EA042631B1 - LARGE-SCALE PRODUCTION OF LIQUID AND SOLID PRODUCTS BASED ON TRICHODERMA - Google Patents

Description

Cross-references to related applications

This application claims the priority and effect of the invention under U.S. Provisional Application Serial Number 62/564,683, filed September 28, 2017, the contents of which are incorporated herein by reference in their entirety.

Prior Art

Soil pathogenic fungi can cause significant damage to crops. These parasites cause, for example, rot (blackleg), root rot, seedling rot and collar rot in a wide variety of host plants. The most common pathogenic fungi of this variety are Rhizoctonia, Pythium, Fusarium, Phytophthora, Sclerotia, Cercospora, Ralstonia, Fragaria, Rhizopus, Botrytis, Colletotrichum, Magnaporthe and many others. The genera Rhizoctonia, Pythium and Sclerotia have an exceptionally wide host range and are capable of infecting many common commercial crops such as beans, tomatoes, cotton, peanuts, potatoes, lettuce and ornamental flowering plants.

The most common ways to control these pathogenic fungi include the use of chemical control agents; however, these chemicals can be expensive and they can be harmful to human health and the environment. In addition, they can disturb the plant microenvironment, for example by changing the surrounding ecosystem.

An alternative to the use of chemicals is the use of biological control agents that are naturally present in the ecosystem. For example, some types of Trichoderma fungi have antagonistic properties against various pests. Some of these fungi are useful when introduced into the soil, where they can multiply and grow in close association with plant roots. They are able to partially protect the roots from the invasion of other plant pathogenic fungi and other microbial and animal pests, and also contribute to the stimulation of plant growth.

Trichoderma can provide stable and long-term colonization of root surfaces by penetrating the epidermis and nearby subsurface cells. These root-microorganism associations induce significant changes in the plant's proteome and metabolism. They produce and/or secrete various compounds that elicit local or systemic defense reactions resulting in a lack of pathogenicity for plants.

In addition, plants defend themselves against numerous classes of plant pathogens by reactions that are similar to systemic acquired resistance and systemic resistance caused by rhizobacteria. Trichoderma spp. can effectively control diseases caused by certain soil plant pathogens. For example, Trichoderma harzianum, Trichoderma hamatum and Trichoderma viride species have fungicidal activity against Sclerotium spp., Rhizoctonia, Solani, Pythium spp., Fusarium spp., Cercospora spp., Ralstonia spp., Fragaria spp., Rhizopus spp., Botrytis spp. , Colletotrichum spp., Magnaporthe spp. and many others. In addition, some strains of Trichoderma are able to effectively suppress the growth of some viral and bacterial plant and soil pathogens, as well as provide significant nematocidal effects.

In addition to protecting plants from pathogens and pests, root colonization by Trichoderma spp. often enhances root growth and development, crop productivity, abiotic stress tolerance and nutrient bioavailability.

Despite the potential effectiveness of using Trichoderma strains to improve plant health, the lack of highly efficient technology for large-scale production of these organisms creates certain difficulties for commercialization. The most common way to grow Trichoderma is to grow them on standard solid media, and prior art methods are too expensive and impractical to be commercially adapted. On the other hand, methods for growing Trichoderma in liquid media, i.e. in depth cultures, are laboratory or small scale methods and cannot provide Trichoderma in the quantities necessary to make them commercially viable (for example, to process hundreds, thousands, or even millions of acres of agricultural land).

Propagation of Trichoderma using a large-scale submerged culture method or a combination of submerged and solid media cultures would be most suitable for commercial production; however, there are no known commercial processes of this kind that are large scale and cheap. Therefore, there is a need for improved methods for the production of Trichoderma fungi that can be scaled up for commercial use.

The essence of the invention

The present invention relates to the large scale production of liquid and dry microbial products for commercial use. More specifically, materials and methods are provided for efficiently cultivating fungi such as Trichoderma and/or obtaining their by-products on a large scale. Methods for using these microorganism-based products are also provided. The advantage of the present invention is that it can be used as an environmentally friendly (green) method for the production of microorganisms on a large scale and at low cost without releasing harmful chemicals into the environment.

The present invention provides systems for the efficient production and use of beneficial microorganisms, as well as for the production and use of substances such as metabolites derived from these microorganisms and the fermentation medium in which they are produced. The organisms of the present invention include, for example, yeast, fungi, bacteria, archaea, and plant cells. In preferred embodiments of the present invention, the microorganisms are fungi. Even more preferably, the microorganisms are Trichoderma fungi, including but not limited to Trichoderma harzianum, Trichoderma viride and/or Trichoderma hamatum.

In particular embodiments, the present invention provides microorganism-based compositions containing the Trichoderma clade of fungi and/or by-products of their growth. Trichoderma-based products may, for example, be in liquid or dry form. Advantageously, in an embodiment of the present invention, microbial products can be in the form of an inoculant that can be scaled up to industrial concentrations for commercial applications using deep fermentation, solid phase fermentation, and/or combinations or hybrids thereof.

Trichoderma-based products may contain the microorganisms themselves and/or by-products of their growth. Microorganisms can be viable, active or in an inactive form. They may be in the form of vegetative cells, spores, conidia, mycelium, hyphae, and/or combinations thereof. Optionally, the compositions may contain a fermentation medium in which the microorganisms have thrived, as well as residual and/or added nutrients that promote microbial growth.

In addition, Trichoderma products of the present invention can be developed, for example, as biofertilizers and/or biopesticides that can be used in applications including, for example, horticulture, horticulture, greenhouse production, as well as large-scale farming and reforestation. . The product can also be used, for example, for seed treatment, land reclamation, for intensive production, for promoting plant root health and/or for stimulating plant growth.

In preferred embodiments, the present invention provides for the cultivation of both liquid forms of microbial products and solid microbial products from a single seed culture. In particular embodiments of the present invention, the microorganism-based products are Trichoderma-based products as described herein.

Methods for the production of microorganisms may include deep fermentation, solid phase fermentation, or hybrids and/or combinations thereof. In an embodiment of the present invention, the methods can be used to produce an inoculum for the production of microbial products on an industrial scale.

In some embodiments, the present invention provides methods for producing liquid and solid microorganism-based products (e.g., Trichoderma-based products) from a single seed crop in commercial quantities, which include:

(a) obtaining an inoculant in the form of alginate-agar beads from a Trichoderma seed culture;

(b) cultivating the inoculant in the form of alginate agar beads in a liquid nutrient culture medium in a reactor until the desired density of microorganisms in the beads is obtained;

(c) collecting the inoculant in the form of alginate-agar beads from the liquid culture medium;

(d) preparing a liquid form of the Trichoderma product, wherein cultured alginate-agar bead inoculants are used to inoculate the deep fermentation reactor, and/or preparing a solid Trichoderma product, where cultured alginate-agar bead inoculants are used for inoculation reactor for solid phase fermentation.

More specifically, in an embodiment of the present invention, the methods include (a) preparing a Trichoderma-based inoculant in the form of alginate granules containing a pre-seed, nutrients, sodium alginate, and agar. The inoculant in the form of alginate granules can be obtained by combining a sterile liquid nutrient medium with a sterile mixture of 1% agar and 2% sodium alginate and 5% homogeneous inoculum suspension to obtain an inoculum solution.

A drip sprayer and a peristaltic pump are then used to drop the inoculum solution into a mixing tank containing a 1% calcium chloride solution. During the spraying process, droplets of seed solution (inoculum) form gel granules containing nutrients and Trichoderma culture, enclosed in an alginate-agar mass. After the granules are formed, the liquid remaining in the mixing tank can be removed and drained into

- 2 042631 liquid waste disposal system.

In an embodiment of the present invention, the methods include (b) cultivating an inoculant in the form of alginate agar beads in a liquid nutrient culture medium until a desired microorganism density is obtained within the beads and/or on the surface of the beads. In some embodiments of the present invention, inoculants in the form of alginate agar beads are collected from a mixing tank and then cultured in a reactor containing a sufficient volume of a suitable liquid nutrient medium until a high concentration of Trichoderma mycelium is obtained, distributed inside and on the surface of the alginate agar beads. In some embodiments of the present invention, a portion of the Trichoderma also enters the liquid nutrient medium from the granules.

In an exemplary embodiment of the present invention, the seed culture for obtaining granules of the inoculant can be obtained from a culture obtained using deep fermentation in a suitable liquid culture medium with continuous aeration and continuous agitation. During this step, temperature and pH are maintained at constant or substantially constant levels (ie, temperature is maintained in the range of about 28° C. to about 30° C.; pH is in the range of about 5.0 to about 6.5). The seed culture can be grown for any period of time sufficient to achieve the desired concentration and/or density of the microorganism, and then homogenized to obtain a suspension of the seed culture.

In an embodiment of the present invention, the method comprises (c) harvesting inoculants in the form of alginate-agar beads from the liquid culture medium after the desired mycelial density has been achieved. These inoculant granules can contain a high concentration of Trichoderma inside and on the surface. The pellets can be used for seeding scale crops immediately after harvest, or the pellets can be processed for short and/or long term storage.

In some embodiments, implementation of the present invention, the method after stage (c) and before stage (d) further includes processing pellets for storage. This may include placing the collected inoculants in the form of alginate-agar beads in a cryopreservation solution so that the inoculants can be stored in a freezer or refrigerator without compromising the viability of the microorganisms. The cryopreservation solution preferably contains water and glycerol in a ratio of, for example, 50%/50%. This solution with inoculant granules placed in it can be stored for extended periods of time at temperatures ranging, for example, in the range from 80°C to -10°C, or for shorter periods of time in a standard refrigerator at temperatures lying, for example , in the range from -10°C to 4.0°C, without reducing the efficiency of the inoculant culture. In some embodiments, implementation of the present invention, the granules are stored in groups, for example, from 1 granule to 50 granules, in hermetically sealed vials.

In an embodiment of the present invention, the method further comprises (d) preparing a scaled liquid form of the Trichoderma product and/or preparing a scaled dry or solid form of the Trichoderma product. In some embodiments of the present invention, step (d) includes the use of inoculant pellets to seed the scaled culture into a deep fermentation reactor, a solid phase reactor, or a hybrid or modified form of the reactor, depending on whether a liquid or solid product is desired.

In some embodiments of the present invention, preparing a product in liquid form comprises seeding a deep fermentation reactor containing a liquid nutrient medium with alginate agar granules of the inoculant of the present invention.

In a particular embodiment of the present invention, inoculant granules are added to a liquid nutrient medium, for example, in a reactor, the volume (working volume) of which is in the range from 200 gallons (757.1 l) to 250 gallons (946.4 l), when essentially constant agitation and constant aeration at a temperature ranging from about 28°C to about 30°C. The pH of the medium during the entire fermentation process is maintained in the range from about 5.0 to about 6.5. Cultivation is carried out for a period of time ranging from 3 days to 10 days, or until the density of conidia obtained from the inoculant is at least 5 x 10 conidia per ml of liquid medium.

In some embodiments, the implementation of the present invention, the preparation of the product in liquid form may include a simple cultivation of residual microorganisms that remain in the liquid medium from stage (b) after the granules of the inoculant have been collected according to stage (c). Residual microorganisms can be cultured in the second reactor or in the same reactor where step (b) proceeded.

In an embodiment of the present invention, the preparation of a liquid microorganism product further comprises increasing the concentration of the microorganism to 1 billion propagules per milliliter and, if necessary, adding additional additives, preservatives and/or pH adjusters. The ready-to-use liquid product can then be filled into containers (eg, 1 gallon (3.785 L) containers), sealed, and labeled for a variety of uses, including commercial activities.

- 3 042631

In some embodiments, the implementation of the present invention, the method includes the preparation of a scaled solid product based on Trichoderma using solid phase fermentation or its hybrid form or modification. Alginate-agar pellet inoculants can be mixed with a solid or semi-solid substrate such as vermiculite or foodstuffs (eg cornmeal, rice, pasta or beans). The substrate is preferably moistened in a suitable nutrient medium, after which the mixture can be cultured in an incubator for a period of time ranging from about 3 days to about 10 days or more, or from about 5 days to about 6 days. The substrate and culture can then be mixed and/or ground and dried to provide a Trichoderma based product in powder form for a variety of applications including commercial use.

In some embodiments, the present invention also provides methods for producing a metabolite and/or growth by-product of fungi, the method comprising cultivating the fungi under conditions favorable for growth and production of the metabolite and/or growth by-product, and optionally purifying the metabolite and/or by-product growth. In particular embodiments, the metabolite and/or growth by-product is an enzyme, biopolymer, acid, solvent, biosurfactant, amino acid, nucleic acid, peptide, protein, lipid, and/or carbohydrate.

Information confirming the possibility of carrying out the invention

The present invention relates to the large scale production of liquid and dry microbial products for commercial use. More specifically, materials and methods are provided for efficiently cultivating fungi such as Trichoderma and/or by-products of their growth on a large scale. Methods for using these microorganism-based products are also provided.

The present invention provides systems for the efficient production and use of beneficial microorganisms, as well as for the production and use of substances such as metabolites derived from these microorganisms and the fermentation medium into which they are produced. The organisms of the present invention include, for example, yeast, fungi, bacteria, archaea, and plant cells. In preferred embodiments of the present invention, the microorganisms are fungi. Even more preferably, the microorganisms are Trichoderma fungi, including, but not limited to, Trichoderma harzianum, Trichoderma viride and/or Trichoderma hamatum.

In particular embodiments, the present invention provides materials and methods for culturing liquid and solid microbial products containing Trichoderma clade fungi and/or Trichoderma growth by-products using submerged fermentation, solid phase fermentation, or hybrids and/or combinations thereof. In an embodiment of the present invention, the methods can be used to provide an inoculum for the production of these microbial products on an industrial scale.

Selected definitions.

When used in the context of the present invention, the term composition based on microorganisms means a composition that contains components obtained from the growth of microorganisms or other cell cultures. Accordingly, the microbial composition may contain the microorganism itself and/or by-products of microbial growth. Microorganisms can be in vegetative form, in the form of spores, in the form of mycelium, in any other form of microbial propagule, or in a mixture of these forms. Microorganisms can be planktonic or in biofilm form, or a mixture of the two. Growth by-products can be, for example, metabolites (eg, biosurfactants), cell membrane components, expressed proteins, and/or other cellular components. Microorganisms may be intact or lysed. Cells may be absent or present, for example, at a concentration equal to 1x10 ⁴ , 1x105, 1x10 ⁴ , 1x106, 1x10 ⁷ , 1x10 ⁸ , 1x10 ⁹ , 1x10 ¹⁰ , 1x1011, 1x10 ¹² , 1x10 ¹³ or more cells or propagules per milliliter of composition . As used in the context of the present invention, a propagule is any part of a microorganism from which a new and/or mature organism can develop, including, but not limited to, cells, mycelium, hyphae, cysts, spores (e.g., reproductive spores, conidia, endospores, and /or exospores), buds and seeds.

The present invention also provides microbial products which are products that can be practiced to achieve the desired result. The microbial product may simply be a microbial composition collected from a microbial culture process. Alternatively, the microorganism-based product may contain additional ingredients that have been added. These additional ingredients may include, for example, stabilizers, buffers, carriers (e.g., water or saline solutions), added nutrients to support further microbial growth, non-nutritional growth promoters, and/or agents that aid in the tracking of microorganisms and/or the composition in the medium, to which they are applied. The microbial product may also contain mixtures of microbial compositions. Microorganism-based product can

- 4 042631 also contain one or more components of the composition based on microorganisms, which has been processed in any way, including, but not limited to, filtration, centrifugation, lysis, drying, purification, and the like.

When used in the context of the present invention, the term inoculum or inoculant (plural inoculum) may be included in the term microbial product. When used in the context of the present invention, the term inoculum (inoculant) means a product based on microorganisms, which can be used, for example, as a seed culture for the inoculation of a large-scale fermentation system or method of fermentation. The inoculum can be scaled up in such a fermentation system to produce the desired amounts of microbial compositions and products.

When used in the context of the present invention, the term isolated or purified means a nucleic acid molecule, a polynucleotide, a polypeptide, a protein, an organic compound such as a small molecule (for example, the molecules described below), or another compound that is essentially free of other compounds, for example - cellular material with which they are associated in nature. For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain genes or sequences that flank it in its natural state. A purified or isolated polypeptide does not contain amino acids or sequences that flank it in its natural state. A purified or isolated strain of microorganism is extracted from the environment in which it exists in nature. Accordingly, the isolated strain may exist, for example, as a biologically pure culture or as spores (or other forms of the strain) in a carrier.

In some embodiments, implementation of the present invention, the purified compounds comprise at least 60 wt.% (in terms of dry weight) of the target compound. The preparation preferably contains at least 75 wt.%, more preferably at least 90 wt.%, and most preferably at least 99 wt.% of the target compound. For example, a purified compound is a compound that contains at least 90 wt.%, 91 wt.%, 92 wt.%, 93 wt.%, 94 wt.%, 95 wt.%, 98 wt.%, 99 wt. % or 100 wt.% of the target compound. Purity is measured by any suitable standard method of analysis, for example by column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC).

The term metabolite refers to any substance that is formed as a result of metabolism (for example, a by-product of growth), or to a substance necessary for participation in a particular metabolic process. The metabolite can be an organic compound, ie a starting material (eg glucose), an intermediate (eg acetyl-CoA) or an end product (eg n-butanol) of metabolism. Examples of metabolites may include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, trace elements, amino acids, polymers, and surfactants.

When used in the context of the present invention, the terms scaled up, large scale, commercial scale, and industrial scale can be used interchangeably and refer to products that, according to their volume, concentration, amount, content and/or potency, can be used in industrial applications. and/or commercial applications. For example, a commercial amount of liquid microbial product or dry microbial product dissolved in a carrier liquid may be from 100 gallons (378.5 L) to 10,000 gallons (37854 L) or more. Industrial and/or commercial applications may include, for example, horticulture, horticulture, greenhouse production, agriculture, soil reclamation, bioremediation, reforestation, and pest control.

When used in the context of the present invention, the term collection refers to the extraction of part or all of the composition based on microorganisms from the reservoir, where the reproduction of microorganisms.

The transitional term containing, which is synonymous with comprising or comprising, is an inclusive or non-limiting term, and does not exclude additional, unlisted elements or method steps. In contrast, the transitive expression consisting of excludes any elements, steps, or ingredients not listed in a claim. The transitional expression consisting essentially of limits the scope of the claim to the indicated materials or steps and those materials or steps that do not significantly affect the basic and novel features of the present invention.

Unless specifically stated or obvious from the context, when used in the context of the present invention, the term or should be understood as inclusive. Unless specifically stated or obvious from the context, when used in the context of the present invention, the singular or plural forms should be understood as such.

Unless specifically stated or obvious from the context, when used in the context of the present invention, the term should be roughly understood as lying within the range of normal tolerances in the art, for example, within 2 standard deviations from the mean. Roughly to be understood as lying within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,

0.1%, 0.05% or 0.01% of the specified value.

A listing of chemical groups in any definition of a variable in this publication includes definitions of that variable as any single group or combination of the listed groups. Giving an embodiment of a variable or aspect in this publication includes that the embodiment is any single embodiment or combination with other embodiments or parts thereof.

All sources cited in this publication are incorporated in this publication by reference in their entirety.

Methods for the production of products based on Trichoderma.

The present invention provides methods for cultivating Trichoderma microorganisms and producing microbial metabolites and/or other by-products of microbial growth. In some embodiments, the implementation of the present invention provides methods for the production of both dry and liquid form products based on Trichoderma. Methods for the production of microorganisms may include submerged culture, solid phase fermentation, or hybrids and/or combinations thereof. When used in the context of the present invention, the term fermentation refers to the cultivation and/or growth of cells under controlled conditions. Growth can be aerobic or anaerobic.

In an embodiment, the present invention provides materials and methods for producing biomass (eg, viable cellular material), extracellular metabolites (eg, small molecules and excreted proteins), residual nutrients, and/or intracellular components (eg, enzymes or other proteins).

The microorganism growth vessel (e.g., reactor) used in accordance with the present invention may have functional controls/sensors, or it may be connected to functional controls/sensors to measure important factors in the culture process such as pH, oxygen content, pressure. , temperature, humidity, viscosity and/or microorganism density and/or metabolite concentration.

The reactor vessel may be inoculated with the selected microorganism. Preferably, the reservoir is inoculated with an inoculant prepared according to the present invention, for example the alginate-agar bead inoculant described in this publication. Depending on the size of the tank, the amount of inoculants needed to inoculate a tank for large scale production can range from 1 inoculant bead to 40 or 50 or more inoculant beads.

In another embodiment of the present invention, microbial growth monitoring (eg, cell number measurement and growth phase determination) can also be performed in the tank. Alternatively, a sample can be removed daily from the reservoir and counted by methods known in the art, for example, by seeding dilutions. Inoculation dilutions are a simple method used to determine the number of microorganisms in a sample. The method may also provide a metric against which different media or treatments can be compared.

The method may include oxygenating the growing culture. In an embodiment of the present invention, a slow moving air flow is used to remove low oxygen air and introduce high oxygen air. In the case of deep fermentation, oxygen-rich air may be atmospheric air supplied daily using mechanisms including impellers to mechanically agitate the liquid and air atomizers to introduce gas bubbles into the liquid to dissolve the oxygen in the liquid.

In an embodiment of the present invention, the method includes enriching the culture with a source of nitrogen. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea and/or ammonium chloride. These nitrogen sources can be used independently of each other or in the form of a combination of two or more of them.

The method may further include enriching the culture with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, maltose, potato dextrose, cellulose, starch and/or kelp; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and/or pyruvic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol and/or glycerin; fats and oils such as soybean oil, rice bran oil, olive oil, canola oil, vegetable oil, corn oil, sesame oil and/or linseed oil, and the like. These carbon sources can be used independently of each other or in the form of a combination of two or more of them.

In an embodiment of the present invention, growth factors and trace elements are included in the nutrient medium. This is particularly advantageous when cultivating microorganisms that do not

- 6 042631 able to produce all the vitamins they need. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt, can also be included in the medium. In addition, sources of vitamins, essential amino acids and trace elements may be included, for example in the form of fine or coarse flour such as cornmeal, or in the form of extracts such as yeast extract, potato extract, beef extract, soy extract, banana peel extract. and the like, or in purified forms. You can also include amino acids, for example - amino acids used for protein biosynthesis.

In an embodiment of the present invention, inorganic salts can also be included. Applicable inorganic salts can be potassium dihydrogen phosphate, dipotassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate and/or sodium carbonate. These inorganic salts can be used independently or in the form of a combination of two or more of them.

In some embodiments of the present invention, the culture method may further include adding additional acids and/or antimicrobials to the liquid medium prior to and/or during the culture process to protect the culture from contamination by unwanted microorganisms. In addition, antifoam agents can also be added to prevent foaming and/or foam accumulation due to gas generation during cultivation.

The pH of the mixture must be suitable for the growth of fungi, in particular Trichoderma. In some embodiments, implementation of the present invention, the pH is in the range from about 5.0 to about 7.0, preferably from about 5.0 to about 6.5. Buffers and pH adjusters such as carbonates and phosphates can be used to stabilize the pH around the preferred value. If high concentrations of metal ions are present, it may be necessary to use a chelating agent in a liquid medium.

The method and equipment for cultivating Trichoderma microorganisms and producing microbial by-products can be carried out in the form of batch, quasi-continuous or continuous processes.

In an embodiment of the present invention, the culture method is carried out at a temperature ranging from about 5°C to about 100°C, preferably from about 15°C to about 60°C, more preferably from about 25°C to about 30°C. WITH. In another embodiment of the present invention, the cultivation can be carried out continuously at a constant temperature. In another embodiment of the present invention, the cultivation can be carried out at varying temperatures.

In an embodiment of the present invention, the equipment used in the culture method and process is sterile. The culture equipment, such as a reactor/tank, may be separate from, but connected to, a sterilization unit, such as an autoclave. The culture equipment may also have a sterilization unit that performs in situ sterilization prior to inoculation. Air can be sterilized by methods known in the art. For example, atmospheric air may be passed through at least one filter before entering the reservoir. In other embodiments of the present invention, the medium can be pasteurized or optionally not heated at all, and water with low activity and low pH is used to control bacterial growth.

In an embodiment, the present invention also provides a method for the production of microbial metabolites such as ethanol, lactic acid, beta-glucan, proteins, peptides, intermediate metabolites, polyunsaturated fatty acids, and lipids, the method comprising culturing the microorganism under conditions favorable for growth and expression of the metabolites. . In particular embodiments of the present invention, the metabolite is an enzyme, a biopolymer, an acid, a solvent, a biosurfactant, an amino acid, a nucleic acid, a peptide, a protein, a lipid, and/or a carbohydrate. The concentration of the metabolite produced by the process may be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

In the case of deep fermentation, the content of biomass in the fermentation broth may lie, for example, in the range from 5 g/l to 180 g/l or more. In an embodiment of the present invention, the solids content of the broth is in the range of 10 g/l to 150 g/l.

A by-product of microbial growth produced by Trichoderma may remain within the microorganisms or be secreted into the culture medium. In another embodiment of the present invention, the method for producing a microbial growth by-product may further comprise the steps of concentrating and purifying the desired microbial growth by-product. In a further embodiment of the present invention, the liquid medium may contain compounds that stabilize the activity of the by-product of microbial growth.

In an embodiment of the present invention, the entire microbial culture composition is recovered after culture is complete (e.g., after reaching the desired cell density or

- 7 042631 density of a certain metabolite in the medium). In this batch procedure, after the first batch is collected, a completely new batch is produced.

In another embodiment of the present invention, only a portion of the fermentation product is recovered each time. In this embodiment of the present invention, the viable cell biomass remains in the tank as an inoculant for the new culture batch. The recovered composition may be a cell-free broth or substrate, or it may contain cells. Thus, a quasi-continuous system is created.

The advantage is that the method does not require complex equipment or high energy consumption. Trichoderma can be cultivated on a small or large scale in situ and even used in the form of a mixture with a medium. Similarly, microbial metabolites can also be produced in large quantities in the place where they are required.

Organisms that can be cultured using the present invention may include, for example, yeast, fungi, bacteria, archaea, and plant cells. In preferred embodiments of the present invention, the microorganisms are fungi. Even more preferably, the microorganisms are Trichoderma fungi, including, but not limited to, Trichoderma reesei, Trichoderma harzianum (Trichoderma narcissi), Trichoderma viride, and/or Trichoderma hamatum.

Other fungi can be produced according to the present invention, including Mycorrhizae, ectomycorrhizal fungi, yeasts such as Starmerella bombicola, and even spores of fruiting fungi such as shiitake (Lentinula edodes).

According to the present invention, large scale commercial quantities of Trichoderma based products can be grown. Advantageously, Trichoderma can be grown for periods ranging from 3 days to 10 days, or 5 days to 6 days, with yields ranging from 5x108 to 5x109 conidia per ml of liquid culture, using a fermentation a 200 gallon (757.1 L) reactor and producing more than 1x109 conidia per gram of dry product in an incubator such as a pressurized thermostatic reactor.

In some embodiments, the present invention provides methods for producing liquid and solid microbial products (eg, Trichoderma products) from a single seed crop in commercial quantities. Advantageously, the use of inoculants in the form of alginate-agar beads (or inoculant beads, granulated inoculants, beads or inoculants) of the present invention allows the reactor to be inoculated with a significantly higher concentration of cells than would be possible with a standard liquid inoculant. High concentration means, for example, at least 101, 10 ² , 103, 10 ⁴ , 105, 106, 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ or more cells or propagules of the desired microorganism per unit (mass or volume).

In preferred embodiments of the present invention, the methods include:

(a) obtaining an inoculant in the form of alginate-agar beads from a Trichoderma seed culture;

(b) cultivating the inoculant in the form of alginate agar beads in a liquid nutrient culture medium in a reactor until the desired density of microorganisms in the beads is obtained;

(c) collecting the inoculant in the form of alginate-agar beads from the liquid culture medium;

(d) preparing a liquid form of the Trichoderma product, wherein cultured alginate-agar bead inoculants are used to inoculate the deep fermentation reactor, and/or preparing a solid Trichoderma product, where cultured alginate-agar bead inoculants are used for inoculation reactor for solid phase fermentation.

More specifically, in an embodiment of the present invention, the methods include (a) obtaining a Trichoderma-based inoculant in the form of alginate-agar pellets containing the pre-seed, nutrients, sodium alginate, and agar. An inoculum (eg, a 5% homogenous inoculum suspension) can be dissolved in sterile growth medium and combined with a mixture of agar and sodium alginate to form an inoculum solution.

The concentration of alginate in the inoculum solution may range from about 0.1% to about 3.0%, preferably from about 0.5% to about 2.5%, more preferably about 2.0%. The agar concentration may range from about 0.1% to about 2.0%, preferably from about 0.5% to about 1.5%, more preferably about 1.0%. In an embodiment of the present invention, the alginate agar can be autoclaved and/or heated prior to mixing with the inoculum.

A drip sprayer and a peristaltic pump are then used to drop the inoculum solution into a mixing tank containing the calcium chloride solution. During the spraying process, inoculum droplets form gel granules containing nutrients and a Trichoderma culture enclosed in an alginate-agar mass. In some embodiments of the present invention, from about 5 kg to about 10 kg of inoculant in the form of alginate granules is obtained from a single batch. After the granules are formed, the liquid remaining in the mixing tank can be removed and drained into the liquid waste disposal system.

In some embodiments of the present invention, the CaCl ₂ solution may be a solution with a concentration ranging from about 1.0% to about 5.0%, preferably a solution with a concentration ranging from about 1.0% to about 2, 0%.

In an embodiment of the present invention, the mixing tank is a mobile rotary tank equipped with a motor. The tank may have a volume ranging from about 2 cubic feet (56.63 L) to about 4 cubic feet (113.3 L) or up to about 6 cubic feet (169.9 L) or more, and may be manufactured from polyethylene, any other polymeric material or metal. The rotary tank may contain, for example, 5 gallons (18.93 L) to 10 gallons (37.85 L) of a 1% calcium chloride solution.

In an exemplary embodiment of the present invention, the seed culture for obtaining granules of the inoculant can be obtained from a culture obtained using deep fermentation in a suitable liquid culture medium (see, for example, the publication of Elad et al. (1982), the contents of which are incorporated herein by references) with continuous aeration and continuous stirring. The temperature and pH during this stage is maintained at constant or substantially constant levels (for example, the temperature is maintained in the range from about 28°C to about 30°C; pH is maintained in the range from about 5.0 to about 6.5). The seed culture can be grown for any period of time sufficient to achieve the desired concentration and/or density of the microorganism, and then homogenized to obtain a suspension of the seed culture.

In an embodiment of the present invention, the methods include (b) cultivating an inoculant in the form of alginate agar beads in a liquid nutrient culture medium until a desired microorganism density is obtained within the beads and/or on the surface of the beads. In some embodiments of the present invention, inoculants in the form of alginate agar beads are collected from a mixing tank and then cultured in a reactor containing a sufficient volume of suitable liquid nutrient medium until a high concentration of Trichoderma mycelium is obtained growing inside each alginate agar bead and distributed over the surface of the beads. . In some embodiments of the present invention, the culture parameters for step (b), such as temperature, medium, and pH, may be the same as those used to produce the initial seed culture. In some embodiments of the present invention, a number of Trichoderma cells also grow in the liquid nutrient medium, separate from the inoculant granules.

In an embodiment of the present invention, the method includes step (c) during which the inoculant in the form of alginate-agar beads is collected from the liquid culture medium. These inoculant beads may contain a high concentration of Trichoderma inside and/or on the surface. The pellets can be used for seeding scale crops immediately after harvest, or the pellets can be processed for short and/or long term storage. In some embodiments, implementation of the present invention, the granules after collection is placed in a container, for example - in a test tube or vial.

In some embodiments, implementation of the present invention, the method after stage (c) and before stage (d) may further include processing pellets for storage. This treatment may include suspending the collected inoculants in the form of alginate-agar beads in a cryopreservation solution so that the inoculants can be stored in a freezer or refrigerator without compromising the viability of the microorganisms. Preferably, storage is in a test tube, flask, cylinder, vial or petri dish, or other similar standard laboratory container.

In some embodiments of the present invention, the cryopreservation solution contains water and a cryoprotectant. Cryoprotectants are known antifreeze agents that are capable of protecting cells and other biological tissues from damage caused by freezing and ice formation. Many species of animals and plants living in cold climates produce natural cryoprotectants to protect their organisms and cells. Isolated and synthetic cryoprotectants are also used for the preservation of biological materials intended for biological research and contained in food products.

Examples of cryoprotectants that can be used in the present invention include, but are not limited to, glycols such as ethylene glycol, propylene glycol and glycerol, dimethyl sulfoxide (DMSO), trehalose, 2-methyl-2,4-pentanediol (MPD), and sucrose.

In preferred embodiments of the present invention, the cryopreservation solution contains water and glycerin, with the percentage of glycerol ranging from 35% to 75%, preferably about 50%.

This solution, with inoculant granules placed therein, can be stored for extended periods of time in a freezer, for example at a temperature ranging from about -80°C to about 0°C, preferably from about -80°C to about - 20°C. Storage at these

- 9 042631 temperatures can last as long as you like while maintaining the effectiveness and viability of the biological material in solution, for example, for 1 month, 6 months or 1 year, 2 years, 3 years, 4 years, 5 years or even 10 years or more.

In some embodiments of the present invention, when a shorter shelf life of, for example, 1 month or less is desired, containers containing inoculant granules suspended in glycerol solution can be stored in a refrigerator at a temperature ranging from about -15° C to about 4°C, or from about -10°C to about 4°C, or from about 0°C to 4°C.

In some embodiments, implementation of the present invention, the beads are stored in groups containing a solution for cryopreservation, for example, from 1 pellet to 50 pellets in a sealed container.

In other embodiments of the present invention, the pellets are not collected from the fermentation reactor according to step (c), but instead the cryopreservation solution is poured directly into the fermentation reactor used in step (b), and the reactor itself is used to store and preserve the entire batch of pellets. The temperature inside the reactor can be adjusted accordingly.

In an embodiment of the present invention, the method further comprises the step of (d) preparing an increased amount of a liquid form of the Trichoderma product and/or preparing an increased amount of a dry or solid form of the Trichoderma product. In some embodiments of the present invention, step (d) includes the use of inoculant pellets to inoculate an increased amount of culture in a deep fermentation reactor, in a solid phase fermentation reactor, or in a hybrid or modified form of the reactor, depending on whether a liquid or solid product is desired.

In some embodiments of the present invention, preparing a product in liquid form comprises seeding a deep fermentation reactor containing a liquid nutrient medium with alginate-agar granules of the inoculant of the present invention. In some embodiments of the present invention, if, for example, the inoculant pellets were preserved using a glycerol solution, the pellets can be removed directly from the freezer or refrigerator where they were stored and used to seed the reactor. Advantageously, the methods of the present invention allow for the inoculation of several large scale fermentation reactors (e.g. having a volume ranging from 100 gallons (378.5 L) to 2000 gallons (7571 L) or up to 10000 gallons (37854 L) or more ) using a single seed crop.

In a preferred embodiment of the present invention, large-scale (scale) production is carried out in a new, portable and distributable reactor. Fermentation using this system is carried out in the form of a batch process without agitation, but with stirring and aeration. In an embodiment of the present invention, the system comprises a large volume tank. The reactor may further comprise an agitation system comprising first and second piping systems, with the first piping system located on the left vertical side of the tank and the second piping system located on the right vertical side of the tank. Each piping system can be equipped with pumps capable of moving culture fluid from the bottom of the tank up through the piping and back to the top of the tank at rates up to about 200 gallons (757.1 liters) per minute. These piping systems can operate continuously throughout the fermentation process to agitate the culture.

This single chamber reactor may include an air distributor supplied with filtered air from a blower capable of delivering 2 liters of air per liter of culture per minute. Filtered air for distribution can be generated by a large volume water pumping system containing pumps equipped with additional filters.

The system reactor preferably has an operating volume ranging from 200 gallons (757.1 L) to 2000 gallons (7571 L), but it can be less (for example, from 100 gallons (378.5 L) to 200 gallons (757. 1 L)) or more (for example, up to 10,000 gallons (37,845 L) or more). However, the size and configuration of the reactors may vary (depending, for example, on the desired final industrial volume of the microbial product). The system can be used to produce cultures of various strains and species of micro-organisms, and has virtually no limits on the total amount of micro-organism product that can be produced.

In some embodiments, the implementation of the present invention to reduce the cost of production of culture and ensure the scalability of production, fermentation systems are not sterilized using traditional methods. Instead, an empty tank sanitization method is used which includes cleaning the interior surfaces with a 2-3% hydrogen peroxide solution and rinsing with bleach and high pressure hot water. Additionally, to reduce the likelihood of significant contamination, the water used to obtain a cultivated culture is filtered through a filter with a mesh diameter of 0.1 μm. The components of the culture medium are decontaminated by heat treatment at a temperature ranging from 85°C to 90°C, or dissolved in a 3% hydrogen peroxide solution (the ratio of the volumes of dry components and H2O is 1:3).

In some embodiments of the present invention, the medium for use in the advanced production step of Trichoderma is a liquid base nutrient medium containing potato dextrose broth or glucose as a carbon source. The environment may also contain an additional source of carbon and a source of nitrogen. The additional carbon source may be selected from glucose, sucrose, maltose, fructose, cellulose, starch and laminarin. Optionally, the medium may also contain malt extract.

Various sources of nitrogen can be used in the liquid base culture medium, although nitrates or nitrites are preferably used. In preferred embodiments of the present invention, ammonium nitrate is used as the nitrogen source.

The liquid base culture medium may also contain suitable amounts of minerals and trace elements such as MgSO ₄ , FeCl 2 , MnSO ₄ , ZnSO ₄ , KCl and K ₂ HPO ₄ . You can also add other trace elements and minerals.

In some embodiments of the present invention, the liquid base nutrient medium contains yeast extract as a source of vitamins. In order to obtain an organic product, antibacterial substances such as antibiotics should not be included in the nutrient medium. Instead, natural components with antibacterial properties (e.g., biosurfactants such as sophorolipids and rhamnolipids and/or hop acids or hop cones) should be used, provided they do not adversely affect the microorganism produced by the methods of the present invention (e.g., various types of Trichoderma).

In an exemplary embodiment of the present invention, the liquid base culture medium for large-scale production of Trichoderma in the reactor systems of the present invention contains the components in the amounts indicated in the table from Example 1 below.

The fermentation temperature for large scale production of Trichoderma based products should be in the range of about 25°C to about 32°C, preferably from about 28°C to about 30°C. The pH should be in the range from about 5.0 to about 6.5, preferably from about 5.5 to about 6.0. pH stabilization during fermentation is not critical, however the pH should not fall below 4.5. If necessary, the pH can be adjusted or maintained during fermentation using manual or automatic methods known in the art, such as using automatic pH controllers to add alkaline components. Preferred alkalis used for pH adjustment include, but are not limited to, NaOH and KOH.

Preferably, the culture is maintained for a period ranging from 3 days to 10 days or more, or 5 days to 6 days, until the density of conidia obtained from the inoculant alginate granules is in the range of about 5x108 to 5x109 conidia per milliliter of liquid culture .

In some embodiments, the implementation of the present invention, the production of a liquid form of the product may include a simple cultivation of residual microorganisms that have survived in the remaining liquid medium after the collection of granules of the inoculant according to stage (c). Residual microorganisms can be cultured in the second reactor or in the same reactor where step (b) was carried out.

In an embodiment of the present invention, obtaining a microorganism-based liquid product further comprises increasing the concentration of the microorganism to 1 billion propagules per milliliter and, if necessary, adding additional additives, preservatives and/or pH adjusters. The ready-to-use liquid product can then be filled into containers (eg, 1 gallon (3.785 L) containers), sealed, and labeled for a variety of uses, including commercial applications.

In some embodiments, the implementation of the present invention, the method includes the production of a large amount of solid product based on Trichoderma using solid phase fermentation or its hybrid or modified form. Inoculants in the form of alginate-agar granules can be mixed with a solid or semi-solid substrate such as vermiculite or foods (eg cornmeal, paste, rice or beans). The substrate is preferably moistened in a suitable nutrient medium. For example, the trays can be sprayed regularly (eg, once a day, every other day, once a week) with sterile growth media throughout cultivation.

The mixture can be cultured in an incubator for a period of time ranging from about 3 days to about 10 days or more, or from about 5 days to about 6 days. The substrate and culture can then be mixed and/or ground and dried to provide a Trichoderma based product in powder form for a variety of applications including commercial applications.

In some embodiments of the present invention, if, for example, the inoculant pellets were preserved using a glycerol solution, the pellets can be removed directly from the freezer or refrigerator where they were stored and used to inoculate solid

- 11 042631 or semi-solid substrate.

In particular embodiments of the present invention, the production of a solid commercial product based on, for example, Trichoderma may include mixing the harvested pellets with a substrate and growth medium and incubating the mixture in trays. In some embodiments, the trays are incubated in proofers or similar heaters.

In an exemplary embodiment of the present invention that uses vermiculite, the vermiculite is heat sterilized in an oven at 150° C. overnight. Approximately 3 or 4 parts of sterilized vermiculite are thoroughly mixed with 1 part of alginate-agar beads. The mixture is spread in a thin layer on the trays. Then, cultivation can be carried out for a period of time ranging from about 5 days to about 6 days or up to about 2 weeks, with atmospheric air aeration.

After completion of the cultivation process, the temperature in the incubator is raised to about 40°C, and drying can be carried out for a period ranging from about 3 days to about 4 days using dry air supply and moist air vacuum. The dried microbial product can be ground, milled or micronized to the desired particle size. The concentration of propagules must be at least 1x10 ⁹ conidia per gram of dry product, and it can be as high as 1x101°, 1x1011, 1x10 ¹² or even 1x10 ¹³ propagules per gram.

The dry microbial product can then be mixed with dry diatomaceous earth and commercial compost to redistribute residual moisture and to standardize the final product. The concentration of propagules after mixing may be, for example, about 1x106 conidia per gram. This final dry product based on Trichoderma can then be packaged in labeled plastic bags and sealed for commercial sale.

In some embodiments, implementation of the present invention, the final dry product based on Trichoderma may contain sources of carbon, protein and/or minerals.

Local production of products based on microorganisms.

In some embodiments of the present invention, the microorganism growth apparatus produces high density fresh microorganisms and/or targeted microbial growth by-products at the desired scale. The microbial growth facility may be located at or near the site of use of the microorganism. The plant produces compositions based on microorganisms with a high density of microorganisms through periodic, quasi-continuous or continuous cultivation.

The microorganism cultivation facilities of the present invention may be located at the site where the microbial product will be applied (eg, in a fish farm). For example, a microbial growth facility may be located less than 300 miles, 250 miles, 200 miles, 150 miles, 100 miles, 75 miles, 50 miles, 25 miles, 15 miles, 10 miles, 5 miles, 3 miles, or 1 mile. from the place of application (where 1 mile corresponds to 1.609 km).

Since the microbial product is produced on site or near the site of use and there is no need for stabilization, conservation, long-term storage, or large-scale transport processes of standard production, a much higher microorganism density can be obtained, resulting in a much smaller volume of microbial product for application. in place. This allows the use of a smaller bioreactor (eg, smaller fermentation tank, lower loadings of starter material, nutrients, pH adjusters and antifoam agents, etc.), making such a system cost effective. In addition, local production contributes to the transportability of the product.

The local production of a microorganism-based product also facilitates the inclusion of a culture medium in the product. The medium may contain agents formed during fermentation which are particularly well suited for topical use.

Locally produced, high-density resistant microbial cultures are more effective in the field than cultures that have been subjected to vegetative cell stabilization or have been in the supply chain for some time. The microbial products of the present invention are particularly advantageous over traditional products in which cells are separated from metabolites and nutrients present in the fermentation culture medium. Reduced transport time ensures that fresh batches of microorganisms and/or their metabolites are produced and delivered at the right time and in the amount needed at the application site.

The microbial growth apparatus of the present invention provides fresh microorganism-based compositions containing the microorganism itself, metabolites of the microorganism, and/or other components of the medium in which the microorganism has been grown. Optionally, the compositions may contain a high density of vegetative cells, inactivated cells, or a mixture of vegetative cells, inactivated cells, spores, mycelium, and/or other microbial propagules. The advantage is that the compositions can be adapted for use in a particular location. In an embodiment of the present invention, the microbial growth facility is located at or near the manufacturing site where the microbial products will be used.

Advantageously, these microbial growth facilities provide a solution to the current problem of geographically dispersed industrial scale producers whose product quality suffers from processing delays in previous production steps, supply chain constraints, improper storage and other unforeseen circumstances. which prevent the timely delivery and use of, for example, a product with a high content of viable cells and/or propagules and accompanying media and metabolites in which the microorganisms were originally grown.

Advantageously, in preferred embodiments of the present invention, the systems of the present invention retain the ability of naturally occurring local microorganisms and their metabolic by-products to attack plant pathogenic bacteria. Indigenous microorganisms can be identified, for example, based on salt tolerance, ability to grow at high temperatures, and using genetic sequence identification. In addition, microorganism growers provide manufacturing flexibility through their ability to tailor microbial products to enhance synergy with geographic areas of destination.

The culturing time in particular tanks may, for example, range from 1 day to 2 weeks or longer. The culture product can be harvested by any of a variety of different methods.

Local production and delivery within, for example, 24 hours after fermentation provides pure compositions with a high density of microorganisms and significantly lower transport costs. With the promise of rapid advances in the development of more effective and potent microbial inoculants, consumers will greatly benefit from this ability to quickly deliver microbial-based products.

The microbial products of the present invention can be used in a variety of unique applications due to, for example, the ability to efficiently deliver to remote locations: 1) fresh fermentation broth with active metabolites; 2) a mixture of microorganisms and fermentation broth; 3) composition with living cells or spores, mycelium, conidia or other microbial propagules; 4) compositions with a high density of microorganisms, including living cells and/or spores, mycelium, conidia or other microbial propagules; 5) products based on microorganisms, ready for use; and 6) microbial based products.

Description of exemplary embodiments of the invention

A better understanding of the present invention and its many advantages can be obtained from the following examples given for the purpose of illustration. The following examples illustrate some of the methods, uses, embodiments, and embodiments of the present invention. They should not be considered as limiting the present invention. You can make many changes and modifications of the present invention.

Example 1 Seed production and method for counting propagules.

An inoculum of Trichoderma was prepared at pH 5.5 using the medium composition shown in the table.

Composition of the medium for the production of Trichoderma

Component Quantity (g/l) Glucose thirty Yeast extract 2.8 Liquid Potato Extract 0.5 (ml/l) NH4NO3 1.0 KN ₂ RO ₄ 1.0 MgSO ₄ 7H ₂ O 0.5 KCI 0.5 FeSO ₄ 7H ₂ O 0.01 ZnSO ₄ 7H ₂ O 0.01 CuSO ₄ 5H ₂ O 0.005

The flasks were inoculated with conidia and actively growing hyphae of the fungus from a Petri dish. To obtain a homogeneous seed culture, the granules with mycelium were destroyed using glass bowls.

- 13 042631 ricks. Shake flasks were incubated at 30° C. for 3-4 days at a shaking frequency of 200 1/min. After 3-4 days, mycelial granules of Trichoderma were formed for large-scale fermentation. The entire contents of the flasks containing the fermented substrate and biomass were completely homogenized using glass beads for a predetermined time of 180 seconds to obtain conidia and mycelium fragments. After homogenization, serial dilutions were performed and the number of micropropagules was determined.

Example 2. Obtaining a culture in the form of alginate-agar granules.

Alginate beads containing Trichoderma were prepared by combining the liquid medium from the table above with a mixture of 1% agar and 2% sodium alginate. This mixture was combined with a 5% homogenous inoculum suspension from Example 1. After thorough mixing, the entire mixture was slowly added dropwise to an autoclaved 100 mM calcium chloride solution with constant stirring. Immediately formed alginate granules with fungal particles inside. The alginate granules were then removed from the solution and the remaining liquid was destroyed.

Example 3 Production of conidia in liquid culture.

Conidia are collected from a biologically pure culture of Trichoderma harzianum, which is grown in a reactor. The nutrient medium composition contains: glucose (30 g/l), yeast extract (2.8 g/l), KH2PO4 (1.0 g/l), MgSO4-7H2O (0.5 g/l), KCl (0. 5 g/l), FeSO4-7H2O (0.01 g/l), ZnSO4-7H2O (0.01 g/l), CuSO ₄ -5H ₂ O (0.005 g/l). The initial pH during cultivation is 5.5, and the temperature is in the range from 25°C to 28°C. The culture volume is approximately 100 gallons (378.5 L). After culturing for 5 days, the yield is greater than about 5x108 to about 5x109 conidia per milliliter of liquid culture.

Example 4 Solid culture of Trichoderma in vermiculite substrate.

Vermiculite and diatomaceous earth are subjected to heat sterilization at 150° C. overnight in a heating oven. Three to four parts of vermiculite are mixed with one part of diatomaceous earth and either with one part of Trichoderma inoculant granules or with 150 ml of inoculum suspension. The components are mixed with 1 liter of nutrient medium. This mixture is thinly spread over the tray and incubated at 30°C for 4-6 days in a proofer with atmospheric air aeration. Conidia are first detected on day 4.

Yield from one tray before drying and processing is approximately 652.60 grams. After completion of the cultivation process, the temperature in the incubator can be raised to about 40°C, and drying can be carried out for a period of time ranging from about 3 days to about 4 days using dry air supply and humid air vacuum. After drying and thorough grinding, up to 4 pounds (1.814 kg) or more of Trichoderma based product can be obtained from each tray. The dried microbial product can be ground, ground or micronized to the desired particle size and then mixed with dry diatomaceous earth and commercial compost to redistribute residual moisture and standardize the final product. The concentration of propagules should be at least 1x106 conidia per gram of dry product, preferably at least 1x109.

This final dry product based on Trichoderma can then be packaged in labeled plastic bags and sealed for commercial distribution. The product can be dissolved in water for various applications.

Example 5 Solid state fermentation of fungal spores in a cornmeal substrate.

To grow spores of fungi such as Trichoderma spp., 250 g of nixtamalized cornmeal is mixed with deionized water and sterilized in a stainless steel steam boiler, then sealed with a lid and tied with ribbons. These cornmeal media pans are aseptically inoculated with a fungal seed culture. The pots are then incubated in a proofer at 30° C. for 10 days. After 10 days about 1x109 propagules/g are collected.

Example 6 Solid state fermentation of fungal spores in a paste based substrate.

For growing Trichoderma spp. 250 grams of dry cornmeal paste is mixed with deionized water mixed with 1000 liters of water, distributed over stainless steel steam boilers. The steam boilers, paste and water are then autoclaved and hermetically sealed with sterilized lids and boiler bands. The paste-water substrate is then aseptically inoculated with a Trichoderma seed culture. The boilers are incubated in a proofer at 30°C for 8 days. After 8 days, approximately 1x109 propagules/g Trichoderma are harvested.

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Claims (15)

CLAIM 1. A method for producing a liquid form of a Trichoderma product and a solid form of a Trichoderma product, which includes: (a) obtaining an inoculant in the form of alginate-agar beads from a Trichoderma seed culture, comprising combining a 5% homogeneous seed culture suspension with a sterile liquid nutrient medium, 1% agar and 2% sodium alginate to obtain an inoculant solution and using a drip spray device to supplying the inoculant solution in drops to a mixing tank containing a 1% solution of calcium chloride, while the drops form inoculants in the form of alginate-agar granules containing nutrients and microorganism particles enclosed in the alginate-agar mass; (b) cultivating the inoculant in the form of alginate agar beads in a liquid nutrient culture medium in a reactor until the desired density of microorganisms in the beads is obtained, the desired increased density of microorganisms in the alginate beads being 1 x 10 ⁹ conidia per gram of alginate beads; (c) collecting the inoculant in the form of alginate-agar beads from the liquid culture medium; (d) preparation of a liquid form of the Trichoderma product, in which cultured inoculants in the form of alginate-agar beads are used to inoculate the deep fermentation reactor, and preparation of a solid product of Trichoderma, in which cultured inoculants in the form of alginate-agar beads are used for inoculation reactor for solid phase fermentation. 2. The method according to claim 1, wherein the Trichoderma microorganism is selected from Trichoderma reesei, Trichoderma harzianum, Trichoderma viride and Trichoderma hamatum. 3. The method according to claim 1, characterized in that step (b) comprises collecting alginate-agar beads and then culturing them in a reactor containing a sufficient volume of a suitable liquid nutrient medium until a high concentration of Trichoderma mycelium is obtained, distributed inside and on the surface of the alginate - agar granules. 4. The method according to claim 1, characterized in that stage (b) is carried out for a period of time from 1 day to 10 days, with continuous aeration and continuous stirring, at a temperature of from 28°C to 30°C and at a pH of 5 .0 to 6.5. 5. The method according to claim 1, characterized in that after step (c) but before step (d) the method comprises treating the inoculant in the form of alginate-agar pellets for storage, the treatment may include collecting the inoculant in the form of alginate-agar pellets from reactor and placing the pellets in a cryopreservation solution containing glycerol and water in a ratio of 50%/50%. 6. The method according to claim 5, characterized in that the solution for cryopreservation is in a container selected from a flask, a test tube, a vial and a Petri dish. 7. Method according to claim 6, characterized in that the container contains up to 50 granules of the inoculant. 8. The method according to claim 6, characterized in that the container is placed in a freezer at a temperature of -80°C to -10°C or the container is placed in a refrigerator at a temperature of -10°C to 4°C. 9. The method according to claim 1, characterized in that obtaining a liquid form of the product based on Trichoderma includes culturing the inoculant in a reactor system with a working volume equal to at least 757 liters, for a period of time from 1 day to 10 days, with continuous aeration and continuous stirring, at a temperature of from 28°C to 30°C and at a pH of 5.0 to 6.5, and preferably the concentration of Trichoderma is increased in liquid culture to 1x10 ⁹ propagules per milliliter and optionally preservatives, additives and / or regulators are added pH. 10. The method according to claim 1, characterized in that obtaining a solid form of the product based on Trichoderma includes mixing the collected inoculant in the form of alginate-agar granules with a liquid nutrient medium and a solid or semi-solid substrate to obtain a mixture; distribution of the mixture in a thin layer on heat-resistant trays; culturing the mixture on trays in an incubator at 30°C for a period of time from 3 days to 10 days with constant aeration with atmospheric air. 11. Method according to claim 10, characterized in that the trays are regularly sprayed with sterile nutrient medium during cultivation. 12. The method according to claim 10, characterized in that the incubator is a proofer. 13. The method according to claim 10, characterized in that the substrate is vermiculite, and the ratio of vermiculite and alginate-agar granules in the mixture is 3:1 or 4:1, or the substrate is a food product selected from cornmeal, rice, beans and pastes. 14. The method according to claim 10, characterized in that it further comprises raising the temperature of the incubator to 40°C; drying the trays for a period of 3 days to 4 days using dry air supply and humid air vacuum to obtain a dried product; grinding the dried product in a crusher to the desired particle size; and mixing the crushed dried product with dry diatomaceous earth and commercial compost, - 15 042631 and preferably the method further comprises packing the dry product mixed with diatomaceous earth and commercial compost into hermetically sealed bags for commercial use, more preferably the concentration of Trichoderma in the dried product after mixing with diatomaceous earth and commercial compost is at least 1x10 ⁶ conidia per gram. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2