Changes in soil enzymatic activity under oil pollution. Changes in the Enzymatic Activity of Soils under Oil Pollution Nature of Soil Enzymes
The processes of metabolism and energy during the decomposition and synthesis of organic compounds, the transition of hard-to-digest nutrients into forms easily accessible to plants and microorganisms occur with the participation of enzymes.
The enzyme invertase (α-fructofuranosidase) catalyzes the breakdown of various carbohydrates into glucose and fructose molecules.
Many data confirm the relationship between the activity of invertase with the biological activity of the soil, the content of organic matter in it, the yield of field crops and the changes that occur in the soil during agricultural use (Khaziev F.Kh., 1972; Galstyan A.Sh., 1978; Vasilyeva L.I., 1980).
With an increase in the depth of plowing, the activity of invertase in top layer soil decreased slightly, which is explained by the depletion of this soil layer, since during deep plowing, the main amount of plant residues is embedded in the lower layers. The accumulation of most of the post-harvest residues in the upper soil layer during non-moldboard tillage causes a decrease in invertase activity in the 30–40 cm layer by 5–15% by the end of the plant growing season.
On a fertilized background, the activity of invertase increased by an average of 5% only after plowing. According to non-moldboard methods of tillage, fertilizers did not affect the activity of this enzyme.
The action of urease is associated with the hydrolytic cleavage of the bond between nitrogen and carbon (CO-IN) in the molecules of nitrogen-containing organic compounds. Therefore, many researchers note a positive correlation between urease activity and nitrogen and humus content in soils. However, urease activity depends not only on the total amount of humus, but on its quality, correlating mainly with the value of the ratio of carbon to nitrogen (C: 14). Organic matter with the widest carbon to nitrogen ratio corresponds to the highest urease activity; with a decrease in the carbon to nitrogen ratio, the activity of the enzyme also decreases. This, according to V.D. Mukha and L.I. Vasilyeva, points to the regulatory effect of urease on the processes of transformation of nitrogen-containing organic compounds in the soil. In our studies, among the variants of mouldboard tillage, the highest urease activity was manifested by plowing to a depth of 20–22 cm. Deepening the tillage led to a significant decrease in the activity of this enzyme. So, at the beginning of the vegetation of plants, plowing to 35–37 cm in a soil layer of 0–40 cm produced 20% less ammonia than by plowing to a normal depth of 20–22 cm (average for 1980–1982, mg YN 3 per 1 g of air-dry soil).
The intensity and direction of the processes of transformation of organic matter in the soil is also determined by the activity of the redox enzymes polyphenol oxidase and peroxidase. Polyphenol oxidase is involved in the conversion of organic compounds of the aromatic series into humus components (Mishustin E.N. et al., 1956, Kononova M.M., 1963, 1965). In the decomposition of humic substances, a large place is given to peroxidase and catalase (Nikitin D.I., 1960). Researchers note a high positive correlation between humus decomposition and peroxidase activity and an almost functional negative correlation with polyphenol oxidase activity (Chunderova A.I., 1970, Dulgerov A.N., 1981). The opposite orientation of the functions of peroxidase and polyphenol oxidase and a single object of their application made it possible for A.I. Chunderova to propose the concept of “humus accumulation coefficient”, the value of which is determined by the ratio of soil polyphenol oxidase activity to peroxidase activity.
According to our research, an increase in the plowing depth from 20-22 cm to 35-37 cm and the use of non-moldboard tillage with a flat cutter, a plow without mouldboards, a chisel, a paraplow-type tool, SibIME tines, as well as when cultivating the soil of the No- til" led to an increase in peroxidase activity by 4-6% and a decrease in polyphenol oxidase activity by 4-5% (Table 15). The coefficient of humus accumulation in this case decreased by 8-10%.
15. Activity of peroxidase and polyphenol oxidase in 0-40 cm soil layer under peas, mg purpurgallin per 100 g air-dry
soil in 30 min. (1980-1982)
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Studies have established a relationship between the humus accumulation coefficient and the ratio of the number of microorganisms assimilating mineral nitrogen to the number of microorganisms assimilating nitrogen of organic compounds (KAA: MPA). The correlation coefficient between the two indicators is -0.248±0.094. An increase in the first indicator in many cases leads to a decrease in the latter and vice versa, which confirms the existence of a relationship between the structure of microbial cenosis and the direction of the process of biochemical transformation of soil organic matter. The ratio of these two coefficients, apparently, can characterize the orientation of the cultural soil-forming process.
This allows us to conclude that the transformation of soil organic matter, due to the activity of peroxidase and polyphenol oxidase, shifts towards increased humus decomposition with deepening plowing and tillage without reversing the layer (Fig. 5).
- ? Row4
- ? RyadZ
- ? Row2
- ? Row1
Rice. 5. Influence various ways and the depth of the main treatment for peroxidase activity in the soil layer of 0-40 cm in the period of 2-4 pairs of true leaves in sunflower, mg purpurallin per 1 g of air-dry soil (1989-1991)
A certain place in the direction and intensity of biochemical processes occurring in the soil is occupied by the enzyme catalase. As a result of its activating action, hydrogen peroxide is split into water and free oxygen. It is believed that catalase, along with peroxidase, can participate in peroxidase-type reactions, during which reduced compounds undergo oxidation. In the experiments of the Research Institute of Agriculture of the Central ChP im. V.V. Dokuchaev did not establish the dependence of catalase activity on the depth or methods of basic tillage. However, with an increase in the plowing depth over 25-27 cm, as well as tillage without reversing the layer, a significant increase in catalase activity was noted compared with plowing to a depth of 20-22 cm and 25-27 cm.
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Introduction
1. Literature review
1.1 Understanding soil enzymes
1.2 Enzymatic activity of soils
1.3 Methodological approaches to determining the enzymatic activity of soils
1.3.1 Allocation of the experimental area
1.3.2 Features of the selection and preparation of soil samples for analysis
1.4 Influence of various factors (temperature, water regime, sampling season) on the enzymatic activity of soils
1.5 Changing communities of microorganisms in soils
1.6 Methods for studying the activity of soil enzymes
Conclusion
List of sources used
Introduction
Under conditions of increased anthropogenic load on the planet's biosphere, the soil, being an element of the natural system and being in dynamic balance with all other components, is subject to degradation processes. Fluxes of substances, getting into the soil as a result of anthropogenic activity, are included in natural cycles, disrupting the normal functioning of the soil biota, and, as a result, the entire soil system. Among the various biological evaluation criteria anthropogenic influence on soils, the most efficient and promising are biochemical indicators that provide information about the dynamics of the most important enzymatic processes in the soil: the synthesis and decomposition of organic matter, nitrification, and other processes.
Available information on enzymatic activity various types soils are currently insufficient and require further study. This makes it very relevant in theoretical and practical terms to study the issues raised in this work.
Object of study: soil enzyme activity.
Target term paper: Study of soil enzymes and enzymatic activity of soils.
In accordance with the purpose of the study, the following tasks:
1. Give a general idea of soil enzymes and the enzymatic activity of soils.
2. Consider methodological approaches to determining the enzymatic activity of soils.
3. Determine the influence of various natural factors on the enzymatic activity of soils
4. Study the issue of the presence and change of communities of microorganisms in soils
5. List and describe methods for studying the activity of soil enzymes.
1 . Literature review
1.1 Understanding soil enzymes
It is difficult to imagine that enzymes, highly organized protein molecules, could be formed in the soil outside of a living organism. Soil is known to have a known enzymatic activity.
Enzymes are catalysts for chemical reactions of a protein nature, differing in the specificity of their action in relation to the catalysis of certain chemical reactions.
Enzymes are biosynthesis products of living soil organisms: woody and herbaceous plants, mosses, lichens, algae, fungi, microorganisms, protozoa, insects, invertebrates and vertebrates, which are represented in nature by certain aggregates - biocenoses.
The biosynthesis of enzymes in living organisms is carried out due to genetic factors responsible for the hereditary transmission of the type of metabolism and its adaptive variability. Enzymes are the working apparatus by which the action of genes is realized. They catalyze thousands of chemical reactions in organisms, from which, as a result, cellular metabolism is composed. Thanks to enzymes chemical reactions in the body are carried out at a high speed.
To date, more than 150 of the two thousand known enzymes have been obtained in crystalline form. Enzymes are divided into six classes:
1. Oxireductases - catalyze redox reactions.
2. Transferases - catalyze the reactions of intermolecular transfer of various chemical groups and residues.
3. Hydrolases - catalyze the reactions of hydrolytic cleavage of intramolecular bonds.
4. Lyases - catalyzing the reactions of adding groups to double bonds and the reverse reactions of detachment of such groups.
5. Isomerases - catalyze isomerization reactions.
6. Ligases - catalyze chemical reactions with the formation of bonds due to ATP (adenosine triphosphoric acid).
When living organisms die and rot, some of their enzymes are destroyed, and some, getting into the soil, retain their activity and catalyze many soil chemical reactions, participating in the processes of soil formation and in the formation of a qualitative sign of soils - fertility.
IN different types Soils under certain biocenoses have formed their own enzymatic complexes, which differ in the activity of biocatalytic reactions.
An important feature of the enzymatic complexes of soils is the orderliness of the action of the existing groups of enzymes. It manifests itself in the fact that the simultaneous action of a number of enzymes representing different groups is ensured. Enzymes exclude the accumulation of excess of any compounds in the soil. Excess accumulated mobile simple compounds (for example, NH 3) in one way or another they temporarily bind and send to cycles, culminating in the formation of more complex compounds. Enzymatic complexes can be represented as some kind of self-regulating systems. Microorganisms and plants play the main role in this, constantly replenishing soil enzymes, since many of them are short-lived.
The number of enzymes is indirectly judged by their activity over time, which depends on the chemical nature of the reacting substances (substrate, enzyme) and on the interaction conditions (concentration of components, pH, temperature, composition of the medium, the action of activators, inhibitors, etc.).
Enzymes belonging to the classes of hydrolases and oxidoreductases are involved in the main processes of soil humification, so their activity is a significant indicator of soil fertility. Therefore, let us briefly dwell on the characteristics of enzymes belonging to these classes.
Hydrolases include invertase, urease, phosphatase, protease, etc.
Invertase - catalyzes the reactions of hydrolytic cleavage of sucrose into equimolar amounts of glucose and fructose, also acts on other carbohydrates (galactose, glucose, rhamnose) with the formation of fructose molecules - an energy product for the life of microorganisms, catalyzes fructose transferase reactions. Studies by many authors have shown that the activity of invertase better than other enzymes reflects the level of fertility and biological activity of soils 3, p. 27.
Urease - catalyzes the reactions of hydrolytic cleavage of urea into ammonia and carbon dioxide. In connection with the use of urea in agronomic practice, it must be borne in mind that urease activity is higher in more fertile soils. It rises in all soils during periods of their greatest biological activity - in July-August.
Phosphatase (alkaline and acid) - catalyzes the hydrolysis of a number of organophosphorus compounds with the formation of orthophosphate. Phosphatase activity is the higher, the less mobile forms of phosphorus are in the soil; therefore, it can be used as an additional indicator when determining the need for phosphate fertilizers to be applied to soils. The highest phosphatase activity is in the rhizosphere of plants.
Proteases are a group of enzymes that break down proteins into polypeptides and amino acids, which are subsequently hydrolyzed to ammonia, carbon dioxide and water. In this regard, proteases are of great importance in the life of the soil, since they are associated with a change in the composition of organic components and the dynamics of nitrogen forms, which are easily assimilated by plants.
The class of oxidoreductases includes catalase, peroxidase and polyphenol oxidase, etc.
Catalase - as a result of its action, the breakdown of hydrogen peroxide, which is toxic to living organisms, occurs:
H2O2 > H2O + O2
Vegetation has a great influence on the catalase activity of mineral soils. Soils under plants with a powerful deep penetrating root system are characterized by high catalase activity. A feature of catalase activity is that it changes little down the profile, has an inverse relationship with soil moisture and a direct relationship with temperature.
Polyphenol oxidase and peroxidase in soils play the main role in the processes of humus formation.
Polyphenol oxidase catalyses the oxidation of polyphenols to quinones in the presence of free atmospheric oxygen. Peroxidase catalyzes the oxidation of polyphenols in the presence of hydrogen peroxide or organic peroxides. At the same time, its role is to activate peroxides, since they have a weak oxidizing effect on phenols. Further condensation of quinones with amino acids and peptides can occur with the formation of a primary humic acid molecule, which can further become more complex due to repeated condensations.
The ratio of polyphenol oxidase activity (S) to peroxidase activity (D), expressed as a percentage, is related to the accumulation of humus in soils, therefore this value is called the conditional coefficient of humus accumulation (K):
Consider the varieties of soil enzymes.
The class of oxidoreductases includes catalyzing redox reactions.
In the vast majority of biological oxidations, it is not the addition of oxygen to the oxidized molecule, but the removal of hydrogen from the oxidized substrates. This process is called dehydrogenation and is catalyzed by dehydrogenase enzymes.
There are aerobic dehydrogenases, or oxidases, and anaerobic dehydrogenases, or reductases. Oxidases transfer hydrogen atoms or electrons from the oxidized substance to atmospheric oxygen. Anaerobic dehydrogenases donate hydrogen atoms and electrons to other hydrogen acceptors, enzymes or carriers, without transferring them to oxygen atoms. Numerous organic compounds that enter the soil with plants and animals undergo oxidation: proteins, fats, carbohydrates, fiber, organic acids, amino acids, purines, phenols, quinones, specific organic substances such as humic and fulvic acids, etc.
As a rule, anaerobic dehydrogenases are involved in redox processes in the cells of animals, plants, and microorganisms, which transfer the hydrogen split off from the substrate to intermediate carriers. In the soil environment, mainly aerobic dehydrogenases are involved in redox processes, with the help of which the substrate hydrogen is transferred directly to atmospheric oxygen, i.e. the hydrogen acceptor is oxygen. The simplest redox system in soils consists of an oxidizable substrate, oxidases, and oxygen.
A feature of oxidoreductases is that, despite the limited set of active groups (coenzymes), they are able to accelerate big number variety of redox reactions. This is achieved due to the ability of one coenzyme to combine with many apoenzymes and each time form an oxidoreductase specific to one or another substrate.
Another important feature of oxidoreductases is that they speed up chemical reactions associated with the release of energy, which is necessary for synthetic processes. Redox processes in soil are catalyzed by both aerobic and anaerobic dehydrogenases. By chemical nature, these are two-component enzymes, consisting of a protein and an active group, or coenzyme.
An active group can be:
NAD + (nicotinamide adenine dinucleotide),
NADP + (nicotinamide adenine dinucleotide phosphate);
FMN (flavin mononucleotide);
FAD (flavin adenine dinucleotide), cytochromes.
About five hundred different oxidoreductases have been found. However, the most common oxidoreductases are those containing NAD + as an active group.
By combining with protein and forming a two-component enzyme (pyridine protein), NAD + enhances its ability to recover. As a result, pyridine proteins become able to take away from substrates, which can be carbohydrates, dicarboxylic and keto acids, amino acids, amines, alcohols, aldehydes, specific soil organic compounds (humic and fulvic acids), etc., hydrogen atoms in the form of protons (H +) . As a result, the active group of the enzyme (NAD +) is reduced, and the substrate passes into an oxidized state.
The mechanism of binding two hydrogen atoms, i.e. two protons and two electrons, is as follows. The active group of dehydrogenases, which accepts protons and electrons, is the pyridine ring. When NAD + is reduced, one proton and one electron are attached to one of the carbon atoms of the pyridine ring, i.e. one hydrogen atom. The second electron is attached to the positively charged nitrogen atom, and the remaining proton passes into the environment.
All pyridine proteins are anaerobic dehydrogenases. They do not transfer the hydrogen atoms removed from the substrate to oxygen, but send them to another enzyme.
In addition to NAD +, pyridine enzymes may contain nicotinamide adenine dinucleotide phosphate (NADP +) as a coenzyme. This coenzyme is a derivative of NAD + , in which the hydrogen of the OH - group of the second carbon atom of the adenosine ribose is replaced by a phosphoric acid residue. The mechanism of substrate oxidation with the participation of NADP* as a coenzyme is similar to NAD + .
After addition of hydrogen, NADH and NADPH have significant reducing potential. They can transfer their hydrogen to other compounds and reduce them, while they themselves turn into an oxidized form. However, hydrogen attached to anaerobic dehydrogenase cannot be transferred to air oxygen, but only to hydrogen carriers. Such intermediate carriers are flavin enzymes (flavoproteins). They are two-component enzymes, which may contain phosphorylated vitamin B 2 (riboflavin) as an active group. Each molecule of such an enzyme has a riboflavin phosphate (or flavin mononucleotide, FMN) molecule. Thus, FMN is a compound of the nitrogenous base of dimethylisoalloxazine with the residues of the five-carbon alcohol ribitol and phosphoric acid. FMN is capable of accepting and donating two hydrogen (H) atoms at the nitrogen (N) atoms of the isoalloxazine ring.
Transferases are called transfer enzymes. They catalyze the transfer of individual radicals, parts of molecules and whole molecules from one compound to another. Transfer reactions usually proceed in two phases. In the first phase, the enzyme splits off the atomic group from the substance involved in the reaction and forms a complex compound with it. In the second phase, the enzyme catalyzes the addition of a group to another substance participating in the reaction, and is itself released in an unchanged state. The class of transferases includes about 500 individual enzymes. Depending on which groups or radicals are transferred by transferases, there are phosphotransferases, aminotransferases, glycosyltransferases, acyltransferases, methyltransferases, etc.
Phosphotransferases (kinases) are enzymes that catalyze the transfer of phosphoric acid residues (H2P03). The donor of phosphate residues, as a rule, is ATP. Phosphate groups are transferred to alcohol, carboxyl, nitrogen-containing, phosphorus-containing and other groups of organic compounds. Phosphotransferases include the ubiquitous hexokinase, an enzyme that accelerates the transfer of a phosphoric acid residue from an ATP molecule to glucose. This reaction begins the conversion of glucose into other compounds.
Glycosyltransferases accelerate the transfer of glycosyl residues to molecules of monosaccharides, polysaccharides or other substances. These are enzymes that provide reactions for the synthesis of new carbohydrate molecules; the coenzymes of glycosyltransferases are nucleoside diphosphate sugar (NDP sugar). From them, in the process of synthesis of oligosaccharides, the glycosyl residue is transferred to the monosaccharide. Currently, about fifty NDP sugars are known. They are widely distributed in nature, synthesized from phosphate esters of monosaccharides and the corresponding nucleoside triphosphates.
Acyltransferases transfer acetic acid residues CH3CO - as well as other fatty acid residues to amino acids, amines, alcohols and other compounds. These are two-component enzymes, which include coenzyme A. The source of acyl groups is acyl coenzyme A, which can be considered as an active group of acyltransferases. When acetic acid residues are transferred, acetyl coenzyme A is involved in the reaction.
The class of hydrolases includes enzymes that catalyze the hydrolysis and sometimes the synthesis of complex organic compounds with the participation of water.
A subclass of esterases includes enzymes that accelerate the reactions of hydrolysis of esters, alcohols with organic and inorganic acids.
The most important subclasses of esterases are ester hydrolases carboxylic acids and phosphatase. The hydrolysis reactions of fats (triglycerides), as a result of which glycerol and higher fatty acids are released, are accelerated by glycerol ester hydrolase lipase. A distinction is made between simple lipases, which catalyze the release of higher fatty acids from free triglycerides, and lipoprotein lipases, which hydrolyze protein-bound lipids. Lipases are single-component proteins with a molecular weight of 48,000 to 60,000. Yeast lipase has been well studied. Its polypeptide chain consists of 430 amino acid residues and is folded into a globule, in the center of which is the active site of the enzyme. The leading role in the active center of lipase is played by the radicals of histidine, serine, dicarboxylic acids and isoleucine.
The activity of lipases is regulated by their phosphorylation-dephosphorylation. Active lipases are phosphorylated, inactive ones are dephosphorylated.
Phosphatases catalyze the hydrolysis of phosphate esters. Phosphatases acting on esters of phosphoric acid and carbohydrates are widely distributed. Such compounds include, for example, glucose-6-phosphate, glucose-1-phosphatase, fructose-1,6-diphosphate, etc. The corresponding enzymes are called glucose-6-phosphatase, glucose-1-phosphatase, etc. They catalyze the elimination of a phosphoric acid residue from phosphorus esters:
Phosphodiester phosphatases - deoxyribonuclease and ribonuclease catalyze the cleavage of DNA and RNA to free nucleotides.
A subclass of hydrolases includes glycosidases accelerating the hydrolysis of glycosides. In addition to glycosides containing monohydric alcohol residues as aglycones, oligo- and polysaccharides are substrates on which glycosidases act. Of the glycosidases acting on oligosaccharides, maltose and sucrose are the most important. They hydrolyse maltose and sucrose.
Of the glycosidases acting on polysaccharides, amylases are the most important. Feature amylase - lack of absolute specificity of action. All amylases are metalloproteins containing Zn 2+ and Ca 2+ . The active sites of amylases are formed by histidine, aspartic and glutamic acids, and tyrosine radicals. The latter performs the function of binding the substrate, and the first tricatalytic. Amylases accelerate the reactions of hydrolysis of glycosyl bonds in the starch molecule with the formation of glucose, maltose or oligosaccharides.
Of no small importance is cellulase, which catalyzes the breakdown of cellulose, inulase, which breaks down the polysaccharide inulin, and aglucosidase, which converts the disaccharide maltose into two glucose molecules. Some glycosidases can catalyze the transfer of glycosyl residues, in which case they are called transglycosidases.
Proteases (peptide hydrolases) catalyze the hydrolytic cleavage of peptide CO-NH bonds in proteins or peptides to form smaller molecular weight peptides or free amino acids. Among peptide hydrolases, there are endopeptidases (proteinases), which catalyze the hydrolysis of internal bonds in a protein molecule, and exopeptidases (peptidases), which provide cleavage of free amino acids from the peptide chain.
Proteinases are divided into four subclasses.
1. Serine proteinases, the active center of these enzymes includes a serine residue. The sequence of amino acid residues on the site of the polypeptide chain in serine proteinases is the same: aspartic acid-series-glycine. The hydroxyl group of serine is characterized by high processes. The second active functional group is the imidazole of the histidine residue, which activates the hydroxyl of serine as a result of the formation of a hydrogen bond.
2. Thiol (cysteine) proteinases have a cysteine residue in the active center, sulfhydryl groups and an ionized carboxyl group have enzymatic activity.
3. Acid (carboxylic) proteinases, optimum pH<5, содержат радикалы дикарбоновых кислот в активном центре.
4. Metalloproteinases, their catalytic action is due to the presence of Mg 2+, Mn 2+, Co 2+, Zn 2+, Fe 2+ in the active center. The strength of the bond between the metal and the protein part of the enzyme can be different. The metal ions included in the active center take part in the formation of enzyme-substrate complexes and facilitate the activation of substrates.
An important feature of proteinases is the selective nature of their action on peptide bonds in a protein molecule. As a result, an individual protein is always cleaved under the influence of a certain proteinase into a strictly limited number of peptides.
5. Peptide hydrolases that cleave amino acids from a peptide, starting from an amino acid with a free NH2 group, are called aminopeptidases, those with a free COOH group are called carboxypeptidases. Complete the hydrolysis of the protein dipeptidase, splitting the dipeptides into amino acids.
6. Amidases catalyze the hydrolytic cleavage of the bond between carbon and nitrogen: deamination of amines. This group of enzymes includes urease, which carries out the hydrolytic cleavage of urea. oxidative enzyme
7. Urease - a single-component enzyme (M = 480 thousand). A molecule is a globule and consists of eight equal subunits. It has absolute substrate specificity, it acts only on urea.
It should be noted that in order to detect free enzymes in the soil, it is necessary first of all to free it from living organisms, that is, to carry out complete or partial sterilization. An ideal factor that sterilizes the soil for the needs of enzymology should kill living cells without disturbing their cellular structure, and at the same time, not affect the enzymes themselves. It is difficult to say whether all currently used methods of sterilization meet these requirements. Most often, the soil for the needs of enzymology is sterilized by adding toluene as an antiseptic, by treating the soil with ethylene oxide, or, which is now increasingly practiced, by killing microorganisms of various kinds with ionizing radiation. The further technique for determining the catalytic properties of the soil does not differ from the methods for determining the activity of enzymes of plant or animal origin. A certain concentration of the substrate for the enzyme is added to the soil, and after incubation, the reaction products are studied. Analyzes of many soils carried out by this method have shown that they contain free enzymes with catalytic activity.
1.2 Enzymatic activity of soils
Enzymatic activity of soils [from lat. Fermentum - leaven] - the ability of the soil to exert a catalytic effect on the processes of transformation of exogenous and its own organic and mineral compounds due to the enzymes present in it. Characterizing the enzymatic activity of soils, they mean the total indicator of activity. The enzymatic activity of various soils is not the same and is associated with their genetic characteristics and a complex of interacting environmental factors. The level of soil enzymatic activity is determined by the activity of various enzymes (invertases, proteases, ureases, dehydrogenases, catalase, phosphatases), expressed by the amount of decomposed substrate per unit of time per 1 g of soil.
The biocatalytic activity of soils depends on the degree of their enrichment with microorganisms and on the type of soils. Enzyme activity varies across genetic horizons, which differ in humus content, types of reactions, redox potential, and other parameters along the profile.
In virgin forest soils, the intensity of enzymatic reactions is mainly determined by the horizons of the forest litter, and in arable soils, by arable layers. All biologically less active genetic horizons below the A or An horizons have low enzyme activity. Their activity slightly increases with soil cultivation. After the development of forest soils for arable land, the enzymatic activity of the formed arable horizon sharply decreases compared to the forest litter, but as it is cultivated, it increases and, in highly cultivated soils, approaches or exceeds the indicators of the forest litter.
Enzymatic activity reflects the state of soil fertility and internal changes that occur during agricultural use and an increase in the level of farming culture. These changes are found both when virgin and forest soils are brought into cultivation, and when they are used in various ways.
Throughout Belarus, up to 0.9 t/ha of humus is lost annually in arable soils. As a result of erosion, 0.57 t/ha of humus is irrevocably carried away from the fields every year. The reasons for soil dehumification are increased mineralization of soil organic matter, lagging behind the processes of new formation of humus from mineralization due to insufficient intake of organic fertilizers into the soil and a decrease in the enzymatic activity of the soil.
Biochemical transformations of soil organic matter occur as a result of microbiological activity under the influence of enzymes. enzymatic activity soil microorganism
Enzymes play a special role in the life of animals, plants and microorganisms. Soil enzymes are involved in the breakdown of plant, animal and microbial residues, as well as in the synthesis of humus. As a result, nutrients from compounds that are difficult to digest are converted into easily accessible forms for plants and microorganisms. Enzymes are distinguished by high activity, strict specificity of action and great dependence on various environmental conditions. Thanks to their catalytic function, they allow a huge number of chemical reactions to proceed quickly in the body or outside it.
Together with other criteria, the enzymatic activity of soils can serve as a reliable diagnostic indicator for determining the degree of soil cultivation. As a result of research 4, p. 91 established the relationship between the activity of microbiological and enzymatic processes and the implementation of measures that increase soil fertility. Soil cultivation, fertilization significantly change the ecological environment for the development of microorganisms.
Currently, several thousand individual enzymes have been found in biological objects, and several hundred of them have been isolated and studied. It is known that a living cell can contain up to 1000 different enzymes, each of which accelerates one or another chemical reaction.
Interest in the use of enzymes is also caused by the fact that the requirements for increasing the safety of technological processes are constantly increasing. Being present in all biological systems, being both products and tools of these systems, enzymes are synthesized and function under physiological conditions (pH, temperature, pressure, the presence of inorganic ions), after which they are easily excreted, undergoing destruction to amino acids. Both products and waste products of most processes involving enzymes are non-toxic and easily degradable. In addition, in many cases, the enzymes used in industry are obtained in an environmentally friendly way. Enzymes differ from non-biological catalysts not only in safety and increased biodegradability, but also in specificity of action, mild reaction conditions, and high efficiency. The efficiency and specificity of the action of enzymes makes it possible to obtain target products in high yield, which makes the use of enzymes in industry economically viable. The use of enzymes contributes to the reduction of water and energy consumption in technological processes, reduces CO2 emissions into the atmosphere, reduces the risk of environmental pollution by by-products of technological cycles.
With the use of advanced agricultural technology, it is possible to change in a favorable direction microbiological processes not only in the arable, but also in the sub-arable layers of the soil.
With the direct participation of extracellular enzymes, the decomposition of soil organic compounds occurs. So, proteolytic enzymes break down proteins into amino acids.
Urease breaks down urea into CO2 and NH3. The resulting ammonia and ammonium salts serve as a source of nitrogen nutrition for plants and microorganisms.
Invertase and amylase are involved in the breakdown of carbohydrates. Enzymes of the phosphate group decompose organophosphorus compounds in the soil and play an important role in the phosphate regime of the latter.
To characterize the general enzymatic activity of the soil, the most common enzymes characteristic of the vast majority of soil microflora are usually used - invertase, catalase, protease and others.
In the conditions of our republic, many studies have been carried out 16, p. 115 on the study of changes in the level of fertility and enzymatic activity of soils under anthropogenic impact, however, the data obtained do not provide an exhaustive answer to the nature of the changes due to the difficulty of comparing the results due to the difference in experimental conditions and research methods.
In this regard, the search for an optimal solution to the problem of improving the humus state of the soil and its enzymatic activity in specific soil and climatic conditions based on the development of resource-saving methods of basic soil cultivation and the use of soil-protective crop rotations that help preserve the structure, prevent soil overconsolidation and improve their quality and restore soil fertility at minimal cost, very relevant.
1.3 Methodological approaches to the determination of enzymaticsoil activity
1.3.1 Isolation experimentallythsiteand mapping
Trial site - a part of the study area, characterized by similar conditions (relief, homogeneity of the soil structure and vegetation cover, the nature of economic use).
The test site should be located in a typical location for the study area. On an area of 100 sq. m, one test site with a size of 25 m is laid. In case of heterogeneity of the relief, the sites are selected according to the elements of the relief.
They outline a preliminary plan for laying the main sections and half-sections in such a way that they characterize the soils of all occurring landforms and differences in soil cover.
The loop method is used in areas with complex terrain and a dense geographical network. With this method, the area under study is divided into separate elementary sectors, taking into account the features of changes in the relief or hydrographic network. The sector is surveyed from one center by performing loop-like routes in the radial direction.
Taking into account the features of the relief and the hydrographic network in one particular area, survey routes can be planned in a combined way, i.e. part of the site is examined by the method of parallel crossings of the territory, and part by the method of loops.
Along the routes, the points of laying the cuts are planned in such a way that all the main differences in the relief and vegetation are covered, i.e. the distances between the sections are not limited, therefore, in some, as a rule, difficult in relief places, the sections may be denser, while in other, relatively homogeneous, areas, the location of the sections may be rare.
Next comes the work associated with soil mapping and a detailed study of soils, starting with a reconnaissance survey of the site (quarter). During the reconnaissance survey, they get acquainted with the boundaries of the site and, in general, with the object of research, which is bypassed along clearings, sights, and roads. In the most characteristic places, cuts are laid, the location of which is applied to the plan. According to the results of reconnaissance surveys, the routes and places for laying soil cuts are finally corrected.
After the reconnaissance survey, they begin the survey itself, during which it is necessary to have a plan for laying soil sections and a clean copy of the outline of the taxation description. A general idea of soil varieties and initial markings of the boundaries of soil contours are obtained on the basis of a study of the main and control sections. Clarification of the boundaries of the distribution of the soil contour is carried out using digging. At the same time, in the field diary for each section, a form for describing the soil section is filled in. A field study of the distribution of soils is carried out after laying and linking sections to establish the classification of the given soil. According to the results of a field assessment of the soil cover and all other elements of the landscape, a separate, relatively homogeneous or monotonous-variegated area is distinguished as a soil contour.
The basis for identifying the boundaries between the contours of various soils is the identification of patterns between soils, relief and vegetation. Changes in soil formation factors lead to changes in soil cover. With a clear change in the relief, plant formations and parent rocks, the boundaries of soil differences coincide with the boundaries on the ground. In turn, the ease of fixing the boundaries on the map and the accuracy of identifying soil contours depend on the accuracy of the topographic base. However, in nature, most often one has to deal with unclear boundaries, a gradual transition. In this case, to establish the boundaries of soil contours, the laying of a large number of pits is required, as well as rich practical experience and good observation skills. When performing the actual survey in the field, on the basis of the plan copied from the taxation plan, an outline of the soils of the study area is drawn up.
It should be remembered that there are no strict boundaries between soil differences in nature, since the replacement of one soil difference by another occurs gradually through the accumulation of some features and the loss of others. Therefore, soil surveying only makes it possible to convey, to a greater or lesser extent, the schematic outlines of the distribution of soil contours, and the accuracy of identifying their boundaries depends on the survey scale, soil type, and other conditions. The minimum dimensions of soil contours that are subject to mandatory identification on a soil map are determined by technical standards.
1.3.2 Features of the selection and preparation of soil samples for analysis
In order to correctly determine the content of a particular substance in the soil, all agrochemical analyzes must be performed with impeccable accuracy and accuracy. However, even a very thorough analysis will give unreliable results if the soil is not sampled correctly.
Since the sample for analysis is taken very small, and the results of the determination should give an objective characteristic of large amounts of material, attention is paid to the elimination of heterogeneity when sampling the soil. Soil sample averaging is achieved by stepwise selection of initial, laboratory and analytical samples.
A mixed initial sample must be composed of individual samples (original samples) taken within the same soil difference. If the site has a complex soil cover, then a single average sample cannot be taken. There should be as many of them as there are soil differences.
Depending on the configuration of the site, the location of points for taking initial samples on it varies. On a narrow, elongated section, they can be placed along (in the middle) of it. On a wide, close to a square, area, a staggered arrangement of sampling sites is better. On large areas, soil sampling is used along the length of the plot in its middle, in an amount of up to 20 pcs.
Thoroughly mix the taken initial soil sample on a piece of tarpaulin, consistently average and reduce to the desired volume, then pour it into a clean bag or box. This is a laboratory sample, its mass is about 400 g.
A plywood or cardboard label, written in pencil, is placed on top of the box with a laboratory sample, indicating:
1. Names of the object.
2. Site names.
3. Plot numbers.
4. Depths of selection.
5. Sample numbers.
6. Surnames of the person who supervised the work or took the sample.
7. Dates of work.
The same entry is simultaneously made in the journal.
The soil sample delivered from the site in the laboratory is poured onto thick paper or a sheet of clean plywood and knead all the caked clods with your hands. Then foreign inclusions are selected with tweezers, the soil is well mixed, and it is slightly crushed. After such preparation of a laboratory sample, it is again scattered to bring it to an air-dry state, then crushed and passed through a sieve with 2 mm holes.
The room for drying the soil must be dry and protected from the access of ammonia, acid fumes and other gases.
To determine the enzymatic activity, soil dried in the open air is usually taken; Wet samples should be dried in the laboratory at room temperature. Care must be taken to ensure that the sample does not contain undecomposed plant residues. Soil clods are crushed and sifted through a sieve with a mesh size of 1 mm. When studying the enzymatic activity of a fresh (wet) sample, the complete removal of plant residues should be given even more attention. Simultaneously with the study of activity, soil moisture is also determined, the result obtained is recalculated for 1 g of absolutely dry soil.
1.4 Influence of various factorson the enzymatic activity of soils
An important factor on which the rate of the enzymatic reaction (and the catalytic activity of the enzyme) depends is the temperature, the effect of which is shown in Figure 1. It can be seen from the figure that with an increase in temperature to a certain value, the reaction rate increases. This can be explained by the fact that as the temperature rises, the movement of molecules accelerates and the molecules of the reacting substances have more opportunities to collide with each other. This increases the likelihood that a reaction between them will occur. The temperature that provides the highest rate of reaction is called optimal temperature.
Each enzyme has its own optimum temperature. In general, for enzymes of animal origin, it lies between 37 and 40C, and for plant - between 40 and 50C. However, there are exceptions: b-amylase from germinated grain has an optimal temperature at 60C, and catalase - within 0-10C. As the temperature rises above the optimum, the rate of the enzymatic reaction decreases, although the frequency of molecular collisions increases. This happens due to denaturation, i.e. loss of the native state of the enzyme. At temperatures above 80C, most enzymes completely lose their catalytic activity.
The decrease in the rate of an enzymatic reaction at temperatures above the optimum depends on the denaturation of the enzyme. Therefore, an important indicator characterizing the ratio of an enzyme to temperature is its thermolability, i.e. the rate of inactivation of the enzyme itself with increasing temperature.
Figure 1 - Effect of temperature on the rate of hydrolysis of starch by amylase
At low temperatures (0C and below), the catalytic activity of enzymes drops to almost zero, but denaturation does not occur. With an increase in temperature, their catalytic activity is restored again.
Also, the enzymatic activity of soils is affected by humidity, the content of microorganisms, and the ecological state of soils.
1.5 Changing communities of microorganisms in soils
Soil microorganisms are very numerous and diverse. Among them are bacteria, actinomycetes, microscopic fungi and algae, protozoa, and living beings close to these groups.
The biological cycle in the soil is carried out with the participation of different groups of microorganisms. Depending on the type of soil, the content of microorganisms varies. In garden, garden, arable soils, there are from one million to several billion microorganisms per 1 g of soil. The soil of each garden plot contains its own microorganisms. They participate with their biomass in the accumulation of soil organic matter. They play a huge role in the formation of available forms of mineral nutrition for plants. The importance of microorganisms in the accumulation of biologically active substances in the soil, such as auxins, gibberellins, vitamins, amino acids, which stimulate the growth and development of plants, is exceptionally great. Microorganisms that form mucus of a polysaccharide nature, as well as a large number of fungal filaments, take an active part in the formation of soil structure, gluing dusty soil particles into aggregates, thereby improving the water-air regime of the soil.
The biological activity of the soil, the number and activity of soil microorganisms are closely related to the content and composition of organic matter. At the same time, the most important processes of soil fertility formation, such as mineralization of plant residues, humification, the dynamics of mineral nutrition elements, the reaction of soil solution, the transformation of various pollutants in the soil, the degree of accumulation of pesticides in plants, the accumulation of toxic substances in the soil and the phenomenon of soil fatigue. The sanitary and hygienic role of microorganisms is also great in the transformation and neutralization of heavy metal compounds.
A promising direction for the restoration and maintenance of fertility and biological intensification of agriculture is the use of organic waste processing products with the participation of earthworm vermicomposts that are in symbiosis with microorganisms. In natural soils, the decomposition of litter is carried out by earthworms, coprophages and other organisms. But microorganisms are also involved in this process. In the intestines of worms, more favorable conditions are created for them to perform any functions than in the soil. Earthworms, in alliance with microorganisms, turn various organic wastes into highly effective biological fertilizers with a good structure, enriched with macro- and microelements, enzymes, active microflora, providing a prolonged (long-term, gradual) effect on plants.
So, by ensuring the development of microorganisms in the soil, the yield increases and its quality improves. After all, microorganisms develop, i.e. divide every 20-30 minutes and, in the presence of sufficient nutrition, form a large biomass. If a bull weighing 500 kg per day forms 0.5 kg - 1 kg, then 500 kg of microorganisms per day is biomass, and 500 kg of plants create 5 tons of biomass. Why is this not observed in the soil? And because for this, microorganisms need food, and on the other hand, various factors limit, in particular pesticides. On an area of 1 ha, as a result of the vital activity of soil microbes, 7500 m3 of carbon dioxide is released during the year. And carbon dioxide is necessary both as a source of carbon nutrition for plants and for dissolving hard-to-reach salts of phosphoric acid and converting phosphorus into a form available for plant nutrition. Those. where microorganisms work well, there is no need to apply phosphorus fertilizers. But the microorganisms themselves need organic matter.
In the balance of soil organic matter, the role of cultivated plants is great. The accumulation of humus in soils is facilitated by perennial grasses, especially legumes. After their harvesting, phytomass remains in the soil, which is enriched with nitrogen due to its fixation by nodule bacteria from the air. Row and vegetable crops (potatoes, cabbage, etc.) reduce the humus content in the soil, because leave a small amount of plant residues in the soil, and the applied system of deep tillage provides an intensive supply of oxygen to the arable layer and, as a result, provides a strong mineralization of organic matter, i.e. his loss.
When analyzing soils, the number of individual physiological groups of microorganisms is often taken into account. This is done by the so-called titer method, in which liquid selective (elective) nutrient media for certain groups of microorganisms are contaminated with different dilutions of a soil suspension. By establishing the degree of dilution after holding in a thermostat, which showed the presence of the desired group of microorganisms, it is then possible to determine the number of its representatives in the soil by simple recalculation. In this way, they find out how rich the soil is in nitrifiers, denitrifiers, cellulose-decomposing and other microorganisms.
To characterize the type of soil and its condition, not only the indicators of the number of different groups of microorganisms are important, but also the analysis of the state in the soil of their individual species. With rare exceptions, even the physiological groups of microorganisms are very broad. The external environment can drastically change the species composition of soil microorganisms, but has little or no effect on the number of their physiological groups. Therefore, when analyzing the soil, it is important to strive to establish the state of individual types of microorganisms.
Among soil microorganisms there are representatives of different systematic units that can assimilate not only easily digestible organic compounds, but also more complex substances of aromatic nature, which include such compounds characteristic of the soil as humus substances.
All soils on Earth were formed from very diverse rocks that come to the surface, which are usually called parent rocks. Loose sedimentary rocks mainly act as soil-forming rocks, since igneous and metamorphic rocks come to the surface relatively rarely.
The founder of scientific soil science, V. V. Dokuchaev, considered the soil as a special body of nature, as distinctive as a plant, animal or mineral. He pointed out that different soils form under different conditions and that they change over time. According to the definition of V. V. Dokuchaev, soil should be called “daytime”, or surface horizons of rocks, naturally altered by the influence of a number of factors. The type of soil is composed depending on: a) the parent rock, b) climate, c) vegetation, d) the relief of the country, and e) the age of the soil-forming process.
Developing the scientific foundations of soil science, V. V. Dokuchaev noted the enormous role of living organisms, and, in particular, microorganisms, in soil formation.
The period of creativity of V. V. Dokuchaev coincided with the time of the great discoveries of L. Pasteur, which showed the great importance of microorganisms in the transformation of various substances and in the infectious process. At the end of the last and at the beginning of this century, a number of important discoveries were made in the field of microbiology, which were of fundamental importance for soil science and agriculture. It was found, in particular, that the soil contains a huge number of different microorganisms. This gave reason to think about the essential role of the microbiological factor in the formation and life of the soil.
Simultaneously with V. V. Dokuchaev, another outstanding soil scientist P. A. Kostychev worked 24, p. 72. In the monograph “Soils of the Chernozem Region of Russia, Their Origin, Composition and Properties” (1886), he wrote that geology is of secondary importance in the question of chernozem, because the accumulation of organic matter occurs in the upper layers of the earth, geologically diverse, and chernozem is the question of the geography of higher plants and the question of the physiology of lower plants that decompose organic matter. PA Kostychev conducted a series of experiments to elucidate the role of individual groups of microorganisms in the creation of soil humus.
Academician V. I. Vernadsky, a student of V. V. Dokuchaev, made a great contribution to the concept of the role of the biological factor in the transformation of the Earth and in the process of soil formation. He believed that the main factor in the migration of chemical elements in the upper part of the earth's crust are organisms. Their activity affects not only organic, but also mineral substances of the soil and subsoil layers.
Already from the initial stages of the transformation of rocks into soil, the role of microorganisms in the processes of weathering of minerals emerges very clearly. The outstanding scientists V. I. Vernadsky and B. B. Polynov considered the weathering of rocks as a result of the activity of plant, mainly lower organisms. To date, this point of view has been confirmed by a large amount of experimental material.
Usually the first settlers of rocks are scale lichens, which form leaf-like plates, under which a small amount of fine earth accumulates. Lichens, as a rule, are in symbiosis with non-spore-forming saprophytic bacteria.
In relation to a number of elements, lichens act as their accumulators. In fine earth under lithophilic vegetation, the amount of organic matter, phosphorus, iron oxide, calcium, and magnesium sharply increases.
Among other plant organisms that settle on parent rocks, microscopic algae, in particular blue-green and diatoms, should be noted. They accelerate the weathering of aluminosilicates and also usually live in association with non-spore-forming bacteria.
Algae obviously play a significant role as autotrophic accumulators of organic substances, without which the vigorous activity of saprophytic microorganisms cannot proceed. The latter produce various compounds that cause the weathering of minerals. Many blue-green algae are nitrogen fixers and enrich the destructible rock with this element.
The main role in the weathering process is probably played by carbon dioxide, mineral and organic acids produced by various microorganisms. There are indications that certain keto acids have a strong dissolving effect. The possibility of participation in the weathering of humus compounds is not excluded.
It should be noted that many bacteria form mucus, which facilitates close contact of microorganisms with the rock. The destruction of the latter occurs both under the influence of the products of the vital activity of microorganisms, and as a result of the formation of complex compounds between the substance of the mucus and the chemical elements that make up the crystal lattices of minerals. The weathering of rocks in nature should be considered as a unity of two opposite processes - the decay of primary minerals and the emergence of secondary minerals. New minerals can arise when microbial metabolites interact with each other.
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Introduction...3
1. Literature review...5
1.1 The concept of the enzymatic activity of soils ... 5
1.2 Effect of heavy metals on enzymatic activity
1.3. The influence of agrochemical agents on the enzymatic activity of soils ... 23
2. Experimental part...32
2.1 Objects, methods and conditions for conducting research ... 32
2.2. Effect of agrochemical backgrounds on the enzymatic activity of soddy-podzolic soil contaminated with lead...34
2.2.1. Agrochemical characteristics of the soil when contaminated with lead and its content in the soil of the experiment ... 34
2.2.2. Influence of agrochemical backgrounds on the yield of spring grain crops in the heading phase on lead-contaminated soil...41
2.2.3. Influence of agrochemical backgrounds on the enzymatic activity of soil contaminated with lead...43
2.3. Effect of agrochemical backgrounds on the enzymatic activity of soddy-podzolic soil contaminated with cadmium...54
2.3.1. Agrochemical characteristics of the soil when contaminated with cadmium and its content in the soil of the experiment ... 54
2.3.2. Influence of agrochemical backgrounds on the yield of spring grain crops in the heading phase on soil contaminated with cadmium...60
2.3.3. Influence of agrochemical backgrounds on the enzymatic activity of soil contaminated with cadmium...62
2.4. Effect of agrochemical backgrounds on the enzymatic activity of soddy-podzolic soil contaminated with zinc...69
2.4.1. Agrochemical characteristics of the soil when contaminated with zinc and its content in the soil of the experiment ... 69
2.4.2. Influence of agrochemical backgrounds on the yield of spring grain crops in the heading phase on soil contaminated with zinc...75
2.4.3. Influence of agrochemical backgrounds on enzymatic activity
soil contaminated with zinc...76
2.5. Effect of agrochemical backgrounds on the enzymatic activity of soddy-podzolic soil contaminated with copper...82
2.5.1. Agrochemical characteristics of the soil when contaminated with copper and its content in the soil of the experiment ... 83
2.5.2. Influence of agrochemical backgrounds on the yield of spring grain crops in the heading phase on soil contaminated with copper...89
2.5.3. Influence of agrochemical backgrounds on enzymatic activity
soil contaminated with copper...90
Conclusion...96
Conclusions...99
References...101
Application
Introduction
Introduction.
The use of agrochemical agents in the agroecosystem is the most important condition for the development of modern agriculture. This is dictated by the need to maintain and improve the level of soil fertility, and, as a result, obtaining high and stable yields.
Agrochemical agents perform a number of ecological functions in the agrocenosis (Mineev, 2000). One of the most important functions of agricultural chemistry is to reduce the negative consequences of local and global technogenic pollution of agroecosystems with heavy metals (HM) and other toxic elements.
Agrochemical agents reduce the negative effect of HMs in several ways, including their inactivation in the soil and strengthening of the physiological barrier functions of plants that prevent HMs from entering them. If there is a lot of information in the literature on the issue of HM inactivation in the soil (Ilyin, 1982, etc., Obukhov, 1992, Alekseev, 1987, etc.), then there are isolated studies on the enhancement of the barrier functions of plants. Due to the enhancement of physiological barrier functions under the action of agrochemical agents, much less HM enters plants at the same content on different agrochemical backgrounds (Solov'eva, 2002). The strengthening of barrier functions is accompanied by the optimization of plant nutrition, and as a result, the improvement of the biological situation in the soil.
This ecological function, namely, the improvement of the biological activity and structure of the microbial cenosis of the soil contaminated with HMs under the action of agrochemical agents, does not yet have sufficient experimental substantiation.
It is known that some indicators of biological activity in the event of a stress situation in the soil change earlier than
other soil characteristics, for example, agrochemical ones (Zvyagintsev, 1989, Lebedeva, 1984). Soil enzymatic activity is one such indicator. Numerous studies have established the negative effect of heavy metals on the activity of enzymes. At the same time, it is known that agrochemical agents have a protective effect on the enzymatic activity of the soil. We tried to consider this problem as a whole and to determine whether the environmental protective properties of agrochemical agents are manifested in relation to the enzymatic activity of the soil when contaminated with biogenic and abiogenic metals. This side of agrochemical means can be detected only if the same amount of HMs is present in different variants of the experiment, and this is possible only with the same indicators of soil acidity. We have not been able to find such experimental data in the literature.
1. LITERATURE REVIEW
1.1. The concept of the enzymatic activity of soils.
All biological processes associated with the transformation of substances and energy in the soil are carried out with the help of enzymes that play an important role in the mobilization of plant nutrients, as well as determining the intensity and direction of the most important biochemical processes associated with the synthesis and decomposition of humus, hydrolysis of organic compounds. and the redox regime of the soil (, 1976; 1979 and others).
The formation and functioning of soil enzymatic activity is a complex and multifactorial process. According to the system-ecological concept, it is a unity of ecologically determined processes of entry, stabilization, and manifestation of enzyme activity in the soil (Khaziev, 1991). These three links are defined as blocks of production, immobilization and action of enzymes (Khaziev, 1962).
Enzymes in the soil are metabolic products of the soil biocenosis, but opinions about the contribution of various components to their accumulation are contradictory. A number of researchers (Kozlov, 1964, 1966, 1967; Krasilnikov, 1958; and others) believe that the main role in the enrichment of the soil with enzymes belongs to the root secretions of plants, others (Katsnelson, Ershov, 1958, etc.) - soil animals, the majority (Galstyan, 1963; Peive, 1961; Zvyagintsev, 1979; Kozlov, 1966; Drobnik, 1955; Hofmann and Seegerer, 1951; Seegerer, 1953; Hofmann and Hoffmann, 1955,1961; Kiss et al., 1958, 1964, 197 1; Sequi, 1974; and others) are of the opinion that the enzymatic pool in the soil consists of intracellular and extracellular enzymes, mainly of microbial origin.
Soil enzymes are involved in the breakdown of plant, animal and microbial residues, as well as in the synthesis of humus. As a result of enzymatic processes, nutrients from hard-to-digest
compounds are converted into easily accessible forms for plants and microorganisms. Enzymes are distinguished by exceptionally high activity, strict specificity of action, and great dependence on various environmental conditions. The latter feature is of great importance in the regulation of their activity in the soil (Khaziev, 1982 and
Enzymatic activity of soils according to (1979)
is made up of:
a) extracellular immobilized enzymes;
b) extracellular free enzymes;
c) intracellular enzymes of dead cells;
d) intracellular and extracellular enzymes formed under artificial conditions of the experiment and not characteristic of this soil.
It has been established that each enzyme acts only on a well-defined substance or a similar group of substances and a well-defined type of chemical bond. This is due to their strict specificity.
By their biochemical nature, all enzymes are high-molecular protein substances. The polypeptide chain of proteins - enzymes is located in space in an extremely complex way, unique for each enzyme. With a certain spatial arrangement of the functional groups of amino acids in molecules6).
Enzymatic catalysis begins with the formation of an active intermediate, the enzyme-substrate complex. The complex is the result of the attachment of a substrate molecule to the catalytically active site of the enzyme. In this case, the spatial configurations of the substrate molecules are somewhat modified. new oriented
the placement of reacting molecules on the enzyme ensures the high efficiency of enzymatic reactions that reduce the activation energy (Khaziev, 1962).
For the catalytic activity of the enzyme, not only the active center of the enzyme is responsible, but also the entire structure of the molecule as a whole. The rate of an enzymatic reaction is regulated by many factors: temperature, pH, enzyme and substrate concentrations, and the presence of activators and inhibitors. Organic compounds can act as activators, but more often various microelements (Kuprevich, Shcherbakova, 1966).
The soil is able to regulate the enzymatic processes occurring in it in connection with changes in internal and external factors through factor or allosteric regulation (Galstyan 1974, 1975). Under the influence of chemical compounds introduced into the soil, including fertilizers, allosteric regulation occurs. Factor regulation is due to the acidity of the environment (pH), chemical and physical composition, temperature, humidity, water-air regime, etc. The influence of soil specifics, humus content and biomass and other factors on the activity of enzymes used to characterize the biological activity of soils is ambiguous (Galstyan 1974; Kiss 1971; Dalai 1975; McBride 1989; Tiler 1978).
The enzymatic activity of the soil can be used as a diagnostic indicator of the fertility of various soils, because the activity of enzymes reflects not only the biological properties of the soil, but also their changes under the influence of agroecological factors (Galstyan, 1967; Chunderova, 1976; Chugunova, 1990, etc.).
The main pathways for enzymes to enter the soil are extracellular enzymes of microorganisms and plant roots secreted during their lifetime and intracellular enzymes that enter the soil after the death of soil organisms and plants.
The release of enzymes into the soil by microorganisms and plant roots usually has an adaptive character in the form of a response to the presence or absence of a substrate for the action of the enzyme or a reaction product, which is especially pronounced with phosphatases. With a lack of mobile phosphorus in the medium, microorganisms and plants sharply increase the release of enzymes. It is on this relationship that the use of soil phosphatase activity as a diagnostic indicator of the supply of plants with available phosphorus is based (Naumova, 1954, Kotelev, 1964).
Enzymes, getting from various sources into the soil, are not destroyed, but remain in an active state. It must be assumed that enzymes, being the most active component of the soil, are concentrated where the vital activity of microorganisms is most intense, that is, on the interface between soil colloids and soil solution. It has been experimentally proven that enzymes in the soil are mainly in the solid phase (Zvyagintsev, 1979).
Numerous experiments carried out under conditions of inhibition of enzyme synthesis in microbial cells using toluene (Drobnik, 1961; Beck and Poshenrieder, 1963), antibiotics (Kuprevich, 1961; Kiss, 1971), or irradiation (McLaren et al., 1957) indicate that that the soil contains a large amount of "accumulated enzymes", sufficient to carry out the transformation of the substrate for some time. Among these enzymes, invertase, urease, phosphatase, amylase, etc. can be named. Other enzymes are much more active in the absence of an antiseptic, which means that they accumulate slightly in the soil (a - and P-galactosidase, dextranase, levanase, malatesterase, etc.). The third group of enzymes does not accumulate in the soil, their activity is manifested only during the outbreak of microbial activity and is induced by the substrate. Received to date
experimental data indicate a difference in the enzymatic activity of soils of different types (Konovalova, 1975; Zvyagintsev, 1976; Khaziev, 1976; Galstyan, 1974, 1977, 1978; and others).
The most well-studied soil enzymes are hydrolases, which represent an extensive class of enzymes that carry out hydrolysis reactions of various complex organic compounds, acting on various bonds: ester, glucosidic, amide, peptide, etc. Hydrolases are widely distributed in soils and play an important role in their enrichment. mobile and sufficient nutrients for plants and microorganisms, destroying high-molecular organic compounds. This class includes the enzymes urease (amidase), invertase (carbohydrase), phosphatase (phosphohydrolase), etc., whose activity is the most important indicator of soil biological activity (Zvyagintsev, 1980).
Urease is an enzyme involved in the regulation of nitrogen metabolism in the soil. This enzyme catalyzes the hydrolysis of urea to ammonia and carbon dioxide, causing hydrolytic cleavage of the bond between nitrogen and carbon in organic molecules.
Of the enzymes of nitrogen metabolism, urease has been studied better than others. It is found in all soils. Its activity correlates with the activity of all major enzymes of nitrogen metabolism (Galstyan, 1980).
In the soil, urease is found in two main forms: intracellular and extracellular. The presence of free urease in the soil allowed Briggs and Segal (Briggs et al., 1963) to isolate the enzyme in crystalline form.
Part of the extracellular urease is adsorbed by soil colloids that have a high affinity for urease. Communication with soil colloids protects the enzyme from decomposition by microorganisms and promotes its accumulation in the soil. Each soil has its own stable level of urease activity, determined by the ability of soil colloids,
mainly organic, show protective properties (Zvyagintsev, 1989).
In the soil profile, the humus horizon shows the highest activity of the enzyme; further distribution along the profile depends on the genetic characteristics of the soil.
Due to the widespread use of urea as a nitrogen fertilizer, the issues associated with its transformations under the action of urease are practically significant. The high urease activity of most soils prevents the use of urea as a universal source of nitrogen nutrition, since the high rate of urea hydrolysis by soil urease leads to local accumulation of ammonium ions, an increase in the reaction of the medium to alkaline values, and, as a result, the loss of nitrogen from the soil in the form of ammonia ( Tarafdar J. C, 1997). By breaking down urea, urease prevents its isomerization into phototoxic ammonium cyanate. Although urea itself is partially used by plants, however, as a result of the active action of urease, it cannot be stored in the soil for a long time. In the studies of a number of scientists, the volatilization of urea nitrogen in the form of ammonia from the soil at high urease activity was noted, and when various urease inhibitors were introduced into the soil, the hydrolysis of urea slowed down and the losses were less (Tool P. O., Morgan M. A., 1994). The rate of urea hydrolysis in soil is affected by temperature (Ivanov and Baranova, 1972; Galstyan, 1974; Cortez et al., 1972, etc.), soil acidity (Galstyan, 1974; Moiseeva, 1974, etc.). Soil saturation with carbonates has a negative effect (Galstyan, 1974), the presence of arsenic, zinc, mercury, sulfate ions, copper and boron compounds in significant amounts, from organic compounds aliphatic amines, dehydrophenols and quinones significantly inhibit urease (Paulson, 1970, Briggsatel ., 1951).
Invertase activity is one of the most stable indicators, showing the clearest correlations with influencing factors. Studies (1966, 1974) established a correlation between invertase and the activity of other soil carbohydrases.
Invertase activity has been studied in many soils and discussed in several review papers (Aleksandrova and Shmurova, 1975; Kuprevich and Shcherbakova, 1971; Kiss et al., 1971, etc.). The invertase activity in the soil decreases along the profile and correlates with the humus content (Pukhitskaya and Kovrigo, 1974; Galstyan, 1974; Kalatozova, 1975; Kulakovskaya and Stefankina, 1975; Simonyan, 1976; Toth, 1987; etc.). Correlation with humus may be absent with a significant content of aluminum, iron, and sodium in the soil. The close relationship of invertase activity with the number of soil microorganisms and their metabolic activity (Mashtakov et al., 1954; Katsnelson and Ershov, 1958; Kozlov, 1964; Chunderova, 1970; Kiss, 1958; Hofinann, 1955, etc.) indicates the advantage in the soil invertases of microbial origin. However, this dependence is not always confirmed (Nizova, 1970); invertase activity is a much more stable indicator and may not be directly related to fluctuations in the number of microorganisms (Ross, 1976).
According to a report (1974), soils with a heavy granulometric composition have a higher enzymatic activity. However, there are reports that invertase is markedly inactivated upon adsorption by clay minerals (Hofmann et al., 1961; Skujins, 1976; Rawald, 1970) and soils with a high content of montmorillonite have low invertase activity. The dependence of invertase activity on soil moisture and temperature has not been sufficiently studied, although many authors attribute seasonal changes in activity to hydrothermal conditions.
The influence of temperature on the potential activity of invertase was studied in detail (1975), establishing the optimum at a temperature of about 60°C, the enzyme inactivation threshold after heating the soil at 70°C, and complete inactivation after three hours of heating at 180°C.
Many authors have considered the invertase activity of soils depending on growing plants (Samtsevich and Borisova, 1972; Galstyan, 1974; Ross 1976; Cortez et al., 1972, etc.). The development of the meadow process, the formation of a thick sod under the grassy cover contributes to an increase in invertase activity (Galstyan, 1959). However, there are works in which the effect of plants on invertase activity has not been established (Konovalova, 1975).
In soils, there is a large amount of phosphorus in the form of organic compounds, which comes with the dying remains of plants, animals and microorganisms. Phosphoric acid is released from these compounds by a relatively narrow group of microorganisms that have specific phosphatase enzymes (Chimitdorzhieva et al., 2001).
Among the enzymes of phosphorus metabolism, the activity of orthophosphoric monophosphoesterases has been most fully studied (Aleksandrova, Shmurova, 1974; Skujins J. J., 1976; Kotelev et al., 1964). Phosphatase producers are mainly cells of soil microorganisms (Krasilnikov and Kotelev, 1957, 1959; Kotelev et al., 1964).
Phosphatase activity of the soil is determined by its genetic characteristics, physicochemical properties and the level of agricultural culture. Among the physical and chemical properties of the soil, acidity is especially important for phosphatase activity. Soddy-podzolic and gray forest soils, which have an acidic reaction, mainly contain acidic phosphatases; in soils with a slightly alkaline reaction, alkaline phosphatases predominate. It should be noted that the optimum activity of acidic
phosphatase is located in the weakly acidic zone, even when the soils are strongly acidic (Khaziev, 1979; Shcherbakov et al., 1983, 1988). This fact confirms the importance of liming acidic soils for accelerating the hydrolysis of complex organic phosphates and enriching the soil with available phosphorus.
The observed characteristic distribution of phosphatases in soils depending on their acidity is due to the composition of the microflora. Microbial communities that are adapted to certain environmental conditions function in the soil, which secrete enzymes that are active under these conditions.
The total phosphatase activity of the soil depends on the content of humus and organic phosphorus, which is a substrate for the enzyme.
Chernozems are characterized by the highest phosphatase activity. In soddy-podzolic and gray forest soils, phosphatase activity is low. The low activity of these acidic soils is due to the stronger adsorption of phosphatases by soil minerals. Due to the low content of organic matter in such soils, the adsorbing surface of minerals is more exposed than in high-humus chernozems, where clay minerals are covered with humified organic matter.
Phosphatase activity is dynamic during the growing season. In the active phases of plant growth at high soil temperatures and sufficient moisture in the summer months, the phosphatase activity of soils is maximum (Evdokimova, 1989).
In some soils, a correlation was noted between phosphatase activity and the total number of microorganisms (Kotelev et al., 1964; Aliev and Gadzhiev, 1978, 1979; Arutyunyan, 1975, 1977; and others) and the number of microorganisms that mineralize organic phosphorus compounds (Ponomareva et al. , 1972), in others - the relationship of phosphatase activity with the number
microorganisms have not been established (Ramirez-Martinez, 1989). The influence of humus is manifested in the nature of the change in the activity of the enzyme along the profile, when comparing soils with different degrees of humus content and carrying out soil cultivation measures (Aleksandrova and Shmurova, 1975; Arutyunyan, 1977). The studies of many authors indicate a direct dependence of the phosphatase activity of soils on the content of organic phosphorus in the soil (Gavrilova et al., 1973; Arutyunyan, Galstyan, 1975; Arutyunyan, 1977; and others).
Let us consider in more detail the general regularities of the formation of the phosphatase pool of soils.
A significant part of the total phosphorus in the soil is organophosphorus compounds: nucleic acids, nucleotides, phytin, lecithin, etc. Most of the organophosphates found in the soil are not directly absorbed by plants. Their absorption is preceded by enzymatic hydrolysis by phosphohydrolases. The substrates of soil phosphatases are specific humic substances, including phosphorus of humic acids, as well as non-specific individual compounds represented by nucleic acids, phospholipids and phosphoproteins, as well as metabolic phosphates. The former accumulate in the soil as a result of the biogenesis of humic substances, the latter, as a rule, enter the soil with plant residues and accumulate in it as products of intermediate metabolic reactions.
The role of higher plants in the formation of the phosphatase pool of soils used in agriculture is lower than that of microorganisms and is mainly associated with the entry of crop residues and root excretions into the soil, which is confirmed by the data of and (1994), who studied the effect of various agricultural crops on hydrolytic activity
and redox enzymes; phosphatases, invertases, proteases, ureases, catalase on thin peat soil. The activity of phosphatase was approximately the same under all crops: barley, potatoes and black fallow, and only slightly more under perennial grasses, while the activity of other enzymes varied significantly depending on the nature of soil use.
, (1972) noted an increase in phosphatase activity in the rhizosphere of wheat and legumes, which may be associated with both an increase in the number of microorganisms in the rhizosphere and extracellular phosphatase activity of roots. From an agrochemical point of view, the final result is important - the growth of the enzymatic pool of soils with an increase in the power of plant root systems.
The depletion of agrocenoses in plants leads to a decrease in the rhizosphere effect and, as a result, to a decrease in the activity of soil phosphatase. A significant decrease in the phosphatase activity of soils during the cultivation of monoculture was noted. The inclusion of soils in crop rotation creates conditions for improving hydrolytic processes, which leads to an increase in the metabolism of phosphorus compounds. (Evdokimova, 1992)
(1994) studied soddy-podzolic soils formed under natural (forest) vegetation of different composition and determined the distribution of phosphatase activity in the soil profile, the ratio between labile and stable forms of enzymes, and their spatial and temporal variability. It has been established that in soils formed under natural forest vegetation, genetic horizons differ in phosphatase activity, the distribution of which in the profile closely correlates with the humus content. According to the data, the highest phosphatase activity was observed in the litter layer, then it decreased several times in the humus-accumulative layer and dropped sharply in the soil layer.
below 20 cm in the soil under the spruce forest (forest vegetation). Under the meadow vegetation, there is a slightly different distribution: the maximum activity in the sod horizon is 1.5–2 times lower in the humus-accumulative horizon, and a further significant decrease is observed only after 40–60 cm. Based on the above, we can conclude that the maximum contribution to the formation The phosphatase pool under natural vegetation is supplied by microorganisms and plant residues as a substrate, while root secretions and postmortem intracellular enzymes play a somewhat smaller role.
The intensity of biochemical processes in the soil and the level of its fertility depends both on the conditions for the existence of living organisms that supply enzymes to the soil, and on the factors that contribute to the fixation of enzymes in the soil and regulate their actual activity.
1.2. Influence of heavy metals and trace elements on the enzymatic activity of soils.
One of the promising directions for using enzymatic activity for diagnosing the biological properties of soils is to identify the level of soil contamination with HMs.
Heavy metals, entering the soil in the form of various chemical compounds, can accumulate in it to high levels, which pose a significant danger to the normal functioning of soil biota. A large amount of data has been accumulated in the literature indicating the negative impact of soil pollution with HMs on soil biota. When the chemical balance in the soil is disturbed, a stressful situation arises. There is evidence that biological indicators react earlier than agrochemical ones to changing conditions that affect various soil properties (Lebedeva,
Bibliography
According to the type of catalyzed reactions, all known enzymes are divided into six classes:
1. Oxidoreductases catalyzing redox reactions.
2. Hydrolases catalyzing reactions of hydrolytic cleavage of intramolecular bonds in various compounds.
3. Transferases that catalyze the reactions of intermolecular or intramolecular transfer of a chemical group and residues with the simultaneous transfer of energy contained in chemical bonds.
4. Ligases (synthetases) that catalyze the reactions of the connection of two molecules, coupled with the splitting of the firophosphate bonds of ATP or another similar triphosphate.
5. Lyases catalyzing the reactions of non-hydrolytic cleavage or addition of various chemical groups of organic compounds to double bonds.
6, Isomerases catalyzing the reactions of transformation of organic compounds into their isomers.
Oxidoreductases and hydrolases, which are very important in soil biodynamics, are widely distributed in the soil and studied in some detail.
Catalase
(H 2 O 2: H 2 O 2 -oxidoreductase)
Catalase catalyzes the decomposition of hydrogen peroxide with the formation of water and molecular oxygen:
H 2 O 2 + H 2 O 2 O 2 + H 2 O.
Hydrogen peroxide is formed during the respiration of living organisms and as a result of various biochemical reactions of the oxidation of organic substances. The toxicity of hydrogen peroxide is determined by its high reactivity, which is exhibited by singlet oxygen, *O 2 . Its high reactivity leads to uncontrolled oxidation reactions. The role of catalase is that it destroys hydrogen peroxide, poisonous to organisms.
Catalase is widely distributed in the cells of living organisms, including microorganisms and plants. Soils also exhibit high catalase activity.
Methods for determining soil catalase activity are based on measuring the rate of decomposition of hydrogen peroxide during its interaction with soil by the volume of released oxygen (gasometric methods) or by the amount of undecomposed peroxide, which is determined by permanganometric titration or colorimetric method with the formation of colored complexes.
E.V. Dadenko and K.Sh. Kazeev, it was found that catalase activity of all enzymes decreases to the greatest extent during storage of samples, so its determination must be carried out in the first week after sampling.
Method A.Sh. Galstyan
Analysis progress. To determine the activity of catalase, a device is used from two burettes connected by a rubber hose, which are filled with water and balance its level. Maintaining a certain level of water in the burettes indicates the achievement of temperature equilibrium in the device. A sample (1 g) of soil is introduced into one of the compartments of a double flask. 5 ml of a 3% hydrogen peroxide solution are poured into the other compartment of the flask. The flask is tightly closed with a rubber stopper with a glass tube, which is connected to the measuring burette with a rubber hose.
The experiment is carried out at a temperature of 20 ° C, since at a different temperature the reaction rate will be different, which will distort the results. In principle, it is not the temperature of the air that is important, but the temperature of the peroxide, it should be 20 0 C. If the air temperature is much higher than 20 0 C (in summer), it is recommended to carry out the analysis in the basement or in another cool room. Recommended in such cases, the use of a water bath with a temperature of 20 ° C is hardly effective.
The beginning of the experiment is noted by a stopwatch or hourglass at the moment when the peroxide is mixed with the soil, and the contents of the vessel are shaken. The mixture is shaken during the whole experiment, trying not to touch the flask with your hands, holding it by the stopper. The released oxygen displaces water from the burette, the level of which is noted after 1 and 2 minutes. The recommendation to determine the amount of oxygen every minute for 3 minutes due to the straightforwardness of the peroxide decomposition reaction only increases the time spent on analysis.
This technique allows one researcher to analyze the catalase activity of more than 100 samples per day. It is convenient to carry out the analysis together, using 5-6 vessels. At the same time, one person is directly involved in the analysis and monitors the level of the burette, and the second monitors the time, records data and washes the vessels.
Dry heat sterilized (180°C) soil served as control. Some soils, compounds and minerals have a high activity of inorganic peroxide decomposition catalysis even after sterilization - up to 30-50% of the total activity.
Catalase activity is expressed in milliliters of O 2 released in 1 min from 1 g of soil.
Reagents: 3% H 2 O 2 solution. The concentration of perhydrol must be periodically checked, the working solution is prepared immediately before analysis. To determine the concentration of perhydrol on an analytical balance, 1 g of H 2 O 2 is weighed in a volumetric flask with a capacity of 100 ml, the volume is adjusted to the mark and shaken. Place 20 ml of the resulting solution in 250 ml conical flasks (3 repetitions), add 50 ml of distilled water and 2 ml of 20% H 2 SO 4 . Then titrated with 0.1 N. KMnO 4 solution. 1 ml of KMnO 4 solution corresponds to 0.0017008 g of H 2 O 2 . After establishing the concentration of perhydrol, a 3% solution is prepared by diluting with distilled water. The KMnO 4 titration solution is prepared from fixanal and kept for several days to establish the titer.
Dehydrogenases
(substrate: NAD(P)-oxidoreductase).
Dehydrogenases catalyze redox reactions by dehydrogenating organic substances. They go according to the following scheme:
AN 2 + V A + VN 2
In the soil, the substrate for dehydrogenation can be non-specific organic compounds (carbohydrates, amino acids, alcohols, fats, phenols, etc.) and specific (humic substances). Dehydrogenases in redox reactions function as hydrogen carriers and are divided into two groups: 1) aerobic, transferring mobilized hydrogen to air oxygen; 2) anaerobic, which transfer hydrogen to other acceptors, enzymes.
The main method for detecting the action of dehydrogenases is the reduction of indicators with a low redox potential, such as methylene blue.
To determine the activity of soil dehydrogenases, colorless tetrazolium salts (2,3,5-triphenyltetrazolium chloride - TTX) are used as hydrogen, which are reduced to red formazan compounds (triphenylformazan - TFF).
Analysis progress. A weighed portion (1 g) of the prepared soil is carefully placed through a funnel on the bottom of a test tube with a capacity of 12-20 ml and mixed thoroughly. Add 1 ml of 0.1 M dehydrogenation substrate solution (glucose) and 1 ml of freshly prepared 1% TTX solution. The test tubes are placed in an anaerostat or a vacuum desiccator. The determination is carried out under anaerobic conditions, for which the air is evacuated at a rarefaction of 10-12 mm Hg. Art. for 2-3 minutes and put in a thermostat for 24 hours at 30 °C. When incubating soil with substrates, toluene is not added as an antiseptic, since; it strongly inhibits the action of dehydrogenases. Sterilized soil (at 180°C for 3 h) and substrates without soil served as controls. After incubation, 10 ml of ethyl alcohol or acetone are added to the flasks, shaken for 5 minutes. The resulting colored TPP solution is filtered and colorimetric. With very intense coloring, the solution is diluted with alcohol (acetone) 2-3 times. Use 10 mm cuvettes and a light filter with a wavelength of 500-600 nm. The amount of formazan in mg is calculated from the standard curve (0.1 mg in 1 ml). The activity of dehydrogenases is expressed in mg TTP per 10 g of soil for 24 hours. The error of determination is up to 8%.
Reagents:
1) 1% solution of 2,3,5-triphenyltetrazolium chloride;
2) 0.1 M glucose solution (18 g of glucose is dissolved in 1000 ml of distilled water);
3) ethyl alcohol or acetone;
4) triphenylformazan for the standard scale. To compile a calibration curve, prepare a series of solutions in ethyl alcohol, acetone or toluene with a concentration of formazan (from 0.01 to 0.1 mg formazan in 1 ml) and photocolorimetric as described above.
In the absence of formazan, it is obtained by reducing TTX with sodium hydrosulfite (ammonium sulfite, zinc powder in the presence of glucose). The initial concentration of the TTX solution is 1 mg/ml. Crystalline sodium hydrosulfite is added to 2 ml of the initial TTX solution at the tip of the lancet. The precipitated formazan is taken up in 10 ml of toluene. This volume of toluene contains 2 mg of formazan (0.2 mg/ml). Further dilution prepares working solutions for the scale.
Invertase
(β-fructofuranosidase, sucrase)
Invertase is a carbohydrase, it acts on the β-fructofuranosidase bond in sucrose, raffinose, gentianose, etc. This enzyme most actively hydrolyzes sucrose with the formation of reducing sugars - glucose and fructose:
invertase
C 12 H 22 O 11 + H 2 O C 6 H 12 O 6 + C 6 H 12 O 6
sucrose glucose fructose
Invertase is widely distributed in nature and occurs in almost all types of soils. Very high invertase activity was found in mountain meadow soils. Invertase activity clearly correlates with humus content and soil fertility. It is recommended when studying the effect of fertilizers to evaluate their effectiveness. Methods for determining the activity of soil invertase are based on the quantitative accounting of reducing sugars according to Bertrand and on changes in the optical properties of a sucrose solution before and after exposure to the enzyme. The first method can be used to study an enzyme with a very wide amplitude of activity and substrate concentration. Polarimetric and photocolorimetric methods are more demanding on the concentration of sugars and are unacceptable for soils with a high content of organic matter, where colored solutions are obtained; therefore, these methods are of limited use in soil studies.
1A study of the enzymatic activity of the soil in the agrosystems of the Upper Volga region, formed in long-term stationary experiments on soddy-podzolic and gray forest soils, was carried out in order to assess their ecological state. In the soddy-podzolic soil of forest ecosystems, the average level of invertase activity is 21.1 mg glucose/1 g soil, and in the soil of agrosystems it is 8.6 mg glucose/1 g soil. Agricultural use reduced the activity of invertase by an average of 2.5 times. A particularly strong depression of invertase is seen on zero backgrounds, where agrotechnical measures are taken annually to grow crops, without the introduction of fertilizer materials. The average activity of urease in the soil of agroecosystems was 0.10 mg N-NH4/1g of soil, it was slightly higher in the soil of forest ecosystems - 0.13 mg N-NH4/1g, which is primarily due to the genetic characteristics of soddy-podzolic soils and their level of fertility. On the gray forest soil, the studied level of agrogenic load did not have a negative impact on the activity of soil enzymes, but, on the contrary, there is a tendency to increase their activity on arable land, which is accompanied by the mobilization of the overall activity of biological processes in the soil compared to the fallow. The intensity of the impact on the soil cover by various technological methods was manifested in a decrease in the indicators of enzymatic activity only in soddy-podzolic soils. In ecological terms, these results can be considered as a sign of the response of the soil cover to external anthropogenic pressures.
agricultural landscapes
invertase
catatase
sod-podzolic
gray forest soils
enzymatic activity
1. Witter A.F. Soil cultivation as a factor in the regulation of soil fertility /A.F. Witter, V.I. Turusov, V.M. Garmashov, S.A. Gavrilov. - M.: Infra-M, 2014. - 174 p.
2. Dzhanaev Z.G. Agrochemistry and biology of soils in the south of Russia / Z.G. Dzhanaev. - M.: Publishing House of Moscow State University, 2008. - 528 p.
3. Zvyagintsev D.G. Soil biology / D.G. Zvyagintsev, N.A. Babieva. - M., 2005. - 520 p.
4. Zinchenko M.K. Enzymatic potential of agrolandscapes of gray forest soil of the Vladimir opol'e / M.K. Zinchenko, S.I. Zinchenko // Successes of modern natural science. - 2015. - No. 1. - S. 1319-1323.
5. Zinchenko M.K. The reaction of soil microflora of gray forest soil to the long-term use of fertilizer systems of different levels of intensification / M.K. Zinchenko, L.G. Stoyanova // Achievements of science and technology of the agro-industrial complex. - 2016. - No. 2. - T. 30. - P. 21-24.
6. Emtsev V.T. Microbiology: a textbook for universities / V.T. Yemtsev. – M.: Bustard, 2005. – 445 p.
7. Enkina O.V. Microbiological aspects of conservation of fertility of Kuban chernozems / O.V. Enkina, N.F. Korobsky. - Krasnodar, 1999. - 140 p.
8. Methods of soil microbiology and biochemistry; [ed. D.G. Zvyagintsev]. - M.: Publishing House of Moscow State University, 1991. - 292 p.
9. Khaziev F.Kh. Enzymatic activity of soils of agrocenoses and prospects for its study / F.Kh. Khaziev, A.E. Gulko // Soil Science. - 1991. - No. 8. - S. 88-103.
10. Khaziev F.Kh. Methods of soil enzymology / F.Kh. Khaziev. – M.: Nauka, 2005. – 254 p.
The agroecological functions of soils are expressed by certain quantitative and qualitative parametric characteristics, the most important of which are biological indicators. The processes of decomposition of plant residues, the synthesis and mineralization of humus, the transformation of hard-to-reach forms of nutrients into forms digestible for plants, the course of ammonification, nitrification and fixation of free nitrogen in the air are due to the activity of soil microorganisms.
The processes of metabolism and energy during the decomposition and synthesis of organic compounds, the transition of hard-to-digest nutrients into forms that are easily accessible to plants and microorganisms, occur with the participation of enzymes. Therefore, soil enzymatic activity is the most important diagnostic indicator of the impact of anthropogenic load on soil systems. This is especially true for agroecosystems with annual agrotechnical impact on the soil. Determining the activity of soil enzymes is very important for determining the degree of influence of agrotechnical measures and agrochemical agents on the activity of biological processes, in order to judge the rate of mobilization of the main organogenic elements.
Research goal was to assess the ecological state of soils in the agrosystems of the Upper Volga region in terms of enzymatic activity. The objects of study were soddy-podzolic soils of varying degrees of podzolization and gray forest soils on adjacent virgin and cultivated landscapes.
Materials and methods of research
Since objective data on soil fertility and its biological activity can be obtained in long-term stationary experiments, soil samples were taken for the study in variants of long-term stationary experiments located on the basis of the Kostroma Research Institute of Agriculture, the Ivanovo Agricultural Academy, and the Vladimir Research Institute of Agriculture. As a result, enzyme activity was analyzed in soddy podzolic light loamy soil (Experiment 1, Kostroma), soddy medium podzolic light loamy soil (Experiment 2, Ivanovo); sulfur forest medium loamy soil (Experiment 3, Suzdal).
In order to be able to identify the degree of influence of various kinds of anthropogenic load on the soils of agroecosystems, we studied the reference soil samples of undisturbed ecosystems adjacent to the experimental plots. The virgin variants of soddy-podzolic soils were areas under a pine forest with an admixture of hardwoods. Gray forest soils of a long-term fallow are formed under broad-leaved forests with abundant forbs in the ground cover.
The gray forest soils of the Vladimir opolye are characterized by an average accumulation of organic matter. The content of humus in horizon A1 (A p) is 1.9 - 4%; the humus horizon is thin (17-37 cm). The value of acidity, characteristic for these soils, is less than for soddy-podzolic soils, weakly acidic soils predominate (рН=5.2-6.0). Therefore, the gray forest soils of the Vladimir region are characterized by more favorable agrochemical indicators compared to soddy-podzolic soils. A stationary field experiment on gray forest soil was established in 1997 to study the effectiveness of adaptive landscape farming systems (ALAS). On the studied variants, for the rotation of a 6-field crop rotation, the following is introduced: on a zero background - manure 40t/ha (at a time); on average - N 240 R 150 K 150; high-intensity mineral - N 510 R 480 K 480; high-intensity organomineral - manure 80t / ha (at a time) + N 495 R 300 K 300.
The content of humus in the soil of the experimental plot of the Ivanovo Agricultural Academy is 1.92%; pH xl - 4.6-6.4; P 2 O 5 - 170-180 mg / kg of soil, K 2 O - 110-170 mg / kg of soil. The thickness of the arable layer is 21-23 cm. The experiment was started in 1987. Soil samples were taken in a four-field crop rotation against a normal background (N 30 P 60 K 60) according to two methods of tillage - moldboard plowing to a depth of 20-22 cm (S) and moldboardless flat-cutting to 20-22 cm (PO).
The fertility of the soddy-podzolic soil of the long-term stationary experiment of the Kostroma Research Institute of Agriculture during the sampling period was characterized by the following average indicators: humus content 1.39-1.54%; pH xl - 4.6-6.4; P 2 O 5 - 105-126 mg/kg; K 2 O - 104-156 mg / kg. A long-term stationary field experiment to study the effect of lime on soil properties and crop yields was established in 1978. The studies were carried out in the rotation of a seven-field crop rotation. For this work, soil samples were selected in the variants N 45 P 45 K 45 - zero background; - normal and Ca 2.5 (N 135 R 135 K 135) - intense. The ameliorant in the experiment was dolomite flour, introduced once at the laying of the experiment at a dose of 25 t/ha in physical weight for the Ca 2.5 (NPK) 3 variant. In the variant Ca 0.5 + Ca 0.5 (NPK) 1, the ameliorant was used fractionally, for the first time - at the start of the experiment at a dose of 5 t/ha, 0.5 hydrolytic acidity; again - at the end of the fourth rotation in 2007 in autumn, under plowing, at a dose of 3.2 t/ha, 0.5 of the requirement for hydrolytic acidity.
In soil samples, the following was determined: catalase activity by the gasometric method according to Galstyan, invertase activity by the method of I.N. Romeiko, S.M. Malinovskaya and urease activity by the method of T.A. Shcherbakova. The activity of these soil enzymes is directly related to the conversion of carbon, nitrogen and redox processes, and therefore characterizes the functional state of soil microorganisms. A comprehensive determination of these parameters makes it possible to more accurately determine the direction of changes in the activity of the enzymatic pool of soil varieties.
Biochemical studies of enzyme activity were carried out in the period from 2011-2013. in the soil layer of 0-20 cm, since the main biological activity and the highest biogenicity are inherent in the upper layers of the soil profile, maximally enriched in organic matter, with the most favorable hydrothermal and air regime for microflora.
Research results and discussion
The most important link in the carbon cycle in nature is the stage of enzymatic conversion of carbohydrates in the soil environment. It ensures the movement of organic material entering the soil in large quantities and the energy accumulated in it, as well as its accumulation in the soil in the form of humus, since pre-humus components are formed in this case.
Plant residues entering the soil are 60% carbohydrates. Mono-, di- and polysaccharides (celluloses, hemicelluloses, starch, etc.) were found in the soil. It is obvious that agroecological impacts leading to changes in the physicochemical and biological state of soils affect the activity of carbohydrate metabolism enzymes. Data on soil invertase activity are presented in Table 1.
Table 1
Invertase activity in soils of agroecosystems
|
Sampling location |
Agroecosystems |
Invertase activity, mg glucose/1g soil in 40 hours |
|
|
Sod-podzolic light loamy soil |
Kostroma |
Forest (control) |
|
|
Zero background N 45 R 45 K 45 |
|||
|
Normal Ca 0.5 + Ca 0.5 (N 45 R 45 K 45) |
|||
|
Intensive Ca 2.5 (N 135 R 135 K 135) |
|||
|
Sod-medium podzolic light loamy |
Forest (control) |
||
|
Normal N 30 R 60 K 60 |
|||
|
Normal N 30 R 60 K 60 |
|||
|
Deposit (control) |
|||
|
*Zero background |
|||
|
N 30-60 R 30 - 60 K 30-60 |
|||
|
high intensity mineral N 120 R 120 K 120 |
|||
|
N 120 R 120 K 120; Manure 80t/ha + N 90 |
Note: In the table, the doses of fertilizers on gray forest soil are given during the study period.
It was revealed that in the soddy-podzolic soil of forest ecosystems, the average level of invertase activity is 21.1 mg glucose/1 g of soil, and in the soil of agrosystems - 8.6 mg glucose/1 g of soil. That is, the agricultural use of arable land had a significant impact on the activity of invertase, reducing it by an average of 2.5 times.
A particularly strong depression of invertase is seen on zero backgrounds, where agrotechnical measures are taken annually to grow crops, without the introduction of fertilizer materials. This may be due to a slight influx of mortmass in the form of root crop residues, as well as a change in physicochemical properties as a result of tillage.
The agrotechnical use of gray forest soil does not significantly reduce the activity of carbohydrate metabolism compared to the fallow soil. In areas of long-term fallows, the average invertase activity is 50.0 mg of glucose per 1 g of soil for 40 hours, which is 9% higher than the average on arable land. The variation of the enzyme values in the gray forest soil of agrosystems over 2 years of research (2012-2013) was V = 7.6%, with an average XS = 45.8 mg glucose / 1 g of soil for 40 hours. The influence of fertilizer systems on the activity of invertase is most pronounced against the average background. In this variant, the enzyme activity indicators were significantly higher (НСР 05 = 2.9) than in other intensification backgrounds. Therefore, when using medium doses of fertilizers, favorable conditions are created for the transformation of organic compounds of the aromatic series into humus components. This is confirmed by the data on the content of organic carbon, since the maximum reserves of humus have been accumulated against an average background of 3.62%.
One of the informative indicators of the enzymatic activity of the soil is the activity of urease. Many researchers consider urease activity as an indicator of the self-cleaning ability of soil contaminated with organic xenobiotics. The action of urease is associated with the hydrolytic cleavage of the bond between nitrogen and carbon (CO-NH) in the molecules of nitrogen-containing organic compounds. In agroecosystems, the rapid increase in urease activity also indicates the ability to accumulate ammonia nitrogen in the soil. Therefore, many researchers note a positive correlation between urease activity and nitrogen and humus content in soils.
The fact that the described soddy podzolic soils are poorly supplied with the initial organic substrate is evidenced by the low activity of this enzyme (Table 2). In our studies, the average activity of urease in the soil of agroecosystems was 0.10 mg N-NH 4 /1g of soil, it is slightly higher in the soil of forest ecosystems - 0.13 mg N-NH 4 /1g, which is primarily due to genetic features soddy-podzolic soils and their level of fertility.
table 2
Urease activity in soils of agrosystems
|
Sampling location |
Agroecosystems |
Urease activity, mg N-NH 4 /1g of soil in 4 hours |
|
|
Sod-podzolic light loamy soil |
Kostroma |
Forest (control) |
|
|
Zero background N 45 R 45 K 45 |
|||
|
Normal Ca 0.5 + Ca 0.5 (N 45 R 45 K 45) |
|||
|
Intensive Ca 2.5 (N 135 R 135 K 135) |
|||
|
Sod-medium podzolic light loamy |
Forest (control) |
||
|
Normal N 30 R 60 K 60 |
|||
|
Gray forest medium loamy soil |
Deposit (control) |
||
|
Zero background |
|||
|
N 30-60 R 30 - 60 K 30-60 |
|||
|
high intensity mineral N 120 R 120 K 120 |
|||
|
High intensity organomineral N 120 R 120 K 120; Manure 80t/ha + N 90 |
At the level of natural biotopes, urease activity is preserved in experiment 1, where agrotechnical measures, in addition to the use of mineral fertilizers, included liming the soil. Against the background of a decrease in the content of hydrogen and aluminum ions in the soil - absorbing complex, stabilization of the enzyme activity is observed.
Cultivation of soddy-podzolic soils without the systematic introduction of lime materials, even with the use of medium doses of mineral fertilizers, by moldboard plowing and flat-cut loosening (Experiment 2) leads to a decrease in urease activity compared to their natural counterparts.
Studies on gray forest soils show that, on average, the level of urease activity in these soils is 2.5 times higher than in soddy-podzolic soils, which is due to the genesis of soil formation and the level of their fertility. This is evidenced by the data of both the natural biotopes of the deposit and the soils of agroecosystems. Many researchers have found that urease activity is directly proportional to the amount of organic carbon in the soil.
At the level of natural biotopes, the indicator of urease activity was noted against a highly intense organomineral background - 0.34 mg N-NH 4 /1g soil (Experiment 3). Against a highly intense organomineral background, the level of urease activity was increased relative to other agroecosystems and fallows. This is due, first of all, to the fact that during the period of research on this option, 80 t/ha of manure was introduced, which enriched the soil with fresh organic matter, urea and stimulated the development of the urobacteria complex. As in the soil of a long-term fallow, with long-term use of organomineral fertilizers, organic matter is formed with the widest ratio of carbon to nitrogen (C:N). This type of organic matter corresponds to the highest urease activity. The observed trend indicates the ability of the soil of these ecosystems to intensive accumulation of ammonia nitrogen. Significantly lower (at HSR 05 = 0.04) enzyme activity in the soil of other agroecosystems. The lowest indicator (0.21 mgN-NH 4 /1g) was noted against a high-intensity mineral background, where only high doses of mineral fertilizers were applied for 18 years. It can be assumed that when using only mineral fertilizers, due to the lack of a specific energy substrate, the ecological and trophic group of bacteria producing urease decreases in the soil microbial pool.
Considering the enzymatic activity of soils, attention should be paid to the oxidation of the products of hydrolysis of organic compounds with the formation of pre-humic substances. These reactions take place with the participation of oxidoreductases, an important representative of which is catalase. Catalase activity characterizes the processes of biogenesis of humic substances. The values of indicators of catalase activity in soddy-podzolic soils demonstrate spatial and temporal variability, but in general they exhibit fluctuations in the range of 0.9-2.8 ml O 2 /1 g of soil per minute. (Table 3). In the agroecosystems of soddy-podzolic soils formed in the Ivanovo and Kostroma regions, the indicators of catalase activity are at the level of their natural counterparts (forest soils). That is, the degree of anthropogenic load did not have a significant impact on the processes of biogenesis of humic substances. They proceed with the same intensity both in the soils of these agrosystems and in the soil of natural biotopes. This is a positive trend, since the formation of agroecosystems on soddy-podzolic soils with a light granulometric composition, without the use of organic fertilizers, can cause an increase in catalase activity. The increase in enzyme activity characterizes the intensive transformation of humic substances in the soil towards their mineralization, in order to provide cultivated crops with nutrients. The activation of these processes can lead to a decrease in the humus content in the soil and a decrease in the potential fertility of the soil.
Table 3
Catalase activity in soils of agricultural systems
|
Sampling location |
Agroecosystems |
Catalase activity, ml O 2 / 1 g of soil per minute |
|
|
Sod-podzolic light loamy soil |
Kostroma |
Forest (control) |
|
|
Zero background N 45 R 45 K 45 |
|||
|
Normal Ca 0.5 + Ca 0.5 (N 45 R 45 K 45) |
|||
|
Intensive Ca 2.5 (N 135 R 135 K 135) |
|||
|
Sod-medium podzolic light loamy |
Forest (control) |
||
|
Normal N 30 R 60 K 60 (OV) |
|||
|
Normal N 30 R 60 K 60 |
|||
|
Gray forest medium loamy soil |
Deposit (control) |
||
|
Zero background |
|||
|
N 30-60 R 30 - 60 K 30-60 |
|||
|
high intensity mineral N 120 R 120 K 120 |
|||
|
High intensity organomineral N 120 R 120 K 120; Manure 80t/ha + N 90 |
The coefficient of variation in the values of catalase activity in the gray forest soil of agroecosystems is V = 18.6%. In general, fluctuations are found in the range of 1.8-2.9 ml O 2 /1 g of soil. At the current level of anthropogenic load on arable land, there is a tendency to activate redox processes in comparison with the fallow soil. The highest activity of these processes is observed with the use of medium doses of fertilizers, which is characterized by a significant increase in catalase activity (at HSR 05 = 0.4) against an average background of intensification. This is due to the sufficient enrichment of the soil with organic matter and the improvement of its transformation regime due to an increase in the number and mobilization activity of the microbial pool of arable land.
To assess the degree of agrogenic influence on the activity of various enzymes and to determine the total enzymatic activity of each agroecosystem in comparable units, we used the method of O.V. Enkina. It is possible to more accurately judge the level of enzymatic activity of individual agricultural backgrounds by interpreting extensive experimental material if we compare their activity with the control (in our case, with the soil of natural ecosystems), taking their enzymatic activity indicators as 100%. That is, the degree of influence of anthropogenic load on various groups of enzymes is reflected by the ratio of their activity indicators in agrosystems to natural analogues (Table 4).
As a result of the research, it was found that in most soddy-podzolic soils of the region, the level of enzymatic activity of agrolandscapes is lower than in their natural counterparts. The enzymatic potential of the soddy-podzolic soil in experiment 1 (Kostroma) decreased by 31% compared to the control, and in experiment 3 (Ivanovo) - by 24%. On the studied backgrounds in these experiments, medium and high doses of mineral fertilizers were applied. Long-term use of mineral fertilizers, especially nitrogen fertilizers, often disrupts the ecological background for the reproduction of beneficial microorganisms on soils with low potential fertility. This, as a rule, occurs due to the acidification of the soil solution, the presence of aluminum and iron ions in the soil-absorbing complex, the root secretions of plants, which cause active reproduction of microscopic fungi, contributing to an increase in the biological toxicity of the soil. In this case, negative changes are accompanied not only by a restructuring of the microbiocenosis structure, but also by a decrease in the enzymatic activity of the soil, and the loss of potential and effective fertility.
Table 4
The level of enzymatic activity of soils of agrosystems (in % of the soil of natural ecosystems)
|
Sampling location |
Agroecosystems |
Catalase |
Invertase |
Average enzyme activity |
|
|
Kostroma |
Forest (control) |
||||
|
Zero background |
|||||
|
Normal |
|||||
|
Intensive |
|||||
|
Average by experience,% |
|||||
|
Forest (control) |
|||||
|
Normal |
|||||
|
Normal |
|||||
|
Average by experience,% |
|||||
|
High Intensity Mineral |
|||||
|
High intensity organomineral |
|||||
|
Average by experience,% |
Thus, the main indicators of the enzymatic activity of soddy-podzolic soils, related to their effective fertility, are higher in natural ecosystems than in arable soils.
With an increased level of soil fertility, the influence of agrogenic factors on the enzymatic potential of the soil is somewhat smoothed out. This is what we observe in the agrosystems of gray forest soil. It was found that during the 3 years of research, the highest enzymatic potential was formed against an average background of 108%. Average doses of mineral and organic fertilizers (40 t/ha of manure once every 6 years) led to an increase in catalase and invertase activity of the soil, which characterizes the activation of the synthesis of humic substances.
Conclusion
It has been established that the studied level of agrogenic load on gray forest soil did not have a negative impact on the activity of soil enzymes, but, on the contrary, there is a tendency to increase their activity on arable land, which is accompanied by an intensification of the overall activity of biological processes in the soil compared to the fallow. The intensity of the impact on the soil cover by various technological methods was manifested in a decrease in the indicators of the enzymatic activity of soddy-podzolic soils. In ecological terms, these results can be considered as a sign of the response of the soil cover to external anthropogenic pressures.
In order to rationally use and protect soil fertility, indicators of enzymatic activity should be used in biomonitoring and biodiagnostics of soils. This is especially important when carrying out production tasks in agriculture.
Bibliographic link
Zinchenko M.K., Zinchenko S.I., Borin A.A., Kamneva O.P. ENZYMATIVE ACTIVITY OF AGRARIAN SOILS OF THE UPPER VOLGA REGION // Modern problems of science and education. - 2017. - No. 3.;URL: http://science-education.ru/ru/article/view?id=26458 (date of access: 01.02.2020). We bring to your attention the journals published by the publishing house "Academy of Natural History"
