Microbial protein hydrolysate composition and method for producing the same

Abstract

Protein hydrolysate compositions and methods for making the same are disclosed. The protein hydrolysate compositions have a protein-rich organic content. The protein hydrolysate compositions may be substantially free of exogenous chelating agents, chaotropic agents, and surfactants. The protein hydrolysate compositions may have a low ash content. The protein hydrolysate compositions are produced by processing biomass, e.g., microbial biomass, through a combination of physical, chemical, and / or enzymatic treatments. The protein hydrolysate may be procured from CCk via microbial biomass as a carbon source. Also disclosed are methods for using the protein hydrolysate compositions, e.g., as biostimulants.

Description

Technical Field

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 62 / 901,169 filed on September 16, 2019 and U.S. Provisional Application No. 62 / 943,754 filed on December 4, 2019, the entire disclosures of both of which are incorporated herein by reference.

[0003] The present disclosure relates to the field of protein hydrolysates produced from biological sources, and methods for manufacturing them and formulating them into various end - products. In particular, the present disclosure relates to a novel method for converting proteins containing biomass generated from renewable resources, such as biological processes designed to recover carbon dioxide emissions and other waste carbon conversion or transformation processes, into protein hydrolysates.

Background Art

[0004] Proteins are nutrients necessary for human and animal growth, and for supporting and maintaining life. Therefore, proteins are an important component of many foods and animal feeds. Furthermore, proteins and protein hydrolysates can serve, for example, as biostimulants or plant nutrients to promote the growth of plants in soil. Additionally, proteins and protein hydrolysates can be used as a nutrient source for the growth of microorganisms. Protein hydrolysates have also been found to stimulate the growth and activity of plant microbiota and beneficial microorganisms in soil. Hydrolyzed proteins (e.g., protein hydrolysates) have been found to be particularly useful as a nutrient source. This process breaks down protein molecules into smaller peptide and amino acid molecules that are more easily digested and absorbed.

[0005] Proteins or biomass-containing proteins produced by microorganisms may be particularly useful for generating protein hydrolysis products for consumption and / or use as biostimulants. Microbial carbon and nitrogen-containing feedstocks can be used to convert proteins or protein-containing biomass. Chemically autotrophic microorganisms can be advantageously used to recover carbon dioxide from the atmosphere, from carbon dioxide emission sources, or from carbon dioxide-producing conversion processes. By generating valuable products from these microbial cultures, it becomes possible to obtain more value from carbon fixation processes.

[0006] Numerous prior art methods exist for hydrolyzing proteins, including various chemical and enzymatic methods. However, these prior art methods are not entirely effective for many reasons, including their inability to hydrolyze consumable or biostimulant products to the desired level, the presence of harmful pesticide residues or by-products in those products, their inability or impracticality to scale up to large-scale commercial processes, and their inability to use highly scalable supply materials such as CO2. [Overview of the Initiative]

[0007] In one embodiment, the present disclosure provides a process for producing bio-derived protein hydrolysis products. In a particular embodiment, the process includes: (1) culturing microorganisms in the presence of a carbon source in an aerobic or microaerobic bioprocess for growing protein-containing biomass, wherein the microorganisms include Cupriavidus necator and the carbon source includes carbon dioxide; (2) collecting the protein-containing biomass into a suspension composition; (3) if the pH of the suspension composition is not in a first target pH range, adjusting the pH of the suspension composition to a first target pH range, thereby forming an alkaline suspension composition, wherein the first target pH range is at least about 10; (4) heating the alkaline suspension composition to a first temperature of at least about 40°C for a first time of at least about 5 minutes; and (5) adding a neutralizing agent to the alkaline suspension composition to form a neutralized suspension composition. The method comprises the steps of (6) optionally adding a protease to the neutralized suspension composition and incubating the neutralized suspension composition at a second temperature range for a second time to hydrolyze the protein, thereby forming a hydrolyzed protein suspension, wherein the second temperature range is at least about 40°C and the second time is at least about 1 hour, and (7) recovering a supernatant containing the hydrolyzed protein from the hydrolyzed protein suspension.

[0008] In some embodiments, the process comprises (a) adjusting the pH of a biomass suspension composition to a pH within a first target pH range of at least about 10, wherein the biomass suspension produced in step (a) or the pH-adjusted suspension composition is an alkaline suspension composition, and (b) heating the alkaline suspension composition at a first temperature of at least about 40°C for a first time of at least about 5 minutes to produce an alkaline hydrolysate suspension containing hydrolyzable microbial proteins.

[0009] In some embodiments, the process further comprises, after step (b), (i) neutralizing the alkaline suspension composition by adding a neutralizing agent to the alkaline suspension composition to form a neutralized suspension composition, wherein the pH of the neutralized suspension composition is within a second target pH range of about 6.5 to about 9.5, and (ii) adding a protease to the neutralized suspension composition at a second temperature of at least about 40°C for a second time of at least about 1 hour to further hydrolyze microbial proteins to form a protease hydrolysate suspension containing hydrolyzed microbial proteins, wherein the process further comprises (c) separating a liquid supernatant from solid material in the alkaline hydrolysate suspension or protease hydrolysate suspension, wherein the supernatant contains soluble hydrolyzed microbial proteins. In certain embodiments, step (b) comprises applying a pressure of at least about 15 psi to the alkaline suspension for at least a portion of a first time. For example, adjusting the pH in step (b) may involve adding one or more bases selected from potassium hydroxide, ammonium hydroxide, ammonia, calcium hydroxide, and sodium hydroxide.

[0010] According to a particular embodiment, the process includes: (1) culturing microorganisms in the presence of a carbon source in an aerobic or microaerobic bioprocess for growing protein-containing biomass, wherein the microorganisms include Cupriavidus necator and the carbon source includes carbon dioxide; (2) collecting the protein-containing biomass into a suspension composition; (3) if the pH of the suspension composition is not within a first target pH range, adjusting the pH of the suspension composition to a first target pH range, thereby forming an acidic suspension composition, wherein the first target pH range is approximately 0.5 or less to approximately 3; (4) heating the acidic suspension composition to a first temperature of at least approximately 40°C for a first time of at least approximately 5 minutes; and (5) adding a neutralizing agent to the acidic suspension composition to form a neutralized suspension composition. The method comprises the steps of: (6) optionally adding a protease to the neutralized suspension composition and incubating the neutralized suspension composition at a second temperature range for a second time to hydrolyze the protein, thereby forming a hydrolyzed protein suspension, wherein the second temperature range is at least about 40°C and the second time is at least about 1 hour; and (7) recovering a supernatant containing the hydrolyzed protein from the hydrolyzed protein suspension.

[0011] In some embodiments, the process includes (a) adjusting the pH of a biomass suspension composition to a pH within a first target pH range of about 0.5 to about 3, as necessary, wherein the biomass suspension produced in step (a) or the pH-adjusted suspension composition is an acidic suspension composition; and (b) heating the acidic suspension composition at a first temperature of at least about 40°C for a first time of at least about 5 minutes to produce an acidic hydrolysate suspension containing hydrolyzable microbial proteins. In some embodiments, the process includes, after step (b), (i) neutralizing the acidic suspension by adding a neutralizing agent to the acidic suspension composition to form a neutralized suspension composition, wherein the pH of the neutralized suspension composition is within a second target pH range of about 5 to about 8; and (ii) adding a protease to the neutralized suspension composition at a second temperature of at least about 40°C for a second time of at least about 1 hour to further hydrolyze microbial proteins to form a protease hydrolysate suspension containing hydrolyzable microbial proteins. In certain embodiments, the process further comprises (c) separating the liquid supernatant from the solid material in the acidic hydrolysate suspension or protease hydrolysate suspension, wherein the supernatant comprises soluble hydrolyzable microbial proteins. In certain embodiments, step (b) comprises applying a pressure of at least about 15 psi to the acidic suspension for at least a portion of a first time. In certain embodiments, adjusting the pH in step (b) may involve adding one or more acids selected from phosphoric acid, sulfuric acid, nitric acid, formic acid, acetic acid, carbonic acid, and hydrochloric acid.

[0012] According to a particular embodiment, the process includes (1) culturing microorganisms to grow biomass, (2) collecting the protein-containing biomass into a suspension composition, (3) optionally adjusting the pH of the suspension composition to at least about 10, preferably about 10 to about 12, (4) optionally adding a chelating agent and / or surfactant to the composition, and (5) heating the composition to at least about 40°C for at least 10 minutes, preferably 40°C to about 130°C for about 10 minutes to about 8 hours, and optionally adding The process includes: (6) pressurizing the composition at a pressure greater than about 15 psi, preferably at a pressure of about 20 psi to about 50 psi, in at least a portion of the heat; (7) adding a neutralizing agent to the composition to adjust the pH to a range of about 7.5 to about 9.5, preferably in the range of about 8.5 to about 9; (8) optionally adding a protease to the composition to further hydrolyze the protein; (9) optionally drying the supernatant and freeze-drying the hydrolyzed protein.

[0013] According to a particular embodiment, the process includes (1) culturing microorganisms to grow biomass, (2) collecting the protein-containing biomass into a suspension composition, (3) adjusting the pH of the suspension composition to about 3 or less, preferably about 1 to about 1.5, if necessary, (4) optionally adding a chelating agent and / or surfactant to the composition, and (5) heating the composition to at least about 40°C for at least 1 minute, preferably 40°C to about 130°C for about 10 minutes to about 8 hours, and optionally adding The process includes: (6) pressurizing the composition at a pressure greater than about 15 psi, preferably at a pressure of about 20 psi to about 50 psi, in at least a portion of the heat; (7) adding a neutralizing agent to the composition to adjust the pH to a range of about 5 to about 7, preferably about 6 to about 6.5; (8) optionally adding a protease to the composition to further hydrolyze the protein; (9) optionally drying the supernatant and freeze-drying the hydrolyzed protein.

[0014] In certain embodiments, any process described herein may further include the formation of hydrolyzable microbial proteins for use as biostimulants. In certain embodiments, any process described herein may include applying hydrolyzable microbial proteins, compositions thereof, or formulations thereof to seeds, plants, or soil, and showing that plants grown in contact with the composition exhibit increased growth in the presence of the composition compared to the absence of the composition.

[0015] Also described herein are protein hydrolysates derived from proteins, including proteins or sources or proteinaceous materials, and methods for producing them. The disclosure includes protein hydrolysate compositions derived from microbial sources and methods for producing them. The protein hydrolysate compositions have a protein-rich organic content. In some embodiments, the protein hydrolysate compositions are substantially free of exogenous chelating agents, chaotropic reagents and / or surfactants. The protein hydrolysate compositions of the disclosure have diverse agricultural or horticultural uses, for example, as biostimulants. The protein hydrolysate compositions of the disclosure may be used for nutritional or pharmaceutical applications in animals and humans. The protein hydrolysate compositions of the disclosure may be used as nutrient sources for cells, including prokaryotic and eukaryotic cells. Sources of protein hydrolysate of the disclosure include microbial sources, for example, photoautotrophic, chemoautotrophic, or oxyhydrogen microbial cultures grown in a bioreactor. The bioreactor may be configured to culture oxyhydrogen microorganisms using carbon waste or low-value sources such as CO2. Therefore, the protein hydrolysate compositions of this disclosure can be sustainably produced from carbon waste or low-value sources such as CO2. The protein hydrolysate compositions can be sustainably produced from other carbon sources, including CO2, CH4, CO and / or greenhouse gases (GHGs) or gases that are pollutants, such as air pollution.

[0016] A method for producing a protein hydrolysis product composition is also disclosed.

[0017] The method includes processing a proteinaceous material in combination with physical, chemical, and / or enzymatic treatment. The proteinaceous material may include cellular biomass such as microbial biomass. The method may include, for example, subjecting a suspension of the proteinaceous material, such as cellular biomass having a pH of about 11.0 or higher, or a pH of less than about 3, to a suitable temperature of at least about 40°C for a suitable time. In some embodiments, the method includes subjecting an alkaline or acidic biomass suspension to overpressure. In certain embodiments, the heat treatment produces an extracted suspension, and the method includes contacting the extracted suspension with a neutralizing buffer to lower the pH to 9.5 or lower (alkaline hydrolysis conditions) or raising the pH to about 6 or higher (acidic hydrolysis conditions) to produce a neutralized suspension, and optionally contacting the neutralized suspension with a protease to produce a protein hydrolysis product composition in a soluble fraction. In some embodiments, the protease is an alkaline protease. In some embodiments, the protease is an acidic protease. In some embodiments, the protease is a metalloprotease.

[0018] In some embodiments, the protein hydrolysis product compositions of the present disclosure have a protein-rich organic content, and the compositions are substantially free of exogenous chelating agents, chaotropic reagents, and surfactants. In certain embodiments, the protein hydrolysis product is of a microbial source. In certain embodiments, the composition is substantially free of sodium and / or chloride. In certain embodiments, the protein hydrolysis product composition has a polypeptide size distribution of 25 kD or less. In some embodiments, the total nitrogen content of the composition is about 5% (w / w) or more. In some embodiments, the phosphate content of the composition is about 5% (w / w) or more. In some embodiments, the potassium content of the composition is about 5% (w / w) or more. In some embodiments, the sodium content of the composition is about 1% (w / w) or less. In some embodiments, the chloride content of the composition is about 1% (w / w) or less.

[0019] In some embodiments, the protein hydrolysis product composition is freeze-dried.

[0020] A method for producing a protein hydrolysis product composition according to the present disclosure comprises adjusting the pH of a biomass suspension to 11.0 or higher to produce an alkaline suspension, and subjecting the alkaline suspension to a temperature of at least about 40°C under conditions sufficient to produce a protein hydrolysis product composition. In some embodiments, the method comprises subjecting the alkaline suspension to overpressure.

[0021] In certain embodiments, the method includes subjecting an alkaline suspension to a temperature of at least about 40°C, neutralizing the suspension to a pH of 9.5 or lower to produce a neutralized suspension, and contacting the neutralized suspension with a protease to produce a protein hydrolysis product composition in the soluble fraction of the suspension. In some embodiments, the protease is an alkaline protease. In some embodiments, the method includes purifying the suspension and freeze-drying the soluble fraction.

[0022] In some embodiments, it includes adjusting the pH by contacting a suspension containing biomass with a base, and the base includes one or more of potassium hydroxide, calcium hydroxide, calcium oxide, ammonium hydroxide or ammonia.

[0023] The method for producing the protein hydrolysate composition of the present disclosure includes adjusting the pH of the biomass suspension to 3 or less to produce an alkaline acidic suspension, and subjecting the alkaline acidic suspension to a temperature of at least about 40°C under conditions sufficient to produce a protein hydrolysate composition. In some embodiments, the method includes subjecting the acidic suspension to overpressure.

[0024] In certain embodiments, the method includes subjecting the acidic suspension to a temperature of at least about 40°C, then neutralizing the suspension to a pH of about 5 or more to produce a neutralized suspension, and contacting the neutralized suspension with a protease to produce a protein hydrolysate composition in the soluble fraction of the suspension. In some embodiments, the protease is an acidic protease. In some embodiments, the method includes purifying the suspension and lyophilizing the soluble fraction.

[0025] In some embodiments, the method includes adjusting the pH by contacting a suspension containing biomass with an acid, and the acid includes phosphoric acid or sulfuric acid.

[0026] In some embodiments, the biomass is microbial biomass.

[0027] In some embodiments, it includes subjecting the microbial biomass suspension to a temperature and / or temperature plus overpressure for a time of about 5 minutes to about 90 minutes.

[0028] In some embodiments, the protein hydrolysate composition is substantially free of exogenous chelating agents, chaotropic reagents, and / or surfactants. In some embodiments, the protein hydrolysate composition comprises an organic inclusion rich in protein. In some embodiments, the protein hydrolysate composition comprises a nitrogen, phosphorus, and potassium (NPK) content of at least 5% (w / w) of each element. In some embodiments, the protein hydrolysate composition is substantially free of sodium and / or chloride.

[0029] In some embodiments, the method comprises separating an insoluble fraction of the suspension from a soluble fraction and extracting a polymer composition from the insoluble fraction. In some embodiments, the polymer composition is a polyhydroxyalkanoate (PHA), such as a polyhydroxybutyrate (PHB) composition.

[0030] Also provided are protein hydrolysate compositions produced using the methods of the present disclosure. Also provided are plant supplements comprising the protein hydrolysate compositions of the present disclosure. The plant supplements are adapted for plants, for example, to promote nutrients and / or growth. The protein hydrolysate compositions of the present disclosure are also used, for example, as nutritional supplements for animals or cells.

[0031] In one embodiment, a method for producing a protein hydrolysis product is provided, the method comprising: culturing microorganisms in the presence of a carbon source in an aerobic or microaerobic bioprocess for growing protein-containing biomass, wherein the microorganisms include Cupriavidus necator and the carbon source includes carbon dioxide; collecting the protein-containing biomass into a suspension composition; if the pH of the suspension composition is not within a first target pH range, adjusting the pH of the suspension composition to a first target pH range, thereby forming an alkaline suspension composition, wherein the first target pH range is at least about 10; heating the alkaline suspension composition to a first temperature of at least about 40°C for a first time of at least about 5 minutes; and adding a neutralizing agent to the alkaline suspension composition to form a neutralized suspension composition. The method comprises the steps of: the pH of the neutralized suspension composition being in a second target pH range, wherein the second target pH range is about 6.5 to about 9.5; adding a protease to the neutralized suspension composition and incubating the neutralized suspension composition at a second temperature range for a second time to hydrolyze the protein, thereby forming a hydrolyzed protein suspension, wherein the second temperature range is at least about 40°C and the second time is at least about 1 hour; and recovering a supernatant containing the hydrolyzed protein from the hydrolyzed protein suspension.

[0032] In one embodiment, extracting proteins from biomass in a suspension composition involves suspending biomass in a biocompatible liquid medium. For example, the biocompatible liquid medium may contain water or a buffer. In some embodiments, the biomass is suspended in the liquid medium by vortexing, homogenization, stirring, or sonication.

[0033] In some embodiments, the first target pH range is at least about 11, or about 10 to about 13, or about 10.5 to about 13, or about 10.5 to about 12.5, or about 10.5 to about 11.5.

[0034] In some embodiments, the step of adjusting the suspension composition to a first target pH range includes adding a base to the suspension composition, the base comprising one or more potassium hydroxide (KOH), ammonium hydroxide (NH4OH), ammonia (NH3), calcium hydroxide (Ca(OH)2), and sodium hydroxide (NaOH), for example, selected from the group consisting of potassium hydroxide (KOH), ammonium hydroxide (NH4OH), ammonia (NH3), calcium hydroxide (Ca(OH)2), and / or sodium hydroxide (NaOH).

[0035] In some embodiments, the second target pH range is at least about 7 to about 9.5, or about 8 to about 9.5, or about 8.5 to about 9.5, or about 9 to about 9.5.

[0036] In some embodiments, the neutralizing agent comprises one or more potassium phosphate, ammonium phosphate, sodium citrate, citric acid, sodium phosphate, phosphoric acid, phosphate buffer, Tris, HEPES, glycine, carbon dioxide, bicarbonate, and carbonic acid, for example, comprising potassium phosphate, ammonium phosphate, sodium citrate, sodium citrate phosphate, phosphoric acid, phosphate buffer, Tris, HEPES, glycine, carbon dioxide, bicarbonate, and / or carbonic acid, selected from the group consisting of potassium phosphate, ammonium phosphate, phosphoric acid, phosphate buffer, sodium citrate, citric acid, sodium phosphate, Tris, HEPES, glycine, carbon dioxide, bicarbonate, and carbonic acid.

[0037] In one embodiment, a method for producing a protein hydrolysis product is provided, the method comprising: culturing microorganisms in the presence of a carbon source in an aerobic or microaerobic bioprocess for growing protein-containing biomass, wherein the microorganisms include Cupriavidus necator and the carbon source includes carbon dioxide; collecting the protein-containing biomass into a suspension composition; if the pH of the suspension composition is not within a first target pH range, adjusting the pH of the suspension composition to a first target pH range, thereby forming an acidic suspension composition, wherein the first target pH range is approximately 0.5 or less to approximately 3; heating the acidic suspension composition to a first temperature of at least approximately 40°C for a first time of at least approximately 5 minutes; and adding a neutralizing agent to the acidic suspension composition to form a neutralized suspension composition, wherein the neutralized suspension The method comprises the steps of: the pH of the composition being in a second target pH range, wherein the second target pH range is about 5 to about 6.5 or about 8; optionally adding a protease to the neutralized suspension composition and incubating the neutralized suspension composition at a second temperature range for a second time to further hydrolyze the protein suspension, thereby forming a hydrolyzed protein suspension, wherein the second temperature range is at least about 40°C and the second time is at least about 1 hour; and recovering a supernatant containing the hydrolyzed protein from the hydrolyzed protein suspension.

[0038] In one embodiment, extracting proteins from biomass in a suspension composition involves suspending the biomass in a biocompatible liquid medium. For example, the biocompatible liquid medium may contain water or a buffer. In some embodiments, the biomass is suspended in the liquid medium by vortexing, homogenization, stirring, or sonication.

[0039] In some embodiments, the first target pH range is approximately 3 or less or approximately 0.5 to approximately 3.

[0040] In some embodiments, adjusting the pH of the suspension composition to a first target pH range involves adding an acid to the suspension composition, the acid comprising one or more phosphoric acid (H3PO4), sulfuric acid (H2SO4), nitric acid (HNO3), formic acid (HCOOH), acetic acid (CH3COOH), hydrochloric acid (HCl), and carbonic acid, such as carbon dioxide (CO2).

[0041] In some embodiments, the second target pH range is at least about 73 to about 6, or about 3 to about 9.

[0042] In some embodiments, the neutralizing agent comprises one or more calcium hydroxide (Ca(OH)2), calcium oxide, calcium carbonate (CaCO3), potassium hydroxide (KOH), a phosphate buffer, ammonium bicarbonate, ammonium carbonate, ammonium hydroxide (NH4OH), and ammonia.

[0043] In some embodiments, the protein hydrolysis product is used as a biostimulant and / or phytonutrient (e.g., a nutrient necessary or beneficial in plant growth and / or metabolism) or a precursor thereof, and the step of adjusting the pH of the suspension composition to a first target pH range includes adding a base (alkaline hydrolysis conditions) or an acid (acid hydrolysis conditions) to the suspension composition, wherein the base or acid contains one or more phytonutrients selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K), for example, nitrogen (N), phosphorus (P), and / or potassium (K). In some embodiments, the protein hydrolysis product is used as a biostimulant and / or phytonutrient or a precursor thereof, and the step of adjusting the pH of the suspension composition to a first target pH range includes adding a base or an acid to the suspension composition, wherein the base or acid does not contain any elements harmful to plant growth. In some embodiments, the protein hydrolysis product is used as a biostimulant and / or plant nutrient or precursor thereof, and the step of adjusting the pH of the suspension composition to a first target pH range includes adding a base or acid to the suspension composition, wherein the base does not contain levels of sodium or chloride that would inhibit plant growth.

[0044] In some embodiments, the method further includes the step of adding a chelating agent and / or surfactant to the suspension composition, after adjusting the pH of the suspension composition to a first target pH range. For example, a chelating agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) may be added, for example, ethylenediaminetetraacetic acid (EDTA) and / or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments, the amount of chelating agent added to the suspension composition is in the range of about 0.1 mM to about 10 mM, or about 0.5 mM to about 8 mM, or about 1 mM to about 7 mM, or about 3 mM to about 5 mM. In some embodiments, the protein hydrolysis product is used as a biostimulant and / or plant nutrient or precursor thereof, and the chelating agent and / or surfactant contains one or more of nitrogen (N), phosphorus (P), and potassium (K), for example, a plant nutrient selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K). In some embodiments, the chelating agent and / or surfactant does not contain any elements harmful to plant growth. For example, a surfactant selected from the group consisting of sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20, and / or Pluronic PF-68 may be added, comprising one or more of the following: sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20, and / or Pluronic PF-68.In some embodiments, the amount of surfactant added to the suspension composition relative to the dry weight of the biomass in the suspension is in the range of about 1% to about 25%, or about 2% to about 20%, or about 4% to about 15%, or about 5% to about 12%, or about 8% to about 12%. In some embodiments, the surfactant is nontoxic to plants or animals and / or biodegradable.

[0045] In some embodiments, the alkaline or acidic suspension composition is heated to a first temperature of about 40°C to about 150°C for a first time of about 5 minutes to about 24 hours. For example, the first temperature may be at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, at least about 90°C, at least about 100°C, at least about 105°C, at least about 110°C, about 40°C to about 150°C, about 60°C to about 150°C, about 70°C to about 140°C, about 90°C to about 135°C, about 100°C to about 130°C, about 100°C to about 125°C, about 105°C to about 125°C, or about 110°C to about 125°C. For example, the first time could be at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 3 hours, at least about 5 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, about 5 minutes to about 90 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 70 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 50 minutes, about 10 to about 40 minutes, about 10 to about 30 minutes, about 20 to about 60 minutes, about 30 to about 60 minutes, about 1 hour to about 24 hours, about 1 hour to about 18 hours, about 1 hour to about 12 hours, or about 1 hour to about 8 hours. In some embodiments, the step of heating the alkaline or acidic suspension composition to a first temperature is performed on the alkaline or acidic suspension under pressure. For example, the pressure of the alkaline or acidic suspension for at least part of the first time may be at least about 15 psi, at least about 20 psi, at least about 25 psi, at least about 30 psi, at least about 35 psi, at least about 40 psi, at least about 45 psi, about 15 psi to about 50 psi, about 15 psi to about 40 psi, about 15 psi to about 35 psi, about 15 psi to about 30 psi, about 15 psi to about 25 psi, or about 15 psi to about 20 psi.In some embodiments, the pressure of the alkaline or acidic suspension in at least part of a first time is at least about 15 psi, at least about 20 psi, at least about 25 psi, at least about 30 psi, at least about 35 psi, at least about 40 psi, at least about 45 psi, about 15 psi to about 50 psi, about 15 psi to about 40 psi, about 15 psi to about 35 psi, about 15 psi to about 30 psi, about 15 psi to about 25 psi, or about 15 psi to about 20 psi, and the at least part of the first time is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0046] In some embodiments, the protein hydrolysis product is used as a biostimulant and / or a phytonutrient or its precursor, and the neutralizer includes a phytonutrient selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K), for example, nitrogen (N), phosphorus (P), and potassium (K). In some embodiments, the protein hydrolysis product is used as a biostimulant and / or a phytonutrient or its precursor, and the neutralizer does not contain any elements harmful to plant growth.

[0047] In some embodiments, the method includes a further step of adding a chaotropic reagent to the neutralized suspension composition. In some embodiments, the chaotropic reagent is added before or together with the protease, thereby allowing the protease hydrolysis to occur in the presence of the chaotropic reagent. For example, the chaotropic reagent may include one of urea, thiourea, and guanidinium chloride, e.g., urea, thiourea and / or guanidinium chloride, e.g., selected from the group consisting of urea, thiourea and guanidinium chloride. In some embodiments, the chaotropic reagent is added in amounts ranging from about 0.1 M to about 2 M, about 0.5 M to about 1.5 M, or about 0.8 M to about 1.2 M. In some embodiments, the protein hydrolysis product is used as a biostimulant and / or plant nutrient or its precursor, and the chaotropic reagent contains one or more of nitrogen (N), phosphorus (P), and potassium (K), for example, nitrogen (N), phosphorus (P), and / or potassium (K), or a plant nutrient selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K). In some embodiments, the protein hydrolysis product is used as a biostimulant and / or plant nutrient or its precursor, and the chaotropic reagent does not contain any elements harmful to plant growth.

[0048] In some embodiments, the protease comprises one of endoprotease, exoprotease, alkaline protease, serine alkaline protease, bacterial alkaline protease, and subtilisin A, for example, comprising endoprotease, exoprotease, alkaline protease, serine alkaline protease, bacterial alkaline protease, and subtilisin A, selected from the group consisting of endoprotease, exoprotease, alkaline protease, serine alkaline protease, bacterial alkaline protease, and subtilisin A. In other embodiments, the protease comprises an acidic protease. In further embodiments, the protease comprises a metalloprotease.

[0049] In some embodiments, the second temperature range is approximately 40°C to 70°C, approximately 45°C to 65°C, or approximately 50°C to 60°C.

[0050] In some embodiments, the second time is a minimum of about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 18 hours, or about 24 hours, and a maximum of about 4 hours, about 8 hours, about 12 hours, about 24 hours, about 36 hours, or about 48 hours.

[0051] In some embodiments, at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% of the polypeptide in the recovered supernatant has an atomic mass of less than about 25 kD, less than about 20 kD, less than about 15 kD, less than about 10 kD, less than about 5 kD, less than about 3 kD, less than about 2 kD, about 0.1 kD to about 30 kD, about 1 kD to about 30 kD, about 1 kD to about 25 kD, about 5 kD to about 25 kD, about 0.1 kD to about 2 kD, about 0.1 kD to about 5 kD, about 0.1 kD to about 10 kD, or about 5 kD to about 20 kD.

[0052] In some embodiments, the method further includes a further step of purifying the suspension by centrifugation or filtration to remove undissolved material from the hydrolyzed protein suspension.

[0053] In some embodiments, the method further includes a further step of purifying the suspension or solution to remove molecules above a specific molecular weight (MW) cutoff, for example, by filtration (e.g., ultrafiltration). In such embodiments, the molecular weight cutoff may be greater than about 20 kD, greater than about 15 kD, greater than about 10 kD, greater than about 5 kD, or greater than about 2 kD.

[0054] In some embodiments, the method further includes the steps of drying the recovered supernatant and freeze-drying the hydrolyzed protein. For example, the freeze-dried protein hydrolyzed product composition may have a water content of about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, or about 2% to about 5%.

[0055] In some embodiments, the method further includes (1) adjusting the pH of the suspension composition to a first target pH range, followed by adding a chelating agent and / or surfactant and / or chaotropic reagent to the suspension composition; (2) adding the chaotropic reagent to the neutralized suspension composition; and (3) removing substantially all of the surfactant, chelating agent and / or chaotropic reagent after the formation of a hydrolyzed protein suspension. In some embodiments, the surfactant, chelating agent and / or chaotropic reagent are removed by gel filtration chromatography, membrane filtration, and / or dialysis.

[0056] In some embodiments, at least a portion of the protein is hydrolyzed by heating an alkaline or acidic suspension composition to a first temperature for a first time.

[0057] In some embodiments, the suspension composition includes a lysate.

[0058] In some embodiments, the step of extracting proteins from biomass into a suspension composition includes subjecting the biomass to dissolution. [Brief explanation of the drawing]

[0059] [Figure 1A] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 1B] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 2] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 3A] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 3B] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 4] This is a schematic diagram of a method for producing a protein hydrolysis product according to embodiments of the present disclosure. [Figure 5] This is a schematic diagram of an integrated process for producing protein hydrolysis products from microbial culture systems. [Figure 6] This document shows the protein gel analysis of protein hydrolysis products prepared according to embodiments of this disclosure. [Figure 7] This shows the protein gel analysis of the fraction of protein hydrolysis products that were not subjected to the enzymatic hydrolysis process. [Figure 8A] This document shows the protein gel analysis of protein hydrolysis products prepared according to embodiments of this disclosure. [Figure 8B] This document shows the protein gel analysis of protein hydrolysis products prepared according to embodiments of this disclosure. [Figure 9] This document shows the protein gel analysis of protein hydrolysis products prepared according to embodiments of this disclosure. [Figure 10] From left to right: (a) Turnips treated with water, (b) NPK fertilizer, and (c) Protein hydrolysate. [Figure 11] From left to right: (a) Lettuce treated with commercial fish and seaweed hydrolysates, (b) acid hydrolysates, and (c) base hydrolysates. [Modes for carrying out the invention]

[0060] Methods for producing protein hydrolysis products and hydrolysates are provided herein. For example, protein hydrolysis products can be produced from microorganisms as described herein.

[0061] Unless otherwise defined herein, all technical and academic terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. Singleton, et al., Dictionary of Microbiology and Molecular Biology, seconded., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, HarperPerennial, NY (1991) provide one of the techniques along with a general dictionary of many of the terms used herein. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the methods, systems, and compositions described herein.

[0062] In the practice of the present invention, unless otherwise specified, the prior art of those skilled in molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry is used. Such techniques are all described in literature such as, for example, Molecular Cloning: ALAboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Current Protocols in Molecular Biology (FMAusubeletal., eds., 1994); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transfer and Expression: ALAboratory Manual (Kriegler, 1990).

[0063] The numerical ranges provided herein include the numbers that define those ranges.

[0064] Unless otherwise specified, nucleic acids are written from left to right, in the 5' to 3' direction, and amino acid sequences are written from left to right, in the amino to carboxy direction.

[0065] I. Definition As used herein and in the claims, “a,” “an,” and “the” refer to multiple things unless the context clearly indicates otherwise; therefore, as used herein and in the claims, the indefinite articles “a,” “an,” and “the” should be understood to mean “at least one” unless the context clearly indicates otherwise.

[0066] As used herein, the term “about” when referring to measurable values ​​such as quantity, duration, etc., means that an error of ±5%, ±1%, or ±0.1% from a particular value is acceptable for carrying out the disclosed method or is relevant to the disclosed composition.

[0067] "Acetogen" refers to microorganisms that produce acetates and / or short-chain organic acids with a maximum C4 chain length as products of anaerobic respiration.

[0068] "Eosinophils" refer to a type of extremophilic bacteria that grows under highly acidic conditions (usually pH 2.0 or lower).

[0069] The term “amino acid” means a molecule containing both an amine group and a carboxyl group bonded to a carbon, indicated as the alpha-carbon. Preferred amino acids include, but are not limited to, both D-isomers and L-isomers of native amino acids, as well as unnatural forms prepared by organic synthesis or other metabolic routes. In some embodiments, a single “amino acid” may have multiple side chain moieties present per extended aliphatic or aromatic backbone. As used herein, the term amino acid is intended to include amino acid analogs unless the context clearly indicates otherwise.

[0070] As used herein and in the appended claims, the “and / or” phrase should be understood to mean “one or both” of the linked elements, i.e., elements that are sometimes constitutively and sometimes disjunctly. Other elements may exist, either relating to or unrelated to the elements specifically identified by the “and / or” phrase, unless explicitly stated otherwise.

[0071] Therefore, as a non-restrictive example, when used with open-ended language such as "including", a reference to "A and / or B" may mean, in one embodiment, A excluding B (optionally including elements other than B), in another embodiment, B excluding A (optionally including elements other than A), and in yet another embodiment, both A and B (optionally including other elements), and so on.

[0072] The term "biomass" refers to the material produced by the growth and / or reproduction of cells. Biomass may include, but is not limited to, cells and / or intracellular contents, as well as extracellular material, including compounds secreted by cells.

[0073] The term “bioreactor” or “fermenter” refers to a sealed or partially sealed container for growing and maintaining cells. While cells are held in a liquid suspension, this is not essential. In some embodiments, instead of being held in a liquid suspension, cells may instead be in contact with, and / or maintained in, or within, other non-liquid substrates, including but not limited to solid growth support materials.

[0074] "Bio-stimulant" or "biostimulant" means a compound that can stimulate plant growth and development, for example, increase and enhance the microbial activity of agricultural crops and soil. The term means any substance that may be applied to plants, seeds, soil, and growing media and that enhances the efficiency of plant nutrient use or provides other direct or indirect benefits to plant development or stress responses.

[0075] The term “carbon fixation” refers to an enzymatic reaction or metabolic pathway that converts carbon (including, but not limited to, CO2, CO, and CH4) in gaseous form under ambient conditions into carbon-based biochemicals that are liquid or solid under ambient conditions, or dissolved or retained in suspensions, aqueous solutions.

[0076] "Carbon source" refers to the type of molecule that microorganisms use to induce the carbon necessary for organic biosynthesis.

[0077] "Carboxidotrophic" refers to microorganisms that are resistant to or can oxidize carbon monoxide. In preferred embodiments, carboxydotrophic microorganisms can utilize CO as a carbon source and / or as a source to reduce electrons for biosynthesis and / or respiration. "Chemical autotrophic" refers to organisms that obtain energy through the oxidation of chemical electron donors by chemical electron acceptors and synthesize all the organic compounds that the organism needs for survival and growth from carbon dioxide.

[0078] In the claims and specification, all conjunctions such as “comprising,” “including,” “carrying,” “having,” “containing,” “involved,” and “holding” are understood to be open-ended, meaning they include but are not limited to them. Only the conjunctions “consisting of” and “essentially consisting of” are considered closed or semi-closed conjunctions, respectively.

[0079] The term "culture" refers to growing a population of cells, such as microbial cells, in a liquid or solid culture medium under conditions suitable for growth.

[0080] The term "derived from" encompasses the terms "originating from," "obtained from," "available from," "isolated from," and "created from," and generally indicates that the origin of a particular material is found in another particular material, or that it possesses characteristics that can be described by referring to another particular material.

[0081] "Energy source" refers to either an electron donor oxidized by oxygen during aerobic respiration, or a combination of an oxidized electron donor and an electron acceptor reduced during anaerobic respiration.

[0082] "Extremophilic bacteria" refers to microorganisms that can grow under extreme physical or geochemical conditions (e.g., high or low temperatures, high pH, ​​or high salinity) compared to the conditions on the Earth's surface or in the oceans that most organisms found on or near the Earth's surface can generally tolerate.

[0083] The term "vaporization" refers to a generally high-temperature process that converts carbon-based materials into a mixture of gases, including hydrogen, carbon monoxide, and synthesis gas, which also includes carbon dioxide, syngas, or generator gas. This process generally involves partial combustion and / or the application of externally generated heat along with controlled oxygen addition, and / or vapor where there is insufficient oxygen for the complete combustion of the carbon-based material.

[0084] "Halophilic bacteria" refers to extremophilic bacteria that grow in environments with very high salt concentrations.

[0085] "Chemical autotrophy" refers to organisms that cannot synthesize all the organic compounds they need for survival and growth from carbon dioxide, and must utilize organic compounds for growth. Heterotrophic organisms cannot produce their own food; instead, they obtain it by ingesting food and energy from plants or animals and metabolizing organic matter, rather than fixing carbon from non-organic sources such as carbon dioxide.

[0086] "Hydrogen-oxidizing agent" refers to microorganisms that use H2 reduced during intracellular reduction equivalent production and / or respiration as an electron donor.

[0087] "Hyperthermophiles" refer to a type of extremophilic bacteria that survives in extremely hot environments, typically growing at temperatures of around 60°C (140°F) or higher.

[0088] The term "knallgas" refers to a mixture of molecular hydrogen and oxygen gas. "Knallgas microorganisms" refers to microorganisms that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration to produce intracellular energy carriers such as adenosine 5'-triphosphate (ATP). The terms "oxyhydrogen" and "oxyhydrogen microorganisms" can be used synonymously with "knallgas" and "knallgas microorganisms," respectively. Knallgas microorganisms generally use hydrogen via hydrogenase to produce NAD. + They use some electrons donated from H2 to reduce (and / or other intracellular reducing equivalents), and some electrons from H2 used for aerobic respiration. Knallgas microorganisms autotrophically fix CO2 through pathways that commonly include, but are not limited to, the Calvin cycle or the reverse citric acid cycle. ["Thermophilic bacteria", Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0089] The term "lysate" means a liquid containing a mixture and / or a solution of cell-containing substances obtained from cell lysis. In some embodiments, the methods described herein include purifying a chemical substance or mixture of chemical substances within a cell lysate. In some embodiments, the methods include purifying amino acids and / or proteins within a cell lysate.

[0090] The term "lysis" means the rupture of the plasma membrane and, if present, the cell wall of a cell, thereby releasing a substantial amount of intracellular material into the extracellular space. Lysis can be carried out by electrochemical, mechanical, osmotic, thermal, or viral methods. In some embodiments, the methods described herein involve carrying out the lysis of cells or microorganisms in accordance with the description herein in order to separate a chemical substance or mixture of chemical substances from the contents of a bioreactor. In some embodiments, the methods involve carrying out the lysis of cells or microorganisms in accordance with the description herein in order to separate amino acids or mixtures of amino acids and / or proteins from a bioreactor or cell growth medium.

[0091] "Methanogenic bacteria" refers to microorganisms that produce methane as a by-product of anaerobic respiration.

[0092] "Methylotrope" refers to a microorganism that can use a reduced one-carbon compound, such as methanol or methane, as a carbon source and / or electron donor for its growth.

[0093] The terms "microorganism" and "microbe" refer to tiny, single-celled organisms.

[0094] "Mixed nutrition" refers to organisms that can utilize a mixture of different energy sources and carbon (e.g., H2 and sugars).

[0095] The term "molecule" means any obvious or distinguishable structural unit of a substance that contains one or more atoms, including, for example, hydrocarbons, lipids, polypeptides, and polynucleotides.

[0096] "Oligopeptides" refer to peptides containing a relatively small number of amino acid residues, such as approximately 2 to 20 different amino acids.

[0097] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as set forth above. For example, when dividing items within a list, “or” or “and / or” should be interpreted as inclusive, that is, including a list of numbers or elements that include at least one, but two or more, and any additional items not included in any additional list. Only terms that clearly indicate contrasting meanings, such as “one of” or “exactly one,” or, when used in the claims, “consisting of,” would mean that only one element of the list of numbers or elements is included. In general, where there are terms of exclusivity such as “either,” “one of,” “only one of,” “exactly one,” or “essentially consisting of,” the term “or” should be interpreted only as indicating an exclusive choice ("one of the two but not both"), and where used in the claims, it should have the usual meaning as used in the field of patent law.

[0098] The term "organic compound" refers to any chemical compound containing carbon atoms in gaseous, liquid, or solid form, with the exception of the following, which are considered non-organic: carbides, carbonates, monocarbonate, cyanides, and allotropes of pure carbon such as diamond and graphite.

[0099] A "peptide" refers to a chain of two or more amino acids, for example, approximately 2 to 50 amino acids, where the carboxyl group of each amino acid is connected to the adjacent amino group by an R-OC-NH-R' type bond.

[0100] As used herein, the term “polynucleotide” means a macromolecular form of nucleotides of any length and any three-dimensional structure, and of single-stranded or multi-stranded (e.g., single-stranded, double-stranded, triple-stranded, etc.), and includes deoxyribonucleotides, ribonucleotides, and / or modified nucleotides or bases or analogs thereof, including deoxyribonucleotides or analogs or modified forms of ribonucleotides. Because the genetic information is denatured, two or more codons may be used to encode a particular amino acid, and the present invention encompasses polynucleotides that encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, as long as the polynucleotide retains the desired functionality under the conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, phosphorothioates). Labels may also be combined for detection or recovery purposes, e.g., radioactive or non-radioactive labels or anchors (e.g., biotin). The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides may be native or unnatural. In this specification, the terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably. Polynucleotides may include RNA, DNA, or both and / or modified forms and / or analogues. The sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphate diester bonds may be replaced by alternative linking groups. Alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR.substituted.2 ("amidate"), P(O)R, P(O)OR', CO or CH.substituted.2 ("formate acetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), and optionally an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in polynucleotides need to be identical. Polynucleotides can be linear or cyclic, or contain a combination of linear and cyclic parts.

[0101] As used herein, “polypeptide” means a composition comprising amino acids, which is recognized as a protein to those skilled in the art. Conventional one-letter or three-letter codes are used herein for amino acid residues. The terms “polypeptide” and “protein” are used interchangeably herein to mean a polymer of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, or may be interrupted with non-amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified by, for example, disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or other operations or modifications (such as conjugation with a labeling component). The definition also includes, for example, the inclusion of one or more analogues of amino acids (e.g., non-natural amino acids), and other modifications well known in the art.

[0102] The term “precursor to” or “precursor to” refers to an intermediate for the manufacture of one or more components of the final product.

[0103] "Generator gas" typically refers to a gas mixture containing H2, CO, and CO2 in varying proportions, with a calorific value ranging from half to one-tenth that of natural gas per unit volume under standard conditions. Generator gas can be produced in various ways from a variety of feed materials, including vaporization, steam reforming, or automated reforming of carbon-based feed materials. In addition to H2, CO, and CO2, generator gas may contain other components, including but not limited to methane, hydrogen sulfide, condensable gases, tar, and ash, depending on the production process and feed materials. The proportion of N2 in the mixture can be high or low depending on whether air was used as an oxidizer in the reactor and whether the heat in the reactor was provided through direct combustion or indirect heat exchange.

[0104] The term "manufacture" includes both intracellular and extracellular production of compounds, as well as secretion of compounds from cells.

[0105] "Psyrophilic bacteria" refers to a type of extremophilic bacteria that can grow and reproduce at low temperatures, typically below approximately 10°C.

[0106] As used herein, the terms “recovery,” “isolation,” “purification,” and “separation” mean material removed from at least one naturally bound component (e.g., protein, nucleic acid, or cell). For example, these terms may mean material that is substantially or essentially devoid of components that would normally be present in its natural state, such as in a pristine ecosystem.

[0107] The phrase "substantially absent" any given component means that, if present, the component is present in a functionally negligible amount, i.e., does not significantly negatively impact the intended performance or the functionality of any process or product. Typically, substantially absent means that the component is present in less than approximately 1% by weight (including less than approximately 0.5% by weight, less than approximately 0.1% by weight, and zero% by weight).

[0108] "Sulfur-oxidizing agent" refers to a microorganism that uses a reducing sulfur compound containing H2S as an electron donor for intracellular reducing equivalent and / or production in respiration.

[0109] "Syngas" or "synthesis gas" refers to a type of gas mixture, similar to a generator gas containing H2 and CO, but more specifically formulated in terms of H2 and CO content and ratio, and the level of impurities for the synthesis of certain types of chemical products, such as methanol or Fischer-Tropsch diesel, but not limited to these. Syngas generally contains H2, CO, and CO2 as its main components and can be produced by established methods including steam reforming of methane, liquid petroleum gas, or biogas, or by vaporization of any organic, combustible carbon-based material, including but not limited to biomass, waste organic matter, various polymers, peat, and coal. The hydrogen component of syngas can be increased through the reaction of vapor and CO during water-gas conversion reactions, and consequently, the CO2 in the syngas mixture increases.

[0110] "Thermophiles" refer to a type of extremophilic bacteria that grow at relatively high temperatures for survival, typically between approximately 45°C and 122°C.

[0111] "Wild type" refers to naturally occurring microorganisms.

[0112] "Yield" refers to the amount of product produced from a supply material (e.g., sugar) relative to the amount of product that would be produced if all the supply material were converted into the product. For example, amino acid yield is expressed as the percentage of amino acids produced relative to the theoretical yield if 100% of the supply material were converted into amino acids.

[0113] II. Method In general terms, the methods of this disclosure may include increasing or decreasing the pH of a proteinaceous suspension of microbial biomass, such as a biomass suspension, for example, a suspension of biomass produced by the growth of chemically autotrophic microorganisms.

[0114] The initial biomass suspension may contain a suitable amount of biomass in the culture medium, for example, microbial biomass in the growth medium. In some embodiments, the amount of biomass (dry weight / reaction volume (w / v)) is about 0.1% or more, for example, about 0.2% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, and about 4% or more. In some embodiments, the amount of biomass (dry weight / reaction volume (w / v)) is about 8% or less, about 6% or more, and about 5% or less, and in some embodiments, each of the aforementioned ranges of biomass may be at least about 0.01%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 2%, or at least about 3%. In some embodiments, the amount of biomass (dry weight / reaction volume (w / v)) is in the range of about 0.1% to about 8%, for example, about 0.2% to about 8%, about 0.5% to about 6%, about 1% to about 6%, about 2% to about 6%, and includes about 3% to about 5%.

[0115] In some embodiments, cells in the biomass are subjected to dissolution at the beginning of the process, for example, before the pH is increased or decreased, to facilitate the extraction of proteins from the biomass into a suspension composition.

[0116] The alkaline or acidic suspension is subjected to heating for a suitable time to produce a protein hydrolysis product composition. The suspension may be concentrated, dried (e.g., lyophilized), or used directly as a liquid suspension. In certain embodiments, the alkaline or acidic suspension is subjected to heating and high pressure, for example, by autoclaving the alkaline or acidic suspension, to produce a protein hydrolysis product composition. In some embodiments, after heating / pressure treatment, the suspension is neutralized with a buffer.

[0117] In certain embodiments, the pH is sufficiently lowered (alkaline suspension) or raised (acidic suspension), and then the suspension is enzymatically treated with a protease. After enzymatic hydrolysis, a protein hydrolysis product composition is produced.

[0118] In certain embodiments, the hydrolyzed proteins in the protein hydrolysis product are mainly located in the soluble fraction of the suspension. The resulting suspension can be purified, for example, by centrifugation to obtain a supernatant fragment containing the hydrolyzed proteins. A. Alkaline hydrolysis

[0119] Referring to Figures IA and 1B, the methods of the present disclosure may include a suspension of cellular biomass as a starting material. The suspension may be obtained by suspending (e.g., by homogenization) the cellular biomass in water or a suitable liquid medium such as a buffer or culture (growth) medium. Any preferred method for suspending the biomass in a medium may be used, including but not limited to vortexing, homogenization, stirring, sonication, etc. The biomass may be dry biomass (e.g., lyophilized biomass) or wet biomass before being suspended in the medium.

[0120] The pH of the biomass suspension can be increased by 110 or 130 by adding a base to the suspension. In some embodiments, the pH of the suspension is increased to about 10.0 or higher, for example, about 11 or higher, about 11.1 or higher, about 11.2 or higher, about 11.3 or higher, about 11.4 or higher, about 11.5 or higher, about 11.6 or higher, about 11.7 or higher, about 11.8 or higher, about 11 or higher, and about 12.0 or higher.

[0121] In some embodiments, the pH of the suspension is raised to about 10.0 to about 14.0, for example, about 11.0 to about 14.0, about 10.0 to about 13.0, about 10.5 to about 13.0, about 10.5t to about 12.5, about 10.5 to about 11.5, about 11.0 to about 13.5, about 11.0 to about 13.0, about 11.0 to about 12.5, about 11.0 to about 12.0, about 11.5 to about 13.0, about 11.5 to about 12.5, about 11.5 to about 12.4, about 11.6 to about 12.4, about 11.7t to about 12.4, and about 11.8 to about 12.3. Any preferred base may be used to raise the pH of the suspension. Suitable bases include, but are not limited to, potassium hydroxide (KOH), ammonium hydroxide (NH4OH), ammonia (NH3), calcium hydroxide (Ca(OH)2), and / or sodium hydroxide (NaOH). In certain embodiments, the base is KOH. In certain embodiments, one or more bases containing plant nutrients are used, including, but are not limited to, nitrogen (N), phosphorus (P), and / or potassium (K). In certain embodiments, bases containing elements that inhibit plant growth are avoided, including, but are not limited to, sodium (Na).

[0122] The alkaline biomass suspension may be subjected to high temperatures of 120, 140. In some embodiments, the suspension is subjected to temperatures of about 40°C or higher, for example, about 50°C or higher, about 60°C or higher, about 70°C or higher, about 80°C or higher, about 90°C or higher, about 100°C or higher, about 105°C or higher, about 110°C or higher, about 11.8°C or higher, and about 121°C or higher. In some embodiments, the suspension is subjected to temperatures of about 40°C to about 150°C, for example, about 60°C to about 150°C, about 70°C to about 140°C, about 80°C to about 140°C, about 90°C to about 135°C, about 100°C to about 130°C, about 100°C to about 125°C, about 105°C to about 125°C, and about 10°C to about 125°C. In some embodiments, the suspension is subjected to a temperature of about 110°C. The suspension may be subjected to high temperatures using any preferred method.

[0123] In some embodiments, the alkaline biomass suspension is subjected to high pressure of 120, 140 (i.e., at least 14.7 psi). In some embodiments, the suspension is subjected to pressures of 15 psi or more, e.g., about 20 psi or more, about 25 psi or more, about 30 psi or more, about 35 psi or more, or about 40 psi or more, and in some embodiments, to pressures of about 40 psi or less, e.g., about 30 psi or less, about 25 psi or less, about 20 psi or less, about 18 psi or less, or about 15 psi or less. In some embodiments, the suspension is subjected to pressures in the range of about 14.7 psi to about 40 psi, e.g., about 15 psi to about 40 psi, about 15 psi to about 35 psi, about 15 psi to about 30 psi, about 15 psi to about 25 psi, or about 15 psi to about 20 psi. The suspension may be subjected to high temperature and high pressure using any preferred method. In some embodiments, the suspension is autoclaved to increase the temperature and pressure.

[0124] In some embodiments, the suspension is subjected to high pressure and exposed to high temperature for only a portion of the time. In some embodiments, the suspension is subjected to high pressure for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the time it is exposed to high temperature.

[0125] The alkaline biomass suspension may be subjected to high temperature, or high temperature and pressure, for a suitable period of time. In some embodiments, the suspension is subjected to high temperature, or high temperature and pressure, for about 5 minutes or more, for example, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, or about 25 minutes or more, and in some embodiments, each of the above times may be capped at about 90 minutes, about 80 minutes, about 70 minutes, about 60 minutes, about 50 minutes, about 40 minutes, or about 30 minutes. In some embodiments, the suspension is subjected to high temperature, or high temperature and pressure, for a period of time including about 5 minutes to about 90 minutes, for example, about 5 minutes to about 80 minutes, about 5 minutes to about 70 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 40 minutes, 10 minutes to about 30 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to 60 minutes. In some embodiments, the suspension is subjected to high temperature, or high temperature and high pressure, for a period of time including about 1 hour or more, for example, about 3 hours or more, about 5 hours or more, or about 8 hours or more, and in some embodiments, each of the above periods may be capped at about 24 hours, about 18 hours, about 12 hours, or about 8 hours. In some embodiments, the suspension is subjected to high temperature, or high temperature and high pressure, for a period of time including about 1 hour to about 24 hours, for example, about 1 hour to about 18 hours, about 1 hour to about 12 hours, or about 1 hour to about 8 hours.

[0126] In some embodiments, the pH of the suspension is reduced by adding a neutralizing agent, such as a neutralizing buffer, after heating and heat treatment.150 In some embodiments, the pH of the suspension is reduced to about 6.5 or higher, for example, including about 6.7 or higher, about 6.9 or higher, about 7.0 or higher, about 7.1 or higher, about 7.2 or higher, about 7.3 or higher, about 7.4 or higher, about 7.5 or higher, about 7.6 or higher, about 7.7 or higher, about 7.8 or higher, about 7.9 or higher, and about 8.0 or higher, and in some embodiments, each of the aforementioned pH ranges is capped at 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5.

[0127] In some embodiments, the pH is reduced to a range of about 6.5 to about 9.5, for example, including about 7.0 to about 9.5, about 7.3 to about 9.4, about 7.5 to about 9.4, about 7.7 to about 9.3, about 7.8 to about 9.2, about 7.9 to about 9.1, about 8.0 to about 9.0, and about 8.5 to about 9.0.

[0128] The pH can be lowered using any of the preferred neutralizing agents. Preferred neutralizing agents include, but are not limited to, potassium phosphate buffers, ammonium phosphate buffers, sodium citrate buffers, sodium phosphate buffers, Tris buffers, HEPES buffers, and / or glycine buffers. In some embodiments, the neutralizing agent is potassium phosphate buffer. In some embodiments, the neutralizing agent includes, but is not limited to, ammonium phosphate, sodium citrate, potassium citrate, citric acid, sodium phosphate, potassium phosphate, or phosphate. In some embodiments, the pH is lowered using CO2 and / or bicarbonates and / or carbonic acid. In certain embodiments, ammonia, ammonium hydroxide, calcium hydroxide, or calcium oxide is used as a base, and CO2 is used for neutralization. In certain embodiments, calcium hydroxide or calcium oxide is used as a base, and phosphate or phosphate buffer is used for neutralization. In some embodiments, the pH is lowered using organic acids. In certain embodiments, one or more neutralizing agents or buffers containing phytonutrients, including but not limited to nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the neutralizing or buffering agent contains sodium (Na) and / or chloride (Cl), but does not contain any other elements that inhibit plant growth. In certain embodiments, the neutralizing or buffering agent is not added after the heating / pressure step.

[0129] After alkaline hydrolysis, the proteins in the composition are broken down into polypeptides of various sizes.

[0130] According to some embodiments, after alkaline hydrolysis, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, of polypeptides in the suspension, for example, the soluble fraction of the suspension, has atomic masses of less than about 30 kD, less than about 25 kD, less than about 20 kD, less than about 15 kD, less than about 10 kD, less than about 5 kD, less than about 3 kD, or less than 2 kD, and in some embodiments, each of the aforementioned masses may be in the range of at least about 0.1 kD, at least about 0.5 kD, at least about 1 kD, or at least about 5 kD. According to some embodiments, polypeptides in a suspension make up at least 60% or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, for example, the soluble fraction of the suspension has atomic masses of about 0.1kD to about 30kD, 1kD to about 30kD, about 1kD to about 25kD, about 5kD to about 25kD, about 0.1kD to about 10kD, about 0.1kD to about 5kD, about 0.1kD to about 2kD, and about 5kD to about 20kD.

[0131] B. Acidic hydrolysis Referring to Figures 3A and 3B, the methods of the present disclosure may include a suspension of cellular biomass as a starting material. The suspension may be obtained by suspending (e.g., by homogenization) the cellular biomass in water or a suitable liquid medium such as a buffer or culture (growth) medium. Any preferred method for suspending the biomass in a medium may be used, including but not limited to vortexing, homogenization, stirring, sonication, etc. The biomass may be dry biomass (e.g., lyophilized biomass) or wet biomass before being suspended in the medium.

[0132] The pH of the biomass suspension can be raised to 310, 330 by adding an acid to the suspension. In some embodiments, the pH of the suspension is lowered to about 0.5 to about 3. Any suitable acid can be used to lower the pH of the suspension. Suitable acids may include, but are not limited to, one or more phosphoric acid (H3PO4), sulfuric acid (H2SO4), nitric acid (HNO3), formic acid (HCOOH), acetic acid (CH3COOH), carbon dioxide (CO2), and hydrochloric acid (HCl). In certain embodiments, one or more acids containing phytonutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, acids containing elements that inhibit plant growth are avoided, and these include, but are not limited to, chloride (Cl).

[0133] Acidic biomass suspensions can be subjected to high temperatures 320, 340. In some embodiments, the suspension is subjected to temperatures including about 40°C or higher, for example, about 50°C or higher, about 60°C or higher, about 70°C or higher, about 80°C or higher, about 90°C or higher, about 100°C or higher, about 105°C or higher, about 110°C or higher, and about 121°C or higher. In some embodiments, the suspension is subjected to temperatures of about 40°C to about 150°C, for example, about 60°C to about 150°C, about 70°C to about 140°C, about 80°C to about 140°C, about 90°C to about 135°C, about 100°C to about 130°C, about 100°C to about 125°C, about 105°C to about 125°C, and about 10°C to about 125°C. In some embodiments, the suspension is subjected to a temperature of about 110°C. The suspension can be subjected to high temperatures using any preferred method.

[0134] In some embodiments, the acidic biomass suspension is subjected to high pressures of 320, 340 (i.e., at least 14.7 psi). In some embodiments, the suspension is subjected to pressures of 15 psi or more, e.g., about 20 psi or more, about 25 psi or more, about 30 psi or more, about 35 psi or more, or about 40 psi or more, and in some embodiments, to pressures of about 40 psi or less, e.g., about 30 psi or less, about 25 psi or less, about 20 psi or less, about 18 psi or less, or about 15 psi or less. In some embodiments, the suspension is subjected to pressures in the range of about 14.7 psi to about 40 psi, e.g., about 15 psi to about 40 psi, about 15 psi to about 35 psi, about 15 psi to about 30 psi, about 15 psi to about 25 psi, or about 15 psi to about 20 psi. The suspension may be subjected to high temperature and high pressure using any preferred method. In some embodiments, the suspension is autoclaved to increase the temperature and pressure. In some embodiments, the suspension is subjected to high pressure and exposed to high temperature for only a portion of the time. In some embodiments, the suspension is subjected to high pressure for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the time it is exposed to high temperature.

[0135] The acidic biomass suspension may be subjected to high temperature, or high temperature and pressure, for a suitable period of time. In some embodiments, the suspension is subjected to high temperature, or high temperature and pressure, for about 5 minutes or more, for example, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, or about 25 minutes or more, and in some embodiments, each of the above times may be capped at about 90 minutes, about 80 minutes, about 70 minutes, about 60 minutes, about 50 minutes, about 40 minutes, or about 30 minutes. In some embodiments, the suspension is subjected to high temperature, or high temperature and pressure, for about 5 minutes to about 90 minutes, for example, about 5 minutes to about 80 minutes, about 5 minutes to about 70 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 40 minutes, 10 minutes to about 30 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to 60 minutes. In some embodiments, the suspension is subjected to high temperature, or high temperature and high pressure, for a period of time including about 1 hour or more, for example, about 3 hours or more, about 5 hours or more, or about 8 hours or more, and in some embodiments, each of the above periods may be capped at about 24 hours, about 18 hours, about 12 hours, or about 8 hours. In some embodiments, the suspension is subjected to high temperature, or high temperature and high pressure, for a period of time including about 1 hour to about 24 hours, for example, about 1 hour to about 18 hours, about 1 hour to about 12 hours, or about 1 hour to about 8 hours.

[0136] In some embodiments, the pH of the suspension is increased by adding a neutralizing agent, such as a neutralizing buffer, after heating and heat treatment.350 In some embodiments, the pH is increased to about 6 or about 7. The pH may be increased using any preferred neutralizing agent. Preferred neutralizing agents include, but are not limited to, bicarbonate buffers or phosphate buffers. In some embodiments, the neutralizing agent includes, but is not limited to, calcium hydroxide (Ca(OH)2), calcium carbonate (CaCO3), potassium hydroxide (KOH), or ammonium hydroxide (NH4OH). In certain embodiments, one or more neutralizing or buffering agents containing phytonutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, sodium (Na) and / or chloride (Cl) are included, but are not limited to, elements that inhibit plant growth. In certain embodiments, the neutralizing or buffering agent is not added after the heating / pressure step.

[0137] After acidic hydrolysis, the proteins in the composition are broken down into polypeptides of various sizes. According to some embodiments, after acidic hydrolysis, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, of polypeptides in the suspension, for example, the soluble fraction of the suspension, has an atomic mass of less than about 30 kD, less than about 25 kD, less than about 20 kD, less than about 15 kD, less than about 10 kD, less than about 5 kD, less than about 3 kD, or less than 2 kD, and in some embodiments, each of the aforementioned mass ranges may be at least about 0.1 kD, at least about 0.5 kD, at least about 1 kD, or at least about 5 kD. According to some embodiments, polypeptides in a suspension make up at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, for example, the soluble fraction of the suspension has atomic masses of about 0.1kD to about 30kD, 1kD to about 30kD, about 1kD to about 25kD, about 5kD to about 25kD, about 0.1kD to about 2kD, about 0.1kD to about 5kD, about 0.1kD to about 10kD, and about 5kD to about 20kD.

[0138] C. Protease treatment In some embodiments, for example, in embodiments where the alkaline or acidic hydrolysis described above is incomplete or peptides with the desired peptide size range and / or characteristics are not obtained, one or more proteases may be added to a neutralized suspension and incubated under conditions suitable for hydrolysis of additional biomass. The protease may be any preferred protease. The protease may be an endoprotease and / or an exoprotease. In some embodiments, the protease is an alkaline protease. In some embodiments, the alkaline protease is a serine alkaline protease. In some embodiments, the alkaline protease is a bacterial alkaline protease. In some embodiments, the alkaline protease includes, but is not limited to, subtilisin A. In some embodiments, the protease is an acidic protease, such as an aspartic acid or glutamic acid protease. In some embodiments, the protease is a metalloprotease. In certain embodiments, a combination of one or more types of proteolytic enzymes is used, such as exoproteases, endoproteases, neutral proteases, carboxypeptidases, and / or aminopeptidases. In such certain embodiments, one or more of the exoproteases, endoproteases, neutral proteases, carboxypeptidases, and aminopeptidases are derived from Aspergillus oryzae. In certain embodiments, an endoprotease enzyme preparation derived from Bacillus subtilis is used. In certain embodiments, glutaminase is used. In such certain embodiments, glutaminase is derived from Aspergillus oryzae.

[0139] In certain limited embodiments, a microorganism is hydrolyzed with at least one enzyme capable of hydrolyzing microbial (e.g., bacterial) proteins to free amino acids and / or short peptides. In certain embodiments, enzymatic hydrolysis includes hydrolysis with a cleansing enzyme. In certain embodiments, enzymatic hydrolysis includes hydrolysis with a mixture of the enzyme and the culture medium in which the enzyme was prepared. In certain embodiments, enzymatic hydrolysis includes hydrolysis with an enzyme derived from plants and / or animals and / or bacteria and / or archaea and / or fungi. In certain embodiments, enzymatic hydrolysis includes hydrolysis with a mixture of one or more enzymes derived from plants, animals, bacteria, archaea, and / or fungi. In certain embodiments, the hydrolytic enzyme is produced by a microbial strain described herein. In certain embodiments, the hydrolytic enzyme is produced from a microorganism grown on a Cl substrate and / or H2 and / or syngas feed material. In certain embodiments, bacterial cells can be hydrolyzed with one or more proteases, lipases, and amylases. In certain embodiments, the enzymatic hydrolysis comprises one or more proteolytic enzymes derived from microorganisms, plants, fungi, and / or animals. In certain embodiments, the method comprises the use of an alkaline protease.In certain embodiments, enzymatic hydrolysis involves hydrolyzing pancreatin together with at least one enzyme selected from papain, bromelain, ficin, bacterial proteases, fungal proteases, neutral proteases produced by Bacillus sp. Alcalase 2.4L, Bacillus B. licheniformis, and / or Subtilisin carlesberg, Esperase from B. lentus, Nutrase from B. amyloliquifacus, Protamex from Bacillus sp., Therolysin / therolase from B. thermoproteolyticus, Flavouzyme from Aspergillus oryzae, Protease N from B. subtilis, trypsin, chymotrypsin, keratinase, pepsin, subtilisin, and / or rennin. As will be well known to those skilled in the art, pancreatin contains digestive enzymes, proteases, and lipases.

[0140] Enzymatic hydrolysis of microbial (e.g., bacterial) cells involves combining the enzyme and microbial (e.g., bacterial) cells in any preferred amount under any preferred conditions. In certain embodiments, the number of reactants, reaction conditions, and sequence of reaction steps are selected to achieve ideal enzyme activity. In certain embodiments, the pressure, temperature, pH, and time conditions for enzymatic hydrolysis are such that maximum or preferred levels of enzyme activity are achieved. In certain embodiments, performing enzymatic hydrolysis involves combining the enzyme and microbial (e.g., bacterial) cells in a weight ratio of enzyme in the range of about 0.1 to about 10 g per 100 g of nitrogen content of the microbial (e.g., bacterial) cells. In other specific embodiments, enzymatic hydrolysis is performed using an azocasein assay with an enzyme storage solution at a concentration of about 0.05 vol% to about 0.5 vol% having about 70,000 units of activity. In many embodiments, any preferred method may be used to improve the efficiency of enzymatic hydrolysis of microbial (e.g., bacterial) cells. In certain embodiments, enzymatic hydrolysis involves combining an enzyme with microbial cells and stirring the combined enzyme and microbial cells by any preferred method. The enzymatic treatment can be carried out by any apparatus known to those skilled in the art, such as a reactor equipped with temperature control and stirring.

[0141] The suspension may be incubated with the protease at a pH and temperature suitable for the preferred or optimal catalytic activity of the specific protease used, and for a suitable time to achieve the desired amount of proteolysis. In some embodiments, the suspension is incubated at approximately 55°C for a suitable time, for example, approximately 3 hours or more, approximately 6 hours or more, approximately 12 hours or more, approximately 18 hours or more, or approximately 24 hours or more. In some embodiments, the suspension is incubated overnight at approximately 55°C with the bacterial alkaline protease.

[0142] In certain embodiments, the protease is inactivated after incubation. In certain embodiments, the protease is inactivated by heat treatment. For example, the heat treatment may involve raising the temperature to about 95°C for about 10 minutes.

[0143] Protease hydrolysis breaks down the protein in the composition into polypeptides of various sizes. According to some embodiments, after proteolysis, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, of polypeptides in the suspension, for example, the soluble fraction of the suspension, has atomic masses of less than about 30 kD, less than about 25 kD, less than about 20 kD, less than about 15 kD, less than about 10 kD, less than about 5 kD, less than about 3 kD, or less than 2 kD, and in some embodiments, each of the aforementioned mass ranges may be at least about 0.1 kD, at least about 0.5 kD, at least about 1 kD, or at least about 5 kD. According to some embodiments, polypeptides in a suspension make up at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, for example, the soluble fraction of the suspension has atomic masses of about 0.1kD to about 30kD, 1kD to about 30kD, about 1kD to about 25kD, about 5kD to about 25kD, about 0.1kD to about 2kD, about 0.1kD to about 5kD, about 0.1kD to about 10kD, and about 5kD to about 20kD.

[0144] D. Pre-processing Prior to the protein hydrolysis step, in certain embodiments, the biomass is subjected to one or more pretreatment steps, including but not limited to cell lysis and / or delipidation, i.e., lipid extraction. In certain embodiments, delipidation is carried out using one or more solvents, including but not limited to methanol, ethanol, isopropyl alcohol, hexane, acetone, propylene carbonate, dichloromethane, and / or chloroform. In certain embodiments, delipidation is carried out using one or more bases, including but not limited to ammonium hydroxide, ammonia, sodium hydroxide, and / or potassium hydroxide. In certain embodiments, the delipidated biomass is subjected to one or more protein hydrolysis methods described herein. In certain embodiments, the delipidated biomass is subjected to enzymatic hydrolysis, which in some embodiments requires relatively small pH adjustments. In such certain embodiments, the pH is adjusted only to about 9 to about 5, during which the enzymatic hydrolysis is carried out. In certain embodiments, enzymatic hydrolysis is carried out on the delipidated, unadjusted biomass after the completion of the delipidation step.

[0145] E. Purification / Separation In some embodiments, after hydrolytic treatment (e.g., alkaline or acidic hydrolysis, optionally including protease treatment), the suspension (hydrolyzate) is purified to remove undissolved material from the suspension (e.g., separation of soluble and insoluble fragments). The suspension may be purified using any preferred method such as centrifugation or filtration. In some embodiments, after the suspension has been purified (e.g., by centrifugation), the supernatant is separated from the pellet.

[0146] In some embodiments, a purified liquid composition containing hydrolyzed protein (e.g., a soluble fraction such as the supernatant of a separated suspension) is dried (e.g., lyophilized) to produce a dried or substantially dried composition. In some embodiments, the lyophilized composition has a water content of about 10% or less (e.g., including about 8% or less, about 6% or less, about 5% or less, and about 3% or less). In some embodiments, the lyophilized protein hydrolysate product composition has a water content of about 1% to about 10% (e.g., including about 1% to about 8%, about 1% to about 6%, and about 2% to about 5%).

[0147] In some embodiments, the purified liquid composition (e.g., a soluble fraction such as the supernatant of a separated suspension) is dehydrated or concentrated to reduce its water content. In some embodiments, the concentrated composition has a water content of about 80% or less (e.g., including about 75% or less, about 50% or less, about 40% or less, and about 30% or less), and in some embodiments, each of the above-mentioned ranges of water content may be at least about 20%, at least about 25%, at least about 30%, at least about 40%, or at least about 50% (to the extent that the above-mentioned ranges exceed the lower limit). In some embodiments, the dehydrated product is dried using heat and / or evaporation by methods such as spray drying, drum drying, oven drying, vacuum drying, vacuum oven drying, drying under an inert gas such as N2, and solar evaporation. In some embodiments, the purified product is first dehydrated in a rotary evaporator to remove, for example, about 50% to about 65% of the moisture. In some embodiments, further dehydration is achieved by freeze-drying, for example, the freeze-dried protein hydrolysate product composition has a water content of about 1% to about 10% (e.g., including about 1% to about 8%, about 1% to about 6%, and about 2% to about 5%).

[0148] In some embodiments, the method of the present disclosure involves processing an insoluble fragment of a suspension, for example, after alkaline or acidic hydrolysis or protease treatment (e.g., alkaline protease, acidic protease, or metalloproteinase) to obtain a co-product. In some embodiments, the treated suspension is separated into soluble and insoluble fragments (e.g., by centrifugation to produce a supernatant fragment containing a protein hydrolysis product composition) and pellet fragments.

[0149] The pellet fragments may be further processed to extract co-products. The co-products may be fragments of microorganisms that yield biomass. In some embodiments, the co-products are biopolymers such as polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoates may include, but are not limited to, polyhydroxybutyric acid (PHB).

[0150] F. Treatment with additional chemicals A chelating agent may be included in any embodiment of the biomass hydrolysis method disclosed herein. A chelating agent may be added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure (see, for example, Figures 2 and 4 (210, 410)). Any suitable chelating agent may be used, including natural chelating agents (e.g., amino acids) and synthetic chelating agents. Suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Any suitable amount of chelating agent (e.g., enough to sequester undesirable metal ions in the suspension) may be added to the biomass suspension. In some embodiments, the amount of chelating agent added to the biomass suspension is in the range of about 0.1 mM to about 10 mM (e.g., including about 0.5 mM to about 8 mM, about 1 mM to about 7 mM, and about 3 mM to about 5 mM). In some embodiments, the amount of chelating agent in the biomass suspension is approximately 5 mM. In certain embodiments, the chelating agent is not added to the biomass suspension before it is subjected to high heat or high heat / pressure. In certain embodiments, synthetic chelating agents are not added to the biomass suspension before it is subjected to high heat or high heat / pressure. In certain embodiments, one or more chelating agents containing one or more plant nutrients, including but not limited to nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the chelating agent does not contain any plant growth inhibitors, including but not limited to sodium (Na) and / or chloride (Cl).

[0151] For example, a surfactant may be included in any embodiment of the biomass hydrolysis method disclosed herein to improve the solubility of the suspension. In some embodiments, the surfactant is added to the biomass suspension before subjecting the suspension to high heat or high heat / high pressure (see Figures 2 and 4 (210, 410)). Any preferred surfactant, including natural surfactants (i.e., obtained directly from natural sources) or synthetic surfactants, may be added to the biomass suspension. Preferred surfactants include, but are not limited to, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20, and Pluronic PF-68. Any preferred amount of surfactant may be added to the biomass suspension. In some embodiments, the amount of surfactant in the biomass suspension may range from about 1% to about 25% (e.g., including about 2% to about 20%, about 4% to about 15%, about 5% to about 12%, and about 8% to about 12%) relative to the dry weight (w / w) of the biomass in the suspension. In some embodiments, the amount of surfactant added to the biomass suspension is about 10% (w / w) of the dry weight (w / w) of the biomass in the suspension. In certain embodiments, surfactants are not added to the biomass suspension before it is subjected to high heat or high heat / pressure. In certain embodiments, synthetic surfactants are not added to the biomass suspension before it is subjected to high heat or high heat / pressure. In certain embodiments, one or more surfactants containing one or more plant nutrients, including but not limited to nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, chelating agents are avoided from containing surfactants containing substances that inhibit plant growth, including but not limited to sodium (Na) and / or chloride (Cl). In certain embodiments, the added surfactants are not harmful to plants or animals and / or are biodegradable and / or have no adverse effects on the environment.

[0152] Chaotropic reagents may be included in any embodiment of the biomass hydrolysis method disclosed herein, for example, to assist in the denaturation or modification of protein molecules. In some embodiments, the chaotropic reagent is added before subjecting the suspension to high heat or high heat / high pressure. In some embodiments, the chaotropic reagent is added to a neutralized suspension containing a natural or synthetic chaotropic reagent (see, e.g., Figures 2 and 4 (240, 440)). In some embodiments, protease hydrolysis is carried out in the presence of the chaotropic reagent in the neutralized suspension. Any suitable amount of the chaotropic reagent may be added to the neutralized suspension. Suitable chaotropic reagents include, but are not limited to, urea, thiourea, and guanidinium chloride. The chaotropic reagent may be added to the neutralized suspension in any suitable amount. In some embodiments, the amount of chaotropic reagent in the neutralized suspension is in the range of about 0.1 M to about 2 M (e.g., including about 0.5 M to about 1.5 M and about 0.8 M to about 1.2 M). In some embodiments, the amount of chaotropic reagent in the neutralized suspension is about 1 M. In certain embodiments, no chaotropic reagent is added to the biomass suspension. In certain embodiments, no synthetic chaotropic reagent is added to the biomass suspension. In certain embodiments, one or more chaotropic reagents containing plant nutrients, including but not limited to nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the chaotropic reagent does not contain substances that inhibit plant growth, including but not limited to sodium (Na) and / or chloride (Cl).

[0153] In some embodiments, the methods of the Disclosure include removing surfactants, chelating agents, and / or chaotropic reagents. In some embodiments, the surfactants, chelating agents, and / or chaotropic reagents are removed after being treated with a protease (e.g., an alkaline protease, an acidic protease, or a metalloproteinase) (see, for example, Figures 2 and 4 (250, 450)), or, in embodiments that do not involve protease treatment as described herein, for example, by buffer exchange. Any preferred method may be used to remove the surfactants, chelating agents, and / or chaotropic reagents, including but not limited to gel filtration chromatography, membrane filtration, and / or dialysis. In some embodiments, the protein hydrolysis product compositions of the Disclosure are prepared without the use of surfactants, chelating agents, or chaotropic reagents.

[0154] III. Biomass Sources The methods of the present disclosure may be used to process a biomass suspension derived from any suitable proteinaceous material source. The organisms from which the protein hydrolysate product composition is derived may be multicellular or unicellular. In some embodiments, the protein hydrolysate product composition of the present disclosure has a microbial source. The microbial organisms from which the protein hydrolysate product composition is derived may be photoautotrophic, heterotrophic, methane-assimilating, methanol-assimilating, carboxydotrophic, or chemoautotrophic organisms. The microbial organisms may be wild-type or genetically modified. Referring to Figure 5, the biomass may be collected, for example, from a culture of suitable microorganisms in a fermenter or bioreactor 510. The collected biomass may be used in any suitable method, such as a centrifuge, to separate cell aggregates from the culture medium. The collected biomass may be used in method 520 of the present disclosure to produce a protein hydrolysate product composition 530. In some embodiments, the collected biomass may be spray-dried or freeze-dried to produce dried biomass, which may then be used to produce a protein hydrolysate product composition according to the method of the present disclosure.

[0155] In some embodiments, the microorganism is selected from *Rhodococcus* or *Gordonia*. In some embodiments, the microorganism is *Rhodococcus* opacus. In some embodiments, the microorganism is *Rhodococcus* opacus (DSM43205) or *Rhodococcus* sp. (DSM3346). In some embodiments, the microorganism is selected from the genera *Ralstonia*, *Cupriavidus*, or *Hydrogenobacter*. In some embodiments, the microorganism is *Cupriavidus* necator or *Cupriavidus* metallidurans. In some embodiments, the microorganism is *Rhodococcus* opacus, *Hydrogenovibrio* marinus, *Rhodopseudomonas* capsulata, *Hydrogenobacter* thermophilus, and *Rhodobacter* sphaeroides. In some non-limiting embodiments, the strain of *Cupriavidus* necator is DSM531 or DSM541. In some embodiments, the microorganism is a strain belonging to the family *burkholderiaceae*. In some embodiments, the microorganism is a strain belonging to the genus Cupriavidus or Ralstonia. In some embodiments, the microorganism includes the species Cupriavidus necator. In some embodiments, the microorganism is a strain of the species Cupriavidus necator DSM53. In some embodiments, the microorganism includes the species Cupriavidus necator. In some embodiments, the microorganism is a strain of the species Cupriavidus metallidurans DSM2839. In some embodiments, the microorganism includes the species Xanthobacter autotrophicus. In some embodiments, the microorganism is a strain of the species Xanthobacter autotrophicus DSM432.

[0156] In some embodiments, a microbial consortium is used as a source of biomass in the methods described herein. The consortium may include one or more microbial species or strains, or microorganisms having the characteristics of one or more microbial species described herein.

[0157] In some embodiments, the microorganisms described herein can accumulate about 50% by weight or more of protein in the total cell mass. In some embodiments, the microorganisms described herein can accumulate about 60% by weight or more of protein in the total cell mass. In some embodiments, the microorganisms can accumulate about 70% by weight or more of protein in the total cell mass. In some embodiments, the microorganisms can accumulate about 80% by weight or more of protein in the total cell mass. In some non-limiting embodiments, the microorganisms exhibiting these characteristics are Cupriavidus necator DSM531 or 541.

[0158] In some embodiments, the microorganisms described herein can spontaneously grow in H2 / CO2 and / or syngas, and the microorganisms can spontaneously accumulate polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) in a total cell mass of about 50% or more. In some embodiments, the microorganisms spontaneously possess the ability to direct high carbon flow rates through acetyl-CoA metabolic intermediates, thereby leading to fatty acid biosynthesis, in addition to many other synthetic pathways, such as PHA (e.g., PHB synthesis and / or amino acid biosynthesis). In some embodiments, the microorganism exhibiting these characteristics is Cupriavidus necator (e.g., DSM531 or DSM541).

[0159] In some embodiments, wild-type microorganisms are used in the methods described herein that, when grown under the same conditions, can produce mutant or manipulated strains of the microorganism that produce PHA such as PHB, and less PHA (e.g., PHB) than the wild-type strain. In some embodiments, the mutant or manipulated strain is unable to produce a detectable amount of PHA (e.g., PHB). In certain embodiments, PHA (e.g., PHB)-negative mutants are used. In such particular embodiments, the species is Cupriavidus necator. In such particular embodiments, the strain is Cupriavidus necator DSM541.

[0160] In some non-limiting embodiments, the microorganism is Corynebacterium autotrophicum. In some non-limiting embodiments, the microorganism is Corynebacterium autotrophicum and / or Corynebacterium glutamicum. In some embodiments, the microorganism is Hydrogenovibrio marinus. In some embodiments, the microorganism is Hydrogenovibrio marinus.

[0161] In some embodiments, the microbial cells include microorganisms selected from one or more genera of Cupriavidus sp., Rhodococcus sp., Hydrogenovibrio sp., Rhodopseudomon ass sp., Hydrogenobacters sp., Gordonia sp., Arthrobacters sp., Streptomycetes sp., Rhodobacters sp., and / or Xanthobacter.

[0162] In some embodiments, the microorganism is a cell of the phylum Actinobacteria. In some embodiments, the microorganism is a strain of the suborder corynebacterineae (corynebacterium, gordoniaceae, mycobacteriaceae, and nocardiaceae). In some embodiments, the microorganism is a cell of the phylum Actinobacteria. In some embodiments, the microorganism is selected from one or more of the classifications Corynebacterium, Gordonia, Rhodococcus, Mycobacterium, and Tsukamurella. In some embodiments, the microorganism is a cell of the genus Rhodococcus.In some embodiments, the cells are Rhodococcus opacus, Rhodococcus aurantiacus; Rhodococcus baikonurensis; Rhodococcus boritolerans; Rhodococcus equi; Rhodococcus coprophilus; Rhodococcus corynebacterioides; erythropolis;Rhodococcus fascians;Rhodococcus globerulus;Rhodococcus gordoniae;Rhodococcus jostii;Rhodococcus koreensis;Rhodococcus kroppenstedtii;Rhodococcus maanshanensis;Rhodococcus marinonascens phenolicus;Rhodococcus polyvorum;Rhodococcus This includes strains of Rhodococcus sp. such as pyridinivorans, Rhodococcus rhodochrous, Rhodococcus rhodnii (synonym: Nocardiarhodnii), Rhodococcus ruber (synonym: Streptothrixrubra), Rhodococcus sp. RHA, Rhodococcus triatomae, Rhodococcus tukisamuensis, Rhodococcus wratislaviensis (synonym: Tsukamurellawratislaviensis), Rhodococcus yunnanensis, and Rhodococcus zopfii. In some embodiments, the microorganism is Rhodococcus opacus strain DSM43205 or DSM43206. In some embodiments, the microorganism is Rhodococcus sp. strain DSM3346.

[0163] In some embodiments, the composition comprises microorganisms (e.g., microorganisms of any genus or species described herein) that can spontaneously grow in H2 / CO2 and / or syngas, and the microorganisms can spontaneously accumulate lipids at a rate of at least about 10%, 20%, 30%, 40%, or 50% or more of the cellular biomass. In some embodiments, the microorganisms (e.g., microorganisms of any genus or species described herein) are naturally capable of delivering high carbon flows into the fatty acid biosynthesis pathway. In some embodiments, microorganisms exhibiting these properties are Rhodococcus opacus (e.g., DSM43205 or DSM43206 or DSM44193) or Cupriavidus necator (e.g., DSM531 or DSM541).

[0164] In some embodiments, the microorganism is acidic hydrogen or a knallgas strain. In some embodiments, the microorganism or composition containing the microorganism contains one or more knallgas microorganisms from the following: Aquifex pyrophilus, Aquifex aeolicus, or other Aquifex sp.; Cupriavidus necator or Cupriavidus metallidurans or other Cupriavidus sp.; Corynebacterium autotrophicum or other Corynebacterium sp.; Gordonia desulfuricans, Gordonia polyisoprenivorans, Gordonia rubripertincta, Gordonia hydrophobica, Gordonia westfalica, or other Gordonia sp.; Nocardia autotrophica, Nocardia opaca, or other Nocardia sp.; Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas Purple non-sulfur bacteria including, but not limited to, Rhodopseudomonasviridis, Rhodopseudomonassuljoviridis, Rhodopseudomonasblastica, Rhodopseudomonasspheroides, Rhodopseudomonasacidophila, or other Rhodopseudomonassp.; Rhodobacter sp., Rhodospirillum rubrum, or other Rhodospirillum sp.; Rhodococcus opacus or other Rhodococcus sp.; Rhizobium japonicum or other Rhizobium sp.; Thiocapsa roseopersicina or other Thiocapsa sp.Pseudomonas facilis, Pseudomonas flava, Pseudomonas putida, Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, Pseudomonas palleronii, Pseudomonas pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophile, or other Pseudomonas sp.; Hydrogenomonas pantotropha, Hydrogenomonas eutropha, Hydrogenomonas facilis, or other Hydrogenomonas sp.; Hydrogenobacter thermophiles, Hydrogenobacter halophilus, Hydrogenobacter hydrogenophilus, or other Hydrogenobacter sp.; Hydrogenophilus islandicus or other Hydrogenophilus sp.; Hydrogenovibrio marinus or other Hydrogenovibrio sp.; Hydrogenothermus marinus or other Hydrogenothermus sp.; Helicobacter pylori or other Helicobacter sp.; Xanthobacter autotrophicus, Xanthobacter jlavus, or other Xanthobacter sp.; Hydrogenophaga jlava, Hydrogenophaga palleronii, Hydrogenophaga pseudojlava, or other Hydrogenophaga sp.; Bradyrhizobium japonicum or other Bradyrhizobium sp.; Ralstonia eutropha or other Ralstonia sp.Alcaligenes eutrophus, Alcaligenes facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Alcaligenes paradoxus, Alcaligenes ruhlandii, or other Alcaligenes sp.; Amycolata sp.; Aquaspirillum autotrophicum or other Aquaspirillum sp.; Arthrobacter strain11 / X, Arthrobacter methylotrophus, or other Arthrobacter sp.; Azospirillum lipoferum or other Azospirillum sp.; Variovorax paradoxus or other Variovorax sp.; Acidovorax facilis, or other Acidovorax sp.; Bacillus schlegelii, Bacillus tusciae, or other Bacillus sp.; Calderobacterium hydrogenophilum or other Calderobacterium sp.; Derxia gummosa or other Derxia sp.; Flavobacterium Autothermophilum or other Flavobacterium sp.; Microcyclus aquaticus or other Microcyclus sp.; Mycobacterium gordoniae or other Mycobacterium sp.; Paracoccus denitrificans or other Paracoccus sp.; Persephonella marina, Persephonella guaymasensis, or other Persephonella sp.; Renobacter vacuolatum or other Renobacter sp.; Seliberia carboxydohydrogena or other Seliberia sp.A consortium of organisms including, but not limited to, Streptomycetes coelicojlavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetes thermocarboxydus, and other Streptomycetes sp.; Thermocrinis ruber or other Thermocrinis sp.; Wautersia sp.; Anabaena oscillarioides, Anabaena spiroides, Anabaenacylindrica, or other Anabaena sp., as well as cyanobacteria, and green algae, including, but not limited to, Arthrospira platensis, Arthrospira maxima, or other Arthrospira sp.; Scenedesmus obliquus or other Scenedesmus sp.; Chlamydomonas reinhardii or other Chlamydomonas sp., Ankistrodesmus sp., and Rhaphidium polymorphium or other Rhaphidium sp., and microorganisms and / or acid-hydrogen microorganisms.

[0165] In some embodiments, a microorganism or a composition containing a microorganism includes one or more of the following genera: Cupriavidus; Xanthobacter; Dietzia; Gordonia; Mycobacterium; Nocardia; Pseudonocardia; Arthrobacter; Alcanivorax; Rhodococcus; Streptomyces; Rhodopseudomonas; Rhodobacter; and Acinetobacter; as well as a consortium of microorganisms and / or an organism containing one or more of these microorganisms.

[0166] In some embodiments, the microorganism or microorganism-containing microorganism comprises one or more of the following: Arthrobacter methylotrophus DSM14008; Rhodococcus opacus DSM44304; Rhodococcus opacus DSM44311; Xanthobacter autotrophicus DSM431; Rhodococcus opacus DSM44236; Rhodococcus ruber DSM43338; Rhodococcus opacus DSM44315; Cupriavidus metallidurans DSM2839; Cupriavidus necator DSM531; Cupriavidus necator DSM541; Rhodococcus aetherivorans DSM44752; Gordonia desulfuricans DSM44462; Gordonia polyisoprenivorans DSM44266; Gordonia polyisoprenivorans DSM44439; Gordonia rubripertincta DSM46039; Rhodococcus percolatus DSM44240; Rhodococcus opacus DSM43206; Gordonia hydrophobica DSM44015; Rhodococcus zopfii DSM44189; Gordonia westfalica DSM44215, Xanthobacter autotrophicus DSM1618; Xanthobacter autotrophicus DSM2267; Xanthobacter autotrophicus DSM3874; Streptomycetes coelicoflavus DSM41471; Streptomycetes griseus DSM40236; Streptomycetes sp. DSM40434; Streptomycetes xanthochromogenes DSM40111; Streptomycetes thermocarboxydus DSM44293; Rhodobacter sphaeroides DSM158.In some embodiments, the microorganism or composition containing a microorganism includes a consortium of microorganisms and / or one or more of these microorganisms, or organisms including any genera or species of microorganism described herein.

[0167] Numerous different microorganisms are characterized by their ability to grow on carbon monoxide as an electron donor and / or carbon source (i.e., carboxydotrophic microorganisms). In some cases, carboxydotrophic microorganisms can also use H2 as an electron donor and / or grow mixotrophically. In some cases, carboxydotrophic microorganisms are facultative chemoautotrophic organisms (Biology of the Prokaryotes, edited by J. Lengeler, G. Drews, H. Schlegel, John Wiley & Sons, Jul 10, 2009, the whole of which is incorporated herein by reference). In some embodiments, the microorganism or composition containing the microorganism comprises one or more carboxydotrophic microorganisms from among Acinetobacter sp.; Alcaligenes carboxydus or other Alcaligenes sp.; Arthrobacter sp.; Azomonas sp.; Azotobacter sp.; Bacillus schlegelii or other Bacillus sp.; Hydrogenophaga pseudoflava or other Hydrogenophaga sp.; Pseudomonas carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas compransoris, Pseudomonas gazotropha, Pseudomonas thermocarboxydovorans, or other Pseudomonas sp.; Rhizobium japonicum or other Rhizobium sp.; and Streptomyces G26, Streptomyces thermoautotrophicus, or other Streptomyces sp. In some embodiments, the microorganism or composition containing the microorganism includes a consortium of microorganisms and / or organisms, such as one or more of the carboxydotropic microorganisms described above.

[0168] In certain embodiments, carboxydotrophic microorganisms are used. In certain embodiments, carboxydotrophic microorganisms capable of chemoautotrophic growth are used. In certain embodiments, carboxydotrophic microorganisms capable of using H2 as an electron donor in respiration and / or biosynthesis are used.

[0169] Acetobacterium Acetobacter sp., Acetobacterium Acetobacterium, Acetobacterium. sp.;Acetogenium sp.;Achromobacter sp.;Acidianus sp.;Acinetobacter sp.;Actinomadura sp.;Aeromonas sp.;Alcaligenes sp.;Alcaliqenes sp.;Aquaspirillum sp.;Arcobacter sp.;Aureobacterium sp.;Bacillus sp.; sp.;Carboxydothermus sp.;Clostridium sp.;Comamonas sp.;Cupriavidus sp.;Dehalobacter sp.;Dehalococcoide sp.;Dehalospirillum sp.;Desulfobacterium sp.;Desuljomonile sp.;Desulfotomaculum sp.;Desuljovibrio sp.;Desulfurosarcina sp.; sp.;Ectothiorhodospirasp.;Enterobacter sp.;Eubacterium sp.;Ferroplasma sp.;Halothibacillus sp.;Hydrogenobacter sp.;Hydrogenomonas sp.;Leptospirillum sp.;Metallosphaera sp.;Methanobacterium sp.;Methanobreccuvi sp.; sp.;Methanococcoides sp.;Methanogenium sp.;Methanolobus sp.;Methanomicrobium sp.;Methanoplanus sp.;Methanosarcina sp.;Methanospirillum sp.;Methanothermus sp.;Methanothrix sp.;Micrococcus sp.;Nitrobacter sp.;Nitrobactera sp., Nitrosococcus sp.; Nitrospina sp., Nitrospira sp., Nitrosolobus sp.; Nitrosomonas sp.; Nitrosospira sp.; Nitrosovibrio sp.; sp.;Ralstonia sp.;Rhodobacter sp.;Rhodococcus sp.;Rhodocyclus sp.;Rhodomicrobium sp.;Rhodopseudomonas sp.;Rhodospirillum sp.;Shewanella sp.;Siderococcus sp. sp.;Thiomicrospira sp.;Thioploca sp.;Thiosphaera The microorganisms include: sp.; Thiothrix sp.; Thiovulum sp.; sulfur oxidizers; hydrogen oxidizers; ionic oxidizers; acetogens; and methanogenic bacteria; a consortium of microorganisms including chemosynthetic autotrophs; chemosynthetic autotrophs originating from at least one of the following: hydrothermal vents, geothermal outlets, hot springs, springs, underground aquifers, salt lakes, concentrated salt formations, mines, acidic mine drainage, mine dressing waste, oil wells, petroleum industrial wastewater, coal seams, deep subsurface; sewage and wastewater treatment plants, geothermal power plants, geothermal vent fields, geothermal soil; and obligate and / or facultative chemoautotrophic microorganisms including one or more extremophilic bacteria selected from hyperthermophiles, eosinophils, halophiles, and psychrophiles. In some embodiments, the microorganisms or compositions containing microorganisms include a consortium of microorganisms and / or organisms containing chemoautotrophic microorganisms, including one or more of the above-mentioned chemoautotrophic microorganisms.

[0170] In some embodiments, extremophilic bacteria microorganisms are provided that can withstand extreme environmental conditions such as temperature, radiation, pressure, gravity, vacuum, dryness, salinity, pH, oxygen partial pressure, and chemical substances. Hyperthermophiles such as Pyrolobus fumarii; thermophiles such as Synechococcus lividis; mesophiles and psychrophiles such as Psychrobacter, and / or hyperthermophilic sulfur metabolic groups such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., and Acidianus sp.; radiation-resistant organisms such as Deinococcus radiodurans; pressure-resistant organisms including piezophiles or barophiles; drought-tolerant and cryptobiotic organisms such as Artemia salina; microorganisms and fungi; salt-tolerant organisms including halophilic bacteria such as Halobacteriacea and Dunaliella salina; pH-tolerant organisms including alkaliphilic bacteria such as Natronobacterium, Bacillus firmus OF4, Spirulina spp., and eosinophils (such as Ideyukogome and Ferroplasma sp.); gas-tolerant organisms that can withstand pure CO2, including Ideyukogome; and Ferroplasma This includes metal-tolerant organisms such as acidarmanus and Ralstonia sp.

[0171] In certain embodiments, the microorganisms provided herein include cell lines selected from eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophilic bacteria, yeasts, fungi, proteobacteria, their modified organisms, and synthetic organisms. In certain embodiments, the genus Spirulina is used.

[0172] In certain embodiments, green non-sulfur bacteria are used, including but not limited to the genera Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseijlexus, and Thermomicrobium.

[0173] In certain embodiments, green sulfur bacteria are used, including but not limited to the genera Chlorobium, Clathrochloris, and Prosthecochloris.

[0174] In certain embodiments, purple sulfur bacteria are used, including but not limited to the genera Allochromatium, Chromatium, Halochromatium, Jsochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis.

[0175] In certain embodiments, purple non-sulfur bacteria are used, including but not limited to the genera Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.

[0176] In some embodiments, the microorganism is a methane-utilizing bacterium and / or a methylotrope. In some embodiments, the microorganism is of the genus Methylococcus. In some embodiments, the microorganism is Methylococcus capsulatus. In some embodiments, the microorganism is a methane-utilizing bacterium and / or a methylotrope. In some embodiments, the microorganism is of the genus Methylobacterium. In some embodiments, the microorganism is obtained from one or more of the genera Methylobacterium zatmanii, Methylobacterium extorquens, and Methylobacterium chloromethanicum. In some embodiments, compositions are provided in which the microorganism is a hydrogen-oxidizing chemical autotroph and / or a carboxydotrope and / or a methylotrope and / or a methane-utilizing bacterium.

[0177] In certain embodiments, microorganisms grow heterotrophically using polycarbonate organic molecules as carbon sources, including but not limited to glucose and / or fructose, and but not limited to sugars. In some embodiments, microorganisms can grow on untreated natural glycerol and / or glucose and / or sucrose and / or sugar juice and / or high-fructose corn syrup and / or corn starch and / or cellulosic biomass and / or methanol and / or acetate, with the latter serving as sole electron donors and carbon sources. In some embodiments, microorganisms can grow mixed trophically on organic carbon sources and use inorganic electron donors or carbon sources.

[0178] In certain embodiments, the microorganisms provided herein include one or more of the following: eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophilic bacteria, archaea, yeasts, fungi, proteobacteria, modified organisms thereof, and synthetic organisms.

[0179] In certain embodiments, the microorganisms depend on specific environmental conditions provided by a bioprocess that is native and / or non-GMO and / or non-pathogenic and / or not present in the surrounding environment.

[0180] In certain embodiments, a microorganism or consortium of microorganisms is independent of an environmental sample and is enriched with a desired microorganism using methods well known in the field of microbiology, such as growth in the presence of a target electron donor including, for example, hydrogen, CO, syngas and / or methane, and / or electron acceptors (including, but not limited to, one or more of oxygen, nitrates, ferric, and / or CO2) and / or environmental conditions (e.g., temperature, pressure, dissolved oxygen (DO), salinity, presence of impurities and contaminants).

[0181] In certain embodiments, the microorganism or microbial consortium includes bacteria, fungi (e.g., yeasts), and / or other microbial cells used to process sugar-supplying materials into useful organic compounds such as proteins and amino acids in a heterotrophic fermentation system.

[0182] In certain embodiments, a microorganism or a consortium of microorganisms includes useful microorganisms. In certain embodiments, a microorganism or a consortium of microorganisms includes Food and Drug Administration Certified Assurance (GRAS) microorganisms and / or organisms. In certain embodiments, a microorganism or organism or consortium of microorganisms includes, but is not limited to, one or more of the following yeasts: Candida humilis; Candida milleri; Debaryomyces hansenii; Kazachstania exigua (Saccharomyces exiguous); Saccharomyces cerevisiae; Saccharomyces florentinus; Torulaspora delbrueckii; Trichosporon beigelli; and / or - fungi including, but not limited to, one or more of the following: Aspergillus oryzae; Aspergillus sojae; Fusarium venenatum A3 / 5; Neurospora intermedia var. oncomensis; Rhizopus oligosporus; Rhizopus oryzae; Aspergillus luchuensis; and / or - Bacillus amyloliquefaciens; Bacillus subtilis; Bifidobacterium animalis(lactis);Bifidobacterium bifidum;Bifidobacterium breve;Bifidobacterium longum;Lactobacillus acidophilus;Lactobacillus brevis;Lactobacillus casei;Lactobacillus delbrueckii subsp.bulgaricus;Lactobacillus fermentum;Lactobacillus helveticus;Lactobacillus kefiranofaciens;Lactobacillus lactis;Lactobacillus plantarum;Lactobacillus rhamnosus;Lactobacillus reuteri;Lactobacillus sakei;Lactobacillus sanfranciscensis lactis, Streptococcus lactis subsp. This includes bacteria containing one or more of the *Thermophilus* species, but not limited to them.

[0183] Proteins containing biomass from which protein hydrolysis product compositions are derived can be produced by a consortium of different microbial and / or multicellular organism species.

[0184] In some embodiments, the consortium includes one or more of the following: oxyhydrogen microorganisms, carboxydotrophytes, methane-assimilating bacteria, methylotrophytes, chemoautotrophs, photoautotrophs, and heterotrophs.

[0185] In some embodiments, the protein hydrolysate also contains one or more vitamins produced by the organism from which the hydrolysate was induced. In some non-limiting embodiments, the microorganism is Cupriavidus necator DSM531 or DSM541. In some non-limiting embodiments, the vitamins are B vitamins, including but not limited to Bl, B2 and / or B12.

[0186] Any suitable method can be used to culture the microorganisms. The microorganisms can grow under any suitable conditions in an environment suitable for biomass growth and production. In some embodiments, the microorganisms can grow under autotrophic culture conditions, heterotrophic culture conditions, or both autotrophic and heterotrophic culture conditions. Heterotrophic culture may include a suitable source of carbon and energy, such as one or more sugars (e.g., glucose, fructose, sucrose, etc.). Autotrophic cultures may include C1 chemistry such as carbon monoxide, carbon dioxide, methane, methanol, formate, and / or formic acid, and / or mixtures containing C1 chemistry, including but not limited to a variety of syngas compositions or a variety of generator gas compositions, for example, lignocellulose energy crops, crop residues, bagasse, sawdust, forest residues, food waste, general waste, sewage sludge, carpet waste, biogas, landfill gas, general natural gas, or petroleum coke through vaporization, partial oxidation, pyrolysis, or steam reforming of the aforementioned low-value or waste carbon sources that can be used as a carbon and energy source by oxyhydrogen microorganisms or hydrogen-oxidizing microorganisms or carbon monoxide-oxidizing microorganisms. Preferred methods for culturing the microorganisms used in the methods of the present invention and producing biomass are described, for example, in U.S. Patents 9,157,058 and 9,556,462, and PCT International Patent WO2018 / 144965, which are incorporated herein by reference. In some embodiments, the organisms may grow photosynthetically in bioreactors, hydroponic systems, greenhouses, or cultivated land, or they may be recovered from waste or natural products. In certain embodiments, the protein-containing biomass used to produce protein hydrolysis products is from microorganisms grown on one or more C1 substrates. In certain embodiments, the microorganisms grow on CO2 and / or CO2 in dissolved form in aqueous solutions (e.g., CO2(aq), bicarbonates, carbonates) as a sole carbon source.In certain embodiments, microorganisms grown on CO2 and / or dissolved CO2 include microbial cells selected from one or more genera of Cupriavidus, Rhodococcus, Hydrogenovibrio, Rhodopseudomonas, Hydrogenobacter, Gordonia, Arthrobacter, Streptomycetes, Rhodobacter, and / or Xanthobacter.

[0187] In certain embodiments, the protein containing biomass derived from microorganisms grown on CO2 and / or dissolved CO2 includes microbial cells selected from one or more of the autotrophic (e.g., chemoautotrophic, photoautotrophic), carboxydotrophic, methane-assimilating, methanol-assimilating, and / or heterotrophic microorganisms described above. In certain embodiments, the protein containing biomass derived from microorganisms grown on CO2 and / or dissolved CO2 includes Cupriavidus necator cells. In such certain embodiments, the Cupriavidus necator strain may include, but is not limited to, DSM428, DSM531, or DSM541. In certain embodiments, the protein containing biomass derived from microorganisms grown on CO2 and / or dissolved CO2 includes microbial cells of PHB-negative mutants or knockouts of wild-type strains that naturally produce PHB. In the aforementioned embodiments, the mutant strains that cannot produce PHB are derived from wild-type strains that can produce PHB, using known methods. In certain embodiments, the PHB-negative mutant or knock strain produces less PHB than the wild-type strain, or an undetectable amount of PHB. In such certain embodiments, the PHB-negative mutant is Cupriavidus necator DSM541.

[0188] In some embodiments, an organism or consortium can be grown mixotrophically. In some such mixotrophic growth conditions, the growth substrate contains H2 gas along with one or more carbon sources.

[0189] IV. Composition Disclosed herein are protein hydrolysis product compositions prepared using the methods of this disclosure. The protein components of the composition may include free amino acids, oligopeptides, and / or polypeptides of about 25 kD or less (e.g., about 20 kD or less, about 15 kD or less, about 10 kD or less, about 5 kD or less, about 3 kD or less, or about 2 kD or less). The protein content of the composition can be analyzed by measuring the total amino acid content using any preferred method (e.g., liquid chromatography). In some embodiments, the high protein organic content is about 10% by weight or more, about 30% by weight or more, about 40% by weight or more of the organic content. The total organic content includes amounts of amino acids in the ranges of approximately 50% by weight or more, approximately 60% by weight or more, approximately 70% by weight or more, approximately 80% by weight or more, and approximately 90% by weight or more. In some embodiments, the high protein organic content includes amounts of amino acids in the ranges of approximately 10% by weight to approximately 98% by weight of the organic content, for example, approximately 20% by weight to approximately 98%, approximately 30% by weight to approximately 98%, approximately 40% by weight to approximately 98%, approximately 50% by weight to approximately 95%, approximately 60% by weight to approximately 95%, approximately 70% by weight to approximately 95%, and approximately 75% by weight to approximately 95%. The total organic content can be measured using any preferred method.

[0190] In some embodiments, the protein hydrolysis product contains an amount of amino acids of about 10% by weight or more of the total dry weight (w / w), for example, about 20% by weight or more, about 30% by weight or more, about 40% by weight or more, about 50% by weight or more, about 60% by weight or more, about 70% by weight or more, about 80% by weight or more, about 85% by weight or more, or about 90% by weight or more of the total dry weight (w / w). In some embodiments, the protein hydrolysis product has a total dry weight (w / w) of about the same as or greater than the amino acid content (w / w) of the starting biomass. In some embodiments, the protein hydrolysis product has an organic matter content of about 10% by weight or more of the biomass content, for example, about 20% by weight or more, about 30% by weight or more, about 40% by weight or more, about 50% by weight or more, about 60% by weight or more, about 70% by weight or more, about 80% by weight or more, or about 90% by weight or more.

[0191] In some embodiments, the protein hydrolysis product has an ash content of about 40% by weight or less of the biomass content, for example, about 30% by weight or less, about 20% by weight or less, about 10% by weight or less, about 7% by weight or less, or about 5% by weight or less. In some embodiments, the protein hydrolysis product has an ash content of about 10% by weight of the dry biomass content. In some embodiments, the ash content of the protein hydrolysis product is about 5%. In certain embodiments, the ash content of the protein hydrolysis product is less than 1% higher than the ash content of the starting biomass. In certain embodiments, the ash content of the protein hydrolysis product is less than or equal to the ash content of the starting biomass. The ash content of the protein hydrolysis product or biomass can be determined by known methods, such as placing the protein hydrolysis product or biomass sample in a tare crucible and performing an ash cycle in a muffle furnace.

[0192] In some embodiments, the protein hydrolysate composition is free from or substantially free from chelating agents (e.g., EDTA, EGTA, etc.), chaotropic reagents (e.g., urea, phenol, etc.), and / or surfactants (e.g., SDS, etc.). In some embodiments, the protein hydrolysate composition is free from or substantially free from synthetic chelating agents, chaotropic reagents, and / or surfactants. In some embodiments, the protein hydrolysate composition is produced from a microbial source without using any chelating agent, chaotropic reagent, and / or surfactant, or a protein hydrolysate composition free from chelating agents, chaotropic reagents, and / or surfactants in detectable amounts (e.g., free from exogenous chelating agents, chaotropic reagents, and / or surfactants in detectable amounts).

[0193] In some embodiments, the protein hydrolysis product composition has a nitrogen content of about 5% (w / w) or more (for example, about 6% (w / w) or more, about 7% (w / w) or more, about 8% (w / w) or more, about 9% (w / w) or more, about 10% (w / w) or more, about 13% (w / w) or more), and in some embodiments, about 20% (w / w) or less, about 18% (w / w) or less, about 15% (w / w) or less, 13% (w / w) The nitrogen content is less than or equal to about 11% (w / w). In some embodiments, the protein hydrolysis product composition has a nitrogen content ranging from about 5% (w / w) to about 20% (w / w), for example, about 6% (w / w) to about 18% (w / w), about 7% (w / w) to about 15% (w / w), and about 8% (w / w) to about 15% (w / w). The nitrogen content can be measured using any suitable method.

[0194] In some embodiments, the protein hydrolysis product composition has a phosphate content expressed in P2O5 equivalents of about 5% (w / w) or more, for example, about 6% (w / w) or more, about 8% (w / w) or more, or about 10% (w / w) or more, and in some embodiments, it has a phosphate content of about 15% (w / w) or less, for example, about 13% (w / w) or less, about 11% (w / w) or less, or about 10% (w / w) or less. In some embodiments, the protein hydrolysis product composition has a phosphate content expressed in P2O5 equivalents ranging from about 5% (w / w) to about 15% (w / w), for example, in the range of about 6% (w / w) to 13% (w / w), or about 6% (w / w) to about 11% (w / w). The phosphate content can be measured using any suitable method.

[0195] In some embodiments, the protein hydrolysate composition has a potassium content expressed in K2O equivalents of about 5% (w / w) or more, for example, about 7% (w / w) or more, about 10% (w / w) or more, about 12% (w / w) or more, or about 15% (w / w) or more, and in some embodiments, it has a potassium content of about 20% (w / w) or less, for example, about 19% (w / w) or less, about 18% (w / w) or less, about 16% (w / w) or less, or about 15% (w / w) or less. In some embodiments, the protein hydrolysate composition has a potassium content expressed in K2O equivalents ranging from about 5% (w / w) to about 20% (w / w), for example, in the range of about 7% (w / w) to about 19% (w / w), or about 10% (w / w) to about 19% (w / w). The potassium content can be measured using any suitable method.

[0196] In some embodiments, the protein hydrolysis product composition has an NPK (nitrogen, phosphorus, potassium) content of at least 5:5:5 by weight.

[0197] In some embodiments, the protein hydrolysate composition has a sodium content of about 1% (w / w) or less, for example, about 0.8% (w / w) or less, about 0.6% (w / w) or less, about 0.4% (w / w) or less, and about 0.3% (w / w). In some embodiments, the protein hydrolysate composition has a chloride content of about 1% (w / w) or less, for example, about 0.8% (w / w) or less, about 0.6% (w / w) or less, about 0.4% (w / w) or less, and about 0.3% (w / w). The protein hydrolysate composition may be substantially free of manganese, calcium, copper, and / or zinc. In some embodiments, the protein hydrolysate composition does not contain detectable amounts of manganese, calcium, copper, and / or zinc. In some embodiments, the protein hydrolysate composition contains one or more of manganese, calcium, copper, and / or zinc.

[0198] Protein hydrolysis product compositions may be in liquid form, suspension, or slurry, or may be substantially dry. In some embodiments, the protein hydrolysis product composition is in liquid form. In some embodiments, the protein hydrolysis product composition is a dry powder, for example, a lyophilized powder. In some embodiments, the protein hydrolysis product composition is a slurry, suspension, or emulsion.

[0199] V. Use of protein hydrolysate compositions Methods of using the protein hydrolysate compositions described herein are also disclosed. The protein hydrolysate compositions of this disclosure may be used in a variety of agricultural or horticultural settings. In some embodiments, the protein hydrolysate is used as a biostimulant and / or plant nutrient or precursor thereof. As a biostimulant, the protein hydrolysate composition may promote the growth of plants or livestock, or other microorganisms or organisms or cell cultures, when provided or applied. In some embodiments, the protein hydrolysate composition is provided to plants by application to the foliage or soil. In some embodiments, the protein hydrolysate composition is provided to plants by application to the foliage or soil. In some embodiments, the protein hydrolysate composition is provided using hydroponics, hydroculture, or aquaponics. In some embodiments, the protein hydrolysate produced according to the present invention may be used as a substitute for animal-derived protein hydrolysate. In some embodiments, the protein hydrolysate provides a vegan substitute for fish hydrolysate or fish emulsion.

[0200] Protein hydrolysate compositions can serve as substitutes for biostimulant products and / or components or precursors of plant nutrients. In some embodiments, protein hydrolysate compositions are combined with preservatives. Any suitable preservative may be used. Suitable preservatives include, but are not limited to, citric acid, benzoic acid, propylene glycol, propionic acid, sorbic acid, zinc sulfate, iron sulfate, copper sulfate, and / or silver chloride. In some embodiments, protein hydrolysate compositions are combined with fertilizers. In some embodiments, protein hydrolysate compositions are combined with trace metals, including, but not limited to, one or more of iron, copper, zinc, boron, manganese, calcium, molybdenum, and magnesium. In some embodiments, protein hydrolysate compositions are combined with herbicides, insecticides, and / or fungicides. Suitable herbicides, insecticides, and / or fungicides include, but are not limited to, thiophanate-methyl, chlorothalonil, captan, piperalin, phenalimol, metalaxyl, ethoxythialdiazole, pyretin, algicidal agents, oryzalin, aldoxylaryl polyethoxyethanol, glyphosate, and naphthalene.

[0201] In some embodiments, protein hydrolysis product compositions are provided to promote the growth of edible crops. In such particular embodiments, the edible crop comprises at least one member selected from fruits, vegetables, tubers, and grains. Examples of agricultural products include vegetables such as broccoli, cauliflower, artichokes, peas, beans, kale, cabbage, spinach, arugula, beet greens, bok choy, Swiss chard, nai sprouts, white turnip leaves, endive, lettuce, mustard, green vegetables, watercress, garlic, leeks, gai Ian, chives, Brussels sprouts, capers, kohlrabi, celery, rhubarb, cardoon, Chinese celery, lemongrass, asparagus, bamboo shoots, galangal root, ginger, soybeans, mung beans, urad, carrot parsnips, beets, turnips, radishes, rutabaga, turnips, burdock, onions, charlottes, leeks, garlic, green beans, lentils, and snow peas; tomatoes, cucumbers, eggplants, zucchini, pumpkins, melons, bell peppers, eggplants, tomate Fruits such as yogurt, chayote, okra, breadfruit, avocado, blackcurrant, redcurrant, gooseberry, guava, lucuma, chili pepper, pomegranate, kiwi, grape, cranberry, blueberry, orange, lemon, lime, grapefruit, blackberry, raspberry, boysenberry, pineapple, fig, mulberry, hedge apple, apple, rosehip, and strawberry; nuts such as almonds, pecans, walnuts, peanuts, pine nuts, and pistachios; tubers such as potatoes, sweet potatoes, cassava, yams, and dahlias; and grains or cereals such as corn, rice, wheat, barley, sorghum, millet, oats, rye, fonio, buckwheat, and quinoa.

[0202] In other embodiments, the crop is an ornamental crop. In such particular embodiments, the ornamental crop includes at least one member selected from turfgrass, trees, shrubs, and flowers. In other embodiments, the crop is a mushroom or fungus. Examples of fungal crops include, but are not limited to, Agaricus bisporus (peony, crimini, portabella), Coprinus quadrifidas, Lepistanuda, and Pleurotus ostreatus (agaricus). In particular embodiments, the protein hydrolysis products described herein are applied to mushrooms or fungi such as one or more of Agaricus bisporus, Coprinus quadrifidas, Lepistanuda, and Pleurotus ostreatus.

[0203] In some embodiments, protein hydrolysate compositions are used as a nutrient source for other organisms, e.g., animals, humans, or cells (prokaryotes or cells), or as a precursor of such nutrient source or components forming such nutrient source. In certain embodiments, hydrolysates prepared as described herein are used as supplements for cell culture. Certain embodiments disclosed herein relate to hydrolysates applied in sports medicine, and in particular, in certain non-limiting embodiments, the consumption of the hydrolysate is made to be absorbed by the body more quickly than the unchanged protein, and thus nutrients are delivered to muscle tissue to the greatest extent possible. In certain embodiments, the hydrolysate is rich in antioxidants, and / or L-aspartic acid, and / or manganese and / or selenium. Certain embodiments relate to the use of microbial hydrolysates as ingredients in pet food, including but not limited to food for mammals (dogs, cats, rabbits, rodents, horses, etc., or poultry such as chickens and turkeys). In certain embodiments, the hydrolysates can also be used as high-value-added nutritional additives for animal feed, more specifically for livestock (e.g., cattle, sheep, goats, pigs), or aquaculture (e.g., fish, shellfish), or insects (e.g., bees), or invertebrates (e.g., earthworms), or heterotrophic microorganisms (e.g., yeast: E. coli), or for animal feed for livestock, pets, or human consumption.

[0204] Preferred uses of the protein hydrolysis product compositions of this disclosure are described, for example, in PCT International Publication No. WO2018 / 144965, which is incorporated herein by reference in its entirety.

[0205] Other features of this disclosure will become clear in full from the following descriptions of embodiments. The following embodiments are for illustrative purposes only and do not limit the present invention. [Examples]

[0206] Example 1: Protein hydrolysis product produced from Cupriavidus necator culture Production of protein hydrolysis products from microbial cultures Cupriavidus necator strain was grown in growth medium with glucose as a carbon source. After growth, the total cellular biomass was isolated from the growth medium and dried. A portion of the dried biomass was processed as follows: 1.2 g of dried biomass was suspended in 100 ml of water. 2. The suspended biomass was homogenized using a T25 Turrax stick at 15,000 rpm for 1 minute. 3.10N KOH strain was added to raise the pH. 4. The alkaline biomass solution was autoclaved at 121°C for 30 minutes with slow exhaust. 5. After the autoclaving cycle, the autoclaved solution was cooled to room temperature. 6. The solution was neutralized with 1M KH2PO4 buffer at pH 5.8, lowering the pH to approximately 8.8. 7.100 μl of 1 mg / ml bacterial alkaline protease (produced in 10 mM Tris buffer, pH 7.4) was added to the neutralized solution. 8. The resulting reaction product was digested overnight in a shaking water bath at 55°C. 9. After enzymatic hydrolysis, the reaction product was centrifuged and the supernatant was separated from the pellet.

[0207] Content analysis of protein hydrolysis products The supernatant and pellet fragments were analyzed using SDS-PAGE gel (Figure 6). The supernatant fragments contained most protein and polypeptide fragments, which were less than 15 kD.

[0208] Figure 6: Fragment gel images of the pellet (lane 1) and supernatant (lane 2) of the enzymatic hydrolysis product. Lane 3 contains the marker.

[0209] The supernatant from Example 1 was freeze-dried to produce a powder, and the following was analyzed. 1.Nitrogen content 2. Ash and humidity levels 3. Phosphorus, phosphoric acid, potassium, and potassium content 4. Amino acid content (including all 20 amino acids, such as cysteine, methionine, and tryptophan) 5. Complete material balance by process yield and weight

[0210] Example 2: Protein hydrolysis product produced from Cupriavidus necator culture The protein hydrolysis product was prepared from the C. necator strain culture of Example 1. It was added to biomass suspended in KOH to obtain a pH of 11.3.

[0211] The supernatant was freeze-dried to produce a powder, which was then analyzed for the following: 1.Nitrogen content (Table 1) 2. Ash and humidity levels (Table 1) 3. Phosphorus, phosphoric acid, potassium, and potassium content (Tables 1 and 2) 4. Amino acid content (all 20 amino acids, including cysteine, methionine, and tryptophan) (Table 2) 5. Complete material balance by process yield and weight [Table 1] [Table 2]

[0212] This protocol yielded protein hydrolysis products from bacterial cell biomass. The protein hydrolysis product (PH) contained a complex mixture of amino acids, proteins, and inorganic nutrients including NPKs. PH had a very high % amino acid (wrt organic matter) content. Organic matter in PH accounted for 80%, and 92% of this organic matter was amino acids.

[0213] Example 3: Production of protein hydrolysis products from a second C. necator culture. The second portion of the dried biomass derived from the C. necator strain culture was treated as in Example 1. KOH was added to the suspended biomass until the final volume was 50 mM.

[0214] The obtained supernatant was freeze-dried and analyzed as in Example 2. The results of the analysis are shown in Tables 3 and 4. [Table 3] [Table 4]

[0215] Example 4: Production of protein hydrolysis products from large-scale Cnecator cultures Approximately 310 g of total cellular biomass was obtained from C. necator. The process described in Example 1 was scaled up to process a large biomass. The dried biomass was suspended in water at a ratio of 20 mg / ml. KOH was added to the suspended biomass to a concentration of 50 mM. The pH of the suspension after adding KOH was 12.3. After autoclaving, the pH was 10.6-10.9. The autoclaved suspension was neutralized with phosphate buffer (KH2PO4) to a pH of 8.9-9.0. The pH after overnight hydrolysis was 8.01.

[0216] Example 5: Effect of pH (1) The amino acid and organic matter content was compared between protein hydrolysis products derived from two different substrate treatments. The protein hydrolysis product from Example 2 (pH titrated to 11.3 before autoclaving) had higher organic matter and amino acid content compared to the protein hydrolysis product prepared using KOH added to 50 mM (pH approximately 12) before autoclaving. Table 5 shows the amino acid and nitrogen content measured relative to the dry weight of the protein hydrolysis product, respectively. [Table 5]

[0217] Example 6: Effects of pH (2) The pH of the biomass suspension obtained from C. necator culture was adjusted to pH 9.6 with IONNaOH. The alkaline suspension was autoclaved at 121°C for 20 minutes with slow exhaust. The hydrolyzed suspension was centrifuged, and the supernatant and pellet fragments were assayed for protein content by SDS-PAGE (Figure 7). Incompletely fragmented proteins were found in the supernatant.

[0218] Figure 7, a gel image, shows the protein distribution between pellets and supernatant fragments of five independent samples. Sample 1: lanes 2-4, Sample 2: lanes 5-7, Sample 3: l-lanes 8-10, Sample 4: lanes 11-13 (lanes for each sample correspond to input, pellet, and supernatant fragment, respectively), Sample 5: l-lanes 14 and 15 (l-lanes correspond to pellet and supernatant fragment, respectively). 5g of wet biomass (lanes 2-13) or dry biomass (lanes 14-15) was processed. Samples 1, 3, and 5 were treated with base, while samples 2 and 4 were not treated with base. Lane 1: Marker.

[0219] Example 7: Effects of neutral buffering agent, surfactant, chelating agent, and chaotropic reagent Sample sets were prepared, each containing 0.3 g of dried biomass, which was suspended and homogenized. The samples were treated with 1 ONNaOH added to 0.1 N, autoclaved at 110°C for 10 minutes, cooled under fast exhaust, neutralized with 1 M potassium phosphate buffer to pH 5.8 (unless otherwise specified), and digested overnight at 55°C with bacterial alkaline protease.

[0220] All samples were treated with enzymes overnight, then centrifuged at 11,000 rpm for 15 minutes, and the supernatant was separated from the pellet. Different fragments were analyzed by SDS-PAGE, Lowry assay, and OPA assay.

[0221] To test the effect of surfactant treatment, several samples were treated with ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20, or Pluronic PF-68 added to biomass suspensions before autoclaving (Figures 8A, 8B). The surfactant was added at a final amount of 10% relative to the dry weight of the biomass. To test the effect of neutralizing buffers, several samples were neutralized with IM Tris buffer pH 7.4 instead of potassium phosphate buffer (Figures 8A, 8B).

[0222] Figure 8A gel image shows the protein distribution between pellet and supernatant fragments of samples treated with Tris buffer and different surfactants. Lane 1: Marker, Lane 2: SDS (pellet); Lane 3: SDS (supernatant); Lane 4: No surfactant (pellet); Lane 5: No surfactant (supernatant); Lane 6: Ammonium lauryl sulfate (pellet); Lane 7: Ammonium lauryl sulfate (supernatant); Lane 8: Triton X-100 (pellet); Lane 9: Triton X-100 (supernatant); Lane 10: Tween 80 (pellet); Lane 11: Tween 80 (supernatant); Lane 12: Tween 20 (pellet); Lane 13: Tween 20 (supernatant); Lane 14: Pluronic PF-68 (pellet); Lane 15: Pluronic PF-68 (supernatant).

[0223] Figure 8B gel image shows the protein distribution between pellet and supernatant fragments of samples treated with potassium phosphate buffer and different surfactants. Lane 1: Marker, Lane 2: SDS (pellet); Lane 3: SDS (supernatant); Lane 4: No surfactant (pellet); Lane 5: No surfactant (supernatant); Lane 6: Ammonium lauryl sulfate (pellet); Lane 7: Ammonium lauryl sulfate (supernatant); Lane 8: Triton X-100 (pellet); Lane 9: Triton X-100 (supernatant); Lane 10: Tween 80 (pellet); Lane 11: Tween 80 (supernatant); Lane 12: Tween 20 (pellet); Lane 13: Tween 20 (supernatant); Lane 14: Pluronic PF-68 (pellet); Lane 15: Pluronic PF-68 (supernatant).

[0224] To test for different bases, several samples were treated with KOH instead of NaOH before autoclaving (Figure 9). To test the effect of chelating agents, several samples were treated with EDTA at a final concentration of 5 mM before autoclaving (Figure 9).

[0225] Figure 9 gel images show the protein distribution between pellet and supernatant fragments of samples treated with NaOH or KOH, and different combinations of EDTA and urea. NaOH-treated sample (left): Lane 1: Marker, Lane 2: EDTA, urea (pellet); Lane 3: EDTA, urea (supernatant); Lane 4: EDTA (pellet); Lane 5: EDTA (supernatant); Lane 6: urea (pellet); Lane 7: urea (supernatant); Lane 8: Control (pellet); Lane 9: Control (supernatant); Lane 10: Marker (low range); KOH-treated sample (right): Lane 1: Marker, Lane 2: EDTA, urea (pellet); Lane 3: EDTA, urea (supernatant); Lane 4: EDTA (pellet); Lane 5: EDTA (supernatant); Lane 6: urea (pellet); Lane 7: urea (supernatant); Lane 8: Control (pellet); Lane 9: Control (supernatant); Lane 10: Marker (low range).

[0226] Conclusion: KOH is suitable as a basic reagent. Treatment with urea and EDTA increased the total protein and supernatant protein yields. Chaotropic and chelating agents such as urea or EDTA can be removed from the resulting supernatant fragment by buffer exchange.

[0227] Example 8: Biostimulant effect of protein hydrolysis products The biostimulant effects of the protein hydrolysates of this disclosure were tested on a variety of plants, including turfgrass, radishes, lettuce, and mushrooms. Plant or mushroom growth was compared with and without the application of the protein hydrolysates. In some cases, the protein hydrolysates were combined with other components to create plant supplements and applied to plants. Plants could be grown in soil or hydroponically. The protein hydrolysates could be supplied in soil or applied to the leaves. The biostimulant effect could be measured by growth density (turfgrass), weight, nitrogen uptake, or chlorophyll content, etc.

[0228] Example 9: Protein hydrolysis product produced from Cupriavidus necator culture Cupriavidus necator strain was cultured in growth medium with fructose as a carbon source. After growth, the entire cellular biomass (WCB) was isolated from the growth medium and dried. The WCB was processed as follows: 1.32 g of dried WCB was suspended in 350 ml of water. 2. The mixture was homogenized for 1 minute at 13,200 RPM using an UltraTurrax stick. 3.13.51 ml of phosphoric acid (H3PO4) was added at an 85% concentration (14.8 M). 4. The suspension was transferred to the pressure tube. 5. The acidic biomass suspension was autoclaved at 121°C and 15 psi for 1 hour. 6. After autoclaving, the suspension was cooled to room temperature. The pH of the suspension after autoclaving was 1.5. 7. The solution was neutralized with a sterilized acidic suspension of 2.2 g Ca(OH)2 per 50 ml. Neutralization was carried out overnight at 4°C, and the pH after neutralization was 5.5. 8. The neutralized suspension was centrifuged at 9603 RPM for 20 minutes at 4°C. 9. The supernatant was collected. 10. The supernatant and pellet were frozen at -80°C for 3 hours, and then transferred to a freeze-dryer (LabconcoFreezone, 4.SL, -SOC). 11. The sample was freeze-dried until completely dry.

[0229] Gel analysis Lyophilized acid hydrolysis samples ("test") were analyzed by SDS-PAGE (Novex 4-20% Tris-glycine) and compared with lyophilized samples (control) that had not been subjected to acid hydrolysis or calcium hydroxide neutralization. The acid hydrolysates were denser and exhibited a brighter band than the pellet fragments, indicating that a significant amount of protein was extracted and solubilized in the supernatant.

[0230] Content analysis of protein hydrolysis products Freeze-dried acid hydrolysis samples ("Test") were analyzed for the following items and compared with freeze-dried samples that had not been subjected to acid hydrolysis or calcium hydroxide neutralization ("Control"). 1. Moisture, ash, and nitrogen (Table 6) 2. Mass balance 3. Nitrogen balance 4.% Nitrogen Solubility

[0231] Table 6 shows the percentage moisture, ash, and nitrogen of the freeze-dried dry mass. [Table 6]

[0232] Example 10: Production of low-ash hydrolysate from defatted whole-cell biomass Several methods were attempted to increase the amino acid content and decrease the inorganic or ash content of protein hydrolysis products. These methods were tested on defatted biomass.

[0233] To degrease the whole cellular biomass, the biomass was suspended in ethanol at a ratio of 3.75 mL per gram of biomass, and then 0.5 mL of NH4OH per gram of biomass was added. This solution was stirred on a magnetic stirring plate for 30 minutes and then filtered through Whatman 4 filter paper. Next, the lipid-containing filtrate (EtOH / NH4OH) was dried in a sand bath at 50°C. The remaining defatted biomass was air-dried overnight in a fume hood and then oven-dried at 40°C for 4-6 hours. This dried, defatted biomass was then used in subsequent hydrolysis reactions. The following modifications were attempted with the defatted biomass.

[0234] Enzyme-only digestion: A 2% solid solution was prepared using defatted biomass and distilled water. This solution was homogenized, adjusted to pH 9 with 1 ONKOH, and bacterial alkaline protease was added. The solution was incubated overnight in a shaking water bath at 55°C. The hydrolysates were then centrifuged at 26,000 x g for 30 minutes to separate the supernatant and pellet fragments, which were then freeze-dried.

[0235] Precipitation of inorganic substances using solid Ca(OH)2: A 2% solid solution was prepared with defatted biomass and distilled water. This solution was homogenized, the pH was adjusted to 11 or 12 with Ca(OH)2, autoclaved at 110°C for 10 minutes, and then the pH was adjusted to 9 with H3PO4. Next, bacterial alkaline protease enzymes were added and incubated overnight in a shaking water bath at 55°C. The hydrolysates were then centrifuged at 26,000xg for 30 minutes to separate the supernatant and pellet fragments, which were then lyophilized.

[0236] Table 7 shows the mass, nitrogen yield, and ash content for each condition. The enzyme digestion sample had the lowest ash content, while the pellets of the chemical hydrolysis sample had a high ash content (approximately 22-32%) due to the presence of precipitated inorganic matter. Protein hydrolysis products derived from the chemical hydrolysis supernatant had a lower ash content compared to the starting material, as observed in Table 7, and a higher amino acid content, as observed in Table 8. [Table 7] [Table 8-1] [Table 8-2]

[0237] Example 11: Culture of low-ash protein hydrolysate produced from Cupriavidus necator Cupriavidus necatorPHB-negative mutant (DSM541) was cultured in growth medium with CO2 as a carbon source. After growth, the total cellular biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows.

[0238] Degreased whole cell biomass (WCB) was mixed with ammonium hydroxide and methanol (1:1:0.4, WCB:NH4OH:MeOH), and the mixture was placed in a tightly sealed container and stirred in a fume hood for 1 hour to degrease (extract lipids). The recovered filtrate contained the extracted lipids. The residue on the filter was degreased and dried overnight in a 40°C incubator.

[0239] Protein hydrolysis on delipidized material with H3PO4 and Ca(OH)2: A solid loaded with 8% defatted dry mass was rehydrated with the required amount of ID water, then vigorously stirred at 15000 rpm for 1 minute using a turret stick, and strong phosphoric acid (14.8 M) was added to a final reaction concentration of 0.5 M. The reaction was carried out in a pressure tube with the screw cap tightly closed, then autoclaved at 121°C, 15 psi for 1 hour, and the reaction product was cooled in a fume hood. Calcium hydroxide was added to neutralize the pH to 8-9, and the pH was confirmed by a pH meter. The protein hydrolysis product (supernatant) was centrifuged at 10000xg for 20 minutes at 7°C. The resulting liquid protein hydrolysis product was freeze-dried to produce a dry powder.

[0240] The measured ash content of the hydrolysates was 7.04%. The ash content was also measured by placing a minimum of 300 mg of protein hydrolysate powder in a tared crucible and running an ash cycle in a muffle furnace, and by external laboratory analysis (SGS, North America).

[0241] Example 12: Culture of low-ash protein hydrolysates produced from Cupriavidus necator Cupriavidus necator strain (-PHB) was cultured in growth medium with CO2 as a carbon source. After growth, the total cellular biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows.

[0242] Degreased whole cell biomass (WCB) was mixed with ammonium hydroxide and methanol (1:1:0.4, WCB:NH4OH:MeOH), and the mixture was placed in a tightly sealed container and stirred in a fume hood for 1 hour to degrease (extract lipids). The recovered filtrate contained the extracted lipids. The residue on the filter was degreased and dried overnight in a 40°C incubator.

[0243] Protein hydrolysis with H3PO4 and CO2: A solid loaded with 2% defatted dry mass was used, rehydrated with the required amount of ID water, and then thoroughly stirred with a turret stick at 15,000 rpm for 1 minute. The pH of the reaction mixture was increased by adding a 28%-30% (pre-made) NH4OH solution in a fume hood. The mixture was transferred to a pressure tube (size: 120 mL) with a working volume of 50 mL and autoclaved at 10°C for 10 minutes with slow exhaust. The pH of the solution after autoclaving was 10.82. The pH was reduced to 9 by bubbling CO2 by inserting a cannula / 18G needle for 10-20 minutes. Enzymatic digestion was carried out overnight with bacterial alkaline protease at 55°C and 110 rpm. The supernatant and pellet (110 rpm rich natural pellet) containing soluble hydrolyzed protein were separated by centrifugation at 20,000 x g for 20 minutes at 5°C. The protein hydrolysis products were freeze-dried.

[0244] The measured ash content of the protein hydrolysate was 5%. The ash content was also measured by placing a minimum of 300 mg of protein hydrolysate powder in a tared crucible and running an ash cycle in a muffle furnace, and by external laboratory analysis (SGS, North America).

[0245] The results are shown in Table 9. [Table 9-1] [Table 9-2]

[0246] Example 13: Culture of low-ash protein hydrolysates produced from Cupriavidus necator Cupriavidus necatorPHB-negative mutant (DSM541) was cultured in growth medium with either CO2 or sugar as a carbon source. After growth, the total cellular biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows.

[0247] Defatting and drying of whole-cell biomass: Whole-cell biomass was defatted by treatment with ammonium hydroxide and ethanol (1:4:0.5 w / v). The biomass and solvent slurry was stirred in a tightly capped glass bottle for 30 minutes and vacuum filtered through Whatman 4 filter paper in a fume hood. The recovered filtrate contained lipid fragments, and the defatted residue was air dried overnight and then dried in an incubator at 40 °C for 4 - 6 hours.

[0248] Protein hydrolysis of defatted biomass with Ca(OH)2 and H3PO4: The dried defatted biomass was resuspended in DI water to a final concentration of 2%. The biomass solution was mixed with an IKA Ultra-Turax at 15000 rpm and resuspended completely and homogeneously. Ca(OH)2 was added to the biomass solution to bring the pH to 11. The solution was transferred to a glass culture bottle, autoclaved at 110 °C for 10 minutes, and then cooled to room temperature. The solution was neutralized to pH 9 using H3PO4. Bacterial alkaline protease (Sigma P8038) was added to the solution at a concentration of active units / g biomass. The biomass solution was placed in a shaking water bath at 55 °C overnight. After digestion for 16 - 24 hours, the slurry was incubated in a water bath at 95 °C for 10 minutes to inactivate the enzyme. Next, the biomass slurry was cooled to room temperature, and the protein hydrolysis product (supernatant fragment) was separated by centrifugation at 26000 xg for 30 minutes at 7 °C. The obtained protein hydrolysis product solution was frozen in an -80 °C freezer and then freeze-dried. The moisture, ash, and nitrogen content of the dry powder were identified, and the protein profile was analyzed via SDS-PAGE analysis. There were no proteins exceeding 2000 Daltons in the protein hydrolysis product, and the obtained ash content was 10.5%.

[0249] Example 14: Demonstration of the biostimulant effect in protein hydrolysis products produced using acidic hydrolysis in Brassica rapa species After incubation using phosphoric acid, the protein hydrolysate produced by neutralizing with calcium hydroxide to precipitate phosphate ions and neutralizing the solution to pH 5 was tested for its impact as a biostimulant using Brassica rapa (turnip) seeds according to OECD GUIDELINES FOR THE TESTING OF CHEMICALS 208 and ISO 11269-1, 2.

[0250] Ten seeds were sown in 8×8×7.5 cm pots filled with approximately 300 g of soil (10% peat, 20% topsoil sieved through a 2 mm mesh, 70% sand). All pots were watered to 70% of the soil water-holding capacity. When 50% of the control seeds had germinated, thinning was done to leave 5 seeds per pot. Water / treatment was applied either on the day after sowing (day 1), after germination (day 7), or on both days 1 and 7. A total of nine treatment groups, including a control to which water and boric acid were applied, were tested with and without basal fertilizer (NPK 150-90-60) for each treatment group. All treatments receiving the protein hydrolysate treatment showed 3.5-fold overall biomass growth compared to the water control and the control group treated with NPK fertilizer. The treatment group receiving the protein hydrolysate application only on day 7 had the maximum overall growth.

[0251] Figure 10 shows, from left to right: (a) water, (b) NPK fertilizer, (c) turnips treated with the protein hydrolysate.

[0252] Example 15: Demonstration of the biostimulant effect of protein hydrolysates produced via acidic and alkaline hydrolysis Two protein hydrolysates were tested as biostimulants for lettuce plants. The first protein hydrolysate was prepared using acidic hydrolysis as described herein. The second protein hydrolysate was prepared using alkaline hydrolysis as described herein. Both were tested for their biostimulant impact in lettuce (Lactucasaliva). All procedures were as described in Xu, C., et al. (2017) HorTechnology 27(4):539-543. Lettuce seedlings were germinated in peat moss and then transferred to pots containing sandy loam. The plants were watered, rotated twice a week, and exposed to 700 μmol photons / m². 2 They were grown under a 14-hour photoperiod with the specified exposure level.

[0253] To compare protein hydrolysis products, water treatment and commercial fish and seaweed hydrolysis treatments were used as controls.

[0254] A Hoagland solution containing 0.4 g Hoagland salt mixture / L was used as the standard nutrient solution for all treatments and applied to the soil on the day seedlings were transplanted. All treatments were normalized so that the nitrogen in the starting soil was at a rate of 0.0165 gN / 750 ​​g, and all results report the average of four plants from each treatment group.

[0255] Figure 11 shows, from left to right: (a) lettuce treated with commercial fish and seaweed hydrolysates, (b) acid hydrolysates, and (c) base hydrolysates.

[0256] Specific embodiments of this disclosure are described herein in detail so that those skilled in the art can practice the entire scope of the invention. However, it should be understood that possible variations of this disclosure that are not described in detail are also included in the invention and the appended claims. Therefore, these descriptions herein are added for illustrative purposes only and are not intended to limit the invention. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials and configurations described herein are for illustrative purposes only, and actual parameters, dimensions, materials and configurations will vary depending on the particular purpose or the purpose for which the teachings herein are used. Those skilled in the art will understand or confirm that many of the equivalents of the specific embodiments disclosed herein are used, as well as conventional experimental methods. Therefore, it should be understood that the embodiments described herein are for illustrative purposes only and are included in the scope of the appended claims and equivalents, and that the invention may be practiced outside the detailed descriptions and claims. The invention relates to each individual feature, system, article, material, kit, composition and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, compositions and / or methods is within the scope of the present invention, provided that they are not contradictory to each other.

[0257] All publications, patents, and patent applications described herein are incorporated in whole by reference for all purposes, and each individual publication, patent, and patent application is incorporated in the same way, as it is clearly and individually described, when it is incorporated in whole by reference for a certain purpose.

Claims

1. 1. A method for producing a protein hydrolysate comprising the steps of: (a) culturing microbial cells in a culture medium, thereby producing a microbial biomass comprising microbial proteins; (b) harvesting the microbial biomass; (c) producing a biomass suspension composition from the harvested microbial biomass, wherein the biomass suspension composition comprises the microbial protein; and (d) optionally adjusting the pH of the biomass suspension composition to a pH within a first target pH range of at least 11.5, wherein the biomass suspension composition produced in step (C) or the pH adjusted suspension composition produced in step (d) is an alkaline suspension composition; and (e) heating the alkaline suspension composition to a first temperature of at least 110° C. for a first period of at least 5 minutes to produce an alkaline hydrolysate suspension comprising hydrolyzed microbial proteins; The method comprising:

2. i) step (e) comprises applying a pressure of at least 15 psi (103.4214 kPa) to the alkaline suspension composition for at least a portion of a first period of time; or ii) adjusting the pH in step (d) comprises adding one or more bases selected from potassium hydroxide, ammonium hydroxide, ammonia, calcium hydroxide and sodium hydroxide; The method of claim 1.

3. After step (e), further (i) neutralizing the alkaline suspension composition by adding a neutralizing agent to the alkaline suspension composition, thereby forming a neutralized suspension composition, wherein the pH of the neutralized suspension composition is within a second target pH range of 6.5 to 9.5; 3. The method of claim 1 or 2, comprising: (ii) adding a protease to the neutralized suspension composition at a second temperature of at least 40° C. for a second period of at least 1 hour, thereby further hydrolyzing the microbial proteins and forming a protease hydrolysate suspension comprising hydrolyzed microbial proteins.

4. i) neutralizing the alkaline suspension composition of step (i) may comprise neutralizing the alkaline suspension composition with ammonium phosphate, sodium citrate, potassium citrate, citric acid, sodium phosphate, phosphoric acid, a phosphate buffer, CO ２ adding one or more neutralizing agents selected from: ii) the protease in step (ii) is an endoprotease, an exoprotease, or a serine alkaline protease; The method according to claim 3.

5. moreover, 4. The method of claim 3, further comprising the step of: (f) separating a liquid supernatant from solid material in the protease hydrolysate suspension, the supernatant comprising soluble hydrolyzed microbial proteins.

6. i) the microbial biomass produced in step (a) constitutes at least 0.1% w / v of the culture medium; or ii) the microbial biomass is subjected to lysis after step (b) and before step (c); or iii) a chelating agent is added to the biomass suspension composition prior to step (e), optionally wherein the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); or iv) a surfactant is added to the biomass suspension prior to step (e), or v) a chaotropic agent is added to the biomass suspension prior to step (e), optionally wherein the chaotropic agent is selected from urea, thiourea, and guanidinium chloride; or vi) the microbial cells are selected from Cupriavidus, Rhodococcus, Hydrogenovibrio, Rhodopseudomonas, Hydrogenobacter, Gordonia, Arthrobacter, Streptomycetes, Rhodobacter, and / or Xanthobacter cells, optionally comprising Cupriavidus cells, and further optionally comprising Cupriavidus cells comprising Cupriavidus necator and / or Cupriavidus metallidurans cells, or vii) the culture medium contains CO as a carbon source ２ Contains, or viii) the microbial cell comprises a mutant strain that is unable to produce PHB, the mutant strain being derived from a wild-type strain that is able to produce PHB, and optionally the mutant strain is Cupriavidus necator DSM541; The method according to any one of claims 1 to 5.

7. i) drying the supernatant to produce a dry or substantially dry composition comprising the hydrolyzed microbial proteins; or ii) processing the solid material to produce a co-product, optionally wherein the co-product comprises a biopolymer, further optionally wherein the biopolymer comprises a polyhydroxyalkanoic acid (PHA), further optionally wherein the PHA comprises polyhydroxybutyric acid (PHB); or iii) at least 60% of the soluble hydrolyzed microbial proteins comprise an atomic mass of less than 30 kD; The method according to claim 5.

8. 6. A protein hydrolysate composition comprising hydrolyzed microbial proteins produced according to any of claims 1 to 5, said protein hydrolysate composition having a pH of at least 11.5 and a temperature of at least 110°C, and optionally i) the hydrolyzed microbial proteins comprise free amino acids, oligopeptides, and / or polypeptides with an atomic mass of 25 kD or less; or ii) the hydrolyzed microbial protein comprises an amount of amino acids, based on organic content (w / w), of 10% by weight or more; or iii) the hydrolyzed microbial protein comprises an amount of amino acids of 10% or more by weight, based on total dry weight (w / w); or iv) the hydrolyzed microbial protein has an ash content of 40% or less by weight based on the dry biomass content (w / w); The protein hydrolysate composition.

9. The protein hydrolysate composition of claim 8, i) use as a compound for stimulating plant growth and development and / or as a plant nutrient or a precursor thereof, or ii) use in a method for stimulating plant growth, the method comprising applying the composition of claim 14 to a seed or soil, wherein a plant grown in contact with the composition exhibits increased growth in the presence of the composition than in the absence of the composition; or iii) as a nutritional additive in animal feed, other than for humans; or iv) As a nutrient source, or a precursor to that nutrient source, or a precursor to a component that forms that nutrient source, for animals other than humans.

10. The method of claim 1, wherein the first temperature is between 110°C and 140°C.

11. The method of claim 1, wherein the first period of time is one hour or more.

12. The method of claim 2, wherein the one or more bases include calcium hydroxide.

13. The method of claim 3 or 4, wherein the neutralizing agent comprises phosphoric acid.

14. The method of claim 5, wherein step (f) comprises centrifugation or filtration.

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