Extraction and Characterization of Humic and Fulvic Acids from Latosols under Tea Cultivation in South India
Humic substances are considered as the most important constituents of soils. They form the largest fraction of soil organic matter and play a dominant role in improving soil productivity. Humic substances promote plant growth and improve the organic matter status of soil and soil fertility. The common method of extracting of humic substances is by treatment with alkali; dilute sodium hydroxide is generally used. Initially, the soil should be washed with 1 N HCl solution. Humic acid showed broad absorption centered around at 3360, 1406, 1233 and 1060 cm-1 regions. The phenolic (OH), amide, methyl, free-NH- bond, carboxylic and carbonyl group are present in the humic and fulvic acids. The absorption around 1715 cm-1 region was identified to be due to C=O stretching of carbonyl or ketonic groups. The organic matter content of humic and fulvic acids are highly influenced by physical parameters of soil. The organic matter content in humic acid was higher (61%) in Munnar soils, followed by Gudalur and Anamallais soils 56 and 55%, respectively. The organic matter content in fulvic acids is lower when compared to humic acid. The organic matter of the soil is positively correlated with organic matter of humic acid. The available magnesium content in fulvic acid is negatively correlated with potassium content of fulvic acid. The organic matter of the soil is negatively correlated with exchangeable K of humic acid and the same trend observed between available K and organic matter of humic acid.
Humic substances are considered as the most important constituents of soils.
They form the largest fraction of soil organic matter and play the dominant
role in improving soil productivity. Humic substances are formed by the decomposition
of plant and animal residues by micro organisms. Organic minerals are intrinsic
and essential components of all soil. It functions as storehouse of nutrients
like nitrogen, phosphorus, sulphur, potassium, calcium, magnesium, zinc, copper,
iron, etc. and hence treated as important parameter for productivity and quality
of tea. The humic substances play an important role in increasing productivity
and soil fertility. The organic part of the soil consists of a complex system
of substances, the dynamics of which is determined by the continuous admission
of organic residues of plant and animal origin into the soil and their continuous
transformation under the action of chiefly biological factors, but also to certain
extent by chemical and physical factors. In developed soils, the second group
forms a large part of the total reserve of organic matter up to 80-90% (Stevenson,
1994; Saikh and Chandra, 1999). The nitrogen dose
may be adjusted based on organic matter status of tea fields, which is an integrated
approach for sustainable productivity of tea in South India. Humic substances
are reported to promote plant growth and improve the organic matter status of
soil and soil fertility (Szajdak and Osterberg, 1996;
Szajdak et al., 2003; Madrid
et al., 2004; Verma and Palani, 1997). The
main aims of the present study were (1) develop methodology for extraction of
humic substances from tea soil, (2) characterise humic and fulvic acid fractions,
(3) find out the nutrients present in humic and fulvic acids and (4) conduct
correlation study between soil properties nutrient contents/organic matter of
extracted acid fractions.
MATERIALS AND METHODS
The study was conducted in three regions viz., Anamallais, Vandiperiyar
and Munnar. The Anamallais is located on the western face of Western Ghats at
elevation ranging from 1000 to 1400 m above Mean Sea Level (MSL) with 3000 to
3500 mm of annual rainfall. Munnar is at an elevation of 1500- 2000 m above
MSL with 3952 mm annual rainfall. The other study site, Vandiperiyar, is in
Kerala state at an elevation of 1000 m above MSL with 1921 mm annual rainfall.
The mean minimum temperatures of the study site varied between 4 and 10°C,
while, the maximum temperatures ranged from 16 to 32°C. The experiment was
conducted at UPASI Tea Research Institute, Valparai during 2007-2009.
Soil Sampling and Analysis
From each region 15 to 20 soil samples were taken, using sampling auger
(Eijkelkamp, ME 52C09). The individual soil samples collected from each region
were mixed together by hand on a polythene sheet. The samples were air dried
and passed through 2 mm sieve.
Extraction of Humic and Fulvic Acid
Humic substances were extracted by treatment with alkali; dilute NaOH is
used. Initially, the soil was washed with 1 N HCl solution (Mukherjee
and Ghosh, 1984). Soil sample was taken in the conical flask and tio NaOH
solution was added at 1:20. All conical flasks were shaken for 24 h using rotatory
shaker (Model: Pelican equipments-ROTEKLES). The temperature was maintained
at 25°C at 150 rpm. After 24 h, the solution was centrifuged at 6000 rpm
and the supernatant was adjusted to the pH between 1 and 2. The solution was
settled for another 24 h, the insoluble humic substances were precipitated.
The precipitate was collected and the supernatant was again adjusted to pH between
4.5 and 5 using NaOH solution. The fulvic acid fraction got was settled after
24 h. Both the humic substances and fulvic acid fractions were washed with distilled
water to remove impurities. Finally, black coloured fine crystals of humic acid
and brown coloured crystals of fulvic acid fractions were obtained.
Infrared spectra of the humic and fulvic acid substances were recorded at room temperature on a Shimadzu double beam IR spectrometer within a scanning range of 4000-400 cm-1 using pellets containing KBr as matrix.
Soil pH was measured in 1:2 soil: water mixture using Orion pH meter (Orion,
950), electrical conductivity was measured using conductivity meter (Systronics,
304), organic matter by Wakley and Black (1934) procedure,
available phosphorus (P) by Bray and Kurtz (1945) method,
potassium (K) by Hanway and Heidal (1952) procedure
and soil particle distribution (sand, silt and clay) by international pipette
Statistical analysis were carried out by the standard method (Gomez
and Gomez, 1984). The correlation coefficients were worked out using SPSS
RESULTS AND DISCUSSION
The physico-chemical properties of the Anamallais, Munnar and Gudalur soils were analysed and the results are furnished in Table 1. The organic matter content was higher (97 g kg-1) in Munnar soils, while the phosphorus content was higher (125 mg kg-1) in Anamallais and potassium content was higher (460 mg kg-1) at Munnar. Such difference existed in available magnesium and calcium also (Table 1). The analysis of mechanical property indicated that clay fraction dominated in the soils of Anamallais while the sand portion was predominant in the soils of Munnar.
Infrared spectra of humic and fulvic acid are shown in Fig.
1 and 2. In humic acid show broad absorption centered
around at 3360, 1406, 1233 and 1060 cm-1 regions (Garcia
et al., 1992; Ayuso et al., 1996).
The Si-O bending, metal-oxygen stretching absorption is occurred at 590 cm-1.
The phenolic (OH), amide, methyl, free-NH- bond, carboxylic and carbonyl group
are presented in the humic and fulvic acids (Sharmah and
Bordoloi, 1993). The absorption are around at 1715 cm-1 region
is due to the C=O stretching of carbonyl or ketonic groups (Haworth,
1971). The absorption are around at 1630 cm-1 region is due to
the C=C skeletal vibrations, H-boned quinines and COO¯ groups bonded to
metal ions (Dorado et al., 2003).
|| Infrared spectrum of fulvic acid fractions
|| Relative intensity of major IR absorption bands of humic
and fulvic acids soils
|| Chemical properties of humic and fulvic acids
The 1200 cm-1 band is assigned to C-O stretching of phenols, carboxylic
acids, ethers and OH- deformation of COOH (Stevenson,
1994). The relative intensity of major IR absorption bands of humic and
fulvic acids of soil are shown in Table 2.
The organic matter content in humic acid was higher (61%) in Munnar soils,
followed by Gudalur and Anamallais soils 56 and 55%, respectively (Table
3). The organic matter content in fulvic acids was lower when compared to
|| Correlation coefficients worked out between nutrient content
in humic and fulvic acid and physical properties of soil
|HA: Humic acid; FA: Fulvic acid; OM: Organic matter; *Significant
at 5% level; **:Significant at 1% level
The organic matter in humic acid was the lowest in the Anamallais (55%) while
fulvic acid it was the highest in Anamallais (24%). Ammoniacal nitrogen content
was varied between 0.839 and 2.011% in both humic and fulvic acids. The soils
of Gudalur had higher quantum of ammoniacal nitrogen. The available magnesium
content in humic acid was higher when compared to fulvic acid. The available
magnesium content in humic acid was higher (0.129%) in Gudalur soils, followed
by Munnar and Anamallais soils 0.124 and 0.111%, respectively.
The organic matter of the soil is positively correlated with organic matter
of humic acid (r = 0.9973; p = 0.01), it revealed that when the soil is having
high organic matter it increased high organic carbon content in humic acid (Basak
and Ghosh, 1999). The organic matter content of humic and fulvic acids are
highly influenced by physical parameters of soil (Table 4).
The proportion of sand is positively correlated (r = 0.933; p=0.01) whereas
silt and clay are negatively correlated (r = -0.933; p=0.01 and r = -0.933;
p=0.01) with organic matter of humic acid. A similar trend was observed in fulvic
acid (Saikh and Chandra, 1999). The organic matter of
the soil is negatively correlated with exchangeable K of humic acid (r = -0.9987
at 1% level), the same trend observed between available K and organic matter
of humic acid. The correlation coefficient was positive in between the humic
and fulvic acid in the case of calcium (r=0.984). The available calcium content
in humic and fulvic acids are almost same, it varies between 0.227 to 0.459%.
The available magnesium content in fulvic acid is negatively correlated with
potassium content of fulvic acid (Dorado et al.,
2003). The higher potassium input in tea soils enhances the antagonism with
magnesium, (Venkatesan et al., 2006; Jayaganesh
and Venkatesan, 2006) the similar kind of trend is observed in the case
of fulvic acids.
Humic substances are formed by the decomposition of plant and animal residues by micro organisms. The content of humic acid was higher in Munnar soils, followed by Gudalur and Anamallais while fulvic acid constituents lower in comparison to humic acid. The study confirmed the importance of soil organic matter in improving soil health due to the presence of constituents such as humic and fulvic acids.
The authors thank Dr. N. Muraleedharan, Director, UPASI Tea Research Foundation for his constant encouragement throughout the study. Our thanks are also due to Dr. P. Sivaswamy, Professor, ANJA College, Sivakasi for helping to take IR spectrum. The financial assistance provided by the Department of Science and Technology, New Delhi is gratefully acknowledged.
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