EA045948B1 - DISTRIBUTED SYSTEMS FOR EFFECTIVE PRODUCTION AND APPLICATION OF COMPOSITIONS BASED ON MICROORGANISMS - Google Patents

Description

Cross reference to related application

This application claims benefit to US Provisional Application Serial No. 62/385,057, filed September 8, 2016, which is incorporated herein by reference in its entirety.

State of the art to which the invention relates

Around the world, agriculture, forestry, livestock, aquaculture and other production of food, food additives, flavors, fragrances, fiber and natural building materials are becoming increasingly difficult due to environmental challenges including pest resistance to common chemicals, extreme temperatures , drought, floods, emerging plant pathogens and harmful insects, the spread of transboundary pests, consumer preference for low chemical residue and non-GMO foods, and multiple regulations that limit the widespread use of common chemical inputs such as herbicides, pesticides, and fertilizers to limit the serious impact of these chemical materials on the environment. The severity and unpredictability of these factors makes farms, forests, pastures and other areas of production very vulnerable and creates a need for greater functional resilience and adaptability. Better resource management and the ability to increase output with fewer inputs can also provide a stronger foundation for farms, forests and pastures.

Given the limited availability of suitable arable land, land suitable for forests, the environmental sensitivity of lands adjacent to these areas, the concerns of communities in which arable land exists, livestock and forest processes, the predominance of rain-fed agricultural products and additional withdrawals from rivers, lakes and aquifers to meet future irrigation needs, increasing shortages of natural nutrients such as phosphate, micronutrients, etc., the focus of everyone's attention is squarely on responsible increase in agricultural, forestry and livestock yields, productive use of water when determined by yield per unit of land area, and per megaliter of water, and per dollar of capital invested - all of which are fundamental to achieving food, fiber and structural materials security over the coming decades.

To promote crop yields and protect crops from pathogens, pests and diseases, farmers have relied heavily on the use of synthetic chemicals and chemical fertilizers; however, if abused or misused, these substances can leach into surface water, leach into groundwater, and evaporate into the air. As sources of water and air pollution, these substances are increasingly being studied, making their responsible use an environmental and commercial requirement. Even when used properly, over-reliance on and long-term use of certain chemical fertilizers and pesticides damages soil ecosystems, reduces stress tolerance, and impairs the growth and vitality of plants and animals.

As synthetic contact nematicides and soil fumigants are increasingly studied, and as new nematicides, herbicides, plant growth regulators, insecticides, bactericides and fungicides, and other crop chemical systems are declining due to increasing regulatory restrictions, environmentally friendly biological pesticides, microbial growth promoters, microbes , which increase soil nutrient levels and help efficiently use water, are becoming more important alternatives, particularly those that provide similar levels of effectiveness as conventional pesticides, fumigants, plant growth regulators, surfactants and fertilizers. Economic and environmental conditions create opportunities for biological treatment of weeds, insects and diseases caused by parasitic plant nematodes, insects, mites, bacteria and fungi. As a result, there is a significant need for more environmentally friendly solutions that make it worth investing in the research and development of new bio-based pesticides.

$1.5 billion a year is spent annually on soybean cyst nematode alone. The use of contact nematicides placed in the seed furrow at sowing is the main method of application. Due to toxicity to nearby animals such as birds, above-ground center pivot irrigation with liquid forms of toxic compounds such as nemakur, temik, furadan, dasinate and mocap are becoming unpreferred.

Since the 1960s, methyl bromide has been used by growers to effectively sterilize fields before planting, primarily for nematode control, but also for disease treatment and weed control; however, because this toxic compound is used in gaseous form, more than half the amount injected into the soil may end up in the atmosphere. As it rises into the atmosphere, it contributes to the thinning of the ozone layer. It was banned in developed countries in 2005 under the Montreal Protocol, which is an international treaty signed in 1987 to protect the stratospheric ozone layer.

- 1 045948

Under the ban, the treaty allows limited use of methyl bromide on strawberries, almonds and other crops for which there are no alternatives for both effective and affordable nematode, disease and weed control. The extent of permitted use is decreasing every year and is likely to end soon. Therefore, finding alternatives to methyl bromide is a priority for the USDA, which has provided a $5 million grant to support research to identify alternatives since 2010. However, neither product provides the broad spectrum control that methyl bromide provides.

The emergence of regulatory mandates to control the availability and use of chemicals, and consumer demands for chemical residue-free, sustainably grown products that have minimal impact on the environment in which the product and fiber are grown, has influenced the industry and led to a change in thinking in the industry. on how to solve many problems. Obviously, immediate cessation of the use of chemical reagents is not practical. Farmers faced with an imminent shift to alternative products seek acceptable alternatives with varying degrees of success. While complete elimination of chemicals is not currently feasible, farmers are increasingly using biological controls as acceptable components of Integrated Nutrient Management and Integrated Pest Management programs.

Nutrition, growth and proper functioning of plants depend on the number and distribution of healthy populations of natural microflora, which in turn are influenced by soil fertility, loosening, humidity, temperature, aeration, organic materials and many other factors. Prolonged drought, variable rainfall and other environmental changes, including the spread of nematodes and other pests and weeds, act on these factors and affect the diversity of soil microflora and plant health.

Also, chemical residues from fertilizers applied to foliage or soil, improperly processed animal waste used as fertilizers, pesticides, fungicides, herbicides, surfactants and other synthetic products can alter microbial profiles and reduce the growth and viability of plants that in turn affect animals that consume these plants or products produced from these plants.

The use of biopesticides and other biological agents is largely limited by difficulties in production, transportation, application, cost and effectiveness. For example, many microorganisms are difficult to grow and then use in industrial agriculture and forestry systems in sufficient quantities to be beneficial. Compounding this problem is the reduction in viability and/or potency due to processing, formulation, storage, stabilization before distribution, sporulation of vegetative cells as a means of stabilization, transportation and application. Moreover, once applied, biological products may not be effective for any of a number of reasons, including, for example, insufficient initial cell density, failure to effectively compete with existing microflora in a particular area, and application to soil and/or other environmental conditions in which the microorganism cannot grow or even survive - especially if it is not introduced into the environment in high enough concentrations to ensure competitive growth with those microorganisms already present in the environment into which the biological products are released, or fed with nutrients that are components of the innovative biological fertilizers

Mild pesticides, such as pesticides derived from bacteria, yeast or fungi, or their metabolites, are used; however, they are usually weaker and can pass quickly through the soil without the residual pest control effect that results from the application of persistent live biological products that continue to produce active metabolites. In fact, the addition of the entire medium (the entire fermentation product) may be important to activate and maintain the effectiveness of the biological product.

Currently, agriculture, livestock and forestry are limited to finding ways to improve microorganisms from distant producers whose product quality suffers due to previous delays in processing, methods used to stabilize the product for later distribution, supply chain constraints, improper storage and other factors that impede the timely delivery and administration of viable microbial products with high cell counts.

Thus, there is a need for systems and compositions for the efficient production and use of microorganisms and their by-products so that farmers can globally sustainably grow more food and nutritional supplements, forests to sustainably produce more fibers and building materials to better utilize dwindling and rare materials. natural resources and promoting operational sustainability and profitability.

Microorganisms and their by-products are useful in many fields, in addition to agriculture, animal husbandry and forestry. These other uses include, but are not limited to

- 2 045948 include the restoration of soil, water and other natural resources, the mining and petroleum business, animal feed, recycling and waste management, food and beverage production and processing, and healthcare. In each of these areas, a readily available source of microbial cells, spores or mycelium, as well as media for the fermentation of these microorganisms, can be highly beneficial. There is currently a need for systems that provide such microorganism-based products in a manner that produces specialized, high-viability products that are readily available to address problems that arise at a specific time and place.

Brief description of the invention

The present invention relates to 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.

In addition, the present invention relates to microorganism-based products, as well as the use of these products, which can be used to achieve beneficial results in many areas, including, for example, to improve bioremediation and mining; waste disposal and processing; improving the health of livestock and other animals; increasing the yield of oil and gas and cleaning equipment for processing and transporting oil and gas; and improving plant health and productivity through the application of one or more microorganism-based products.

In the context of agriculture, in accordance with the present invention, it is possible to obtain and effectively distribute and apply microorganism-based products having advantageous characteristics for a particular crop at a particular point in the growing season and in a particular region.

The systems of the present invention facilitate the production and use of specialized products based on microorganisms that have high concentrations of vegetative cells, reproductive spores, fungal mycelium and/or other microbial propagules, as well as the environment in which these microbes are grown. The by-products of microbial growth in this environment can be highly beneficial in a wide range of agricultural, forestry, herding and other applications. Microorganism-based products can be tailored to provide specific, targeted solutions to specific problems in a specific location within a few days or less.

The microbial products of the present invention can be used in a variety of unique situations due to, for example, the ability to effectively provide: 1) fresh fermentation medium with active metabolites; 2) a mixture of cells, spores and/or mycelia and fermentation medium; 3) compositions with vegetative cells, spores and/or mycelium; 4) compositions with a high cell density, including vegetative cells, spores and/or mycelium; 5) products based on microorganisms in a short time; and 6) microorganism-based products to remote regions.

These microorganism-based products can be used in applications including, but not limited to, crops, livestock, forestry, turfgrass, grazing, aquaculture, mining, waste management, environmental remediation, and healthcare.

In specific embodiments, the systems of the present invention provide scientific solutions that increase agricultural productivity by, for example, increasing crop viability; increasing culture yield; increasing resistance to insects and diseases; control of insects, nematodes, diseases and weeds; improving plant nutrition; improving nutritional value in agricultural, forestry and pasture soils; and ensuring improved and more efficient use of water.

In one embodiment, the system of the present invention includes a first aspect that provides an apparatus having the ability to produce a variety of microorganism-based compositions; a second aspect for distributing microorganism-based products produced by the installation; and optionally a third aspect that promotes the application of microorganism-based products in the field. As used in this context, field conditions include, but are not limited to, agriculture. Thus, field conditions can be any place where the microorganism-based products of the present invention will be used.

Additional aspects of the system may include the initial identification and development of microorganisms, as well as monitoring the number and movement of microorganisms and/or their metabolites after application.

In one embodiment, the present invention relates to a microorganism cultivation apparatus having a plurality of microorganism cultivation vessels for culturing microorganisms and producing microorganism by-products. Preferably, installation

- 3 045948 includes at least 2, 25, 50, 75, 110, 250, 500 or more (including any number between 2 and 500 or more) cultivation containers, where these containers are independently controlled and can be used for the cultivation of different microorganisms and then transporting the products from these containers for cultivation either as a single organism product or as a mixture of two or more of these products.

The microorganism cultivation apparatus of the present invention produces fresh microorganism-based compositions containing the microorganisms themselves, metabolites of the microorganisms and/or other components of the medium in which the microorganisms are grown. If desired, the compositions may have a high density of vegetative cells, spores, mycelia, other propagules, or mixtures thereof.

Advantageously, the compositions can be adapted for use in a specific area, for example, with certain crops, and to solve specific problems. In one embodiment, the microbial growth facility is located in or near an area in which microbial products will be applied to plants, to the plant environment, or otherwise used.

Advantageously, the modular system used in a microbial growth facility allows production to be scaled up or down at low setup or dismantling costs, which in some embodiments will allow the facility to be moved from one area to another. A plant for growing microorganisms does not require highly trained specialists for production and one operator can manage multiple systems for growing microorganisms. Because products are produced close to the consumer, the time and product damage associated with engineering and distribution are reduced.

Advantageously, these microbial growth facilities provide a solution to the pressing problem of dependence on distant industrial-scale producers whose product quality is degraded due to previous processing delays, supply chain constraints, inadequate storage, and other factors that impede the timely delivery and use of viable microbial products with high the number of cells and their associated media and metabolites in which the cells were originally grown. In the case of agriculture, this ability to significantly reduce the time between production and application allows farmers to grow more, have higher profits and improve sustainable farm management in the face of the impacts of climate change, limited natural resources, changing regulatory requirements, consumer preferences and other emerging market challenges. .

Advantageously, in preferred embodiments, the systems of the present invention harness the power of naturally occurring local microorganisms and their metabolic by-products to nourish, enhance and protect agricultural ecosystems, and the communities and environments in which these ecosystems exist. Enhancing local microbial populations may also be beneficial in other applications including, but not limited to, environmental remediation (e.g., oil spills), livestock, aquaculture, forestry, rangeland, turfgrass, horticultural products, disposal and recycling waste, mining, oil production, and healthcare, including in remote regions.

In one embodiment, the system includes an aspect that provides storage (usually short-term) and/or distribution of microorganism-based products. This aspect of the system may include, for example, a distribution center. In one embodiment, microorganism-based compositions and/or products are stored in a microbial growth facility. In certain embodiments, the distribution center may include an inventory center used to store microbial products prior to transportation to the point of use.

In one embodiment, the distribution center may further include a mechanical and/or technical support department that provides mechanical and technical support to customers who, for example, use microorganism-based products. The center can provide support through on-site diagnostics, analysis and troubleshooting of microbial products and/or their applications.

In one embodiment, the system of the present invention includes the additional aspect of providing field application of microorganism-based products. In the case of agriculture, the microorganism-based compositions of the present invention can be applied, for example, through an irrigation system, as a spray, as a seed treatment, on a soil surface, on a plant surface, and/or on a pest surface. Mechanical application using conventional tools or robotic means using either aerial or ground drones can also be used.

Advantageously, the systems of the present invention provide consumers, using new technologies involving cost-effective approaches, fresh, effective compositions at

- 4 045948 based on microorganisms with a high cell count, which may include metabolites and/or other by-products produced during the cultivation process. Moreover, these microorganism-based products can be tailored and produced quickly to meet the immediate needs of a specific farm or other consumer.

Thus, the distributed systems of the present invention facilitate the customized production of microorganisms, mixtures of microorganisms, fermentation media, and mixtures thereof, to meet the needs of consumers in a specific geographic region with specific needs for increased yield, efficiency, and other goals.

Brief description of drawings

Figure - Block diagram of a system for the efficient production and use of microorganisms and microbial growth by-products.

Detailed description

The present invention relates to systems for the efficient production and use of beneficial microorganisms, as well as for the production and use of substances, such as metabolites, produced by these microorganisms.

In a preferred embodiment, the present invention relates to microorganism-based compositions, as well as methods of using the compositions to promote plant health, soil microbial diversity, plant nutrition, soil nutritional capacity, optimization of soil moisture, soil aeration, soil water retention capacity and reduction of plant sensitivity to pests, diseases and weeds. This is achieved by improving the plant's natural defences, nutrient content, soil microbial health, and direct control of plant pests, diseases and weeds. This plant enhancing effect results from the application of one or more microbial products of the present invention to the plant and/or its environment.

Thus, in certain embodiments, the present invention provides compositions and methods for improving a plant's defense against external attacks using one or more microorganisms and/or microorganism-based compositions. The nature of external attacks can be biotic or abiotic. Non-limiting examples of biotic attacks include plant pests, herbivores, insects, plant disease vectors, bacteria, fungi, viruses, and invasive plants such as weeds. Abiotic attacks can be caused, for example, by changes in the plant's environment. In some embodiments, external attacks are abiotic as a result of extreme physical conditions, which include, but are not limited to, changes in temperature, light exposure, salt content, nutritional content, water quality, water availability, aeration, soil quality, and other factors affecting growth of this plant.

In one embodiment, the system of the invention includes an efficient chain for the production, distribution and use of microorganism-based products. Products may contain microorganisms themselves, as well as by-products of their growth. In preferred embodiments, the system includes installations and equipment that enable the production, distribution and use of microorganism-based products. These installations may include, but are not limited to, production, storage and distribution installations.

As used herein, a microorganism-based composition means a composition that contains components produced by the growth of microorganisms or other cell cultures. Thus, the microorganism-based composition may contain the microorganisms themselves and/or by-products of the growth of microorganisms. Microbes can be in a vegetative state, in the form of spores, in the form of mycelium, in the form of any other propagule, or in the form of a mixture of these. Microorganisms can be in the form of plankton or biofilm. Growth by-products may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. Microorganisms may also be intact or lysed. In preferred embodiments, the microorganisms in the microorganism-based composition are present with the medium in which they were grown. The cells may be present at a concentration of, for example, 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , 1 x 10 ⁹ , 1 x 10 ¹⁰ or 1 x 10 11 or more propagules per milliliter of composition. As used herein, a propagule is any part of a microorganism from which a new and/or mature organism can develop, including, but not limited to, cells, spores, mycelium, buds and seeds.

In addition, the present invention relates to microorganism-based products, which are products that are intended to be used in practice to achieve a desired result. The microbial product may simply be a microbial composition collected through a microbial culture process.

Alternatively, the microorganism-based product may contain additional added ingredients. These additional ingredients may include, for example, stabilizers, buffers, suitable vehicles such as water, saline solutions or any other suitable vehicle, to

- 5 045948 added nutrients to support further growth of microorganisms, non-nutritive growth enhancers such as plant hormones and/or agents that allow the microorganisms and/or composition to be traced in the environment into which they are introduced. The microorganism-based product may also contain mixtures of microorganism-based compositions. The microbial product may also contain one or more components of the microbial composition that have been processed in some manner, such as, but not limited to, filtration, centrifugation, lysis, drying, purification, or the like.

In accordance with the present invention, it is possible to produce and effectively distribute microorganism-based products having advantageous characteristics, for example, for a particular crop at a particular time in the growing season and in a particular region. Thus, in one embodiment, the systems of the present invention provide scientific solutions that increase agricultural productivity, for example, by increasing crop viability, enhancing insect and disease resistance, and providing improved responses to stress related to water, salt, aeration, or nutritional deficiencies.

Distributed systems for the production and use of microorganism-based products

The present invention relates to a system for producing microorganisms and/or microorganism-based products for use in certain conditions and/or environments, where the system includes:

(i) a microorganism growth apparatus comprising a plurality of modular growth vessels in which the same or different microorganisms can grow under the same or different growth conditions to produce a microorganism-based composition in each growth vessel, where the medium for growth, oxygen saturation, pH, shaking and/or temperature can be independently and/or jointly controlled for each of said containers; and wherein the microorganism-based composition is collected, thereby obtaining a microorganism-based product, which may optionally have additional components added to obtain a microorganism-based product, which can then be provided to the consumer; and (ii) a transport component for transporting said microorganism-based product(s) produced in the microbial growth apparatus, wherein said transport component comprises containers and/or channels for transporting the microorganism-based product(s) from the microbial growth apparatus to distribution center or area where the microorganism-based product will be used.

As used herein, harvest refers to removing part or all of the microorganism-based composition from the growth container. The figure is a block diagram of components according to one embodiment of the present invention. Although the components are shown sequentially for ease of explanation, there is no restriction on the sequence other than as set forth in the accompanying claims. In addition, some components may be optional.

In one embodiment, the present invention relates to systems for identifying and developing agriculturally useful strains of microorganisms; growing such microorganisms to produce, locally and on a desired scale, microorganism-based compositions having desired characteristics when applied to plants or their environment; making microorganism-based products easily and effectively available to consumers; and when using such products to ensure plant health, yield, vitality and marketability.

In one embodiment, a system for efficiently producing and using microorganisms includes: 1) identifying and/or developing microorganisms of interest; 2) obtaining a fermentable and genetically stable strain of microorganisms; 3) growing a specific strain of microorganisms either in pure form or with other microorganisms; 4) distribution of microorganisms and/or their by-products of their growth to the consumer; 5) introducing microorganisms into the area to be used.

The system of the present invention may include an aspect responsible for product development, including, for example, the development, identification and/or characterization of microorganisms and/or metabolites of microorganisms. Product development may be carried out in a local scientific laboratory or in collaboration with another research center.

Microorganisms can be identified, engineered, optionally modified, and tested for their various properties (eg, pest control properties, ability to produce a metabolite of interest, ability to grow in a particular environment, nutrient requirements, etc.). The system of the present invention may also include an innovation center equipped and staffed to meet the distinct and varied needs of consumers in specific areas.

Consumers may be, for example, farmers, foresters, herders, decorators, turf specialists, mining and/or drilling operators, local conservation officers, park and other natural resource specialists,

- 6 045948 waste processors, livestock pen operators and other livestock or agricultural operators, and bioremediation centers.

Installations for growing microorganisms

In preferred embodiments of the present invention, the microbial growth apparatus produces high-density fresh microorganisms and/or microbial growth by-products of interest at the desired scale. The installation 6 for growing microorganisms may be located in or near the application area. The plant produces compositions based on microorganisms at high densities through batch, pseudo-continuous or continuous cultivation.

The distributed microbial cultivation facilities of the present invention may be located in a location where the microbial product will be used (e.g., a farm, forest, pasture, feedlot, mine, waste treatment site, park, remediation center, or aquaculture facility), or close to the area of application. For example, a microbial growth facility may be located less than 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile (483, 402, 322, 241, 161, 121, 80, 40, 24, 16, 8, 5 or 1.6) km from the application area.

Since the microorganism-based product is produced locally without the need for the stabilization, preservation, storage and transport processes of microorganisms inherent in conventional microorganism production, a significantly higher density of living microorganisms in the vegetative state, in the form of spores and/or mycelium can be achieved, thereby requiring a smaller volume of microorganism-based product for on-site application applications or allowing the use of microorganisms at significantly higher densities when desired results are achieved. This allows the bioreactor to be scaled down (e.g., smaller fermentation vessel, less feedstock, nutrients, pH controls, and defoamers provided), making the system efficient. Local production of a microorganism-based product also facilitates the inclusion of a growth medium in the product. The growing medium may contain substances produced during fermentation, which are particularly suitable for topical application.

Locally produced persistent cultures with high microbial densities are more effective in the field than cultures that have undergone vegetative cell stabilization, have undergone sporulation, or have been in the supply chain for some time. The microbial products of the present invention are particularly advantageous over conventional products in which the cells, spores or mycelium are separated from the metabolites and nutrients present in the fermentation growth medium. Reduced transport times allow the production and delivery of fresh batches of microorganisms and/or their metabolites at a time and in volume in accordance with local needs.

In preferred embodiments, the installation 6 includes production systems that are modular in design and cost-effectively allow rapid and unlimited scaling. Moreover, microbial growth facilities support the production of multiple strains of microbes to meet the diverse and changing needs of consumers who are geographically close to the production facility.

A single microbial growth facility (eg, building) may contain, for example, 2, 5, 10, 20, 50, 75, or 100 or more microbial growth containers. Containers can have different volumes and can use different growing media and growing conditions and principles. Growing containers can be added or removed as needed. They can be moved to a new area. Preferably, each growth container has its own control and measurement systems for at least temperature and pH. In addition to monitoring and controlling temperature and pH, each container may optionally have the ability to monitor and control, for example, dissolved oxygen, shaking, foaming, microbial culture purity, production of desired metabolites, and the like.

In one embodiment, one type of microorganism is grown in a container. In alternative embodiments, multiple types of microorganisms can be grown in a single container and can grow together without adverse effects on growth or the final product. In one container, for example, 2-3 or more different microorganisms can be grown simultaneously.

The growing container may have a volume, for example, from 5 liters to 2000 liters or more. Typically, the containers have a volume of from 10 to 1500 liters, and preferably from 100 to 1000 liters, and more preferably from 250 to 750 liters, or from 400 to 600 liters. These grow containers can use a variety of growing systems.

These containers can be made, for example, from glass, polymers, metals, metal alloys and combinations thereof. Before growing microbes, the container can be disinfected or sterilized.

Advantageously, the use of multiple independent growing containers has the advantage of limited damage and economic loss if one of the containers

- 7 045948 has a mechanical problem and/or one of the batches is spoiled.

The cultivation time in separate containers can be, for example, from 1 to 7 days or more. The culture product can be collected in any of a number of different ways.

Because the microorganism-based product is produced at or near the point of use without the need for the stabilization, preservation, long-term storage and lengthy transportation processes associated with conventional production, a significantly higher density of living microorganisms can be achieved, thereby requiring a significantly smaller volume of microorganism-based product for on-site use. This allows the use of a smaller scale bioreactor (e.g. smaller fermentation vessel, smaller supplies of starting materials, nutrients, pH controls and defoamers, etc.), ensures there is no reason for cell stabilization or separation from the culture medium, and promotes compactness of the product.

The microbial growth facility provides production versatility by being able to tailor microbial products to improve interaction with finite geographic areas, regardless of topography, crop or animal type, soil profile, climate, pests, etc. For example, fermentation times typically range from one to six days, facilitating a rapid response to seasonal problems that threaten crops.

The production of multiple strains of microorganisms in the microbial cultivation plants of the present invention facilitates the provision of a wide range of products to consumers. The system is capable of providing specialized microorganisms for the needs and current conditions of a given area and operation.

Local production and delivery within, for example, 24 hours of fermentation results in pure compositions with high cell density and significantly lower transportation costs. Given the prospect of rapid advances in the development of more effective and potent microbial inocula, this ability to rapidly deliver innovations that can increase microbial yield and effectiveness is of significant benefit to consumers. In the case of agriculture, this can lead to improved quality of, for example, high-value crops, lawn grasses and ornamental plants.

Methods for producing microorganisms

The present invention relates to methods for cultivating microorganisms and producing microbial metabolites and/or other byproducts of microbial growth. In addition, the present invention relates to cultivation processes that are suitable for cultivating microorganisms and producing microbial metabolites on a desired scale. These culture processes include, but are not limited to, submerged culture/fermentation, surface culture, solid state fermentation (SSF) and/or combinations thereof, and can be carried out as aerobic or anaerobic processes.

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

Microbial culture systems typically use fermentation via submerged culture; however, surface cultivation and hybrid systems can also be used. As used herein, fermentation refers to growing cells under controlled conditions. Growth can be aerobic or anaerobic.

The microorganism cultivation vessel used in accordance with the present invention may be any fermenter or culture reactor for industrial use. The cultivation process is carried out in a container, which can be, for example, conical or tube-shaped. In one embodiment, the container may optionally have functional controls/sensors or may optionally be coupled with functional controls/sensors to quantify important factors in the culture process such as pH, oxygen, pressure, temperature, agitator power, humidity, viscosity and/or microbial density and/or metabolite concentrations. In some embodiments, cultivation may proceed without stabilizing or monitoring fermentation conditions.

In a further embodiment, the container may also be capable of monitoring the growth of microorganisms within the container (eg, determining cell counts and growth phases). Alternatively, a sample may be taken from the container daily and evaluated by methods known in the art, such as by dilution culture. Dilution culture is a simple method used to estimate the number of bacteria in a sample. The method may also provide an index by which different environmental conditions or treatments can be compared.

In one embodiment, the method includes supplementing the cultivation with a nitrogen source.

- 8 045948

The nitrogen source may be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea and/or ammonium chloride. These nitrogen sources can be used independently or in combination of two or more.

The method can provide oxygenation of the culture for cultivation. In one embodiment, slow air movement is used to remove low-oxygen air and introduce oxygenated air. Oxygenated air may be ambient air provided daily by mechanisms including agitators to mechanically agitate the liquid and air distributors to introduce gas bubbles into the liquid to dissolve oxygen in the liquid.

In addition, the method may include supplementing the cultivation with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol and/or maltose; 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, corn oil, sesame oil and/or flaxseed oil; etc. These carbon sources can be used independently or in combination of two or more.

In one embodiment, growth factors and micronutrients for microorganisms are included in the medium. This is particularly advantageous when growing microorganisms that are unable to produce all the vitamins they require. Inorganic nutrients may also be included in the medium, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt.

In one embodiment, inorganic salts may also be included. Suitable inorganic salts may be potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferric chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate and/or sodium carbonate. These inorganic can be used independently or in combination of two or more.

In some embodiments, the culture method may further include adding additional acids and/or antimicrobial agents to the liquid medium prior to and/or during the culture process. Antimicrobials or antibiotics are used to protect the crop from contamination. In addition, defoamers can also be added to prevent the formation and/or accumulation of foam when gas is generated during cultivation.

The pH of the mixture must be suitable for the microorganism of interest. Buffers and pH adjusters such as carbonates and phosphates can be used to stabilize the pH around the preferred value. When metal ions are present in high concentrations, the use of a chelating agent in a liquid medium may be necessary.

The method and equipment for cultivating microorganisms and producing microbial by-products may correspond to batch, pseudo-continuous or continuous processes.

Microorganisms can be grown in planktonic form or as a biofilm. In the case of biofilm, the container may contain a substrate on which microorganisms can grow in the form of a biofilm. The system may also have, for example, the ability to apply stimuli (such as shear stress) that improve and/or enhance biofilm growth characteristics.

In one embodiment, the method of cultivating microorganisms is carried out at a temperature of from about 5° to about 100°C, preferably from 15 to 60°C, more preferably from 25 to 50°C. In a further embodiment, the cultivation can be carried out continuously at a constant temperature. In another embodiment, temperatures may vary during cultivation.

In one embodiment, the equipment used in the culture method and process is sterile. Culture equipment such as a reactor/vessel may be separate from, but connected to, a sterilization system such as an autoclave. The culture equipment may also have a sterilization system that performs in situ sterilization prior to inoculation. The air can be sterilized by methods known in the art. For example, ambient air may pass through at least one filter before entering the container. In other embodiments, the medium may be pasteurized, or optionally no heating at all, where low water activity and low pH may be used to control bacterial growth.

In one embodiment, the present invention relates to a method for producing microbial metabolites such as proteins, peptides, metabolic intermediates, polyunsaturated fatty acids and lipids. The content of metabolites produced in this way can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

- 9 045948

The biomass content of the fermentation medium can be, for example, from 5 g/L to 180 g/L or more. In one embodiment, the solids content of the medium is from 10 g/L to 150 g/L.

In one embodiment, a method for producing a microorganism metabolite of the present invention includes the steps of: 1) mixing hydrophobic and hydrophilic particles to produce a matrix-forming solid; 2) bringing said matrix-forming solid into contact with a medium that is inoculated with the microorganism of interest, thereby producing a microreactor matrix; 3) growing said microorganism in said microreactors; and 4) collecting said metabolite produced by said microorganism.

The microbial growth byproduct produced by the microorganisms of interest may be retained in the microorganisms or secreted into the liquid medium. In another embodiment, the method for producing a microbial growth byproduct may further include the steps of concentrating and purifying the microbial growth byproduct of interest. In a further embodiment, the liquid medium may contain compounds that stabilize the activity of the microbial growth by-product.

In one embodiment, after completion of the culture, the entire composition for cultivating microorganisms is removed (eg, when the desired cell density or density of a particular metabolite in the medium is achieved). In this batch procedure, after the first batch is harvested, a completely new batch is started to be cultivated.

In another embodiment, only a portion of the fermentation product is recovered at any one time. In this embodiment, the biomass with viable cells remains in the container as an inoculum for culturing a new batch. The composition that is recovered may be a cell-free medium or may contain cells. Thus, a pseudo-continuous system is created.

Obtaining products based on microorganisms

One of the microorganism-based products of the present invention is simply a fermentation medium containing the microorganism and/or microbial metabolites produced by the microorganism and/or any residual nutrients. The fermentation product can be used directly without extraction or purification. If desired, extraction and purification can be easily accomplished using standard extraction methods or technologies described in the literature.

Microorganisms in a microorganism-based product may be in active or inactive form. Products based on microorganisms can be used without further stabilization, preservation and storage. Advantageously, immediate application of these microorganism-based products maintains high microbial viability, reduces the possibility of contamination by foreign agents and unwanted microorganisms, and maintains the activity of microbial growth by-products.

The microorganisms and/or the medium resulting from the cultivation of the microorganisms can be removed from the growth vessel and transported, for example through a pipeline, for immediate use.

In other embodiments, the composition (microorganisms, medium, or both microorganisms and medium) may be placed in containers of a suitable size, taking into account, for example, the intended use, the intended method of use, the size of the fermentation vessel, and any method of transport from the microorganism growth facility to application area. Thus, the containers in which the microorganism-based composition is placed may have a volume, for example, from 1 gallon to 1000 gallons (3.8 L to 3800 L) or more. In other embodiments, the containers have a volume of 2 gallons (7.2 L), 5 gallons (19 L), 25 gallons (95 L), or larger.

When collecting a microorganism-based composition from growth containers, additional components may be added as the collected product is placed into containers and/or piped (or otherwise transported for use). Additives may be, for example, buffers, carriers, other microbial compositions produced in the same or a different plant, viscosity modifiers, preservatives, microbial growth nutrients, plant growth nutrients, tracking agents, pesticides, herbicides, animal feed, foodstuffs and other ingredients specialized for the intended use.

Advantageously, in accordance with the present invention, the microorganism-based product may contain a medium in which the microorganisms are grown. The product may constitute, for example, at least by weight, 1%, 5%, 10%, 25%, 50%, 75% or 100% of the medium. The amount of biomass in the product by weight can be, for example, any amount from 0% to 100%, including all percentages in between.

Optionally, the product can be stored before use. Preferably, the storage time is short. Thus, the storage time can be less than 60 days, 45 days, 30 days,

- 10 045948 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day or 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at a cold temperature, such as, for example, less than 20°C, 15°C, 10°C or 5°C. On the other hand, the biological surfactant composition can generally be stored at ambient temperature.

The microorganism-based products of the present invention may be, for example, microbial inocula, biopesticides, nutrient sources, remediation agents, health products, and/or biological surfactants.

Distribution of microorganism-based products

The system does not necessarily include a microbial product distribution aspect. In one embodiment, this aspect includes a distribution center 3, which may optionally include an inventory center 4. The inventory center 4 may be responsible for storing and further distributing microorganism-based products. The inventory center may provide consumer services, including, for example, advising consumers regarding available and available microbial inoculum.

In one embodiment, the distribution center 3 may provide services relating to the sale, storage and transport of microorganism-based products. The distribution center 3 can also provide mechanical and technical support.

Application of microorganism-based products

In another aspect, the methods and systems of the present invention may include methods, systems and devices for using microorganism-based products.

In the case of agriculture, the compositions may be applied, for example, to an irrigation system, sprayed from backpacks or similar devices, applied by ground or aerial robotic devices such as a drone, and/or applied to seeds. Application to seeds can be carried out, for example, by coating the seeds or applying the composition to the soil at the same time as sowing the seeds. This may be done automatically, for example, by providing a device or irrigation system that applies the microorganism-based composition with and/or adjacent to the seeds at or around the time of sowing the seeds. Thus, the microorganism-based composition can be applied within, for example, 5, 4, 3, 2 or 1 days before or after sowing or simultaneously with sowing the seeds.

In some agricultural embodiments, the compositions described herein, as either a dry or liquid formulation, are applied by seed treatment or to the soil surface, to the plant surface, and/or to a pest or weed surface.

In certain embodiments, the compositions described herein are applied to the soil surface without the involvement of mechanical means. The beneficial effect of soil application can be activated by rain, sprinkler irrigation, flood irrigation or drip irrigation and then transferred, for example, to target pests to ensure that their population levels are reduced to acceptable thresholds, or to plant roots to influence root microbial control or facilitate microbial uptake product into the vascular system of the crop or plant into which the microbial product is introduced. In an illustrative embodiment, the compositions described herein can be effectively applied through a center pivot irrigation system or by spraying into a seed furrow.

Reference herein to application of a composition to or near a pest or plant, or to the environment of a pest or plant, means that the application is such that the composition sufficiently contacts the pest or plant to achieve the desired result (e.g., killing the pest, increasing yield , preventing plant damage, regulation of genes and/or hormones, etc.). Typically, it may be within, for example, 10, 5, 3, 2 or 1 ft (3, 1.5, 0.9, 0.6 or 0.3 m) or less of a pest, weed or other desired targets.

The microbial product can also be applied to promote colonization of the roots and/or rhizosphere, as well as the plant's vascular system, to promote plant health and vitality. In this way, nutrient-fixing microorganisms such as rhizobium and/or mycorrhiza, as well as other endogenous (already present in the soil) as well as exogenous microorganisms or their by-products, can be stimulated to control pests, weeds or diseases, or otherwise thereby improving growth, health and/or yield. The microbial product may also support the plant vascular system, for example, by entering and colonizing said vascular system and delivering metabolites and nutrients important for plant health and productivity, or metabolites with pest control properties.

Advantageously, the method does not require complex equipment or high energy consumption. Microorganisms of interest can be cultured on a small or large scale in situ and used while still mixed with their environment. Likewise, metabolites of microorganisms can be

- 11 045948 receive in large quantities in the required area.

Advantageously, microorganism-based products can be produced in remote regions. In one embodiment, microorganism-based products can be used for human nutrition and/or the prevention and/or treatment of diseases. Installations for growing microorganisms can operate autonomously using, for example, solar, wind and/or hydroelectric energy.

Microorganism-based products can be applied directly to animal waste and/or used in a waste treatment facility. Microorganism-based products can also be applied directly to a contaminated environment, such as an oil spill or hazardous waste area. Microbial products can also be added to the ore to isolate metals, minerals or other substances of interest. Microorganism-based products can also be injected into oil wells and/or pipes, tanks and other equipment associated with oil wells and/or oil processing. Thus, the scope of application includes drilling equipment, petroleum storage equipment, transfer pipeline, tankers and oil refineries.

Microorganism inocula

In one embodiment, the microorganism-based product is an inoculum of microorganisms. When applied to, for example, seed, plant or soil in row crops, silvicultural operations, managed pastures, horticultural crops, controlled turf grasses, animal waste and/or animal feed, the inoculum becomes integral to the qualities of the treated soil or managed environment and stimulates the healthy growth of naturally occurring beneficial microorganisms that are beneficial to that soil or environment, or to the plants and animals that are grown, fed, or otherwise interact with that soil and environment. Once applied to the soil, the inoculum of microorganisms of the present invention improves the mineralization of organic matter, increases nitrogen fixation required for photosynthesis, increases the availability of phosphorus to crops by limiting its leakage into the environment, produces beneficial plant signaling metabolites, stimulates the growth of root mass by facilitating the uptake of water and key nutrients, increase soil fertility and/or increases the amount of biomass.

In one embodiment, inocula can be individually tailored by crop or geography to facilitate persistent colonization by beneficial microorganisms, making this technology ideal for early control of certain crops grown in vastly different soil ecosystems. The ability to individually adapt microorganisms to meet the needs of different soil ecosystems becomes even more important as we understand how complex microbial communities respond to extreme temperatures, prolonged drought, variable rainfall and other factors resulting from climate change and intensive agriculture.

Due to the high density of vegetative cells, spores, mycelia, and/or other microbial propagules, in certain embodiments of the present invention, the microbial products of the present invention are uniquely advantageous due to their ability to colonize the environment, such as soil, and interact favorably with existing microflora . Due to the exceptionally high number of cells, spores and/or mycelia, the microorganism-based products of the present invention allow long-term survival of microorganisms in soil (or other suitable environment). This survival can be further increased and prolonged, for example, by providing the microorganisms with nutrients.

In one embodiment of the present invention, the survival and persistence of microorganisms is monitored by tracking and/or quantifying the microorganisms and/or their movement in the soil or other environment.

Biological control agents

In another embodiment, the microorganism-based product is a biological control agent. Compared to conventional synthetic chemical pesticides, which can pollute the environment and adversely affect non-target plants and animals, biopesticides are non-toxic, safe to use and can be highly specific. Biopesticides, which are best used as a preventive, rather than curative, tool to control weeds, diseases, nematodes and insects and other pests, allow farmers to reduce their traditionally high dependence on chemical pesticides and herbicides without affecting crop yield. Biopesticides help create an environment in which pests are unable to establish and thrive, a key benefit given that pest proliferation is associated with adverse weather. The use of biopesticides also allows farmers to reduce soil contamination for rotational crops, toxicity to non-target plants and animals, crop toxicity, development of pesticide resistance, and yield and leakage in chemicals.

- 12 045948 environmentally sensitive areas, water sources, etc. and other consequences of the use of chemical pesticides.

Resistance to chemical pesticides is a major problem because resistant pests and insects threaten agricultural productivity and, once resistance develops, become costly to control.

Soil changes

In one embodiment, the microbial product is intended to amend soil for use in improving the health, growth and yield of plants and/or crops, such as in agriculture, horticulture, greenhouse cultivation, landscaping, and the like. The present invention is also used to improve one or more soil qualities, thereby increasing the effectiveness of the soil for agricultural, domestic and horticultural purposes.

As used herein, enhancement means improvement or increase. For example, enhancing plant health means improving the plant's ability to grow and develop, including the plant's ability to resist pests and/or disease, and the plant's ability to survive drought and/or waterlogging. Increased plant growth means an increase in the size and/or weight of the plant, increasing the ability of the plant to achieve the required size and/or weight. Enhanced yield means improving the end products produced by plants in cultivation, for example, by increasing the number of fruits per plant, increasing fruit size, and/or improving fruit quality (e.g., flavor, texture).

As used herein, a soil amendment or soil improver is any compound, material, or combination of compounds or materials that is added to soil to improve the physical properties of the soil. The soil amendment may include organic and inorganic matter and may further include, for example, fertilizers, pesticides and/or herbicides. Nutrient-rich, well-draining soil is essential for plant growth and health, and thus soil amendments can be used to enhance plant growth and health by altering the nutrient and moisture content of the soil. Soil amendment can also be used to improve many different soil qualities, including, but not limited to, soil structure (eg, preventing compaction); increasing nutrient concentration and storage capacity; increasing water retention in dry soils; and increasing drainage in waterlogged soils.

In preferred embodiments, the methods of the invention include the application of a microorganism-based product, i.e. means for modifying the soil, based on microorganisms, into the soil in which a plant or crop grows or will be sown. A microorganism-based product may contain a microorganism and/or a by-product of the growth of a microorganism. In preferred embodiments, the growth byproduct is a sophorolipid biological surfactant.

In some agricultural embodiments, the compositions described herein, either in a dry or liquid formulation, are applied as a seed treatment to either the soil surface or the plant surface (eg, the plant root surface).

In certain embodiments, the compositions described herein are applied to the soil surface without the involvement of mechanical means. The beneficial effect of soil application can be activated by rain, sprinkler, flood or drip irrigation and then transmitted, for example, to plant roots to influence the root microbiome or facilitate uptake of the microbial product into the vascular system of the crop and plant to which the microbial product is applied. In an illustrative embodiment, the compositions described herein can be effectively applied through a center pivot irrigation system or by spraying into a seed furrow.

The microbial product can also be applied to stimulate colonization of the roots and/or rhizosphere, as well as the plant's vascular system, to improve plant health and vitality. In this way, nutrient-fixing microorganisms such as rhizobium and/or mycorrhiza, as well as other endogenous (already present in the soil) as well as exogenous microorganisms or their by-products, can be stimulated to improve crop growth, health and/or yield. The microbial product may also support the plant vascular system, for example, by entering and colonizing said vascular system and providing metabolites and nutrients important for plant health and productivity.

The soil can be cultivated at any time during the plant cultivation process. For example, the microbial product may be applied to the soil prior to sowing seeds or at any time thereafter during the development and growth of the plant or plants.

In some embodiments, the method includes bringing soil into contact with a microorganism in combination with biological surfactants that they produce. Microorganisms may be either living (or viable) or inactive at the time of application. In addition, methods may include adding microbial growth promoting materials during application (eg, adding nutrients to promote plant growth). IN

- 13 045948 In one embodiment, the nutrient sources may include, for example, nitrogen, nitrate, phosphorus, magnesium and/or carbon.

In other embodiments, the method includes simply adding biological surfactants to soil without microorganisms.

In one embodiment, the method includes applying a microbial product containing the yeast Starmerella bombicola and/or its growth by-products to the soil, where the composition allows enhanced absorption of water and/or nutrients from the soil. In one embodiment, the microorganism of the microorganism-based product is Wickerhamomyces anomalus. In another embodiment, the microorganism-based product contains SLP without microorganisms. Advantageously, the methods improve plant health, growth and/or yield without the use of harsh chemicals.

The present invention can be used to improve any number of qualities in any type of soil, for example, clay, sand, silt, peaty, calcareous, loamy and/or combinations thereof. Moreover, the methods and compositions can be used to improve the quality of dry, waterlogged, porous, depleted, compacted soil and/or combinations thereof.

In one embodiment, the method can be used to improve drainage and/or water dispersal in waterlogged soils. In one embodiment, the method can be used to increase water retention in dry soil.

In one embodiment, the method can be used to increase nutrient retention in porous and/or depleted soils.

Microorganism-based products can be used either alone or in combination with other compounds to effectively enhance plant health, growth and/or yield. For example, commercial and/or natural fertilizers, pesticides, herbicides and/or other soil amendments may be applied along with the microbial product. In certain embodiments, microorganism-based products can be used to enhance the effectiveness of other compounds, for example, by promoting retention of the compound in the soil or providing a more uniform distribution of the compound in the soil.

In other applications, desired soil attributes can be achieved by adding various materials to the soil, including, for example, bone meal, alfalfa, corn gluten, potassium carbonate and/or manure from various animals, including horses, cows, pigs, chickens, bats, sheep Other additional elements that may be added include, but are not limited to, mineral nutrients such as magnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur, iron, copper and zinc. The exact materials and quantities can be determined by a soil specialist.

Biological surfactants

In one embodiment, the microorganism-based product contains a biological surfactant. Microbial biological surfactants are compounds produced by various microorganisms such as bacteria, fungi and yeast. Biological surfactants constitute an important class of secondary metabolites that are found in many microorganisms, such as Starmerella spp., Pichia spp., Pseudomonas spp. (P. aeruginosa, P. putida, P. florescens, P. fragi, P. syringae); Flavobacterium spp.; Bacillus spp. (B. subtilis, B. pumillus, B. cereus, B. licheniformis); Candida species (C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. torulopsis); Rhodococcus sp.; Arthrobacter spp.; campylobacter spp.; cornybacterium spp. etc. Safe, effective microbial biological surfactants reduce surface and interfacial tension between molecules of liquids, solids and gases. These dynamics can be used to promote plant health, increase yields, control soil aeration, and responsibly use available irrigation water sources.

Thus, in one embodiment, the microbial product improves the health and productivity of plants subject to water stress.

Biological surfactants are unique in that they are produced through the fermentation of microorganisms, but have the same properties as chemical surfactants, in addition to other attributes that their synthetic counterparts do not possess. Biological surfactants reduce the tendency of water to pool, they increase the adhesion or wettability of surfaces, resulting in greater hydration of the entire rhizosphere, and they reduce the volume of water that might otherwise escape below the root zone through microchannels created by drip and drip systems. microirrigation. This wettability also ensures better root health as there are fewer areas of desiccation (or extreme dryness) to inhibit proper root growth and increased availability of applied nutrients as chemical nutrients and micronutrients are more readily available and distributed.

The more uniform distribution of water in the rhizosphere of the crop, which becomes possible as a result of increased wettability, also prevents the accumulation or trapping of water above the maximum limit.

- 14 045948 optimal penetration levels, thereby mitigating anaerobic conditions that inhibit the free exchange of oxygen and carbon. After effective application of biological surface matter, a more porous or breathable rhizosphere is formed and roots become more resistant to some soil-borne diseases. The combination of a properly hydrated and aerated rhizosphere also increases the sensitivity of soil pests and pathogens (such as nematodes, and soil-borne fungi and their spores) to chemical pesticides and biopesticides. Biological surfactants can be used for a wide range of beneficial applications, including disease and pest control.

Biological surfactants produced in accordance with the present invention can be used for other non-agricultural purposes, including, for example, cleaning pipes, reactors and other devices or surfaces.

The biological surfactants of the present invention include, for example, low molecular weight glycolipids (GL), lipopeptides (LP), flavolipids (FL), phospholipids and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes and polysaccharide-protein-fatty acid complexes.

Biological surfactants are biodegradable and can be easily and inexpensively produced using individual organisms on renewable substrates. Most biological surfactant-producing organisms produce biological surfactants in response to the presence of a source of hydrocarbons (eg, oils, sugar, glycerol, etc.) in the growth medium. Other media components, such as iron concentration, can also significantly influence the production of biological surfactants.

In one embodiment, the microbial biological surfactant is a glycolipid such as rhamnolipid, sophorolipids (SLP), trehalose lipid, and mannosylerythrol lipid (MEL).

Microbial factories

The microorganisms and their products obtained according to the present invention can be used to obtain a wide range of useful products, including, for example, biopesticides, biological surfactants, ethanol, nutritional compounds, therapeutic proteins such as insulin, compounds useful as vaccines , and other biopolymers. The microorganisms used as these microbial factories can be natural, mutant or recombinant.

Microorganisms grown in accordance with the present invention

Microorganisms grown in accordance with the systems and methods of the present invention may be, for example, bacteria, yeast and/or fungi. These microorganisms may be natural or genetically modified microorganisms. For example, microorganisms can be transformed with certain genes to exhibit certain characteristics.

In specific embodiments, the microorganisms are bacteria, including gram-positive and gram-negative bacteria. The bacteria may be, for example, Bacillus firmus, Bacillus laterosporus, Bacillus megaterium, Bacillus amyloliquifaciens, Azobacter vinelandii, Pseudomonas chlororaphis subsp. aureofaciens (Kluyver), Agrobacterium radiobacter, Azospirillumbrasiliensis, Azobacter chroococcum, Rhizobium, Sphingomonas paucimobilis, Ralslonia eulropha, Clostridium (C. butyricum, C. tyrobutyricum, C. acetobutyricum, Clostridium NIPER 7 and C. beijerinckii) and/or Rhodospirill um rubrum.

In another embodiment, the microorganism is a yeast organism or a fungus. Yeast and fungal species suitable for use in accordance with the present invention include Candida, Saccharomyces cerevisiae, Saccharomyces boulardii sequela and Saccharomyces torula, Issalchenkia, Kluyveromyces, Pichia, Wickerhamomyces, Starmerella, Mycorrhiza, Mortierella, Phycomyces, Blakeslea, Thraustochytrium, Phythium, Entomophthora , Aureobasidium pullulans, Fusarium venenalum, Aspergillus, Trichoderma reesei and/or Rhizopus spp.

In one embodiment, the yeast is killer yeast. As used herein, killer yeast means a strain of yeast characterized by its secretion of toxic proteins or glycoproteins to which the strain itself is resistant. Exotoxins secreted by killer yeast can cause the death of other strains of yeast, fungi, or bacteria. For example, microorganisms that can be controlled by killer yeasts include Fusarium and other filamentous fungi. Examples of killer yeasts in accordance with the present invention are killer yeasts that can be safely used in the food and fermentation industries, for example, to make beer, wine and bread; killer yeasts, which can be used to control other microorganisms that may contaminate such production processes; killer yeast, which can be used in biocontrol for food preservation; killer yeast that can be used to treat fungal infections in both humans and

- 15 045948 and plants; and killer yeast, which can be used in recombinant DNA technology. Such yeast may include, but are not limited to them, WickerHamomyces, Pichia (for example, Pichia Anomala, Pichia Guailliermondii, Pichia Kudrizvzevi) Hansenula, Saccharmyces, Hanseniaspora, such as Hanseniaspora Uvarum, Uvarum, Ustilag o Maydis, Debaryomyces Hansenii, Candida, Cryptococcus, Kluyveromyces, Toriopsis, Ustilago, Williopsis, Zygosaccharomyces such as Zygosaccharomyces bailii, and others.

In other embodiments, the microorganism may be a cell culture, such as, for example, an insect, nematode, or mammalian cell culture.

Target plants

Plants that may benefit from the use of the products and methods of the present invention include: row crops (e.g., corn, soybeans, sorghum, peanuts, potatoes, etc.), field crops (e.g., alfalfa, wheat, cereals, etc.) .etc.), tree crops (e.g. walnuts, almonds, pecans, hazelnuts, pistachios, etc.), citrus crops (e.g. orange, lemon, grapefruit, etc.), fruit crops (e.g. apples , pears, etc.), lawn crops, ornamental crops (e.g. flowers, climbing plants, etc.), vegetables (e.g. tomatoes, carrots, etc.), vine crops (e.g. grapes, strawberries , blueberries, blackberries, etc.), forest trees (e.g. pine, spruce, eucalyptus, poplar, etc.), commercial grasslands (any mixture of plants used to support herbivores).

Benefits may take the form of, for example, increased yield, quality, disease and pest resistance, reduced stress (eg salt, drought, heat, etc.) and improved water use.

The following plants that may benefit from the products and methods of the invention include all plants that belong to the superfamily Viridiplantae, in particular monocotyledons and dicotyledons, including forage or forage legumes, ornamental plants, food crops, trees or shrubs of Acer spp. , Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, oilseed turnip]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera ), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis , Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum , Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., and others.

The following examples of plants include, but are not limited to, corn (Zea mays), Brassica sp. (e.g. B. napus, B. rapa, B. juncea), in particular those Brassica species that are suitable as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum ( Sorghum bicolor, Sorghum vulgare), bristle grass (e.g. pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius ), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea),

- 16 045948 cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp. ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea) , papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetables, ornamental plants and coniferous plants.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (such as Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the Cucumis genus such as cucumber (C. sativus ), cantaloupe (C. cantalupensis) and cantaloupe (C. melo). Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Coniferous plants that can be used to practice the embodiments include, for example, pine trees such as lodgepole pine (Pinus taeda), Elliott pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta) and radiata pine (Pinus radiata); California fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); sequoia (Sequoia sempervirens); true firs such as white fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as arborvitae (Thuja plicata) and Alaska cedar (Chamaecyparis nootkatensis). Plants in embodiments include crop plants (eg, corn, alfalfa, sunflower, kale, soybeans, cotton, safflower, peanuts, sorghum, wheat, millet, tobacco, etc.) such as corn and soybeans.

Lawn grasses include, but are not limited to: Poa annua; annual ryegrass (Lolium multiflorum); bluegrass (Roa compressa); red fescue (Festuca rubra); thin bentgrass (Agrostis tenuis); marsh bentgrass (Agrostis palustris); wheatgrass (Agropyron desertorum); desert wheatgrass (Agropyron cristatum); tough fescue (Festuca longifolia); meadow bluegrass (Rhoa pratensis); hedgehog (Dactylis glomerate); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); white bentgrass (Agrostis alba); common bluegrass (Poa trivialis); grooved fescue (Festuca ovine); awnless brome (Bromus inermis); tall fescue (Festuca arundinacea); timothy grass (Phleum pretense); dog bentgrass (Agrostis canine); distans (Puccinellia distans); Smith's wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); Augustine grass (Stenotaphrum secundatum); Japanese zoysia (Zoysia spp.); noticeable buckwheat (Paspalum notatum); compressed axonopus (Axonopus affinis); snaketail eremochloa (Eremochloa ophiuroides); Kikuyu (Pennisetum clandesinum); vaginal paspalum (Paspalum vaginatum); sweet bison (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); Bouteloua curtipendula.

Plants of interest include cereal plants that provide seeds of interest, oilseeds and leguminous plants. Seeds of interest include seeds of cereal crops such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oilseed plants include cotton, soybean, safflower, sunflower, cabbage, maize, alfalfa, palm, coconut, flax, castor bean, olive, etc. Legumes include beans and peas. Legumes include cyamopsis, carob, fenugreek, soybean, bean, cowpea, golden bean, lima bean, green bean, lentil, snap pea, etc.

Target pests

The products and methods of the present invention can be used to reduce damage caused by a wide range of pests.

Examples of such pests include arthropods, including, for example, Lepidoptera (for example, Plutellidae, Noctuidae, Pyralidae, Tortricidae, Lyonetiidae, Carposinidae, Gelechiidae, Crambidae, Arctiidae and Lymantiida ), Hemiptera (for example, Cicadellidae, Delphacidae, Psyllidae, Aphididae, AlayRodidae, Orthezidae , Miridae, Tingidae, Pentatomidae and Lygaiedae), Coleoptera (e.g. Scarabaeidae, Elateridae, Coccinellidae, Cerambycidae, Chrysomelidae and Curculionidae), Diptera (e.g. Muscidae, Calliphoridae, Sarcophagidae, Anthomyiidae, Tephritidae, Opomyzoidea and Carnoidea), Orthoptera (e.g. Acrididae, Catantopidae and Pyrgomorphidae), Thysanoptera (e.g. Thripidae, Aeolothripidae and Merothripidae), Tylenchida (e.g. Aphelenchoididae and Neotylechidae), Collembola (e.g. Onychiurus and Isotomidae), Acarina (e.g. Tetranychidae, Dermanyssidae, Acaridae and Sarcoptidae), Styl ommatophora (e.g. Philomycidae and Bradybaenidae), Ascaridida (e.g. Ascaridida and Anisakidae), Opisthorchiida, Strigeidida, Blattodea (e.g. Blaberidae, Cryptocercidae and Panesthiidae) and Thysanura (e.g. Lepismatidae, Lepidotrichidae and Nicoletiidae).

Examples of pests classified as Lepidoptera include Chilo suppressalis Walker, Cnaphalocrocis medinalis, Parnara guttata, Sesamia inferens, Mythimna separata, Naranga aenescens Moore, Spodoptera litura, Etiella zinckenella, Etiella behrii, Matsumuraeses falcana, Leguminivora glycinivorella, Pleuroptya naafis, Agrotis seget um, Agrotis ipsilon, Helcystogramma triannulellum, Xestia c-nigrum, Helicoverpa assulta, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Plutella xylostella, Pieris rapae, Pieris brassicae, Hel

- 17 045948 lulla undalis and Autographa nigrisigna.

Examples of pests classified as Hemiptera include Nilaparvata lugens, Sogatella furcifera, Laodelphax stratella, Nephotettix cincticeps, Recilia dorsalis, Stenotus rubrovittatus, Trigonotylus caelestialium, Leptocorisa chinensis, Nezara antennata, Nezara viridula, Lagynotomus elongatus, Scotinophara lurida, Eys Arcoris annamita, Eysarcoris lewisi, Eysarcoris ventralis, Togo hemipterus Scott, Cletus punctiger, Piezodorus hybneri, Halyomorpha halys, Dolycoris baccarum, Neotoxoptera formosana, Rhopalosiphum padi, Rhopalosiphum maidis and Aphis glycines.

Examples of pests classified as Coleoptera include Rice Lissorhoptrus oryzophilus, Oulema oryzae, Echinocnemus squameus, Melanotus legatus, Melanotus fortnumi, Anomala cuprea, Popillia japonica, Maladera castanea, Epilachna varivestis, Paraluperodes nigrobilineatus, Epilachna vigintioctomaculata, Henosepilach na vigintioctopunctata, Harmonia axyridis, Anomala rufocuprea , Anomala testaceipes, Aulacophora indica and Phyllotreta striolata.

Examples of pests classified as Diptera include Chlorops oryzae, Hydrellia griseola, Sitodiplosis mosellana, Delia platura, Asphondylia yushimai, Melanagromyza sojae, Liriomyza trifolii, Liriomyza sativae, Liriomyza huidobrensis and Liriomyza bryoniae.

Examples of pests classified as Orthoptera include Ohua yezoensis and Ohua japonica. Examples of pests classified as Thysanoptera include Stenchaetothrips biformis and Thrips palmi. Examples of pests classified as Tylenchida include Meloidogyne, Nematoda, and Heterodera. Examples of pests classified as Collembola include Onchiurus psuedamatus yagii and Onychiurus matsumotoi. Examples of pests classified as Acarina include Penthaleus major, Tetranychus urticae, Tetranychus kanzawai, Tyrophagus putrescentiae and Tarsonemus bilobatus. Examples of pests classified as Stylommatophora include Helix and Philomycidae. Examples of pests classified as Ascaridida include Ascaris lumbricoide. Examples of pests classified as Opisthorchiida include Metagonimus yokogawai. Examples of pests classified as Strigeidida include Schistosoma japonicum. Examples of pests classified as Blattodea include Blattella germanica, Periplaneta fuliginosa, Periplaneta americana and Blatta lateralis. Examples of pests classified as Thysanura include Ctenolepisma and Lepisma.

In one embodiment, the present invention relates to methods and products for controlling nematodes. Nematodes are a class of roundworms or threadworms of the genus Nemathelminthes. Nematodes are also known as roundworms. Examples of members of the class are cyst nematodes of the genus Heterodera, blunt root nematodes of the genus Trichodorus, bulb and stem nematodes of the genus Ditylenchus, golden nematode Heterodera rostochiensis, root nodule nematodes of the genus Meloidogyne, root-damaging nematodes of the genus Pratylenchus, citrus nematodes genus Tylenchulus, stinging nematodes of the genus Belonalaimus, and parasitic plant nematodes of genera such as Naccobus and Radopholus. In an illustrative embodiment, the method and products inhibit the formation of root galls caused by nematodes such as root-nodule nematodes and cyst nematodes.

Target plant diseases

Examples of plant diseases that can be controlled using the present invention include the following:

Wheat diseases: fusarium rot (Fusarium graminearum, F. avenacerum, F. culmorum, Microdochium nivale), typhullosis (Typhula sp., Micronectriella nivalis), loose smut (Ustilago tritici, U. nuda), loose smut (Tilletia caries), blight leaves (Mycosphaerella graminicola) and septoria (Leptosphaeria nodorum);

Corn diseases: smut (Ustilago maydis) and brown spot (Cochliobolus heterostrophus);

Citrus diseases: melanosis (Diaporthe citri), scab (Elsinoe fawcetti) and mold (Penicillium digitatum, P. italicum);

Apple tree diseases: gray mold (Monilinia mali), powdery mildew (Podosphaera leucotricha), Alternaria (apple pathogen Alternaria alternata), scab (Venturia inaequalis), bitter rot (Colletotrichum acutatum) and root collar rot (Phytophtora cactorum);

Pear diseases: scab (Venturia nashicola, V. pirina), black spot (pear pathogen Alternaria alternata Japanese), rust (Gymnosporangium haraeanum) and late blight fruit rot (Phytophtora cactorum);

Peach tree diseases: brown rot (Monilinia fructicola), scab (Cladosporium carpophilum) and Phomopsis sp.;

Grape diseases: anthracnose (Elsinoe ampelina), berry rot (Glomerella cingulata), black spot (Guignardia bidwellii), downy mildew (Plasmopara viticola) and gray mold (Botrytis cinerea);

Diseases of Japanese persimmon: anthracnose (Gloeosporium kaki) and leaf spot (Cercospora kaki, Mycosphaerella nawae);

Pumpkin diseases: anthracnose (Colletotrichum lagenarium), target leaf spot (Corynespora cassiicola), black rot (Mycosphaerella melonis), fusarium wilt (Fusarium oxysporum), downy mildew (Pseudoperonospora cubensis) and late blight (Phy

- 18 045948 tophthora sp.);

Tomato diseases: Alternaria solani, leaf mold (Cladosporium fulvum) and late blight (Phytophthora infestans);

Cruciferous diseases: Alternaria blight (Alternaria japonica), white spot (Cercosporella brassicae) and downy mildew (Peronospora parasitica);

Diseases of rapeseed: sclerotinia sclerotiorum and gray leaf spot (Alternaria brassicae);

Soybean diseases: purple seed cercospora (Cercospora kikuchii), sphaceloma (Elsinoe glycines), pod and stem rot (Diaporthe phaseolorum var. sojae), rust (Phakopsora pachyrhizi) and brown rot of stems (Phytophthora sojae);

Diseases of adzuki beans: gray mold (Botrytis cinerea) and sclerotinia (Sclerotinia sclerotiorum);

Common bean diseases: gray mold (Botrytis cinerea), seed sclerotinia (Sclerotinia sclerotiorum) and common bean anthracnose (Colletotrichum lindemthianum);

Peanut diseases: leaf spot (Cercospora personata), brown leaf spot (Cercospora arachidicola) and southern sclerotium rot (Sclerotium rolfsii);

Potato diseases: brown spot (Alternaria solani) and late blight (Phytophthora infestans);

Cotton diseases: Fusarium wilt (Fusarium oxysporum);

Tobacco diseases: brown spot (Alternaria longipes), anthracnose (Colletotrichum tabacum), downy mildew (Peronospora tabacina) and tobacco late blight (Phytophthora nicotianae);

Sugar beet diseases: cercospora leaf rot (Cercospora beticola), leaf spot (Thanatephorus cucumeris), tanapherous root rot (Thanatephorus cucumeris) and aphanomyces root rot (Aphanomyces cochlioides).

Rose diseases: black spot (Diplocarpon rosae) and powdery mildew (Sphaerotheca pannosa).

Diseases of chrysanthemums and plants of the aster family: downy mildew (Bremia lactucae) and leaf spot (Septoria chrysanthemi-indici);

Diseases of various plants: diseases caused by Pythium spp. (Pythium aphanidermatum, Pythium debarianum, Pythium graminicola, Pythium irregulare, Pythium ultimum), gray mold (Botrytis cinerea), sclerotial rot (Sclerotinia sclerotiorum) and blackleg (Rhizoctonia solani) caused by Rhizoctonia spp.;

Daikon diseases: Alternaria leaf spot (Alternaria brassicicola);

Lawn grass diseases: sclerotial rot (Sclerotinia homeocarpa), brown blight and rhizoctonia (Rhizoctonia solani);

Banana disease: cercospora blight (Mycosphaerella fijiensis, Mycosphaerella musicola, Pseudocercospora musae); And

Diseases of seeds or diseases in the early stages of growth of various plants caused by bacteria of the genus Aspergillus, genus Penicillium, genus Fusarium, genus Tricoderma, genus Thielaviopsis, genus Rhizopus, genus Mucor, genus Phoma and genus Diplodia.

Diseases may be root-borne, foliar-borne, present in the plant's vascular system, or insect-borne, and include all bacterial, viral, and fungal plant pathogens.

The plant disease control composition of the present invention can be applied to agricultural lands, such as cultivated lands, flooded fields, lawns and orchards, or non-agricultural lands. The plant disease control composition of the present invention can control plant diseases in agricultural lands and forests in which plants are grown.

Target weeds

Since weeds can be controlled using the methods and compositions of the present invention, the following examples are provided.

Weeds of the family Urticaceae: Urtica urens; weeds of the family Polygonaceae: Polygonum convolvulus, Polygonum lapathifolium, Polygonum pensylvanicum, Polygonum persicaria, Polygonum longisetum, Polygonum aviculare, Polygonum arenastrum, Polygonum cuspidatum, Rumex japonicas, Rumex crispus, Rumex obtusifolius and Rumex acetosa; weeds of the Portulacaceae family: Portulaca oleracea; weeds of the family Caryophyllaceae: Stellaria media, Cerastium holosteoides, Cerastium glomeratum, Spergula arvensis and Silene gallica; weeds of the family Molluginaceae: Mollugo verticillata; weeds of the Chenopodiaceae family: Chenopodium album, Chenopodium ambrosioides, Kochia scoparia, Salsola kali and Atriplex spp.; weeds of the Maranthaceae family: Amaranthus retroflexus, Amaranthus viridis, Amaranthus lividus, Amaranthus spinosus, Amaranthus hybridus, Amaranthus palmeri, Amaranthus rudis, Amaranthus patulus, Amaranthus tuberculatos, Amaranthus blitoides, Amaranthus deflexus, Amaranthus quitensis, Alternanthe ra philoxeroides, Alternanthera sessilis and Alternanthera tenella; weeds of the Papaveraceae family: Papaver rhoeas and Argemone mexicana; weeds of the Brassicaceae family: Raphanus raphanistrum, Raphanus sativus, Sinapis arvensis, Capsella bursa-pastoris, Brassica juncea, Brassica campestris, Descurainia pinnata, Rorippa islandica, Rorippa sylvestris, Thlaspi arvense, Mya

- 19 045948 grum rugosum, Lepidium virginicum and Coronopus didymus; weeds of the family Capparaceae: Cleome affinis; weeds of the Fabaceae family: Aeschynomene indica, Aeschynomene rudis, Sesbania exaltata, Cassia obtusifolia, Cassia occidentals, Desmodiumtortuosum, Desmodiumadscendens, Trifolium repens, Pueraria lobata, Vicia angustifolia, Indigofera hirsute, Indigofera truxillensis and Vigna sinensis; weeds of the Oxalidaceae family: Oxalis corniculata, Oxalis strica and Oxalis oxyptera; weeds of the Geraniaceae family: Geranium carolinense and Erodium cicutarium; weeds of the family Euphorbiaceae: Euphorbia helioscopia, Euphorbia maculate, Euphorbia humistrata, Euphorbia esula, Euphorbia heterophylla, Euphorbia brasiliensis, Acalypha australis, Croton glandulosus, Croton lobatus, Phyllanthus corcovadensis and Ricinus communis; weeds of the family Malvaceae: Abutilon theophrasti, Sida rhombiforia, Sidacordifolia, Sidaspinosa, Sidaglaziovii, Sida santaremnensis, Hibiscus trionum, Anoda cristata and Malvastrum coromandelianum; weeds of the Sterculiaceae family: Waltheria indica; weeds of the Violaceae family: Viola arvensis and Viola tricolor; weeds of the Cucurbitaceae family: Sicyos angulatus, Echinocystis lobata and Momordica charantia; weeds of the Lythraceae family: Lythrum salicaria; weeds of the Apiaceae family: Hydrocotyle sibthorpioides; weeds of the Sapindaceae family: Cardiospermum halicacabum; weeds of the family Primulaceae: Anagallis arvensis; weeds of the Asclepiadaceae family: Asclepias syriaca and Ampelamus albidus; weeds of the Rubiaceae family: Galium aparine, Galium spurium var. echinospermon, Spermacoce latifolia, Richardia brasiliensis and Borreria alata; weeds of the Convolvulaceae family: Ipomoea nil, Ipomoea hederacea, Ipomoea purpurea, Ipomoea hederacea var. integriuscula, Ipomoea lacunose, Ipomoea triloba, Ipomoea acuminate, Ipomoea hederifolia, Ipomoea coccinea, Ipomoea quamoclit, Ipomoea grandifolia, Ipomoea aristolochiafolia, Ipomoea cairica, Convolvulus arvensis, Calystegia hederacea, Calystegia japonica, Merremia he deacea, Merremia aegyptia, Merremia cissoids and Jacquemontia tamnifolia; weeds of the Boraginaceae family: Myosotis arvensis; weeds of the Lamiaceae family: Lamium purpureum, Lamium amplexicaule, Leonotis nepetaefolia, Hyptis suaveolens, Hyptis lophanta, Leonurus sibiricus and Stachys arvensis; weeds of the Solanaceae family: Datura stramonium, Solanum nigrum, Solanum americanum, Solanum ptycanthum, Solanum sarrachoides, Solanum rostratum, Solanum aculeatissimum, Solanum sisymbriifolium, Solanum carolinense, Physalis angulata, Physalis subglabrata and Nicandra physaloides; weeds of the Scrophulariaceae family: Veronica hederaefolia, Veronica persica and Veronica arvensis; weeds of the Plantaginaceae family: Plantago asiatica; weeds of the Asteraceae family: Xanthium pensylvanicum, Xanthium occidentale, Helianthus annuus, Matricaria chamomilla, Matricaria perforate, Chrysanthemum segetum, Matricaria matricarioides, Artemisia princeps, Artemisia vulgaris, Artemisia verlotorum, Solidago altissima, Taraxacum officinale, Galinsoga ciliate, Galinsoga parvifl ora, Senecio vulgaris, Senecio brasiliensis, Senecio grisebachii, Conyza bonariensis, Conyza Canadensis, Ambrosia artemisiaefolia, Ambrosia trifida, Bidens pilosa, Bidens frondosa, Bidens subalternans, Cirsium arvense, Cirsium vulgare, Silybum marianum, Carduus nutans, Lactuca serriola, Sonchus oleraceus, Sonchus asper, Wedelia glauca , Melampodium perfoliatum, Emilia sonchifolia, Tagetes minuta, Blainvillea latifolia, Tridax procumbens, Porophyllum ruderale, Acanthospermum australe, Acanthospermum hispidum, Cardiospermum halicacabum, Ageratum conyzoides, Eupatorium perfoliatum, Eclipta alba, Erechtites hieracifolia, Gamocha eta spicata, Gnaphalium spicatum, Jaegeria hirta, Parthenrum hysterophorus , Siegesbeckia orientalis and Soliva sessilis; weeds of the Liliaceae family: Allium canadense and Allium vineale; weeds of the Commelinaceae family: Commelina communis, Commelina bengharensis and Commelina erecta; weeds of the Roaceae family: Echinochloa crus-galli, Setaria viridis, Setaria faberi, Setaria glauca, Setaria geniculata, Digitaria ciliaris, Digitaria sanguinalis, Digitaria horizontalis, Digitaria insularis, Eleusine indica, Poa annua, Alospecurus aequalis, Alopecurus myosuroides, Avena fatua, Sorghum halepense , Sorghum vulgare, Agropyron repens, Lolium multiflorum, Lolium perenne, Lolium rigidum, Bromus secalinus, Bromus tectorum, Hordeum jubatum, Aegilops cylindrica, Phalaris arundinacea, Phalaris minor, Apera spica-venti, Panicum dichotomiflorum, Panicum texanum, Panicum maximum, Brachiaria platyphylla , Brachiaria ruziziensis, Brachiaria plantaginea, Brachiaria decumbens, Brachiaria brizantha, Brachiaria humidicola, Cenchrus echinatus, Cenchrus pauciflorus, Eriochloa villosa, Pennisetum setosum, Chloris gayana, Eragrostis pilosa, Rhynchelitrum repens, Dactyloctenium aegyptium, Ischaemum rugosum, Oryza sativa , Paspalum notatum, Paspalum maritimum, Pennisetum clandestinum, Pennisetum setosum and Rottboellia cochinchinensis; weeds of the Suregaceae family: Cyperus microiria, Cyperus iria, Cyperus odoratus, Cyperus rotundus, Cyperus esculentus and Kyllinga gracillima; and weeds of the Equisetaceae family: Equisetum arvense and Equisetum palustre; and so on.

Examples

It should be understood that the examples and embodiments described herein are presented for illustrative purposes only, and that various modifications or changes thereto may be suggested by one skilled in the art and are within the spirit and scope of this application.

Example 1: Fermentation of Starmerella bombicola for the production of sophorolipids (SLP) in a 110 L spread reactor.

Uses a portable airlift fully enclosed reactor controlled by PLC with water filtration, temperature control element and built-in air blower. The reactor has a working volume of 90 L for growing S. bombicola to produce SLP.

In preferred embodiments, the nutrients for SLP production are glucose, urea, yeast extract, canola oil, magnesium sulfate and phosphate

- 20 045948 potassium.

The reactor is inoculated with 8 liters of liquid culture grown in flasks. The duration of the cultivation cycle for the production of SLP is 7-8 days at 25°C and pH 3.5, with samples taken twice a day.

The final concentration of SLP is approximately 10% of the working volume, in this case approximately 9 liters of product containing 300-400 grams of SLP per liter.

Example 2: Fermentation of Wickerhamomyces and/or Pichia yeasts for the production of sophorolipids in a 450 L spread reactor.

Use a mobile airlift reactor controlled by PLC with water filtration, temperature control element and air blower for sufficient aeration. This process can be carried out by batch culture method. The reactor has a working volume of 400 L for growing Wickerhamomyces or Pichia to produce SLP.

In preferred embodiments, the nutrients for SLP production are glucose, urea, yeast extract, canola oil, magnesium sulfate and potassium phosphate.

Inoculation of this reactor requires up to 5% of the liquid seed culture of the working volume. The duration of the cultivation cycle is 7 days at a temperature of 25°C and pH 3.5, with samples taken twice a day.

The final SLP concentration is approximately 20-25% of the working volume, in which case more than 90 L of product is formed.

Example 3: Fermentation of Wickerhamomyces and/or Pichia yeasts for cellular and single-cell protein production in a 900 liter distributed reactor.

A portable reactor is used, divided into two containers, under the control of a central airlift to ensure mixing of the two containers simultaneously. The reactor has a working volume of 600 L for growing Wickerhamomyces and/or Pichia for cell production.

In a preferred embodiment, the cell production nutrients are glucose or baker's sugar, urea, yeast extract, magnesium sulfate and potassium phosphate.

The reactor is inoculated with 2% seed culture. Fermentation continues for 48-72 hours without stabilizing the pH, and at a temperature of 26 to 32°C.

The final cell concentration is 100 g wet mass per liter. The concentration of wet biomass can reach 90 kilograms per cycle with protein concentrations up to 45 kilograms.

Example 4: Fermentation of Wickerhamomyces and/or Pichia yeasts for cellular and single-cell protein production in a 2000 liter distributed reactor.

A portable reactor is used, divided into two square containers with 2 loops for mass exchange between them. The reactor has a working volume of 2000 L for growing Wickerhamomyces and/or Pichia for cell production.

In a preferred embodiment, the cell production nutrients are glucose or baker's sugar, urea, yeast extract, magnesium sulfate and potassium phosphate.

The reactor is inoculated with 2% seed culture. Fermentation continues for 48-72 hours without stabilizing the pH, and at a temperature of 26 to 32°C.

The final cell concentration is 100 g wet mass per liter. The concentration of wet biomass can reach 200 kilograms per cycle with protein concentrations up to 100 kilograms.

Example 5: Synergistic fermentation of a combined culture of Acinetobacter venetianus and Bacillus subtilis in a 900 L spread reactor.

A portable reactor is used, divided into two containers, under the control of a central airlift to ensure mixing of the two containers simultaneously. The reactor has a working volume of 600 L for growing Acinetobacter/Bacillus subtilis for the production of biological surfactant.

In a preferred embodiment, the nutrients for producing the biological surfactant are glucose, molasses powder, sucrose, potassium phosphate, sodium phosphate, potassium chloride, magnesium sulfate, calcium chloride, urea, ammonium chloride and yeast extract.

A reactor containing 600 liters of nutrient medium with a pH of 6.8-7.0 is inoculated with 60 liters of a seed culture containing both Acinetobacter and Bacillus subtilis. Fermentation continues for 24 hours without pH stabilization and at a temperature of 28 to 30°C.

The final cell concentration is up to 1 billion cells of the combined culture with an approximately equal distribution of both microorganisms.

Example 6: Fermentation of Starmerella bombicola for the production of sophorolipids in a 14 L portable dispense reactor.

- 21 045948

This reactor is an autoclavable glass container in a jacket with air purging and a stirrer. It is equipped with a probe for determining dissolved oxygen, pH, temperature and foam; It has a built-in control station with color touch screen, built-in pumps, gas flow controllers, and pH/DO foam/level controllers.

The working volume of the reactor is 10 l.

The nutrient medium contains glucose, yeast extract, urea and vegetable oil. The inoculum may be a 1 to 2 day old culture of Starmerella bombicola in an amount of approximately 5-10% of the total culture volume. Cultivation and collection of the finished product continue for 5-14 days. The final production of sophorolipid can reach 1-2 kilograms per cycle.

Example 7: Fermentation of Pseudozyma aphidis to produce mannosylerythritol lipid (MEL) in a 14 L portable dispense reactor.

This reactor is an autoclavable jacketed glass vessel with air purging and a Rushton stirrer. It is equipped with a probe for determining dissolved oxygen, pH, temperature and foam; It has a built-in control station with color touch screen, built-in pumps, gas flow controllers, and pH/DO foam/level controllers. The working volume of the reactor is 10 liters.

Composition of the nutrient medium: sodium nitrate, potassium phosphate, magnesium sulfate, yeast extract and vegetable oil. The inoculum may be a 1 to 2 day old culture of Pseudozyma aphidis in an amount of approximately 5-10% of the total culture volume. Duration of cultivation and sample collection: 9-15 days. General MEL production: 800-1000 grams.

Example 8: Cultivation of Bacillus subtilis in a distributed hybrid system

This system is a hybrid system between liquid phase fermentation and solid phase fermentation. In this system, a large number of individual nutrient-rich hydrophilic particles are suspended in a matrix of hydrophobic particles. The hydrophilic particles may be, for example, carbon black, perlite, vermiculite or diatomaceous earth. The hydrophobic particles can be, for example, polymer-coated sand (hydrophobic sand), perlite, vermiculite.

Confinement of hydrophilic particles within hydrophobic particles creates a sterile microculture environment. Also, the hydrophobic matrix allows efficient gas exchange for crops. Working volume: 500 ml - 1 metric ton.

Composition of the nutrient medium: nutrient broth, M9 medium, tryptic soy broth. Types of Inoculum and Amount: The inoculum may be 12-16 hours old and may constitute approximately 1-5% of the total culture volume. Duration of cultivation: 1-3 days. Final CFU count: 1-2 billion cells/spores per gram.

Example 9: Cultivation of Trichoderma harzianum in a distributed hybrid system

A similar method was developed for culturing Mycorrhiza using hydrophobic sand and diatomaceous earth as described in Example 8. This system has 1 part by weight diatomaceous earth saturated with nutrient medium homogenized in 6 parts hydrophobic sand. Powdered inoculum was then added. This mixture was allowed to incubate under optimal conditions for fungal growth. The sample was counted every day.

Working volume: from 100 ml to 1 metric ton. Composition of the nutrient medium: potato broth with dextrose and soy peptone. Types of inoculum and its quantity: dry spores or mycelial propagules. Duration of cultivation: 7-10 days. Total mass production: up to 10 ⁹ propagules/gram.

Example 10: Preparation of a soil amendment based on sophorolipids

For the present invention, a natural mixture of sophorolipids was synthesized by fermentation of S. bombicola in a fermentation medium containing 100 g/L glucose, 10 g/L yeast extract, 1 g/L urea and 100 ml/L canola oil in water. After fermentation for 5-7 days, approximately 500 g/L of sophorolipids precipitated as a brown layer at the bottom of the fermentation vessel.

The sophorolipid layer was collected and diluted 4 times to an SLP concentration of 125 g/L. The pH value was adjusted to 6.5-7.0 using sodium hydroxide. Advantageously, the sophorolipid broth also contains some residual yeast cells and micronutrient metabolites and media components. This ensures the presence of high concentrations of mannoprotein as the outer surface protein of the yeast cell wall (mannoprotein is a highly effective bioemulsifier, capable of achieving an emulsification index of up to 80%); the presence of biopolymer beta-glucan (emulsifier) in the walls of yeast cells; presence of sophorolipids in culture; and the presence of solvents and/or other metabolites (eg, lactic acid, ethanol, etc.).

This fermentation process can produce two different products: one containing a pure layer of SLP and one containing a whole broth containing a culture of S. bombicola and SLP.

All patents, patent applications, provisional applications and publications mentioned or cited

Claims (5)

Bathed in the present description are incorporated herein by reference in their entirety, including all figures and tables, unless they are inconsistent with the precise wording of the present description. The description in this specification of any aspect or embodiment of the invention using terms such as containing, having, including or containing, with respect to an element or elements, is intended to substantiate a similar aspect or embodiment of the invention which consists of, essentially consists of or essentially contains that particular element or elements unless otherwise indicated or unless the context contradicts it (e.g., a composition described herein containing a particular element is to be understood as describing a composition consisting of that element unless otherwise indicated or the context does not contradict this). As used herein, the term comprising includes essentially consisting of and consisting of. The term essentially consisting of, as used herein, limits the scope of the ingredients and steps to specific materials or steps, and materials and steps that do not significantly affect the essential and novel feature(s) of the present invention, for example, compositions and methods for stimulating plant growth. The term essentially consisting of, as used herein, limits the scope of the ingredients and steps to specific materials and steps, and materials and steps that do not significantly affect the essential and novel feature(s) of the present invention. CLAIM 1. A system for obtaining a product based on microorganisms and making the product based on microorganisms available to the consumer, where the said system contains: (i) a microorganism growth apparatus comprising at least 10 independent modular growth vessels in which the same or different microorganisms can grow under the same or different growth conditions to produce a microorganism-based composition in each growth container, wherein the growth medium, oxygenation, pH, shaking and/or temperature can be independently controlled for each of said growth containers; wherein the microorganisms are selected from Starmerella bombicola, Trichoderma harzianum, Bacillus subtilis, Pseudozyma aphidis, Acinetobacter venetianus, Wickerhamomyces and Pichia; and wherein the microorganism-based composition is collected from the growth containers, thereby obtaining a microorganism-based product, which can then be provided to the consumer; and (ii) a transport component for transporting said microbial product produced in a microbial growth facility, wherein said transport component comprises 2 gallon to 1000 gallon (7.6 to 3785 L) containers and/or transport piping microorganism-based product from the microorganism-based product to a distribution center or area where the microorganism-based product will be used, where the microorganism-based product in (i) is located less than 300 miles (482,803 m) from the area where the microorganism-based product microorganisms will be used where the microorganism-based product contains the microorganism and the environment in which the microorganisms were grown, and where the microorganism-based product does not require stabilization. 2. The system of claim 1, wherein each of said at least 10 modular grow tanks has its own temperature, dissolved oxygen, and pH controls. 3. The system of claim 1, wherein the microbial growth facility of (i) is located within 100 miles (160,934 m) of the location where the microbial product will be used. 4. A method for producing a microorganism-based product and making the microorganism-based product available to the consumer, wherein the method includes growing microorganisms in a microorganism growing system according to claim 1, containing at least ten independent modular cultivation tanks to obtain the composition based on the microorganisms from each growth vessel, where the microorganism growing facility is located less than 300 miles (482,803 m) from the application area, the microorganisms being selected from Starmerella bombicola, Trichoderma harzianum, Bacillus subtilis, Pseudozyma aphidis, Acinetobacter venetianus, Wickerhamomyces and Pichia wherein the same or different microorganisms may be grown in each growth vessel under the same or different growth conditions, wherein the growth conditions, which may differ between growth vessels, include one or more of growth medium, oxygenation, pH , shaking and temperature, wherein the microorganism-based product contains microorganisms, as well as the environment in which the microorganisms were grown, and wherein the microorganism-based product does not require stabilization. 5. The method according to claim 4, where the cell mass of microorganisms in the transported product is based on -