms that the behavior of organic
matter is diverse and inconsistent correlations between organic matter and
aggregate stability is the rule rather than the exception. But the relationship
between aggregate stability and organic matter is dependent on several things i.e.,
total organic matter, one or more of the organic matter constituents, disposition of
the organic matter in the aggregates, the aggregate sizes being studied etc.
However a detailed and more extensive investigational work may be needed to
come to a better conclusion.
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