Microbe protein hydrolysate composition and production method

Abstract

To provide a method for producing protein hydrolysate, and a protein hydrolysate composition.SOLUTION: There is provided a method for producing a protein hydrolysate, comprising the steps of: (a) producing microbial biomass; (b) harvesting the microbial biomass; (c) producing a biomass suspension composition from the harvested microbial biomass; (d) adjusting a 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 time period of at least 5 minutes, thereby producing an alkaline hydrolysate suspension that comprises hydrolyzed microbial protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] The present disclosure relates to the field of protein hydrolysates produced from biological sources, and methods for producing and incorporating the same into various end products. In particular, the present disclosure relates to novel methods for converting protein containing biomass produced from renewable resources, such as biological processes designed to capture carbon dioxide emissions, and other waste-to-carbon conversion or transformation processes, into protein hydrolysates. [Background technology]

[0004] Protein is a nutrient necessary for human and animal growth and for supporting and maintaining life. As such, protein is an important component of many foods and animal feeds. Furthermore, proteins and protein hydrolysates can serve as biostimulants or plant nutrients to promote the growth of, for example, plants in soil. Furthermore, 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 beneficial microorganisms in plant microbiomes and 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, which are more easily digested and absorbed.

[0005] Proteins or protein-containing biomass produced by microorganisms can be particularly useful for producing protein hydrolysates for consumption and / or use as biostimulants. Microorganisms can be used to convert carbon- and nitrogen-containing feedstocks into proteins or protein-containing biomass. Chemoautotrophic microorganisms can be advantageously used to capture carbon dioxide from the atmosphere, from carbon dioxide emission sources, or from carbon dioxide-producing conversion processes. Producing valuable products from these microbial cultures can enable greater value to be obtained from carbon fixation processes.

[0006] Numerous prior art methods exist for hydrolyzing proteins, including various chemical and enzymatic methods, which are not completely effective for a number of reasons, including the inability to hydrolyze consumable or biostimulant products to the desired level, the presence of harmful pesticide residues or by-products in those products, the inability or impracticality of scaling up to large-scale commercial processes, and the inability to use highly scalable feedstocks such as CO2. Summary of the Invention

[0007] In one aspect, the present disclosure provides a process for producing biologically derived protein hydrolysates. In certain embodiments, the process comprises: (1) culturing a microorganism in the presence of a carbon source in an aerobic or microaerobic bioprocess to grow a protein-containing biomass, the microorganism being Cupriavidus ne (2) harvesting 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 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 period of at least about 5 minutes; and (5) adding a neutralizing agent to the alkaline suspension composition to form a neutralized suspension composition. (6) optionally further adding a protease to the neutralized suspension composition and incubating the neutralized suspension composition at a second temperature range for a second period of 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 period of 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 includes: (a) adjusting the pH of a biomass suspension composition, if necessary, to a pH within a first target pH range of at least about 10, wherein the biomass suspension or pH-adjusted suspension composition produced in step (a) 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 period of at least about 5 minutes to produce an alkaline hydrolysate suspension comprising hydrolyzed 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 period of at least about 1 hour to further hydrolyze the microbial proteins and form a protease hydrolysate suspension comprising hydrolyzed microbial proteins, the process further comprising (c) separating a liquid supernatant from the alkaline hydrolysate suspension or solid material in the protease hydrolysate suspension, wherein the supernatant comprises 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 the first time period. For example, adjusting the pH in step (b) may include adding one or more bases selected from potassium hydroxide, ammonium hydroxide, ammonia, calcium hydroxide, and sodium hydroxide.

[0010] According to certain embodiments, the process comprises (1) culturing a microorganism in the presence of a carbon source in an aerobic or microaerobic bioprocess to grow a protein-containing biomass, the microorganism being Cupriavidus. (2) culturing a 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 about 0.5 or less to about 3; (4) heating the acidic suspension composition to a first temperature of at least about 40°C for a first time period of at least about 5 minutes; (5) forming a neutralized suspension composition by adding a neutralizing agent to the acidic suspension composition, wherein the pH of the neutralized suspension composition is within a second target pH range, wherein the second target pH range is about 5 to about 6.5, or about 8.0; and (6) optionally further adding a protease to the neutralized suspension composition to form a neutralized suspension composition. (6) incubating the neutralized suspension composition at a second temperature range for a second period of 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 period of 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, as needed, to a pH within a first target pH range of about 0.5 to about 3, wherein the biomass suspension or the pH-adjusted suspension composition produced in step (a) 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 period of at least about 5 minutes to produce an acidic hydrolysate suspension comprising hydrolyzed 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, thereby forming 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 period of at least about 1 hour, thereby further hydrolyzing the microbial proteins and forming a protease hydrolysate suspension comprising hydrolyzed microbial proteins. In certain embodiments, the process further comprises (c) separating a liquid supernatant from solid material in the acidic hydrolysate suspension or the protease hydrolysate suspension, wherein the supernatant comprises soluble hydrolyzed 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 the first time period. In certain embodiments, adjusting the pH in step (b) can comprise 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 certain embodiments, the process includes the steps of (1) culturing a microorganism to grow a biomass; (2) harvesting the protein-containing biomass in a suspension composition; (3) optionally adjusting the pH of the suspension composition to a pH of 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 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. (6) adding a neutralizing agent to the composition to adjust the pH to within the range of about 7.5 to about 9.5, and preferably about 8.5 to about 9; (7) optionally adding a protease to the composition to further hydrolyze the proteins; (8) recovering a supernatant from the suspension containing the hydrolyzed proteins; and (9) optionally drying the supernatant and lyophilizing the hydrolyzed proteins.

[0013] According to certain embodiments, the process includes the steps of (1) culturing a microorganism to grow a biomass; (2) harvesting the protein-containing biomass in a suspension composition; (3) optionally adjusting the pH of the suspension composition to a pH of about 3 or less, preferably about 1 to about 1.5; (4) optionally adding a chelating agent and / or surfactant to the composition; and (5) heating the composition at a temperature of at least about 40° C. for at least 1 minute, preferably between 40° C. and about 130° C. for about 10 minutes to about 8 hours. (5) adding a neutralizing agent to the composition to adjust the pH to a range of about 5 to about 7, and preferably about 6 to about 6.5; (6) adding a neutralizing agent to the composition to adjust the pH to a range of about 5 to about 7, and preferably about 6 to about 6.5; (7) optionally adding a protease to the composition to further hydrolyze the proteins; (8) recovering a supernatant containing the hydrolyzed proteins from the suspension; and (9) optionally drying the supernatant to remove the hydrolyzed proteins. and freeze-drying the protein.

[0014] In certain embodiments, any process described herein may further comprise forming hydrolyzed microbial proteins for use as biostimulants. In certain embodiments, any process described herein may comprise applying a hydrolyzed microbial protein, composition, or formulation thereof to seeds, plants, or soil, wherein plants grown in contact with the composition exhibit increased growth in the presence of the composition compared to when the composition is absent.

[0015] Also described herein are protein hydrolysates derived from proteins, including proteins or sources or proteinaceous materials, and methods for producing the same. The present disclosure includes protein hydrolysate compositions derived from microbial sources and methods for producing the same. 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 agents, and / or surfactants. The protein hydrolysate compositions of the present disclosure have a variety of agricultural or horticultural uses, such as as biostimulants. The protein hydrolysate compositions of the present disclosure can be used for nutritional or pharmaceutical purposes in animals and humans. The protein hydrolysate compositions of the present disclosure can be used as a nutrient source for cells, including prokaryotic and eukaryotic cells. Sources of the protein hydrolysates of the present disclosure include microbial sources, including, for example, photoautotrophic, chemoautotrophic, or acidohydrogen microbial cultures grown in bioreactors. The bioreactor can be configured to cultivate the acidohydrogen microorganisms using waste or low-value sources of carbon, such as CO2. Thus, the protein hydrolysate compositions of the present disclosure can be sustainably produced from waste or low-value sources of carbon, such as CO. The protein hydrolysate compositions can be sustainably produced from CO, CH, CO, and / or other carbon containing greenhouse gases (GHGs) or gases that are pollutants, such as, for example, air pollution.

[0016] Methods for producing the protein hydrolysate compositions are also disclosed.

[0017] The method includes processing the 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 proteinaceous material, such as cellular biomass, having a pH of about 11.0 or greater, or having a pH of less than about 3, to a temperature of at least about 40°C for a suitable period of time. In some embodiments, the method includes subjecting the 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 less (alkaline hydrolysis conditions) or to raise the pH to about 6 or greater (acidic hydrolysis conditions) to produce a neutralized suspension, and optionally contacting the neutralized suspension with a protease to produce a protein hydrolysate 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 hydrolysate compositions of the present disclosure have a protein-rich organic content, and the compositions are substantially free of exogenous chelating agents, chaotropic agents, and surfactants. In certain embodiments, the protein hydrolysate is of microbial origin. In certain embodiments, the compositions are substantially free of sodium and / or chloride. In certain embodiments, the protein hydrolysate composition has a size distribution of polypeptides of 25 kD or less. In some embodiments, the total nitrogen content of the composition is about 5% (w / w) or greater. In some embodiments, the composition In some embodiments, the composition has a phosphate content of about 5% (w / w) or more. In some embodiments, the composition has a potassium content of about 5% (w / w) or more. In some embodiments, the composition has a sodium content of about 1% (w / w) or less. In some embodiments, the composition has a chloride content of about 1% (w / w) or less.

[0019] In some embodiments, the protein hydrolysate composition is lyophilized.

[0020] Methods of producing a protein hydrolysate composition of the present disclosure include adjusting the pH of a biomass suspension to 11.0 or greater 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 hydrolysate composition. In some embodiments, the method includes subjecting the alkaline suspension to an overpressure.

[0021] In certain embodiments, the method includes subjecting the alkaline suspension to a temperature of at least about 40° C., followed by neutralizing the suspension to a pH of 9.5 or less to produce a neutralized suspension, and contacting the neutralized suspension with a protease to produce a protein hydrolysate composition in a soluble fraction of the suspension. In some embodiments, the protease is an alkaline protease. In some embodiments, the method includes clarifying the suspension and lyophilizing the soluble fraction.

[0022] In some embodiments, the method includes contacting the suspension comprising the biomass with a base to adjust the pH, wherein the base comprises one or more of potassium hydroxide, calcium hydroxide, calcium oxide, ammonium hydroxide, or ammonia.

[0023] Methods of producing a protein hydrolysate composition of the present disclosure include adjusting the pH of a 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 superatmospheric pressure.

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

[0025] In some embodiments, the method comprises contacting the suspension comprising the biomass with an acid to adjust the pH, wherein the acid comprises phosphoric acid or sulfuric acid.

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

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

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

[0029] In some embodiments, the method includes separating an insoluble fraction of the suspension from a soluble fraction and extracting a polymeric composition from the insoluble fraction, hi some embodiments, the polymeric composition is a polyhydroxyalkanoic acid (PHA), e.g., a polyhydroxybutyric acid (PHB) composition.

[0030] Also provided is a protein hydrolysate composition produced using the method of the present disclosure. Also provided is a plant supplement comprising the protein hydrolysate composition of the present disclosure. The plant supplement can also be applied to plants, for example, to promote nutrients and / or growth. The protein hydrolysate composition of the present disclosure can also be used as a nutritional supplement for animals or cells, for example.

[0031] In one aspect, a method for producing a protein hydrolysate is provided, the method comprising culturing a microorganism in the presence of a carbon source in an aerobic or microaerobic bioprocess to grow a protein-containing biomass, the microorganism comprising Cupriavidus necator, and the carbon source comprising carbon dioxide; harvesting the protein-containing biomass into a suspension composition; and, 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, the first target pH range being at least about 10; heating the alkaline suspension composition to a first temperature of at least about 40° C. for a first time period of at least about 5 minutes; and adding a neutralizing agent to the alkaline suspension composition to form a neutralized suspension composition. wherein the pH of the neutralized suspension composition is in a second target pH range, the second target pH range being from 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 period of 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 period of time is at least about 1 hour; and recovering a supernatant containing the hydrolyzed protein from the hydrolyzed protein suspension.

[0032] In one embodiment, harvesting protein from biomass in a suspension composition comprises suspending the biomass in a biocompatible liquid medium. For example, the biocompatible liquid medium may comprise water or a buffer. In some embodiments, the biomass is suspended in the liquid medium by vortexing, homogenizing, stirring, or sonicating.

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

[0034] In some embodiments, adjusting the suspension composition to the first target pH range comprises adding a base to the suspension composition, wherein the base comprises one or more of 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), such as potassium hydroxide (KOH), ammonium hydroxide (NH4OH), ammonia (NH3), calcium hydroxide (Ca(OH)2), and 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 of potassium phosphate, ammonium phosphate, sodium citrate, citric acid, sodium phosphate, phosphoric acid, phosphate buffer, Tris, HEPES, glycine, carbon dioxide, bicarbonate, and carbonate, e.g., potassium phosphate, ammonium phosphate, sodium citrate, sodium citrate phosphate, phosphoric acid, phosphate buffer, Tris, HEPES, glycine, carbon dioxide, bicarbonate, and / or carbonate, e.g., 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 carbonate.

[0037] In one aspect, a method for producing a protein hydrolysate is provided, the method including culturing a microorganism in the presence of a carbon source in an aerobic or microaerobic bioprocess to grow a protein-containing biomass, the microorganism comprising Cupriavidus necator, and the carbon source comprising carbon dioxide; harvesting the protein-containing biomass into a suspension composition; and, 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 acidic suspension composition, the first target pH range being from about 0.5 or less to about 3; heating the acidic suspension composition to a first temperature of at least about 40° C. for a first time period of at least about 5 minutes; and adding a neutralizing agent to the acidic suspension composition to form a neutralized suspension composition, the neutralized suspension composition being formed. the pH of the composition is in a second target pH range, said second target pH range being about 5 to about 6.5, or about 8; optionally further hydrolyzing said protein suspension by adding a protease to said neutralized suspension composition and incubating said neutralized suspension composition at a second temperature range for a second period of time, thereby forming a hydrolyzed protein suspension, wherein said second temperature range is at least about 40°C and said second period of time is at least about 1 hour; and recovering a supernatant containing said hydrolyzed protein from said hydrolyzed protein suspension.

[0038] In one embodiment, harvesting protein from biomass in a suspension composition comprises suspending the biomass in a biocompatible liquid medium. For example, the biocompatible liquid medium may comprise water or a buffer. In some embodiments, the biomass is suspended in the liquid medium by vortexing, homogenizing, stirring, or sonicating.

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

[0040] In some embodiments, adjusting the pH of the suspension composition to the first target pH range comprises adding an acid to the suspension composition, the acid comprising one or more of phosphoric acid (H3PO4), sulfuric acid (H2SO4), nitric acid (HNO3), formic acid (HCOOH), acetic acid (CH3COOH), hydrochloric acid (HCl), and carbonic acid, e.g., 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 is one or more of calcium hydroxide (Ca(OH)), calcium oxide, calcium carbonate (CaCO), potassium hydroxide (KOH), phosphate buffer, ammonium bicarbonate, ammonium carbonate, ammonium hydroxide (NH4OH ), and ammonia.

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

[0044] In some embodiments, the method further includes adding a chelating agent and / or surfactant to the suspension composition after adjusting the pH of the suspension composition to the 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), including one or more of ethylenediaminetetraacetic acid (EDTA) and / or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), may be added. In some embodiments, the amount of chelating agent added to the suspension composition is within 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 hydrolysate is used as a biostimulant and / or plant nutrient or precursor thereof, and the chelating agent and / or surfactant comprises a plant nutrient selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K), e.g., comprising one or more of nitrogen (N), phosphorus (P), and / or potassium (K). In some embodiments, the chelating agent and / or surfactant does not contain 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 Pluronic PF-68 may be added, including one or more of sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20 and Pluronic PF-68.In some embodiments, the amount of surfactant added to the suspension composition relative to the dry weight of 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 non-toxic 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 period 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., or 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 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, or about 110°C to about 125°C. For example, the first time period can 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, from about 5 minutes to about 90 minutes, from about 5 minutes to about 80 minutes, from about 5 minutes to 70 minutes, from about 10 minutes to about 60 minutes, from about 10 minutes to about 50 minutes, from about 10 minutes to about 40 minutes, from about 10 minutes to about 30 minutes, from about 20 minutes to about 60 minutes, from about 30 minutes to about 60 minutes, from about 1 hour to about 24 hours, from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, or from 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 carried out with the alkaline or acidic suspension under pressure. For example, the pressure of the alkaline or acidic suspension during at least a portion of the first period can 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, between about 15 psi and about 50 psi, between about 15 psi and about 40 psi, between about 15 psi and about 35 psi, between about 15 psi and about 30 psi, between about 15 psi and about 25 psi, or between about 15 psi and about 20 psi.In some embodiments, the pressure of the alkaline or acidic suspension during at least a portion of a first period 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, between about 15 psi and about 50 psi, between about 15 psi and about 40 psi, between about 15 psi and about 35 psi, between about 15 psi and about 30 psi, between about 15 psi and about 25 psi, or between about 15 psi and about 20 psi, and the pressure during at least a portion of the first period 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% of the first period.

[0046] In some embodiments, the protein hydrolysate is used as a biostimulant and / or plant nutrient or precursor thereof, and the neutralizing agent comprises a plant nutrient comprising one or more of nitrogen (N), phosphorus (P), and potassium (K), e.g., selected from the group consisting of nitrogen (N), phosphorus (P), and potassium (K). In some embodiments, the protein hydrolysate is used as a biostimulant and / or plant nutrient or precursor thereof, and the neutralizing agent does not comprise elements harmful to plant growth.

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

[0048] In some embodiments, the protease comprises one of an endoprotease, an exoprotease, an alkaline protease, a serine alkaline protease, a bacterial alkaline protease, and subtilisin A, e.g., an endoprotease, an exoprotease, an alkaline protease, a serine alkaline protease, a bacterial alkaline protease, and subtilisin A, e.g., an endoprotease, an exoprotease, an alkaline protease, a serine alkaline protease, a 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 from about 40°C to about 70°C, from about 45°C to about 65°C, or from about 50°C to about 60°C.

[0050] In some embodiments, the second period of time is at least about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 18 hours, or about 24 hours, and at most 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 polypeptides in the recovered supernatant have 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, between about 0.1 kD and about 30 kD, between about 1 kD and about 30 kD, between about 1 kD and about 25 kD, between about 5 kD and about 25 kD, between about 0.1 kD and about 2 kD, between about 0.1 kD and about 5 kD, between about 0.1 kD and about 10 kD, or between about 5 kD and about 20 kD.

[0052] In some embodiments, the method further comprises the additional step of clarifying the suspension through centrifugation or filtration to remove undissolved material in the hydrolyzed protein suspension.

[0053] In some embodiments, the method further comprises the step of clarifying the suspension or solution to remove molecules above a particular molecular weight (MW) cutoff, for example, via filtration (e.g., ultrafiltration). In such embodiments, the molecular weight cutoff can 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 comprises the additional steps of drying the collected supernatant and lyophilizing the hydrolyzed protein. For example, the lyophilized protein hydrolysate 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 comprises the steps of: (1) adding a chelating agent and / or surfactant and / or chaotropic agent to the suspension composition after adjusting the pH of the suspension composition to a first target pH range; (2) adding a chaotropic agent to the neutralized suspension composition; and (3) removing substantially all of the surfactant, chelating agent and / or chaotropic agent after forming the hydrolyzed protein suspension. In some embodiments, the surfactant, chelating agent and / or chaotropic agent are removed through gel filtration chromatography, membrane filtration, and / or dialysis. do.

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

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

[0058] In some embodiments, the step of harvesting protein from a biomass into a suspension composition comprises subjecting the biomass to lysis. [Brief explanation of the drawings]

[0059] [Figure 1A] 1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 1B] 1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 2]1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 3A] 1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 3B] 1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 4] 1 is a schematic diagram of a method for producing a protein hydrolysate according to an embodiment of the present disclosure. [Figure 5] FIG. 1 is a schematic diagram of an integrated process for producing protein hydrolysates from a microbial culture system. [Figure 6] 1 shows a protein gel analysis of protein hydrolysates prepared according to embodiments of the present disclosure. [Figure 7] 1 shows a protein gel analysis of a fraction of a protein hydrolysate that has not been subjected to an enzymatic hydrolysis step. [Figure 8A] 1 shows a protein gel analysis of protein hydrolysates prepared according to embodiments of the present disclosure. [Figure 8B] 1 shows a protein gel analysis of protein hydrolysates prepared according to embodiments of the present disclosure. [Figure 9] 1 shows a protein gel analysis of protein hydrolysates prepared according to embodiments of the present disclosure. [Figure 10] From left to right: Turnips treated with (a) water, (b) NPK fertilizer, (c) protein hydrolysate. [Figure 11] Shown from left to right: (a) lettuce treated with commercial fish and seaweed hydrolysates, (b) acid hydrolysates, and (c) base hydrolysates. DETAILED DESCRIPTION OF THE INVENTION

[0060] Provided herein are protein hydrolysates and methods for producing the hydrolysates. For example, protein hydrolysates can be produced from microorganisms as described herein.

[0061] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Marsh, eds. kham, The Harper Collins Dictionary of Biology, HarperPerennial, NY (1991), provides one of the techniques with a general dictionary of many of the terms used in this invention. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods, systems, and compositions described herein.

[0062] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of those of ordinary skill in the art. Such techniques include those described in, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Current Protocols in Molecular Biology (F.M.A. Subb. ed., 1994); PCR: The Polymerase Chain Reaction (PCR) These techniques are fully described in such publications as The Chain Reaction (Mullis et al., eds., 1994) and Gene Transfer and Expression: A Laboratory Manual (Kriegler, 1990).

[0063] Numerical ranges provided herein are inclusive of the numbers defining the range.

[0064] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0065] I. Definition As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise; therefore, as used in this specification and the claims, the indefinite articles "a," "an," and "the" should be understood to mean "at least one" unless the context clearly dictates otherwise.

[0066] As used herein, the term "about" when referring to a measurable value such as an amount, duration, etc., means encompassing an error of ±5%, ±1%, or ±0.1% from the particular value, which error is suitable for practicing the disclosed methods or associated with the disclosed compositions.

[0067] "Acetogen" means a microorganism that produces acetate and / or short-chain organic acids of up to C4 chain length as a product of anaerobic respiration.

[0068] "Eosinophil" refers to a type of extremophilic bacteria that grows under highly acidic conditions (usually pH 2.0 or less).

[0069] The term "amino acid" refers to a molecule containing both an amine group and a carboxyl group attached to the carbon designated as the alpha-carbon. Suitable amino acids include, but are not limited to, both D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring 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. Unless the context clearly dictates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.

[0070] As used in this specification and the appended claims, the phrase "and / or" should be understood to mean "either or both" of the connected elements, i.e., elements that are in some cases conjunctively and in some cases disjunctively indicated. Other elements, related or unrelated to the elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" clause, unless expressly stated to the contrary.

[0071] Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can 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); in yet another embodiment, both A and B (optionally including other elements); etc.

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

[0073] The term "bioreactor" or "fermentor" refers to a sealed or partially sealed vessel for growing and maintaining cells. The cells may, but need not, be maintained in liquid suspension. In some embodiments, rather than being maintained in liquid suspension, the cells may alternatively be grown and / or maintained in contact with, on, or within other non-liquid substrates, including, but not limited to, solid growth support materials.

[0074] "Bio-stimulant" or "biostimulant" refers to a compound that stimulates plant growth and development, e.g., agricultural crops, and can increase and enhance microbial activity in soil. The term refers to any substance that can be applied to plants, seeds, soil, or growing media and that increases plant nutrient use efficiency or provides other direct or indirect benefits to plant development or stress response.

[0075] The term "carbon fixation" process, reaction, or pathway refers to an enzymatic reaction or metabolic pathway that converts forms of carbon (including, but not limited to, CO, CO, and CH) that are gaseous under ambient conditions into carbon-based biochemicals that are liquid or solid under ambient conditions, or that are dissolved or held in suspension, aqueous solution.

[0076] "Carbon source" refers to the type of molecule from which a microorganism derives the carbon required for organic biosynthesis.

[0077] "Carboxydotrophic" refers to a microorganism that is tolerant of or capable of oxidizing carbon monoxide. In a preferred embodiment, a carboxydotrophic microorganism can utilize CO as a carbon source and / or a reducing source of electrons for biosynthesis and / or respiration. "Chemical autotrophy" refers to an organism that obtains energy through the oxidation of chemical electron donors by chemical electron acceptors and synthesizes all of the organic compounds the organism needs for survival and growth from carbon dioxide.

[0078] In the claims and the specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," etc., are to be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0079] The term "culture" refers to the growth of a population of cells, e.g., microbial cells, in a liquid or solid medium. This means growing the cells under conditions suitable for the growth.

[0080] The term "derived from" encompasses the terms "sourced from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that the origin of a particular material is found in, or has characteristics that can be described with reference to, other particular materials.

[0081] By "energy source" is meant either an electron donor that is oxidized by oxygen during aerobic respiration, or a combination of an oxidized electron donor and an electron acceptor that is reduced during anaerobic respiration.

[0082] "Extremophilic bacteria" means microorganisms capable of growth under extreme physical or geochemical conditions (e.g., high or low temperature, pH, or high salinity) compared to the conditions at the Earth's surface or in the oceans typically tolerated by most organisms found on or near the Earth's surface.

[0083] The term "gasification" refers to a generally high-temperature process that converts carbonaceous materials into a mixture of gases containing hydrogen, carbon monoxide, and carbon dioxide, also known as synthesis gas, syngas, or producer gas. This process generally involves partial combustion and / or the application of externally generated heat with the controlled addition of oxygen and / or steam in the absence of sufficient oxygen for complete combustion of the carbonaceous material.

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

[0085] "Chemical autotrophy" refers to an organism that cannot synthesize all of the organic compounds it needs to survive and grow from carbon dioxide and must utilize organic compounds for growth. Heterotrophic organisms cannot produce their own food and instead obtain food and energy by ingesting plant or animal matter and metabolizing organic matter, rather than fixing carbon from non-organic sources such as carbon dioxide.

[0086] "Hydrogen-oxidizer" refers to a microorganism that uses reduced H2 as an electron donor in the production of intracellular reducing equivalents and / or in respiration.

[0087] "Hyperthermophile" refers to a type of extremophilic bacteria that thrives in very hot environments, typically above about 60°C (140°F).

[0088] The term "knallgas" refers to a mixture of molecular hydrogen and oxygen gases. "Knallgas microorganisms" refer 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 interchangeably with "knallgas" and "knallgas microorganisms," respectively. Knallgas microorganisms generally convert molecular hydrogen via hydrogenases to NAD. + Knallgas microorganisms typically fix CO2 autotrophically through pathways including, but 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" refers to a liquid containing a mixture and / or solution of cell contents obtained from cell lysis. In some embodiments, the methods described herein involve purifying a chemical or mixture of chemicals within a cell lysate. In some embodiments, the methods involve purifying amino acids and / or proteins within a cell lysate.

[0090] The term "lysis" means that the plasma membrane and, if present, the cell wall of a cell are ruptured, thereby leaking a substantial amount of intracellular material into the extracellular space. Lysis can be performed by electrochemical, mechanical, osmotic, thermal, or viral methods. In some embodiments, the methods described herein involve lysing cells or microorganisms as described herein to separate a chemical or mixture of chemicals from the contents of a bioreactor. In some embodiments, the methods involve lysing cells or microorganisms as described herein to separate an amino acid or mixture of amino acids and / or proteins from the bioreactor or cell growth medium.

[0091] "Methanogen" means a microorganism that produces methane as a product of anaerobic respiration.

[0092] "Methylotroph" means a microorganism that can use reduced one-carbon compounds, such as, but not limited to, 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] "Mixotrophic" refers to an organism that can use a mixture of different energy sources and carbon (e.g., H2 and sugars).

[0095] The term "molecule" means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes, for example, carbohydrates, lipids, polypeptides, and polynucleotides.

[0096] The term "oligopeptide" refers to a peptide containing a relatively small number of amino acid residues, for example, from about 2 to about 20 different amino acids.

[0097] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" described above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one, but also including two or more of the list of numbers or elements, and optionally additional unlisted items. Only terms clearly indicating the opposite, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall mean that exactly one element of the list of numbers or elements is included. Generally, as used herein, the term "or" shall be interpreted only as indicating exclusive alternatives ("either / or," "one of," "only one of," "exactly one," "consisting essentially of," and the like, and when used in the claims, shall have its ordinary meaning as used in the field of patent law.

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

[0099] By "peptide" is meant two or more amino acids linked in a chain, e.g., from about 2 to about 50 amino acids, in which the carboxyl group of each amino acid is connected to the amino group of the next amino acid by a bond of the type R-OC-NH-R'.

[0100] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and any three-dimensional structure, and single- or multi-stranded (e.g., single-, double-, triple-, etc.), containing deoxyribonucleotides, ribonucleotides, and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because genetic information is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides encoding specific amino acid sequences. 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, phosphorothioate). Labels may also be incorporated for detection or recovery purposes, such as radioactive or non-radioactive labels or anchors (e.g., biotin). The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides may be natural or non-natural. As used herein, the terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably. A polynucleotide can comprise RNA, DNA, or both and / or modified forms and / or analogs thereof. The sequence of nucleotides can be interrupted by non-nucleotide components. One or more phosphodiester bonds can 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 substituted or unsubstituted alkyl (1-20C), optionally with an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. The linkages in all polynucleotides need not be identical. Polynucleotides can be linear or circular, or contain a combination of linear and circular portions.

[0101] As used herein, "polypeptide" refers to a composition comprising amino acids, recognized by those skilled in the art as a protein. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation or modification (such as conjugation with a labeling component). The definition also includes, for example, one or more analogs of an amino acid (e.g., unnatural amino acids), as well as other modifications known in the art.

[0102] The term "precursor to" or "precursor of" refers to an intermediate in the preparation of one or more components of a final product.

[0103] "Producer gas" means a gas mixture containing varying proportions of H2, CO, and CO2, which typically has a heating value in the range of one-half to one-tenth that of natural gas per unit volume under standard conditions. Producer gas is produced by vaporization, steam reforming, or the autogenous conversion of carbonaceous feedstocks. It can be produced in a variety of ways from a variety of feedstocks, including mechanical reforming. In addition to H2, CO, and CO2, producer gas can contain other components, including, but not limited to, methane, hydrogen sulfide, condensable gases, tar, and ash, depending on the production process and feedstock. The percentage of N2 in the mixture can be higher or lower depending on whether air is used as the oxidant in the reactor and whether reactor heat is provided through direct combustion or indirect heat exchange.

[0104] The term "produce" includes both intracellular and extracellular production of a compound, including secretion of a compound from a cell.

[0105] "Psychrophile" refers to a type of extremophilic bacteria that is capable of growing and reproducing at low temperatures, typically temperatures below about 10°C.

[0106] As used herein, the terms "recovered," "isolated," "purified," and "separated" refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated. For example, these terms can refer to material that is substantially or essentially free from components that normally accompany it in its natural state, such as, for example, in an intact ecosystem.

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

[0108] "Sulfur-oxidizer" refers to a microorganism that uses reduced sulfur compounds, including but not limited to H2S, as an electron donor for the production of intracellular reducing equivalents and / or sulfur compounds in respiration.

[0109] "Syngas" or "synthesis gas" refers to a type of gas mixture, similar to producer gas containing H2 and CO, but more specifically tailored in terms of H2 and CO content and ratios and impurity levels for the synthesis of specific types of chemical products, such as, but not limited to, methanol or Fischer-Tropsch diesel. Syngas generally contains H2, CO, and CO2 as major components and can be produced through established processes including steam reforming of methane, liquid petroleum gas, or biogas, or through the vaporization of any organic, combustible carbonaceous material, including, but not limited to, biomass, waste organics, various polymers, peat, and coal. The hydrogen content of syngas can be increased through the reaction of steam with CO during a water-gas shift reaction, resulting in a concomitant increase in CO2 in the syngas mixture.

[0110] "Thermophiles" refer to a type of extremophilic bacteria that grow at relatively high temperatures to survive, typically from about 45°C to about 122°C.

[0111] "Wild-type" means a naturally occurring microorganism.

[0112] "Yield" means the amount of product produced from a feed material (e.g., sugar) relative to the amount of material that would be produced if all of the feed material were converted to 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 feed material were converted to amino acids.

[0113] II. Method In general terms, the methods of the present disclosure can involve increasing or decreasing the pH of a proteinaceous suspension, such as a biomass suspension, e.g., a suspension of microbial biomass, e.g., biomass produced by chemoautotrophic microbial growth.

[0114] The starting biomass suspension can include a suitable amount of biomass in a medium, e.g., a microbial biomass in a growth medium. In some embodiments, the amount of biomass (dry weight / reaction volume (w / v)) is about 0.1% or more, e.g., about 0.2% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, including 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, including about 5% or less, and in some embodiments, each of the foregoing ranges of biomass can 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)) ranges from about 0.1% to about 8%, e.g., from about 0.2% to about 8%, from about 0.5% to about 6%, from about 1% to about 6%, from about 2% to about 6%, including from about 3% to about 5%.

[0115] In some embodiments, cells within the biomass are subjected to lysis at the beginning of the process, for example, before raising or lowering the pH, to facilitate the collection of proteins from the biomass into a suspension composition.

[0116] The alkaline or acidic suspension is heated for a suitable period of time to produce a protein hydrolysate composition. The suspension can be concentrated, dried (e.g., lyophilized), or used directly as a liquid suspension. In certain embodiments, the alkaline or acidic suspension is subjected to heat and high pressure to produce the protein hydrolysate composition, for example, by autoclaving the alkaline or acidic suspension. In some embodiments, after heat / 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 hydrolysate composition is produced.

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

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

[0120] The pH of the biomass suspension can be raised to 110, 130 by adding a base to the suspension. In some embodiments, the pH of the suspension is raised to about 10.0 or greater, e.g., about 11 or greater, about 11.1 or greater, about 11.2 or greater, about 11.3 or greater, about 11.4 or greater, about 11.5 or greater, about 11.6 or greater, about 11.7 or greater, about 11.8 or greater, about 11 or greater, including about 12.0 or greater.

[0121] In some embodiments, the pH of the suspension is raised to about 10.0 to about 14.0, e.g., about 11.0 to about 14.0, about 10.0 to about 13.0, about 10.5 to about 13.0, about 10.5 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 11.7 to about 12.4, including about 11.8 to about 12.3. Any suitable 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 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 not limited to, sodium (Na).

[0122] The alkaline biomass suspension can be subjected to elevated temperatures. In some embodiments, the suspension is subjected to a temperature of about 40°C or higher, e.g., 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, including about 121°C or higher. In some embodiments, the suspension is subjected to a temperature of about 40°C to about 150°C, e.g., 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, or 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 elevated temperatures using any suitable method.

[0123] In some embodiments, the alkaline biomass suspension is subjected to high pressure (i.e., at least 14.7 psi). In some embodiments, the suspension is subjected to a pressure of 15 psi or greater, e.g., about 20 psi or greater, about 25 psi or greater, about 30 psi or greater, about 35 psi or greater, or about 40 psi or greater, and in some embodiments, 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 a pressure ranging from about 14.7 psi to about 40 psi, e.g., from about 15 psi to about 40 psi, from about 15 psi to about 35 psi, from about 15 psi to about 30 psi, from about 15 psi to about 25 psi, or from about 15 psi to about 20 psi. The suspension can be subjected to high temperature and pressure using any suitable 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 elevated temperature for only a portion of the time, ie, the suspension is exposed 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 that it is exposed to elevated temperature.

[0125] The alkaline biomass suspension can 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, e.g., about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, or about 25 minutes or more. In some embodiments, each of the above periods can 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 for about 5 minutes to about 90 minutes, e.g., 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, or about 20 minutes to about 60 minutes. The suspension is subjected to high temperature, or high temperature and high pressure for a time period including about 1 hour or more, e.g., about 30 to 60 minutes, inclusive. In some embodiments, the suspension is subjected to high temperature, or high temperature and high pressure for a time period including about 1 hour or more, e.g., about 3 hours or more, about 5 hours or more, or about 8 hours or more, in some embodiments, each of the above-mentioned times can 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 time period including about 1 hour to about 24 hours, inclusive, e.g., 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, after the temperature and heat treatment, the pH of the suspension is reduced by adding a neutralizing agent, e.g., a neutralizing buffer. 150 In some embodiments, the pH of the suspension is reduced to about 6.5 or greater, including about 6.7 or greater, about 6.9 or greater, about 7.0 or greater, about 7.1 or greater, about 7.2 or greater, about 7.3 or greater, about 7.4 or greater, about 7.5 or greater, about 7.6 or greater, about 7.7 or greater, about 7.8 or greater, about 7.9 or greater, or about 8.0 or greater, and in some embodiments, each of the foregoing 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 lowered to about 6.5 to about 9.5, including, for example, 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 suitable neutralizing agent. Suitable neutralizing agents include, but are not limited to, potassium phosphate buffer, ammonium phosphate buffer, sodium citrate buffer, sodium phosphate buffer, Tris buffer, HEPES buffer, and / or glycine buffer. 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 phosphoric acid. In some embodiments, the pH is lowered using CO2 and / or bicarbonate and / or carbonic acid. In certain embodiments, ammonia, ammonium hydroxide, calcium hydroxide, or calcium oxide is used as the base, and CO2 is used for neutralization. In certain embodiments, calcium hydroxide or calcium oxide is used as the base, and phosphoric acid or a phosphate buffer is used for neutralization. In some embodiments, the pH is lowered using an organic acid. In certain embodiments, one or more neutralizing agents or buffers containing plant nutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the neutralizing agent or buffer does not contain elements that inhibit plant growth, including, but not limited to, sodium (Na) and / or chloride (Cl). In certain embodiments, the neutralizing agent or buffer is not added after the heat / pressure step.

[0129] After alkaline hydrolysis, the proteins in the composition are degraded 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 the polypeptides in the suspension, e.g., the soluble fraction of the suspension, have 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 about 2 kD, and in some embodiments, each of the foregoing masses is 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, 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 the polypeptides in the suspension, e.g., the soluble fraction of the suspension, have an atomic mass of about 0.1 kD to about 30 kD, 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 10 kD, about 0.1 kD to about 5 kD, about 0.1 kD to about 2 kD, or about 5 kD to about 20 kD.

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

[0132] The pH of the biomass suspension can be increased 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 can include, but are not limited to, one or more of phosphoric acid (HPO), sulfuric acid (HSO), nitric acid (HNO), formic acid (HCOOH), acetic acid (CHCOOH), carbon dioxide (CO), and hydrochloric acid (HCl). In certain embodiments, one or more acids containing plant nutrients are used, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K). In certain embodiments, acids containing elements that inhibit plant growth are avoided, including, but not limited to, chloride (Cl).

[0133] The acidic biomass suspension can be subjected to elevated temperatures 320, 340. In some embodiments, the suspension is subjected to temperatures of about 40°C or higher, including 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 a temperature of about 40°C to about 150°C, including 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, or about 110°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 elevated temperatures using any suitable 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 a pressure of 15 psi or greater, e.g., about 20 psi or greater, about 25 psi or greater, about 30 psi or greater, about 35 psi or greater, or about 40 psi or greater, and in some embodiments, to a pressure 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 a pressure ranging from about 14.7 psi to about 40 psi, e.g., from about 15 psi to about 40 psi, from about 15 psi to about 35 psi, from about 15 psi to about 30 psi, from about 15 psi to about 25 psi, or from about 15 psi to about 20 psi. The suspension can be subjected to high temperature and pressure using any suitable method. In some embodiments, the suspension is autoclaved to elevate the temperature and pressure. In some embodiments, the suspension is subjected to elevated pressure and exposed to elevated temperature for only a portion of the time. In some embodiments, the suspension is subjected to elevated 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%, at least about 95 ... The membrane is exposed to high pressure for 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.

[0135] The acidic biomass suspension can be subjected to elevated temperature or elevated temperature and pressure for a suitable period of time. In some embodiments, the suspension is subjected to elevated temperature or elevated temperature and pressure for about 5 minutes or more, e.g., about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, or about 25 minutes or more. In some embodiments, each of the above periods can 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 elevated temperature or elevated temperature and pressure for about 5 minutes to about 90 minutes, e.g., 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, about 10 minutes to about 30 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In some embodiments, the suspension is subjected to elevated temperature or elevated temperature and pressure for a period of time of about 1 hour or more, including about 3 hours or more, about 5 hours or more, about 8 hours or more, and in some embodiments, each of the aforementioned 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 elevated temperature or elevated temperature and pressure for a period of time of about 1 hour to about 24 hours, including 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, after the temperature and heat treatment, the pH of the suspension is increased by adding a neutralizing agent, e.g., a neutralizing buffer. In some embodiments, the pH is increased to about 6 or about 7. The pH can be increased using any suitable neutralizing agent. Suitable neutralizing agents include, but are not limited to, bicarbonate buffer or phosphate buffer. In some embodiments, the neutralizing agent includes, but is not limited to, calcium hydroxide (Ca(OH)), calcium carbonate (CaCO), potassium hydroxide (KOH), or ammonium hydroxide (NHOH). In certain embodiments, one or more neutralizing agents or buffers containing plant nutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the neutralizing agent or buffer does not contain elements that inhibit plant growth, including, but not limited to, sodium (Na) and / or chloride (Cl). In certain embodiments, no neutralizing agent or buffer is added after the heating / pressure step.

[0137] After acid hydrolysis, the protein in the composition is degraded into polypeptides of various sizes.According to some embodiments, after acid 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 the polypeptides in the suspension, for example, the soluble fraction of the suspension, have 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 about 2 kD; in some embodiments, each of the aforementioned mass ranges can 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, 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 the polypeptides in the suspension, e.g., the soluble fraction of the suspension, have an atomic mass of about 0.1 kD to about 30 kD, 1 kD to about 30 kD, about 1 kD to about 25 kD, about 5 kD to about 25, 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.

[0138] C. Protease Treatment In some embodiments, for example, alkaline or acidic hydrolysis as described above is incomplete or does not yield peptides having the desired peptide size range and / or characteristics. In some embodiments, one or more proteases may be added to the neutralized suspension 160, 360 and incubated under conditions suitable for hydrolysis of additional biomass. The protease may be any suitable 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 or glutamic protease. In some embodiments, the protease is a metalloprotease. In certain embodiments, a combination of one or more types of proteolytic enzymes, such as exoproteases, endoproteases, neutral proteases, carboxypeptidases, and / or aminopeptidases, is used. In certain such embodiments, one or more of the exoproteases, endoproteases, neutral proteases, carboxypeptidases, and aminopeptidases are derived from Aspergillus oryzae. In certain such embodiments, an endoprotease enzyme preparation derived from Bacillus subtilis is used. In certain such embodiments, glutaminase is used. In certain such embodiments, the glutaminase is derived from Aspergillus niger.

[0139] In certain limiting embodiments, the microorganisms are hydrolyzed with at least one enzyme capable of hydrolyzing microbial (e.g., bacterial) proteins into free amino acids and / or short peptides. In certain embodiments, the enzymatic hydrolysis includes hydrolysis with a clarifying enzyme. In certain embodiments, the enzymatic hydrolysis includes hydrolysis with a mixture of enzymes and an enzyme-prepared medium. In certain embodiments, the enzymatic hydrolysis includes hydrolysis with enzymes derived from plants and / or animals and / or bacteria and / or archaea and / or fungi. In certain embodiments, the 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 enzymes are produced by the microbial strains described herein. In certain embodiments, the hydrolytic enzymes are produced from microorganisms grown on a Cl substrate and / or H2 and / or syngas feedstock. In certain embodiments, bacterial cells may be hydrolyzed with one or more of a protease, lipase, and amylase. In certain embodiments, the enzymatic hydrolysis involves one or more proteolytic enzymes of microbial, plant, fungal and / or animal origin, hi certain embodiments, the method involves the use of alkaline proteases.In certain embodiments, the enzymatic hydrolysis involves hydrolysis with at least one enzyme selected from pancreatin, 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 known to those skilled in the art, pancreatin includes digestive enzymes, proteases, and lipases.

[0140] Enzymatic hydrolysis of microbial (e.g., bacterial) cells involves combining enzymes and microbial (e.g., bacterial) cells in any suitable amounts and under any suitable conditions. In certain embodiments, the number of reactants, reaction conditions, and order of reaction steps are determined by the ideal enzyme. The enzymatic hydrolysis is performed using an enzyme stock solution at a concentration of about 0.05% to about 0.5% by volume, having an activity of about 70,000 units, and using an azocasein assay. In certain embodiments, any suitable method can be used to improve the efficiency of the enzymatic hydrolysis of microbial (e.g., bacterial) cells. In certain embodiments, the enzymatic hydrolysis involves combining the enzyme with the microbial (e.g., bacterial) cells and agitating the combined enzyme and microbial cells by any suitable method. The enzymatic treatment can be performed using any equipment known to those skilled in the art, such as a reactor equipped with temperature control and agitation.

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

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

[0143] Protease hydrolysis breaks down the proteins 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 the polypeptides in the suspension, for example, the soluble fraction of the suspension, have 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 about 2 kD; in some embodiments, each of the aforementioned mass ranges can 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, 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% of the polypeptides in the suspension, e.g., the soluble fraction of the suspension, have an atomic mass of about 0.1 kD to about 30 kD, 1 kD to about 30 kD, about 1 kD to about 25 kD, about 5 kD to about 25, 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.

[0144] D. Pre-processing Prior to the protein hydrolysis step, in certain embodiments, the biomass is subjected to one or more pre-treatment steps, such as, 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, and in some embodiments, The pH of the biomass is adjusted to a pH of about 9 to about 5, which is the pH at which enzymatic hydrolysis is performed. In certain such embodiments, the pH is adjusted only to a pH of about 9 to about 5, at which point enzymatic hydrolysis is performed. In certain embodiments, enzymatic hydrolysis is performed on the delipidated biomass after the delipidation step is complete, without pH adjustment.

[0145] E. Purification / Isolation In some embodiments, the hydrolytic treatment (e.g., alkaline or acid hydrolysis, optionally including protease treatment) is followed by clarification of the suspension (hydrolysate) to remove undissolved material in the suspension (e.g., separation of soluble and insoluble fragments). The suspension may be clarified using any suitable method, such as centrifugation, filtration, etc. In some embodiments, after the suspension is clarified (e.g., centrifuged), the supernatant is separated from the pellet.

[0146] In some embodiments, a clarified 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 dry or substantially dry composition. In some embodiments, the lyophilized composition has a water content of about 10% or less (e.g., about 8% or less, about 6% or less, about 5% or less, including about 3% or less). In some embodiments, the lyophilized protein hydrolysate composition has a water content of about 1% to about 10% (e.g., about 1% to about 8%, about 1% to about 6%, including about 2% to about 5%).

[0147] In some embodiments, the clarified 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., about 75% or less, about 50% or less, about 40% or less, about 30% or less), and in some embodiments, each of the above water content ranges can 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 such ranges exceed the lower limit). In some embodiments, the dehydrated product is dried using heat and / or evaporation using 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 clarified 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 lyophilization, e.g., the lyophilized protein hydrolysate composition has a water content of about 1% to about 10% (e.g., including about 1% to about 8%, about 1% to about 6%, about 2% to about 5%).

[0148] In some embodiments, the disclosed methods process the insoluble fraction of the suspension, e.g., after alkaline or acid hydrolysis or protease (e.g., alkaline protease, acid protease, or metalloprotease) treatment, to obtain a co-product. In some embodiments, the treated suspension is separated into soluble and insoluble fractions (e.g., by centrifugation to produce a supernatant fraction containing the protein hydrolysate composition) and a pellet fraction.

[0149] The pellet fractions can be further processed to extract co-products. The co-products can be fractions of the microorganisms from which the biomass is obtained. In some embodiments, the co-products are biopolymers such as polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoates can include, but are not limited to, polyhydroxybutyrate (PHB).

[0150] F. Treatment with additional drugs Any of the embodiments of the biomass hydrolysis methods disclosed herein may include a chelating agent. A chelating agent may be added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure (see, e.g., 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., an amount sufficient to sequester undesired 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., about 0.5 mM to about 8 mM, about 1 mM to about 7 mM, about 3 mM to about 5 mM). In some embodiments, the amount of chelating agent in the biomass suspension is about 5 mM. In certain embodiments, no chelating agent is added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure. In certain embodiments, no synthetic chelating agent is added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure. In certain embodiments, one or more chelating agents are used that contain one or more plant nutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K). In certain embodiments, the chelating agent does not contain substances that inhibit plant growth, including, but not limited to, sodium (Na) and / or chloride (Cl).

[0151] A surfactant may be included in any embodiment of the biomass hydrolysis method disclosed herein, for example, to improve the solubility of the suspension. In some embodiments, a surfactant is added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure (see Figures 2 and 4 (210, 410)). Any suitable surfactant may be added to the biomass suspension, including natural surfactants (i.e., obtained directly from natural sources) or synthetic surfactants. Suitable 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 suitable 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., about 2% to about 20%, about 4% to about 15%, about 5% to about 12%, about 8% to about 12%) based on 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) based on the dry weight (w / w) of biomass in the suspension. In certain embodiments, no surfactant is added to the biomass suspension before subjecting the suspension to high heat or high heat / pressure. In certain embodiments, no synthetic surfactant is added to the biomass suspension before subjecting the suspension 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 including surfactants containing substances that inhibit plant growth, including, but not limited to, sodium (Na) and / or chloride (Cl). In certain embodiments, the added surfactant is not harmful to plants or animals, and / or is biodegradable, and / or has no adverse effects on the environment.

[0152] A chaotropic agent can be included in any embodiment of the biomass hydrolysis methods disclosed herein, for example, to help denature or modify protein molecules. In some embodiments, the chaotropic agent is added before subjecting the suspension to high heat or high heat / pressure. In some embodiments, the chaotropic agent is added to a neutralized suspension containing a natural or synthetic chaotropic agent (see, e.g., Figures 2 and 4 (240, 440)). In some embodiments, protease hydrolysis is carried out in the presence of the chaotropic agent in the neutralized suspension. Any suitable amount A chaotropic agent may be added to the neutralized suspension. Suitable chaotropic agents include, but are not limited to, urea, thiourea, and guanidinium chloride. The chaotropic agent may be added to the neutralized suspension in any suitable amount. In some embodiments, the amount of chaotropic agent in the neutralized suspension ranges from about 0.1 M to about 2 M (e.g., about 0.5 M to about 1.5 M, including about 0.8 M to about 1.2 M). In some embodiments, the amount of chaotropic agent in the neutralized suspension is about 1 M. In certain embodiments, no chaotropic agent is added to the biomass suspension. In certain embodiments, no synthetic chaotropic agent is added to the biomass suspension. In certain embodiments, one or more chaotropic agents containing plant nutrients, including, but not limited to, nitrogen (N), phosphorus (P), and / or potassium (K), are used. In certain embodiments, the chaotropic agent 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 present disclosure include removing detergents, chelating agents, and / or chaotropic agents. In some embodiments, detergents, chelating agents, and / or chaotropic agents are removed after treatment with a protease (e.g., alkaline protease, acid protease, or metalloprotease) (see, e.g., Figures 2 and 4 (250, 450)), or in other embodiments not involving protease treatment as described herein, e.g., via buffer exchange. Any suitable method can be used to remove detergents, chelating agents, and / or chaotropic agents, including, but not limited to, gel filtration chromatography, membrane filtration, and / or dialysis. In some embodiments, the protein hydrolysate compositions of the present disclosure are produced without the use of detergents, chelating agents, or chaotropic agents.

[0154] III. Biomass Sources The methods of the present disclosure can be used to process biomass suspensions derived from any suitable source of proteinaceous material. The organism from which the protein hydrolysate composition is derived can be multicellular or unicellular. In some embodiments, the protein hydrolysate composition of the present disclosure has a microbial source. The microbial organism from which the protein hydrolysate composition is derived can be a photoautotrophic, heterotrophic, methanotrophic, methanolotrophic, carboxydotrophic, or chemoautotrophic organism. The microbial organism can be wild-type or genetically modified. Referring to FIG. 5, biomass can be harvested from a culture of a suitable microorganism, for example, in a fermenter or bioreactor 510. The harvested biomass can be used in any suitable method, such as a centrifuge, to separate the cell mass from the culture medium. The harvested biomass can be used in the methods 520 of the present disclosure to produce the protein hydrolysate composition 530. In some embodiments, the collected biomass is spray-dried or freeze-dried to produce a dried biomass, which can then be used to produce the protein hydrolysate composition according to the methods of the present disclosure.

[0155] In some embodiments, the microorganism is selected from the genera Rhodococcus or Gordonia. In some embodiments, the microorganism is Rhodococcus opacus. In some embodiments, the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus sp. (DSM 3346). 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 In some non-limiting 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 comprises the species Cupriavidus necator. In some embodiments, the microorganism is a strain of the species Cupriavidus necator DSM53. In some embodiments, the microorganism comprises the species Cupriavidus necator. In some embodiments, the microorganism is a strain of the species Cupriavidus metallidurans DSM2839. In some embodiments, the microorganism comprises the species Xanthobacter autotrophicus. In some embodiments, the microorganism is a strain of the species Xanthobacter autotrophicus DSM432.

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

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

[0158] In some embodiments, the microorganisms described herein can thrive on H2 / CO2 and / or syngas, and the microorganisms can naturally accumulate polyhydroxybutyrate (PHB) or polyhydroxyalkanoates (PHAs) at about 50% or more of the total cell mass. In some embodiments, the microorganisms naturally have the ability to direct high carbon fluxes through acetyl-CoA metabolic intermediates, thereby directing them toward 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 capable of producing PHAs, such as PHB, and mutant or engineered strains of microorganisms that produce less PHA (e.g., PHB) than wild-type strains when grown under the same conditions are used in the methods described herein. In some embodiments, the mutant or engineered strains are unable to produce detectable amounts of PHA (e.g., PHB). In certain embodiments, PHA (e.g., PHB)-negative mutants are used. In certain such embodiments, the species is Cupriavidus necator. In certain such embodiments, the strain is Cupriavidus necatorDSM541.

[0160] In some non-limiting embodiments, the microorganism is Corynebacterium 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 comprise a microorganism selected from one or more genera of Cupriavidus sp., Rhodococcus sp., Hydrogenovibrio sp., Rhodopseudomonas 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 following classes: 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 pyridinivorans; Rhodococcus rhodochrous; Rhodococcus rhodnii; (synonym: Nocardiarhodnii); Rhodococcus ruber (synonym: Streptothrixrubra); Rhodococcus sp. RHAl; Rhodococcus triatomae; Rhodococcus tukisamuensis; Rhodococcus wratislaviensis (synonym: Tsukamurellawratislaviensis); Rhodococcus yunnanensis; and strains of Rhodococcus sp., such as Rhodococcus zopfii. In some embodiments, the microorganism is Rhodococcus opacus strain DSM 43205 or DSM 43206. In some embodiments, the microorganism is Rhodococcus sp. strain DSM 3346.

[0163] In some embodiments, the composition comprises a microorganism (e.g., a microorganism of any microbial genus or species described herein) capable of growing natively on H2 / CO2 and / or syngas, and the microorganism comprises at least about 10%, 20%, 30%, or 40% by weight of cellular biomass. The microorganism can naturally accumulate lipids at 40%, 40% by weight, 50% by weight, or more. In some embodiments, the microorganism (e.g., a microorganism of any genus or species described herein) naturally has the ability to drive a high carbon flux through the fatty acid biosynthetic pathway. In some embodiments, the microorganism exhibiting these characteristics is Rhodococcus opacus (e.g., DSM43205, DSM43206, or DSM44193) or Cupriavidus necator (e.g., DSM531 or DSM541).

[0164] In some embodiments, the microorganism is an oxytocin or knallgas strain. In some embodiments, the microorganism or composition comprising the microorganism comprises one or more of the following knallgas microorganisms: 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 Purple non-sulfur bacteria, including, but not limited to, Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas assuljoviridis, Rhodopseudomonas blastica, Rhodopseudomonas asspheroides, Rhodopseudomonas acidophila, or other Rhodopseudomonas sp.; 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 pallero. nii, 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 strain 11 / 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., Streptomycetes coelicojlavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetes thermocarboxydus, and other Streptomycetes sp.; Thermocrinis ruber or other Thermocrinis sp.; Watersia sp.; Anabaenaoscillarioides, Anabaenaspiroides, Anabaenacylindrica, or other Anabaena sp.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 consortia of organisms including microorganisms and / or oxyhydrogen microorganisms.

[0165] In some embodiments, the microorganism or composition comprising a microorganism comprises 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 consortia of microorganisms and / or organisms comprising one or more of these microorganisms.

[0166] In some embodiments, the microorganism or microorganisms comprising the microorganism include one or more of the following: Arthrobacter methylotrophus DSM 14008; Rhodococcus opacus DSM 44304; Rhodococcus opacusDSM44311; necatorDSM541;Rhodococcus aetherivoransDSM44752;Gordonia desulfuricansDSM44462;Gordonia polyisoprenivoransDSM44266;Gordonia polyisoprenivoransDSM44439;Gordonia rubripertinctaDSM46039;Rhodococcus percolatusDSM44240;Rhodococcus opacusDSM43206;Gordonia hydrophobica DSM 44015; Rhodococcus zopfii DSM 44189; Gordonia westfalica DSM 44215, Xanthobacter autotrophicus DSM 1618; Xanthobacter autotrophicus DSM 2267; Xanthobacter autotrophicus DSM 3874; Streptomycetes coelicoflavus DSM 41471; Streptomycetes griseus DSM 40236; Streptomycetes sp. DSM 40434; Streptomycetes xanthochromogenes DSM 40111; Streptomycetes thermocarboxydus DSM 44293; Rhodobacter sphaeroides DSM 158. In some embodiments, the microorganism or composition comprising a microorganism comprises a consortium of microorganisms and / or organisms comprising one or more of these microorganisms or any of the genera or species of microorganisms described herein.

[0167] Many 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 H as an electron donor and / or grow mixotrophically. In some cases, carboxydotrophic microorganisms are facultative chemoautotrophic growth organisms (Biology of the Prokaryotes, edited by J. Lengeler, G. Drews, H. Schlegel, John Wiley & Sons, Jul. 10, 2009, incorporated herein by reference in its entirety). In some embodiments, the microorganism or composition comprising the microorganism is one or more of 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 comprising a microorganism comprises a microorganism and / or a consortium of organisms that includes a carboxydotrophic microorganism, such as one or more of the carboxydotrophic 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] In some embodiments, the microorganism or composition comprising the microorganism is Acetoanaerobium sp.; 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.; Beggiatoa sp.; Butyribacterium 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.;Ectothiorhodospirasp.;Enterobacter sp.;Eubacterium sp.;Ferroplasma sp.;Halothibacillus sp.;Hydrogenobacter sp.;Hydrogenomonas sp.;Leptospirillum sp.;Metallosphaera sp.;Methanobacterium sp.;Methanobrevibacter sp.;Methanococcus sp.;Methanococcoides sp.;Methanogenium sp.;Methanolobus sp.;Methanomicrobium sp.;Methanoplanus sp.;Methanosarcina sp.;Methanospirillum sp.; Methanothermus sp.; Methanothrix sp.; Micrococcus sp.; Nitrobacter sp.; Nitrobacteraceae sp., Nitrococcus sp., Nitrosococcus sp.; Nitrospina sp., Nitrospira sp., Nitrolobus sp.; Nitrosomonas sp.; sp.;Nitrospina sp.;Oleomonas sp.;Paracoccus sp.;Peptostreptococcus sp.;Planctomycetes sp.;Pseudomonas sp.;Ralstonia sp.;Rhodobacter sp.;Rhodococcus sp.;Rhodocyclus sp.;Rhodomicrobium sp.;Rhodopseudomonas sp.;Rhodospirillum sp.;Shewanella sp.;Siderococcus sp.;Streptomyces sp.;Sulfobacillus sp.;Sulfolobus sp.;Thermothrix sp., Thiobacillus sp.; Thiomicrospira sp.; Thioploca sp.; Thiosphaera sp.; Thiothrix sp.; Thiovulum sp.; sulfur oxidizers; hydrogen oxidizers; ion oxidizers; acetogens; and microbial consortia including methanogens and chemolithoautotrophs, hydrothermal vents, geothermal outlets, hot springs, springs, underground aquifers, salt lakes, concentrated salt formations, mine shafts, acid mine drainage, mine dressing wastes, oil wells, and petroleum industry wastewater. , coal seams, the deep subsurface, sewage and wastewater treatment plants, geothermal power plants, geothermal solvation vent fields, geothermal soils, and extremophilic bacteria selected from hyperthermophiles, acidophiles, halophiles, and psychrophiles. In some embodiments, the microorganism or composition comprising the microorganism comprises a consortium of microorganisms and / or organisms comprising chemoautotrophic microorganisms comprising one or more of the above chemoautotrophic microorganisms.

[0170] In some embodiments, extremophilic bacterial microorganisms are provided that can tolerate extremes in a variety of environmental factors, such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, chemicals, etc. These include hyperthermophiles such as Pyrolobus fumarii; thermophiles such as Synechococcus lividis; mesophiles and psychrophiles such as Psychrobacter, and / or extreme thermophilic sulfur-metabolizing groups such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., and Acidianus sp.; Deinococcus radiodurans; pressure-tolerant organisms, including piezophiles or barophiles; desiccation-tolerant and xerophilic organisms, such as Artemia salina; microorganisms and fungi; halotolerant organisms, including halophiles, such as Halobacteriacea and Dunaliella salina; pH-tolerant organisms, including alkaliphiles, such as Natronobacterium, Bacillus firmus OF4, Spirulina spp., and acidophiles (such as Ideyukogome and Ferroplasma sp.); gas-tolerant organisms, which can tolerate pure CO2, including Ideyukogome; and metal-tolerant organisms, including metalotolerants, such as Ferroplasma 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, yeast, fungi, proteobacteria, engineered organisms thereof, 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 those of the genera Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseijlexus, and Thermomicrobium.

[0173] In certain embodiments, green sulfur bacteria are used, including but not limited to those of 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, Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum Purple non-sulfur bacteria are used, including but not limited to the genera um, Rhodovibrio, and Roseospira.

[0176] In some embodiments, the microorganism is a methanotroph and / or methylotroph. In some embodiments, the microorganism is a Methylococcus genus. In some embodiments, the microorganism is Methylococcus capsulatus. In some embodiments, the microorganism is a methanotroph and / or methylotroph. In some embodiments, the microorganism is a Methylobacterium genus. In some embodiments, the microorganism is obtained from one or more of Methylobacterium zatmanii, Methylobacterium extorquens, and Methylobacterium chloromethanicum. In some embodiments, compositions are provided in which the microorganism is a hydrogen-oxidizing chemoautotroph and / or a carboxydotroph and / or a methylotroph and / or a methanotroph.

[0177] In certain embodiments, the microorganisms grow heterotrophically using multi-carbon organic molecules as a carbon source, such as, but not limited to, sugars, such as, but not limited to, glucose and / or fructose. In some embodiments, the microorganisms can grow on unprocessed 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 as the sole electron donor and carbon source. In some embodiments, the microorganisms can grow mixotrophically on an organic carbon source and use an inorganic electron donor or carbon source.

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

[0179] In certain embodiments, the microorganisms are naturally occurring and / or non-genetically modified (non-GMO) microorganisms and / or non-pathogenic and / or depend on specific environmental conditions provided by bioprocesses that are not present in the surrounding environment.

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

[0181] In certain embodiments, the microorganism or consortium of microorganisms includes bacteria, fungi (e.g., yeast) and / or other microbial cells used to process sugar feedstock into useful organic compounds such as proteins and amino acids in heterotrophic fermentation systems.

[0182] In certain embodiments, the microorganism or consortium of microorganisms comprises beneficial microorganisms. In certain embodiments, the microorganism or consortium of microorganisms comprises "GRAS" (German for Regulatory Approval) microorganisms and / or organisms. In certain embodiments, the microorganism or organism or consortium of microorganisms comprises Candida humilis; Candida milleri; Debaryomyces hansenii; Kazachstania exigua (Saccharomyces exiguous); Saccharomyces cerevisiae; Saccharomyces fl orentinus; Torulaspora delbrueckii; Trichosporon beigelli; and / or fungi including, but not limited to, one or more of: 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.diacetylactis);Leuconostoc;Leuconostoc carnosum;Leuconostoc cremoris;Leuconostoc mesenteroides;Pediococcus;Propionibacterium freudenreichii; Arthrospira (Spirulina) platensis; Streptococcus faecalis; Streptococcus thermophilus.

[0183] The protein containing biomass from which the protein hydrolysate composition is derived may be produced by a consortium of different microorganisms and / or species of multicellular organisms.

[0184] In some embodiments, the consortium comprises one or more of an acidohydrogen microorganism, a carboxydotroph, a methanotroph, a methylotroph, a chemoautotroph, a photoautotroph, and a heterotroph.

[0185] In some embodiments, the protein hydrolysate also contains one or more vitamins produced by the organism from which the hydrolysate was derived. In some non-limiting embodiments, the microorganism is Cupriavidus necator DSM531 or DSM541. In some non-limiting embodiments, the vitamin is a B vitamin, including but not limited to B1, B2, and / or B12.

[0186] Any suitable method can be used to culture the microorganisms. The microorganisms can be grown under any suitable conditions in an environment suitable for biomass growth and production. In some embodiments, the microorganisms can be grown under autotrophic culture conditions, heterotrophic culture conditions, or both autotrophic and heterotrophic culture conditions. Heterotrophic cultures can include suitable carbon and energy sources, such as one or more sugars (e.g., glucose, fructose, sucrose, etc.). Autotrophic cultures can be grown on a mixture containing C1 chemistry, including, but not limited to, carbon monoxide, carbon dioxide, methane, methanol, formate, and / or formic acid, and / or various syngas compositions or various producer gas compositions. The biomass sources may include, for example, lignocellulosic energy crops, crop residues, bagasse, sawdust, forest residues, food waste, municipal solid waste, sewage, carpet waste, biogas, landfill gas, natural gas, or petroleum coke through gasification, partial oxidation, pyrolysis, or steam reforming of the aforementioned low-value or waste carbon sources, which can be used by oxyhydrogen microorganisms or hydrogen-oxidizing or carbon monoxide-oxidizing microorganisms as carbon and energy sources. Suitable methods for culturing microorganisms and generating biomass for use in the methods of the present invention are described, for example, in U.S. Pat. Nos. 9,157,058 and 9,556,462 and PCT International Patent Publication No. WO 2018 / 144965, which are incorporated herein by reference. In some embodiments, the organisms may be grown photosynthetically in bioreactors, hydroponic systems, greenhouses, cultivated land, or recovered from waste or natural sources. In certain embodiments, the protein-containing biomass used to produce the protein hydrolysate is from microorganisms grown on one or more C1 substrates. In certain embodiments, the microorganisms are grown on CO and / or CO dissolved in aqueous solution (e.g., CO(aq), bicarbonate, carbonate) as the sole carbon source. In certain embodiments, the microorganisms grown on CO and / or dissolved CO comprise microbial cells selected from one or more of the genera 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 CO and / or dissolved CO comprises microbial cells selected from one or more of the autotrophic (e.g., chemoautotrophic, photoautotrophic), carboxydotrophic, methanotrophic, methanolotrophic, and / or heterotrophic microorganisms described above. In certain embodiments, the protein-containing biomass derived from microorganisms grown on CO and / or dissolved CO comprises Cupriavidus necator cells. In certain such embodiments, Cupriavidus necator strains may include, but are not limited to, DSM428, DSM531, or DSM541. In certain embodiments, the protein-containing biomass derived from microorganisms grown on CO and / or dissolved CO comprises microbial cells of a PHB-negative mutant or a knockout of a wild-type strain that naturally produces PHB. In certain such embodiments, the mutant strain unable to produce PHB is derived from a wild-type strain capable of producing PHB using known methods. In certain embodiments, the PHB-negative mutant or knock-in strain produces less or undetectable amounts of PHB than the wild-type strain. In certain such embodiments, the PHB-negative mutant is Cupriavidus necator DSM541.

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

[0189] IV. Composition Disclosed herein are protein hydrolysate compositions produced using the disclosed methods. The proteinaceous component of the composition can 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 suitable method (e.g., liquid chromatography). In some embodiments, the high protein organic content is about 10% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, by weight of the organic content. The total organic content may comprise an amount of amino acids ranging from about 10% to about 98% by weight, e.g., from about 20% to about 98% by weight, from about 30% to about 98% by weight, from about 40% to about 98% by weight, from about 50% to about 95% by weight, from about 60% to about 95% by weight, from about 70% to about 95% by weight, or from about 75% to about 95% by weight. The total organic content may be measured using any suitable method.

[0190] In some embodiments, the protein hydrolysate comprises an amount of amino acids of about 10% or more by weight, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, or about 90% or more by weight of the total dry weight (w / w). In some embodiments, the protein hydrolysate has a total dry weight (w / w) equal to or greater than the amino acid content (w / w) of the starting biomass. In some embodiments, the protein hydrolysate has an organic matter content of about 10% or more by weight, e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more by weight of the biomass content.

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

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

[0193] In some embodiments, the protein hydrolysate composition has a nitrogen content of about 5% (w / w) or more (e.g., 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) or less. In some embodiments, the protein hydrolysate composition has a nitrogen content of about 5% (w / w) to about 20% (w / w), e.g., about 6% (w / w) to about 18% (w / w), about 7% (w / w) to about 15% (w / w), or about 8% (w / w) to about 15% (w / w). Nitrogen content can be measured using any suitable method.

[0194] In some embodiments, the protein hydrolysate composition comprises about 5% (w / w) or more The protein hydrolysate composition has a phosphate content, expressed as PO equivalents, of about 6% (w / w) or more, about 8% (w / w) or more, about 10% (w / w) or more, and in some embodiments, about 15% (w / w) or less, e.g., about 13% (w / w) or less, about 11% (w / w) or less, including about 10% (w / w) or less. In some embodiments, the protein hydrolysate composition has a phosphate content, expressed as PO equivalents, in the range of about 5% (w / w) to about 15% (w / w), e.g., about 6% (w / w) to 13% (w / w), 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 as KO equivalents, of about 5% (w / w) or more, e.g., about 7% (w / w) or more, about 10% (w / w) or more, about 12% (w / w) or more, about 15% (w / w) or more, and in some embodiments, a potassium content of about 20% (w / w) or less, e.g., about 19% (w / w) or less, about 18% (w / w) or less, about 16% (w / w) or less, including about 15% (w / w) or less. In some embodiments, the protein hydrolysate composition has a potassium content, expressed as KO equivalents, in the range of about 5% (w / w) to about 20% (w / w), e.g., about 7% (w / w) to about 19% (w / w), or about 10% (w / w) to about 19% (w / w). Potassium content can be measured using any suitable method.

[0196] In some embodiments, the protein hydrolysate composition has an NPK (nitrogen, phosphate, potassium) content of at least 5:5:5 wt %.

[0197] In some embodiments, the protein hydrolysate composition has a sodium content of about 1% (w / w) or less, e.g., about 0.8% (w / w) or less, about 0.6% (w / w) or less, about 0.4% (w / w) or less, or about 0.3% (w / w). In some embodiments, the protein hydrolysate composition has a chloride content of about 1% (w / w) or less, e.g., about 0.8% (w / w) or less, about 0.6% (w / w) or less, about 0.4% (w / w) or less, or 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] The protein hydrolysate composition can be in liquid form, a suspension or slurry, or can be substantially dry. In some embodiments, the protein hydrolysate composition is in liquid form. In some embodiments, the protein hydrolysate composition is a dry powder, such as a lyophilized powder. In some embodiments, the protein hydrolysate composition is a slurry, suspension, or emulsion.

[0199] V. Uses of Protein Hydrolysate Compositions Methods of using the protein hydrolysate compositions described herein are also disclosed. The protein hydrolysate compositions of the present disclosure can be used in various agricultural or horticultural settings. In some embodiments, the protein hydrolysate is used as a biostimulant and / or plant nutrient or a precursor thereof. As a biostimulant, the protein hydrolysate composition can 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 foliar or soil application. In some embodiments, the protein hydrolysate composition is provided to plants by foliar or soil application. In some embodiments, the protein hydrolysate composition is provided using hydroponics, aquaponics, or aquaponics. In some embodiments, protein hydrolysates produced according to the present invention can be used in place of animal-derived protein hydrolysates, hi some embodiments, the protein hydrolysates provide a vegan substitute for fish hydrolysates or fish emulsions.

[0200] The protein hydrolysate composition can substitute for a component or precursor of a biostimulant product and / or plant nutrient. In some embodiments, the protein hydrolysate composition is combined with a preservative. Any suitable preservative can be used. Suitable preservatives include, but are not limited to, citric acid, benzoic acid, propylene glycol, propionic acid, sorbic acid, zinc sulfate, ferrous sulfate, copper sulfate, and / or silver chloride. In some embodiments, the protein hydrolysate composition is combined with a fertilizer. In some embodiments, the protein hydrolysate composition is 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, the protein hydrolysate composition is combined with a herbicide, insecticide, and / or fungicide. Suitable herbicides, insecticides, and / or fungicides include, but are not limited to, thiophanate methyl, chlorothalonil, captan, piperalin, fenarimol, metalaxyl, ethoxythialdiazole, pyretin, algicides, oryzalin, aldxylarypolyethoxyethanol, glyphosate, and naphthalene.

[0201] In some embodiments, the protein hydrolysate composition is provided to promote the growth of food crops, hi certain such embodiments, the food crops include 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, chard, cai sam, white turnip greens, endive, lettuce, mustard, greens, watercress, garlic, chives, gaiIan, leeks, Brussels sprouts, capers, kohlrabi, celery, rhubarb, cardoon, Chinese celery, lemongrass, asparagus, bamboo shoots, galangal root, ginger, soybeans, mung beans, urad, carrots, parsnips, beets, turnips, radishes, rutabaga, turnips, burdock, onions, shallots, leeks, garlic, green beans, lentils, and snow peas; tomatoes, cucumbers, tomato, eggplant, zucchini, pumpkin, melon, bell peppers, eggplant, tomato Fruits such as yuzu, cucumber, okra, breadfruit, avocado, black currant, red currant, gooseberry, guava, lucuma, chili pepper, pomegranate, kiwi, grapes, 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 potato, sweet potato, cassava, yam, and dahlia; and grains or cereals such as corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio, buckwheat, and quinoa.

[0202] In other embodiments, the crop is an ornamental crop. In certain such embodiments, the ornamental crop comprises 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 quadrifidus, Lepistanuda, and Pleurotus ostreatus (agaricus). In certain embodiments, the protein hydrolysates described herein are derived from Agaricus bisporus, Applied to mushrooms, or fungi, such as one or more of Coprinus quadrifidus, Lepistanuda, and Pleurotus ostreatus.

[0203] In some embodiments, the protein hydrolysate composition is used as a nutrient source for other organisms, such as animals, humans, or cells (prokaryotic or cellular), or as a precursor to such a nutrient source or components that form such a nutrient source. In certain embodiments, the hydrolysate produced as described herein is used as a cell culture supplement. Certain embodiments disclosed herein relate to hydrolysates for sports medicine applications, particularly in certain non-limiting embodiments, where consumption of the hydrolysate results in faster absorption by the body than intact protein, thus maximizing nutrient delivery to muscle tissue. In certain embodiments, the hydrolysate is enriched in antioxidants, L-aspartic acid, manganese, and / or selenium. Certain embodiments relate to the use of microbial hydrolysates directly or as ingredients in pet foods, including, but not limited to, mammalian (e.g., dog, cat, rabbit, rodent, horse, etc., or poultry (e.g., chicken, turkey, etc.)) foods. In certain embodiments, the hydrolysate may be used as a value-added nutritional additive for animal feed, more particularly 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 livestock or pets, or animal feed for human consumption.

[0204] Suitable uses of the protein hydrolysate compositions of the present 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 the present disclosure will become more fully apparent from the following examples, which are intended to illustrate, but not limit, the present invention. [Example]

[0206] Example 1: Protein hydrolysates produced from Cupriavidus necator cultures Production of protein hydrolysates from microbial cultures Cupriavidus necator strains were grown in growth medium with glucose as the carbon source. After growth, the whole cell biomass was isolated from the growth medium and dried. A portion of the dried biomass was processed as follows: 1.2 g of dry biomass was suspended in 100 ml of water. 2. The suspended biomass was homogenized with a T25 Turrax stick at 15000 rpm for 1 minute. 3.10N KOH stock 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 1 M KH2PO4 buffer, pH 5.8, to reduce the pH to approximately 8.8. 7. 100 μl of 1 mg / ml bacterial alkaline protease (prepared in 10 mM Tris buffer, pH 7.4) was added to the neutralized solution. 8. The resulting reaction was digested overnight in a shaking water bath at 55°C. 9. After enzymatic hydrolysis, the reaction was centrifuged and the supernatant was separated from the pellet.

[0207] Protein hydrolysate content analysis The supernatant and pellet fractions were analyzed on an SDS-PAGE gel (Figure 6). It contained most of the proteins and polypeptide fragments, which were less than 15 kD.

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

[0209] The supernatant from Example 1 was lyophilized to produce a powder that was analyzed for: 1.Nitrogen content 2. Ash and humidity levels 3. Phosphorus, phosphate, potassium and potash content 4. Amino acid content (all 20 amino acids, including cysteine, methionine, and tryptophan) 5. Process Yield and Complete Mass Balance by Weight

[0210] Example 2: Protein hydrolysates produced from Cupriavidus necator cultures Protein hydrolysates were produced from the C. necator strain culture of Example 1. KOH was added to the suspended biomass to obtain a pH of 11.3.

[0211] The supernatant was freeze-dried to produce a powder that was analyzed for: 1.Nitrogen content (Table 1) 2. Ash and humidity levels (Table 1) 3. Phosphorus, phosphate, potassium and potash content (Tables 1 and 2) 4. Amino acid content (all 20 amino acids, including cysteine, methionine, and tryptophan) (Table 2) 5. Process Yield and Complete Mass Balance by Weight [Table 1] [Table 2]

[0212] This protocol produced protein hydrolysates from bacterial cell biomass. Protein hydrolysates (PH) contained a complex mixture of amino acids, proteins, and inorganic nutrients, including NPK. PH had a very high % amino acids (wrt organic matter). The organic matter in this PH was 80%, and 92% of this organic matter was amino acids.

[0213] Example 3: Producing protein hydrolysates from a secondary C. necator culture A second portion of the dried biomass from the C. necator strain culture was treated as in Example 1. KOH was added to the suspended biomass to a final volume of 50 mM.

[0214] The resulting supernatant was lyophilized 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: Producing protein hydrolysates from large-scale Cnecator cultures Whole cell biomass weighing approximately 310 g was obtained from C. necator. The process described in Example 1 was scaled up to process large scale 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 the addition of KOH was 12.3. After air sterilization, the pH was 10.6-10.9. The autoclaved suspension was neutralized with phosphate buffer (KH2PO4) to a pH of 8.9-9.0. After overnight hydrolysis, the pH was 8.01.

[0216] Example 5: Effect of pH (1) The amino acid and organic matter contents were compared between the protein hydrolysates from the two different substrate treatments. The protein hydrolysate from Example 2 (pH titrated to 11.3 before autoclaving) had higher organic matter and amino acid contents compared to the protein hydrolysate made using KOH added to 50 mM (pH about 12) before autoclaving. In Table 5, the amino acid and nitrogen contents are each measured relative to the dry weight of the protein hydrolysate. [Table 5]

[0217] Example 6: Effect of pH (2) The pH of the biomass suspension obtained from C. necator culture was adjusted to pH 9.6 with 1% NaOH. The alkaline suspension was autoclaved at 121°C for 20 min with slow evacuation. The hydrolysis suspension was centrifuged, and the supernatant and pellet fractions were assayed for protein content by SDS-PAGE (Figure 7). The supernatant contained incompletely resolved protein.

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

[0219] Example 7: Effects of Neutral Buffers, Detergents, Chelating Agents, and Chaotropic Agents A set of samples was prepared, each containing 0.3 g of dry biomass, suspended and homogenized, treated with 1N NaOH at 0.1 N, autoclaved at 110°C for 10 minutes, cooled by fast evacuation, neutralized with 1M potassium phosphate buffer at pH 5.8 (unless otherwise noted), and digested with bacterial alkaline protease overnight at 55°C.

[0220] All samples were centrifuged at 11,000 rpm for 15 minutes after overnight enzyme treatment, and the supernatant was separated from the pellet. The different fragments were analyzed by SDS-PAGE, Lowry assay, and OPA assay.

[0221] To test the effect of surfactant treatment, some samples were treated with ammonium lauryl sulfate, Triton X-100, Tween 80, Tween 20, or Purified Water before autoclaving. Ionic PF-68 was added to the biomass suspension (Figures 8A and 8B). The surfactant was added at a final amount of 10% based on the dry weight of the biomass. To test the effect of the neutralizing buffer, some samples were neutralized with 1M Tris buffer, pH 7.4, instead of potassium phosphate buffer (Figures 8A and 8B).

[0222] Figure 8A shows the distribution of proteins among the pellet and supernatant fractions of samples treated with Tris buffer and different detergents. Lane 1: Marker; Lane 2: SDS (pellet); Lane 3: SDS (supernatant); Lane 4: No detergent (pellet); Lane 5: No detergent (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 shows the distribution of proteins among the pellet and supernatant fractions of samples treated with potassium phosphate buffer and different detergents. Lane 1: Marker; Lane 2: SDS (pellet); Lane 3: SDS (supernatant); Lane 4: No detergent (pellet); Lane 5: No detergent (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 different bases, KOH was used in place of NaOH in some samples before autoclaving (Figure 9).To test the effect of chelating agents, EDTA was added to a final concentration of 5 mM in some samples before autoclaving (Figure 9).

[0225] Figure 9 shows the distribution of proteins between the pellet and supernatant fractions of samples treated with NaOH or KOH and different combinations of EDTA and urea. NaOH-treated samples (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 samples (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 a suitable basic reagent. Urea and EDTA treatment increased total protein and supernatant protein yields. Chaotropic and chelating agents such as urea or EDTA can be removed from the resulting supernatant fraction by buffer exchange.

[0227] Example 8: Biostimulant effect of protein hydrolysates The biostimulant effects of the protein hydrolysates of the present disclosure were tested on a variety of plants, such as turfgrass, radishes, lettuce, etc., and mushrooms. The growth of the plants or mushrooms was compared with and without application of the protein hydrolysate. In some cases, the protein hydrolysate was combined with other ingredients to create a plant supplement and applied to the plant. The plants may be grown in soil or hydroponically. The protein hydrolysate may be provided to the soil or as a foliar application. Biostimulant efficacy may be measured by growth density (turfgrass), weight, nitrogen uptake, or chlorophyll content.

[0228] Example 9: Protein hydrolysates produced from Cupriavidus necator cultures Cupriavidus necator strain was cultured in growth medium with fructose as a carbon source. After growth, the whole cell 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. Homogenized with UltraTurrax stick at 13,200 RPM for 1 minute. 3.13.51 ml of phosphoric acid (H3PO4) was added to a concentration of 85% (14.8M). 4. The suspension was transferred to a 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 an autoclaved 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 (Labconco Freezone, 4.SL, -SOC). 11. The samples were lyophilized until dry.

[0229] Gel analysis The lyophilized acid hydrolyzed sample ("Test") was analyzed by SDS-PAGE (Novex 4-20% Tris-glycine) and compared to a lyophilized sample (Control) that had not been subjected to acid hydrolysis or calcium hydroxide neutralization. The acid hydrolyzate was denser than the pellet fragment and exhibited a bright band, indicating that a significant amount of protein had been extracted and solubilized in the supernatant.

[0230] Protein hydrolysate content analysis The freeze-dried acid hydrolyzed samples ("Test") were analyzed for the following items and compared to a freeze-dried sample ("Control") that had not been subjected to acid hydrolysis or calcium hydroxide neutralization: 1. Moisture, Ash, and Nitrogen (Table 6) 2. Mass Balance 3. Nitrogen balance 4.% Nitrogen Solubility

[0231] The percent moisture, ash, and nitrogen of the freeze-dried dry mass are shown in Table 6. [Table 6]

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

[0233] Whole-cell biomass was delipidated by suspending the biomass in ethanol at a ratio of 3.75 mL per gram of biomass, followed by the addition of 0.5 mL of NH4OH per gram of biomass. The solution was stirred on a magnetic stir plate for 30 minutes and then filtered through Whatman 4 filter paper. The lipid-containing filtrate (EtOH / NH4OH) was then dried in a sand bath at 50 °C. The remaining defatted biomass was air-dried in a fume hood overnight and then oven-dried at 40 °C for 4–6 hours. This dried, defatted biomass was then used in the subsequent hydrolysis reaction. The following modifications were attempted with the defatted biomass:

[0234] Enzyme-only digestion: A 2% solids solution was made with defatted biomass and distilled water. This solution was homogenized, adjusted to pH 9 with 10 KOH, bacterial alkaline protease was added, and incubated overnight in a shaking water bath at 55°C. The hydrolysate was then centrifuged at 26,000 x g for 30 minutes, and the supernatant and pellet fractions were separated and lyophilized.

[0235] Solid Ca(OH)2 was used to precipitate minerals: a 2% solids solution was made with defatted biomass and distilled water. This solution was homogenized, the pH adjusted to 11 or 12 with Ca(OH)2, autoclaved at 110°C for 10 minutes, and then adjusted to pH 9 with H3PO4. Bacterial alkaline protease enzyme was then added and incubated overnight in a shaking water bath at 55°C. The hydrolysate was then centrifuged at 26,000 x g for 30 minutes, and the supernatant and pellet fractions were separated and lyophilized.

[0236] Table 7 shows the mass, nitrogen yield, and ash content for each condition. The enzyme-digested samples had the lowest ash content, while the pellets from the chemically hydrolyzed samples had a higher ash content (approximately 22-32%) due to the presence of precipitated inorganic matter. Protein hydrolysates from the chemically hydrolyzed supernatants 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: Low-ash protein hydrolysate produced from Cupriavidus necator culture Cupriavidus necator PHB-negative mutant (DSM541) was cultured with CO2 as a carbon source in the growth medium. After growth, the whole cell biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows:

[0238] The delipidated WCB of whole-cell biomass was defatted (lipids extracted) by mixing it with ammonium hydroxide and methanol (1:1:0.4, WCB:NH4OH:MeOH) and stirring the mixture in a tightly capped container in a fume hood for 1 hour. The collected filtrate contained the extracted lipids. The residue on the filter was delipidated and dried overnight in an incubator at 40°C.

[0239] Protein hydrolysis on defatted mass with H3PO4 and Ca(OH)2: Using a solid loaded with 8% defatted dry mass, rehydrate with the required amount of ID water. Then, vigorously stir with a TurretStick at 15,000 rpm for 1 minute. 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 a tightly closed screw cap. It was then autoclaved at 121°C and 15 psi for 1 hour. The reaction was then allowed to cool in a fume hood. Calcium hydroxide was added to neutralize the pH to 8-9, and the pH was confirmed with a pH meter. The protein hydrolysate (supernatant) was centrifuged at 10,000 x g for 20 minutes at 7°C. The resulting liquid protein hydrolysate was lyophilized to produce a dry powder.

[0240] The measured ash content of the hydrolysate was 7.04%. Ash content was also determined 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: Low-ash protein hydrolysate produced from Cupriavidus necator culture Cupriavidus necator strain (-PHB) was cultured in growth medium with CO as a carbon source. After growth, the whole cell biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows:

[0242] The delipidated WCB of whole-cell biomass was defatted (lipids were extracted) by mixing it with ammonium hydroxide and methanol (1:1:0.4, WCB:NH4OH:MeOH) and stirring the mixture in a tightly capped container in a fume hood for 1 hour. The collected filtrate contained the extracted lipids. The residue on the filter was delipidated and dried overnight in an incubator at 40 °C.

[0243] Protein hydrolysis with H3PO4 and CO2: Using a 2% defatted dry mass loading, the solid was rehydrated with the required amount of 1D water and then vigorously stirred with a TurretStick at 15,000 rpm for 1 min. The pH of the reaction mixture was increased by adding 8%-30% (premade) 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 min with slow exhaust. The pH of the solution after autoclaving was 10.82. The pH was lowered to 9 by bubbling CO2 through a cannula / 18G needle for 10-20 min. Enzymatic digestion was performed with bacterial alkaline protease at 55 °C and 110 rpm overnight. The supernatant containing the soluble hydrolyzed protein and the pellet (rich native pellet at 110 rpm) were centrifuged at 20,000 x g for 20 min at 5 °C. The protein hydrolysate was freeze-dried.

[0244] The measured ash content of the protein hydrolysate was 5%. Ash content was 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 also by external laboratory analysis (SGS, North America).

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

[0246] Example 13: Low-ash protein hydrolysate produced from Cupriavidus necator culture Cupriavidus necator PHB-negative mutant (DSM541) was cultured in growth medium with either CO or sugar as the carbon source. After growth, the whole cell biomass was isolated from the growth medium and freeze-dried. A portion of the dried biomass was processed as follows:

[0247] Defatting of Whole-Cell Biomass. Dry 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 for 30 minutes in a tightly capped glass bottle and vacuum filtered through Whatman 4 filter paper in a fume hood. The collected filtrate contained lipid fragments, and the defatted retentate was air-dried overnight and then dried in a 40°C incubator for 4-6 hours.

[0248] Protein hydrolysis of defatted biomass with Ca(OH)2 and H3PO4: Dry defatted biomass was resuspended in DI water to a final concentration of 2%. The biomass solution was mixed with an IKA Ultra-Turax at 15,000 rpm to ensure complete and homogeneous resuspension. The biomass solution was adjusted to pH 11 by adding Ca(OH)2. The solution was transferred to a glass media bottle and autoclaved at 110°C for 10 minutes, then allowed to cool 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 activity units / g biomass. The biomass solution was placed in a shaking water bath at 55°C overnight. After 16-24 hours of digestion, the enzyme was inactivated by incubating the slurry in a 95°C water bath for 10 minutes. The biomass slurry was then cooled to room temperature, and the protein hydrolysate (supernatant fraction) was separated by centrifugation at 26,000 x g for 30 minutes at 7°C. The resulting protein hydrolysate solution was frozen in a -80°C freezer and then lyophilized. The moisture, ash, and nitrogen contents of the dried powder were determined, and the protein profile was analyzed via SDS-PAGE analysis. No proteins greater than 2,000 Daltons were present in the protein hydrolysate, and the resulting ash content was 10.5%.

[0249] Example 14: Demonstration of biostimulant effect in protein hydrolysates produced using acidic hydrolysis in Brassicarapa species Protein hydrolysates prepared by incubation with phosphoric acid, followed by neutralization with calcium hydroxide to precipitate the phosphate ions, and then neutralizing the solution to pH 5, were tested in accordance with OECD Guidelines for the Testing of Chemicals 2008 and The impact as a biostimulant was tested using Brassicarapa (turnip) species according to ISO 11269-1, 2.

[0250] Ten seeds were sown in 8 x 8 x 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's water-holding capacity. Once 50% of the control seeds had germinated, the pots were plowed out, leaving five seeds per pot. Water / treatments were applied either after sowing (day 1), after germination (day 7), or both days 1 and 7. A total of nine treatments, including a water and boric acid control, were tested with and without basal fertilizer (NPK 150-90-60). All treatments receiving the protein hydrolysate treatments showed a 3.5-fold increase in overall biomass compared to the water control and the NPK fertilizer-treated control. The treatment receiving protein hydrolysate only on day 7 had the greatest overall growth.

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

[0252] Example 15: Demonstration of 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 tested was produced using acidic hydrolysis as described herein. The second protein hydrolysate tested was produced using alkaline hydrolysis as described herein. Both were tested for their impact as biostimulants in lettuce (Lactuca saliva). 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 soil. Plants were watered, rotated twice a week, and exposed to 700 μmol photons / m 2 The plants were grown under a 14-hour photoperiod with a light exposure of 1000μg / ml.

[0253] To compare the protein hydrolysates, the water treatment and the commercial fish and seaweed hydrolysate treatment were used as controls.

[0254] A base nutrient solution of 0.4 g Hoagland salt mixture / L Hoagland solution was used for all treatments and applied to the soil on the day seedlings were transferred. All treatments were normalized to a starting soil nitrogen rate of 0.0165 g N / 750 ​​g, and all results are reported as the average of four plants from each treatment group.

[0255] FIG. 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 the present disclosure have been described in detail herein to enable those skilled in the art to practice the full scope of the present invention. However, it should be understood that possible variations of the present disclosure not specifically described are also encompassed within the scope of the present invention and the appended claims. Accordingly, these descriptions are provided by way of example only and are not intended to limit the present invention. More generally, all parameters, dimensions, materials, and configurations described herein are for illustrative purposes, and those skilled in the art will readily appreciate that actual parameters, dimensions, materials, and configurations will vary depending on the particular purpose or purpose for which the teachings of the present disclosure are used. Those skilled in the art will recognize or be able to ascertain, using as little as routine experimentation, many equivalents to the specific embodiments disclosed herein. Accordingly, the above-described embodiments are for illustrative purposes only, and the accompanying drawings are not intended to be limiting unless otherwise specified. It is understood that the present invention may be practiced other than as specifically described and claimed, within the scope of the appended claims and their equivalents. The present invention is not limited to each individual feature, system, article, material, kit, composition, and / or method described herein. Further, any such features, systems, articles, materials, kits, compositions and / or The combination of two or more of the methods may include any of the features, systems, articles, materials, kits, compositions, and the like. As long as the methods and / or techniques are not mutually exclusive, they are within the scope of the present invention.

[0257] All publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety for all purposes, and each individual publication, patent, and patent application is specifically and individually indicated to be incorporated by reference in its entirety for any purpose.

Claims

1. 1. A method for producing a protein hydrolysate, comprising: (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) adjusting the pH of the biomass suspension composition, if necessary, 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 time 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, 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 of 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, wherein the supernatant comprises 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), and optionally 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 the microbial cells comprise Cupriavidus cells, and further optionally the Cupriavidus cells comprise 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 cannot produce PHB, the mutant strain being derived from a wild-type strain that can 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 of claim 5.

8. 6. A protein hydrolysate composition comprising hydrolyzed microbial protein produced according to any of claims 1 to 5, wherein the protein hydrolysate composition has 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 contains an amount of amino acids of 10% by weight or more, based on organic content (w / w); or iii) the hydrolyzed microbial protein contains an amount of amino acids of 10% or more by total dry weight (w / w); or iv) the hydrolyzed microbial protein has an ash content of 40% by weight or less, based on the dry biomass content (w / w); The protein hydrolysate composition.

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

10. The method of any one of claims 1 to 7, wherein the first temperature is from 110°C to 140°C.

11. The method of any one of claims 1 to 7 and 10, wherein the first period of time is at least 1 hour.

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

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

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

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