FORMULATIONS OF BIOSTIMULANTS, A PERSONAL EXPERIENCE.

INTRODUCTION

Generally in agriculture we are exposed to facing stress situations, and on this, we point out that any environmental change that affects the optimum growth rate and preventing the plant from reaching its full productive potential. Stress can be caused by biotic factors, such as phytoparasites, animals, insects or pathogens (bacterial or fungal). It can also be caused by abiotic factors of the environment, such as salinity, deficiencies and toxicity due to mineral nutrients, high or low temperatures and water deficits or excesses. These authors confirm our theory that, in addition to altering the growth pattern, one of the main symptoms of plants under stress conditions is the development of chlorosis, which reflects the degradation of the photosynthetic apparatus. And it is there where biostimulants are of marked interest, which leads to their application and incorporation into nutritional plans.

In the metabolism of plants described as the sequence of biochemical reactions, respiration is observed, which is the reverse of photosynthesis, when hydrolysis of sugars occurs to give, when complete, carbon dioxide and water, with energy release. The stages that comprise the respiratory process are glycolysis, the cycle of tricarboxylic acids (Krebs cycle) and oxidative phosphorylation (electron transport system). The various stages of respiration serve for the formation of several organic products necessary for the plants, so the respiratory rate depends on the presence of an available substrate. Stressed plants that have low reserves of starch, fructans or sugar, breathe at low rates. Plants with a deficiency in sugars often breathe more quickly when they are given such sugars. Energy demand and oxygen availability are directly related to the respiratory rate. This is the key that triggers the recovery of the plant.

Now, exogenous applications of preformed nutrients through foliar fertilization can contribute to the quality and increase of crop yields, and we know that many soil fertilization problems can easily be solved by foliar fertilization. Although this form of nutrition through the leaves is not the normal form, the specific function of the leaf is to produce the carbohydrates, but due to its anatomical characteristics it presents morphoanatomical characteristics for an immediate incorporation of the nutrients to the photosynthates and the subsequent translocation of these to the places of the plant of greater demand.

Without a doubt, if you ask the agricultural specialists, all apply biostimulants, to increase their yields. It is estimated that the biostimulant market grows at an annual rate of 10%, since it has become an important agricultural input for the sustainable production of crops. Thus, the Peruvian consultant Maximixe (2015) predicted that the trends of the fertilizer market in Peru would grow 8% annually, due to the growth of agricultural production by 3.7% annually, and by the expansion of the cultivable areas with the carrying out irrigation projects such as Olmos and Majes Siguas II.

II. AGRICULTURAL FORMULATIONS

When formulating agricultural bioestimulants, we ensure that they contain molecules and / or microorganisms whose functionality lies, depending on whether it is applied to the aerial parts of the plants or the rhizosphere, even in the treatment of seeds to ensure a high vigor of emergence in germination and early stages of development. In all of them, the objective is achieved by stimulating processes that, without their application, develop naturally, but with them they develop in a much more accelerated way, thanks to which the absorption and efficiency of nutrients can be improved, tolerance to biotic stress and abiotic.

The biostimulant market includes amino acids, phytochemical extracts of plants (including algae) and extracts of microorganisms, as well as cellular components that integrate biochemical pathways and activation of physiological responses such as carboxylic acids from glucogenic pathways, etc., are the different types of biostimulants of common use. The cost of producing one of them depends on the biotechnological complexity it uses and the budget available for research and development in continuous improvement.

What can we offer for agriculture for the active ingredient?

• Liquid organic matter

• The carboxylates that integrate metabolic pathways

• Free amino acids and protein hydrolysates

• Physiologically activated energy carbohydrates

• Plant extracts with biological activity

• Seaweed metabolites and their biotrained broths

II.a. BIO TRANSFORMATION

In these types of products it is proposed to replace part of the synthetic chemical nutrition with products or waste from the same ecosystem, the nutrients are released from the elaboration of biochemical transformations by microorganisms and phytochemical reactions, and in some cases with organic chemistry techniques. With the use of livestock waste, organic matter of high protein content, carbohydrate and fat, all of them with a high susceptibility to be hydrolyzed keeping phosphorylated monomers easily absorbed. The waste from the agroindustry whose origins are organic waste from discarding stand out.

The production process of a biostimulant begins with the production of microbial inoculum that is faced with a varied type of substrate, in biotechnological processes of anaerobic and aerobic fermentation, the final product of this process is the obtaining of an excellent organic amendment and fertilizer with a good beneficial microbial population. Phytochemical extraction is a tool to concentrate molecules considered as natural products with biological activity

The final products obtained are natural substances with biostimulant function (stimulates growth, flowering, rooting, fruit setting and ripening) and also fertilizer (provides organic nutrients to plants).

Organic fertilizers start from organic substrates and undergo a series of biotransformations in substances that can be assimilated by plants. Our method for the elaboration of organic fertilizers takes special care not to affect the microbiological activity in the decomposition.

Our procedure contemplates the use of organic sources with defined compositions in order that the final product possesses an ideal proportion of nutritious elements for the crop to be treated. It is a biotechnological production process, using enzymes produced in the same substrates that give rise to biostimulants, in a two-stage process for obtaining a liquid product rich in soluble and highly absorbent phytonutrients, for use as fertilizers and stimulants of germination, rooting, growth, flowering, fruit setting and maturation of plants and their fruits. It can be applied in its different variants to any type of plant, time of plant development, soil and leaf form to the crop, and potentially useable in organic agriculture.

The productive processes are divided into anaerobic and aerobic fermentatives:

a. An anaerobic fermentative phase with microorganisms that produce hydrolytic enzymes using organic residues and CaCl2 at concentrations that favor the excretion of enzymes. The fermentative processes developed with microbial treatments, we have, that due to the little control that is exerted on the microbial growth that is nonspecific, causes that in the final product two types of products are obtained: Soluble substances in liquid form with biostimulant capacity and fertilizer, and a solid by-product with fertilizing capacity, this reductive phase produces methane, ammonia, phosphine, hydrogen sulphide and borane.

b. An aerobic fermentation phase: production by direct enzymatic hydrolysis, are much more efficient and result in liquid products that have a greater biostimulant capacity, greater bioavailability and functionality at the time of application. Being the optimal culture medium of Bacillus licheniformis, an excretory microorganism of enzymes. In the fermentative phase, a production of a liquid phase enriched in hydrolytic activity of microbial origin must be achieved. The bioreactor must be at neutral pH, Ta optimum for the development of the microorganism (35-370C) and aeration during 240-380 hours / m3, depending on the volume of the batch produced. A solution of the modified substrate with a high content of enzymes excreted by the microorganism is obtained, which is used in the hydrolytic phase of the process as an enzy-matic base to increase the productivity of the biostimulant. Enzymes that hydrolyze ester bonds, enzymes that attack glycosidic bonds and enzymes that break down nitrogen bonds

Therefore, carbon dioxide, nitrates, phosphates, sulfate and borate are produced in this phase of complete oxidation. Finally, the solid - liquid separation is carried out by filtration and then decantation, at the concentration and acidifying the medium to lower the pH, facilitating storage, transport and, above all, because in these conditions it is completely stable. The resulting non-solubilized solids can be used as conventional organic fertilizer and the resulting liquid is the product with biostimulant capacity for use as fertilizer

for use as fertilizers and stimulants of germination, rooting, growth, flowering, fruit setting and maturation of their plants and fruits.

A protein can be hydrolyzed by enzymes or by the action of acids and alkalis, but physiologically it is not advisable to use alkalis for losing its nature, acid and enzymatic hydrolysis, they maintain the levorotatory characteristic of amino acids, the difference between them is the ability to form free amino acids, the enzymes are very specific for certain bonds, so it generates a limited amount of free amino acids, acid hydrolysis greatly exceeds the enzymatic.

To obtain our amino acids, we perform protein hydrolysis in a bioreactor, with control of agitation, pH, temperature and time of the process. The substrate dissolves in water, the microbial strain producing the enzyme protease is added, initiating the hydrolysis. As it progresses there is a decrease in pH due to the breakdown of the peptide bonds, which develops in a set of simultaneous reactions of breakage of bonds, giving different types of amino acids, a subsequent acid hydrolysis allows us to reach the structure base of an amino acid that is the simplest amino acid, Glycine, this is the magician that allows the best stability to the metal chelate complex, of our metalosates, for having a load balance, it is much easier to take and drop the metal than when there are more competing ionic forces, such as when chelating with a racemic mixture of different amino acids. At the same time, then, the amine structure serves to form other vegetable amino acids and further proteins, and the metal has been transported and made available to the biochemistry of the plant, to fulfill its physiological and nutritional function.

To obtain our formulas based on seaweed extracts as a biostimulant, we use seaweed powder from the Peruvian coast Lessonia (Aracantos), and Macrocystis (Sargasso) in addition to Fucus and Laminaria, micronized, desalinated and filtered, and subjected to microbial biotransformation , to recover a wide range of biological bioelicitors or phenolic molecules with biological activity, which integrate the metabolic pathways of growth regulation such as auxins, cytokinins and gibberellins.

II.b CHELATES OF METALS WITH AMINO ACIDS:

According to the physical and chemical properties they share, the elements can be classified into three major categories: metals, metalloids and non-metals. The metals form ionic compounds similar to salts with non-metallic compounds. In Agriculture we are mainly interested in: Potassium, Calcium, Magnesium, Manganese, Iron, Zinc, Molybdenum, Cobalt, as an example we will take Potassium.

Potassium is a metal because it is positioned in period 4 and group I (alkaline) of the periodic table, this means that it has the configuration of the last valence layer ns ^ 1, tends to give 1 electron to get the maximum stability (8 electrons in the last layer), that is, it tends to make monopositive cations, has low electronegativity and tends to react with non-metals by ionic bond: Therefore, it forms complexes with amino acids, as a ligand.

This complex of an atom or central ion that acts as Lewis acid is coordinated by a ligand that acts as a Lewis base. Called ligand monodentado that is the case of the amino group of the ionized amino acid that leads to the formation of a chelate. To name a complex, ligands are named before the metal. If the complex is an anion, its name ends in ato. Therefore the vulgar to call them metalosatos that means chelate of a metal with amino acid.

Most foliares are concentrated liquids and the organic molecules of the formulation require dilution in water, and these as the chain of the compound becomes longer, becomes less soluble in water. Water is an amphiprotic solvent and therefore can, for example, ionize an amine by giving it a proton or an acid by accepting a proton from it. This reaction is illustrated as follows:

The number of acids and bases that can be ionized by water is limited, and it is likely that most of those that dissolve do so by hydrogen bonding rather than by ionization and solvation of the ions.

III: CARBOXYLATES OF METALS

The carboxylic acids that we use in formulations are tartaric, fumaric, lactic, citric, malic and maleic acids. The compounds containing the carboxyl group are acids and are called carboxylic acids.

A carboxylic acid yields protons by heterolytic cleavage of O-H bond giving a proton and a carboxylate ion. The carboxylic acids form hydrogen bonds with water, a carboxylic acid can be dissociated in water to give a proton and a carboxylate ion. The equilibrium constant Ka is called the acidity constant,

there is where you can enter a substituent that stabilizes the carboxylate ion, with a negative charge, increases the dissociation and produces a stronger acid. In this way the electronegative atoms increase the strength of an acid. This inductive effect can be very large if one or more groups that attract electrons are present in the alpha carbon atom.

The Krebs cycle (also known as the tricarboxylic acid cycle or the citric acid cycle) is a metabolic cycle of fundamental importance in all cells that use oxygen during the cellular respiration process9. In these aerobic organisms, the Krebs cycle is the ring of conjunction of the metabolic pathways responsible for the degradation and desasimilation of carbohydrates, fats and proteins in carbon dioxide and water, with the formation of chemical energy.

The citric acid cycle, considered the funnel of metabolism, consists of eight enzymatic reactions, all of them mitochondrial in eukaryotes. The citric acid cycle is the central route of aerobic metabolism: it is the final oxidative pathway in the catabolism of carbohydrates, fatty acids and amino acids, and it is also an important source of biosynthetic pathway intermediaries. In many cells the coupled action of the citric acid cycle and the electron transport chain are responsible for most of the energy produced.

The Krebs cycle is the common central route for the degradation of the acetyl residues (of 2 C atoms) that are derived from carbohydrates, fatty acids and amino acids. It is a universal route, catalyzed by a multienzyme system that accepts the acetyl groups of acetyl-CoA as fuel, degrading it up to CO2 and hydrogen atoms, which are driven to the O2 that is reduced to form H2O (in the electron transport chain ).

IV. EXOGENOUS INCORPORATION OF FRUCTANOS.

When we provide sugars in our formulations, especially during the period of rapid expansion of the size of the fruit, soluble sugars are responsible for most of the increase in the biomass of the fruit. In addition, some authors point out that these sugars decrease as oil synthesis begins and during the ripening of the fruit, this suggests that these carbohydrates play an important role, not only in the metabolic processes associated with fruit development, but also that also in the respiratory processes associated with the maturation and physiology of the fruit during postharvest.

One of the critical periods during the development of the fruit is the competition that is established between it and the vegetative development by photosynthates in the early stages of growth. When the greatest demands of these products are registered by the fruit and the shoots, therefore for the successful production of fruits, after flowering, it depends on an adequate availability of carbohydrates. and it must be ensured that carbohydrate levels increase at the same time as sugars, sugars are not the main source of carbohydrate reserves, but they are the immediate source of energy for the plant.

  

V. EXOGENOUS INCORPORATION OF POLYPHENOLS

It is possible to incorporate crude extracts of phenolic molecules from the metabolic pathway of the shikimic, in biochemical union that allow it to translocate easily inside the plant, the phenolic acids are intermediaries of the biosynthesis of phytoalexins, to obtain an immediate and sustained response against the infection of phytopathogens and activate the physiological response to stress. To do this, we collect, dried, weed up the remains of flower petals to ruin powder, to proceed with the extraction of the phytoconstituents, with the successive use of different suitable solvents such as ethanol, hexane, dichloromethane, acetone, etc, and in hot water.