KR20230104202A - How to produce livestock with a reduced carbon footprint - Google Patents

Abstract

The present invention provides compositions and methods for producing livestock with a reduced carbon footprint. Microbe-based soil treatment compositions reduce greenhouse gas emissions due to livestock feed production and consequently improve livestock animal health and productivity.

Description

How to produce livestock with a reduced carbon footprint

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/108,392, filed on November 1, 2020, and U.S. Provisional Patent Application No. 63/119,284, filed on November 30, 2020, both of which are incorporated herein by reference in their entirety. are incorporated herein by reference.

Gases that trap heat in the atmosphere are called “greenhouse gases” or “GHGs,” and include carbon dioxide, methane, nitrous oxide, and fluorinated gases ((EPA Report 2016, 6). “Carbon Footprint "footprint" is the total GHG emissions caused directly or indirectly by a person, organization, event or product. Different GHGs may be emitted during the lifetime, or life cycle, of a product, each with a greater or lesser ability to trap heat in the atmosphere. This difference is the global warming potential of each gas. , GWP) is calculated in terms of carbon dioxide equivalents (CO ₂ e), providing a single unit for easy comparison of carbon footprints (Michigan 2020).

Recent measurements from monitoring stations around the world and measurements of old air bubbles trapped in the ice layers of Antarctica and Greenland suggest that global atmospheric concentrations of greenhouse gases have increased significantly over the past few hundred years (EPA report 2016, eg, 6, 15 ).

Especially since the start of the Industrial Revolution in the 1700s, human activities have increased the amount of greenhouse gases in the atmosphere through activities such as burning fossil fuels and deforestation. Large amounts of greenhouse gases emitted into the atmosphere remain there for long periods ranging from 10 to thousands of years. Over time, these gases are removed from the atmosphere by chemical reactions or by emission sinks such as oceans and vegetation that absorb GHGs from the atmosphere.

World leaders are attempting to curb the growth of greenhouse gas emissions or reduce the carbon footprint of various activities through treaties and other international agreements. One such initiative is the use of a carbon credits system. Carbon credits are a collective term for tradable certificates or permits that represent the right to emit one ton of carbon dioxide or equivalent greenhouse gases. In a typical carbon credits system, a governing body sets a quota for the amount of GHG emissions an operator can produce. If these quotas are exceeded, operators must purchase additional allowances from other operators who have not used up their carbon credits.

The goal of the carbon credits system is to encourage companies to invest in more green technology, machinery and practices to benefit from these credit trading. Under the Kyoto Protocol of the United Nations Framework Convention on Climate Change (UNFCCC), many countries have agreed to be internationally bound by greenhouse gas reduction policies, including emissions trading. The United States is not bound by the Kyoto Protocol, and although the United States does not have a central national emissions trading system, some states, such as California and the Northeast, are beginning to adopt such systems.

One industry sector with a high carbon footprint is food production, i.e. agriculture and livestock production, which emit mainly carbon dioxide, nitrous oxide and methane.

In particular, ruminants such as cattle, sheep, buffalo, goats, deer and camels contribute to methane production due to their unique digestive systems. Ruminants have four stomach compartments: the reticulum, rumen, omasum, and abomasum. In particular, the rumen serves as a vessel for anaerobic fermentation of biofilm-forming methanogenic bacteria that produce gaseous by-products such as carbon dioxide and methane. More than about 130 to 250 gallons of rumen gas produced by fermentation can be excreted from a single cow each day.

Other animals, including non-ruminants, also contribute to intestinal GHG production. For example, pigs, rodents, monkeys, horses, mules, donkeys, rhinos, hippos, bears, poultry and certain other birds also contain methanogenic bacteria in their digestive systems.

In addition to enteric fermentation, livestock manure can also be a source of GHG emissions. Manure contains two components during storage and processing that can lead to GHG emissions: organic matter that can be converted to methane emissions, and nitrogen that indirectly leads to nitrous oxide emissions. Manure is stored in septic tanks, artificial storage ponds and storage tanks, where methane is released when methanogenic bacteria break down the organic matter in the manure. Also, nitrogen in the form of ammonia (NH ₃ ) is released during storage and processing of manure and urine. Ammonia can then be transformed into nitrous oxide. (Gerber et al., 2013).

Methods exist to reduce intestinal and manure-based methane emissions in livestock, including, for example, microbial elimination of the digestive system and even vaccination against methanogens, but these strategies can reduce the number of beneficial gut microbes; The method may be short-lived due to the adaptation of microorganisms to it. Another known strategy is to manipulate enteric fermentation by redirecting hydrogen ions from methanogens to directly inhibit or reduce methanogens, for example methanogens and protozoa, particularly for cattle grazing pastures. Include dietary changes. Most anti-methanogenic compounds are expensive, short-lived, have inconsistent results, require high concentrations, do not contain _H2 acceptors, do not affect methanogens in biofilm form, and are readily Includes compounds that are destroyed and/or excreted.

Additional strategies include improved animal management practices. It is believed that there is a direct link between GHG emission intensity and animal efficiency. The higher the animal's productivity (eg meat and/or dairy production), the lower the environmental impact (per unit of product). For example, more efficient animals retain more dietary nitrogen protein and excrete less nitrogen in feces and urine. Some methods to increase efficiency include selective breeding practices and genetic modification to reduce the nutritional requirements of animals. However, selective breeding methods can be unpredictable and time-consuming, and the effects of genetic modification on animal health and welfare are unknown (Gerber et al., 2013).

Another important aspect of the livestock sector is feed production and processing, which accounts for nearly half of industrial GHG emissions. These greenhouse gas emissions arise, for example, from land use change, manufacture and use of fertilizers and pesticides, manure excreted and applied to fields, agricultural machinery and operations, feed processing and feed transport.

Nearly 60% of the world's biomass (eg, corn and other feed) harvested worldwide enters livestock subsystems as feed or barn material. Soil carbon dioxide emissions are generated as a result of soil carbon dynamics (eg, plant residue decomposition, soil organic matter mineralization, land use change, etc.), synthetic fertilizer and pesticide manufacture, and fossil fuel use in on-farm agricultural operations. Nitrous oxide is released when organic and inorganic fertilizers are applied to the soil. These fertilizers are susceptible to loss by leaching and denitrification prior to crop uptake (Grossi et al., 2019).

Livestock is important, for example in meat and dairy production, but growing concerns about climate change and the growing need to reduce GHG emissions require improving approaches to raising livestock while reducing the carbon footprint of the livestock sector.

The present invention provides an environmentally friendly method for the production of livestock. The present invention further provides microbial-based products and uses of these products to achieve beneficial results in many settings within the livestock operating chain, including, for example, feed production aspects and animal husbandry aspects. Advantageously, the present invention uses organic, non-GMO ingredients and environmentally friendly methods to reduce the carbon footprint of livestock production.

In certain embodiments, the methods of the present invention promote livestock feeding and production in a manner that reduces GHG emissions resulting from livestock feeding and production, including GHGs such as carbon dioxide, methane and nitrous oxide.

In a preferred embodiment, the method comprises an agricultural aspect of growing a livestock feed with a reduced carbon footprint, and a livestock aspect of feeding the reduced carbon footprint feed to a livestock animal.

In one embodiment, the agricultural aspect comprises applying a microorganism-based soil treatment composition to land of farmland comprising land cleared by natural fire and manipulated fire, wherein the soil treatment composition is applied to plants of the farmland and/or provide one or more direct or indirect benefits to the soil. These benefits include, for example, improved plant health and growth (both aboveground and belowground), improved plant protein and/or nutrient content, reduced fertilizer use, enhanced carbon sequestration in the soil, improved soil microbiome diversity, improved soil moisture and/or or improving nutrient dispersion, and/or increasing uptake of soil nutrients by plant roots.

In certain embodiments, the soil treatment composition includes one or more beneficial microorganisms. In a preferred embodiment, the beneficial microorganisms are nonpathogenic, soil-crowding fungi, yeasts and/or bacteria, which are capable of producing one or more of the following: surfactants such as lipopeptides and/or glycolipids; bioactive compounds with antibacterial and immunomodulatory effects; polyketide; mountain; peptide; anti-inflammatory compounds; enzymes such as proteases, amylases and/or lipases; A source of amino acids, vitamins and other nutrients.

In a preferred embodiment, the microorganism is , for example, Trichoderma species, Bacillus species , Wickerhamomyces anomalus , Myxococcus xanthus , Pseudomonas chlororapis ( Pseudomonas chlororaphis ), Starmerella bombicola ( Starmerella bombicola ), Saccharomyces boulardii ( Saccharomyces boulardii ), Pichia occidentalis ( Pichia occidentalis ), Pichia kudriavzevii ( Pichia kudriavzevii ), Mellojima Gilly Ermondi ( Meyerozyma guilliermondii ), mycorrhizal fungi, nitrogen fixers (eg Azotobacter vinelandii ) and/or potassium mobilizers (eg Frateuria aurantia ) ), non-pathogenic bacteria, yeasts and/or fungi selected from

The species and proportions of microorganisms and other components in the composition may depend on, for example, the geographic region in which the treatment will occur, the species of livestock animal(s) that will consume the plant, the health of the farmland at the time of treatment, and other factors. Thus, the composition can be tailored for any given location.

In certain exemplary embodiments, the soil treatment composition includes a first microorganism and a second microorganism, by-products of their growth, and optionally one or more nutrient sources. In certain exemplary embodiments, the first microorganism is Trichoderma harzianum and the second microorganism is Bacillus amyloliquefaciens (eg, B. amyloliquefaciens) NRRL B-67928).

In one embodiment, the soil treatment composition further comprises microbial growth by-products, which may include, for example, a fermentation medium in which the microorganisms are cultured, and/or any nutrients left over from cultivation. Growth by-products may further include metabolites or other biochemical substances produced as a result of cell growth, including, for example, biosurfactants, enzymes and/or solvents.

The method may further include the step of applying (application) a substance to enhance microbial growth during application (e.g., germination enhancers, prebiotics and/or agents to promote plant and/or microbial growth). nutrients added). In one embodiment, the nutrient sources may include, for example, sources of magnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur, iron, copper, zinc, protein, vitamins and/or carbon. In one embodiment, prebiotics may include, for example, one or more of kelp extract, fulvic acid, chitin, humate and humic acid.

In some embodiments, the method of the present invention further comprises applying additional agricultural ingredients, such as herbicides, fertilizers, insecticides and/or soil improvers. Preferably, the additional ingredients are non-toxic and environmentally friendly. The exact material and amount thereof can be determined by a soil scientist having the benefit of this disclosure.

In certain embodiments, the soil treatment composition is contacted with plant parts. In certain embodiments, the composition is contacted with one or more roots of a plant. The composition may be applied to the roots, for example by directly spraying or wetting the roots, and/or may be applied to the roots indirectly, for example by administering the composition to the soil in which the plants are growing (eg, the rhizosphere). there is. The composition may be applied to the seed of the plant before or at the time of planting, or to any other part of the plant and/or its surroundings.

The method of the present invention may utilize standard methods and equipment used for the maintenance of agricultural land. For example, the soil treatment composition can be applied in liquid form using an irrigation system. Additionally, the composition may be applied using a manual spreader, such as a broadcast spreader, drop spreader, handheld spreader, or handheld sprayer.

In some embodiments, agricultural land includes grasses including, for example, bluegrass, Bermuda grass, fescue, buffalo grass, and the like. Other forages such as forbs, weeds and shrubs as well as forage crops such as corn, oats and barley may also be treated with the soil treatment composition. In some embodiments, in some instances, the composition may be applied to a paddock where an engineered landslide has been performed to promote vegetation.

In one embodiment, the method can be used to reduce the carbon footprint of grain-finished livestock as well as grain-fed livestock production, wherein the grain produced according to the agricultural aspect of the method is carbon-reducing grains, and/or their by-products (eg, distiller's dried grains with solubles, DDGS).

In one embodiment, agricultural aspects of the method may increase the feed value of grasses compared to conventionally grown, high carbon footprint grains produced for livestock feeding and/or processing.

The present invention increases the marketability of pasture- or grass-fed livestock, reduces land use change, reduces the manufacture and use of fertilizers and pesticides, and feeds by feeding livestock grains produced using carbon footprint reduction methods. The carbon footprint of the livestock feed industry can be reduced by reducing the operation of agricultural machinery that burns fossil fuels, including those used for processing and transporting food.

This can be achieved, for example, by enhancing plant carbon utilization and storage in farmland, increasing soil carbon sequestration, reducing soil-based GHG emissions, improving agricultural nitrogen-based fertilization practices, improving soil microbial biodiversity, and improving agricultural soil management.

In certain embodiments, enhanced plant carbon utilization may include, for example, increased leaf, increased stem and/or trunk diameter in a plant, enhanced root growth, and/or increased plant per unit area. It can be in the form of a number.

In certain embodiments, increased soil sequestration may include, for example, increased growth (eg, length and density) of plant roots, increased uptake by microorganisms of organic compounds excreted by plants (from plant roots). exudates), and improved microbial colonization of the soil.

In certain embodiments, the method can reduce the amount of GHGs such as methane, carbon dioxide and/or nitrous oxide/precursors thereof emitted from the soil.

In certain embodiments, improved agricultural fertilization practices, soil biodiversity, and/or soil management may be in the form of reducing nitrogen-rich fertilizers and inoculating the rhizosphere of plants with one or more beneficial microorganisms. For example, in a preferred embodiment, the microbes of the soil treatment composition can colonize the rhizosphere and provide a number of benefits including protection, hydration and nutrient supply to plants with roots growing therein. Thus, the method may reduce nitrous oxide emissions by replacing some or all of the fertilizers, pesticides and/or other soil conditioners with one or more beneficial soil microorganisms.

In certain embodiments, the method further comprises a livestock farming mode, which includes making available to livestock animals a plant produced according to the agricultural mode for the livestock animal to consume the plant. In one embodiment, livestock animals are placed on land in farmland treated according to an agricultural modality for grazing pastures. In one embodiment, plants are harvested from treated farmland and provided to animals as reduced-carbon footprint feed, grain and/or other forms of loose feed.

In one embodiment, a combination of feeding methods is used, as is customary, for example, when producing grain-finished livestock.

In one embodiment, the livestock aspect of the method reduces the carbon footprint of livestock farming by improving livestock animal health and/or productivity in a manner that reduces livestock digestion, manure, and GHG emissions from large-scale production.

These benefits may include, for example, improved feed efficiency, improved animal health and fertility, improved quantity and nutritional quality of meat and dairy products, and reduced dependence on feed crops with a high carbon footprint, such as conventionally produced maize. One of the specific and important benefits of improved feed efficiency is the reduction of ammonia and nitrous oxide produced in the digestive system and animal waste products as a result of increased feed nitrogen consumption. In addition, improved feed efficiency can lead to improved animal productivity, meaning that fewer feed and/or GHG emitting animals are required to produce a given amount of product.

In one embodiment, the animal husbandry aspect may further include reducing the amount of GHG excreted therefrom while increasing degradation of the manure by microorganisms by directly applying the soil treatment composition to livestock manure. In some embodiments, applying the composition to manure further contributes to reducing the carbon footprint, as the manure can be used in place of synthetic nitrogen-rich fertilizer for agricultural land that will eventually become feed for livestock animals.

In some embodiments, the methods of the present invention may be used by livestock producers and/or livestock feeders to reduce the use of carbon credits. Thus, in certain embodiments, the method can be used to reduce methane, carbon dioxide and/or other noxious atmospheric gases, and/or precursors thereof (e.g., nitrogen) by livestock producers and/or livestock feeders using standard techniques in the art. and/or ammonia). It may further include performing measurements to evaluate the effectiveness of the method.

Advantageously, in certain embodiments, the present invention promotes the environmental sustainability of producing and consuming meat, dairy and other animal products; Improving the nutrient content of agricultural soils; promotion of improved soil moisture and water use efficiency; improving soil microbiome diversity; reduced fertilizer use; Increased feed value of grasses with reduced grain dependence; Reduction of intestinal GHG emissions from livestock and manure; Improved feed-to-muscle conversion; improving the productivity of livestock animals (eg, improving the quantity and nutritional quality of meat and milk); and provide solutions to improve others.

1 schematically illustrates a flow diagram of a system according to one embodiment of the present invention.

The present invention provides an environmentally friendly method for producing livestock with a reduced carbon footprint, the method comprising an agricultural and/or animal husbandry aspect, wherein:

The agricultural aspects improve soil nutrient and water content and dispersibility, improve plant health and growth, improve plant protein content, reduce nitrogen-rich fertilizer use, and/or improve soil and/or plant material. producing agricultural land on which plants for livestock feeding are grown or will be grown, using technology that enhances the carbon sequestration of livestock;

The livestock farming aspect includes making available to livestock animals a plant produced in the farmland described above so that the livestock animal consumes the plant,

The agricultural aspects result in reduced greenhouse gas emissions compared to traditional farming techniques, and in preferred embodiments, the livestock farming aspects improve the health and productivity of livestock animals.

Advantageously, the present invention can utilize organic, non-GMO ingredients and environmentally friendly methods to reduce the carbon footprint of livestock production.

“Carbon footprint” can be defined as a measure of the total amount of carbon dioxide (CO ₂ ) and other GHGs emitted directly or indirectly by human activities or accumulated over the entire life cycle of a product or service. As an example, a product that needs to be transported over many miles by truck (eg, harvested fodder grain) may have a larger carbon footprint than an alternative product that does not require transportation (eg, pasture-grown grass).

Carbon footprints can be calculated using Life Cycle Assessment (LCA) methods or limited to immediate emissions from fossil fuel energy use. Life-cycle assessment (also known as LCA, life-cycle analysis, ecological balance, and cradle-to-grave analysis) is the investigation and evaluation of the environmental impacts that occur or are necessary as a result of the existence of a particular product or service. The carbon footprint lifecycle concept is all-encompassing, meaning that it includes all possible causes of carbon emissions. That is, all direct (on-site, internal) and indirect (off-site, external, resident, upstream, downstream) emissions must be considered.

Generally, the carbon footprint is expressed in terms of CO ₂ equivalents. A carbon dioxide equivalent is a quantity that, for a given mixture and quantity of GHG, accounts for the quantity of CO ₂ that has the same global warming potential (GWP) when measured over a specified time scale (usually 100 years). Thus, the carbon dioxide equivalent reflects time-integrated radiative forcing. The carbon dioxide equivalent of a gas is obtained by multiplying the gas's mass by its GWP. Commonly used units are:

a) United Nations Climate Change Panel IPCC: 1 billion metric tons CO ₂ equivalent (GtCO ₂ eq);

b) Industry: million metric tons of 　carbon　dioxide equivalents (MMTCDE);

c) Vehicles: carbon 　dioxide equivalents (g)/km (gCDE/km).

For example, methane has a GWP of 21 and nitrous oxide has a GWP of 310. This means that 1 million metric tons of methane and nitrous oxide emissions, respectively, are equivalent to 21 million metric tons and 310 million metric tons of carbon dioxide emissions.

A variety of methods for calculating or estimating a carbon footprint exist in the art and can be used in the present invention.

Advantageously, in a preferred embodiment, the present invention may be useful for reducing the carbon footprint of livestock production, including reducing the carbon footprint of producing feed-based, feed-based and/or grain-based feeds for livestock. .

"Carbon footprint reduction" means a reduced change (negative change) in the amount of carbon dioxide and other GHGs emitted per unit time over the entire life cycle of producing livestock feed and feeding livestock. Negative changes in CO ₂ and/or other GHG emissions, for example, by at least 0.25%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

In some embodiments, the term “carbon footprint” is interchangeable herein with the terms “carbon intensity” and “emission intensity”. Emissions intensity is a measure of the emission rate of a given GHG for the “intensity” of a particular activity or industrial process (eg fuel burning, livestock production, dishwasher production). Emissions intensity may include, for example, emissions relative to amount of fuel burned, number of livestock animals produced, amount of industrial product produced, total distance traveled, and/or number of economic units created. there is.

Emission intensity is measured over the entire life cycle of a product. For example, a fuel's emission intensity measures all greenhouse gas emissions emitted along the supply chain for that fuel, including all emissions from exploration, mining, collection, production, transportation, distribution, distribution, and combustion of that fuel. calculated by counting

selected definition

As used herein, “agriculture” refers to the cultivation and breeding of plants, algae and/or fungi for food, fiber, biofuels, pharmaceuticals, cosmetics, supplements, ornamental purposes and other uses. According to the present invention, agriculture may also include horticulture, landscaping, gardening, plant conservation, forestry and reforestation, pasture and prairie restoration, orchards, arboretum, and agronomy. Agriculture includes the management, monitoring and maintenance of soil.

As used herein, a “biofilm” (biofilm) is a complex aggregate of microorganisms, such as bacteria, in which cells are attached to each other and/or to each other's surfaces. The cells of a biofilm are physiologically distinct from the planktonic cells of the same organism, which are single cells that can float or swim in a liquid medium.

As used herein, “conventional” agriculture and livestock production utilizes one or more of genetically modified organisms (GMOs), concentrated animal feeds (CAFOs), and synthetic chemicals such as fertilizers, pesticides, and/or herbicides. Conventional agriculture and livestock production are generally resource and energy intensive and can contribute significantly to GHG emissions.

As used herein, "digestive system" refers to the organ system within the body of an animal that enables digestion or consumption of food and its conversion to energy and waste products. The digestive system may include, for example, the oral cavity, esophagus, crop, gizzard, gastrocnemius, stomach, rumen, hive, midgut, stomach, pancreas, liver, small intestine, large intestine (colon), caecum, appendix, and/or anus. can Additional organs or parts related to digestion and specific to a particular animal are also included.

As used herein, "enhancement" means an improvement or increase. For example, improving plant health includes increasing seed germination and/or emergence, improving the ability to fight pests and/or diseases, and improving the ability of plants to grow and thrive, including improving the ability to survive environmental stressors such as drought and/or flooding. means improvement of Enhanced plant growth and/or enhanced plant biomass is an increase in plant size and/or mass both above and below ground (e.g., increased canopy/foliar volume, height, trunk calipers, branch length, shoot length, protein content, root size/density and/or overall growth index) and/or the ability of the plant to reach a desired size and/or mass. Improved yield is achieved by plants in crops, for example by increasing the number and/or size of fruits, leaves, roots and/or tubers per plant and/or improving the quality of fruits, leaves, roots and/or tubers. Improving the final product produced (eg improving taste, texture, sweetness, chlorophyll content and/or color).

As used herein, “agricultural land” includes any land on which plants are grown, cultivated, and/or managed for human benefit. Agricultural land includes:

Pastures or land containing predominantly grasses, legumes and non-grass herbaceous plants on which livestock graze;

meadows, generally ungrazed land that may be used to harvest hay or other animal feed;

rangelands, including uncontrolled or unmanaged grasslands, scrublands, woodlands, wetlands and deserts, where livestock or wild animals are grazed; and

agricultural cropland.

As used herein, "feed" means any plant material that is harvested or otherwise cut for feeding domestic animals. Feed may include, but is not limited to, grasses, forbs, shrubs, alfalfa, hay, straw, legumes, nuts, seeds, fruits, vegetables and/or crop residues.

As used herein, "feed" means any plant material that grows in the tracts of agricultural land and is consumed by, or at least capable of being ingested by, livestock animals.

As used herein, “grain-fed” livestock means that the livestock animal consumes grain as part of its regular diet throughout its life. Grain may comprise, for example, at least 10%, at least 25%, at least 50%, at least 75%, at least 85%, at least 95% or 100% of the animal's total feed intake. Grains include, but are not limited to, corn, oats, barley, wheat, sorghum, milo, and soybeans.

In some embodiments, grain-eating livestock are “grain-finished,” meaning that livestock animals spend most of their lives grazing on grasslands and/or eating grass and forage-based feed, but then spend the last 4 to 6 months means eating a predominantly grain-based diet (eg, greater than 50%, 60%, 75%, or 90% of caloric intake). These grain-based diets often consist of high-energy grains such as corn, wheat, and milo, but in some cases, animals also consume a variety of other feed sources in addition to feedstock grains such as potato skins, sugar beets, and hay.

As used herein, a “grass-fed” livestock refers to a livestock that eats only grass and forage throughout its life, beginning after weaning.

As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, protein, organic compound such as a small molecule (eg, those described below), or other compound is substantially different from It relates to a compound, eg, cellular material, which is inherently related. For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) has no genes or sequences flanking it in its naturally occurring state. A purified or isolated polypeptide has no amino acids or sequences surrounding it in its naturally occurring state. Purified or isolated microbial strains are removed from the environment in which they exist in nature. Thus, an isolated strain may exist, for example, as a biologically pure culture or as a spore (or other form of strain) associated with a carrier.

In certain embodiments, the purified compound is at least 60% by weight of the compound of interest. Preferably, the formulation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is a compound that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) by weight of the desired compound. . Purity is determined by any suitable standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

"Metabolite" means any substance produced by metabolism (eg, a growth by-product) or substance required to participate in a particular metabolic process. Metabolites can be starting materials, intermediates, or organic compounds that are end products of metabolism. Examples of metabolites include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, trace elements, amino acids, polymers, polyketides, and surfactants.

As used herein, a “methanogen” is a microorganism that produces methane gas as a by-product of metabolism. Methanogens are archaea that can be found in the digestive system and metabolic waste products of ruminant and non-ruminant animals (eg pigs, poultry and horses). Examples of methanogenic substances include Methanobacterium species (eg M. formicicum ), Methanobrevibacter species (eg M. luminantium (M. ruminantium) ), Methanococcus species (eg, M. paripaludis ), Methanoculeus species (eg, M. burgensis ( M. bourgensis ) , Methanoforens species (eg M. stordalenmirensis ) , Methanofollis liminatans , Methanogenium wolfei) , Methanomicrobium species (eg, M. mobile ), Methanopyrus kandleri , Methanoregula boonei , Methanosaeta ( Methanosaeta species (eg, M. concilii , M. thermophile ), Methanosarcina species (eg, M. barkeri ) ) , M. mazeii ) ), Methanosphaera stadtmanae , Methanospirillium hungatei , Methanothermobacter species and/or Methanotrix Including but not limited to Methanothrix sochngenii .

As used herein, the term “plant” refers to woody, ornamental or ornamental, crops or cereals, fruits or vegetables, fruit or vegetable plants, flowers or trees, macroalgae or microalgae, phytoplankton and photosynthetic algae (eg For example, but is not limited to, any species of the green alga Chlamydomonas reinhardtii ) . "Plant" also includes unicellular plants (eg, microalgae) and a plurality of plant cells (eg, volvox) that broadly differentiate into colonies or structures present at any stage of plant development. Such structures include, but are not limited to, fruits, seeds, shoots, roots, stems, leaves, flowers, and the like. The plant may also stand alone, for example in a lawn or garden, or it may be one of a number of plants, for example as part of an orchard, forest or crop.

The term “plant tissue” refers to plants including those present in roots, shoots, leaves, pollen, seeds, and tumors or galls, as well as those present in cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.) of differentiated and undifferentiated tissues. A plant tissue may be a plant, organ culture, tissue culture, or cell culture. As used herein, the term “plant part” refers to a plant structure or plant tissue.

Ranges provided herein are to be understood as shorthand for all values within that range. For example, a range of 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 . It should be understood to include any number, combination of numbers, or sub-ranges from the group. With respect to sub-ranges, "overlapping sub-ranges" extending from either endpoint of the range are specifically contemplated. For example, sub-ranges encompassed by the exemplary range of 1 to 50 include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 50 in the other direction. 30, 50 to 20, and 50 to 10.

As used herein, "decrease" means a negative change, and "increase" means a positive change, wherein a positive or negative change is at least 0.25%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , is a change by 95% or 100%.

As used herein, "soil treatment", "soil conditioner" or "soil conditioner" is any compound, substance, or combination of compounds or substances added to soil to improve the properties of the soil and/or rhizosphere. . Soil conditioners may include organic and inorganic substances, and may further include, for example, fertilizers, pesticides, and/or herbicides. Because nutrient-rich and well-drained soil is essential for plant growth and health, soil conditioners can be used to enhance plant biomass by altering the nutrient and water content of the soil. Soil amendment may also include soil structure (eg, preventing compaction); improving nutrient concentration and storage capacity; improved water retention in dry soil; It can be used to improve various qualities of soil, including but not limited to improving drainage of flooded soil.

As used herein, "surfactant" refers to a compound that lowers the surface tension (or interfacial tension) between phases. Surfactants act, for example, as detergents, wetting agents, emulsifying agents, foaming agents, and dispersing agents. A "biosurfactant" is a surfactant produced by a living organism.

The transitional term “comprising”, which is synonymous with “comprising” or “including,” is inclusive or open-ended and does not exclude additional unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in a claim. The transitional phrase "consisting essentially of" limits the scope of a claim to those materials or steps specified that "do not materially affect the basic and novel characteristics" of the claimed invention. Use of the term "comprising" contemplates other embodiments that "consist of" or "consist essentially of" the recited element(s).

Unless specifically stated or apparent from context, the term “or” as used herein is to be understood inclusive. Unless specifically stated or apparent from context, the terms "a", "an" and "an" as used herein are to be understood in the singular or plural.

Unless specifically stated or apparent from context, the term "about" as used herein is to be understood to be within a range of normal tolerance in the art, eg, within 2 standard deviations of the mean. “About” means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of a stated value. can be understood

Recitation of a list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes the disclosure of the embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

All references cited herein are incorporated herein by reference in their entirety.

How to produce livestock with a reduced carbon footprint

In certain embodiments, methods are provided for promoting livestock feeding and production in a manner that reduces GHGs resulting from livestock feeding and production, including GHGs such as carbon dioxide, methane and nitrous oxide.

In a preferred embodiment, the method comprises an agricultural aspect and a livestock aspect.

agricultural mode

In one embodiment, agricultural aspects of the present invention include producing livestock feed using a technology that reduces total GHG emissions as compared to GHG emissions from feed production that does not use such technology.

More specifically, in certain embodiments, the agricultural aspect of the method comprises a microorganism-based soil treatment comprising one or more beneficial microorganisms and/or their growth by-products in an area of agricultural land where plants suitable for feeding livestock are or will be grown. comprising applying the composition, wherein the soil treatment composition provides one or more direct or indirect benefits to plants and/or soil within the farmland, wherein the one or more benefits contribute to reducing GHG emissions from cultivation and/or management of the farmland. .

These benefits include, for example, improved plant health and growth (both aboveground and belowground), improved plant protein and/or nutrient content, improved soil microbiome diversity, improved soil moisture and/or nutrient dispersibility, and/or plant roots. may include increased nutrient absorption by

In one embodiment, agricultural aspects of the present invention are directed to enhancing plant carbon utilization and storage in farmland, increasing carbon sequestration in soil, reducing soil GHG emissions, improving agricultural nitrogen-based fertilization practices, improving soil microbial biodiversity and agricultural soil management. Improvements reduce the carbon footprint of livestock feed production.

In certain embodiments, improved vegetable carbon utilization may be in the form of, for example, increased leaves in a plant, increased stem and/or trunk diameter, enhanced root growth, and/or increased number of plants per unit area. .

In certain embodiments, increased soil carbon sequestration is due to, for example, increased growth (eg, length and density) of plant roots, increased uptake by microorganisms of GHG precursors/organic compounds secreted by plants. (including secretions from plant roots), and increased microbial colonization of the soil.

In some embodiments, reducing soil GHG emissions includes reducing methane, carbon dioxide and/or nitrous oxide/precursors thereof emitted from the soil. For example, in some embodiments, this may be achieved through a reduction in water stress and/or an increase in the plant's water use efficiency. For example, sufficient soil moisture lowers soil temperature and increases nutrient transport to plants, both of which contribute to reduced soil respiration leading to GHG emissions and reduced free GHG precursor molecules in the soil. Additionally, in some exemplary embodiments, the methods may facilitate the movement of water through the soil, which may lead to deoxygenation of the soil and avoid water overflow and pooling that may promote the growth of anaerobic methanogenic microorganisms.

In certain embodiments, improved agricultural fertilization practices, improved soil biodiversity, and/or improved soil management may be in the form of inoculating the rhizosphere of a plant with one or more beneficial microorganisms. For example, in a preferred embodiment, the microbes of the soil treatment composition can colonize the rhizosphere and provide a number of benefits including protection, hydration and nutrient supply to plants with roots growing therein. Thus, the method may replace or reduce the use of nitrogen-rich fertilizers, pesticides, and/or other soil conditioners that produce nitrogen and nitrous oxide precursors such as ammonia.

In certain embodiments, the protein content of the plant is increased as a result of treatment according to the present method. Protein content can be measured using traditional laboratory assays known in the art.

Many livestock animals that consume grain-based diets require protein supplementation to meet the animal's nutritional requirements. Protein deficiency can cause loss of appetite, weight loss, stunted growth, infertility and reduced milk production. Thus, increased protein content is beneficial for improving the feed efficiency of domestic animals by reducing the amount of feed needed to achieve a desired level of productivity.

Advantageously, the agricultural aspect serves to reduce the reliance on conventionally grown grains produced and transported to feed livestock. In some embodiments, this, in turn, increases the feed value of grasses, increases available land space for planting carbon-sequestering pastures and forests, and/or feeds grass-fed meat and dairy products and other low-carbon footprint animals. It can improve the marketability of the product.

In some embodiments, the methods may reduce the carbon footprint of grain-eating and/or grain-finished livestock production, where such methods are necessary and/or more economical than producing grain-fed livestock. For example, most poultry are grain-fed livestock.

Types of plants for which this method may be useful include any plant species capable of being consumed by one or more domesticated animals. annual ryegrass ( Lolium multiflorum ) ; Canadian bluegrass ( Poa compressa ) ; Chewings fescue ( Festuca rubra ); Colonial bentgra (colonial bentgra) (Agrostis tenuis 9 Agrostis tenuis ); Creeping bentgrass ( Agrostis palustris ); Crested wheatgrass ( Agropyron desertorum ); fairway wheatgrass ( Agropyron cristatum ); hard fescue ( Festuca longifolia ); Kentucky bluegrass ( Poa pratensis ); Orchardgrass ( Dactylis glomerate ); Perennial ryegrass ( Lolium perenne ); Red fescue ( Festuca rubra ) ); Redtop ( Agrostis alba ); Rough bluegrass ( Poa trivialis ); Sheep fescue ( Festuca ovine ) ); smooth bromegrass ( Bromus inermis ); tall fescue ( Festuca arundinacea) ; Timothy ( Phleum pretense ) )); Velvet bentgrass ( Agrostis canine); Weeping alkaligrass ( Puccinellia distans); Western wheatgrass (Agrostis canine); Ti ( Agropyron smithii)); Bermuda grass ( Cynodon species); St. Augustine grass ( Stenotaphrum secundatum); Joysia grass (Joysia ( Zoysia species);Bahia grass ( Paspalum notatum); carpet grass ( Axonopus affinis); centipede grass ( Eremochloa ophiuroides); kikuyu grass ( Pennisetum clandesinum) ; seashore paspalum ( Paspalum vaginatum ) ; Floratam ( Stenotaphrum secundatum "Floratam")); blue gramma ( Bouteloua gracilis ) ; buffalo grass ( Buchloe dactyloids) ; Sideot Gramma ( Boteloa curtipendula ( Bouteloua curtipendula )); foxtail; bromegrass; orchardgrass; quackgrass; and canarygrass.

Additional plants include, for example, pigeon pea, Queensland arrowroot, comfrey, sweet potato, nasturtium, choco, alfalfa, duckweed, trefoil, brassica spp, clover, millet, sorghum, soybean, mulberry, maize, oats, barley, wheat, These include cottonseed, safflower, sunflower, soybean, peanuts, peanuts, apples, acorns, and sprouted legumes.

animal husbandry

In certain embodiments, the method further comprises an animal husbandry aspect, wherein livestock are fed with plants produced according to the agricultural aspect, and optionally, the microorganism-based soil treatment composition according to the agricultural aspect is used to feed the animal. It includes processing the manure.

More specifically, in certain embodiments, animal husbandry aspects include making available to livestock animals a plant produced according to agricultural aspects for the livestock animals to consume the plants. In one embodiment, livestock animals are placed on farmland treated according to agricultural practices for grazing pastures.

In one embodiment, plants are harvested from treated farmland and provided to animals as reduced-carbon footprint feed, grain and/or other forms of loose feed. Preferably, the distance required to transport harvested plants to livestock is minimal, eg less than 10 miles. Even more preferably, in some embodiments, the harvested plants are harvested from the same farmland on which livestock roam and/or graze.

In one embodiment, a combination feeding method is used. For example, in some embodiments, livestock is grain-finished, where agricultural land grazed and sources of grain and other feed finished by livestock animals are treated according to an agricultural aspect of the present method.

In some embodiments, plant material produced according to agricultural practices is processed prior to being provided to domestic animals. For example, in some embodiments, plant material is fermented.

In one embodiment, the processed plant material is a leaf-based feed that is a product of silage, fermentation and storage, which can be used as a preserved livestock feed source during the winter months.

In one embodiment, the processed plant material is a by-product of fermenting grain to produce alcohol. In one embodiment, the processed grain is grain consumed by the brewer and is an insoluble by-product of brewing barley. In one embodiment, the processed plant material is unfermented grain residue of distilled grains, such as distiller grain, maize and rice. Grain from the distillers can be further processed by drying to produce soluble dry distilled grain (DDGS), commonly used as a high protein feed additive for livestock.

A "domestic" animal, as used herein, is a "domesticated" animal that has been influenced, bred, domesticated, and/or controlled by humans over successive generations such that a reciprocal relationship exists between the animal and humans. means species. In particular, livestock animals include animals raised in agricultural or industrial environments to produce commodities such as food, textiles, and labor. The types of animals included in the term livestock include alpacas, llamas, pigs, horses, mules, donkeys, camels, dogs, ruminants, chickens, turkeys, ducks, geese, guinea pigs, and squabs. It may include, but is not limited to.

In certain embodiments, the livestock animal is a “ruminant” or mammal that utilizes a compartmentalized stomach suitable for fermenting plant foods prior to digestion with the help of a specialized gut microbiome. Ruminants include, for example, cattle, sheep, goats, ibex, giraffe, deer, elk, moose, caribou, reindeer, antelope, gazelle, impala, wildebeest, and some kangaroos.

In a specific exemplary embodiment, the livestock animal is a bovine animal, which is a ruminant belonging to the bovine subfamily of the bovine family. Small animals may include domesticated and/or wild species. Specific examples include water buffalo, anoa, tamarau, aurok, banteng, gua, gayal, yak, kupri, beef cattle and dairy cows (eg, Bos taurus , Bos indicus) , Bull, bullock, zebu, saola, bison, buffalo, wisent, bongo, kudu, qwell, imbabala, kudu, nyala, sitatunga, and eland Including, but not limited to.

In certain embodiments, a domesticated animal experiences an increase in growth, muscularity, fertility, and/or milk production as a result of ingesting the plant according to the present methods. This may be due, for example, to an increased protein content of the plant.

In certain embodiments, products from domesticated animals are more nutritious for human consumption when their diets include more grasses treated according to the present methods than other grains and feeds. Grass-fed beef, for example, is known to contain more omega-3 fatty acids, which are important for brain and heart health.

soil treatment composition

The method utilizes a "microbe-based" composition, which means a composition comprising components produced as a result of the growth of a microorganism or other cell culture. Thus, a microbe-based composition may include the microbes themselves and/or by-products of the growth of microbes. Microorganisms may be in the vegetative state, in the form of spores or conidia, in the form of hyphae, in other forms of reproduction, or in the form of a mixture thereof. Microorganisms may be in the form of planktonic or biofilms, or mixtures thereof. By-products of growth can be, for example, metabolites, cell membrane components, proteins, and/or other cellular components. Microorganisms may be intact or lysed. In a preferred embodiment, the microorganism is present in a microorganism-based composition with a growth medium in which it is grown. Microorganisms can be, 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 , 1 per milliliter or at least per gram of the composition. x 10 ¹¹ , 1 x 10 ¹² or 1 x 10 ¹³ or more CFU.

The microorganisms of the present composition can be obtained through a small-scale to large-scale culture process. Such culturing processes include, but are not limited to, cultivation/fermentation in water, solid state fermentation (SSF), and combinations thereof.

The composition can be, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, or 100% growth medium by weight. The amount of biomass in the composition, by weight, can be, for example, 0% to 100%, 10% to 75%, or 25% to 50%, including all percentages there between.

The fermentation product can be used directly with or without extraction or purification. If desired, extraction and purification can be readily accomplished using standard extraction and/or purification methods or techniques described in the literature.

Microorganisms useful according to the present invention may be, for example, strains of non-plant-pathogenic, soil-crowding bacteria, yeasts and/or fungi. A microorganism may be in an active or inactive form, or in a reproductive form of vegetative cells, spores, and/or any other form. Microorganisms can be natural microorganisms or genetically modified microorganisms. For example, microorganisms can be transformed with specific genes to exhibit specific characteristics. A microorganism may also be a mutant of a desired strain. As used herein, “mutant” refers to a strain, genetic variant or subtype of a reference microorganism, wherein a mutation is one or more genetic changes (e.g., point mutations, misses) compared to a reference microorganism. sense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion). Procedures for preparing mutants are known in the art of microbiology. For example, UV mutagenesis and nitrosoguanidines are widely used for this purpose.

In a preferred embodiment, the beneficial microorganisms of the microbial-based soil treatment composition are non-pathogenic, soil-colonising fungi, yeasts and/or bacteria, which are capable of producing one or more of the following: surfactants such as lipopeptides and/or glycolipids; bioactive compounds with antibacterial and immunomodulatory effects; polyketide; mountain; peptide; anti-inflammatory compounds; enzymes such as proteases, amylases and/or lipases; A source of amino acids, vitamins and other nutrients.

In one embodiment, the microorganism is a yeast or fungus. Yeast and fungal species suitable for use in accordance with the present invention include: Aureobasidium ( eg A. pullulans ), Blakeslea , Candida ( Candida ) (eg, C. apicola ( C. apicola ) , C. bombicola ( C. bombicola ) , C. nodaensis ( C. nodaensis )), Cryptococcus ( Cryptococcus ), Debariomyces ( Debaryomyces ) (For example, D. Hansenii ( D. hansenii )), Entomophthora ( Entomophthora ), Hanseniaspora ( Hanseniaspora ) (eg, H. Uvarum ( H. uvarum )), Hansenula ( Hansenula ), Issatchenkia ), Kluyveromyces ( Kluyveromyces ) ( eg K. papi ( K. phaffii )), Mortierella ( Mortierella ), mycorrhizal fungi, Penicillium ), Picomyces ( Phycomyces ), Pichia ( Pichia ) (eg, P. anomala ( P. anomala ) , P. guilliermondi ( P. guilliermondii ) , P. occidentalis ( P. occidentalis ) , P. kudriad Swallows ( P. kudriavzevii )), Pleurotus species (eg, P. austratus ( P. ostreatus )), Pseudozyma (eg, P. aphidis ( P. aphidis )) ))), Saccharomyces (eg, S. boulardii ( S. boulardii ) , S. cerevisiae ( S. cerevisiae ) , S. Torula ( S. torula )), Star Merella ( Starmerella ) ( For example, S. bombicola ( S. bombicola )), Torulopsis ( Torulopsis ), Trichoderma ( For example, T. reesei ( T. reesei ) , T. harzianum ( T. harzianum ) , T. hamatum ( T. hamatum ) , T. viride ( T. viride )), Ustilago ( Ustilago ) (for example, U. Mydis ( U. maydis )), Wickerhamomyces ( Wickerhamomyces ) (eg, W. anomalus ( W. anomalus )), Williopsis (eg, W. mrakii ( W. mrakii )), Zygosaccharomyces ( eg , Zygosaccharomyces ) (eg For example, Z. bailii ( Z. bailii )) and others.

As used herein, “mycorrhizal fungi” includes any species of fungus that forms a non-parasitic mycorrhizal relationship with the roots of a plant. Fungi can be ectodermal fungi and/or endodermal fungi, including subtypes thereof (eg, arbuscla, ericoid, and orchid mycorrhiza).

Non-limiting examples of mycorrhizal fungi according to the present invention include: Glomeromycota, Basidiomycota, Ascomycota, Zygomycota, Helotiales and Hymenococcus. As well as species belonging to Hymenochaetales, Acaulospora species (eg, A. alpina ( A. alpina ), A. brasiliensis ( A. brasiliensis ) , A. poveata ( A. .foveata )), Amantia ( Amanita ) Species (eg, A. Muscaria ( A. muscaria ), A. phalloides ( A. phalloides )), Amphinesma ( Amphinema ) species (eg, A. Byssoides ( A. byssoides ), A. Diadema ( A. diadema ), A. rugosum ( A. rugosum )), Astraeus ( Astraeus ) Species (eg A. Hygrometricum ( A. hygrometricum )), Bisocorticium ( Bayssocorticium ) Species (eg B. Atrovirens ( B. atrovirens )), B. Sophoria terrestris ( Byssoporia terrestris ) (eg, B. terrestris sartorii ( B. terrestris sartoryi ) , B. terrestris illacinorosea ( B. terrestris lilacinorosea ) , B. terrestris aurrantiaca ( B. terrestris lilacinorosea ) ) terrestris aurantiaca ), B. terrestris sublutea ( B. terrestris sublutea ), B. terrestris parxii ( B. terrestris parksii )), Cairney Leah ( Cairneyella ) species (eg C. variabilis ( C. variabilis )), Canterellus ( Cantherellus ) species (eg C. cibarius ( C. cibarius ), C. minor ( C. minor ) , C. Cinnabarinus ( C. cinnabarinus ) , C. Friesi ( C. friesii )), Cenococcum ( Cenococcum ) species (eg, C. Geophilum ( C. geophilum )), Ceratobasidium ( Ceratobasidium ) Species (eg C. Cornigerum ( C. cornigerum )), Cortinarius ( Cortinarius ) Species (eg C. Austrovenetus ( C. austrovenetus ), C. Capellatus ( C. caperatus ), C. Violaceus ( C. violaceus )), Endogone ( Endogone ) Species (eg, E. Fishformis ( E. pisiformis )), Entrophospora (eg, E. Colombiana ) Species (eg, E. Colombiana ) , Funneliformis ( Funneliformis ) Species (eg, F. mosseae ( F. mosseae )), Gamarada ( Gamarada ) species (eg, G. Debralockiae ( G. debralockiae )), Gigaspora ( Gigaspora ) Species (eg, G. gigantean ( G. gigantean ), G. Margarita ( G. margarita )), Glomus ( Glomus ) Species (eg, G. aggregatum ( G. aggregatum ) , G. brasilianum ( G. brasilianum ), G. clarum ( G. clarum ) , G. deserticola ( G. deserticola ) , G. Entucatum ( G. etunicatum ) , G. fasciculatum ( G. fasciculatum ) , G. intraradices ( G. intraradices ) , G. lamellosum ( G. lamellosum ), G. macrocarpum ( G. macrocarpum ) , G. monosporum ( G. monosporum ) , G. mosesea ( G. mosseae ) , G. versiforme ( G. versiforme )), Gomphidius ( Gomphidius ) Species (eg, G. glutinosus ( G. glutinosus )), Hebeloma ( Hebeloma ) Species (eg, H. cylindrosporum ( H. cylindrosporum )), hydnum ( Hydnum ) Species (eg, H. repandum ( H. repandum )), Hymenoscyphus ( Hymenoscyphus ) Species (eg, H. ericae ( H. ericae )), Inocybe ( Inocybe ) Species (eg, I. Bongardi ( I. bongardii ) , I. Sindonia ( I. sindonia )), Lactarius ( Lactarius ) species (eg L. Hygrophoroides ( L. hygrophoroides )), Lindtneria species (eg L. brevispora ( L. brevispora ) ) , melanogaster ( Melanogaster ) species ( For example, M. Ambiguous ( M. ambiguous )), Meliniomyces ( Meliniomyces ) Species (eg, M. bariabilis ( M. variabilis )), Morchella ( Morchella ) species, Mortierella ( Mortierella ) Species (eg M. polycephala ( M. polycephala )), Oidiodendron ) Species ( eg O. maius ( O. maius )), Paraglomus ( Paraglomus ) Species (eg P. brasilianum ( P. brasilianum )), Paxillus ( Paxillus ) Species (eg, P. inovolutus ( P. involutus )), Penicillium ( Penicillium ) Species (eg, P. pinophilum ( P. pinophilum ) , P. tomili ( P. thomili )), Penziza ( Peziza ) species (eg, P. Whitei ( P. whitei )), Pezoloma ( Pezoloma ) species (eg, P. ericae ) ; Phlebopus species (eg P. marginatus ( P. marginatus )) , Philoderma ( Piloderma ) Species (eg P. croceum ( P. croceum )), Pisolithus ( Pisolithus ) species (eg P. tinctorius ) , Pseudotomentella species (eg P. tristis ), Rhizoctonia Species, Rhizodermea species (eg, R. veluwensis ( R. veluwensis )), Rhizophagus ( Rhizophagus ) Species (e.g., R. Iregularis ( R. irregularis )), Rhizopogon ( Rhizopogon ) Species (eg, R. luteorubescens ( R. luteorubescens ) , R. pseudoroseolus ( R. pseudoroseolus )), Rhizoscyphus ( Rhizoscyphus ) Species (eg, R. ericae ( R. ericae )), Russula ( Russula ) species (eg R. liveescens ( R. livescens )) , Sclerocystis species (eg S. sinuosum ( S. sinuosum )), Scleroderma ( Scleroderma ) Species (eg S. cepa ( S. cepa ) , S. Verrucosum ( S. verrucosum )), Scutellospora ( Scutellospora ) species (eg S. pellucida ( S. pellucida ) , S Heterogama ( S. heterogama )), Sebacina ( Sebacina ) Species (e.g., S. sparassoidea ( S. sparassoidea )), Setcheliogaster ( Setchelliogaster ) Species (eg, S. tenuipes ( S. tenuipes )), Suilus ( Suillus ) species (eg S. luteus ( S. luteus )), Thanatephorus species (eg T. cucumeris ) , Telephora ( Thelephora ) species (eg For , T. terrestris ), Tomentella Species (eg, T. badia ( T. badia ), T. cinereoumbrina ( T. cinereoumbrina ), T. erinalis ( T. erinalis ), T. galzini ( T. galzinii )) , Tomentellopsis ( Tomentellopsis ) Species (eg, T. echinospora ( T. echinospora )), Trechispora ( Trechispora ) species (eg, T. hymenocystis ( T. hymenocystis ), T. stellulata ( T. stellulata ) , T. telephora ( T. thelephora )), Trichophaea ( Trichophaea ) species (eg For example, T. abundans ( T. abundans ), T. woolhopeia ( T. woolhopeia )), Tulasnella ( Tulasnella ) species (eg, T. calospora ( T. calospora )), and Tylospora ( Tylospora ) species (eg, T. fibrillose ( T. fibrillose )).

In certain embodiments, the present invention relates to the phylum Glomeromycota and Glomus, Gigaspora, Acaulospora , Sclerocystis , and Entropos Endoscopic fungi, including fungi from the genus Entrophospora , are used. Examples of endoscopic fungi include , but are not limited to: Glomus aggregatum , Glomus brasilianum , Glomus clarum , Glomus deserticola ( Glomus deserticola ), Glomus etunicatum ( Glomus etunicatum ), Glomus fasciculatum ( Glomus fasciculatum ), Glomus intraradices ( Glomus intraradices ) ( Rhizophagus Iregularis ( Rhizophagus irregularis )), Glomus lamello Breath ( Glomus lamellosum ), Glomus Macroparcum ( Glomus macrocarpum ), Gigaspora Margarita ( Gigaspora margarita ), Glomus Monosporum ( Glomus monosporum ), Glomus Moseea ( Glomus mosseae ) ( Funelliformis moseea ( Funneliformis mosseae )), Glomus Versiform ( Glomus versiforme ), Scutellospora heterogama ( Scutellospora heterogama ), And Sclerocystis ( Sclerocystis ) bell.

In certain embodiments, the microorganism is a bacterium, including gram-positive and gram-negative bacteria. Bacteria can be, for example, Agrobacterium (eg A. radiobacter ( A. radiobacter )), Azotobacter ( Azotobacter ) ( A. vinelandi ( A. vinelandii )) , A. croococcum ( A. chroococcum )), Azospirillum ( Azospirillum ) (eg A. brasiliensis ( A. brasiliensis ) ), Bacillus ( Bacillus ) (eg B. amylorique Paciens ( B. amyloliquefaciens ), B. T circulanseu ( B. circulans ) , B. Firmus ( B. firmus ) , B. Laterosporus ( B. laterosporus ) , B. Lee Keniformis ( B. licheniformis ) , B. megaterium ( B. megaterium ) , B. Musilla Ginosus ( B. mucilaginosus ) , B. coagulanseu ( B. coagulans ) , B. subtilis ( B. subtilis )), Frateuria (eg F. aurantia ( F. aurantia )), Microbacterium ( Microbacterium ) (eg M. laevaniformans ( M. laevaniformans )), Myxo Bacteria (myxobacteria) (e.g., Myxococcus xanthus , Stignatella aurantiaca , Sorangium cellulosum , Minicystis rosea ) ), Pantoea ( Pantoea ) (eg, P. agglomerans ( P. agglomerans )), Pseudomonas ( Pseudomonas ) (eg, P. aeruginosa ( P. aeruginosa ), P. Chlororapis Subspecies Aureofaciens ( P. chlororaphis subsp. aureofaciens ) ( Kluyver ), P. putida ( P. putida )), Rhizobium species, Rhodospirillum (eg, R. rubrum ( R. rubrum ) ) , Sphingomonas ( Sphingomonas ) (eg, S. paucimobilis ) and/or Thiobacillus thiooxidans ( Acidothiobacillus thiooxidans ) .

In one exemplary embodiment, the composition comprises Wickerhamomyces anomalus strain NRRL Y-68030.

In another exemplary embodiment, a composition comprises Bacillus subtilis B4 NRRL B-68031. Advantageously, in some embodiments, strain B4 is capable of producing lipopeptide biosurfactants in improved amounts, particularly surfactin. In some embodiments, B4 is “surfactant overproduction”. For example, the strain may contain at least 0.1 to 10 g/L, such as 0.5 to 1 g/L biosurfactant, or, for example, at least 10%, 25%, 50%, or 50%, compared to other B. subtilis bacteria. %, 100%, 2x, 5x, 7.5x, 10x, 12x, 15x or more. For example, in some embodiments, ATCC 39307 can be used as a reference strain.

In an exemplary embodiment, the composition comprises Trichoderma sp . fungi and Bacillus sp. bacteria. In some embodiments, the Bacillus microorganisms are capable of solubilizing phosphorus compounds in soil.

In certain embodiments, the Trichoderma is T. harzianum and the Bacillus is B. amyloliquefaciens NRRL B-67928.

Cultures of the Vickerhamomyces Anomalus NRRL Y-68030 microorganism were deposited with the Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Ave., SW, Washington, DC, 20250, USA It became. The Deposit was designated by the Depositary as Accession Number NRRL Y-68030 and was deposited on May 10, 2021.

Cultures of the B. subtilis B4 microorganism were deposited with the Agricultural Research Service Northern Regional Research Laboratory (NRRL) located at 1400 Independence Ave., SW, Washington, DC, 20250, USA. The Deposit was designated by the Depositary as Accession Number NRRL B-68031 and was deposited on May 10, 2021.

Cultures of the B. amyloliquefaciens "B. amy" microorganism were prepared by the Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Ave., SW, Washington, DC, 20250, USA. has been deposited in The Deposit was designated by the Depository with accession number NRRL B-67928 and was deposited on February 26, 2020.

Each of these cultures was deposited pursuant to 37 CFR 1.14 and 35 U.S.C. 122 under conditions determined by the Commissioner of Patents and Trademarks to ensure that access to the cultures is available while this patent application is pending. A deposit is available as required by foreign patent law in the country in which the counterpart of this application or its descendants were filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the subject invention in violation of patent rights granted by government action.

In addition, the deposited culture is stored and provided to the public in accordance with the provisions of the Budapest Treaty for the Deposit of Microorganisms. That is, for a period of at least 5 years from the most recent request to provide a sample of the deposit, and in any case at least 30 years, thirty (30) years from the date of deposit or for the enforceable life of the patent capable of issuing a culture disclosure; It is stored with all care taken to keep it as uncontaminated as possible. The depositor acknowledges the obligation to replace the deposited material if the depositor is unable to provide a sample upon request due to the conditions of the deposit. All restrictions on the availability of this deposited culture to the public are irrevocably removed upon grant of a patent disclosing it.

In one embodiment, the composition may comprise 1 to 99% by weight of Trichoderma and 99 to 1% by weight of Bacillus . In some embodiments, the Trichoderma to Bacillus cell count ratio is between about 1:9 and about 9:1, between about 1:8 and about 8:1, between about 1:7 and about 7:1, between about 1:6 and about 1:6. about 6:1, about 1:5 to about 5:1, or about 1:4 to about 4:1.

In one embodiment, the composition comprises about 1 x 10 ⁶ to 1 x 10 ¹² , 1 x 10 ⁷ to 1 x 10 ¹¹ , 1 x 10 ⁸ to 1 x 10 ¹⁰ , to 1 x 10 ⁹ CFU/ml of Trichoderma. includes

In a specific embodiment, the composition contains between about 1 x 10 ⁶ and 1 x 10 ¹² , 1 x 10 ⁷ and 1 x 10 ¹¹ , 1 x 10 ⁸ and 1 x 10 ¹⁰ , and 1 x 10 ⁹ CFU/ml of Bacillus . include

In certain embodiments, the microorganism is one capable of fixing and/or solubilizing nitrogen, potassium, phosphorus and/or other micronutrients in the soil.

In one embodiment, the microorganism is selected from, for example, species of Azospirillum , Azotobacter , Chlorobiaceae , Cyanothese , Frankia , Klebsiella , Rhizobia , Trichodesmium , Bacillus and some archaea nitrogen-fixing microorganisms, or diazotrophs, that become In certain embodiments, the nitrogen-fixing bacterium is Azotobacter vinelandi . In another specific embodiment, the nitrogen-fixing microorganism is a B. amy or B4 strain.

In another embodiment, the microorganism is, for example, a potassium-mobilizing microorganism selected from Wickerhamomyces anomalus , Bacillus musilaginosus , Phrateuria aurantia or Glomus mosaea , or KMB. In certain embodiments, the potassium-mobilizing microorganism is Phrateuria aurantia . In another specific embodiment, the potassium-mobilizing microorganism is Wickerhamomyces anomalus , eg strain NRRL Y-68030.

In certain embodiments, the microorganism is a phosphorus-recruiting microorganism, such as Wickerhamomyces anomalus . These microorganisms aid in nutrient and water mobilization, solubilization and soil uptake by producing beneficial organic acids and biosurfactants. W. anomalus also produces the enzyme phytase, which mobilizes phosphate into a usable form of inorganic phosphorus. W. anomalus also produces ethyl acetate, which in certain embodiments can degrade biofilms such as those formed by many plant vascular bacterial pathogens.

Other specific examples include, but are not limited to , Pleurotus austreatus , Devaryomyces hanseni , Saccharomyces cerevisiae , Saccharomyces boulardi , and Bacillus licheniformis .

In a specific embodiment, one or more of the microorganisms is each from 1 x 10 ⁶ to 1 x 10 ¹² , 1 x 10 ⁷ to 1 x 10 ¹¹ , 1 x 10 ⁸ to 1 x 10 ¹⁰ , or 1 x 10 ⁹ CFU/ml is present at a concentration of

In one embodiment, the microorganisms of the composition comprise about 5 to 20%, or about 8 to 15%, or about 10 to 12% by weight of the total composition.

The species and proportions of microorganisms and other components in the composition can be customized depending on, for example, the plant being treated, the type of soil in which the plant is grown, the state of health of the plant at the time of treatment, and other factors.

In one embodiment, the combination of microorganisms applied to a plant and/or its surrounding environment is tailored for a given plant and/or environment. Advantageously, in some embodiments, combinations of microorganisms act synergistically with each other to improve plant health, growth, and/or yield.

The microorganisms and microorganism-based compositions of the present invention have a number of beneficial properties useful for improving plant health, growth and/or yield. For example, the composition may include products derived from the growth of microorganisms, such as biosurfactants, proteins and/or enzymes in purified or unpurified form.

In one embodiment, the microorganism of the composition is capable of producing a biosurfactant. In another embodiment, the biosurfactant can be produced separately by other microorganisms and added to the composition in purified or unpurified form. Biosurfactants in crude form may include, for example, biosurfactants and other products of cell growth in residual fermentation media resulting from the cultivation of biosurfactant-producing microorganisms. This crude biosurfactant composition is about 0.001% to about 90%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60% , from about 45% to about 55%, or about 50% pure biosurfactant.

Biosurfactants form a major class of secondary metabolites produced by various microorganisms such as bacteria, molds and yeasts. As amphiphilic molecules, microbial biosurfactants reduce the surface and interfacial tension between liquid, solid and gas molecules. In addition, the biosurfactant according to the present invention is biodegradable, has low toxicity, is effective in solubilizing and decomposing insoluble compounds in soil, and can be produced using low cost and renewable resources. They can inhibit the attachment of undesirable microorganisms to various surfaces, prevent biofilm formation, and have strong emulsifying and demulsifying properties. Biosurfactants can also be used to improve wettability and achieve uniform solubilization and/or distribution of fertilizers, nutrients and water in the soil.

Biosurfactants according to the method may be, for example, low molecular weight glycolipids (e.g., sophorolipids, mannosylerythritol lipids, rhamnolipids, and trehalose lipids) , lipopeptides (e.g. surfactin, iturin, fengycin, arthrofactin and lichenysin), flavose lipids, phospholipids (e.g. cardiolipin), and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

The composition may include one or more biosurfactants at a concentration of 0.001% to 10%, 0.01% to 5%, 0.05% to 2%, and/or 0.1% to 1%.

Advantageously, according to the present invention, the soil treatment composition may include a medium in which each microorganism is grown. The composition can be, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, or 100% growth medium by weight.

The fermentation medium may contain active and/or inactive cultures, growth by-products in purified or unpurified form, such as biosurfactants, enzymes and/or other metabolites, and/or any residual nutrients. The amount of biomass in the composition can be any value by weight, for example from about 0.01% to 100%, about 1% to 90%, about 5% to about 80%, or about 10% to about 75%. there is.

In one embodiment, different species of microorganisms are grown separately and then mixed together to make a soil treatment composition. In one embodiment, microorganisms such as B. amyloliquefaciens and M. xanthus can be co-cultured.

In certain embodiments, the soil treatment composition includes a germination enhancer for enhancing germination of spore-forming microorganisms used in the soil treatment composition. In certain embodiments, the germination enhancer is an amino acid such as L-alanine and/or L-leucine. In one embodiment, the germination enhancer is manganese.

In one embodiment, the composition includes one or more fatty acids. The fatty acids may be produced by microorganisms in the composition and/or may be produced separately and included as additional ingredients. In certain preferred embodiments, the fatty acid is a saturated long chain fatty acid having a carbon backbone of 14 to 20 carbons, for example myristic acid, palmitic acid or stearic acid. In some embodiments, combinations of two or more saturated long chain fatty acids are included in the composition. In some embodiments, saturated long-chain fatty acids can inhibit methanogenesis and/or increase cell membrane permeability of methanogens.

In one embodiment, the composition includes vitamins and/or minerals in any combination. The vitamins used in the composition of the present invention include, for example, vitamins A, E, K3, D3, B1, B3, B6, B12, C, biotin, folic acid, pantothenic acid, nicotinic acid, choline chloride, inositol and para-amino-benzoic acid. can include Minerals may include, for example, calcium, magnesium, phosphorus, potassium, sodium, chlorine, sulfur, chromium, cobalt, copper, iodine, iron, manganese, molybdenum, nickel, selenium, zinc, and the like. Other components may include, but are not limited to, antioxidants, beta-glucans, bile salts, cholesterol, enzymes, carotenoids, and many others.

In some embodiments, the composition comprises ingredients known to reduce methane production by methanogens, such as, for example, algae (eg, Asparagopsis taxiformis ) ; kelp; 3-nitrooxypropanol; anthraquinone; ionophores (eg, monensin and/or lasaloside); polyphenols (eg saponins, tannins); Yucca schidigera extract (a plant species producing steroidal saponins); Quillaja saponaria extract (triterpenoid saponin-producing plant species); organosulfur (eg garlic extract); flavonoids (eg, quercetin, rutin, kaempferol, naringin, and anthocyanidins; bioflavonoids from green citrus fruits, rose hips, and black currants); carboxylic acids; and/or terpenes (eg, D-limonene, pinene, and citrus extracts).

In one embodiment, the composition is preferably formulated for application to soil, seeds, whole plants, or plant parts (including but not limited to roots, tubers, stems, stalks, shoots, flowers, and leaves). In certain embodiments, the composition is formulated as, for example, a liquid, dust, granule, microgranule, pellet, wettable powder, flowable powder, emulsion, microcapsule, oil, or aerosol.

In order to improve or stabilize the effect of the composition, it can be used in combination with appropriate auxiliaries and then used as such or after dilution, if necessary. In a preferred embodiment, the composition is formulated as a dry powder or granules that can be mixed with water and other ingredients to form a liquid, concentrated liquid, or liquid product.

In one embodiment, the composition comprises glucose (eg in the form of molasses), glycerol and/or glycerin, or in addition to an osmotic agent, to promote osmotic pressure during storage and transportation of the dry product.

The composition can be used alone or in combination with other compounds and/or methods to effectively enhance plant health, growth and/or yield and/or supplement microbial growth. For example, in one embodiment, the composition contains nutrients and/or trace nutrients to enhance plant and/or microbial growth, such as magnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur, iron, copper and zinc; and/or one or more prebiotics, such as kelp extract, fulvic acid, chitin, humates and/or humic acids. In some embodiments, the microorganisms of the composition produce and/or provide these substances. The exact substance and amount thereof can be determined by a grower or agricultural scientist having the benefit of this disclosure.

The composition may also be used in combination with other agricultural compounds and/or crop management systems. In one embodiment, the composition may optionally include or be applied with, for example, natural and/or chemical insecticides, insect repellents, herbicides, fertilizers, water treatments, non-ionic surfactants and/or soil conditioners. Preferably, however, the composition does not contain and/or is not used with benomyl, dodecyl dimethyl ammonium chloride, hydrogen dioxide/peroxyacetic acid, imazilil, propiconazole, tebuconazole or triflumisole.

When the composition is mixed with compatible chemical additives, it is preferred to dilute the chemicals with water prior to adding the composition.

Additional ingredients such as buffers, carriers, other microbial-based compositions prepared in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, tracers, biocides, other microbes, surfactants, emulsifiers, Lubricants, solubility modifiers, pH modifiers, preservatives, stabilizers and ultraviolet light fastness agents may be added to the composition.

The pH of the microbe-based composition should be suitable for the microbe of interest. In a preferred embodiment, the pH of the composition is about 3.5 to 7.0, about 4.0 to 6.5, or about 5.0.

Optionally, the composition may be stored prior to use. A short storage time is preferred. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. In a preferred embodiment, where viable cells are present in the product, the product is stored at a cold temperature, such as below 20°C, 15°C, 10°C, or 5°C.

However, microbial-based compositions can be used without additional stabilization, preservation and storage. Advantageously, the direct use of these microbial-based compositions preserves the high viability of the microbes, reduces the potential for contamination from foreign agents and undesirable microbes, and retains the activity of by-products of microbial growth.

In other embodiments, the composition may be placed in an appropriately sized container, taking into account, for example, the intended use, the method of application considered, the size of the fermentation vessel, and any mode of transport from the microbial growth facility to the point of use. there is. Thus, the container in which the microbial-based composition is placed can be, for example, from 1 pint to 1,000 gallons or larger. In certain embodiments, the vessel is 1 gallon, 2 gallons, 5 gallons, 25 gallons, or larger.

application mode

Advantageously, in a preferred embodiment, the microorganism-based composition according to the present invention is non-toxic and can be applied (applied) in high concentrations without causing irritation to, for example, the skin or digestive tract of humans or other non-infested animals. can Thus, the present invention is particularly useful where application of the microbial-based composition is in the presence of living organisms such as growers and livestock.

As used herein, “applying” (applying) a composition or product to a site means bringing the composition or product into contact with the site so that the composition or product can affect the site. The effect may be due to, for example, microbial growth and colonization, and/or the action of metabolites, enzymes, biosurfactants, or other by-products of microbial growth. The method of application varies depending on the formulation of the composition and may include, for example, spraying, pouring, sprinkling, injecting, dusting, mixing, dunking, fogging and spraying. Formulations may include, for example, liquids, dry and/or wettable powders, flowable powders, dusts, granules, pellets, emulsions, microcapsules, stakes, oils, gels, pastes and/or aerosols. In an exemplary embodiment, the composition is applied after the composition has been prepared, eg, after dissolving the composition in water.

In some embodiments, prior to application of the composition to a conduit in farmland, the method comprises evaluating the site for local conditions, a desired formulation for the composition tailored to the local conditions (e.g., microbial and/or growth by-products). type, combination and/or ratio), and preparing the composition in the desired formulation.

Local conditions may include, for example, soil conditions (e.g., soil type, species of soil microbiota, amount and/or type of soil organic matter content, amount and/or type of GHG precursor substrates in the soil, fertilizers present or amount and/or type of other soil additives or amendments); crop and/or plant condition (eg, type, number, age and/or health of plants grown); environmental conditions (eg, current climate, season or time of year); site’s GHG emissions and types; application mode and/or rate of composition, and other conditions relevant to the site.

After evaluation, a preferred formulation for the composition can be determined, and thus the composition can be tailored to local conditions. The composition may then be cultured in a microbial growth facility that is preferably within 300 miles, preferably within 200 miles, and even more preferably within 100 miles of the application site.

In some embodiments, local conditions are assessed periodically, eg annually, biennially, or even monthly. In this way, composition formulations can be modified in real time as needed to meet the unique needs of changing local conditions.

In one embodiment, the site to which the composition is applied is the soil (or rhizosphere) in which plants are planted or growing (eg, farmland, fields, orchards, groves, meadows/grasses or forests). The composition of the present invention may be pre-mixed with an irrigation fluid, wherein the composition penetrates through the soil and is delivered to the roots of the plant to affect, for example, the root microbiome.

In one embodiment, the composition is applied to a soil surface with or without water, wherein the beneficial effects of the soil application may be activated by rainfall, sprinkler, flood or drip irrigation.

In one embodiment, the site of application is a plant or plant part. The composition may be applied as a seed treatment or directly to the surface of a plant or plant part (eg, the surface of a root, tuber, stem, flower, leaf, fruit, or flower). In certain embodiments, the composition is contacted with one or more roots of a plant. The composition may be applied to the roots, for example by directly spraying or wetting the roots, and/or may be applied to the roots indirectly, for example by administering the composition to the soil (or rhizosphere) in which the plants are growing. The composition may be applied to the seed of the plant before or at the time of planting, or to any other part of the plant and/or its surroundings.

In one embodiment, the method is used in large-scale settings such as large-scale pastures or crops, the method comprising dosing the composition into a tank connected to an irrigation system used to supply water, fertilizer, pesticide, or other liquid composition. can do. Thus, the plants and/or the soil surrounding the plants can be, for example, soil injection, soil drenching, spraying over the seed furrows, using center pivot irrigation systems, micro-jets, drench sprayers, boom sprayers, sprinklers and/or It can be treated with the composition via a drip irrigation device. Advantageously, the method is suitable for treating hundreds of acres of land.

In one embodiment, the method is used in a smaller scale setting, such as on a smaller farm or cattle ranch, the method may include applying the composition using a hand-held lawn and garden sprayer or spreader and/or or using a portable watering can.

A plant and/or its environment may be treated at any point in the process of growing the plant. For example, the composition may be applied to the soil prior to, simultaneously with, or after planting the seeds into the soil. It may also be applied at any point during the plant's development and growth, including when the plant is in bloom, fruiting, during and/or after leaf shedding.

Reduce your carbon footprint

Advantageously, in certain embodiments, the present invention promotes the environmental sustainability of producing and consuming meat, dairy and other animal products; improving the nutrient content of agricultural and pasture soils; promotion of improved soil moisture and water use efficiency; improving soil microbiome diversity; reduced fertilizer use; reduced dependence on grain for livestock feed; Reduction of intestinal GHG emissions from livestock and manure; improving feed efficiency; improving the nutritional quality of meat and milk; and provide solutions to improve others.

In a preferred embodiment, the methods of the present invention are useful for reducing the carbon footprint of livestock and animal-based product production, such as meat, organs, dairy products, hides, fur, feathers, collagen, eggs, and manure manure.

As used herein, “decrease” means a negative change, and the term “increase” means a positive change, where a negative or positive change is at least 0.01%, 0.1%, 0.25%, 0.5% , 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85 A change by %, 90%, 95%, or 100%.

In some embodiments, the desired reduction is achieved within a relatively short period of time, eg, within 1 week, 2 weeks, 3 weeks or 4 weeks. In some embodiments, the desired reduction is achieved, eg, within 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after adopting the method. In some embodiments, the desired reduction is achieved within 1 year, 2 years, 3 years, 4 years, or 5 years of adopting the method.

In some embodiments, a reduction in carbon footprint according to the present methods is achieved through improved plant carbon utilization and storage as well as increased carbon sequestration in the soil. For example, enhanced vegetable carbon utilization may be in the form of, for example, increased leaf growth, increased stem and/or trunk diameter, improved root growth, and/or increased plant numbers.

Additionally, increased soil sequestration may be associated with, for example, increased plant root growth, increased uptake by microbes of organic compounds excreted by plants (including exudates from plant roots), and improved microbes of the soil and roots. It can be a form of clustering.

In one particular embodiment, the method reduces carbon dioxide in the atmosphere. By increasing plant biomass above and below ground, plants act as carbon sinks by fixing carbon during photosynthesis and storing carbon as biomass. In addition, increased plant root biomass supplies applied and natural microbial biomass by increasing the root structure on which microorganisms can colonize as well as increasing the rate of excretion and the amount of sugars and other nutrients exuded from plant roots. Microorganisms consequently convert plant-based material, increasing the level of carbon stored in the soil. Thus, subsurface stimulated microbial populations (both additional and natural) serve as carbon storage systems. In certain embodiments, the microbial cell biomass is yeast biomass.

In certain embodiments, reduction in carbon footprint is achieved through improved agricultural fertilization practices and improved agricultural soil management.

Improved agricultural fertilization practices may take the form, for example, of reducing nitrogen-rich fertilizers, as well as replacing some or all fertilizers, pesticides, and/or other soil conditioners with compositions comprising one or more environmentally friendly soil microorganisms. there is. Advantageously, reducing fertilizer and other chemical applications reduces the amount of these chemicals that contaminate soil and groundwater when left unabsorbed by plants, further reducing runoff to other water sources. Reducing fertilizer application also reduces soil emissions of nitrous oxide and carbon dioxide from such application.

In some embodiments, improved agricultural fertilization may be in the form of using manure produced by livestock animals to fertilize the farmland on which the plants they eat grow or will grow. In certain embodiments, the soil treatment composition may be mixed with manure while it is being stored. Microorganisms can reduce the amount of GHGs (eg, methane, carbon dioxide and/or nitrous oxide) released from manure while promoting increased decomposition of manure. For example, in one embodiment, when the composition includes a biosurfactant and/or a microorganism that produces the biosurfactant, the composition may exhibit antibacterial properties against methanogenic substances. In another embodiment, when the composition comprises a killer yeast, such as Wickerhamomyces anomalus , the composition can be effective in controlling methanogenic microorganisms due to exotoxins secreted by the killer yeast.

Additionally, in some embodiments, applying the composition to manure enhances the value of manure as an organic fertilizer due to the ability of the microorganisms to inoculate the soil of farmland on which the manure is ultimately applied. Since the livestock producing the manure has increased use of feed nitrogen, the associated waste produces less nitrogen and less ammonia. Thus, the circular effects include reduced carbon footprint fertilizers and reduced need for synthetic nitrogen-rich fertilizers.

The method increases aboveground and belowground biomass of plants, including, for example, increased leaf volume, increased stem and/or trunk diameter, increased root growth and/or density, and/or increased plant number. can make it In one embodiment, this is achieved by improving the overall cheeriness of the rhizosphere in which the roots of the plant are growing, for example by improving the nutrient and/or water retention properties of the rhizosphere.

Thus, the present invention may aid in the vegetation and/or restoration of depleted agricultural land due to anthropogenic causes such as overgrazing by livestock, logging, commercial, urban and/or residential development and/or dumping. In some embodiments, the amount of vegetation is depleted due to fire, disease, or other natural and/or environmental stressors.

Additionally, in one embodiment, the method can be used to inoculate the soil and/or rhizosphere of plants with beneficial microorganisms. The microorganisms of the present microorganism-based composition may promote colonization of the root and/or rhizosphere as well as the vascular system of the plant, for example by aerobic bacteria, yeasts and/or fungi.

In certain embodiments, the method may be used to remove nitrous oxide directly from air and/or soil. For example, certain microorganisms according to the present invention (eg, Diadobacter fermenter ) can reduce nitrous oxide to nitrogen in soil without denitrification. Denitrification is the reduction of nitrate and nitrite to molecular nitrogen. Intermediates of the reduction process include nitrogen oxide products such as nitrous oxide that can leak into the atmosphere.

In one embodiment, promotion of colonization can lead to improved biodiversity of the soil microbiome. As used herein, enhancing biodiversity means increasing the diversity of microbial species in soil. Preferably, improved biodiversity includes increasing the ratio of aerobic bacterial species, yeast species, and/or fungal species to anaerobic microorganisms in the soil.

For example, in one embodiment, the microorganisms of the present composition can colonize the roots, soil and / or rhizosphere, and other nutrient-fixing microorganisms, such as Rhizobium and / or mycorrhizal ( Mycorrhizae ), and plant biomass accumulation It may promote the colonization of other endogenous and/or exogenous microbes that it promotes.

In one embodiment, soil biodiversity and root colonization can be further improved by applying biostimulants or substances that promote increased growth rates of microorganisms to the soil.

In one embodiment, improved soil biodiversity promotes enhanced nutrient solubilization and/or uptake. For example, certain aerobic bacterial species can acidify the soil and solubilize NPK fertilizers into a form usable by plants.

In yet another embodiment, the method can be used to combat and/or inhibit colonization of the rhizosphere by soil microorganisms that may compete with harmful or beneficial soil microorganisms. For example, a higher presence of aerobic microbes in the soil may result in less growth of anaerobic microbes, such as nitrate reducing microbes, which can produce harmful atmospheric by-products such as nitrous oxide.

In one embodiment, the method can be used to enhance penetration of beneficial molecules through the outer layer of root cells, eg, through the root-soil interface of the rhizosphere.

The present invention can be used to improve the various qualities of any type of soil, such as clay, sand, silt, peat, chalky, loam soil and/or combinations thereof. In addition, the methods and compositions can be used to improve the quality of dryness, water temperature, porosity, depletion, compacted soil, and/or combinations thereof. The soil may include soil present in the rhizosphere or soil outside the rhizosphere.

In one embodiment, the method can be used to improve drainage and/or dispersion of water in submerged soil. In one embodiment, the method can be used to improve water retention in dry soil.

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

In one embodiment, the method can be used to improve the structure and/or nutrient content of eroded soil.

In one embodiment, the method can be used to reduce and/or replace chemical or synthetic fertilizers, wherein the composition fixes, solubilizes and/or retains nitrogen, potassium, phosphorus (or phosphate) and/or other trace nutrients in the soil. and/or include microorganisms capable of mobilizing.

In certain embodiments, reduction of noxious atmospheric gases is achieved through reduction of methanogenic microorganisms of both animal and environmental origin. Reduction of methanogenic microbes can be in the form of, for example, enhanced management and treatment of manure and/or organic waste, improved land and crop management.

In one embodiment, the livestock aspects of the method reduce the carbon footprint of livestock farming by improving the health and/or productivity of livestock animals in a manner that reduces GHG emissions from livestock digestion, manure and highly concentrated mass production. .

These benefits may include, for example, improved feed efficiency, improved animal health and fertility, improved quantity and nutritional quality of meat and dairy products, and reduced reliance on feed crops with a high carbon footprint, such as transported grains. One particular key benefit of improving feed efficiency is that the increased feed nitrogen usage reduces ammonia and nitrous oxide produced in the digestive system and animal waste products.

In some embodiments, the methods of the present invention may be used by livestock producers or livestock feeders to reduce the use of carbon credits. Accordingly, in certain embodiments, the present methods are directed to methods of reducing the production of carbon dioxide and/or other noxious atmospheric gases, and/or precursors thereof (eg, nitrogen and/or ammonia) using standard techniques in the art. About. It may further include performing measurements to evaluate the effectiveness of the method.

Measurements can be made at specific points after application of the microbial-based composition to farmland. In some embodiments, the measurement is within about 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 30 days, within 60 days, within 90 days, within 120 days, within 180 days, and/or within 1 year. is performed after

Additionally, measurements can be repeated over time. In some embodiments, the measurement is repeated daily, weekly, monthly, bimonthly, semimonthly, semiannually, and/or annually.

In certain embodiments, the assessment of GHG emissions can take the form of measuring GHG emissions from a site. Gas chromatography and electron capture are commonly used to test samples in a laboratory environment. In certain embodiments, GHG emissions can also be performed in situ using, for example, flux measurements and/or in situ soil exploration. Flux measurements analyze the release of gases from the soil surface to the atmosphere, for example using a chamber surrounding a soil area, and then estimate the flux by observing the accumulation of gases inside the chamber over a period of time. The probe can be used to generate a soil gas profile, starting with measuring the concentration of a gas of interest at a specific depth in the soil and directly comparing the concentration between the probe and the surrounding surface conditions (Brummell and Siciliano 2011, 118).

GHG emissions measurement may include other forms of direct emissions measurement and/or fuel input analysis. Direct emissions measurement may include, for example, identifying polluting operating activities (eg, fuel burning vehicles) and directly measuring the emissions of these activities through Continuous Emissions Monitoring Systems (CEMS). Fuel input analysis calculates the amount of energy resources used (eg, electricity, fuel, wood, biomass, etc.) consumed, determines the content of, for example, carbon in that fuel source, and calculates the amount of that carbon and applying the quantity to the amount of fuel consumed to determine emissions.

In certain embodiments, the carbon content of the site on which the plant is grown, e.g., an agricultural field, crop, turf or turf farm, pasture/steppe or forest, quantifies, for example, above-ground and/or above-ground biomass of the plant. can be measured by Typically, for example, the carbon concentration of a tree is assumed to be about 40-50% of the biomass.

Biomass quantification can take the form, for example, of harvesting plants from a sample area and weighing different parts of the plants before and after drying. Biomass quantification can also be performed using non-destructive observation methods such as measuring plant stem diameter, height, volume and other physical parameters. Remote quantification such as, for example, laser profiling and/or drone analysis may also be used.

In some embodiments, the carbon content of the site may further include sampling and measuring the carbon content of litter, woody debris and/or soil in the sampling area. Soils can be analyzed, in particular, using dry burning to determine, for example, the percentage of total organic carbon (TOC), which uses: a potassium permanganate oxidation assay for activated carbon detection; Volumetric density measurement (weight per unit volume) to convert percent carbon to tonnes/acre.

In some embodiments, measurement of GHG levels from livestock is performed using, for example, gas capture and quantification, chromatography, respiratory chambers (to measure the amount of methane exhaled by an individual animal), and in vitro gas production techniques (controlled laboratory and microbial conditions). methods known in the art, including measuring the amount of methane and/or nitrous oxide released per gram of dry matter resulting from the fermentation of a feed (see, eg, Storm et al., 2012, incorporated herein by reference). can be performed according to The measurement may be performed by testing the animal's microbial population, for example by sampling the milk, feces and/or stomach contents and determining the number of methanogenic microbes present therein using, for example, DNA sequencing and/or cell plating. It may also be provided in a form.

Measurements can be performed at specific points after application of the microbe-based composition. In some embodiments, the measurement is within about 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 30 days, within 60 days, within 90 days, within 120 days, within 180 days, and/or within 1 year. is performed after

Additionally, measurements can be repeated over time. In some embodiments, the measurement is repeated daily, weekly, monthly, bimonthly, semimonthly, semiannually, and/or annually.

Growth of microorganisms according to the present invention

The present invention utilizes methods for the cultivation of microorganisms and the production of microbial metabolites and/or other by-products of microbial growth. The present invention further utilizes a culture process suitable for culturing microorganisms and producing microbial metabolites on a desired scale. Such cultivation processes include, but are not limited to, aquaculture/fermentation, solid state fermentation (SSF) and transformation, hybrids and/or combinations thereof.

As used herein, "fermentation" refers to the cultivation or growth of cells under controlled conditions. Growth can be aerobic or anaerobic. In a preferred embodiment, the microorganism is grown using SSF and/or modified versions thereof.

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

The microbial growth vessel used in accordance with the present invention may be any fermentor or culture reactor for industrial use. In one embodiment, the vessel may have functional controls/sensors to measure important factors in the cultivation process, such as, for example, pH, oxygen, pressure, temperature, humidity, microbial density and/or metabolite concentration, or , can be connected to functional controls/sensors.

In a further embodiment, the container can also monitor the growth of microorganisms inside the container (eg, cell number and growth stage can be measured). Alternatively, a sample may be taken from the container daily and counted using techniques known in the art, such as dilution plating techniques. Dilution plating is a simple technique used to estimate the number of organisms in a sample. This technique can also provide an indicator against which different environments or treatments can be compared.

In one embodiment, the method includes supplementing the crop with a nitrogen source. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride. These nitrogen sources can be used independently or in combination of two or more.

The method can provide oxygen to growing cultures. One implementation uses slow motion of air to remove hypoxic air and introduce oxygenated air. For submerged fermentation, oxygenated air can be supplemented with ambient air daily through a mechanism comprising an impeller for mechanical agitation of the liquid, and an air sparger to supply gas bubbles to the liquid to dissolve the oxygen in the liquid. there is.

The method may further include supplementing the crop with a carbon source. Carbon sources include carbohydrates 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 glycerol; fats and oils such as soybean oil, canola oil, rice bran oil, olive oil, corn oil, sunflower oil, sesame oil, and/or linseed 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 especially desirable when growing microorganisms that cannot produce all the vitamins they need. Inorganic nutrients including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. In addition, sources of vitamins, essential amino acids and trace elements may be, for example, in the form of wheat flour or meal, for example corn meal, or in the form of extracts, for example yeast extract, potato extract, beef extract. , soybean extract, banana peel extract, etc., or in purified form. Amino acids may also be included, such as those useful, for example, in the biosynthesis of proteins.

In one embodiment, inorganic salts may also be included. Usable inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate and /or sodium carbonate. These inorganic salts can be used independently or in combination of 2 or more types.

In some embodiments, the culturing method may further include adding an additional acid and/or antimicrobial agent to the medium prior to culturing and/or during the culturing process. Antibacterial agents or antibiotics are used to protect cultures from contamination.

Additionally, antifoaming agents may also be added to prevent the formation and/or accumulation of foam during submerged cultivation.

The pH of the mixture should be suitable for the microorganism of interest. Buffers and pH adjusting agents such as carbonates and phosphates can be used to stabilize the pH around desired values. If metal ions are present in high concentrations, the use of chelating agents in the medium may be necessary.

Microorganisms can grow either in planktonic form or as biofilms. In the case of a biofilm, the container may have a substrate within which microorganisms can grow in a biofilm state. The system may also have the ability to apply stimuli (eg, shear stress) that promote and/or improve biofilm growth properties, for example.

In one embodiment, the method of culturing the microorganism is performed at about 5 ° to about 100 ° C, preferably, 15 to 60 ° C, more preferably, 25 to 50 ° C. In a further embodiment, culturing can be carried out continuously at a constant temperature. In another embodiment, the culture can be subjected to temperature changes.

In one embodiment, the method and equipment used in the culturing process are sterile. Culture equipment, such as reactors/vessels, may be separate from, or connected to, a sterilization unit, eg, an autoclave. The culture equipment may also have a sterilization unit that sterilizes on site before starting the inoculation. Air can be sterilized by methods known in the art. For example, ambient air may pass through at least one filter before being introduced into the vessel. In another embodiment, the medium can be pasteurized or, optionally, not added at all, wherein the use of low water activity and low pH can be used to control undesirable bacterial growth.

In one embodiment, the present invention comprises culturing a microbial strain of the present invention under conditions suitable for growth and metabolite production; and microbial metabolites, such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, beta-glucans, peptides, metabolic intermediates, polyunsaturated fatty acids and lipids, optionally by purifying the metabolites. Methods of producing microbial metabolites are further provided. The metabolite content produced by the method can be, for example, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

Microbial growth by-products produced by the microorganism of interest may reside on the microorganism or be excreted into the growth medium. The medium may contain compounds that stabilize the activity of microbial growth by-products.

The biomass content of the fermentation medium may be, for example, from 5 g/l to 180 g/l or more, or from 10 g/l to 150 g/l.

The cell concentration is, for example, at least 1 x 10 ⁶ to 1 x 10 ¹³ , 1 x 10 ⁷ to 1 x 10 ¹² , 1 x 10 ⁸ to 1 x 10 ¹¹ , or 1 x 10 ⁹ to 1 x 10 ¹⁰ CFU/ can be ml.

Methods and equipment for culturing microorganisms may be performed in the production of microbial by-products, in batch, semi-continuous or continuous processes.

In one embodiment, all microbial culture compositions are removed upon completion of culturing (eg, upon achieving a desired cell density or specified metabolite density). In this batchwise procedure, an entirely new batch is started when the first batch is harvested.

In another embodiment, only a portion of the fermentation product is removed at one time. In this embodiment, the biomass with viable cells, spores, conidia, hyphae and/or mycelia remains in the vessel as an inoculum for a new culture batch. The composition to be removed may be a cell-free medium or contain cells, spores, or other reproductive propagules, and/or combinations thereof. In this way, a semi-continuous system is created.

Advantageously, the method does not require complicated equipment or high energy consumption. Microorganisms of interest may be cultured in situ on a small or large scale, and may still be mixed with the medium.

Advantageously, the microbial-based product can be produced at a remote location. The microbial growth facility may be operated on the grid utilizing, for example, solar, wind and/or hydroelectric power.

Manufacture of Microbial-Based Products

The present invention also provides a "microbe-based product", which is a product that must actually be applied to achieve a desired result. The present microbial-based product may simply be a microbial-based composition harvested from a microbial culture process. Alternatively, the microbial-based product may include added additional ingredients. Such additional ingredients may be, for example, stabilizers, buffers, suitable carriers such as water, salt solutions or any other suitable carrier, added nutrients to support further microbial growth, non-nutrient growth enhancers and/or the substrate on which they are applied. Agents that facilitate tracking of microorganisms and/or compositions in the environment may be included. Microbe-based products may also include mixtures of microbe-based compositions. A microbial-based product may also include one or more components of a microbial-based composition that have been processed in some way, such as filtration, centrifugation, dissolution, drying, purification, and the like.

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

The microorganisms in the microorganism-based product may be in active or inactive form, or in the form of vegetative cells, gametes, conidia, mycelia, hyphae, or any other form of microbial propagation. A microbe-based product may also contain a combination of any of these types of microbes.

In one embodiment, different strains of microorganisms are grown separately and then mixed together to produce a microorganism-based product. Microorganisms can optionally be mixed with the medium in which they are grown and dried before being mixed.

In one embodiment, the different strains are not mixed together, but applied to the plant and/or its environment as separate microbial-based products.

Microbial-based products can be used without further stabilization, preservation and storage. Advantageously, the direct use of these microbial-based products preserves the high viability of the microbes, reduces the potential for contamination from foreign agents and undesirable microbes, and retains the activity of by-products of microbial growth.

When harvesting a microorganism-based composition from a growth vessel, additional ingredients may be added when the harvested product is placed in the vessel or otherwise transported for use. Additives include, for example, buffers, carriers, other microbial-based compositions prepared in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, surfactants, emulsifiers, lubricants, solubility regulators, tracers, solvents, biocides. biologics, antibiotics, pH modifiers, chelating agents, stabilizers, ultraviolet light-resistant agents, other microorganisms and other suitable additives commonly used in such agents.

In one embodiment, buffers including organic and amino acids or salts thereof may be added. Suitable buffering agents include citrate, gluconate, tartarate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine and mixtures thereof. Phosphoric acid and phosphorous acid or salts thereof may also be used. Synthetic buffers are also suitable for use, but it is preferred to use natural buffers such as the organic and amino acids or salts thereof listed above.

In a further embodiment, the pH adjusting agent comprises potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid or mixtures thereof.

The pH of the microbe-based composition should be suitable for the microbe(s) of interest. In a preferred embodiment, the pH of the composition is about 3.5 to 7.0, about 4.0 to 6.5, or about 5.0.

In one embodiment, aqueous agents such as sodium bicarbonate or sodium carbonate, sodium sulfate, sodium phosphate, sodium phosphate can be included in the formulation.

In certain embodiments, an adhesive material may be added to the composition to prolong the attachment of the product to the plant part. Polymers, such as charged polymers, or polysaccharide-based materials may be used, for example, xanthan gum, guar gum, levan, xylinan, gellan gum, curdlan, pullulan, dextran, and the like.

In a preferred embodiment, commercial grade xanthan gum is used as an adhesive. The concentration of gum should be selected according to the gum content of commercial products. For high purity xanthan gum, 0.001% (w/v - xanthan gum/solution) is sufficient.

In one embodiment, glucose, glycerol and/or glycerin are added to the microbe-based product, which can function as an osmotic pressure regulator during storage and transport, for example. In one embodiment, molasses may be included.

In one embodiment, prebiotics may be added to and/or co-applied to a microbial-based product to enhance microbial growth. Suitable prebiotics include, for example, kelp extract, fulvic acid, chitin, humates and/or humic acids. In certain embodiments, the amount of prebiotics applied is between about 0.1 L/acre and about 0.5 L/acre, or between about 0.2 L/acre and about 0.4 L/acre.

In one embodiment, certain nutrients may be added to and/or concurrently applied to a microbial-based product to enhance microbial inoculation and growth. These may include, for example, soluble potassium (K ₂ O), magnesium, sulfur, boron, iron, manganese and/or zinc. Nutrients may be derived from, for example, potassium hydroxide, magnesium sulfate, boric acid, ferrous sulfate, manganese sulfate and/or zinc sulfate.

Optionally, the product may be stored prior to use. A short storage time is preferred. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. In a preferred embodiment, where viable cells are present in the product, the product is stored at a cold temperature, such as below 20°C, 15°C, 10°C, or 5°C.

Local production of microbe-based products

In certain embodiments of the present invention, a microbial growth facility produces fresh, high-density microorganisms and/or microbial growth by-products of interest at a desired scale. The microbial growth facility may be located at or near the application site. The facility produces high-density microbial-based compositions in batch, semi-continuous or continuous culture.

The microbial growth facility of the present invention may be located at the site where the microbial-based product is to be used (eg, a cattle ranch). For example, the microbial growth facility may be within 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3 or 1 mile from the site of use.

Because the microbial-based product can be produced locally, it does not rely on the microbial stabilization, preservation, storage, and transportation processes of conventional microbial production, and much higher densities of microbes can be produced, thereby enabling on-site application. Requires a lower volume of microbial-based product for use, or allows application of even higher densities of microbes when necessary to achieve desired efficacy. This allows the system to be more efficient with a smaller bioreactor (e.g., a smaller fermentation vessel, a reduced supply of starting materials, nutrients and pH regulators) and eliminates the need to stabilize or separate the cells from the culture medium. Local production of microbial-based products also facilitates incorporation of growth media into the product. The medium may contain agents produced during fermentation, which are particularly suitable for local use.

High-density, robust microbial cultures produced locally are more effective on-site than those left in the supply chain for a period of time. The microorganism-based products of the present invention are particularly advantageous over traditional products in which the cells are separated from the metabolites and nutrients present in the fermentative growth medium. Reduced shipping times allow production and delivery of fresh batches of microbes and/or metabolites at the required time and quantity, depending on local demand.

The microbial growth facility of the present invention produces fresh microbial-based compositions comprising the microbes themselves, microbial metabolites, and/or other components of the medium in which the microbes are grown. If desired, the composition may have a high density of vegetative cells or propagules, or a mixture of vegetative cells and propagules.

In one embodiment, the microbial growth facility is located on, or near, eg, within 300 miles, 200 miles, or even 100 miles of the site (eg, a citrus orchard) where the microbial-based product will be used. Advantageously, this allows the composition to be tailored for use at a particular location. The formulation and efficacy of the microbial-based composition depends on the specific field conditions at the time of application, eg, what type of soil, plant and/or crop is being treated; the season, climate and/or time the composition is applied; and depending on what mode and/or application speed is being utilized.

Advantageously, a decentralized microbial growth facility eliminates upstream processing delays, supply chain bottlenecks, inadequate storage, and timely delivery and application of, for example, viable, high cell-number products and associated media and metabolites in which the cells are originally grown. It provides a solution to the current problem of relying on producers on a wide range of industrial scales, where product quality deteriorates due to other interfering contingencies.

Additionally, by making the composition locally, formulation and efficacy can be adjusted in real time depending on the specific location and conditions present at the time of application. This provides advantages over compositions that are premade in a central location and have, for example, set ratios and formulations that may not be optimal for a given location.

Microbial growth facilities provide manufacturing versatility with the ability to customize microbe-based products to improve synergies with destination regions. Advantageously, in a preferred embodiment, the system of the present invention exploits the ability of naturally occurring local microbes and their metabolic by-products to improve GHG management.

The incubation time for an individual container can be, for example, one day or more. Culture products can be harvested in a variety of ways.

For example, pure, high cell density compositions can be harvested through on-site production and delivery within 24 hours of fermentation, significantly reducing transportation costs. Given the prospect of rapid progress in the development of more effective and potent microbial inoculants, consumers can greatly benefit from this ability to quickly provide corresponding microbial-based products.

Example

A further understanding of the present invention and its many advantages may be obtained from the following examples, given by way of example. The following examples illustrate some of the methods, applications, implementations and variations of the present invention. They should not be considered as limiting the present invention. Numerous changes and modifications may be made in connection with the present invention.

Example 1 - Carbon Footprint Reduction Livestock

1 shows a flow diagram 100 of one embodiment of a method that can produce livestock in a manner that reduces the carbon footprint of the overall operation.

As part of the agricultural aspect of the method ( 05 , 10 , 11 , 11a , 11b , 12 ), a selected microorganism-based soil treatment is applied to farmland 10 . In some optional embodiments, the farmland has previously undergone a defined slash-and-burn treatment ( 05 ) and requires vegetation and/or soil regeneration.

As a result of application of the product ( 10 ), the microorganisms of the product inhabit the rhizosphere and plant roots to synergistically improve the nutrient and water content/dispersity of the soil, promote plant health and growth, and improve plant growth in the field. produce beneficial metabolites that enhance plant protein content ( 11 ). Advantageously, this has resulted in reduced fertilizer use ( 11a ) and improved carbon sequestration in soil and plant matter ( 11b ), both of which contribute to reducing GHGs in the atmosphere ( 30 ).

As part of the animal husbandry aspect of the method ( 12, 20, 20a, 20b ), a livestock animal is provided with plant material grown on agricultural land ( 10 ) treated with a microorganism-based soil treatment composition, and the animal ingests the plant material. make it possible ( 20 ). This may be a form of grazing of farmland with the benefits ( 11 ) provided by the application of the soil treatment composition ( 10 ). Alternatively, or additionally, livestock animals may be provided feed or fodder 12 harvested from agricultural land.

As a result of consuming plant material produced according to the present method ( 20 ), animals may experience improved feed efficiency, which may include, for example, lower feed requirements and more productive results in terms of quantity and quality of meat and dairy products produced. It means that it ends with an animal. This also includes improved nitrogen utilization during digestion, which reduces nitrous oxide and ammonia emissions in livestock manure ( 20a ).

Advantageously, by using grass-fed and/or feed crops produced according to the method for livestock animals, the method can be used to feed high carbon footprint grain crops produced exclusively for livestock feed, such as corn, wheat, barley and oats. can reduce their dependence on ( 20b ). Not only does this help reduce the carbon footprint of livestock farming ( 30 ), it may also help reduce the reliance on antibiotics (eg, ionophores) required by cattle fed grain-based diets.

references

Gerber, PJ, et al . (2013). "Tackling climate change through livestock - A global assessment of emissions and mitigation opportunities." Food and Agriculture Organization of the United Nations, Rome. Viewed April 5, 2019. http://www.fao.org/3/i3437e/i3437e.pdf. ("Gerber et al . 2013").

Grossi, G., et al . (2019). "Livestock and climate change: impact of livestock on climate and mitigation strategies." Animal Frontiers , Volume 9, Issue 1, Pages 69-76. https://academic.oup.com/af/article/9/1/69/5173494. ("Grossi et al . 2019").

Government of Western Australia. (2018). "Carbon farming: reducing methane emissions from cattle using feed additives." https://www.agric.wa.gov.au/climate-change/carbon-farming-reducing-methane- emissions-cattle-using-feed-additives. ("Carbon Farming 2018").

Holtshausen, L. et al. (2009). "Feeding saponin-containing Yucca schidigera and Quillaja saponaria to decrease enteric methane production in dairy cows." J. Dairy Sci. 92:2809-2821.

Ishler, V.A., (2016). "Carbon, Methane Emissions and the Diary Cow." Penn State College of Agricultural Sciences. https://extension.psu.edu/carbon-methane-emissions-and-the-dairy-cow. ("Ishler 2016").

Pidwirny, M. (2006). "The Carbon Cycle" Fundamentals of Physical Geography , 2nd Edition . Viewed October 1, 2018. http://www.physicalgeography.net/fundamentals/9r.html. ("Pidwirny 2006").

Storm, Ida MLD, ALF Hellwing, NI Nielsen, and J. Madsen. (2012). "Methods for Measuring and Estimating Methane Emission from Ruminants." Animals (Basel) . Jun. 2(2): 160-183. doi: 10.3390/ani2020160.

United States Environmental Protection Agency. (2016). "Climate Change Indicators in the United States." https://www.epa.gov/sites/production/files/2016-08/documents/climate_indicators_2016.pdf. ("EPA Report 2016").

United States Environmental Protection Agency. (2016). "Overview of Greenhouse Gases." Greenhouse Gas Emissions. https://www.epa.gov/ghgemissions/overview-greenhouse-gases. ("Greenhouse Gas Emissions 2016").

Center for Sustainable Systems, University of Michigan. (2020). "Carbon Footprint Factsheet." Pub. No. CSS09-05. http://css.umich.edu/factsheets/carbon-footprint-factsheet . ("Michigan 2020").

Claims (29)

A method for producing livestock with a reduced carbon footprint, the method comprising agricultural and animal husbandry aspects:
The agricultural aspect improves soil nutrient and water content and dispersibility, improves plant health and growth, improves plant protein content, reduces nitrogen-rich fertilizer use, reduces soil greenhouse gas emissions and/or cultivating agricultural land on which plants for feeding of said livestock are grown or will be grown, using techniques that reduce or enhance the carbon sequestration of soil and/or plant matter;
The livestock farming aspect includes making available to the livestock animals the plants produced on the farmland so that the livestock animals consume the plants,
Wherein the agricultural aspect reduces the carbon footprint in livestock feed and/or animal-based product production by resulting in reduced greenhouse gas emissions compared to traditional agricultural techniques. According to claim 1,
Techniques for cultivating plants include applying a microorganism-based soil treatment composition comprising one or more soil colonizing microorganisms and/or microbial growth by -products to farmland, wherein the one or more microorganisms are Bacillus species , Trichoderma Species , Pleurotus Species , Saccharomyces Species , Debaryomyces ( Debaryomyces ) Species, Lentinula ( Lentinula ) Species, Wickerhamomyces ( Wickerhamomyces ) Species, Starmerella ( Starmerella ) Species , Meyerozyma Species , Pichia species , Monascus Species, Acremonium ( Acremonium ) Species, and Myxococcus ( Myxococcus ) The method is selected from species. According to claim 2,
The microorganism is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) NRRL B-67928 and Trichoderma harzianum . According to claim 2,
Wherein the microorganism is Wickerhamomyces anomalus NRRL Y-68030, Meyerozyma guilliermondii or Bacillus subtilis NRRL B-68031. According to claim 2,
Wherein the composition further comprises a fermentation medium in which the one or more microorganisms are cultured. According to claim 2,
Wherein the composition comprises a microorganism-free growth by-product. According to claim 2,
The method of claim 1, wherein the microbial growth by-product is a biosurfactant selected from glycolipids and lipopeptides. According to claim 7,
Wherein the glycolipid is selected from sophorolipid, mannosylerythritol lipid, rhamnolipid and trehalose lipid. According to claim 7,
Wherein the lipopeptide is selected from surfactin, iturin, fengycin, arthrofactin and lichenysin. According to claim 1,
The method of claim 1, wherein the livestock animal is placed on farmland to graze plants. According to claim 1,
Wherein the plant is harvested from agricultural land and provided to livestock animals as carbon footprint reducing feed and/or grain. According to claim 11,
Wherein the carbon footprint reduction feed comprises grass, broadleaf herb, shrub, hay, straw, alfalfa, fruit, nut, seed, vegetable and/or crop residue. According to claim 12,
wherein the feed is processed to produce carbon footprint reducing silage, and the reduced carbon-footprint silage is provided to livestock as feed. According to claim 11,
The livestock animal is a grain-fed or grain-finished livestock animal, and the step of providing grain with a reduced carbon footprint to the livestock animal is compared to the step of providing conventionally produced grain to the livestock animal, and the grain-fed livestock animal and reducing the carbon footprint of the grain-finished livestock animal. According to claim 11,
Wherein the carbon footprint reducing grain comprises corn, oats, wheat, barley, sorghum, milo, and/or soybean. According to claim 15,
The carbon footprint reducing grain is processed through distillation or brewing to produce alcohol and a carbon footprint reducing grain byproduct, wherein the carbon footprint reducing grain byproduct is provided to livestock animals as feed. According to claim 16,
Wherein the carbon footprint reduction grain by-product is brewing grain, distillation grain, and/or dried distiller's grain with soluble (DDGS). According to claim 1,
Wherein the agricultural aspect results in an increase in the protein content of the plant. According to claim 1,
wherein the agricultural aspect reduces emissions of carbon dioxide, methane and/or nitrous oxide from the soil. According to claim 1,
wherein said livestock animal provides improved feed efficiency and improved quantity and/or quality of meat and dairy products as a result of ingesting said plant. According to claim 1,
The method of claim 1, wherein the livestock animal has improved feed nitrogen utilization, thereby reducing nitrous oxide and ammonia emissions from livestock excretion. According to claim 1,
and reducing reliance on conventionally grown grain crops to feed livestock. The method of claim 22,
wherein said reduced dependence on conventionally grown grain crops increases the marketability and value of pastured livestock. The method of claim 22,
wherein the reduced dependence on conventionally grown grain crops reduces the carbon footprint of grain-feeding and grain-finished livestock production. According to claim 1,
reducing the carbon footprint in the production of animal-based products selected from meat, intestines, dairy products, leather, fur, collagen, eggs, feathers and manure. According to claim 1,
The one or more microorganisms are colonized in soil and/or roots of plants growing in soil, wherein the colonization comprises:
an increase in leaf volume, stem diameter, trunk diameter, root growth and/or number of said plants in said plants;
increasing the protein content of the plant;
an increase in microbial biomass in the soil;
improved soil biodiversity, and
which results in increased uptake of organic plant exudates by microorganisms. The method of claim 26,
Wherein the improved biodiversity increases the ratio of aerobic bacterial species, yeast species, and/or fungal species in the soil to anaerobic microorganisms in the soil. The method of claim 26,
atmospheric carbon dioxide is reduced by enhancing nutrient carbon utilization and storage. The method of claim 26,
wherein carbon sequestration is enhanced.

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