Fractionation of humic substances in leonardite

Humic substances are not chemically individual compounds but represent a mixture of substances of various origins, differing in molecular weight and chemical structure, yet more or less similar in their properties. Therefore, by applying various techniques, they can be separated into a number of fractions differing in physico-chemical and biological properties.

Fractionation of humic substances is performed: using various solvents, by fractional precipitation from solutions with polyvalent cations at different pH values, by extracting them from raw materials with alkaline solutions of increasing concentration, by using electrophoresis and chromatography, separation on Sephadex, etc. Most researchers perform the separation of humic substances to study their structure, origin, and physico-chemical properties. Our task is to study physiological activity.

Since in most cases the first stage of separation of humic substances is their extraction from raw materials (peat, coal, leonardite, etc.) using a solvent, we decided first to study the physiological activity of fractions of leonardite from the Zamglay deposit in the Chernihiv region, obtained using various solvents.

We obtained benzene, alcohol, water, and alkaline fractions. The latter was separated into fulvic acids, hymatomelanic acids, and humic acids. Their characteristics are given in Table 1.

The physiological activity of fractions at a concentration of 0.005% was tested by three tests: their effect on the growth of barley seedlings, on barley growth increment, and yeast cell proliferation was studied. The results of the experiments are given in Tables 2, 3, and 4.

Analyzing the results of the experiments, the following can be noted:

• All fractions of leonardite organic matter had a noticeable effect on the growth of barley roots. It was slightly stronger in the humic acid fraction and weaker in hymatomelanic acids.
• The manifestation of the stimulating effect of fractions on the growth of the above-ground mass was approximately equal and weaker, which can be explained by the short duration of the experiments, which lasted 10–12 days from the moment of planting the sprouted seeds on the solutions.
• The auxin activity of the fractions, compared to heteroauxin, is relatively small: it is slightly higher in hymatomelanic acids and lower in benzene and alcohol fractions.
• All fractions had a fairly significant effect on yeast cell proliferation, but it was especially great in the water fraction and fulvic acids.

The largest fraction, both by content in leonardite and by quantity upon extraction, is the fraction of humic and fulvic acids. Since they, according to preliminary data, exert the strongest regulating effect on the growth and development of higher plants, it was decided to begin a more complete study of leonardite humic substances with this fraction.

We used two methods to fractionate humic acids. According to the first method, a sample of leonardite was poured with a fivefold volume of 0.1 N KOH solution and infused for a day with constant stirring. After settling, the solution was drained into a bottle, and the sediment was again poured with the same alkali solution and the extraction operation was repeated. After 4–5 extractions, the entire solution was combined and, by changing the pH value and centrifuging, was separated into eight narrower fractions. The conditions for their isolation and characteristics are given in Table 5. It should be noted that when dissolving the first two fractions, the solution turns slightly cloudy.

Determination of the physiological activity of fraction solutions with a concentration of 0.005%, just as in the previous cases, was performed using three tests (Tables 6, 7, and 8).

Comparing the physiological effect of humic acid fractions (Table 6) with the effect of leonardite organic matter fractions shown in Table 2 on the growth of barley seedlings, it can be noted that all fractions affect the growth of the above-ground mass approximately equally. As for the root system, two fractions of humic acids (3 and 4), in two experiments out of three, had a stronger effect on its growth than all the others, the effect of which was approximately equal, but slightly lower than the effect of unfractionated potassium humate (standard). The auxin activity of humic acid fractions, compared to heteroauxin, is insignificant and equal to the fractions discussed above.

The addition of humic acid fraction solutions to Rieder's medium sharply increased yeast reproduction. This is especially noticeable in fraction No. 8, which in its properties approaches fulvic acids. However, unfractionated potassium humate (standard) showed the greatest activity. This is obviously explained by the fact that it includes water-soluble substances and fulvic acids, which showed the best results in the previous experiment (Table 4).

However, it is not possible to establish a dependence of the magnitude of physiological activity of fractions on the content or quantity of certain functional groups. This is complicated not only by the fact that the activity of fractions changes in relation to different tests, but also by the fact that it does not remain stable when repeating experiments with the same tests: in some experiments, the activity of a fraction may be higher than the activity of others, while in other experiments it may be slightly lower. In all likelihood, the physiological activity of fractions of organic matter of leonardite depends not so much on the presence and quantity of functional groups as on the molecular structure of the chemical compounds included in their composition.

As a second method for isolating humic acids, we used the method of sequential extraction from leonardite with a 0.02 N KOH solution. Leonardite was poured with a fivefold volume of alkaline solution and infused for a day with constant stirring. After settling, the solution was centrifuged for half an hour at 3000 rpm and drained into a glass bottle, in which the extracted fraction of humic acids was precipitated with hydrochloric acid. The resulting precipitate was separated by centrifugation and washed with distilled water until a neutral reaction of the wash water, and then dried at a temperature of 40°C.

The remaining leonardite was again poured with a fivefold volume of alkaline solution of the above concentration, and the entire extraction operation was repeated. Thus, we isolated 31 fractions of humic acids. Their characteristics are given in Table 9.

The table shows that the physico-chemical properties of the fractions differ. The first fractions have significantly more active acidic groups and slightly higher optical density than the last ones.

Physiological activity of the fractions was checked in an experiment with winter wheat. Wheat seeds were germinated for a day, and then transplanted onto distilled water (control) and humic acid fraction solutions with a concentration of 0.003%. To obtain humic acid fraction solutions with a concentration of 0.003%, a sample of dry fraction substance in the amount of 30 mg (calculated on absolutely dry ash-free substance) was ground in a mortar with two milliliters of 0.1 N KOH, and then brought to one liter with distilled water.

Wheat seedlings were grown on the solutions for 10 days. Then the experiment was stopped, and the length of the roots and leaves of the plants, as well as their fresh and dry weight, were determined.

Based on the results of the experiment (Table 10), it can be seen that under the influence of humic acid fraction solutions, the length of roots and leaves increased compared to the control, as well as the fresh and dry weight of wheat seedlings. This testifies not only to the enhancement of division and growth of plant cells but also of synthesis processes within them. The influence of individual humic acid fractions on the growth of roots, leaves, and dry matter accumulation by wheat seedlings is not identical. The increase in root length ranges from 20 to 70%, leaves — from 16 to 32%, dry matter accumulation — from 7 to 32%. But the differences in the strength of action between fractions, unlike fractions isolated by changing the pH value and centrifugation (Table 6), are small and, upon repetition of experiments, just as in the cases described above, do not remain stable both in absolute value and in relation to each other, although the stimulating property of the fractions is preserved in all cases.

It is known that weak solutions of humic acids under any conditions influence the course of biochemical processes in plants and are peculiar regulators of their growth and development. This is especially sharply manifested in the presence of nutrients, but under conditions unfavorable for their assimilation.

An example of this can be the experiment we conducted with "Kievskie" variety tomatoes. The unfavorable factor in this experiment was distilled water with a pH of 5.8. The Kossovich nutrient mixture was prepared on this same water and had the same pH value. Tomato seeds were germinated on water. After four days, the sprouted seeds were planted on distilled water, Kossovich mixture, and humic acid fraction solutions, and after another 10 days, measurements of root and stem length were taken and the fresh and dry weight of the seedlings was determined. The results of the experiments are presented in Table 11.

When growing tomato seedlings on distilled water with a pH of 5.8, their root system and above-ground part were severely inhibited. The addition of various fractions of humic acids slightly increased the growth of roots and stems, but the dry weight of the seedlings changed very little due to the lack of necessary nutrients in the solutions for the synthesis of cell proteins.

We see a completely different picture when growing tomato seedlings on Kossovich nutrient mixture with the addition of humic acid fraction solutions, despite the fact that the pH value in the solutions did not change and remained equal to the pH of distilled water. If on the Kossovich mixture the growth of roots, stems, and dry matter increased slightly compared to distilled water, then with the addition of humic acids to the mixture, not only did the growth of roots and stems increase sharply, but the accumulation of dry matter by the seedlings also increased significantly.

Thus, it can be concluded that the organic matter fractions of leonardite possess stimulating properties. The nature and strength of the action of the fractions depend both on the molecular structure of the substances included in their composition, and the characteristics of the organism on which they act, as well as on environmental conditions. The strength of their action increases under unfavorable conditions and decreases under favorable ones. By applying appropriate separation methods, it is possible to obtain narrower fractions or even individual compounds possessing specific physiological activity.