Photolysis and deformation of humic substances under the influence of light, humidity, and temperature

The positive, and in some cases negative, effect of humic substances, including humic preparations, on the nutritional conditions and development of plants has been established by many researchers. The nature of the phenomenon and the mechanism of interaction between humic acids and plants are debatable, but regardless, the question becomes of fundamental importance: why can relatively small doses of humic fertilizers be effective on soils containing significantly higher amounts of the same substances.

A general answer to this question is provided by analogy with the concept of the availability of mineral nutrition elements and nitrogen to plants. In the context of humic acids, this concept is much broader and obviously includes:

• their mobility (solubility) in soils;
• interaction with mineral components;
• size and configuration of molecules;
• quality of the molecules themselves: content of functional groups, paramagnetic centers, and so on.

Therefore, when characterizing the physiological effect of humic substances, it is more correct to speak not about availability, but about their **activity** or the active components of humus.

One reason for the increased activity of humic preparations compared to native soil humic acids is, in our opinion, the changes that occur in them during the preparation process. In addition to the destruction of organo-mineral bonds, the effect of alkaline solutions and other factors may cause:

• hydrolysis of humic acids, their oxidation;
• change the nature of the molecular weight distribution of particles;
• lead to changes in the configuration and conformation of molecules.

Phenomena of this kind, which do not change the compounds' classification as humic acids but affect the details of their structure, can be grouped under the term "**denaturation**," understanding it in a broader sense than is used in protein chemistry. We have investigated some of the denaturation processes of humic acids that occur under the influence of their drying, heating, and illumination.

Deformation of Humic Acid Molecules under the Influence of Humidity and Drying

Differences in the configuration of humate and humic acid molecules in the wet (dissolved) state and in the dry state are clearly visible when measuring relative densities. The decrease in the molar volumes of proteins when dissolved in water is well known. Dry humic acids are practically insoluble in water, so we measured their density first in water ("dry preparation"), and then in weak KOH solutions.

In dilute KOH solutions, the specific volumes of humic acids decrease, and the densities increase, with increasing alkali concentration (Fig. 1). With a further increase in alkali concentration, when virtually all carboxyl groups and all or most phenolic groups are completely neutralized, the density decreases, reaching values lower than in the dry state.

This change in density can be explained by the fact that dry humic acid preparations are not very hydrophilic, and the introduction of a small amount of alkali dramatically changes the state of the substance. The appearance of negative charges due to the formation of carboxylate ions causes the dissolution of part of the preparation. Water molecules orient and compact near the ionogenic groups, which leads to an apparent decrease in specific volume; the measured density value increases. The binding of water is confirmed by the degree of humate hydration, which, according to viscometric data, is at least 1.2–2.5 g of water per 1 g of humic acid.

Upon further neutralization of the carboxyl groups, a large number of negative charges appear; their mutual repulsion contributes to the straightening of the molecule chains, and the substance tends to occupy a larger volume than in the dry state. A similar phenomenon is known for RNA, whose molecule acquires a rigid "rod" structure due to the electrostatic repulsion of neighboring ionized groups. In accordance with these ideas, the composition of humic acids, and the degree of orientation of molecules, cannot be the same in the dry state and in the gel. In gels ("swollen" preparations), humic acid molecules should be arranged more orderly than in dry preparations.

To verify the latter assumption, we obtained diffractograms of gels of some humic acids, and then diffractograms of the same samples after drying them in the air. Dry humic acid preparations do not have sharp reflections; the diffraction pattern is blurred, and the intensity of the reflections is low: its maximum falls in the region of 3.5–3.7 Å (Fig. 2).

The intensities of reflections in gels increase sharply, and the diffractograms differ somewhat from the dry preparations in the nature of the distribution of reflection intensities (Fig. 2). The increase in reflection intensities is undoubtedly due to the greater ordering of the arrangement of molecules. Both polydispersity and deformation of molecules hinder ordering in the dry state. The intensity maximum shifts from 3.5–3.7 Å to 3.3–3.1 Å, suggesting a higher deformability of structure elements with larger repetition periods.

Other data also indicate the deformability of humic acid molecules. Typically, electron micrographs show a spherical shape of humic acid particles. However, viscometric determinations indicate an ellipsoidal conformation of particles with an axis ratio of 1:6. Probably, even when transitioning from humate solutions used in viscometry to dry precipitates (electron microscopy), the deformation of molecules leads to a change in the shape of particles (or associates) from elongated to spherical.

Scans of the surface of dry preparations obtained using a scanning microscope (Fig. 3, 4) can serve as additional confirmation of the deformation of humic and fulvic acids upon drying. The layered structure of the particles is clearly visible in the images. Deep cracks and fractures indicate significant deformation of the organic matter molecules upon drying.

The results obtained allow us to assume that the newly formed humic acids whose molecules are most straightened possess maximum activity in soils. They are more mobile, react vigorously with mineral and organic components, and participate in structure formation. The aging of humic acids that occurs as a result of drying is accompanied by:

• deformation of molecules;
• loss of solubility;
• intramolecular blocking of some functional groups.

All this leads to a decrease in their effect on plants.

Photolysis and Change in Molecular Weight Composition under the Action of Light

Light has a different effect on solutions of humic acids. In our experiments, radiation from mercury-quartz lamps BUV—15 (monochromatic radiation with a wavelength of 2537 Å) and PRK—7 (discrete emission spectrum in the 312–611 nm range) was used. Humic acid solutions, placed in hermetically sealed cylindrical quartz cuvettes, were subjected to light. The solvents were 0.1 N and 0.01 N sodium hydroxide solutions and distilled water. The effect of light was monitored by electronic absorption spectra.

In all variants of the experiments, a significant drop in optical density was found after just 10–15 minutes of continuous irradiation, and after 15–25 hours, in a number of cases, the solutions were practically completely decolorized (Fig. 4). The nature of the spectra hardly changed (Fig. 5), only a change in the slope of the absorption curves was noted.

During the experiments, temperature control was carried out:

• When solutions were irradiated with UV light from the BUV-15 lamp, no change in temperature was noted.
• In experiments with the PRK—7 lamp, which has strong thermal radiation, the temperature of the solutions rose to 40–50°C.

Such heating only slightly affected the color intensity, which is confirmed by experiments in which sodium humate solutions were heated for 4 hours in an ultrathermostat at temperatures from 25° to 95°C (Table 1). Elevated temperatures reduced the optical densities of the solutions, but not enough to attribute the loss of color intensity upon irradiation to this effect.

Sample 1 — Merck humic acid preparation; sample 2 — sod-strongly podzolic soil of the Moscow region, hor. Ar; sample 3 — leached (mountain) chernozem, hor.

The use of ultraviolet radiation does not exclude the possibility of oxidation of humic molecules under the influence of the ozone produced. To eliminate this objection, we conducted a series of experiments irradiating an aqueous solution of fulvic acid and sodium humate with a PRK—7 lamp, which emits radiation in the visible and near-ultraviolet regions. Figure 6 shows that for equal doses of absorbed energy, the optical density of humic substances changes equally under the influence of radiation of any studied wavelength.

The experiments confirm that the effect of ozone, like heating, can only be an accompanying factor, especially considering that ozone is formed only under the action of radiation with a wavelength less than 280 nm. Radiation in the 300–330 nm range promotes the destruction of ozone and its conversion into molecular oxygen.

The main reason for the change in the optical characteristics of humic substances upon irradiation is **photolysis reactions, photochemical destruction**. The decolorization of humic substance solutions upon irradiation is accompanied, as our experiments show, by a change in the molecular weight distribution of particles (Fig. 7).

The gel filtration curves of sodium humates on Sephadexes have two maxima: A (high molecular weight fraction) and B (low molecular weight fraction). After irradiation, the yield of fraction B increases compared to A. Comparison of the elution curves on the gel also indicates a decrease in the molecular weight of humic acid after irradiation:

• The maxima on the elution curves shift toward lower molecular weights.
• The weight-average molecular weight of the low molecular weight fraction decreases after irradiation from 13800 to 2975.
• A new fraction C appears on the elution curve through Sephadex, with a molecular weight of 15600.

The composition of humic substances also changes under the influence of light, as indicated by IR spectra (Fig. 8):

• There are tendencies toward a relative increase in the intensity of the 1620–1630 cm⁻¹ band compared to the –COOH band (1715 cm⁻¹).
• Absorption in the 1000–1100 cm⁻¹ and 3400 cm⁻¹ regions also slightly increases, which can be attributed to the deformation vibrations of alcohol and phenolic hydroxyl groups.

Apparently, the effect of light leads to significant changes in humic substances:

• the size of their molecules decreases;
• oxidizability increases due to an increase in the number of carbonyl groups (possibly quinones), hydroxyl groups of alcohols and phenols;
• the length of conjugated chains (C=C bonds) decreases.

Along with the listed processes, intensive illumination of humic acids causes a sharp increase in **paramagnetic centers** (free radicals) in them. In our experiments, the concentration of PMCs after irradiation of alkaline humate solutions increased from 0.53 to 1.5, with an accompanying decrease in optical densities.

It should be emphasized that photochemical destruction of humic acids occurs not only under the influence of relatively powerful radiation sources. If irradiation with lamps reduces the optical density of humates by 50–70% in 15–20 hours, the same effect is achieved in 4–8 months under daylight. The experimental data consistently indicate the photochemical destruction of humic acids, proceeding as oxidative processes involving free radicals.

The same process must inevitably take place when humic substances are extracted from soils with alkaline solutions and subsequently in the preparation of humic preparations.

Thus, it can be assumed that during the preparation of humic preparations, the **activation of humic substances** occurs, which includes:

• change in the conformation of their molecules;
• release of functional groups;
• partial decomposition of the highest molecular weight fractions into constituent fragments with smaller MW;
• partial oxidation with the accumulation of carbonyl (possibly quinone) groups;
• increase in the concentration of paramagnetic centers.

Molecules activated in this way have a more energetic effect on plant development and their nutritional conditions. The same factors: illumination, temperature, periodicity, and degree of drying, must inevitably lead to differences in many properties of the humic acids of zonal soil types.