JP2023549694A - How to produce livestock with a low carbon footprint - Google Patents

Abstract

The present invention provides compositions and methods for reducing the carbon footprint of livestock production. Microbial-based soil treatment compositions reduce greenhouse gas emissions from livestock feed production and further improve the health and productivity of livestock animals. [Selection diagram] Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application receives priority from U.S. Provisional Application No. 63/108,392, filed November 1, 2020, and U.S. Provisional Application No. 63/119,284, filed November 30, 2020. This content is 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 (8, 6). "Carbon footprint" is the total amount of GHG emissions caused directly and indirectly by an individual, organization, event or product. It is calculated by summing emissions from all stages of a product or service's life (material production, manufacturing, use, and end-of-life). Different GHGs are emitted throughout a product's life, or lifecycle, and each has a greater or lesser ability to trap heat in the atmosphere. These differences are accounted for by calculating the global warming potential (GWP) of each gas, in units of carbon dioxide equivalents ( _CO2e ), and carbon footprints are expressed simply for ease of comparison. One unit (Non-Patent Document 10).

Based on the latest measurements from monitoring stations around the world, as well as measurements of stale air from bubbles trapped in ice layers from Antarctica and Greenland, global atmospheric concentrations of GHGs have increased significantly over the past several hundred years. (Non-patent Document 8, e.g. 6, 15).

In particular, since the beginning of the Industrial Revolution in the 1700s, human activities have contributed to the amount of GHG in the atmosphere, such as by burning fossil fuels, clearing forests, and conducting other activities. Most of the GHGs emitted into the atmosphere exist for long periods ranging from 10 years to several thousand 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 limit the growth of GHG emissions and/or reduce the carbon footprint of various activities through treaties and other agreements between nations. One such attempt is through the use of carbon credit systems. Carbon credits are a collective term for tradable certificates or permits representing the right to emit one ton of carbon dioxide, or GHG equivalent. In a typical carbon credit system, a governing body sets a quota for the GHG emissions that businesses can emit. Beyond these quotas, operators will be required to purchase additional emissions credits from other operators who are not using all their carbon credits.

The goal of the carbon credit system is to encourage businesses to invest in more green technology, machinery and practices in order to profit from trading these credits. Under the Kyoto Protocol of the United Nations Framework Convention on Climate Change (UNFCCC), many countries have agreed to be internationally bound by policies to reduce GHG emissions, including trading in emissions credits. Although the United States is not bound by the Kyoto Protocol and does not have a central national emissions trading system, several states, such as California and a group of northeastern states, have begun to adopt such trading systems.

One particular area of industry with a significant carbon footprint is food manufacturing, ie, agricultural and livestock production, which primarily results in emissions of carbon dioxide, nitrous oxide, and methane.

In particular, ruminant livestock such as cows, sheep, buffalo, goats, deer and camels contribute to methane production due to their unique digestive systems. Ruminants have four gastric compartments: the abomasum, rumen, abomasum, and abomasum. The rumen functions as an anaerobic fermentation vessel, especially for biofilm-forming methanogenic bacteria that produce gaseous byproducts such as carbon dioxide and methane. The approximately 130-250+ gallons of rumen gas produced by fermentation comes from the burps of one dairy 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 that can lead to GHG emissions during storage and processing: organic matter, which can be converted to methane emissions, and nitrogen, which can indirectly lead to nitrous oxide emissions. There is. Methane is released when methanogenic bacteria decompose organic matter in manure when retained in lagoons, tailings ponds, or holding tanks. Additionally, nitrogen in the form of ammonia ( _NH3 ) is excreted from manure during storage and processing. Ammonia is later converted to nitrous oxide (Non-Patent Document 1).

For example, methods exist to reduce intestinal and fecal-based methane emissions in livestock, including defaunation of the digestive system and even vaccination against methanogens, but these strategies are unlikely to be beneficial. Reducing the number of gut microbes, and these methods are short-lived due to microbial adaptation. Other known strategies include for manipulating enteric fermentation, for example by directly inhibiting methanogens and protozoa, or by directing hydrogen ions away from methanogens to reduce methane production. This includes, among other things, food modification for livestock grazing pastures. Most anti-methanogenic compounds are expensive, short-lived, inconsistent, require high concentrations, do not contain _H2 receptors, do not affect methanogens in the form of biofilms, and/or contain compounds that are easily destroyed and/or excreted in the intestine.

Further strategies include improved animal management practices. It is believed that there is a direct relationship between GHG emission intensity and animal efficiency. The more productive the animal is (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 therefore excrete less nitrogen in their feces and urine. Some methods to increase efficiency include breeding and genetic modification to reduce the nutritional requirements of animals. However, breeding can be unpredictable and time-consuming, and the impact of genetic modification on animal health and well-being is unknown (Non-Patent Document 1).

Another important aspect of the livestock sector is feed production and processing, which contributes to almost half of industry's GHG emissions. Such GHG emissions result from, for example, land use change, fertilizer and pesticide manufacturing and use, manure excreted and applied to farms, agricultural machinery and operations, feed processing, and feed transportation.

Approximately 60% of the global biomass harvested around the world (eg, corn and other forage) enters the livestock subsystem as feed or bedding material. Soil carbon dioxide emissions are the result of soil carbon dynamics (e.g. decomposition of plant residues, mineralization of soil organic matter, land use change, etc.), the production of synthetic fertilizers and pesticides, and the use of fossil fuels in agricultural practices on farms. arises as When organic and inorganic fertilizers are applied to soil, nitrous oxide is emitted. These fertilizers are subject to loss through leaching and denitrification prior to crop uptake (Non-Patent Document 2).

Gerber, P.J., 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 M.L.D., A.L.F. Hellwing, N.I. 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)

The livestock industry is important, for example, for the production of meat and dairy products, but increasing concerns about climate change and the need to reduce GHG emissions have led to improvements in feeding livestock while reducing the livestock sector's carbon footprint. A tailored approach is required.

The present invention provides an environmentally friendly method for livestock production. The invention further relates to microbial-based products, as well as the use of these products, to achieve beneficial results in many situations within the livestock chain, including, for example, feed production aspects and husbandry aspects. I will provide a. Advantageously, the present invention utilizes organic, non-GMO ingredients and environmentally friendly methods to reduce the carbon footprint of livestock farming.

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

In a preferred embodiment, the method includes an agricultural aspect in which a low carbon footprint livestock feed is grown and a livestock aspect in which the low carbon footprint feed is fed to a livestock animal.

In one embodiment, the agricultural aspect includes applying a microbial-based soil treatment composition to a plot of agricultural land, including a plot cleared by natural fire or burning, and the soil treatment composition comprises applying a microbial-based soil treatment composition to a and/or provide one or more direct or indirect benefits to the soil. These benefits include, for example, improved plant health and growth (both above and below ground), improved plant protein and/or nutrient content, reduced fertilizer usage, enhanced carbon sequestration in the soil, Mention may be made of improving the diversity of the microbial flora, improving the distribution of water and/or nutrients in the soil, and/or increasing the uptake of soil nutrients by plant roots.

In certain embodiments, the soil treatment composition includes one or more beneficial microorganisms. In a preferred embodiment, beneficial microorganisms include surfactants such as lipopeptides and/or glycolipids; bioactive compounds with antibacterial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory compounds; proteases, amylases, and/or or enzymes such as lipase; and non-pathogenic soil-colonizing fungi, yeasts, and/or bacteria that can produce one or more of amino acids, vitamins, and other nutrients.

In a preferred embodiment, the microorganism is, for example, Trichoderma spp. , Bacillus spp. , Wickerhamomyces anomalus , Myxococcus xanthus , Pseudomonas chlororaphis , Starmerella bombicola , Saccharomyces boulardii , Pichia occidentalis , Pichia kudriavzevii , Meyerozyma guilliermondii , mycorrhizal fungi, nitrogen fixing fungi Agent ( Azotobacter vinelandii ) and/or potassium mobilizing agents (eg, non-pathogenic bacteria, yeasts, and/or fungi selected from Frateuria aurantia ).

The species and proportions of microorganisms and other ingredients in the composition may be determined according to, for example, the geographical area in which the treatment is carried out, the species of domestic animal that consumes the plant, the health of the agricultural land at the time of treatment, and other factors. I can do it. In this way, the composition can be customized for any given location.

In certain exemplary embodiments, the soil treatment composition includes a first microorganism and a second microorganism, growth byproducts thereof, and, optionally, one or more nutrient sources. In a specific exemplary embodiment, 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 can include, for example, the fermentation medium in which the microorganism was cultured, and/or any residual nutrients from the culture. Growth byproducts can further include metabolites or other biochemicals produced as a result of cell growth, including, for example, biosurfactants, enzymes, and/or solvents.

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

In some embodiments, the methods of the invention further include applying additional agricultural ingredients such as, for example, herbicides, fertilizers, pesticides, and/or soil conditioners. Preferably, the additional ingredients are non-toxic and environmentally friendly. The exact materials and amounts thereof can be determined by soil scientists who can benefit from this disclosure.

In certain embodiments, a soil treatment composition is contacted with a portion of a plant. In certain embodiments, the composition is contacted with one or more roots of a plant. The composition can be applied directly to the roots, for example, by spraying or dipping the roots, and/or indirectly, for example, by administering the composition to the soil in which the plant grows (e.g., the rhizosphere). It can be applied to The composition can 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 surrounding environment.

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

In some embodiments, the farmland includes grasses including, for example, bluegrass, bermudagrass, fescue, buffalograss, and the like. Other forages, such as forbs, weeds and shrubs, as well as forage crops, such as corn, oats, and barley, can also be treated with the soil treatment composition. In some embodiments, the composition can be applied to pasture, and in some cases, burning has been conducted to promote vegetation.

In one embodiment, the method can be used to reduce the carbon footprint of producing grain-fed livestock as well as grain-finished livestock, where the grain is produced according to the agronomic aspects of the method. carbon grain and/or its by-products (eg, solubles added dried distillers' grain, DDGS).

In one embodiment, the agronomic aspects of the method can increase the feed value of grass compared to conventionally grown high carbon footprint grains produced for feeding and/or finishing livestock.

By increasing the marketability of pasture or grass-fed livestock and/or by feeding livestock grains produced using low carbon footprint methods, the present invention of the livestock feed industry by reducing utilization changes, reducing the production and use of fertilizers and pesticides, and reducing the operation of fossil fuel-burning agricultural machinery, including those used to process and transport feed. Carbon footprint can be reduced.

This can be achieved, for example, by increasing nutrient carbon utilization and storage in areas of agricultural land, increasing carbon sequestration in soils, reducing soil-based GHG emissions, improving agricultural nitrogen-based fertilization practices, and increasing biodiversity in soil microbiota. improvement, and improved agricultural soil management.

In certain embodiments, increasing nutrient carbon utilization includes, for example, increasing plant leaves, increasing stem and/or stem diameter, increasing root growth, and/or increasing the number of plants per area unit. It can be in the form of

In certain embodiments, increased soil sequestration may include, for example, increased plant root growth (e.g., length and density), increased microbial uptake of organic compounds secreted by the plant (ex. (including substances), and can be in the form of improving soil microbial colonization.

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

In certain embodiments, improved agricultural fertilization practices, soil biodiversity, and/or soil management include reducing nitrogen-rich fertilizers, as well as inoculating the plant rhizosphere with one or more beneficial microorganisms. It can be in the form of For example, in preferred embodiments, the microorganisms of the soil treatment composition colonize the rhizosphere and provide multiple benefits to the plants whose roots are growing therein, including protection, hydration, and nutrition. be able to. Accordingly, the method can reduce nitrous oxide emissions by replacing some or all fertilizers, pesticides, and/or other soil amendments with one or more beneficial soil microorganisms.

In certain embodiments, the method further includes a husbandry aspect that includes making plants produced according to the agricultural aspect available to the livestock animal so that the livestock animal ingests the plant. In one embodiment, livestock animals are placed in areas of agricultural land treated according to agricultural practices for free grazing. In one embodiment, plants are harvested from treated farmland and provided to animals as low carbon footprint fodder, grain and/or other forms of loose feed.

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

In one embodiment, the animal husbandry aspect of the method improves the livestock industry by promoting the health and/or productivity of livestock animals in a way that reduces GHG emissions resulting from livestock digestion, fertilizers, and large-scale production. Reduce your carbon footprint.

These benefits include, for example, improved feed efficiency, which translates into improved animal health and fertility, improved meat and dairy quantities and nutritional quality, and conventionally produced corn, etc. resulting in reduced dependence on high carbon footprint feed crops. One specific and important benefit of improved feed efficiency is an increase in feed nitrogen usage, which results in a reduction in ammonia and nitrous oxide produced in the digestive system, as well as livestock waste. . In addition, improved feed efficiency can result in improved animal productivity, which means less feed and/or GHG emitting animals are required to produce a given amount of product. means.

In one embodiment, the livestock aspect further includes applying the soil treatment composition directly to livestock manure to promote increased microbial degradation of the manure while reducing the amount of GHGs emitted therefrom. can be included. In some embodiments, application of the composition to manure reduces the carbon footprint because the fertilizer can be used in place of synthetic nitrogen-rich fertilizers for agricultural land that ultimately becomes feed for livestock animals. further contribute to

In some embodiments, the methods of the invention can be utilized by livestock producers and/or livestock feed suppliers to reduce carbon credit usage. Accordingly, in certain embodiments, the method includes the method of removing methane, carbon dioxide, and/or other harmful atmospheric gases by livestock producers and/or livestock feeders using standard techniques in the art. and/or their precursors (e.g., nitrogen and/or ammonia).

Among the advantages, in certain embodiments, the present invention promotes, for example, the growth and vigor of forage crops and pastures; improves the nutrient content of agricultural soils; promotes improvements in soil moisture and water use efficiency; Increasing the diversity of soil microbiota; reducing fertilizer use; increasing the feed value of pasture with reduced dependence on grains; reducing enteric GHG emissions from livestock and manure; improving feed-to-muscle conversion solutions to improve the environmental sustainability of producing and consuming meat, dairy, and other animal-based products; by improving the productivity of livestock animals, such as meat and milk yields and nutritional quality; and by other means; provide.

1 is a schematic flowchart of a system according to an embodiment of the invention.

The present invention provides an environmentally friendly method for producing livestock with a low carbon footprint, the method comprising an agricultural aspect and/or a husbandry aspect;
Agricultural aspects improve farmland where plants are growing or will grow for feeding livestock by increasing soil nutrient and moisture content and distribution, promoting plant health and growth, and improving plant protein content. using techniques that increase carbon content, reduce the use of nitrogen-rich fertilizers, and/or enhance carbon sequestration in the soil and/or plants;
Husbandry aspects include making plants produced in agricultural land available to livestock animals so that they ingest the plants;
The agricultural aspects reduce greenhouse gas emissions compared to traditional agricultural techniques, and in preferred embodiments, the animal husbandry aspects result in improved 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 farming.

“Carbon footprint” is defined herein as the 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. Defined as a measure of total quantity. As just one example, a product that requires transportation over many miles by truck (e.g., harvested feed grain) has a larger carbon footprint than an alternative product that does not require transportation (e.g., grass grown in a pasture). Has a print.

Carbon footprints can be calculated using LCA (life cycle assessment) methods or limited to immediately attributable emissions from fossil fuel energy use. Life cycle assessment (also known as LCA, life cycle analysis, ecobalance, cradle-to-grave analysis) is the investigation and evaluation of the environmental impacts of a particular product or service caused or required by its existence. It is. The carbon footprint life cycle concept is all-encompassing, meaning it includes all sources that can cause carbon emissions. In other words, all direct (on-site, internal) and indirect emissions (off-site, external, embodied, upstream, downstream) need to be considered.

Carbon footprint is usually expressed as _CO2 equivalents. A carbon dioxide equivalent is a quantity that represents the amount of _CO2 that has the same global warming potential (GWP) for a given GHG mixture and amount when measured over a particular timescale (generally 100 years). Therefore, carbon dioxide equivalents reflect time-integrated radiative forcing. The carbon dioxide equivalent of a gas is obtained by multiplying the mass of the gas by the GWP. The following units are commonly used:
a) According to the United Nations Climate Change Panel IPCC: billion metric tons of CO ₂ equivalents (GtCO ₂ equivalents);
b) Industry: Million Metric Tons of Carbon Dioxide Equivalent (MMTCDE);
c) For vehicles: g carbon dioxide equivalent/km (gCDE/km).

For example, the GWP of methane is 21 and the GWP of nitrous oxide is 310. This means that emissions of 1 million metric tons of methane and nitrous oxide, respectively, correspond to emissions of 21 and 310 million metric tons of carbon dioxide.

Various methods for calculating or estimating carbon footprints exist in the art and can be used in the present invention.

Advantageously, in a preferred embodiment, the present invention reduces the carbon footprint of producing livestock, including reducing the carbon footprint of producing forage-based, fodder-based, and/or grain-based feed for livestock. Useful for reducing printing.

“Low carbon footprint” means that over the entire life cycle of producing livestock feed and feeding livestock with this feed, the livestock/animal-based product is means a negative change in the amount of carbon dioxide and other GHGs emitted. The negative change in _CO2 and/or other GHG emissions is, for example, 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 used interchangeably herein with the terms "carbon intensity" and "emissions 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 combustion, livestock production, dishwasher production). Emission intensity includes, for example, emissions relative to the amount of fuel burned, the number of livestock animals produced, the amount of industrial products produced, the total distance traveled, and/or the number of economic units produced. It will be done.

Emission intensity is measured over the entire life cycle of a product. For example, fuel emissions intensity measures all emissions emitted along the fuel supply chain, including all emissions emitted during fuel exploration, mining, collection, production, transportation, distribution, distribution, and combustion. Calculated by aggregating GHG emissions.

Selected Definitions As used herein, "agriculture" refers to the cultivation and cultivation of plants, algae, and/or fungi for food, fiber, biofuels, pharmaceuticals, cosmetics, supplements, decorative purposes, and other uses. means breeding. According to the invention, agriculture can also include horticulture, landscaping, horticulture, plant conservation, forestry and reforestation, pasture and grassland restoration, horticulture, arboriculture, arboriculture, and cropping. Additionally, agriculture includes soil care, monitoring and maintenance.

As used herein, a "biofilm" is a complex aggregate of microorganisms, such as bacteria, in which cells adhere to each other and/or to surfaces. Cells in a biofilm are physiologically different from planktonic cells of the same organism, which are single cells that can float or migrate in liquid media.

As used herein, "conventional" agriculture and animal husbandry refers to genetically modified organisms (GMOs), concentrated animal feeding operations (CAFOs), and the synthesis of fertilizers, pesticides, and/or herbicides, etc. Utilizing one or more of the chemicals. Conventional agriculture and livestock farming are typically extremely resource and energy intensive and can contribute to significant GHG emissions.

As used herein, "digestive system" refers to the system of organs within an animal's body that enables digestion, or ingestion of food and its conversion into energy and waste. The digestive system includes, for example, the oral cavity, esophagus, crop sack, gizzard, anterior ventricle, stomach, rumen, abomasum, abomasum, pancreas, liver, small intestine, large intestine (colon), and cecum. , appendix, and/or anus. Additional organs or parts related to digestion and specific to a particular animal are also envisioned.

As used herein, "enhance" means to improve or increase. For example, improving plant health includes increasing seed germination and/or emergence, improving the ability to control pests and/or diseases, and improving the ability to survive environmental stressors such as drought and/or over-irrigation. , which means improved plant growth and reproductive ability. Enhanced plant growth and/or enhanced plant biomass increases plant size and/or mass both above and below the ground (e.g. canopy/leaf volume, height, trunk caliper, branching). length, shoot length, protein content, root size/density and/or overall growth index) and/or improve the ability of the plant to reach a desired size and/or mass. It means that. The enhanced yield can be achieved, 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. by improving the final product produced by plants in a crop (e.g. by improving taste, texture, Brix, chlorophyll content and/or color).

As used herein, "agricultural land" includes any parcel of land where plants are grown, cultivated, and/or managed for human benefit. In the farmland,
Pasture or land containing primarily grasses, legumes and non-herbaceous plants for consumption by livestock;
pasture, which is typically an ungrazed plot of land used for harvesting hay or other animal feed;
Includes grazing land, wild and cultivated grassland, shrubland, woodland, wetlands and deserts consumed by livestock or wildlife; and agricultural crops.

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

As used herein, "forage" means any plant material that grows on a patch of agricultural land and is consumed by, or at least edible 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 can, for example, constitute 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 ration. Grains include, but are not limited to, corn, oats, barley, wheat, sorghum, milo, and soybeans.

In some embodiments, the grain-fed livestock is "grain-finished." That is, domestic animals spend most of their lives grazing and/or consuming grass and forage-based diets, but for the last four to six months of their lives, for example, consume predominantly grain-based diets. (e.g., more than 50%, 60%, 75%, or 90% of caloric intake). This grain-based diet often includes high-energy grains such as corn, wheat, and milo, but in some cases the animal feeds feedlot grains such as potato husks, sugar beets, and hay. They also consume a variety of other feed sources.

As used herein, "grass-fed" livestock means that the livestock animal consumes only grass and forage throughout its entire life, beginning after weaning.

As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, protein, organic compound such as a small molecule (e.g., as listed below) or Other compounds are substantially free of other compounds such as naturally associated cellular materials. For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not include the genes or sequences that flank it in its naturally occurring state. A purified or isolated polypeptide does not include the amino acids or sequences that naturally occur alongside it. A purified or isolated microbial strain is removed from its naturally occurring environment. Thus, the isolated strain exists, for example, as a biologically pure culture or as a spore (or other form of the strain) associated with a carrier.

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

"Metabolite" refers to any substance produced by metabolism (eg, a growth by-product) or necessary to participate in a particular metabolic process. Metabolites are organic compounds that are starting materials, intermediates or 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 byproduct of its metabolism. Methanobacterium is an archaeal bacterium that can be found in the digestive systems and metabolic wastes of ruminants and non-ruminants (eg, pigs, poultry, and horses). Methanobacteria include Methanobacterium spp. (e.g. M. formicicum ), Methanobrevibacter spp. (e.g. M. ruminantium ), Methanococcus spp. (e.g. M. paripaludis ), Methanoculleus spp. (e.g. M. bourgensis ), Methanoforens. spp. (e.g. M. stordalenmirensis ), Methanofollis liminatans , Methanogenium wolfei , Methanomicrobium spp. (e.g. M. mobile ), Methanopyrus kandleri , Methanoregula boonei , Methanosaeta spp. (e.g. M. concilii , M. thermophile ), Methanosarcina spp. ( eg, M. barkeri , M. mazeii ), Methanosphaera stadtmanae , Methanospirillium hungatei , Methanothermobacter spp. , and/or Methanothrix sochngenii .

As used herein, the term "plant" means woody, ornamental or ornamental, crop or cereal, fruit or vegetable, fruit or vegetable plant, flower or tree, macroalgae or microalgae, phytoplankton. and any species of photosynthetic algae (e.g., the green alga Chlamydomonas reinhardtii ). "Plant" also includes unicellular plants (eg, microalgae) and colonies (eg, volvox) or a plurality of plant cells that are largely differentiated into 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. Furthermore, the plant can be free-standing, for example in a lawn or garden, or it can be one of many plants, for example as part of an orchard, forest or crop.

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

The ranges shown here are concise versions of all values within the range. For example, the range 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, 46 , 47, 48, 49 or 50, including any number, combination of numbers, or subrange, and also the value of the intermediate decimal point between the above-mentioned integers, e.g. 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9 are also considered to be included. With respect to subranges, "nested subranges" extending from either endpoint of the range are specifically contemplated. For example, the nested subranges of the exemplary range 1 to 50 are 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction and 50 to 40, 50 to 30, 50 to 20, and 50 in the other direction. ~10.

As used herein, "reduction" means a negative change and "increase" means a positive change, where 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%, 95% or 100%.

As used herein, "soil treatment," "soil amendment," or "soil conditioner" refers to any compound, material, or compound added to soil to enhance the properties of the soil and/or rhizosphere. or a combination of compounds or materials. Soil amendments can include organic and inorganic substances, and can further include, for example, fertilizers, pesticides and/or herbicides. Nutrient-rich, well-drained soil is essential for plant growth and health, and therefore soil amendments are used to increase plant biomass by changing the nutrient and water content of the soil. be able to. Soil amendments also include, but are not limited to, improving soil structure (e.g., prevention of compaction), nutrient concentration and storage capacity, improving water retention in dry soils, and improving drainage in water-containing soils. It can also be used to improve many different qualities of soil, including remediation.

As used herein, "surfactant" refers to a compound that reduces the surface tension (or interfacial tension) between phases. Surfactants act, for example, as detergents, wetting agents, emulsifiers, blowing agents, and dispersants. A "biosurfactant" is a surfactant produced by living organisms.

The transitional phrase "comprising" is synonymous with "having" and "containing," and is inclusive, open-ended, and does not exclude additional, unlisted elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials and steps and to those "that do not materially affect the essential novel features" of the claimed invention. Use of the term "comprising" contemplates other embodiments "consisting of" or "consisting essentially of" the listed elements.

Unless stated otherwise, the term "or" as used herein is considered inclusive. Unless otherwise specified, "a," "and," and "the" as used herein may be considered singular or plural.

Unless otherwise specified, "about" as used herein is considered to be within normal tolerances, eg, within two standard deviations of the mean. "About" means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0. It is considered to be within 0.05% or 0.01%.

In the definitions of variables herein, a recitation of a chemical group includes definitions of the variable as a single group or a combination of recited groups. The recitation of embodiments for variables and aspects herein includes embodiments as a single embodiment or in combination with other embodiments or portions thereof.

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

Low Carbon Footprint Livestock Production Methods In certain embodiments, feeding and producing livestock animals in a manner that reduces GHG emissions resulting from livestock animals, including, for example, GHGs such as carbon dioxide, methane, and nitrous oxide. A method is provided to facilitate.

In preferred embodiments, the method includes agricultural and livestock aspects.

Agricultural Aspects In one embodiment, agricultural aspects of the invention provide for producing livestock feed using technologies that result in a reduction in total GHG emissions when compared to GHG emitted from feed production without these technologies. Including producing.

More specifically, in certain embodiments, the agricultural aspects of the method include adding one or more beneficial microorganisms to an area of agricultural land where plants suitable for feeding livestock grow or will grow. and/or its growth by-products, the soil treatment composition having one or more direct or indirect benefits to plants and/or soil in agricultural land. and the one or more benefits contribute to reducing GHG emissions from the cultivation and/or management of agricultural land.

These benefits include, for example, improved plant health and growth (both above and below ground), improved plant protein and/or nutrient content, improved soil microbial diversity, water and/or Mention may be made of improved distribution of nutrients and/or increased uptake of nutrients by the roots of the plant.

In one embodiment, the agricultural aspects of the method include enhancing nutrient carbon utilization and storage in areas of agricultural land, increasing carbon sequestration in soil, reducing soil GHG emissions, improving agricultural nitrogen-based fertilization practices, improving soil microbial Reduce the carbon footprint of livestock feed production by improving biodiversity in grasslands and improving agricultural soil management.

In certain embodiments, increasing nutrient carbon utilization includes, for example, increasing plant leaves, increasing stem and/or stem diameter, increasing root growth, and/or increasing the number of plants per area unit. It can be in the form of

In certain embodiments, increasing soil carbon sequestration may include, for example, increasing plant root growth (e.g., length and density), increasing microbial uptake of GHG precursors/organic compounds secreted by plants (plant (including secretions from the roots of the soil) and increased microbial colonization of the soil.

In some embodiments, reducing soil GHG emissions includes reducing the amount of methane, carbon dioxide, and/or nitrous oxide/its precursors emitted from the soil. For example, in some embodiments this can be achieved by reducing water stress and/or increasing water use efficiency of the plant. Large amounts of soil moisture result in, for example, lower soil temperatures and increased nutrient transport to plants, both of which contribute to reduced soil respiration resulting in reduced GHG emissions and free GHG precursor molecules in the soil. Further, in some exemplary embodiments, the method can promote water movement through the soil, prevent water flooding and retention that can result in deoxygenation of the soil, and prevent anaerobic methanogenic microbial promote the growth of

In certain embodiments, the improved agricultural fertilizer application, improved soil biodiversity, and/or improved soil management is in the form of inoculating the rhizosphere of the plant with one or more beneficial microorganisms. I can do it. For example, in preferred embodiments, the microorganisms of the soil treatment composition can colonize the rhizosphere and provide multiple benefits to the plants in which the roots are growing, including protection, hydration, and nutrition. . Accordingly, the present method can replace or reduce the use of nitrogen-rich fertilizers, pesticides, and/or other soil amendments 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 methods. Protein content can be measured using conventional laboratory assays known in the art.

Many livestock animals fed grain-based diets require protein supplementation to meet the animal's nutritional needs. Protein deficiency can cause decreased appetite, weight loss, poor growth, infertility, and reduced milk production. Therefore, increasing protein content is beneficial in improving the feed efficiency of livestock animals by reducing the amount of feed required to achieve a desired level of productivity.

Among the advantages, the agricultural aspect contributes to reducing dependence on conventionally grown grains that are produced and transported to feed livestock. In some embodiments, this increases the forage value of grasses, increases the land space available for pasture and forest plantations that sequester carbon, and/or increases the land space available for grassland and other low-carbon The marketability of footprint animal products can be enhanced.

In some embodiments, the methods reduce the carbon footprint of producing grain-fed and/or grain-finishing livestock where such methods are necessary and/or more economical than producing herbivorous livestock. I can do it. For example, most poultry are grain-fed livestock.

Types of plants for which the method may be useful include any plant species that is edible to one or more livestock animals, such as annual bluegrass (Poa annua), annual ryegrass (Lolium multiflorum), Canadian bluegrass (Poa compressa), chewing fescue (Festuca rubra), colonial bentgrass (Agrostis tenuis), creeping bentgrass (Agrostis palustris), crested wheat (Agropyron desertorum), fairway wheat (Agropyron cristatum), hard fescue ( Festuca longifolia), Kentucky bluegrass (Poa pratensis), orchard grass (Dactylis glomerate), perennial grass (Lolium perenne), red fescue (Festuca rubra), red top (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 (Agropyron smithii), Bermuda grass (Cynodon spp.), St. Augustine grass (Stenotaphrum secundatum), Zoysia spp., Bahia grass (Paspalum notatum), Carpet grass (Axonopus affinis), Centipede grass (Eremochloa ophiuroides), Kikuyu grass (Pennisetum clandesinum), Seashore Paspalum vaginatum, floratum (Stenotaphrum secundatum "Floratam"), blue grama (Bouteloua gracilis), buffalo grass (Buchloe dactyloids), side oat grama (Bouteloua curtipendula) foxtail, brome grass, orchard grass, quack grass and canary grass is included.

Further plants include, for example, pigeon pea, Queensland arrow, comfrey, sweet potato, nasturtium, chocolate, alfalfa, duckweed, trefoil, rapeseed, clover, millet, sorghum, soybean, mulberry, corn, oats, barley, wheat, Includes cottonseed, safflower, sunflower, peanuts, peanuts, apples, acorns, and sprouted legumes.

Husbandry Aspects In certain embodiments, the method comprises feeding livestock with plants produced in accordance with an agronomic aspect, and optionally treating animal manure with a microbial-based soil treatment composition in accordance with an agronomic aspect. It further includes animal husbandry aspects, including:

More specifically, in certain embodiments, the husbandry aspect includes making plants produced according to the agricultural aspect available to the livestock animal so that the livestock animal ingests the plant. In one embodiment, livestock animals are placed on farmland treated according to agricultural practices for free grazing.

In one embodiment, plants are harvested from treated farmland and provided to animals as low carbon footprint fodder, 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. In some embodiments, it is further preferred that the harvested plants are obtained from the same agricultural land where livestock roam and/or graze.

In one embodiment, a combination feeding method is utilized. For example, in some embodiments, the livestock is grain-finished, and the pasture on which the livestock animals are finished, as well as the grain and other feed sources, are processed according to the agronomic aspects of the method.

In some embodiments, plant material produced according to agricultural aspects is processed before being provided to livestock animals. For example, in some embodiments, the plant material is fermented.

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

In one embodiment, the processed plant material is a byproduct of fermenting grain to produce alcohol. In one embodiment, the processed grain is brewer's spent grain, which is an insoluble by-product of brewing barley. In one embodiment, the processed plant material is distiller's grain, which is unfermented grain residue, such as distiller's grain, corn or rice. Distillers' grains can be further processed by drying to produce dried distillers' grains (DDGS) containing soluble materials, which is commonly used as a high protein feed additive for livestock.

A "domesticated" animal, as used herein, is an animal that has been "domesticated" and has been influenced by humans over successive generations such that a symbiotic relationship exists between the animal and the human. means a species that has been subjected to, propagated, modified and/or controlled. In particular, domestic animals include animals raised in agricultural or industrial settings to produce goods such as food, fiber and labor. Types of animals included in the term livestock include alpacas, llamas, pigs, horses, mules, donkeys, camels, dogs, ruminants, chickens, turkeys, ducks, geese, guinea pigs, guinea pigs, and squabs. However, it is not limited to these.

In certain embodiments, the domestic animal has a compartmentalized stomach suitable for fermenting plant-based foods before digestion with the aid of a "ruminant" or specialized gut microbiome. It is a mammal that is used. Ruminants include, for example, cows, sheep, goats, ibex, giraffes, deer, elk, moose, caribou, reindeer, antelope, gazelles, impalas, wildebeest and kangaroos.

In certain exemplary embodiments, the domestic animal is a cow, a ruminant belonging to the subfamily Bovidae of the family Bovidae. Cattle include livestock and/or wild species. Specific examples include buffalo, anoa, tamaraw, aurochs, banteng, guar, gayar, yak, kouprey, meat and dairy cattle (e.g. Bos taurus, Bos indicus), bulls, castrations. Includes, but is not limited to, cows, oxen, saola, bison, buffalo, European buffalo, bongo, kudu, kewel, bushbuck, nyala, sitatunga, and yam.

In certain embodiments, ingesting plants according to the present methods results in increased growth, muscularity, fertility and/or milk production in domestic animals. This may be due, for example, to an increase in the protein content of the plant.

In certain embodiments, products from livestock animals are more nutritious for human consumption if their diets include more grass than other grains and forage treated according to the present methods. For example, grass-fed beef is known to contain higher levels of omega-3 fatty acids, which are important for brain and heart health.

Soil Treatment Compositions The present invention utilizes "microbial-based compositions," which are compositions that include components produced as a result of the growth of microbial or other cell cultures. Thus, microbial-based compositions can include the microorganism itself and/or microbial growth by-products. The microorganism can be in vegetative form, in spore or conidial form, in hyphal form, in any other form of propagules, or in mixtures thereof. The microorganisms may be planktonic, in biofilm form, or a mixture of both. Growth by-products may be, for example, metabolites, cell membrane components, proteins, and/or other cell components. Microorganisms may be intact or lysed. In a preferred embodiment, the microorganisms are present in the microorganism-based composition together with the growth medium in which they were grown. For example, the microorganisms may be present in at least 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 gram or per ml of the composition. It may be present at a concentration of CFU of x10 ¹¹ , 1 x 10 ¹² , 1 x 10 ¹³ or more.

The microorganisms of the compositions of the invention can be obtained through cultivation processes ranging from small to large scale. These culture processes include, but are not limited to, submerged culture/fermentation, solid phase fermentation (SSF), and combinations thereof.

The composition may be, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, or 100% growth medium by weight. The amount of biomass in the composition may be, for example, from 0% to 100%, from 10% to 75%, or from 25% to 50%, and all percentages therebetween. is included.

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

Microorganisms useful in the invention can be, for example, non-phytopathogenic, soil-colonizing strains of bacteria, yeasts and/or fungi. Microorganisms may be in active or inactive form, or in the form of vegetative cells, spores and/or any other form of propagules. The microorganism may be a natural or genetically modified microorganism. For example, microorganisms may be transformed with particular genes to exhibit particular characteristics. The microorganism may also be a mutant of the desired strain. As used herein, "variant" means a strain, genetic variant or subtype of a reference microorganism, where a variant has one or more Having a genetic variation (eg, a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion). Procedures for creating mutants are well known in the microbiological art. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

In a preferred embodiment, the beneficial microorganisms of the microbial-based soil treatment composition include surfactants such as lipopeptides and/or glycolipids; bioactive compounds with antimicrobial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory non-pathogenic soil-colonizing fungi, yeasts, and/or bacteria capable of producing chemical compounds; enzymes such as proteases, amylases, and/or lipases; and one or more of amino acids, vitamins, and other nutrients. It is.

In one embodiment, the microorganism is a yeast or fungus. Yeast and fungal species suitable for use according to the invention include Aureobasidium (e.g. A. pullulans ), Blakeslea , Candida (e.g. C. apicola , C. bombicola , C. nodaensis ), Cryptococcus , Debaryomyces (e.g. D. hansenii) . ), Entomophthora , Hanseniaspora (e.g. H. uvarum ), Hansenula , Issatchenkia , Kluyveromyces (e.g. K. phaffii ), Mortierella , mycorrhizal fungi , Penicillium , Phycomyces , Pichia (e.g. P. anomala , P. guilliermondii , P. occidentalis ) , P. kudriavzevii ), Pleurotus spp. (e.g., P. ostreatus ), Pseudozyma (e.g., P. aphidis ), Saccharomyces (e.g., S. boulardii , S. cerevisiae , S. torula ), Starmerella (e.g., S. bombicola) ), Torulopsis , Trichoderma (e.g. T. reesei , T. harzianum , T. hamatum , T. viride ), Ustilago (e.g. U. maydis ), Wickerhamomyces (e.g. W. anomalus ), Williopsis (e.g. W. mrakii) ), Zygosaccharomyces (for example, Z. bailii ), etc.

As used herein, "mycorrhizal fungi" includes any species of fungi that form a non-parasitic mycorrhizal relationship with the roots of plants. The fungi can be ectomycorrhizal and/or endomycorrhizal, including subtypes thereof (eg, arbuscular, ericoid, and orchid mycorrhizae).

Mycorrhizal fungi according to the present invention include the genus Glomus, the phylum Basidiomycota, the phylum Ascomycota, the phylum Zygomycota, the order Bacteriales, the orders Nicotiana, as well as Acaulospora spp. (e.g. A. alpina , A. brasiliensis , A. foveata ), Amanita spp. (e.g. A. muscaria , A. phalloides ), Amphinema spp. (e.g. A. byssoides , A. diadema , A. rugosum ), Astraeus spp. (e.g. A. hygrometricum ), Byssocorticium spp. (e.g. B. atrovirens ), Byssoporia terrestris (e.g. B. terrestris sartoryi , B. terrestris lilacinorosea , B. terrestris aurantiaca , B. terrestris sublutea , B. terrestris parksii ), Cairneyella spp (e.g. C. variabilis ) , Cantherellus spp. (e.g. C. cibarius , C. minor , C. cinnabarinus , C. friesii ), Cenococcum spp. (e.g. C. geophilum ), Ceratobasidium spp. (e.g. C. cornigerum ), Cortinarius spp. (e.g. For example, C. austrovenetus , C. caperatus , C. violaceus ), Endogone spp. (e.g. E. pisiformis ), Entrophospora spp. (e.g. E. colombiana ), Funneliformis spp. (e.g. F. mosseae ), Gamarada spp. ( e.g. G. debralockiae ), Gigaspora spp. (e.g. G. gigantean , G. margarita ), Glomus spp. (e.g. G. aggregatum , G. brasilianum , G. clarum , G. deserticola , G. etunicatum , G. fasciculatum , G. intraradices , G. lamellosum , G. macrocarpum , G. monosporum , G. mosseae , G. versiforme ), Gomphidius spp. (e.g. G. glutinosus ), Hebeloma spp. (e.g. H. cylindrosporum ) , Hydnum spp. (e.g. H. repandum ), Hymenoscyphus spp. (e.g. H. ericae ), Inocybe spp. (e.g. I. bongardii , I. sindonia ), Lactarius spp. (e.g. L. hygrophoroides ), Lindtneria spp. (e.g. L. brevispora ), Melanogaster spp. (e.g. M. ambiguous ), Meliniomyces spp. (e.g. M. variabilis ), Morchella spp. , Mortierella spp. (e.g. M. polycephala ), Oidiodendron spp. (e.g. O. maius ), Paraglomus spp. (e.g. P. brasilianum ), Paxillus spp. (e.g. P. involutus ), Penicillium spp. (e.g. P. pinophilum , P. thomili ), Peziza spp. (e.g. , P. whitei ), Pezoloma spp. (e.g., P. ericae ), Phlebopus spp. (e.g., P. marginatus ), Piloderma spp. (e.g., P. croceum ), Pisolithus spp. (e.g., P. tinctorius ), Pseudotomentella spp. (e.g. P. tristis ), Rhizoctonia spp. , Rhizodermea spp. (e.g. R. veluwensis ), Rhizophagus spp. (e.g. R. irregularis ), Rhizopogon spp. (e.g. R. luteorubescens , R. pseudoroseolus) . ), Rhizoscyphus spp. (e.g. R. ericae ), Russula spp. (e.g. R. livescens ), Sclerocystis spp. (e.g. S. sinuosum ), Scleroderma spp. (e.g. S. cepa , S. verrucosum ), Scutellospora spp. (e.g. S. pellucida , S. heterogama ), Sebacina spp. (e.g. S. sparassoidea ), Setchelliogaster spp. (e.g. S. tenuipes ), Suillus spp. (e.g. S. luteus ), Thanatephorus spp. ( e.g., T. cucumeris ), Thelephora spp. (e.g., T. terrestris ), Tomentella spp. (e.g., T. badia , T. cinereoumbrina , T. erinalis , T. galzinii ), Tomentellopsis spp. (e.g., T. echinospora ), Trechispora spp. (e.g. T. hymenocystis , T. stellulata , T. thelephora ), Trichophaea spp. (e.g. T. abundans , T. woolhopeia ), Tulasnella spp. (e.g. T. calospora ) and Tylospora Examples include, but are not limited to, species belonging to T. spp. (e.g., T. fibrillose ).

In certain embodiments, the invention utilizes endomycorrhizal fungi, including fungi from the phylum Glomeromycota , Glomus , Gigaspora , Acaulospor , Sclerocystis , and Entrophospora . Examples of endomycorrhizal fungi include Glomus aggregatum , Glomus brasilianum , Glomus clarum , Glomus deserticola , Glomus etunicatum , Glomus fasciculatum , Glomus intraradices ( Rhizophagus irregularis ), Glomus lamellosum , Glomus macrocarpum , Gigaspora margarita , Glomus monosporum , Glomus mosseae ( Funneliformis mosseae ), Glomus versiforme , Scutellospora heterogama and Sclerocystis spp .

In certain embodiments, the microorganism is a bacterium, including gram-positive and gram-negative bacteria. Bacteria include, for example, Agrobacterium (e.g. A. radiobacter ), Azotobacter ( A. vinelandii, A. chroococcum ), Azospirillum (e.g. A. brasiliensis), Bacillus (e.g. B. amyloliquefaciens , B. circulans , B. firmus , B. laterosporus , B. licheniformis , B. megaterium , B. mucilaginosus , B. coagulans , B. subtilis ), Frateuria (e.g. F. aurantia), Microbacterium (e.g. M. laevaniformans ), myxobacteria (e.g. Myxococcus xanthus , Stignatella aurantiaca , Sorangium cellulosum , Minicystis rosea ), Pantoea (e.g. P. agglomerans ), Pseudomonas (e.g. P. aeruginosa , P. chlororaphis subsp. aureofaciens ( Kluyver ), P. putida ), Rhizobium spp., Rhodospirillum (e.g. R. rubrum), Sphingomonas (eg S. paucimobilis ) and/or Thiobacillus thiooxidans ( Acidothiobacillus thiooxidans ).

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

In other exemplary embodiments, the composition comprises Bacillus subtilis B4 NRRL B-68031. Advantageously, in some embodiments, the B4 strain is capable of producing enhanced amounts of lipopeptide biosurfactants, particularly surfactin. In some embodiments, B4 is "surfactant overproduction." For example, the strain may contain at least 0.1 to 10 g/L of biosurfactant, such as 0.5 to 1 g/L, or, for example, at least 10%, 25%, 50% as compared to other B. subtilis bacteria. , 100%, 2x, 5x, 7.5x, 10x, 12x, 15x or more. For example, in some embodiments, ATCC39307 can be used as a reference strain.

In an exemplary embodiment, the composition comprises Trichoderma spp. fungi and Bacillus spp. bacteria. In some embodiments, 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.

Wickerhamomyces anomalus NRRL Y-68030 microorganism culture was obtained from Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Av. e, S. W. , Washington, DC, 20250, USA. The deposit was assigned accession number NRRL Y-68030 by the depositary institution and was deposited on May 10, 2021.

Cultures of the B. subtilis B4 microorganism were obtained from the Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Ave, S.A. W. , Washington, DC, 20250, USA. The deposit was assigned accession number NRRL B-68031 by the depositary institution and was deposited on May 10, 2021.

Cultures of the B. amyloliquefaciens " B.amy " microorganism were obtained from the Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Ave, S.A. W. , Washington, DC, 20250, USA. This deposit has been assigned accession number NRRL B-67928 by the depository and was deposited on February 26, 2020.

The main cultures were tested at 37CFR 1.14 and 35U, respectively. S. C122 to which the Commissioner of Patents and Trademarks is determined to be entitled, under conditions that ensure that access to the culture will be available during the pendency of this patent application. The deposit will be available as required by foreign patent law in the countries in which the subject application's counterpart or descendant applications are filed. However, it is to be understood that the availability of a deposit does not constitute a license to practice the invention upon invalidation of patent rights granted by governmental action.

Furthermore, each main culture deposit shall be archived and made available to the public in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, i.e. at least 5 days after the latest request for the provision of a sample of the deposit. stored with all care necessary to keep it viable and free from contamination be done. The depositor acknowledges the obligation to replace the deposit if, due to the terms of the deposit, it is not possible to provide samples when requested. All restrictions on the availability of this Culture Deposit to the public are irrevocably removed upon the granting of a patent disclosing it.

In one embodiment, the composition can include 1-99% Trichoderma and 99-1% Bacillus by weight. In some embodiments, the Trichoderma to Bacillus cell number ratio is about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6. to 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 1×10 ⁶ to 1×10 ¹² , 1×10 ⁷ to 1×10 ¹¹ , 1×10 ⁸ to 1×10 ¹⁰ , or 1×10 ⁹ CFU/ml of Trichoderma including.

In one specific embodiment, the composition has about 1×10 ⁶ to 1×10 ¹² , 1×10 ⁷ to 1×10 ¹¹ , 1×10 ⁸ to 1×10 ¹⁰ , or 1×10 ⁹ CFU/ Contains ml of Bacillus .

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

In one embodiment, the microorganism is a nitrogen-fixing bacterium or diazotroph selected from, for example, Azospirillum , Azotobacter , Chlorobiaceae , Cyanothece , Frankia , Klebsiella , rhizobia , Trichodesmium , Bacillus and some archaeal species. In certain embodiments, the nitrogen-fixing bacterium is Azotobacter vinelandii . In other specific embodiments, the nitrogen-fixing microorganism is a B.amy or B4 strain.

In other embodiments, the microorganism is a potassium mobilizing microorganism selected from, for example, Wickerhamomyces anomalus , Bacillus mucilaginosus , Frateuria aurantia or Glomus mosseae , or KMB. In certain embodiments, the potassium mobilizing microorganism is Frateuria aurantia . In other specific embodiments, the potassium mobilizing microorganism is Wickerhamomyces anomalus , eg, strain NRRL Y-68030.

In certain embodiments, the microorganism is a phosphorus mobilizing microorganism, such as Wickerhamomyces anomalus . The microorganisms produce beneficial organic acids and biosurfactants that aid in the mobilization, solubilization and absorption of nutrients and water in the soil. Additionally, W. anomalus produces the enzyme phytase, which mobilizes phosphate into usable forms of inorganic phosphorus. Additionally, W. anomalus produces ethyl acetate, which, in certain embodiments, can degrade biofilms such as those formed by many plant angiobacterial pathogens.

Other examples include, but are not limited to, Pleurotus ostreatus , Debaryomyces hansenii , Saccharomyces cerevisiae , Saccharomyces boulardii , and Bacillus licheniformis .

In certain embodiments, the one or more microorganisms contain 1×10 ⁶ to 1×10 ¹² , 1×10 ⁷ to 1×10 ¹¹ , 1×10 ⁸ to 1×10 ¹⁰ , or 1×10 ⁹ CFU each. It is present at a concentration of /ml.

In one embodiment, the microorganism of the composition comprises about 5-20%, or about 8-15%, or about 10-12% by weight of the total composition.

The species and proportions of microorganisms and other ingredients in the composition can be customized according to, for example, the plant being treated, the soil type in which the plant is growing, the health of the plant at the time of treatment, as well as other factors. .

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

The microorganisms and microorganism-based compositions of the present invention have many beneficial properties useful for promoting plant health, growth, and/or yield. For example, the composition may contain products resulting from the growth of microorganisms, such as biosurfactants, proteins and/or enzymes, in either purified or crude form.

In one embodiment, the microorganism of the composition is capable of producing biosurfactant. In other embodiments, the biosurfactant can be produced separately by other microorganisms and added to the composition in either purified or crude form. Crude form biosurfactant can include, for example, biosurfactant and other products of cell growth in the remaining fermentation medium resulting from the cultivation of biosurfactant-producing microorganisms. The crude biosurfactant composition may contain 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%. , about 45% to about 55%, or about 50% pure biosurfactant.

Biosurfactants form an important class of secondary metabolites produced by various microorganisms such as bacteria, fungi, and yeast. As amphiphilic molecules, microbial biosurfactants reduce surface and interfacial tension between molecules of liquids, solids, and gases. Furthermore, the biosurfactant according to the invention is biodegradable, has low toxicity, is effective in solubilizing and degrading insoluble compounds in soil, and is produced at low cost and using renewable resources. be able to. They can inhibit the attachment of unwanted microorganisms to various surfaces, prevent the formation of biofilms, and have strong emulsifying and demulsifying properties. Additionally, biosurfactants can be used to improve wettability and achieve uniform solubilization and/or distribution of fertilizers, nutrients, and water in the soil.

Biosurfactants according to the present method include, for example, low molecular weight glycolipids (e.g., sophorolipids, cellobiose lipids, rhamnolipids, mannosylerythritol lipids, and trehalose lipids), lipopeptides (e.g., surfactin, iturin, fengycin, anthrofactin, and lichenisin). ), flavolipids, phospholipids (eg, cardiolipin), and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

The composition comprises 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%. I can do it.

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

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

In one embodiment, different species of microorganisms are grown separately and then mixed together to produce the soil treatment composition. In one embodiment, the microorganism can be co-cultured with B. amyloliquefaciens and M. xanthus .

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

In one embodiment, the composition includes one or more fatty acids. The fatty acids can be microbially produced and/or produced separately and included as an additional component of the composition. In certain preferred embodiments, the fatty acid is a saturated long chain fatty acid with a carbon backbone of 14 to 20 carbons, such as myristic acid, palmitic acid or stearic acid. In some embodiments, a combination of two or more saturated long chain fatty acids is included in the composition. In some embodiments, saturated long chain fatty acids can inhibit methane production and/or increase cell membrane permeability of methanogens.

In one embodiment, the composition includes vitamins and/or minerals in any combination. Vitamins used in the compositions of the 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 may be included. Examples of minerals include calcium, magnesium, phosphorus, potassium, sodium, chlorine, sulfur, chromium, cobalt, copper, iodine, iron, manganese, molybdenum, nickel, selenium, and zinc. Other ingredients include, but are not limited to, antioxidants, beta-glucans, bile salts, cholesterol, enzymes, carotenoids, and many others.

In some embodiments, the compositions include, for example, seaweed (e.g., Asparagopsis taxiformis ), kelp, 3-nitrooxypropanol, anthraquinones, ionophores (e.g., monensin and/or lasalocid), polyphenols (e.g., saponins, tannins). , Yucca schidigera extract (steroidal saponin-producing plant species), Quillaja saponaria extract (triterpenoid saponin-producing plant species), organic sulfur (e.g. garlic extract), flavonoids (e.g. quercetin, rutin, kaempferol, naringin and anthocyanidins; green citrus fruits, bioflavonoids from rose hips and black currants), carboxylic acids, and/or terpenes (eg, d-limonene, pinene, and citrus extracts).

In one embodiment, the composition is preferably for application to soil, seeds, whole plants, or plant parts including, but not limited to, roots, tubers, stems, stems, buds, flowers and leaves. It is formulated into In certain embodiments, the compositions are formulated as, for example, liquids, dusts, granules, microgranules, pellets, wettable powders, flowable powders, emulsions, microcapsules, oils, or aerosols.

In order to improve or stabilize the effectiveness of the composition, the composition can be mixed with suitable adjuvants and then used as is or after dilution, if desired. In preferred embodiments, the composition is formulated as a liquid, a concentrated liquid, or a dry powder or granules that can be mixed with water and other ingredients to form a liquid product.

In one embodiment, the composition comprises glucose (e.g. in the form of molasses), glycerol and/or glycerol as or in addition to the osmotic agent to promote osmotic pressure during storage and transport of the dry product. can include.

The compositions may be used alone or in combination with other compounds and/or methods to effectively promote plant health, growth and/or yield and/or to supplement microbial growth. I can do it. For example, in one embodiment, the composition contains nutrients and/or micronutrients for enhancing plant and/or microbial growth, such as magnesium, phosphorus, nitrogen, potassium, selenium, calcium, sulfur, iron, copper, zinc, etc. , and/or one or more prebiotics such as kelp extract, fulvic acid, chitin, humates and/or humic acids, and/or can be applied simultaneously. In some embodiments, the microorganism of the composition produces and/or provides these substances. The exact ingredients and amounts thereof can be determined by a grower or agricultural scientist having the benefit of this disclosure.

The compositions can also be used in combination with other agricultural compounds and/or crop management systems. In one embodiment, the composition may optionally include, for example, natural and/or chemical pesticides, repellents, herbicides, fertilizers, water treatment agents, non-ionic surfactants and/or soil conditioners. or can be applied together with them. Preferably, however, the composition does not contain and/or is not used with benomyl, dodecyldimethylammonium chloride, hydrogen dioxide/peroxyacetic acid, imadylyl, propiconazole, tebuconazole, or triflumizole.

When the composition is mixed with compatible chemical additives, the chemicals are preferably diluted with water before adding the composition of the invention.

Additional components, such as buffers, carriers, other microbial-based compositions produced in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, tracers, biocides, other microorganisms , surfactants, emulsifiers, lubricants, solubility control agents, pH adjusters, preservatives, stabilizers and super-resistant agents can be added to the composition.

The pH of the microorganism-based composition will be appropriate for the microorganism of interest. In preferred embodiments, the pH of the composition is about 3.5-7.0, about 4.0-6.5, or about 5.0.

Optionally, the composition can be stored prior to use. Preferably, the storage time is short. Therefore, storage time is less than 60 days, less than 45 days, less than 30 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 5 days, less than 3 days, less than 2 days, less than 1 day, Or it may be less than 12 hours. In preferred embodiments, if live cells are present in the product, the product is stored at low temperatures, such as, for example, below 20°C, 15°C, 10°C, or 5°C.

However, microbial-based compositions may be used without further stabilization, preservation, and storage. Among the advantages, the direct use of these microbial-based compositions preserves high microbial viability, reduces the possibility of contamination from foreign agents and undesirable microorganisms, and eliminates microbial growth by-products. activity is maintained.

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

Methods of Application Advantageously, in a preferred embodiment, the microbial-based composition according to the invention is non-toxic and can be applied in high concentrations, for example, without causing irritation to the skin or gastrointestinal tract of humans or other non-harmful animals. It can be applied in Accordingly, the present invention is particularly useful when the application of microbial-based compositions occurs in the presence of living organisms, such as growers or livestock.

As used herein, "applying" a composition or product to a location means applying the composition or product to a location so that the composition or product can have an effect on that location. It refers to bringing something into contact with that place. This effect may be due to, for example, microbial growth and colonization and/or the action of metabolites, enzymes, biosurfactants or other microbial growth by-products. The mode of application depends on the formulation of the composition and can include, for example, spraying, pouring, dusting, injecting, spreading, mixing, dunking, atomizing and misting. Formulations can include, for example, liquids, dry and/or hydrated powders, free-flowing powders, powders, granules, pellets, emulsions, microcapsules, stakes, oils, gels, pastes and/or aerosols. In an exemplary embodiment, the composition is applied after the composition is prepared, for example, by dissolving the composition in water.

In some embodiments, prior to applying the composition to an area of agricultural land, the method includes assessing the site for local conditions, determining a preferred formulation for the composition customized for local conditions (e.g., (types, combinations and/or ratios of microorganisms and/or growth byproducts) and producing compositions using preferred formulations.

Local conditions may include, for example, soil conditions (e.g. soil type, species of soil microbiota, amount and/or type of soil organic content, amount and/or type of GHG precursor substrates in the soil, fertilizers or (amounts and/or types of other soil additives or modifications); crop and/or plant conditions (e.g. type, number, age and/or health of plants being grown); environmental conditions (e.g. current climate the type of GHG emissions at the location; the mode and/or rate of application of the composition, and others relevant to the location.

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

In some embodiments, local conditions are evaluated on a regular basis, eg, once a year, twice a year, or once a month. In this way, the formula 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) where plants are planted or growing (e.g. crops, fields, orchards, woodlands, pastures/grasslands or forests). . Compositions of the invention can be premixed with irrigation fluids, and the compositions can penetrate through the soil and be delivered to, for example, plant roots to affect the root microbiome.

In one embodiment, the composition is applied to the soil surface, with or without water, and the beneficial effects of soil application can be activated by rainfall, sprinklers, flooding, or drip irrigation.

In one embodiment, the site is a plant or a plant part. The composition can be applied as a seed treatment or directly to the surface of a plant or plant part (eg, to the surface of roots, tubers, stems, flowers, leaves, fruits, or flowers). In certain embodiments, the composition contacts one or more roots of a plant. The composition can be applied to the roots directly, for example by spraying or dipping the roots, and/or indirectly, for example by administering the composition to the soil (or rhizosphere) in which the plant grows. Can be applied. The composition can be applied to the seeds of the plant before or at the time of planting, or to any other part of the plant and/or its surrounding environment.

In one embodiment, the method is connected to an irrigation system used to supply water, fertilizer, pesticides or other liquid compositions when used in large-scale environments such as large-scale pastures or agricultural crops. The method may include administering the composition to a tank. Thus, the plants and/or the soil surrounding the plants can be irrigated, for example by soil injection, soil soaking, using center pivot irrigation systems, spraying over seed furrows, microjet, dip sprayers, boom sprayers, sprinklers and/or drip irrigation. Depending on the container, it can be treated with the composition. Advantages include: This method is suitable for treating hundreds of acres of land.

In one embodiment, the method is used in a smaller setting, such as a small farm or livestock ranch, and the method uses a handheld lawn and garden sprayer or spreader and/or a handheld watering system. The method can include applying the composition using a can.

The plants and/or their environment can be treated at any point during the process of cultivating the plants. For example, the composition can be applied to the soil before, at the same time as, or after the seeds are planted. It can also be applied at any subsequent time during plant development and growth, including when the plant is flowering, fruiting, and during and/or after defoliation.

Reducing Carbon Footprint In certain embodiments, the present invention provides benefits such as promoting the growth and vigor of forage crops and pastures; improving the nutrient content of agricultural and pasture soils; promoting improvements in soil moisture and water use efficiency; increasing soil microbial diversity; reducing fertilizer use; reducing dependence on grains for livestock feed; reducing enteric GHG emissions from manure; improving feed efficiency; improving the nutritional quality of meat and milk; and the environment in which meat, milk, and other animal-based products are produced and consumed. Provide solutions that improve sustainability.

In a preferred embodiment, the methods of the invention are useful for reducing the carbon footprint of producing livestock and animal-based products such as meat, offal, dairy products, leather, fur, feathers, collagen, eggs, and manure. It is.

As used herein, the term "reduction" refers to a negative change and the term "increase" refers to 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%, 90%, 95%, or 100%.

In some embodiments, the desired reduction is achieved within a relatively short period of time, such as within one week, two weeks, three weeks, or four weeks. In some embodiments, the desired reduction is achieved within, for example, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after using the method. In some embodiments, the desired reduction is achieved within 1, 2, 3, 4, or 5 years after using the method.

In some embodiments, carbon footprint reduction by the present methods is achieved through enhanced nutrient carbon utilization and storage, as well as increased carbon sequestration in the soil. For example, enhanced nutrient carbon utilization can be in the form of, for example, increased leaf size, increased stem and/or stalk diameter, increased root growth, and/or increased plant number.

Furthermore, increased soil sequestration may result in, for example, increased plant root growth, increased microbial uptake of organic compounds secreted by plants (including secretions from plant roots), and microbial colonization of soil and roots. It can be a form of improvement.

In one particular embodiment, the method reduces atmospheric carbon dioxide. By increasing plant biomass above and below ground, plants act as carbon sinks by fixing carbon during photosynthesis and storing carbon as biomass. Furthermore, increased plant root biomass not only increases the root structures that can be colonized by microorganisms, but also increases the secretion rate and amount of sugars and other nutrients leached from plant roots, providing applied and natural microbial biomass. increase. Microorganisms then convert the plant-based materials into increasing levels of carbon that are stored in the soil. In this way, stimulated microbial populations below ground (both added and natural) further serve as a storage system for carbon. In certain embodiments, the microbial cell biomass is yeast biomass.

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

Improved agricultural fertilization practices include, for example, the reduction of nitrogen-rich fertilizers, as well as the use of one or more environmentally friendly soil microorganisms in some or all fertilizers, pesticides, and/or other soil amendments. It can be in the form of a replacement for a composition containing. Among the benefits: Reducing the application of fertilizers and other chemicals reduces the amount of these chemicals that contaminate soil and groundwater if left unabsorbed by plants, and also reduces the amount of these chemicals that can be added to other water sources. Reduce runoff. Additionally, reducing fertilization reduces soil emissions of nitrous oxide and carbon dioxide resulting from such fertilization.

In some embodiments, improved agricultural fertilization involves the use of fertilizer produced by livestock animals to fertilize agricultural land where livestock plants are growing or will grow. can do. In certain embodiments, the soil treatment composition can be mixed with fertilizer during storage. Microorganisms can promote increased decomposition of manure while reducing the amount of GHGs emitted therefrom, such as methane, carbon dioxide and/or nitrous oxide. For example, in one embodiment, if the composition includes a biosurfactant and/or a microorganism that produces a biosurfactant, the composition can exhibit antimicrobial properties against methanogens. In other embodiments, when the composition includes 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 the fertilizer increases the value of the fertilizer as an organic fertilizer due to its ability to inoculate microorganisms into the agricultural soil to which the fertilizer is ultimately applied. let Livestock that produce manure increase the nitrogen utilization of their feed, so their waste products produce less nitrogen and ammonia, and therefore the circular effect includes lower carbon footprint fertilizers and synthetic nitrogen enrichment. Includes reduced need for fertilizers.

The method improves the above- and below-ground biomass of plants, including, for example, increasing leaf volume, increasing stem and/or stalk diameter, increasing root growth and/or density, and/or increasing plant number. can be increased. In one embodiment, this is achieved by improving the overall comfort 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. .

Accordingly, the present invention can benefit the reforestation and/or restoration of agricultural land that has been depleted due to human 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.

Furthermore, in one embodiment, the method can be used to inoculate the soil and/or the rhizosphere of plants with beneficial microorganisms. The microorganisms of the present microorganism-based compositions can, for example, promote the colonization of roots and/or rhizospheres and the vascular system of plants by aerobic bacteria, yeasts, and/or fungi.

In certain embodiments, the method can be used to directly remove nitrous oxide from air and/or soil. For example, certain microorganisms according to the invention (eg, Dyadobacter fermenters ) are capable of reducing nitrous oxide to nitrogen in soil without denitrification. Denitrification is the reduction of nitrates and nitrites to molecular nitrogen. Intermediates of the reduction process include nitrogen oxide products, such as nitrous oxide, which can escape into the atmosphere.

In one embodiment, enhanced colonization can result in improved biodiversity of the soil microbiome. As used herein, improving biodiversity refers to increasing the diversity of microbial species within soil. Preferably, improved biodiversity comprises increasing the ratio of aerobic to anaerobic bacterial, yeast, and/or fungal species in the soil.

For example, in one embodiment, the microorganisms of the present composition are capable of colonizing the roots, soil and/or rhizosphere, and include other nutrient-fixing microorganisms such as Rhizobium and/or Mycorrhizae , as well as others that promote plant biomass accumulation. colonization of endogenous and/or exogenous microorganisms.

In one embodiment, soil biodiversity and root colonization can be further enhanced by applying to the soil a biostimulant, or a substance that promotes an increase in the growth rate of microorganisms.

In one embodiment, improved soil biodiversity promotes enhanced nutrient solubilization and/or uptake. For example, certain aerobic bacterial species can acidify soil and solubilize NPK fertilizers into plant-available forms.

In yet other embodiments, the method can be used to prevent and/or inhibit colonization of the rhizosphere by soil microorganisms that may be harmful or competitive with beneficial soil microorganisms. For example, if more aerobic microorganisms are present in the soil, fewer anaerobic microorganisms, such as nitrate-reducing microorganisms, can thrive and produce harmful atmospheric byproducts such as nitrous oxide.

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

The present invention is suitable for improving the quality of any number of soil types, such as clay, sandy, silty, peaty, chalky, loamy soils, and/or combinations thereof. It can be used for. Additionally, the methods and compositions can be used to improve the quality of dry, water-containing, porous, depleted, compacted soils, and/or combinations thereof. Soil can include soil present in the rhizosphere or soil outside the rhizosphere.

In one embodiment, the method can be used to improve drainage and/or dissemination of water in water-containing soils. 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, and the composition includes nitrogen, potassium, phosphorus (or phosphates) and/or other fertilizers in the soil. including microorganisms capable of fixing, solubilizing and/or mobilizing micronutrients.

In certain embodiments, the reduction of harmful atmospheric gases is achieved through the reduction of methanogenic microorganisms of both animal and environmental origin. Reduction of methanogenic microorganisms may be in the form of, for example, enhanced management and disposal of manure and/or organic waste, as well as enhanced land and crop management.

In one embodiment, the husbandry aspect of the method is performed by promoting the health and/or productivity of livestock animals in a manner that reduces GHG emissions resulting from livestock digestion, manure, and highly concentrated mass production. , reducing the carbon footprint of the livestock industry.

These benefits include, for example, improved feed efficiency, resulting improved animal health and reproductive performance, improved meat and dairy quantities and nutritional quality, and a higher carbon footprint of transported grain, etc. This may include reducing dependence on forage crops. One specific and important benefit of improved feed efficiency is an increase in feed nitrogen usage, which results in a reduction in ammonia and nitrous oxide produced in the digestive system and in livestock waste. .

In some embodiments, the methods of the invention may be utilized by livestock producers or livestock feed providers to reduce carbon credit usage. Thus, in certain embodiments, the method uses standard techniques in the art to remove carbon dioxide and/or other harmful atmospheric gases, and/or their precursors (e.g., nitrogen and The method may further include performing measurements to evaluate the effectiveness of the method for reducing the production of (or ammonia).

Measurements can be taken at specific points in time after application of the microbial-based composition to an area of agricultural land. In some embodiments, the measurement takes about 1 week or less, 2 weeks or less, 3 weeks or less, 4 weeks or less, 30 days or less, 60 days or less, 90 days or less, 120 days or less, 180 days or less, and/or This will take place after less than a year.

Furthermore, measurements can be repeated over time. In some embodiments, measurements are repeated daily, weekly, monthly, bimonthly, semi-monthly, semi-annually, and/or annually.

In certain embodiments, assessing GHG production can take the form of measuring GHG emissions from the location. Gas chromatography and electron capture are commonly used to test samples in laboratory settings. In certain embodiments, GHG emissions can also be performed on-site using, for example, flux measurements and/or in-situ soil surveys. Flux measurements analyze the emission of gases from the soil surface to the atmosphere, for example using a chamber surrounding an area of soil, and then estimate the flux by observing the accumulation of gases in the chamber over a period of time. . The probe can be used to generate a soil gas profile, initiating a measurement of the concentration of the gas of interest at a particular depth in the soil and making a direct comparison between the probe and ambient surface conditions (Brummel and Siciliano 2011, 118).

GHG emissions measurements may also include other forms of direct emissions measurements and/or fuel input analysis. Direct emissions measurement can include, for example, identifying polluting driving activities (e.g., fuel-burning vehicles) and directly measuring the emissions of those activities via a continuous emissions monitoring system (CEMS). can. Fuel input analysis involves calculating the amount of energy resources used (e.g., the amount of electricity, fuel, wood, biomass, etc. consumed); e.g., determining the carbon content in the fuel source; The method may include applying carbon content to the amount of fuel consumed to determine emissions.

In certain embodiments, the carbon content of the place where the crop is grown, e.g., an agricultural field, a crop, a sod or turf farm, a pasture/grassland or a forest, e.g. quantifies the above-ground and/or below-ground biomass of the crop. It can be measured by Generally, for example, the carbon concentration of trees is assumed to be about 40-50% of the biomass.

Biomass quantification can take the form of, for example, harvesting a plant in a sample area and measuring the weight of different parts of the plant before and after drying. Quantification of biomass can also be performed using non-destructive observational methods, such as, for example, measuring the diameter, height, volume, and other physical parameters of the plant trunk. Remote quantification can also be used, for example laser profiling and/or drone analysis.

In some embodiments, the carbon content of the location can further include sampling and measuring the carbon content of litter, wood debris, and/or soil in the sampling area. In particular, soils can be analyzed using, for example, dry combustion to determine percent total organic carbon (TOC), by potassium permanganate oxidation analysis to detect activated carbon, and percent carbon. can be analyzed by bulk density measurements (weight per unit volume) to convert to tons/acre.

In some embodiments, measuring GHG levels from livestock can be performed using, for example, gas capture and quantification, chromatography, breathing chambers (which measure the amount of methane exhaled by an individual animal), and in vitro gas production. (fermenting the feed under controlled laboratory and microbial conditions and measuring the amount of methane and/or nitrous oxide emitted per gram of dry matter). (see, eg, Non-Patent Document 7, incorporated herein by reference). Measurements can also be made, for example, by sampling milk, feces, and/or stomach contents and determining the number of methanogenic microorganisms present therein, using, for example, DNA sequencing and/or cell plating. , may also be in the form of testing microbial populations in animals.

Measurements can be taken at some point after application of the microbial-based composition. In some embodiments, the measurement takes about 1 week or less, 2 weeks or less, 3 weeks or less, 4 weeks or less, 30 days or less, 60 days or less, 90 days or less, 120 days or less, 180 days or less, and/or It is carried out after one year or less.

Furthermore, measurements can be repeated over time. In some embodiments, measurements are repeated daily, weekly, monthly, bimonthly, semi-monthly, semi-annually, and/or annually.

Growth of Microorganisms According to the Invention The invention utilizes methods of culturing microorganisms and producing 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 at the desired scale. These culture processes include, but are not limited to, submerged culture/fermentation, solid phase fermentation (SSF), modifications, 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, microorganisms are grown using SSF and/or modifications thereof.

In one embodiment, the invention produces 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 present invention provides materials and methods for doing so.

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

In further embodiments, the container can also monitor the growth of microorganisms within the container (eg, cell number and growth phase measurements). Alternatively, daily samples may be taken from the container and subjected to enumeration by 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 metrics with which different environments or treatments can be compared.

In one embodiment, the method includes supplementing the culture 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 alone or in combination of two or more.

The method can provide oxygenation to a growing culture. In one embodiment, slow movement of air is utilized to remove hypoxic air and introduce oxygenated air. For immersion fermentation, oxygenated air is replenished daily via a mechanism that includes an impeller for mechanical agitation of the liquid, and an air sparger to supply air bubbles to the liquid for dissolution of oxygen in the liquid. It may be ambient air that is

The method can further include supplementing the culture with a carbon source. Carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose; acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvate Organic acids; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; soybean oil, canola oil, rice bran oil, olive oil, corn oil, sunflower oil, sesame oil, and/or linseed oil, etc. It can be oils and fats, etc. These carbon sources may be used alone or in combination of two or more.

In one embodiment, microbial growth factors and micronutrients are included in the medium. This is particularly preferred when growing microorganisms that cannot produce all of the vitamins they need. Mineral nutrients including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. Additionally, sources of vitamins, essential amino acids and trace elements may be provided, for example, in the form of flour or meal, such as corn flour, or in the form of extracts, such as yeast extract, potato extract, cow extract, soybean extract, banana peel extract, or May be included in purified form. Various amino acids, such as those useful in protein biosynthesis, may also be included.

In one embodiment, inorganic salts may also be included. Inorganic salts that can be used are 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, and sulfuric acid. It can be copper, calcium chloride, sodium chloride, calcium carbonate, and/or sodium carbonate. These inorganic salts may be used alone or in combination of two or more.

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

Additionally, antifoaming agents may be added to prevent foam formation and/or accumulation during underwater culture.

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

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

In one embodiment, the method for culturing microorganisms is carried out at about 5°C to about 100°C, preferably 15°C to 60°C, more preferably 25°C to 50°C. In further embodiments, culturing may be carried out continuously at a constant temperature. In other embodiments, the culture may be exposed to varying temperatures.

In one embodiment, the method and equipment used in the culture process are sterile. The culture device, such as a reactor/vessel, may be separate from the sterilization unit, eg an autoclave, but may also be connected to it. The culture device may also have a sterilization unit for sterilizing in situ before starting inoculation. Air can be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the container. In other embodiments, the medium may be pasteurized or, optionally, no heat may be applied, and the use of low water activity and low pH is utilized to control undesirable bacterial growth. may be done.

In one embodiment, the present invention provides methods for controlling microbial metabolites such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, beta-glucan, peptides, metabolic intermediates, polyunsaturated fatty acids, and lipids during growth and metabolism. Further provided are methods of producing, and optionally purifying, the metabolite by culturing under conditions suitable for product production. The amount of metabolite produced by the method can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

Microbial growth by-products produced by the microorganism of interest may be retained within the microorganism or secreted into the growth medium. The medium may contain compounds that stabilize the activity of microbial growth byproducts.

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 1×10 ⁶ to 1×10 ¹³ , 1×10 ⁷ to 1×10 ¹² , 1×10 ⁸ to 1×10 ¹¹ , or 1×10 ⁹ to 1×10 ¹⁰ CFU/ml. It may be.

The methods and apparatus for culturing microorganisms and producing microbial byproducts can be carried out in batch, semi-continuous, or continuous processes.

In one embodiment, all of the microbial culture composition is removed upon completion of the culture (eg, upon achieving a desired cell density or density of a particular metabolite). In this batch procedure, a completely new batch is started at the time the first batch is taken.

In other embodiments, only a portion of the fermentation product is removed at any time. In this embodiment, the biomass with live cells, spores, conidia, hyphae and/or mycelium remains in the vessel as inoculum for a new culture batch. The composition to be removed may be a cell-free medium or may include cells, spores, or other reproductive propagules, and/or combinations thereof. In this way, a quasi-continuous system is formed.

Advantages include that the method does not require complex equipment or high energy consumption. The microorganism of interest can be cultivated on-site on a small scale or large scale, and can also be used even if it is mixed with the culture medium.

An advantage is that microbial-based products can be produced remotely. Microbial growth facilities can be operated off-grid, for example by utilizing solar, wind and/or water power.

Preparation of Microbial-Based Products The present invention further provides "microbial-based products," which are products that are applied in practice to achieve desired results. A microbial-based product can simply be a microbial-based composition harvested from a microbial culture process. Alternatively, the microbial-based product may include additional ingredients added. These additional ingredients may include, for example, stabilizers, buffers, water, suitable carriers such as salt solutions, or any other suitable carriers, added nutrients to support further microbial growth, non-nutritive growth. It may include an enhancer and/or an agent that facilitates the tracking of microorganisms and/or compositions in the environment to which it is applied. The microbial-based product may also include a mixture of microbial-based compositions. Microbial-based products also include one or more of the microbial-based compositions that have been processed in some way, such as, but not limited to, filtration, centrifugation, dissolution, drying, purification, etc. It may also contain ingredients.

One microbial-based product of the invention is simply a fermentation medium containing microorganisms and/or microbial metabolites produced by the microorganisms and/or residual nutrients. Fermentation products may be used directly without extraction or purification. Extraction and purification, if desired, 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, reproductive spores, conidia, hyphae, mycelium, or any other form of microbial propagation. Microbial-based products may also contain combinations of any of these forms of microorganisms.

In one embodiment, different microbial strains are grown separately and then mixed together to produce a microbial-based product. The microorganisms can optionally be mixed with the grown and dried medium prior to mixing.

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

Microbial-based products may be used without further stabilization, preservation, and storage. Among the advantages, the direct use of these microbial-based products preserves high microbial viability, reduces the possibility of contamination from foreign agents and undesirable microorganisms, and reduces the activity of microbial growth by-products. maintained.

When harvesting a microbial-based composition from a growth vessel, additional ingredients can be added when the harvested product is placed in the vessel or transported for use. Additives include, for example, buffers, carriers, other microbial-based compositions produced in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, surfactants, emulsifiers, lubricants. agents, solubility control agents, tracers, solvents, biocides, antibiotics, pH modifiers, chelating agents, stabilizers, superresistant agents, other microorganisms and other agents conventionally used in such preparations. It can be a suitable additive.

In one embodiment, a buffer containing an organic acid and an amino acid or a salt thereof can be added. Suitable buffering agents include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactate. , glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine and mixtures thereof. Phosphoric acid and phosphorous acid or their salts may also be used. Although synthetic buffers are suitable for use, it is preferred to use organic and natural buffers such as amino acids and their salts.

In further embodiments, pH adjusting agents include potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid or mixtures.

The pH of the microorganism-based composition will be appropriate for the microorganism of interest. In preferred embodiments, the pH of the composition is about 3.5-7.0, about 4.0-6.5, or about 5.0.

In one embodiment, additional ingredients such as aqueous preparations of salts, eg, sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium biphosphate, can be included in the formulation.

In certain embodiments, adhesive substances can be added to the composition to provide prolonged adhesion of the product to plant parts. For example, charged polymers such as xanthan gum, guar gum, levan, xylinan, gellan gum, curdlan, pullulan, dextran or polysaccharide-based materials can be used.

In a preferred embodiment, commercial grade xanthan gum is used as the adhesive. The concentration of gum shall be selected based on the content of gum in commercial products. If the xanthan gum is of high purity, 0.001% (w/v xanthan gum/solution) is sufficient.

In one embodiment, glucose, glycerol and/or glycerin can be added to the microbial-based product to serve, for example, for osmotic pressure during storage and transportation. In one embodiment, molasses can be included.

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

In one embodiment, certain nutrients are added to and/or applied simultaneously to the microbial-based product to enhance microbial inoculation and growth. These can include, for example, water-soluble potassium carbonate (K2O), magnesium, sulfur, boron, iron, manganese, and/or zinc. Nutrients can be derived from, for example, potassium hydroxide, magnesium sulfate, boric acid, ferrous sulfate, manganese sulfate, and/or zinc sulfate.

Optionally, the product can be stored prior to use. Preferably, the storage time is short. Therefore, storage time is less than 60 days, less than 45 days, less than 30 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 5 days, less than 3 days, less than 2 days, less than 1 day, Or it may be less than 12 hours. In preferred embodiments, if live cells are present in the product, the product is stored at low temperatures, such as, for example, below 20°C, 15°C, 10°C, or 5°C.

On-Site Production of Microbial-Derived Products In certain embodiments of the invention, desired fresh, high-density microorganisms and/or microbial growth by-products are produced at a desired scale in a microbial growth facility. Microbial growth facilities may be located at or near the site of application. This facility produces high-density microbial-based compositions in batch, semi-continuous, or continuous cultures.

The microbial growth facility of the present invention can be located where microbial-based products are used (eg, livestock farms). For example, the microbial growth facility may be less than 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from the site of use.

Microbial-based products can be produced on-site without resorting to the microbial stabilization, preservation, storage, and transportation processes of traditional microbial production, allowing for the production of very high microbial densities; Thereby, only small volumes of microorganism-based products are required for use in field applications, or the application of very high densities of microorganisms is possible where necessary to achieve the desired efficacy. This allows for downsized bioreactors (e.g., smaller fermentation vessels, fewer supplies of starting materials, nutrients and pH regulators), making the system more efficient, and stabilizing cells or removing them from their culture media. The need to separate them can be eliminated. Local production of microbial-based products also facilitates the inclusion of growth media into the products. The medium may contain drugs produced during fermentation that are particularly suitable for local use.

Dense, robust microbial cultures produced locally are more effective in the field than those that have been sitting in the supply chain for some time. The microbial-based products of the present invention are particularly advantageous compared to conventional products in which cells are separated from the metabolites and nutrients present in the fermentation growth medium. The reduced transport time allows the production and distribution of fresh batches of microorganisms and/or their metabolites in the time and quantities required by local requirements.

The microbial growth facility of the present invention produces fresh microbial-based compositions that include the microorganism itself, microbial metabolites, and/or other components of the medium in which the microorganism grows. If desired, the composition can have a high density of vegetative cells, propagules, or a mixture of vegetative cells and propagules.

In one embodiment, the microbial growth facility is located at or near the location where the microbial-based product is used (e.g., a citrus orchard), preferably within 300 miles, more preferably within 200 miles, and even more preferably within 100 miles. placed within. Advantageously, the composition can be tailored for use at a particular location. The formulation and efficacy of microbial-based compositions depend upon application, e.g., the type of soil, plants and/or crops to be treated; the season, climate and/or time of day when the composition is applied; the mode of application utilized; It can be customized to specific local conditions, such as speed and/or speed.

Distributed microbial growth facilities are advantageous in that they provide a solution to the current problems of relying on remote industrial scale producers. Issues include upstream processing delays, supply chain bottlenecks, inadequate storage, timely delivery and application of products with high numbers of living cells, binding media and metabolites in which microorganisms were originally grown. Product quality is affected by other unforeseen circumstances that impede product quality.

Furthermore, by producing the composition on-site, the formulation and potency can be adjusted in real time to the particular location and conditions existing at the time of application. This provides an advantage over compositions that are prefabricated at a central location and have, for example, set ratios or formulations that are not optimal for a particular location.

Microbial growth facilities provide manufacturing versatility by being able to tailor microbial-based products to improve synergy with destination geography. Advantageously, in preferred embodiments, the system of the present invention can reduce the power of naturally occurring local microorganisms and their metabolic by-products to improve GHG management.

The culture time for each container may be, for example, 1 day to 7 days or more. Culture products can be harvested in any of a number of different ways.

For example, on-site production and distribution within 24 hours of fermentation results in a pure, high cell density composition and substantially lower transportation costs. Given the expected rapid progress in the development of more efficient and potent microbial inocula, consumers can benefit from immediate dispensing of microbial-based products.

EXAMPLES The invention and its many advantages may be better understood from the following examples, given by way of illustration. The following examples illustrate some of the methods, applications, embodiments, and variations of the present invention. They do not limit the invention. Many changes and modifications can be made to the invention.

Example 1 - Low Carbon Footprint Livestock Figure 1 shows a flowchart 100 of one embodiment of how livestock can be produced in a manner that reduces the overall carbon footprint of the operation.

As part of the agricultural aspect of the method (05, 10, 11, 11a, 11b, 12), selected microbial-based soil treatments are applied to agricultural land (10). In some optional embodiments, the farmland has been previously burnt (05) and requires revegetation and/or soil regeneration.

As a result of applying the product (10), the product's microorganisms colonize the rhizosphere and plant roots, synergistically increasing nutrient and water content/dispersion in the soil and enhancing plant health and growth. and produce beneficial metabolites (11) that improve the plant protein content of plants grown in agricultural fields. Among the benefits, this contributes to the reduction of GHGs in the atmosphere (30), reduces fertilizer usage (11a) and increases carbon sequestration in soil and plants (11b).

As part of the husbandry aspect of the present method (12, 20, 20a, 20b), livestock animals are treated with a microbial-based soil treatment composition (10) to enable them to consume plant matter (20). Plants grown on cultivated farmland are provided. This can be the formation of free-range grazing of agricultural land with the benefits (11) provided by the application (10) of soil treatment compositions. Alternatively or additionally, livestock animals can be provided with fodder or feed harvested from agricultural fields (12).

As a result of consuming the plant material produced according to the present method (20), the feed efficiency of the animal can be improved, e.g. with respect to the quantity and quality of meat and dairy products produced, the feed requirements This means that the animal's productivity will increase. This also includes improving nitrogen utilization during digestion, resulting in reduced nitrous oxide and ammonia emissions in livestock waste (20a).

Advantageously, by utilizing livestock animals and/or feed crops produced according to the method, the method can reduce the high carbon footprint of crops such as corn, wheat, barley and oats produced solely for livestock feed. Enables reducing dependence on printed grain crops (20b). In addition to helping reduce the carbon footprint of livestock farming (30), this also reduces the dependence on antibiotics (e.g., ionophores) required by cattle fed grain-based diets. It can also be helpful to

Claims (29)

A method for producing livestock with a low carbon footprint, including agricultural and livestock aspects,
The agricultural aspect improves the agricultural land on which plants for feeding livestock are or will grow, by increasing soil nutrient and water content and distribution, promoting plant health and growth, and improving plant Cultivation using techniques that improve protein content, reduce the use of nitrogen-rich fertilizers, reduce soil greenhouse gas emissions, and/or increase carbon sequestration in the soil and/or plants. including doing;
The husbandry aspect includes making plants produced in the farmland available to livestock animals so that the livestock animals ingest the plants;
A method wherein said agricultural aspect reduces greenhouse gas emissions compared to traditional agricultural techniques, thereby reducing livestock feed and/or animal-based product production. The technique for growing said plants comprises applying to said agricultural land a microbial-based soil treatment composition comprising one or more soil-colonizing microorganisms and/or microbial growth by-products, said one or more microorganisms Is BACILLUS SPP. , Trichoderma SPP. , Preurotus Spp. , Saccharomyces Spp. , Debaryomyces Spp. , Lentinula Spp. , Meyerozyma Spp. , Pichia Spp. , Monascus Spp. , Acremonium Spp. The method according to claim 1, selected from Myxococcus spp . 3. The method of claim 2, wherein the microorganisms are Bacillus amyloliquefaciens NRRL B-67928 and Trichoderma harzianum. 3. The method according to claim 2, wherein the microorganism is Wickerhamomyces anomalus NRRL Y-68030, Meyerozyma guilliermondii , or Bacillus subtilis NRRL B-68031. 3. The method of claim 2, wherein the composition further comprises a fermentation medium in which the one or more microorganisms are cultured. 3. The method of claim 2, wherein the composition comprises microbial-free growth byproducts. 3. The method of claim 2, wherein the microbial growth by-product is a biosurfactant selected from glycolipids and lipopeptides. 8. The method according to claim 7, wherein the glycolipids are selected from sophorolipids, mannosylethritol lipids, rhamnolipids and trehalose lipids. 8. The method of claim 7, wherein the lipopeptide is selected from surfactin, iturin, fengycin, anthrofactin and lichenisin. 2. The method of claim 1, wherein the livestock animals are placed on the farmland to consume the plants. 2. The method of claim 1, wherein the plants are harvested from the farmland and provided to the livestock animals as low carbon footprint fodder and/or grain. 12. The method of claim 11, wherein the low carbon footprint forage comprises grasses, forbs, shrubs, hay, straw, alfalfa, fruits, nuts, seeds, vegetables and/or crop residues. 13. The method of claim 12, wherein the fodder is processed to produce a low carbon footprint silage, and the low carbon footprint silage is provided to the livestock animal as feed. The livestock animal is a grain-fed or grain-finishing livestock animal, and providing the livestock animal with grain that has a low carbon footprint compared to providing the livestock animal with conventionally produced grain 12. The method of claim 11, wherein the method reduces the carbon footprint of the grain-fed and grain-finished livestock production. 12. The method of claim 11, wherein the low carbon footprint grain comprises corn, oats, wheat, barley, sorghum, milo, and/or soybean. The low carbon footprint grain is processed by distillation or brewing to produce alcohol and a low carbon footprint grain byproduct, and the low carbon footprint grain byproduct is fed to the livestock animal as feed. 16. The method of claim 15, provided. 17. The method of claim 16, wherein the low carbon footprint grain by-product is brewer's spent grain, distiller's grain, and/or solubles added dried distiller's grain (DDGS). 2. The method of claim 1, wherein the agronomic aspect results in an increase in protein content of the plant. 2. The method of claim 1, wherein the agricultural aspect results in a reduction in carbon dioxide, methane and/or nitrous oxide emissions from soil. 2. The method according to claim 1, wherein as a result of consuming the plant, it leads to improved feed efficiency of the livestock animal and improved meat and dairy product quantity and/or quality. 2. The method of claim 1, which leads to improved feed nitrogen utilization of the domestic animal, resulting in reduced nitrous oxide and ammonia emissions in the animal's waste. 2. The method of claim 1, wherein dependence on conventionally grown grain crops for feeding livestock is reduced. 23. The method of claim 22, wherein reducing dependence on conventionally grown grain crops increases marketability and value of grass-fed livestock. 23. The method of claim 22, wherein the reduced reliance on conventionally grown grain crops reduces the carbon footprint of grain-fed and grain-finished livestock production. 2. The method of claim 1, wherein the method reduces the carbon footprint during the production of animal-based products selected from meat, offal, dairy products, leather, fur, collagen, eggs, feathers, and manure. The one or more microorganisms of said composition colonize the roots of said soil and/or plants growing in said soil, said colonization comprising:
increase in leaf volume, stem diameter, trunk diameter, root growth, and/or number of plants;
increasing protein content in said plant;
increasing microbial biomass in the soil;
2. The method of claim 1, resulting in improved soil biodiversity and increased uptake of organic plant secretions by microorganisms. 27. The method of claim 26, wherein improved biodiversity comprises increasing the ratio of aerobic bacterial, yeast, and/or fungal species in the soil to anaerobic microorganisms in the soil. . 27. The method of claim 26, wherein atmospheric carbon dioxide is reduced by increasing nutrient carbon utilization and storage. 27. The method of claim 26, wherein carbon sequestration is enhanced.

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