Alternative technology of safflower cultivation by sewage sludge use as a fertilizer-ameliorant

. The effect of sewage sludge as a fertilizer-ameliorant in the safflower cultivation was studied. Sewage sludge from Volzhskij wastewater treatment plants prepurified by enzyme-cavitation method was used as a fertilizer-ameliorant in the safflower cultivation. It was found that prepurified sewage sludge is environmentally friendly and has a high fertilizing value. The amino acid composition of safflower seeds was studied. The obtained data indicate that sewage sludge introduction made it possible to significantly increase the safflower nutritional value and the soil microbiological activity. It was found that the greatest increase in the safflower crop yields was observed in the case of sewage sludge introduction at a dose of 10 t/ha with chisel tillage. It can be stated that the technology of safflower cultivation proposed by us, including the application of sewage sludge as a fertilizer-ameliorant in combination with the chisel tillage, significantly affects its productivity. The increase in safflower crop yields over the years of the studies was 8.4-15.3%. In general, it can be concluded that sewage sludge has a complex effect on the soil: improves its water-physical, agrochemical and physico-chemical proper-ties, increases the activity of soil biota and the content of organic matter – one of the main indicators of soil fertility.


Introduction
In the conditions of arid agriculture all over the world, as well as a sharply changing climate, it becomes necessary to grow stress-resistant crops. Recently, along with traditional oilseeds, safflower has become widespread. Ethiopia and Afghanistan are considered the homeland of safflower (Carthamus tinctorius L.). Its valuable consumer and useful properties were well known in Ancient Egypt as far back as the 16th century BC. Safflower used to dye fabrics and its flowers -for cult application. Currently safflower has a very wide application. It is used as a coloring, oilseed, technical, medicinal, fodder, melliferous and ornamental crop [1,2], and also due to its phytomeliorative properties it can be used for the remediation of soil fertility [3,4].
The application of fertilizers to agronomical soil is a worldwide practice used for improving crop yield [5]. Modern strategies of agriculture development led to the significant reduction in the use of chemical and mineral fertilizers, mainly because of their high cost and negative impact on the environment. It becomes necessary to switch to organic fertilizers. Due to the constant production of organic waste material, attention has been paid to the reuse of this waste in agriculture as organic fertilizers [6]. The introduction of environmentally friendly technologies with the use of organic fertilizers on the base of waste for their further application to saving of soil fertility and improve product quality is a very actually.
Sewage sludge is a one of such organic waste material, which is formed during wastewater treatment and problems with its disposal are growing in all over the world. Since sludge is enriched with organic matter, macro and microelements, as well as a complex of microorganisms. There are several ways to dispose of sewage sludges [7]. The recycling of waste sludges to agricultural land is a cost-effective disposal route [8,9]. The composition and physico-chemical properties of sewage sludge may differ depending on the method of processing and wastewater treatment. The sewage sludge may also contain heavy metals, harmful microorganisms or organic pollutants [10 -12]. Therefore, before sewage sludge using in agriculture, it is necessary to carry out complex studies.
There are numerous publications on the use of sewage sludges in agriculture, their properties and features are discussed in works such as [8, 10, 11, 13 -28].
This paper presents the results of complex investigations of various methods of basic tillage and the possibility of sewage sludge application aimed at saving of soil fertility and crop yields increasing. It is proposed to use prepurified sewage sludge after its biological treatment as an organic fertilizer-ameliorant.

Materials and Methods
Sewage sludge from Volzhskij wastewater treatment plants prepurified by enzyme-cavitation method was used for studies. The enzyme-cavitation method was developed by Closed Joint Stock Company "Ekotor" [29].
The enzyme-cavitation method provides for the generation of low-intensity cavitation in the circulating substrate (with a cavitation number of no more than 0.05), as well as intensive enrichment of the substrate with oxygen. As a result, the substrate and active sewage sludge are in a microbubble and enriched environment. This ensures the decom-position and fragmentation of organic matter, the destruction of pathogenic microflora, as well as the release of mineral nutrients.

Amino acid composition of safflower seeds
Mass fraction of amino acids in safflower seeds was determined by the system of capillary electrophoresis "KAPEL-105 M", which has a special cassette for the analysis of amino acids according to GOST R 55569-2013. The method for determining the amino acid composition is based on the decomposition of samples by acid or alkaline (only for tryptophan) hydrolysis with the conversion of amino acids into free forms, the preparation of their phenylthiocarbonyl derivatives, their further separation and quantitative de-termination by capillary electrophoresis. Detection was carried out in the UV region of the spectrum at λ= 254 nm. For the direct quantitative determination of tryptophan without obtaining the phenylthiocarbonyl derivative, the absorption was recorded at λ = 219 nm.
Amino acid score. Amino acid score (AS) was determined from the equation: where C1 -the amount of essential amino acid contained in 1 g of the investigated protein, mg; C2 -the amount of the same essential amino acid contained in 1 g of the reference protein, mg.

The physico-chemical parameters of sewage sludge after biological treatment by enzyme-cavitation method
The pH of the samples was determined using a salt extract from sewage sludge and pH-meter "pH-410" according to GOST R 27979-88.
The moisture content of the sewage sludge was determined by the thermostat-weight method according to GOST R 26713-85.
The content of total and ammonia nitrogen was determined by the Kjeldahl procedure using the "Keltran" complex according to GOST R 26715-85, 26716-85.
The content of total phosphorus was determined spectrophotometrically at λ = 710 nm by spectrophotometer "ПЭ-5400" according to GOST R 26717-85.
The content of total potassium was determined by flame spectrometry using an automatic flame photometer "FPA-2-01" according to GOST R 26718-85.
The content of organic matter was determined by thermogravimetric method according to GOST R 27980-88.
The content of heavy metal cations (Cu, Zn, Cd, Pb, Ni, Hg, As) in the sewage sludge was determined by atomic absorption spectrometry using an atomic absorption spectrometer Thermo ICE 3000 according to Federal Environmental Regula-tions16.2.2:2.3.71-2011.
The residual amount of pesticides in the sewage sludge was determined by gas chromatography using a gas chromatograph "Kristallux 4000 M" according to GOST R 53217-2008.
The microbiological activity of soil. The microbiological activity of the soil was determined using linen canvas by the "application" method [30]. Uniform pits were made on the plots, a white linen canvas (size -20x5 cm) was pressed to the flat vertical side of each of them and covered on the other side, compacting it to its original state. The exposure is determined by the intensity of the decomposition of the canvas, which depends on the fertility of the soil, weather conditions, fertilizers, the nature of the vegetation and other conditions. The canvases are removed from the soil when it becomes apparent that continued exposure will lead to tissue loss during digging. Then weighed canvases, which were previously washed and dried.

Field experiment
The experiments were carried out in 2016-2018 on the base of the Federal State Budget Scientific Institution "Federal Scientific Centre of Agroecology, Complex Melioration and Protective Afforestation of the Russian Academy of Sciences". All experiments were carried out in accordance with the methodology of B.A. Dospekhov [31].
Counting of the crop yields was carried out by plots using the method of direct combining (combine harvester "Sampo"-500). Sewage sludge was introduced onto the surface of the field in the form of a mulch layer in various dosages (0, 5, 10 t/ha) according to the proposed technology of safflower cultivation [32]. The doses of sewage sludge introduction were determined in accordance with the calculated formula of the permissible introduction doses of sludge as fertilizer for agricultural crops (GOST R 17.4.3.07 -2001). The basic tillage of the soil was carried out in the autumn, the tillage variants are given in the experimental scheme. The scheme of the experiment is shown in Figure 1.

Results and discussion
This The microbiological activity of the soil is the most important indicator of the soil fertility level. Introduction of fertilizers is a significant factor that determines the microbiological activity of the soil. Fertilizers have a positive effect on the development of micro-organisms in the soil and on their biological activity. The microbiological activity of the soil is of great importance for the nutrition of the plants growing on this soil. The activity of microorganisms has a great effect on the intensity of protein depolymerization and the formation of free amino acids. [33]. Most of the free amino acids are released into the soil solution after the depolymerization of proteins and peptides of soil organic matter. In addition, cells of microbes and fungi, animal tissues, excretion of microorganisms and plants are also sources of free amino acids. [34]. Amino acids are necessary for the normal passage of plant metabolism and are involved in the regulation of all processes occurring in the plant cell [35,36]. The amino acid composition of safflower seeds grown on light-chestnut soil without of fertilizers introduction was assessed (Table 1). Proteins extracted from the seeds of oil crops such as safflower are practically storage proteins that also determine the crop quality. It was found that the protein of safflower seed contains 8 essential amino acids (lysine, phenylalanine, histidine, leucine, isoleucine, methionine, valine, tryptophan), which take part in protein synthesis and help the body to perform all its functions correctly. However, the presence of essential amino acids does not completely determine the nutritional value of protein. The amount and ratio of all amino acids that make up a complete protein should be optimal. The nutritional value of protein (Table  2) is characterized by such parameter as the amino acid score, which was determined from the equation (1). The amino acid score was determined using a reference protein, the amino acid composition of which was proposed by a joint FAO/WHO/UNU expert consultation [37].  The obtained data ( Table 2) indicate that the content of essential amino acids in safflower seeds is lower than the content of essential amino acids of the reference protein. Tryptophan is an exception. It is one of the most important essential amino acids, the value of the amino acid score of which is determined primarily in the development of protein-enriched products.
It was found that the safflower protein is best combined with appropriate additives to increase its nutritional value. Introduction of fertilizers, as is known [38], strongly affects the vital activity of microorganisms, as well as the concentration of free amino acids in soils. The introduction of corresponding fertilizer-ameliorant will lead to an increase in the content of amino acids in plants, as well as the microbiological activity of the soil. The intake of amino acids from the outside allows the plant to accelerate metabolic processes and not spend additional energy on its own synthesis.
Nowadays in the context of limited possibilities for traditional regulation of production processes in agrobiocenoses caused by the high cost of mineral fertilizers, irrigation water and etc., an increase in the activity of soil biota and the potential crop yields of agricultural crops is possible through the use of excess technogenic resources, namely, household wastewater. Therefore, the main task of the paper was to study the possibility of sewage sludge application aimed at saving of soil fertility and crop yields increasing.

Study of the main parameters of sewage sludge
The sewage sludge application in agriculture is widespread throughout the world. However, sewage sludge treatment technologies and the possibility of its reuse are poorly understood in the Russian Federation and their use is only 10 %. Therefore, investigation of the possibility of sewage sludge application in agriculture is actually task.
The sewage sludge samples from Volzhskij wastewater treatment plants prepurified by enzyme-cavitation method was used for studies. An enzyme-cavitation method for processing of sewage sludge was developed and implemented in Volgograd [29]. The sewage sludge obtained as a result of such purification has a number of fundamental differences in comparison with the sewage sludge obtained as a result of classical purification by the anaerobic digestion method. The sewage sludge prepurified by enzyme-cavitation method has sorption and ion-exchange properties. Sewage sludge contains active silt, where microorganisms accumulate The introduction of such sewage sludge into the soil leads to the accumulation of air and moisture from the atmosphere into the upper most fertile soil layer. The physico-chemical properties of sewage sludge depend on the processing regimes at the treatment plant and on the storage periods on silt maps. Mass fraction of moisture should not exceed 70% according to GOST R 54651-2011. Sewage sludge is a liquid mass with a moisture content of 70% or more. This indicates that sewage sludge with such moisture content is not suitable for use as an organic fertilizer. The physico-chemical parameters of sewage sludge after biological treatment by enzyme-cavitation method were studied and presented in Table 2. The main indicator of soil fertility is organic matter. An increased content of organic matter (32%) in the studied sewage sludge is observed. The presence of total forms of nitrogen (3.3%), phosphorus (4.27%), potassium (0.31%), as well as ammonia nitrogen (0.19%), which are easily available to plant roots and soil biota, is observed in the sewage sludge samples. The results indicate that prepurified sewage sludge reaches the optimum moisture content (11 %) and fully complies with the technical requirements of GOST R 54651-2011. All this testifies to the high fertilizing value of sewage sludge.
The application of sewage sludge as fertilizer is accompanied by the introduction of not only useful compounds into the soil, but also heavy metal ions and pesticides. This leads to soil pollution and degradation. In this regard, it is very important to establish control over the content of heavy metals and pesticides in sewage sludge. A study of con-tent of heavy metals and pesticides in sewage sludge after biological treatment was carried out. It was shown that in the studied samples the maximum permissible concentrations of heavy metals, established in the technical requirements for sewage sludge, are not exceeded. The residual amount of hexachlorocyclohexane (HCH) isomers, 4, 4´-dichlorodiphenyltrichloroethane (DDT) and its metabolites was not found. The corresponding data are presented in Table 4. The obtained data indicate that sewage sludge after biological treatment by enzymecavitation method fully complies with the technical requirements for fertilizers based on sewage sludge, is environmentally friendly and suitable for application as a fertilizerameliorant in the cultivation of crops.

Study of microbiological activity of soil in safflower crops during the introduction of sewage sludge
The microbiological activity of the soil is used to evaluate the activity of the soil biota. The activity of soil microflora depends on the introduction and presence of organic com-pound in the soil. Sewage sludge after biological treatment by enzyme-cavitation method is the source of such organic compound in our studies.
Studies of microbiological activity were carried out during the active phases of safflower growth. It was revealed that the method of basic tillage and different doses of sew-age sludge introduction influenced the microbiological processes in the soil in different ways (Table 5). The studies have shown that the use of disk tillage by HDH-3, as well as the classical moldboard tillage by PN-4-35 plow led to a decrease in the activity of microorganisms in comparison with chisel tillage by Rancho. Classic moldboard tillage leads to the formation of a "plow sole", which in turn prevents the development of the plant root system and leads to the loss of organic matter. However, the use of deep chisel tillage by Rancho additionally promotes the activation of microorganisms. A significant part of plant residues during deep plowing is embedded in the depths of the soil, where their decomposition occurs.
It was found that the highest microbiological activity of the soil is observed in the phase of 5-6 leaves for all experiment variants ( Table 5). The data in table 5 indicate that the lowest indicators of microbiological activity are observed in the experiment variant without the introduction of sewage sludge. The introduction of sewage sludge as a fertilizer at a dose of 5 t/ha led to an increase in microbiological activity for all studied basic tillage up to 357 μg of amino acid/g of canvas. The introduction of sewage sludge at a maximum dose (10 t/ha) was more preferable and contributed to an even greater increase in microbiological activity up to 372 μg of amino acid/g of canvas. The maximum value of the microbiological activity of the soil was observed in a case of using of deep chisel tillage by Rancho with introduction of sewage sludge at a dose of 10 t/ha. Microbiological activity decreased in all experiment variants to the phase of full ripeness, which is directly related to the drying out of the soil. However, the tendency of a positive effect from the introduction of alternative fertilizer in various doses in combination with deep chisel tillage was remained.

The efficiency of technological operations used in the safflower cultivation
The efficiency of technological operations used in the safflower cultivation is deter-mined by its crop yields ( Table 6). The safflower crop yields depended on a number of factors such as weather conditions for the period of our studies, the method of basic till-age, as well as the introduction of sewage sludge in different doses. The highest crop yields of safflower were observed in 2016 for all experiment variants, which is fundamentally related to weather conditions. The amount of precipitation for the growing season in 2016 was 331.6 mm (Hydro-thermal Coefficient of Selyaninov (HTC) = 1,22), which significantly exceeded the average climatic norms for the Lower Volga region. HTC in 2017 was 0,94 (the amount of precipitation for the growing season -243.0 mm). There was a decrease in the safflower yields in 2017 compared to 2016 for all experiment variants. The most unfavourable year was 2018, where the HTC was 0.48 (the amount of precipitation for the growing season -142.1 mm), in this regard, the lowest crop yields of the cultivated crop was observed. The data in Table 6 show that, on average for 2016-2018, the safflower crop yields in a case of using of disk tillage by HDH-3 was lower than in a case of using of moldboard tillage by PN-4-35 plow and significantly lower for chisel tillage by Rancho at corresponding doses of sewage sludge introduction. It was shown that obtaining a higher saf-flower crop yields are possible by sewage sludge introduction in different doses. In the case of fertilizerameliorant introduction at a dose of 5 t/ha the average crop yields of safflower over the years of studies was from 1.23 to 1.42 t/ha depending on the method of the basic tillage. The highest crop yields (1.51 t/ha) were obtained by introduction of sewage sludge at a dose of 10 t/ha in combination with deep chisel tillage. The average increase in safflower crop yields over the years of the studies was 8.4-15.3% for chisel tillage by Rancho with sewage sludge introduction in different doses. It can be stated that the technology of safflower cultivation proposed by us, including the application of sewage sludge as a fertilizer-ameliorant in combination with the chisel tillage, significantly affects its productivity.
Thus, the introduction of non-traditional fertilizer based on sewage sludge radically influenced the increase of the safflower crop yields. This is due to the fact that the prepurified sewage sludge has a complex effect on the soil: improves its water-physical, agro-chemical and physico-chemical properties, increases the activity of soil biota and the con-tent of organic matter -one of the main indicators of soil fertility.

Conclusions
Thus, the effect of methods of basic tillage and doses of sewage sludge introduction on increasing the level of amino acids in plants and crop yields, as well as main parameters of sewage sludge after biological treatment by enzyme-cavitation method were studied. It was found that sewage sludge after biological treatment by enzyme-cavitation method fully complies with the technical requirements for fertilizers based on sewage sludge, is environmentally friendly and suitable for application as a fertilizer-ameliorant in the cultivation of crops. Systematic studies of the possibility of sewage sludge application aimed at saving of soil fertility and crop yields increasing showed that to increase the level of amino acids in plants and microbiological activity of the soil, as well as the safflower crop yields, it is recommended to apply sewage sludge as a fertilizer-ameliorant at a dose of 10 t/ ha in combination with deep chisel tillage by Rancho.