Control of Contaminants during Fermentation

Abstract

Methods and compositions for use in the growth of pink-pigmented facultative methylotrophs (PPFM) and / or methanotrophic bacteria are provided herein, and the fermentation contains hop acids to inhibit the growth of bacterial contaminants.

Description

Technical Field

[0001] Cross - Reference to Related Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 391,007, filed on July 21, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the field of microbial culture. More specifically, methods are provided for controlling contaminants during the commercial fermentation of pink - pigmented facultative methylotrophs (PPFM) such as species of Methylobacterium and Methylorubrum, as well as during the fermentation of methanotrophic bacteria including species of Methylomicrobium, Methylosarcina, and Methylocystis.

Background Art

[0003] Pink - pigmented facultative methylotrophs (PPFM) are α - proteobacteria that have been shown to have beneficial symbiotic relationships with a wide range of agricultural crops. The benefits reported for the treatment of plants by PPFM bacteria include positive effects on nitrogen metabolism and seed germination, inhibition of fungal diseases, reduction of plant damage by insects and / or nematodes, and promotion of plant growth. The use of PPFM bacteria to improve plant growth, plant yield, seed germination, and plant nutritional value has been disclosed. Methanotrophic bacteria, or methanotrophs, are present in various environments and can grow using methane as a carbon source. Methanotrophs can be used in various agricultural and industrial processes, including the production of commercially valuable compounds, bioremediation of contaminants, and mitigation of methane gas.

[0004] To supply large amounts of PPFM and / or methanotroph inoculum for agricultural and / or industrial applications, it is desirable to grow them in a controlled bioreactor or fermenter. However, maintaining sterility during large-scale fermentation can be problematic. In large-scale fermentation, contamination can occur by bacteria from equipment, media components, process water, and other sources of contamination. Contamination during the fermentation process (typically Gram-positive strains) can reduce the final biomass, recovery efficiency, and stability of the formulation, and can also reduce the titer of PPFM and / or methanotrophic bacteria, or in cases where the growth level of contaminants is high, the fermenting bacteria may be completely lost.

[0005] In the production of ethanol for fuel or brewing, typically in the yeast fermentation of mash or molasses, contaminating bacteria mainly by lactic acid bacteria have become a problem. Hop acids are commonly used in beer brewing, and it has been shown that hop α-acids impart bitterness to beer and have an antibiotic / bacteriostatic effect against Gram-positive bacteria without affecting brewing yeast in the optimal pH range for beer brewing of 5.2 - 5.6. The antibacterial effect of hop acids has been shown to decrease as the pH increases from pH 4 to pH 7. US6547971 reports the use of α or β hop acids in an aqueous system to inhibit and / or kill the growth of organisms including Gram-positive and Gram-negative bacteria. SUMMARY OF THE INVENTION

[0006] Methods and compositions for the growth of pink-pigmented facultative methylotrophs (PPFM) and / or methanotrophic bacteria are provided herein, wherein hop acids are used to inhibit the growth of bacterial contaminants.

[0007] In one embodiment, the method disclosed herein includes: a) providing a sterilized fermentation medium containing nutrients suitable for the growth of PPFM and / or methanotrophic bacteria; b) adding hop acid to the sterilized fermentation medium; c) adding an inoculum of PPFM and / or methanotrophic bacteria cells to the fermentation medium to provide a fermentation broth; and d) maintaining the fermentation broth under conditions suitable for the growth of PPFM and / or methanotrophic bacteria, thereby inhibiting the growth of bacterial contaminants. In one embodiment, the pH of the fermentation is greater than pH 6. In one embodiment, the hop acid is present at a concentration sufficient to inhibit the growth of Gram-positive bacteria at pH 6 or higher. In one embodiment of the method, the hop acid contains β-acid at a concentration of at least 7.5 ppm. In one embodiment, the hop acid is a mixture of α-acid and β-acid. In one embodiment, the hop acid contains a higher percentage of β-acid than α-acid. In one embodiment, the hop acid is mainly β-acid. In one embodiment, the hop β-acid is present at a concentration of at least about 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ppm or more during fermentation. In one embodiment of the method, the hop acid is added to the fermentation medium as a 10% dilution in methanol to facilitate handling. In one embodiment of the method, PPFM and / or methanotrophic bacteria are grown to a titer of at least 10 9 CFU per milliliter in the fermentation medium. In one embodiment, the method disclosed herein further includes harvesting the PPFM and / or methanotrophic bacteria grown in the fermentation broth. In other embodiments, the hop acid is used in the culture of PPFM and / or methanotrophic bacteria for the production of chemical products, foods, proteins, and other products. Examples of products by the culture of PPFM and / or methanotrophic bacteria include biochemicals such as PHA, PHB, carotenoids, amino acids, terpenoids, polyketides, formic acid, glyoxylic acid, etc. In such embodiments, the produced product can be harvested separately from the fermentation broth or together with the PPFM and / or methanotrophic bacteria cells generated during fermentation.

[0008] In one embodiment, the fermentation broth provided herein comprises nutrients that support the growth of PPFM and / or methanotrophic bacteria, a population of growing PPFM bacteria, and hop acids. In one embodiment, the population of PPFM bacteria in the fermentation broth is at least 10 6 colony forming units (CFU) per milliliter. In one embodiment, β - hop acid is present at a concentration of at least 7.5 ppm. In one embodiment, the pH of the fermentation broth is greater than 6. In one embodiment, the hop acid is a mixture of α - acid and β - acid. In one embodiment, the fermentation broth contains a higher percentage of β - acid. In one embodiment, the hop acid during fermentation is predominantly β - acid. In one embodiment, hop β - acid is present at a concentration of at least about 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ppm or more during fermentation. In one embodiment, the population of PPFM and / or methanotrophs is at least 10 9 colony forming units per milliliter. In one embodiment, the hop acid is present in a composition containing harvested PPFM and / or methanotrophic cells and provides additional advantages during storage, formulation, and application for further use of such a composition.

Mode for Carrying Out the Invention

[0009] Definition As used herein, the term "and / or" is to be construed as each of two or more specified features or components being specifically disclosed, regardless of the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0010] As used herein, the terms "include", "includes", and "including" are to be construed as having at least the features they refer to or encompassing the items they refer to, and not as excluding additional unspecified features or unspecified items.

[0011] Also, the indefinite articles "a" and "an" preceding an element or component of the present invention are to be non-limiting with respect to the number of instances (i.e., occurrences) of that element or component. Thus, "a" or "an" is to be interpreted as including one or at least one, and the singular word form of an element or component includes the plural form as well, unless the number is clearly intended to be singular.

[0012] As used herein, the term "biological agent" refers to a component of a composition for treating plants or parts of plants that are composed of or derived from microorganisms. Biological agents include biocontrol agents, other beneficial microorganisms, microbial extracts, natural products, plant growth activators, or plant protectants. Non-limiting examples of biocontrol agents include bacteria, fungi, beneficial nematodes, and viruses. In certain compositions, the composition may include a single culture or co-culture of Methylobacterium, or a combination of separately cultured Methylobacterium strains or isolates.

[0013] The term "PPFM" refers to pink-pigmented facultative methylotrophs, which are a type of Gram-negative bacteria that can grow using only carbon compounds such as methanol as the sole carbon and energy source, but can also utilize more complex organic compounds such as sugars, alcohols, and organic acids. "PPFM" refers to bacteria of the genus "Methylobacterium" and the genus "Methylorubrum" that are pink-pigmented facultative methylotrophs. For convenience, although defined as pink-pigmented, the PPFM used in this specification also includes non-pink-pigmented Methylobacterium species and colorless mutants of Methylobacterium or Methylorubrum isolates. For example, PPFM refers to, but is not limited to, bacteria of the species listed below, and new Methylobacterium species that have not yet been reported or described and are characterized as Methylobacterium or Methylorubrum based on phylogenetic analysis. Methylobacterium adhaesivum, Methylobacterium oryzae, Methylobacterium aerolatum, Methylobacterium oxalidis, Methylobacterium aquaticum, Methylobacterium persicinum, Methylobacterium brachiatum, Methylobacterium phyllosphaerae, Methylobacterium brachythecii, Methylobacterium phyllostachyos, Methylobacterium brattatumbullatum), Methylobacterium platani, Methylobacterium cerastii, Methylobacterium pseudosasicola, Methylobacterium currus, Methylobacterium radiotolerans, Methylobacterium dankookense, Methylobacterium soli, Methylobacterium frigidaeris, Methylobacterium specialis, Methylobacterium fujisawaense, Methylobacterium tardum, Methylobacterium gnaphalii, Methylobacterium tarhaniae, Methylobacterium goesingense, Methylobacterium thuringiense, Methylobacterium gossipiicola, Methylobacterium trifolii, Methylobacterium gregans, Methylobacterium variabile, Methylobacterium haplocladii, Methylobacterium aminovorans (Methylorubrum aminovoransaminovorans)), Methylobacterium hispanicum, Methylobacterium extorquens (Methylorubrum extorquens), Methylobacterium indicum, Methylobacterium podarium (Methylorubrum podarium), Methylobacterium iners, Methylobacterium populi (Methylorubrum populi), Methylobacterium isbiliense, Methylobacterium pseudosasae (Methylorubrum pseudosasae), Methylobacterium jeotgali, Methylobacterium rhodesianum (Methylorubrum rhodesianum), Methylobacterium komagatae, Methylobacterium rhodinum (Methylorubrum rhodinum), Methylobacterium longum, Methylobacterium salsuginis (Methylorubrum salsuginis), Methylobacterium marchantiae, Methylobacterium suomienseMethylorubrum suomiense, Methylobacterium mesophilicum, Methylobacterium thiocyanatum (Methylorubrum thiocyanatum), Methylobacterium nodulans, Methylobacterium zatmanii (Methylorubrum zatmanii), Methylobacterium symbiota, or Methylobacterium organophilum.

[0014] As used herein, nutrients suitable for the growth of PPFM bacteria refer to the nutrients used in the fermentation broth for the production of PPFM bacteria. Such nutrients include fermentable sugars or alcohols as a carbon source, including oligosaccharides and monosaccharides that can be used as a carbon source by PPFM in the fermentation process. Carbon sources used in the methods provided herein include, but are not limited to, glycerol, fructose, methanol, ethanol, glutamic acid, sucrose, MSG, betaine, trimethylglycine, aspartic acid, and succinic acid.

[0015] As used herein, the terms "methanotrophic bacteria" or "methanotroph" refer to genera and species of bacteria that can utilize methane as a carbon source for growth. Methanotrophic bacteria include species of the genera Methyloacidimicrobium, Methyloacidiplilum, Methylobacter, Methylocaldum, Methylocapsa, Methylocella, Methylococcus, Methylocystis, Methyloferula, Methylogaea, Methyloglobus, Methylohalobius, Methylomagnum, Methylomarinum, Methylomicrobium, Methylomonas, Methyloparacoccus, Methyloperedens, Methyloprofundus, Methylosarcina, Methylosinus, Methylosoma, Methylosphaera, Methylothermus, and Methylovulum.

[0016] Nutrients suitable for the growth of methanotrophic bacteria as used herein refer to the nutrients used in fermentation broth for the production of methanotrophic bacteria. Such nutrients include C1 substrates such as methane, methanol, formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide, methylated amines, methylated thiols, and methyl halides. In some embodiments, the fermentation broth may contain a single C1 substrate as the sole carbon source, or a mixture of two or more C1 substrates to provide multiple carbon sources.

[0017] The term "contaminant" refers to the presence of microorganisms that have not been intentionally introduced. Microorganisms other than PPFM present in the fermentation broth are considered contaminants.

[0018] The term "substantial contamination" refers to the level of bacterial contamination in the fermentation broth that leads to a decrease in the yield of PPFM.

[0019] The term "fermentation medium" as used herein refers to a composition that promotes the growth of PPFM bacteria. The fermentation medium can be used at any scale, such as small-scale culture or large-scale production fermentation.

[0020] The term "fermentation broth" refers to a composition containing a fermentation medium and a PPFM inoculum.

[0021] The term "hop acid" refers to a product extracted from hops (female flowers of the hop plant Humulus lupulu) by CO2. The extracted hop acid is composed of α-acid (humulone) and β-acid (lupulone). Derivatives of hop acid can be produced, for example, by boiling or chemical reduction, and such derivatives are also considered hop acids herein. Hop α-acids include, but are not limited to, humulone, cohumulone, adhumulone, posthumulone, and prehumulone. Hop β-acids include, but are not limited to, colupulone, lupulone, and adlupulone.

[0022] As used herein, the term "strain" should include all isolates of such a strain.

[0023] The term "seed culture" is a culture of PPFM cells for inoculating a large amount into a fermentation medium to produce a fermentation broth.

[0024] Fermentation This specification provides useful antibacterial agents for fermentation that selectively target contaminating bacteria, mainly Gram-positive bacteria, without inhibiting the growth of PPFM and / or methanotrophic bacteria. Methods and fermentation broths containing PPFM and / or methanotrophic bacteria are disclosed, and such methods and fermentation broths include the use of hop acids that enable the efficient growth of PPFM and / or methanotrophic bacteria while inhibiting the growth of bacterial contaminants. In fermentation processes, undesirable contaminating bacteria can be introduced from biomaterials, process equipment, inoculum cultures, process water, air, or other sources of origin. By controlling contamination during fermentation, PPFM and / or methanotrophic bacteria can grow and produce at higher levels than in the presence of contaminating bacteria, increasing the yield of the desired fermentation product, preventing loss of fermentation yield, or preventing loss of the entire fermentation batch, thus providing a more efficient and economical fermentation process.

[0025] Hop acids are typically used in the brewing industry and are typically known to exhibit better performance under low pH conditions with a pH of about 5.2 to about 5.6. The antibacterial effect of hop acids has been shown to decrease as the pH increases from pH 4 to pH 7. The examples provided herein demonstrate that hop acids not only control Gram-positive contaminants at pH 6.0 and above but also do not exhibit antibacterial or bacteriostatic effects against PPFM or methanotrophic bacteria, as has been reported for various Gram-negative bacterial species heretofore.

[0026] Hop acid is obtained from the female flower cones of the hop plant Humulus lupulus by extraction with carbon dioxide (CO2). The extracted material may be further separated and / or chemically modified to produce hop acid preparations containing various proportions of alpha and beta hop acids, or modified forms of hop acids such as isomerized forms of alpha acids. Various types of hop acid preparations are commercially available from manufacturers such as BetaTec and HopSteiner. In some embodiments, the hop acid product used in the methods and compositions of the present invention predominantly comprises beta hop acid. HopSteiner Beta Bio 45% is a preparation containing 45% natural hop beta acid. BetaTec IsoStab is an aqueous solution of isomerized alpha acid. BetaTec BetaStabXL is an aqueous formulation in which beta acid (8.5 - 9.5% w / w) is emulsified with fatty acids. Of particular interest is the use of products containing high concentrations of beta hop acid, and the use of such products to achieve beta acid concentrations of about 7.5 ppm or greater in the fermentation broths and methods provided herein. Methods for the extraction and purification of hop acids, as well as methods for the concentration of specific types of hop acids containing beta hop acid, are known in the art. The concentration of hop acids can vary depending on the variety of hops, and it is possible to identify varieties known to have a higher proportion of beta hop acid compared to alpha hop acid, and to provide a source of hop acid for use in the methods and compositions of the present invention.

[0027] In one embodiment provided herein, hop acid and PPFM and / or a methane trophic cell inoculum are added to a sterilized fermentation medium to produce a fermentation broth, which is maintained under conditions suitable for the growth of PPFM and / or methane trophic cells. In one embodiment, hop acid is added to the sterilized fermentation medium, followed by the addition of PPFM and / or methane trophic cells to produce a fermentation broth. In other embodiments, hop acid and PPFM and / or methane trophic cells are added to the fermentation medium simultaneously, or PPFM and / or methane trophic cells are added to the medium and then hop acid is added. Thus, the fermentation broth composition of the present invention comprises a fermentation medium, hop acid, and a population of growing PPFM and / or methane trophic cells. After inoculation, the cells grow to form a population of growing PPFM and / or methane trophic cells in the fermentation broth.

[0028] Any type of fermentation medium that can support the growth of PPFM and / or methane trophic bacteria can be used. In some embodiments, the fermentation medium is prepared from readily available components including, but not limited to, inorganic salts such as potassium phosphate and magnesium sulfate, carbon sources such as glycerol, methanol, ethanol, glutamic acid, fructose, sucrose, MSG, betaine, trimethylglycine, aspartic acid, and succinic acid, and amino acid mixtures such as peptone and tryptone. Exemplary synthetic media that can be used for PPFM fermentation include, but are not limited to, ammonium inorganic salt (AMS) medium (Whittenbury et al., 1970), Vogel-Bonner (VB) minimal medium (Vogel and Bonner, 1956), and LB broth ("Luria-Bertani Broth"). In some embodiments, the fermentation medium may contain a solid substance that promotes the growth of PPFM bacteria, such as particulate solids (US10920214). In some embodiments, the fermentation medium can be an emulsion comprising, for example, a continuous aqueous phase containing nutrients for the growth of PPFM and a dispersed phase comprising a non-aqueous liquid that is immiscible or only partially miscible with the continuous phase (US10287544).

[0029] For example, a method for inhibiting the growth of bacterial contaminants in a medium containing pink-pigmented facultative methylotroph (PPFM) bacteria and / or methanotroph bacteria is disclosed, the method comprising adding hop acid to a sterilized fermentation medium containing nutrients suitable for the growth of PPFM and / or methanotroph bacteria, adding an inoculum of PPFM and / or methanotroph bacteria to the fermentation medium to provide a fermentation broth, and maintaining the fermentation broth under conditions suitable for the growth of PPFM and / or methanotroph bacteria, thereby inhibiting the growth of bacterial contaminants.

[0030] In some embodiments, PPFM and / or methanotroph bacteria are grown in a fermentation medium for the production of biodegradable plastics such as PHA and PHB, industrial products or compounds such as glyoxylate and carotenoids, or for the conversion of CO2 into high-value products such as amino acids, terpenoids, polyketides, and formic acid. In some embodiments, PPFM and / or methanotroph cells are harvested after fermentation and further processed to produce dry or liquid inoculum cultures for use in agricultural and / or methane mitigation methods.

[0031] In one embodiment, the pH of the fermentation medium or broth provided herein ranges from about pH 6.0 to about pH 8.0. In one embodiment, the pH of the fermentation medium and broth provided herein ranges from about pH 6.5 to pH 7.5.

[0032] The amount of hop acid required to control contamination in large-scale fermentation varies depending on various factors such as the source and amount of contamination, the medium used in fermentation, the ratio of alpha-hop acid to beta-hop acid in the added hop acid product, the concentration of PPFM and / or methanotroph cells after inoculation, and other fermentation conditions. Those skilled in the art can adjust the amount of hop acid required according to the observed fermentation conditions. In some embodiments, the addition of concentrated hop acid, which may result in a viscous product, is facilitated by dissolving the hop acid product in methanol to provide a 10% dilution.

[0033] The concentration of hop acids useful for inhibiting the growth of bacterial contaminants in PPFM and / or methanotrophic fermentation broth varies depending on the percentage of alpha and beta acids in the hop acid preparation. The examples provided herein show that when hop acids were added to a complex medium agar plate at a concentration of 10 ppm of the product, it was not effective in preventing the growth of bacterial contaminants, and as a result, the final hop acid concentration on the plate was about 1 - 4.5 ppm of hop acid depending on the concentration of the particular product. Hop acid products containing mainly alpha hop acids also could not inhibit the growth of gram-positive contaminants when 100 or 1000 ppm of the hop acid product was added to the plate with a final hop acid concentration of about 10 - 300 ppm. Hop acid products containing mainly beta hop acids have been demonstrated herein to inhibit the growth of all gram-positive bacteria tested when the hop acid product was added to the medium plate at 100 or 1000 ppm, with the final beta hop acid concentration being about 9 - 450 ppm. One product, Beta Bio 45, contains beta hop acids at a concentration of 45%, and when the product concentration is 100 ppm or 1000 ppm, the concentration in the fermentation medium is 45 ppm or 450 ppm. Another product, BetaStab XL, contains 8.5 - 9.5% hop beta acids, and when the product concentration is about 100 or 1000 ppm, the concentration in the fermentation medium is about 9 or 90 ppm. Thus, in the compositions and methods of the present invention, beta hop acids are used for inhibiting contamination of PPFM fermentation broth when provided at a concentration exceeding about 7.5 ppm. Of interest are methods and compositions for the fermentation of PPFM and / or methanotrophs, wherein beta hop acids are provided at a concentration of at least about 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ppm or more (including any integer or fraction therebetween) of beta hop acid.

[0034] The PPFM and / or methanotroph inoculum can be any cell source effective to initiate the fermentation broth culture. Typically, the cells are cryopreserved and revived by growing the inoculum in a synthetic medium. When the inoculum is added to the fermentation medium, a fermentation broth or culture is produced. In some embodiments, contamination in the inoculum can be controlled by adding hop acids dissolved in methanol as described herein. In such cases, the methanol also acts as an antibacterial agent and when the seed culture is used to inoculate a large-scale fermentation, it may not be necessary to add additional hop acids to the fermentation medium separately from the inoculum. Thus, in one embodiment, contamination during fermentation is controlled by including hop acids in the seed culture used to inoculate the fermentation medium.

[0035] Any strain of PPFM and / or methanotrophic bacteria can be used in the methods and fermentation broths provided herein. In some embodiments, PPFM and / or methanotrophic bacteria are useful for inoculating plants to improve plant germination, nitrogen use efficiency, growth, root mass, and yield and / or to provide resistance to various organisms such as bacteria, fungi, insects, nematodes, and plant pests. In some embodiments, PPFM and / or methanotrophs are mutant or genetically engineered strains that produce increased levels of useful industrial compounds, nutrients, or plant growth regulators. U.S. Patent No. 8,153,118 discloses various Methylobacterium isolates that can be used in the methods and compositions provided herein to increase the levels of vitamin B-12 and amino acids. One or more Methylobacterium, e.g., Methylobacterium mutant B12-11 (Accession No. ATCC PTA-1561) that overproduces vitamin B-12, Methylobacterium rhodinum (ATCC#43282) that overproduces the amino acid threonine, Methylobacterium sp. (ATCC#21371) that overproduces the amino acid (L-glutamic acid), Methylobacterium sp. (ATCC#21372) that overproduces the amino acid (L-glutamic acid), Methylobacterium sp. (ATCC#21926) that overproduces the amino acid (L-lysine), Methylobacterium sp. (ATCC#21969) that overproduces the amino acid (L-glutamic acid), Methylobacterium sp. (ATCC#21927) that overproduces the amino acids (L-lysine, L-aspartic acid, L-alanine, L-valine, L-leucine, and L-arginine), and / or Methylobacterium sp. (ATCC#21438) that produces single cell protein are also provided, including fermentation broths, fermentation broth products, and compositions.

[0036] Non-limiting examples of Methylobacterium strains that can be used in the methods provided herein are disclosed in Table 1. Other Methylobacterium strains useful in the specific methods provided herein include variants of the Methylobacterium strains disclosed in Table 1. Also, various combinations of two or more strains or variants of the Methylobacterium strains disclosed in Table 1 are useful for treating plants or parts thereof.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0037] In some embodiments provided herein, the inoculated medium or fermentation broth is cultured under conditions suitable for the growth of PPFM and / or methanotrophic bacteria. Typically, the temperature is from about 30 °C to about 37 °C and the pH is from about 6.5 to about 7.5. To minimize foaming, an antifoaming agent such as dimetpolysiloxane can be added to the medium if necessary. In some embodiments, the PPFM fermentation for the production of the PPFM inoculum is carried out as fed-batch fermentation, which is carried out for 1 to 7 days, and a PPFM concentration of 1×10 8 ~ about 1×10 11 CFU per milliliter is obtained. In some embodiments, the PPFM fermentation for the production of the PPFM inoculum is carried out as fed-batch fermentation and is carried out for 1 to 4 days, 1 to 3 days, or 1 to 2 days. The fermentation for the production of methanotrophic bacteria can be carried out as a batch or continuous culture process. For the growth of methanotrophic bacteria, a C1 carbon source such as methane or methanol is used. Exemplary synthetic media for the growth of methanotrophic bacteria are described, for example, in WO2021071966 and include Higgins minimal nitrate medium (NSM), MM-W1 medium, master mix feed (MMF), medium MMF1.1, medium MMS1.0, and AMS medium. The media and conditions for the culture of PPFM and / or methanotrophic bacteria can be optimized according to the strain(s) of bacteria selected for growth.

[0038] Control of contaminating bacteria can be evaluated by overlaying fermentation broth samples. In some embodiments, PPFM and / or methanotrophic cells are harvested after fermentation and further processed to produce a dry or liquid inoculum culture for use in agricultural, bioremediation, and / or methane mitigation methods. The dry formulation can be prepared, for example, by spray drying, freeze drying, air drying, fluidized bed drying, electrospray drying, or other drying methods.

[0039] In fermentation cultures for commercial production, various culture methods can be applied. For example, in large-scale production, both batch culture and continuous culture methodologies can be used. The classical batch culture method is a closed system where the composition of the medium is set at the start of the culture and is not artificially changed during the culture process. Thus, at the start of the culture process, the desired organism is inoculated into the medium and growth or metabolic activity occurs. Typically, however, a "batch" culture is batch-wise with respect to the addition of carbon sources, and control factors such as pH and oxygen concentration are adjusted. In a batch system, the metabolite and biomass composition of the strain changes continuously until the end of the culture. In batch culture, cells transition from a stationary lag phase through a high logarithmic growth phase and ultimately to a stationary phase where the growth rate decreases or stops. If not processed, the cells in the stationary phase will ultimately die. Cells in the logarithmic growth phase are often responsible for most of the ethanol production.

[0040] A variation of the standard batch system is the fed-batch system. The fed-batch culture process is also suitable for the methods and compositions of the present invention and includes a typical batch system except that substrates are gradually added as the culture progresses. Since it is difficult to measure the actual substrate concentration in a fed-batch system, it is estimated based on changes in measurable factors such as the partial pressure of waste gases such as pH and CO2. The batch culture method and the fed-batch culture method are common and well-known in the art and examples can be found in the field of biotechnology. A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA, or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992)

[0041] The methods and compositions of the present invention can also be used in continuous culture processes. Continuous culture is an open system in which medium is continuously added to a bioreactor while an equal amount of conditioned medium is removed and processed simultaneously. Continuous culture generally maintains cells at a relatively high liquid phase density where the cells are mainly in the logarithmic growth phase. Alternatively, continuous culture can be carried out using immobilized cells, in which case carbon and nutrients are continuously added and valuable products, by-products, or waste are continuously removed from the cell mass. Immobilization of cells can be carried out using a wide range of solid supports composed of natural materials and / or synthetic materials, as is known to those skilled in the art.

[0042] Use of PPFM and / or Methanotroph Fermentation Products PPFM and / or methanotrophs prepared using the media and methods provided herein can be used in agriculture. The microbial material composition is provided to a plant or part of a plant, the soil in which the plant grows, e.g., the nutrient solution in which a plant grows in a hydroponic system, the soil on which a part of a plant such as a seed is deposited, aeroponic applications such as root spraying, or any combination thereof. Treatments or applications can include, but are not limited to, spraying, coating, partially coating, dipping, and / or absorbing the compositions provided herein onto a plant or part of a plant. In certain embodiments, seeds, leaves, plant cuttings, fruits, stems, roots, tubers, or coleoptiles can be dipped, soaked, and / or absorbed in a liquid, semi-liquid, emulsion, or slurry of the compositions provided herein. Also provided herein are plants and parts of plants coated or partially coated with the microbial material composition.

[0043] A part of a plant for treatment with PPFM and / or methanotroph bacteria is selected from the group consisting of leaves, stems, fruits, plant cuttings, flowers, roots, seedlings, tubers, or seeds. The PPFM and / or methanotroph bacterial inoculum is about 1×10 2 , 1×10 3 , 1×10 4 , or 1×10 5From about 1×10 7 to 1×10 8 to 1×10 9 or 1×10 10 CFU of PPFM and / or methanotrophic bacteria are applied to a plant or a part of a plant (e.g., a seed).

[0044] Plants that can be treated with a PPFM and / or methanotrophic bacterium composition include corn, soybeans, Brassica species (e.g., B. napus, B. rapa, B. juncea), alfalfa, rice, rye, wheat, barley, oats, sorghum, miscellaneous grains (e.g., pearl millet (Pennisetum glaucum), foxtail millet (Panicum miliaceum), Italian millet (Setaria italica), finger millet (Eleusine coracana)), sunflower, safflower, tobacco, potato, peanut, cotton, Cannabis species (including but not limited to Cannabis sativa and industrial hemp varieties), sweet potato (Ipomoea batatus), cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, date palm, banana, apple, pear, grape, berry plants (including but not limited to blackberry, raspberry, strawberry, blueberry plants), avocado, fig, guava, kiwi, mango, olive, papaya, cashew nut, macadamia nut, almond, sugar beet, sugar cane, tomato, pepper, lettuce, leafy vegetables (including but not limited to spinach, kale, microgreens of escarole, collard greens, cabbage, beet leaves, cress, romaine lettuce, Swiss chard, arugula, endive, Chinese cabbage, turnip leaves, etc.), green beans, lima beans, peas, lentils, cucurbitaceous vegetables (including but not limited to cucumber, muskmelon, melon, pumpkin, squash, zucchini, etc.).Other leafy vegetable plants cultivated for the production and harvesting of microgreens and / or herbs can also be treated with PPFM, including but not limited to lettuce, cauliflower, broccoli, cabbage, cress, arugula, garlic, onion, leek, amaranth, bok choy, beans, spinach, melon, cucumber, pumpkin, basil, celery, coriander, radish, radicchio, chicory, dill, rosemary, French tarragon, basil, carrot, fennel, beans, peas, mung beans, lentils. In other embodiments, the treated plants are ornamental plants (including but not limited to rhododendron, hydrangea, hibiscus, rose, tulip, rhaponticum, petunia, carnation, poinsettia, penisetum, chrysanthemum, etc.), coniferous trees (including but not limited to pine trees such as lodgepole pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, etc., fir trees such as Douglas fir, western hemlock, Sitka spruce, redwood, silver fir and balsam fir, etc., cedar trees such as western red cedar and Alaska yellow cedar, etc.), and turfgrasses (including but not limited to annual bluegrass, annual ryegrass, Canada bluegrass, fescue, bentgrass, wheatgrass, Kentucky bluegrass, orchardgrass, ryegrass, redtop, bermudagrass, St. Augustinegrass, zoysiagrass, etc.).

[0045] A microbial material composition for agricultural use is provided, and this composition has a titer exceeding about 5×10 7 colony forming units per milliliter, a titer exceeding about 1×10 8 colony forming units per milliliter, a titer exceeding about 5×10 8 colony forming units per milliliter, a titer exceeding about 1×10 9 colony forming units per milliliter, a titer exceeding about 1×10 10 colony forming units per milliliter, and at least about 3×10 10Contains PPFM and / or methanotrophic bacteria in colony-forming unit titer. In certain embodiments, the microbial material composition provided herein is at least about 5×10 7 , 1×10 8 , or 5×10 8 colony-forming units to at least about 4×10 10 colony-forming units, at least about 5×10 8 colony-forming units to at least about 4×10 10 colony-forming units, or at least about 5×10 8 colony-forming units to at least about 6×10 10 colony-forming units in colony-forming unit titer. In certain embodiments, the composition of the microbial inoculum provided herein is at least about 1×10 9 colony-forming units to at least about 3×10 10 colony-forming units, at least about 1×10 9 colony-forming units to at least about 4×10 10 colony-forming units, or at least about 1×10 9 colony-forming units to at least about 6×10 10 colony-forming units in colony-forming unit titer. In certain embodiments, the microbial material composition provided herein is at least about 1×10 10 colony-forming units to at least about 3×10 10 colony-forming units, at least about 1×10 10 colony-forming units to at least about 4×10 10 colony-forming units, or at least about 1×10 10 colony-forming units to at least about 6×10 10Contains PPFM and / or methanotrophic bacteria at a titer of colony forming units. In certain embodiments, the microbial material composition provided herein is at least about 3×10 10 colony forming units per milliliter to at least about 4×10 10 colony forming units per milliliter, or at least about 3×10 10 colony forming units per milliliter to at least about 6×10 10 colony forming units and contains PPFM and / or methanotrophic bacteria. In any of the foregoing compositions, the composition is essentially free of contaminating microorganisms and the PPFM and / or methanotrophic bacteria can include PPFM and / or methanotrophic bacteria that adhere and / or associate with substances to which they do not adhere and / or associate in nature, or any combination thereof.

[0046] In certain embodiments of any of the foregoing compositions and methods, about 5×10 7 , 1×10 8 , or 5×10 8 colony forming units per gram or more, about 1×10 9 colony forming units per gram or more, about 1×10 10 colony forming units per gram or more, at least about 3×10 10 colony forming units and provides a dried preparation or microbial material composition containing PPFM and / or methanotrophic bacteria. In certain embodiments, the microbial material composition provided herein is at least about 5×10 7 , 1×10 8 , or 5×10 8 colony forming units per gram to at least about 3×10 10 colony forming units per gram, at least about 5×10 7 , 1×10 8 , or 5×10 8 colony forming units per gram to at least about 4×10 10 colony forming units per gram, or at least about 5×10 7, 1×10 8 , or 5×10 8 colony forming units ~ at least about 6×10 10 colony forming units per gram contain PPFM and / or methanotrophic bacteria. In certain embodiments, the microbial material composition provided herein is at least about 1×10 9 colony forming units ~ at least about 3×10 10 colony forming units, at least about 1×10 9 colony forming units ~ at least about 4×10 10 colony forming units, or at least about 1×10 9 colony forming units ~ at least about 6×10 10 colony forming units per gram may contain PPFM and / or methanotrophic bacteria. In certain embodiments, the microbial material composition provided herein is at least about 1×10 10 colony forming units ~ at least about 3×10 10 colony forming units, at least about 1×10 10 colony forming units ~ at least about 4×10 10 colony forming units, or at least about 1×10 10 colony forming units ~ at least about 6×10 10 colony forming units per gram contains PPFM and / or methanotrophic bacteria. In certain embodiments, the microbial material composition provided herein is at least about 3×10 10 colony forming units ~ at least about 4×10 10 colony forming units, or at least about 3×10 10 colony forming units ~ at least about 6×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 5×10 13The titer of colony forming units contains PPFM and / or methanotrophic bacteria. In any of the aforementioned microbial materials or compositions, the composition may contain PPFM and / or methanotrophic bacteria attached to a solid substance. In any of the aforementioned microbial material compositions, the composition is essentially free of contaminating microorganisms and contains PPFM and / or methanotrophic bacteria that attach and / or associate with substances to which they do not attach and / or associate in nature, or any combination thereof.

[0047] In some embodiments, the compositions or methods disclosed herein may include one or more PPFM and / or methanotrophic bacterial isolates and additional active ingredients that can be, for example, pesticides, non-biological plant stimulants or microbial stimulants, or a second biological agent. In certain embodiments, the pesticide can be an insecticide, fungicide, herbicide, nematicide, or other biocide. The second biological agent can be a strain that improves yield or controls insects, pests, fungi, weeds, nematodes. In some embodiments, the second biological agent is a second PPFM and / or methanotrophic strain.

[0048] Non-limiting examples of insecticides and nematicides include carbamates, diamides, macrolide lactones, neonicotinoids, organophosphates, phenylpyrazoles, pyrethrins, spinosins, synthetic pyrethroids, tetronic acids, and tetramic acids. In certain embodiments, the insecticides and nematicides include abamectin, aldicarb, aldoxycarb, bifenthrin, carbofuran, chlorantraniliprole, clothianidin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, dinotefuran, emamectin, ethiprole, fenamiphos, fipronil, flubendiamide, fosthiazate, imidacloprid, ivermectin, lambda-cyhalothrin, milbemectin, nitenpyram, oxamyl, permethrin, thioxazafen, spinetoram, spinosad, spirodiclofen, spirotetramat, tefluthrin, thiacloprid, thiamethoxam, and thiodicarb.

[0049] Non-limiting examples of useful fungicides include aromatic hydrocarbons, benzimidazoles, benzothiadiazoles, carboxamides, carboxylic acid amides, morpholines, phenylamides, phosphonates, quinone outside inhibitors (e.g., strobilurins), thiazolidines, thiophanates, thiophene carboxamides, and triazoles. Specific examples of fungicides include acibenzolar-S-methyl, azoxystrobin, benalaxyl, bixafen, boscalid, carbendazim, cyproconazole, dimethomorph, epoxiconazole, fluopyram, fluoxastrobin, flutianil, flutolanil, fluxapyroxad, fosetyl-Al, ipconazole, isopyrazam, kresoxim-methyl, mefenoxam, metalaxyl, metconazole, microbutanil, orysastrobin, penflufen, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, sedaxane, silthiopham, tebuconazole, tifluzamide, thiophanate, tolclofos-methyl, trifloxystrobin, and triticonazole. Non-limiting examples of other biocides include, for example, isothiazolinones, 1,2-benzothiazolin-3-one (BIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CIT), 2-methyl-4-isothiazolin-3-one (MIT), octyl isothiazolinone (OIT), dichlorooctyl isothiazolinone (DCOIT), and butyl benzisothiazolinone (BBIT), 2-bromo-2-nitro-propane-1,3-diol (bronopol), 5-bromo-5-nitro-1,3-dioxane (bronidox), tris(hydroxymethyl)nitromethane, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and alkyl dimethyl benzyl ammonium chloride.

[0050] Non-limiting examples of herbicides include ACCase inhibitors, acetanilides, AHAS inhibitors, carotenoid biosynthesis inhibitors, EPSPS inhibitors, glutamine synthetase inhibitors, PPO inhibitors, PS II inhibitors, and synthetic auxins. Specific examples of herbicides include acetochlor, clethodim, dicamba, flumioxazin, mesotrione, glyphosate, glufosinate, metolachlor, quizalofop, sulfentrazone, sulcotrione, and 2,4-D.

[0051] In some embodiments, the compositions or methods disclosed herein may include PPFM and / or a methanotroph strain and an additional active ingredient selected from the group consisting of clothianidin, ipconazole, imidacloprid, metalaxyl, mefenoxam, thioxazafen, azoxystrobin, thiamethoxam, fluxapyroxad, prothioconazole, pyraclostrobin, and sedaxane.

[0052] In some embodiments, the compositions or methods disclosed herein may include an additional active ingredient that may be a biostimulant for plants or microorganisms. In some embodiments, the additional active ingredient may be a second biological agent. The second biological agent may be a biocontrol agent, other beneficial microorganisms, microbial extracts, plant extracts (plant components), other natural products (e.g., protein hydrolysates and other nitrogen-containing compounds, plant growth activators or stimulants, or plant protectants). Examples of useful additional components include yeast or seaweed or their extracts and powders, humic acids and fulvic acids, amino acids, peptides, chitosan and other biopolymers, plant hormones such as auxins and cytokinins or their derivatives, trace elements, and nucleic acids. In some embodiments, the compositions provided herein contain any combination of these compounds or related compounds. Non-limiting examples of biocontrol agents include bacteria, fungi, beneficial nematodes, and viruses.

[0053] In certain embodiments, the second biological agent is Actinomycetes, Agrobacterium, Arthrobacter, Alcaligenes, Aureobacterium, Azobacter, Azorhizobium, Azospirillum, Azotobacter, Beijerinckia, Bacillus, Brevibacillus, Burkholderia, Chromobacterium, Clostridium, Clavibacter, Comomonas, Corynebacterium, Curtobacterium, Enterobacter, Flavobacterium, Gluconacetobacter, Gluconobacter, Herbaspirillum, Hydrogenophage, Klebsiella, Luteibacter, Lysinibacillus, Mesorhizobium, Methylobacterium, Microbacterium, Ochrobactrum, Paenibacillus, Pantoea, Pasteuria, Phingobacterium, Photorhabdus, Phyllobacterium, Pseudomonas, Rhizobium, Rhodococcus,It can be bacteria of the genera Bradyrhizobium, Serratia, Sinorhizobium, Sphingomonas, Streptomyces, Stenotrophomonas, Variovorax, Xanthomonas, and Xenorhadbus. In certain embodiments, the bacteria are selected from the group consisting of Bacillus amyloliquefaciens, Bacillus cereus, Bacillus firmus, Bacillus lichenformis, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Chromobacterium suttsuga, Pasteuria penetrans, Pasteuria usage, and Pseudomona fluorescens.,

[0054] In certain embodiments, the second biological agent can be a fungus of the genus Acremonium, Alternaria, Ampelomyces, Aspergillus, Aureobasidium, Beauveria, Botryosphaeria, Cladosporium, Cochliobolus, Colletotrichum, Coniothyrium, Embellisia, Epicoccum, Fusarium, Gigaspora, Gliocladium, Glomus, Laccaria, Metarhisium, Muscodor, Nigrospora, Paecilonyces, Paraglomus, Penicillium, Phoma, Pisolithus, Podospora, Rhizopogon, Scleroderma, Trichoderma, Typhula, Ulocladium, and Verticillium. In certain embodiments, the fungus is Beauveria bassiana, Coniothyrium minitans, Gliocladium vixens, Muscodor albus, Paecilomyces lilacinus, or Trichoderma polysporum.

[0055] In a further embodiment, the second biological agent can be a plant growth activator or a plant defense agent including, but not limited to, harpin, Reynoutria sachalinensis, jasmonic acid, lipochito-oligosaccharides, and isoflavones.

[0056] In a further embodiment, the second biological agent includes, but is not limited to, various Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp., and Trichoderma sp. Particularly useful biopesticide microorganisms include various strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and Streptomyces lydicus. Other microorganisms added can be genetically engineered or wild-type isolates available as pure cultures. In certain embodiments, the second biological agent may be provided in the composition in the form of spores.

[0057] In a further embodiment of any of the foregoing compositions or methods, the additional components may include agricultural excipients and / or agricultural adjuvants. Agriculturally acceptable adjuvants used in the compositions include, but are not limited to, components that enhance production efficiency and / or products that enhance the ease of application of the product. Adjuvants that enhance production efficiency include various wetting / spreading agents that promote the adhesion and spreading of the composition on plant parts, adhesives that promote adhesion to plant parts, penetrants that promote contact between the active agent and internal tissues, extenders that extend the half-life of the active agent by inhibiting degradation by the environment, and humectants that increase the density or drying time of the sprayed composition. Wetting / spreading agents used in the compositions include, but are not limited to, nonionic surfactants such as alkyl polyglucosides (APG), polysorbate / sorbitan-based surfactants (e.g., Tween / Span), fatty alcohol ethoxylates, fatty acid ethoxylates, alkylphenol ethoxylates, fatty glycerol esters, block copolymers (e.g., poloxamers), anionic surfactants, cationic surfactants, phospholipids (including but not limited to phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin), amphoteric surfactants such as cocamidopropyl betaine-based surfactants (including but not limited to betaine, sultaine), organosilicate surfactant wetting agents (including but not limited to trisiloxane, polyether siloxane), and / or acidified surfactants. Adhesives used in the compositions may include calcium alginate or other mineral alginates, gelatin, drying oils (such as tung oil, linseed oil), film-forming substances such as polyvinyl alcohol / acrylates, latex-based substances, terpenes / pinolenes, pyrrolidone-based substances. Penetrants may include mineral oils, vegetable oils, esterified vegetable oils, organosilicate surfactants, and acidified surfactants. Extenders used in the compositions may include, but are not limited to, ammonium sulfate, or menthene-based substances. Humectants used in the compositions may include, but are not limited to, glycerol, propylene glycol, and diethyl glycol.Adjuvants that enhance the ease of application of the product include, but are not limited to, dispersants, acidifying agents / buffers, antifoaming agents / defrothing agents, compatibilizing agents, drift reducers, dyes, and water conditioners. Dispersants that enhance the uniform mixing and ease of use and handling of the product include, for example, acrylate polymers or copolymers, lignosulfonates, (alkyl)naphthalenesulfonates. The antifoaming agent / defrothing agent used in the composition may include, but is not limited to, dimethopolysiloxane. The compatibilizing agent used in the composition may include, but is not limited to, ammonium sulfate. The drift reducer used in the composition may include, but is not limited to, polyacrylamide and polysaccharides. The water conditioner used in the composition may include, but is not limited to, ammonium sulfate.

[0058] In certain embodiments, the PPFM composition used to treat plants or plant seeds may contain agriculturally acceptable excipients and / or adjuvants. Such excipients include, but are not limited to, wood flour, clay, activated carbon, diatomaceous earth, particulate inorganic solids, calcium carbonate, etc. Clays and inorganic solids that can be used in the compositions provided herein include, but are not limited to, calcium bentonite, kaolin, pottery clay, talc, graphite, perlite, mica, vermiculite, silica, quartz powder, montmorillonite, and mixtures thereof. In certain embodiments of treating plant seeds with the PPFM composition, the composition further comprises one or more lubricants to ensure smooth flow and separation (singulation) of the seeds within a seeding mechanism, e.g., a planter box. Lubricants used in such compositions include talc, graphite, polyethylene wax-based powders (such as Fluency Agent), protein powders (e.g., soy protein powder), or combinations of protein powders and lipids (e.g., lecithin or vegetable oil). Combinations of such lubricants can also be used, including, for example, mixtures of clay and talc. The lubricant can be applied to the seeds simultaneously with the application of the inoculum or can be mixed with the inoculum prior to applying the composition to the seeds.Agriculturally acceptable adjuvants that promote adhesion to seeds include, but are not limited to, polyvinyl acetate, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetate, polyvinyl pyrrolidone, polyvinyl pyrrolidone-vinyl acetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymers, waxes, latex polymers, cellulose including ethyl cellulose and methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxymethylpropyl cellulose, alginates, dextrin, maltodextrin, polysaccharides, fats, oils, proteins, karaya gum, jagar gum, tragacanth gum, polysaccharide gums, mucilage, gum arabic, shellac, vinylidene chloride polymers and copolymers, soy-derived protein polymers and copolymers, lignin sulfonates, acrylic copolymers, starch, polyvinyl acrylate, zein, gelatin, carboxymethyl cellulose, chitosan, polyethylene oxide, acrylic imide polymers and copolymers, polyhydroxyethyl acrylate, methyl acrylimide monomer, alginates, ethyl cellulose, polychloroprene and syrups or mixtures thereof. Other useful agriculturally acceptable adjuvants that can promote coating include, but are not limited to, polymers and copolymers of vinyl acetate, polyvinyl pyrrolidone-vinyl acetate copolymers, and water-soluble waxes. The various surfactants, dispersants, anti-caking agents, foam control agents and dyes disclosed herein and in U.S. Patent No. 8,181,388 can be adapted for use in the compositions provided herein.

[0059] Deposit Information Samples of the following strains of Methylobacterium sp. were deposited with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture (1815 North University Street, Peoria, Illinois 61604 U.S.A.) based on the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Methylobacterium sp. NRRL B-50929, NRRL B-50930, NRRL B-50931, NRRL B-50932, NRRL B-50933, NRRL B-50934, NRRL B-50935, NRRL B-50936, NRRL B-50937, NRRL B-50938, NRRL B-50939, NRRL B-50940, NRRL B-50941 and NRRL B-50942 were deposited with NRRL on March 12, 2014. Methylobacterium sp. NRRL B-67339, NRRL B-67340 and NRRL B-67341 were deposited with NRRL on November 18, 2016. Methylobacterium sp. NRRL B-67741, NRRL B-67742, NRRL B-67743 were deposited with NRRL on December 20, 2018. Methylobacterium sp. NRRL B-67809 was deposited with NRRL on June 28, 2019. Methylobacterium sp. NRRL B-67892 was deposited with NRRL on November 26, 2019. Methylobacterium sp. NRRL B-67925, NRRL B-67926 and NRRL B-67927 were deposited with NRRL on February 21, 2020.Methylobacterium sp. NRRL B-67929 was deposited with the NRRL on March 3, 2020. Methylobacterium sp. NRRL B-68032, NRRL B-68033, and NRRL B-68034 were deposited with the NRRL on May 20, 2021. Methylobacterium sp. NRRL B-68064, NRRL B-68065, NRRL B-68066, NRRL B-68067, NRRL B-68068, and NRRL B-68069 were deposited with the NRRL on September 9, 2021. Methylobacterium sp. NRRL B-68074 and NRRL B-68075 were deposited with the NRRL on October 6, 2021. Methylobacterium sp. NRRL B-68186, NRRL B-68187, NRRL B-68188, and NRRL B-68189 were deposited with the NRRL on August 3, 2022. Methylobacterium sp. NRRL B-68194, NRRL B-68195, NRRL B-68196, and NRRL B-68197 were deposited with the NRRL on August 30, 2022. Methylobacterium sp. NRRL B-68215, NRRL B-68216, NRRL B-68217, and NRRL B-68218 were deposited with the NRRL on November 2, 2022. Methylobacterium sp. NRRL B-68236, NRRL B-68237, NRRL B-68238, and NRRL B-68239 were deposited with the NRRL on November 23, 2022. Methylobacterium sp. NRRL B-68261 and Methylosarcina sp. NRRL B-68262 were deposited with the NRRL on February 14, 2023. Methylobacterium sp. NRRL B-68260 was deposited with the NRRL on March 9, 2023.Methylosarcina sp. NRRL B-68281, Methylocystis sp. NRRL B-68282, NRRL B-68283, NRRL B-68284, NRRL B-68285 and NRRL B-68286 were deposited with the NRRL on June 7, 2023.

[0060] In accordance with 37 CFR §1.808(b), all restrictions imposed by the depositor on the public disclosure of the deposited materials will be irrevocably removed upon the grant of a patent from this patent application.

[0061] Although the embodiments have been described in detail, it is obvious that modifications and changes can be made without departing from the scope defined by the appended claims.

Example

[0062] Example 1, Tolerance of PPFM and Methanotrophs to Hop Acids A composite medium plate (glutamic acid / phyto-peptone) pH 6.8 with an overlay of PPFM bacteria was prepared by adding several different PPFM strains (100 uL) to 0.7% agar (4 ml) of the same medium. The hop acids tested were Hopsteiner Beta Bio 45% (containing 45% β-acid), BetaTec IsoStab (aqueous solution containing 28.0 - 32.0% isomerized α-acid), BetaTec BetaStabXL (containing 8.5 - 9.5% hop β-acid), and BetaTec FermaHop Pro (containing 9.5 - 10.5% hop acid consisting mainly of α-acid). Solutions (10 uL) of 10, 100, or 1000 ppm of each hop acid product were added to an acrodisk, placed on the overlay, or directly deposited into small circles on the overlay. The final concentrations of hop acids added to the plates of the products tested are shown in Table 2 below.

Table 2

[0063] Several different species of PPFM were tested, and the addition of 10, 100, or 1000 ppm of hop acid did not inhibit the growth of any of the strains.

[0064] To demonstrate the resistance of methanotrophic bacteria to hop acids, 100 μl aliquots of cultures of Methylomicrobium, Methylosarcina, and one or more Methylocystis strains disclosed in Table 1 are plated onto a carbon-free mineral salt medium (such as nitrate mineral salt (NMS), pH 6.8). A solution (10 μL) of a hop acid product at 10, 100, or 1000 ppm is added to an Acrodisc and placed on the plate or deposited directly in a small circle on the plate. The plates are incubated under methane gas, and the growth of colonies indicating the resistance of the methanotrophic strains to hop acids is monitored.

[0065] Example 2, Effect of Hop Acid on Gram-Positive Bacteria As described in Example 1, an overlay plate was prepared and various strains of Gram-positive bacteria were placed in the overlay. The Gram-positive bacteria tested were Bacillus weidmannii, Lysinibacillus fusiformis, or Rhodococcus corynebacterioides. The hop acid products were tested at 10, 100, and 1000 ppm, and the final hop acid concentrations were obtained as shown in Table 2 above. None of the hop acid products tested showed an inhibition zone indicating growth inhibition when applied at 10 ppm or when the final hop acid concentration was about 1 - 4.5 ppm. When applied at 100 or 1000 ppm and the final concentration was 9 - 450 ppm, growth inhibition zones were produced for all Gram-positive bacterial species tested by the high beta acid hop extract product. The hop acid product mainly containing alpha acids did not inhibit the growth of any of the Gram-positive bacterial species tested when applied at 100 or 1000 ppm until the final hop acid concentration reached 10 - 300 ppm.

[0066] Example 3, Effect of Hop Acid on Contaminants in Fermentation Medium Two PPFM strains were grown in the presence or absence of the addition of Lysinibacillus fusiformis (Lf) or Bacillus weidmannii (Bw) contaminants and in the presence or absence of 45 ppm of beta hop acid. After medium sterilization, 100 ppm of Beta Bio 45% was added to a minimal fermentation medium at pH 6.8 containing fructose (PPFM strain 1 experiment) or glycerol (PPFM strain 2 experiment) as the carbon source, and the final concentration of beta hop acid was 45 ppm. The initial PPFM concentration was approximately 10 7 CFU per ml, but the contaminating bacteria were added at a titer of 10 5 per ml. The number of PPFM and contaminating bacteria was measured at the time of inoculation (time 0), 27 hours, and 53 hours after inoculation. The number of contaminants in the medium containing hop acid was also measured 77 hours after inoculation.

Table 3

Table 4

[0067] These tests showed that the number of contaminating bacteria did not increase even after 77 hours of growth. The number decreased to 1×10 4 CFU / ml or less, but was still detectable. Hop acid appears to be bacteriostatic but not bactericidal against the Gram-positive contaminants tested.

[0068] The PPFM strain was present at a concentration of at least 10 7 CFU / ml at the start of the experiment and increased to 10 9 or 10 10 CFU / ml after 53 hours of growth. Six different PPFM strains tested in this way did not show growth inhibition in the fermentation medium containing hop acid.

Claims

1. A method for inhibiting the growth of bacterial contaminants in a medium, comprising pink pigmented facultative methylotroph (PPFM) bacteria and / or methanotroph bacteria, said method comprising: (a) providing a sterilized fermentation medium containing nutrients suitable for the growth of PPFM and / or methanotroph bacteria; (b) adding hop acid to said sterilized fermentation medium; (c) adding an inoculum of PPFM and / or methanotroph bacterial cells to said fermentation medium to provide a fermentation broth; and (d) maintaining said fermentation broth under conditions suitable for the growth of said PPFM and / or methanotroph bacteria, thereby inhibiting the growth of said bacterial contaminants.

2. The method according to claim 1, wherein the pH of said fermentation broth is greater than pH 6.

3. The method according to claim 1 or 2, wherein said bacterial contaminants are Gram-positive bacteria.

4. The method according to any one of claims 1 to 3, wherein said hop acid is present at a concentration sufficient to inhibit the growth of said Gram-positive bacteria at pH 6 or higher.

5. The method according to any one of claims 1 to 4, wherein said hop acid contains β-acid at a concentration of at least 7.5 ppm.

6. The method according to any one of claims 1 to 5, wherein said hop acid contains β-acid at a concentration of at least 45 ppm.

7. The method according to any one of claims 1 to 6, wherein said hop acid contains β-acid at a concentration of about 100 ppm.

8. The method according to any one of claims 1 to 7, wherein said hop acid is a mixture of α-acid and β-acid.

9. The method according to any one of claims 1 to 8, wherein said hop acid contains a higher percentage of β-acid than α-acid.

10. The method according to claim 9, wherein said hop acid is mainly β-acid.

11. The method according to any one of claims 1 to 10, wherein said hop acid is added to said fermentation medium as a 10% dilution in methanol.

12. Growing the PPFMs and / or methanotrophic bacteria to a titer of at least 10 ９ CFU per milliliter, the method according to any one of claims 1 to 11.

13. The method according to any one of claims 1 to 12, further comprising harvesting said PPFM and / or methanotroph bacteria grown in said fermentation broth.

14. The method according to any one of claims 1 to 13, further comprising harvesting one or more chemical products or proteins produced in said fermentation.

15. The method according to any one of claims 1 to 14, wherein said medium contains PPFM bacteria.

16. The method according to any one of claims 1 to 14, wherein the medium contains methanotrophic bacteria.

17. The method according to any one of claims 1 to 14, wherein the medium contains PPFMs and methanotrophic bacteria.

18. A fermentation broth comprising a nutrient, a population of growing pink pigmented facultative methylotrophic (PPFM) bacteria and / or methanotrophic bacteria, and a hop acid, wherein the population of PPFM and / or methanotrophic bacteria has a titer of at least 1 × 10 ７ CFU per milliliter, and the hop acid comprises β-acid at a concentration of at least 7.5 ppm.

19. The fermentation broth according to claim 18, wherein the pH of the fermentation broth is greater than pH 6.

20. The fermentation broth according to claim 18 or 19, wherein the hop acid is a mixture of alpha acid and beta acid.

21. The fermentation broth according to any one of claims 18 to 20, wherein the hop acid contains a higher percentage of beta acid than alpha acid.

22. The fermentation broth according to any one of claims 18 to 21, wherein the hop acid is mainly beta acid.

23. The population of the PPFMs and / or methanotrophs is at least 1×10 ９ CFU per milliliter, and the fermentation broth according to any one of claims 18 to 22.

24. The fermentation broth according to any one of claims 18 to 23, wherein the concentration of the hop beta acid is at least 45 ppm.

25. The fermentation broth according to any one of claims 18 to 24, wherein the hop acid contains beta acid at a concentration of about 100 ppm.

26. The fermentation broth according to any one of claims 18 to 25, wherein the fermentation broth contains PPFMs.

27. The fermentation broth according to any one of claims 18 to 25, wherein the fermentation broth contains methanotrophic bacteria.

28. The fermentation broth according to any one of claims 18 to 25, wherein the fermentation broth contains PPFMs and methanotrophic bacteria.

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