Properties of humic acids from various soils of Ukraine and their physiological activity
Humic substances, despite common structural features, are not chemically individual compounds. Representing a group of aromatic polymers, these substances are heterogeneous in nature and properties. A common feature of all humic acids isolated from various soils is the participation of polyphenols in their formation. Our scientists proposed a formula for humic acids of chernozem, peat, and leonardite, whose common property is the presence of polyphenolic and quinoid groups in their molecules.
The diversity of humic substances is manifested in the varying degrees of condensation of aromatic nuclei in their molecules, in the ratio of aromatic and aliphatic structures, etc. Differences in the nature and properties of humic acids of different origins are apparently reflected in their unequal physiological activity.
Researchers from various countries have pointed to the multifaceted participation of humic substances in physiological and biochemical processes occurring in plants. It has been established that humic substances of different origins in small concentrations have a profound and multifaceted effect on plants. However, humic substances from different soils are not identical. Hence, their effect on plants cannot be completely the same. It is logical to assume that the nature and degree of influence of humic acids from various soils on plant organisms are directly dependent on the composition and properties of these acids. This study is devoted to investigating this question across the major soil types of Ukraine.
Study of the nature and properties of humic acids
Fractionation techniques
Using various fractionation techniques (fractional precipitation with acids and buffer solutions, ultracentrifugation, different types of chromatography, electrophoresis, and others), humic substances can be divided into several fractions. Using electrophoresis on paper strips, humic acid was separated into two fractions, which by color corresponded to gray and brown humic acids. According to researchers, all humic acids are a mixture of these two fractions with a predominance of one or the other.
Using chromatography methods on starch columns and paper, as well as paper electrophoresis, it was possible to separate humic acids from chernozem and sod-podzolic soil into three fractions, one of which is detectable only under ultraviolet light. Since the separation of humic acids by paper strip electrophoresis does not yield sufficient quantities of individual fractions, scientists switched to the continuous electrophoresis method, which allowed the separation of humic acid into 10-15 fractions representing a transition from gray to brown humic acids.
When fractionating humic acids from various soils using circular paper chromatography, their zones differed from each other. The zones of humic acid from chernozem were more compact, while those from sod-podzolic soil had a diffuse appearance.
Fractionation of humic acids by electrophoresis also showed that humic acids from chernozem consist of fractions that are less mobile in an electric field, with the majority remaining at the start and only a small amount moving toward the anode. Humic acids from red soil and sod-podzolic soil exhibited increased mobility and fluorescent ability.
In our work, the extraction of humic acid preparations from various soils was performed from pre-decalcified soil. To study the nature and properties of humic acids, methods of circular chromatography and paper electrophoresis were used.
Fractionation of humic acids by circular chromatography was carried out according to the Kononova and Belchikova method, with the difference that fast-filtering paper was used, reducing the exposure time to two hours. Under daylight, all humic acid chromatograms showed two zones: the first—at the center of the chromatogram, at the site of application—and the second—at a certain distance from the first, in the form of narrow light-brown rings. Humic acids from chernozem produced narrow and more compact rings, those from sod-podzolic soil—diffuse rings, and those from other soils—rings of intermediate appearance. Under ultraviolet light, the chromatograms appeared differently: three zones were observed. Around the dark-colored spot, another ring with fluorescent properties was visible, which for humic acid from chernozem fluoresced orange-yellow, from sod-podzolic soil—bright yellow with a bluish tint, and humic acids from other soils produced fluorescent rings of intermediate colors. The next ring, which appeared light brown under daylight, fluoresced in the same sequence of colors under ultraviolet light.
After one hour of separation on paper strips, two zones emerged: the first, remaining at the start, was grayish in color; the second, at a certain distance from the first, was brownish. Under ultraviolet light, a third zone with fluorescent properties was detected, with humic acids from chernozem fluorescing orange-yellow, those from sod-podzolic soil—yellow, and others—intermediate shades.
Differences in the distribution of substances across zones are illustrated by curves recorded using a densitometer. For humic acids from chernozem and dark-chestnut soil, most of the substance remained at the start, with only a small portion moving toward the anode. The most intense movement was observed in the electrophoregram of humic acid from sod-podzolic soil, where most of the substance moved toward the anode. Humic and fulvic acids from gray podzolic soil occupied an intermediate position.
Thus, fractionation of humic acids by chromatography and electrophoresis confirms the heterogeneity of humic acids, with the ratio of mobile and immobile fractions in humic acids from different soil types being unequal: humic acid from chernozem contains fewer mobile fractions, while that from sod-podzolic soil contains more. The presence of certain fractions in humic substances determines their nature—the degree of condensation of the aromatic nucleus, the ratio of aromatic structures, the presence and arrangement of functional groups, etc.
The optical density and coagulation threshold characterize the degree of dispersion of humic substances and are indirect indicators of their complexity or particle condensation. Optical density was determined according to Kononova's method using a photometer. The coagulation threshold was established by the minimum amount of electrolyte (CaCl₂) required for complete coagulation of humic acid over a specific time interval. The concentration of the working humic acid solution was 0.136 g/l.
| Study objects | Optical density (λ=440 nm) | Amount of CaCl₂ per 1 g of humate, mg-equiv | ||
|---|---|---|---|---|
| Start of coagulation | Precipitation after 30 minutes | Complete coagulation (after 2 and 4 hours) | ||
| Humic acids from: | ||||
| Sod-podzolic soil | 1.32 | 5 | 18 | Solution above precipitate is light brown (20) |
| Gray podzolic soil | 1.92 | 4 | 14 | Solution above precipitate is light brown (18) |
| Podzolic chernozem | 2.32 | 3 | 12 | Solution above precipitate is light brown (16) |
| Ordinary chernozem | 2.52 | 1 | 10 | Solution above precipitate is light brown (18) |
| Dark-chestnut soil | 2.08 | 2 | 13 | Solution above precipitate is light brown (20) |
| Southern carbonate chernozem | 1.68 | 4 | 15 | Solution above precipitate is light brown (18) |
| Brown soil | 1.08 | 4 | 15 | Solution above precipitate is light brown (18) |
Data from Table 1 show that the degree of condensation of aromatic carbon networks in humic and fulvic acids increases from sod-podzolic soils to chernozems, with a simultaneous decrease in their molecules' side aliphatic chains. The opposite ratio of these structures is observed in humic acids when transitioning from chernozems to chestnut and further to brown soils. This position is confirmed by the results obtained when determining the coagulation threshold of humic and fulvic acids. The table data indicate the high dispersity of humic acids from sod-podzolic and brown soils, which suggests a high content of side radicals carrying hydrophilic groups in their molecules.
All this indicates that as we transition from sod-podzolic soils to chernozems, the nature of humic acids becomes more complex: a condensed aromatic nucleus becomes more pronounced while the number of side radicals decreases; the opposite phenomenon is observed when transitioning from ordinary to southern carbonate chernozems and brown soils.
The presence of functional groups (carboxyl and phenolic hydroxyls) determines the acidic properties and exchange capacity of humic and fulvic acids. The total content of functional groups in humic acids was determined by Dragunova's method, and the content of carboxyl groups—by Kukharenko's method, i.e., by soaking humic acid samples in a 0.5 N calcium acetate solution with a pH of 6.8-7. The analysis results are presented in Table 2.
| Study objects | Total functional groups, mg-equiv Ba bound per 1 g of humic acid | Carboxyl groups, mg-equiv Ca bound per 1 g of humic acid | Exchange capacity per 100 g of absolute dry ash-free substance, mg-equiv |
|---|---|---|---|
| Humic acids from: | |||
| Sod-podzolic soil | 5.91 | 2.89 | 289 |
| Gray podzolic soil | 7.17 | 3.21 | 321 |
| Slightly podzolic chernozem | 7.57 | 4.05 | 405 |
| Ordinary chernozem | 8.32 | 5.11 | 511 |
| Dark-chestnut soil | 8.22 | 4.20 | 420 |
| Southern carbonate chernozem | 6.65 | 3.79 | 379 |
| Brown soil | 5.57 | 2.22 | 222 |
The table data show that the content of functional groups capable of exchange reactions increases from sod-podzolic soil to chernozems; it decreases when transitioning to dark-chestnut and brown soils. All the above differences in the composition and properties of humic acids of different origins largely determine their role in soil processes, as well as their influence on plant organisms.
Physiological activity of humic acids from various soils of Ukraine
Soluble salts of humic acids with monovalent metals are absorbed by plants and cause a specific physiological effect, enhancing growth, primarily of the root system, and then the above-ground mass. The influence of humic acids is most pronounced during the initial period of plant development. Our scientists believe that the physiological activity of humic acids is directly related to their specific molecular structure and, above all, the presence of quinoid and polyphenolic groups.
The physiological activity of humic acids was determined by conducting experiments with barley on a depleted Pryanishnikov mixture, from which calcium and iron were excluded, as they coagulate humic acid and render it physiologically inactive. Since the experiments were short-term, it was assumed that the reserve of these elements contained in the seeds would be sufficient. The precipitate in the Pryanishnikov mixture was replaced by a Sørensen buffer solution with a pH of 6.8-7.
The experiment was set up as follows. To the Pryanishnikov mixture (diluted with water 1:1), certain amounts of humic acid solutions were added to achieve a concentration of 0.001%. Seedlings were planted in 700 ml chemical beakers, two plants per beaker. The experiment was conducted in triplicate. Measurements of the experimental plants were taken after two weeks. To verify reproducibility, such experiments were conducted four times.
| Study objects | Experiment 1 | Experiment 2 | |||
|---|---|---|---|---|---|
| Average length of primary roots, mm | Relative to control, % | Average height of above-ground part, mm | Number of secondary roots | Relative to control, % | |
| Control—depleted Pryanishnikov mixture (Background) | 44±1 | 100 | 179 | 16 | 100 |
| Background + humic acid from sod-podzolic soil | 67±5 | 152 | 201 | 36 | 127 |
| Background + humic acid from gray podzolic soil | 87±9 | 198 | 190 | 39 | 158 |
| Background + humic acid from podzolic chernozem | 103±2 | 234 | 201 | 44 | 186 |
| Background + humic acid from ordinary chernozem | 107±3 | 243 | 203 | 39 | 172 |
| Background + humic acid from dark-chestnut soil | 97±4 | 220 | 211 | 41 | 193 |
| Background + humic acid from southern carbonate chernozem | 85±5 | 193 | 195 | 51 | 146 |
| Background + humic acid from brown soil | 86±8 | 195 | 220 | 40 | 141 |
The data show that the highest physiological activity is exhibited by humic acids from chernozems and dark-chestnut soils, which have a more condensed aromatic nucleus. Humic acids from sod-podzolic and brown soils, which are less condensed and more dispersed, exhibit lower physiological activity. Solutions of humic acids from chernozem have a stronger stimulating effect on root respiration than solutions of humic acids from peat.
Scientists hydrolyzed humic acid from peat and found that potassium humate of the non-hydrolyzable residue has greater physiological activity compared to hydrolysates. In their opinion, the nucleus of humic acids plays a leading role in stimulating their effect on biochemical processes and the entire plant organism as a whole.
For a more complete characterization of the effect of humic acids from various soils on plant organisms, an experiment was conducted in sand culture with an indicator crop—tomatoes. Humic acids were applied as potassium humate solutions at a rate of 0.22 g of humic acid per vessel. The experiment was set up as follows: 1. Control—complete Pryanishnikov mixture (background). 2. Background + 0.22 g of humic acid per vessel. The experiment was conducted in triplicate. Harvesting was performed four weeks after germination. In the plants, fresh weight, peroxidase activity by the Pochinok method, reducing sugars by the Hagedorn-Jensen method, chlorophyll by the Guthrie method, and vitamin C by the Murri method were determined. After harvesting in the sand culture, the total number of microorganisms growing on MPA, nitrates with disulfophenolic acid by the Grandval-Lajoie method, and mobile phosphorus by the Truog method were determined. The analysis results are presented in Table 4.
| Study objects | Influence of humic acids on plants | Influence of humic acids on soil | ||||||
|---|---|---|---|---|---|---|---|---|
| Weight of 100 seedlings (fresh matter), g | Relative to control, % | Peroxidase activity, mg cobalt | Chlorophyll content per 100 g fresh matter, mg | Ascorbic acid content per 100 g fresh matter, mg | Total sugars, % | Number of microorganisms on MPA, thousand per 1 g dry soil | NO₃ content per 100 g dry soil, mg | |
| Control—Pryanishnikov mixture (Background) | 32.6 | 100 | 13.00 | 109.58 | 22.4 | 0.50 | 1760 | 4.16 |
| Background + humic acid from: | ||||||||
| Sod-podzolic soil | 40.88 | 125 | 14.16 | 121.76 | 24.8 | 0.58 | 2090 | 4.54 |
| Gray podzolic soil | 47.63 | 146 | 16.20 | 121.76 | 27.2 | 0.77 | 2280 | 4.90 |
| Podzolic chernozem | 48.22 | 148 | 16.20 | 126.42 | 29.6 | 0.85 | 2750 | 6.09 |
| Ordinary chernozem | 49.79 | 153 | 16.20 | 131.50 | 30.4 | 0.95 | 2886 | 6.35 |
| Dark-chestnut soil | 62.46 | 192 | 16.20 | 131.50 | 33.0 | 0.85 | 2640 | 7.14 |
| Southern carbonate chernozem | 41.66 | 128 | 14.60 | 121.76 | 27.0 | 0.90 | 2616 | 6.66 |
| Brown soil | 36.53 | 112 | 14.60 | 121.76 | 25.6 | 0.78 | 1932 | 4.54 |
The table data show that the more "mature" humic acids from chernozems and dark-chestnut soils, in genetic terms, are physiologically more active. Under their influence, the weight of green seedling mass increases, the activity of respiratory enzymes rises, and the accumulation of sugars, ascorbic acid, and chlorophyll in plants increases to a greater extent than under the influence of less condensed humic acids from sod-podzolic and brown soils. In the variants with humic acids from chernozems and dark-chestnut soils, the sand cultures contained more microorganisms and nutrients. The more "mature" humic acids have a greater stimulating effect on the absorption of nutrients by tomatoes, thereby increasing seedling weight and nutrient uptake.
Conclusions
- Humic acids from different soil types possess varying properties, which largely determine the role of humic substances in soil processes, as well as their influence on plant organisms. The more "mature" humic acids from chernozems and dark-chestnut soils, which have a more condensed aromatic nucleus, exert a greater stimulating effect on biological processes in soils and plants compared to the less condensed humic acids from sod-podzolic and brown soils.
- The action of humic acids is apparently associated with their total content of functional groups.
- One of the factors contributing to the greater effectiveness of humic fertilizers on sod-podzolic and brown soils is likely the lower physiological activity of humic acids from these soils.