Bio-derived alternative carbon black and its manufacturing method

Abstract

A method for producing carbon black pigment from microbial biomass is disclosed. In certain aspects, the method includes providing a microbial biomass solution of a plurality of microbial cells in an aqueous solvent, nucleating the plurality of microbial cells by adding a first soluble ion to the microbial biomass solution, initiating crystal formation in and / or on the plurality of microbial cells by adding a second soluble ion to the microbial biomass solution to form a plurality of crystalline shell-coated microbial cells, wherein the charge of the first soluble ion is opposite to the charge of the second soluble ion, and the crystals are formed from precipitation of the first and second ions, and heat-treating the plurality of crystalline shell-coated microbial cells to form a carbonized biomass. Washing the carbonized biomass to form a microbial char.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 021,494, filed May 7, 2020, entitled "Biologically - derived carbon black alternative and method of making the same", the entire contents of which are incorporated herein by reference under 35 U.S.C. § 119(e). The disclosed technology relates generally to the production of pigments and colorants from microbial biomass.

Background Art

[0002] Pigments and colorants are an industry worth over $30 billion annually. However, the production of these compositions involves the generation of toxic bioproducts that can be harmful to human health and the environment. Previous attempts to create pigments from non - toxic biomass have been limited in terms of the ability to produce particle sizes small enough to be suitable for most industrial applications. Therefore, a method for producing pigments / colorants from sustainable resources that are suitable for industrial requirements is sought in the art.

Summary of the Invention

[0003] A method for producing carbon black pigment from microbial biomass is disclosed. In a particular embodiment, the method includes: providing a microbial biomass solution comprising a plurality of microbial cells in an aqueous solvent; nucleating the plurality of microbial cells by adding a first soluble ion to the microbial biomass solution; initiating crystal formation in and / or on the plurality of microbial cells by adding a second soluble ion to the microbial biomass solution to form a plurality of crystal-encrusted microbial cells, wherein the charge of the first soluble ion is opposite to the charge of the second soluble ion, and the crystal is formed from the precipitation of the first and second ions; and heat-treating the plurality of crystal-encrusted microbial cells to form carbonized biomass; and washing the carbonized biomass to form microbechar.

[0004] In certain embodiments, the first ion is an anion and the second ion is a cation. In further embodiments, the first ion is a cation and the second ion is an anion. In exemplary executions of these embodiments, the cation is calcium and the anion is a phosphate ion. According to certain embodiments, the first and second ions are present in stoichiometric ratios.

[0005] In certain embodiments, the nucleation step may include performing a nucleation incubation by incubating a first ion with a plurality of microbial cells for a duration of about 5 minutes to about 2 hours. The incubation step may further include heating the microbial biomass solution to a temperature of about 32°C to about 65°C. The incubation step may further include stirring the microbial biomass solution. In certain embodiments, the stirring step is performed by shear mixing the microbial biomass solution at about 2000 rpm for about 2 minutes.

[0006] In certain embodiments, the crystallization step may further include a crystallization incubation. In a particular run, this step is carried out by incubating a microbial biomass solution for a duration of approximately 5 minutes to approximately 2 hours. In a particular run, the crystallization step results in the formation of multiple crystalline shell-coated microorganisms with cell surface crystallization and / or intracellular crystallization.

[0007] In a particular embodiment, the microbial biomass includes multiple prokaryotic cells, the average cell size of which is less than approximately 50 μm. In a particular embodiment, the microbial biomass is dried at a temperature of about 30°C to about 300°C prior to the heat treatment step, until the moisture content is reduced to less than about 15%. In a particular run, the heat treatment step is carried out until the carbonized biomass has a fixed carbon content of about 20% to about 70%. In a further run, the heat treatment step is carried out until the oxygen concentration is about 10-15%.

[0008] In certain embodiments, the washing of the carbonized biomass is acid washing. In exemplary runs, acid washing is performed by washing the carbonized biomass in a solution having a pH of less than about 2. In certain runs, this washing is performed for a duration of about 1 minute to about 1 hour. In exemplary embodiments, the acid washing and subsequent water washing produce porous microbial char.

[0009] According to a particular embodiment, a grinding step is performed on the microbial charcoal following an acid washing step to form powdered microbial charcoal. In a particular execution, the grinding step is performed until the average particle size diameter of the powdered microbial charcoal is less than approximately 10 μm.

[0010] In certain embodiments, the carbon black pigment produced by the method of the present disclosure has increased fluidity compared to microbial biomass obtained by equivalent heat treatment without the crystallization process. Furthermore, the produced carbon black pigment has increased porosity compared to microbial biomass obtained by equivalent heat treatment without the crystallization process. In further embodiments, the produced carbon black pigment has increased blackness compared to microbial biomass obtained by equivalent heat treatment without the crystallization process.

[0011] In a further embodiment, a method for producing a carbon black pigment designed from microbial biomass is disclosed. This method also includes heat treatment of microbial biomass, which may contain a plurality of crystalline shell-coated microbial cells, to form carbonized biomass, and washing the carbonized biomass by acid washing, the washing step may include lowering the pH of the carbonized biomass to less than about 2 over a time period of about 1 minute to about 1 hour to form microbial char.

[0012] In a further embodiment, it is disclosed that the designed carbon black pigment may contain carbonized biomass derived from microbial biomass, having a particle size of approximately 0.01 microns to approximately 100 microns.

[0013] While several embodiments are disclosed, those skilled in the art will recognize other embodiments of the Disclosure from the following detailed descriptions illustrating exemplary embodiments of the disclosed apparatus, systems, and methods. As is recognized, all disclosed apparatus, systems, and methods are subject to various obvious modifications without departing from the spirit and scope of the Disclosure. Accordingly, the drawings and detailed descriptions should be considered illustrative rather than limiting. [Brief explanation of the drawing]

[0014] [Figure 1]Figure 1A is a scanning electron microscope (SEM) image of carbonized Spirulina biomass (Arthrospira) treated with a soluble anion / cation mixture before carbonization, according to one embodiment. Figure 1B is an SEM image of carbonized Spirulina biomass (Arthrospira) from which all salts have been washed off before carbonization, according to one embodiment. Figure 1C is an SEM image of carbonized Spirulina biomass (Arthrospira) from which the washed, salt-free biomass has been retreated with a soluble anion / cation mixture before carbonization, according to one embodiment. [Figure 2] Figure 2A is an image of pure, untreated carbonized Spirulina biomass (Arthrospira) according to one embodiment. Figure 2B is an SEM image of pure, untreated carbonized Spirulina biomass (Arthrospira) treated with a soluble anion / cation mixture before carbonization, according to one embodiment. [Figure 3] Figure 3A is an SEM image of untreated carbonized yeast (Saccharomyces, Candida, or other yeast genera) biomass according to one embodiment. Figure 3B is an SEM image of carbonized Saccharomyces / Candida / or other yeast genera) biomass treated with a soluble anion / cation mixture before carbonization according to one embodiment. [Figure 4] Table 1 summarizes the results of Example 4. Figure 4A shows carbonized Spirulina biomass in which all salts have been washed off before carbonization, according to one embodiment. Figure 4B shows carbonized Spirulina biomass treated with a soluble anion / cation mixture before carbonization, according to one embodiment. [Figure 5] Figure 5A shows pure, untreated carbonized yeast biomass in which all salts have been washed off before carbonization, according to one embodiment. Figure 5B shows carbonized yeast biomass treated with a soluble anion / cation mixture before carbonization, according to one embodiment. [Figure 6-1] Tables 2 to 4 show that adding salt to biomass before carbonization and then acid washing after carbonization significantly increases the blackness value. [Figure 6-2] Figure 6A is a microscopic image of carbonized biomass treated with a soluble anion / cation mixture before carbonization, according to one embodiment. Figure 6B is a microscopic image of salted carbonized biomass, after salt has been removed by post-carbonization acid washing, according to one embodiment. [Modes for carrying out the invention]

[0015] In this specification, a range may be expressed as "about" one particular value, to and / or "about" another particular value. Where such a range is expressed, further aspects include "from" one particular value, and / or "to" another particular value. Similarly, where a value is expressed as an approximation by the use of the antecedent "about", it is understood that a particular value forms further aspects. Furthermore, it is understood that each endpoint of a range has meaning both in relation to other endpoints and independently of other endpoints. It is also understood that numerous values ​​are disclosed in this specification, and each of those values ​​is disclosed in this specification not only as the value itself, but also "about" that particular value. For example, where the value "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, where 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0016] In this specification and the last claim, references to parts by weight of a particular element or component in a composition indicate a weight relationship between that element or component and any other element or component in the composition or article in which parts by weight are represented. Therefore, in the case of a formulation containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y exist in a weight ratio of 2:5, and this ratio exists regardless of whether additional components are present in the formulation.

[0017] The weight percentage (W%) of an ingredient is based on the total weight of the preparation or composition containing that ingredient, unless otherwise specified. As used herein, the terms “as requested” or “as requested” mean that the event or situation described thereafter may or may not occur, and that the description includes both cases in which such event or situation occurs and cases in which it does not occur.

[0018] As used herein, the term “microbial charcoal” means heat-treated carbonized biomass produced according to the methods disclosed herein. Microbial charcoal can function as a carbon black pigment without further modification. Depending on the intended purpose, microbial charcoal may be further treated by mechanical (e.g., pulverization) or chemical (e.g., acid / base washing) means. Methods for modifying / treating microbial charcoal are disclosed in U.S. Patent Application No. 16 / 677,644 (U.S. Patent Application Publication No. 2020-014069), which is incorporated herein by reference in all respects.

[0019] A method for producing a designed carbon black pigment from microbial biomass by heat treatment of microbial biomass is disclosed herein, wherein the microbial biomass comprises multiple crystalline shell-coated microbial cells. As a result of the heat treatment, carbonized biomass is formed. In a particular execution, the method further comprises grinding the carbonized biomass to a particle size of about 0.01 microns to about 100 microns to form powdered microbial char.

[0020] In certain embodiments, the disclosed method is a method for producing carbon black pigment from microbial biomass, comprising providing a microbial biomass solution containing a plurality of microbial cells in an aqueous solvent, nucleating the plurality of microbial cells by adding a first soluble ion to the microbial biomass solution, initiating crystal formation in and / or on the plurality of microbial cells by adding a second soluble ion to the microbial biomass solution, forming a plurality of crystal-coated microbial cells, wherein the charge of the first soluble ion is opposite to the charge of the second soluble ion, performing a heat treatment on the plurality of crystal-coated microbial cells to form carbonized biomass, washing the carbonized biomass, and grinding the carbonized biomass to a particle size of about 0.01 micron to about 100 microns to form powdered microbial carbon.

[0021] Microbial biomass Biological materials mainly include carbohydrates, proteins, and lipids. When these molecules are subjected to pyrolysis at 500 - 600 °C, they undergo complex depolymerization and dehydration reactions, followed by various cleavage, elimination, and condensation reactions to produce non-condensable volatile substances, condensable vapors (liquid tar after cooling), and carbonaceous char as solid residues.

[0022] Carbohydrates are molecules consisting of carbon, hydrogen, and oxygen. Inside cells, carbohydrates are usually stored as long-chain polysaccharides such as cellulose and hemicellulose (woody plants), starch (plants and algae), and glycogen (cyanobacteria, fungi, bacteria). Glycogen is the main storage form of carbohydrates in cyanobacteria such as Spirulina. It is a large, highly branched polysaccharide of glucose, having a structure similar to starch but with a higher degree of branching. When glycogen is pyrolyzed at high temperatures (above 500 °C), products similar to those obtained from starch are generated. These products include water, carbon monoxide, ethane, formaldehyde, ketene, propene, carbon dioxide, acetaldehyde, formic acid, acrolein, hydroxyacetaldehyde, pyruvaldehyde, hydroxypropane, 2-furaldehyde, furfuryl alcohol, 5-methyl-2-furaldehyde, levoglucosenome, 5-hydroxymethyl-2-furaldehyde, and levoglucosan. Many of these products are lost as volatile substances, but some form carbonaceous carbon of the solid residue.

[0023] Polysaccharides such as glycogen can be broken down into their smaller monosaccharide components by hydrolysis. One method of glycogen hydrolysis involves applying heat and dilute acid to hydrolyze glucose units from the branching of glycogen. This reaction readily occurs at 100°C. When monosaccharide units such as glucose are heated to 160°C, they undergo a process called caramelization, at which point the crystalline sugars melt into clear molten sugar. If the temperature rises to 165°C, the molten sugar becomes pourable, but upon cooling, it becomes a hard, glassy, ​​brittle state. If heated further to 210°C, upon cooling, it becomes a softer, more viscous texture. When glucose is thermally decomposed up to 500°C, the main products can be classified into three main types: (1) low molecular weight compounds, (2) furan / pyran ring derivatives, and (3) anhydrous sugars. Biological substances with high monosaccharide / simple sugar content may exhibit melting properties during the thermal decomposition heating process up to 500°C, and the mushrooming effect of biomass due to the release of volatile substances in the viscous material.

[0024] Proteins are polymer chains of amino acids linked by peptide bonds and are highly susceptible to thermal denaturation at temperatures as low as 40°C. The denaturation process involves cleaving weak bonds within the protein molecule, leading to structural changes. This is often followed by coagulation of denatured proteins, where the protein "solidifies" as a solid or viscous liquid, a process that is irreversible once cooled. When exposed to the extreme temperatures of thermal decomposition, proteins undergo thermal decomposition via pathways of decarboxylation, deamination, cleavage of hydrocarbon residues, dimerization, and cleavage of peptide bonds, forming amides / amines / nitriles, esters, hydrocarbons, and N-heterocyclic compounds, particularly diketopiperazines (DKPs).

[0025] Lipids are also susceptible to thermal decomposition. When heated in the presence of water, the ester bonds of lipids are hydrolyzed, releasing free fatty acids. During thermal decomposition, lipids decompose through pathways including dehydration, decarboxylation, hydrolysis of ester bonds, conjugation of double bonds, polymerization, dehydrogenation and cyclization, aromatization, dehydrogenation, and carbon-carbon cleavage.

[0026] According to a particular embodiment, the microbial biomass for the disclosed method can be derived from a number of microbial sources. In a particular execution, the microbial biomass comprises a plurality of microbial cells. In a particular embodiment, these cells (or groups of cells) have an average size of about 10 nanometers to about 300 micrometers. The starting material may have one or more of the following characteristics: cells derived from unicellular organisms, colonial organisms, multicellular organisms, or filamentous organisms; untreated cells; cells or cellular material exhibiting a spherical shape; cells from which certain cellular components have been removed prior to heat treatment; and / or cells in solution or cells removed from solution.

[0027] Multiple cells constituting a microbial biomass can obtain energy through chemonutrition, heterotrophy, or autotrophy. In certain embodiments, the microorganisms are eukaryotes. In alternative embodiments, the microorganisms are prokaryotes. In certain further embodiments, the multiple microbial cells constituting the microbial biomass are a mixture of any of the aforementioned microorganisms, such as those found in natural aquatic environments.

[0028] Various runs utilize colony-forming bacteria, algae, and cyanobacteria. In various runs, multiple cells of the microbial biomass have aggregate diameters of less than 300 microns. One aspect of this disclosure relates to a run in which the pigment portion is approximately 0.01 to 300 microns. It is understood that this size allows for an increase in the amount of pigment particles dispersed to an acceptable density so that a deep color can be obtained. In various runs, a size of 0.01 to 300 microns can be achieved in several ways. In a particular run, the size can be achieved by growing biological cells of an appropriate size. In another run, the size can be achieved by grinding cells or cell aggregates to an appropriate size (0.01 to 300 microns). In yet another run, cells with a diameter of 0.01 to 300 microns, and both grinding of cells or aggregates, may be used.

[0029] According to a particular implementation, microbial biomass includes multiple microbial cells. Suitable microbial cells for the disclosed microbial methods include heterotrophic, autotrophic, mixed trophic, or extremophilic microorganisms, including microalgae, algae, macroalgae, cyanobacteria, fungi, and bacteria. In a particular implementation, the multiple cells are a mixture of the aforementioned cells. According to a particular embodiment, the microorganism comprising multiple microbial cells is one or more selected from the following: Synechocystis PCC 6803, Synechococcus PCC 6717, Synechococcus PCC 6301, Synechococcus IU 625, Synechococcus PCC 6312, Synechococcus elongatus PCC 7942, Nostoc sp., Synechococcus 6911, Synechococcus leopoliensis, Plankthorax rubescens, Synechococcus PCC 7002, Arthospira platensis PCC 7345, Haematococcus pluvailis, Navicula pelliculosa, Cryptomonas erosa, Rhodomonas minuta, Porphyridium purpureum, Phaeodactylum tricornutum, Nannochloropsis sp., Synechocystis, Synechococcus, Nostoc, Planctrax, Alsospira, Haematococcus, Navicula, Cryptomonas, Rhodomonas, Porphyridium, Phaeodactylum, Nannochloropsis, Volvox Anabena sp., Chlorella sp., Euglena sp., Achnantes sp.), Botryococcus sp., Chaetoceros sp., Chroococcus sp., Cosmarium sp., Microcystis sp., Microspora sp., Pediastrum sp., Scenedesmus sp., Spirogyra sp., Spirulina sp., Zygnema sp., Chlorobium sp., Escherichia sp., Spirillum sp., Chromobacterium * * Candida sp., Leucosporidium sp., Rhodotorula sp., Schizosaccharomyces sp., Dekker sp., Brettanomyces sp.), Synechocystis species, Synechococcus species, Nostoc species, Planctrax species, Alsospira species, Haematococcus species, Navicra species, Cryptomonas species, Rhodomonas species, Porphyridium species, Pheodactylum species, Nannochloropsis species, Volvox species, Anabaena species, Chlorella species, Euglena species, Aknanthes species, Botryococcus species, Chaetoceros species, Chroococcus species, Cosmarium species, Microcystis species, Microspora species, Pediastrum species, Senedesmus species, Spirogyra species, Spirulina species Species of the genera Signema, Chlorobium, Eschericia, Spirillum, Chromobacterium, Jansinobacterium, Streptomyces, Xanthomonas, Salsina, Serratia, Rhizobium, Prevotella, Actinomyces, Staphylococcus, Proteus, Micrococcus, Lugamonas, Pseudomonas, Helicobacter, Saccharomyces, Candida, Leucosporidium, Rhodotorula, Schizosaccharomyces, Deckel, and Brettanomyces. Those skilled in the art will understand that other microorganisms are also possible.

[0030] According to certain embodiments, the diameter of each untreated microbial cell is less than approximately 300 microns. According to certain executions of these embodiments, the microorganisms are Haematococcus, Euglena, and / or Odontella species.

[0031] According to further implementations, the diameter of each untreated microbial cell is less than approximately 10 microns. According to certain implementations of these embodiments, the microorganisms may be one or more of the following: species of the genera Planctrax, Alsospira, Synechocystis, Synechococcus, Nostoc, Planctrax, Alsospira, Haematococcus, Navicra, Cryptomonas, Rhodomonas, Porphyridium, Pheodactylum, Nannochloropsis, Acnanthes, Botryococcus, Chaetoceros, Chroococcus, Cosmarium, Microcystis, Microspora, Pediastrum, Senedesmus, Spirogyra, Spirogyra Species of the genera Rulina, Signema, Chlorobium, Eschericia, Spirillum, Chromobacterium, Jansinobacterium, Streptomyces, Xanthomonas, Sarcina, Serratia, Rhizobium, Prevotella, Actinomyces, Staphylococcus, Proteus, Micrococcus, Lugamonas, Pseudomonas, Helicobacter, Saccharomyces, Candida, Leukosporidia, Rhodotorula, Schizosaccharomyces, Deckel, Brettanomyces, Lactobacillus sp.), Pyrococcus sp., Corynebacterium sp., Aspergillus sp., Bacillus sp., Streptococcus sp., Acetobacter sp., Clostridium sp., Trichoderma sp., Penicillium sp., Prochlorococcus sp., Anabaena sp., Chlorella sp., Thermosynechococcus sp., Chlamydomonas sp., Gloeocapsa sp.), species of the genera Anabaenopsis, Calothrix, Oscillatoria, Gloebacter, Cyanidioschyzon, Crypthecodinium, and / or Galdieria.

[0032] In certain embodiments, the microbial biomass comprises multiple microbial cells including untreated whole-cell microorganisms. In alternative embodiments, the microbial biomass includes destroyed microbial cells (e.g., cells with destroyed cell wall and / or cell membrane integrity). In certain aspects of these embodiments, the microbial biomass includes destroyed microbial cellular components. According to certain executions of these embodiments, one or more microbial components are removed from the microbial biomass. In exemplary executions, lipids, amino acids, carbohydrates, minerals, and / or colorant molecules are removed from the microbial biomass.

[0033] In a particular execution, the cells may have cellular components extracted from the whole biomass. In exemplary embodiments, these cellular components are one or more of carbohydrates, proteins, fats, minerals, nucleic acid substances, and / or any combination thereof.

[0034] In a particular execution, multiple microbial cells constituting the microbial biomass are treated to remove certain cellular components before heat treatment. Exemplary treatments include, but are not limited to, salt addition / removal, cold immersion, sonication, high-pressure homogenization, freeze-thaw, acid extraction, base extraction, organic extraction, inorganic extraction, lysozyme extraction, mechanical extraction, membrane filtration, and / or high-pressure extraction.

[0035] In certain embodiments, microbial biomass is prepared for treatment in a microbial biomass solution. In exemplary executions, microbial biomass is dissolved in an aqueous solvent to prepare a microbial biomass solution. In certain embodiments, the aqueous solvent is water.

[0036] Nucleation and crystal formation In certain embodiments, the disclosed method includes the step of causing crystals to form on the cell surface and / or in the intracellular space of a plurality of microbial cells constituting a microbial biomass. In certain embodiments, the step of causing cell crystal formation includes two steps: a nucleation step and a crystal formation step. In an exemplary execution, the nucleation step is carried out by adding a first soluble ion to a microbial biomass solution, and the crystallization step is carried out by adding a second soluble ion to the microbial biomass solution, wherein the charge of the first soluble ion is opposite to the charge of the second soluble ion (e.g., if the charge of the first ion is positive, the charge of the second ion is positive, and if the charge of the first ion is negative, the charge of the second ion is positive). The first and second ions combine to form insoluble crystals on the cell surface and / or in the cellular space.

[0037] In certain embodiments, the first ion is an anion and the second ion is a cation. In certain alternative embodiments, the first ion is a cation and the second ion is an anion. In exemplary embodiments, the first and second ions are added sequentially to the microbial biomass solution by adding solutions of the first and second ions, respectively. Such solutions of the first and second ions can be prepared by dissolving soluble salts of the ions in an aqueous solvent (e.g., water). In exemplary runs, when the solution of the second ion is added to the microbial biomass, the first and second ions precipitate and crystallize in / on the cells, while the counterions of the first and second ions remain dissolved, retaining high solubility. In certain exemplary runs of these embodiments, the cation is calcium and the anion is a phosphate ion. Calcium and phosphate ion solutions are prepared (according to certain embodiments) by dissolving calcium chloride and sodium phosphate, respectively, in water. When a second ion solution is added, calcium and phosphate ions form crystals in / on the cells, while sodium and chloride ions dissolve in the solution.

[0038] In further embodiments, the first and second ions may be any ion pair that precipitates / crystallizes in the solvent of the microbial biomass solution. Exemplary ion pairs are shown in Table 1, but these should not be construed as limiting. It should be understood that the ions in columns A and B may each be either the first or second ion, depending on a particular embodiment.

[0039] [Table 1]

[0040] In a particular embodiment, the first and second ions exist in stoichiometric ratios. In a further embodiment, the first and second ions exist in a ratio of about 2:1 to about 1:2.

[0041] In some embodiments, a given combination of anions and cations may be added in any order, but in certain embodiments, one must be added before the other. For example, in the case of calcium chloride and sodium phosphate, the order is important in certain embodiments. Independently, these ions are highly soluble in aqueous solution. The addition of two mixtures containing these ions produces insoluble calcium phosphate and highly soluble sodium chloride. The choice between calcium and phosphate ions is important because the microbial cell surface allows for passive diffusion of extracellular calcium ions, while phosphate ions are very widely present in most living cells. Swelling of microbial biomass in calcium chloride solution is beneficial because the microbial cell surface tends to become negatively charged, which attracts positively charged calcium ions into the cell membrane and even into the internal space of the cell. This allows for a high concentration of calcium ions in the cell, which leads to a high degree of crystallinity when sodium phosphate solution is added. However, when attempting to swell algae in sodium phosphate solution, phosphate ions are rejected from the membrane and remain in the extracellular solution. When calcium chloride is added, most of the precipitate is formed extracellularly.

[0042] Returning to the nucleation step, also referred to herein as the nucleation step, the first ion can be added to a microbial biomass solution to bind to the cell surface and intracellular sites, thereby providing nucleation sites for further crystal growth. In certain embodiments, the nucleation step further includes performing a nucleation incubation by incubating the first ion with a plurality of microbial cells for a duration of approximately 1 minute to approximately 24 hours. In further executions, the incubation lasts for approximately 5 minutes to approximately 2 hours. In even further executions, the incubation step is approximately 1 hour.

[0043] In a particular embodiment, the incubation step further includes heating the microbial biomass solution to a temperature of about 32°C to about 65°C. In further embodiments, the nucleation step further includes a stirring step for the purpose of increasing the penetration of the first ion into the cell. In a particular run, the stirring step is performed by shear mixing the microbial biomass solution at about 2000 RPM for about 2 minutes. In an alternative run, the stirring step is performed by sonication.

[0044] Next, considering the crystallization process, following the nucleation process, a second ion is added to the microbial biomass solution, and precipitate / crystal formation proceeds at the nucleation sites on / within the cells. Such crystal formation results in multiple crystalline shell-coated microorganisms. In certain embodiments, the crystallization process further includes performing a crystallization incubation by incubating the microbial biomass solution for a duration of approximately 1 minute to approximately 24 hours. In further runs, the crystallization incubation lasts for a duration of approximately 5 minutes to approximately 2 hours. In even further runs, the incubation process is approximately 1 hour. In certain runs, the crystallization incubation is at approximately 25°C.

[0045] In certain embodiments, multiple crystalline shell-coated microorganisms have crystal formation on the cell surface. In further embodiments, multiple crystalline shell-coated microorganisms have crystal formation inside the cell. In even further embodiments, multiple crystalline shell-coated microorganisms have crystal formation both on the cell surface and inside the cell.

[0046] According to one particular alternative embodiment, another method for growing crystals in a controlled manner is supersaturation and slow cooling (cooling of the solution). The solubility of most compounds increases with increasing temperature. For example, in the case of trisodium phosphate (Na3PO4): 5.4 g / 100 mL (0°C); 12 g / 100 mL (20°C); 14.5 g / 100 mL (25°C); 23.3 g / 100 mL (40°C); 94.6 g / 100 mL (100°C). 94.6 g of trisodium phosphate can be dissolved in 100 ml of water at 100°C. When this solution is cooled to 0°C, 89.2 g of sodium phosphate will precipitate. Calcium chloride also exhibits a similar solubility trend, with increasing temperature resulting in increased solubility: 49.4 g / 100 mL (-25°C), 59.5 g / 100 mL (0°C), 65 g / 100 mL (10°C), 81.1 g / 100 mL (25°C), and 102.2 g / 100 mL (30.2°C). A limited number of compounds exhibit the opposite solubility trend. For example, calcium hydroxide (Ca(OH)2) tends to decrease in solubility with increasing temperature: 1.89 g / L (0°C), 1.73 g / L (20°C), and 0.66 g / L (100°C), in which case increasing temperature leads to precipitation. The supersaturation method can result in crystallization both inside and outside the cell, depending on the chemical species, algal species, and concentration, with microbial cells acting as nucleating agents. The advantages of this method are the reuse of added minerals for subsequent processing and the ease of washing the charcoal due to the addition of highly soluble minerals. An example of processing is carried out as follows: 100 g of calcium chloride is dissolved in 100 mL of DI water at 30°C. Next, 8 wt% of algae is added to the calcium chloride solution and sheared and mixed using a shear mixer at 2000 RPM for 5 minutes, followed by stirring at 30°C for 1 hour. Next, this solution is cooled to 25°C and maintained at that temperature for 10 minutes. The solution containing the precipitate is centrifuged and dried.

[0047] According to another embodiment as a further alternative, another method of crystal formation is controlled drying (solvent evaporation). In an exemplary run, as the solvent evaporates, the solubility of the solvent is maintained unchanged due to the constant environment, but the total dissolution capacity of the soluble compound decreases as the amount of solvent decreases. Upon sufficient evaporation, the solvated compound precipitates as growing crystals. For example, 81 g of calcium chloride can be dissolved in 100 mL of water at 25°C. When 100 mL of water is evaporated to 50 mL at 25°C, 40 g of calcium chloride will precipitate. Microbial cells act as a nucleating agent. The advantages of this method are its scalability and reusability.

[0048] In another embodiment, a further crystallization method involves mixing a poor solvent with the ionic solution (solvent mixing). Each compound has a solvent that it can dissolve in and a solvent in which it cannot. The former is called a good solvent, and the latter is called a poor solvent. Trisodium phosphate has very high solubility in water (also known as a good solvent), while it is insoluble in ethanol (also known as a poor solvent). Therefore, when ethanol is added to an aqueous solution of trisodium phosphate, the solubility of the solution (ethanol + water) gradually decreases as the mixed solution changes from a good solvent to a poor solvent. Crystallization occurs as a result of this change. An example procedure is as follows: 14 g of trisodium phosphate is dissolved in 100 mL of DI water at 25°C. Next, 8 wt% of algae is added to the calcium chloride solution and shear-mixed using a shear mixer at 2000 RPM for 5 minutes, followed by stirring at 25°C for 1 hour. Next, 50 mL of ethanol is added. This mixture is filtered and dried.

[0049] As can be understood, each crystallization method may be used in combination with other crystallization methods. For example, a heated supersaturated solution may be cooled and a poor solvent added to produce crystals. The methods described herein are not exhaustive. Other crystal growth methods include deposition from a gas (e.g., chemical vapor deposition).

[0050] While not bound by theory, crystal formation treatment is thought to result in the following: 1) It acts as a physical barrier, maintaining the separation of cells during the carbonization process and preventing them from fusing together due to melting from high temperatures, resulting in the growth of dendritic crystals on the cell surface. The melting point of most carbohydrates, lipids, and proteins is below 200°C, but the temperatures applied during heat treatment can exceed 500°C. 2) It induces crystal growth in the cell voids, shearing the cell membrane and simultaneously preventing internal folds from bonding together, creating high porosity that contributes to strong blackening. 3) It provides a mineral-based thermal dispersion layer, uniformly carbonizing the biomass from the surface and internal spaces. The thermal conductivity of typical biomass is approximately 0.1 W / mK, while that of typical minerals is an order of magnitude higher (approximately 1 W / mK).

[0051] In one particular alternative embodiment, a dry salt powder may be added directly to the biomass. These added crystals may be either ionic precursors or precipitates, or any other mineral. Preferably, these mineral powders have anti-solidification properties and a melting temperature higher than the carbonization temperature. Examples of these mineral additives, but not limited to, include tricalcium phosphate, dicalcium phosphate, magnesium silicate, silicon dioxide, and / or sodium aluminosilicate. The size of the mineral additives is preferably equal to or an order of magnitude smaller than the biomass particles, and more preferably two orders of magnitude smaller. The size of many microbial biomass particles is in the range of 1 micrometer to several tens of micrometers, and above. Therefore, the size of these additives is preferably in the range of an order of magnitude micrometers. These mineral powders can be applied by various coating methods, but not limited to, fluidized beds, powder coating sprays, and co-grinding using various mills. A dry mineral precipitate is added to the microbial biomass, which may be in solution form or in dry form. These embodiments may include the step of adding minerals, and / or other conversion and binders, to the biomass. These minerals act to stabilize the biomass and / or components present in the biomass so that the size and structure of the biomass do not substantially change during the heat treatment process.

[0052] In exemplary executions of these embodiments, the added minerals constitute about 0.1 to about 70% of the total mass of the sample. In a particular execution, the minerals may be added in any physical state (e.g., gas, liquid, or solid). In a particular embodiment, the minerals cause simple carbohydrates to volatilize more quickly and thus prevent the biomass from sticking together during carbonization.

[0053] While not bound by any particular theory, minerals are thought to be capable of performing one or more of the following functions: i) altering the electromagnetic surface "charge" of particles by changing the functional groups present on the surface of the particles themselves; ii) acting as unique heating units that "burn" through cell walls / membranes in unique regions. That is, minerals absorb thermal energy and dissipate this energy at very high temperatures in very unique regions. This causes specific locations on the biomass surface to become "hot spots" on the surface, where localized areas experience temperature increases and thus unique reactions occur that create new organic species within the small regions surrounding the mineral biomass; iii) acting as "binders" that can modify the generation of various products in biomass during biomass carbonization; iv) participating in reactions that reduce the overall amount of gaseous oxygen species produced. This occurs when various minerals added to biomass undergo reactions that can remove oxygen from organic matter found in the biomass; v) breaking down carbohydrates into other forms of organic matter, thereby reducing the amount of structural transformation that leads to the "melting" of biomass; vi) accelerating the decomposition of organic matter into gaseous species, thereby reducing the amount of biomass that "liquefies" in the sample; and / or vii) imparting elements that cause structural changes to the biomass components.

[0054] Biomass contains organic substances, namely proteins, carbohydrates, and lipids, as well as inorganic elements, which may be both endogenous and exogenous minerals. Minerals include alkali metals, alkaline metals, and transition metals, such as Ca, K, Si, Mg, Al, S, Fe, P, Cl, Na, and some trace metals. These minerals can vary among different species. In woody biomass, ash content from Cl, K, N, S, and Si is lower, while Ca and Mg are present in higher amounts. Minerals are known to stabilize biochar through oxidation resistance during the thermal decomposition process. In particular, alkali metals and alkaline earth metals catalyze the decomposition and char formation reactions of biomass. Pretreatment to reduce ash content often results in a lower char yield. Fe impregnation can affect the degree of aromatic condensation and the porosity of the biochar. Similarly, certain alkali metals, such as K and Na, can react with chlorine to form gaseous compounds such as KOH, KCl, K2SO4, NaCl, and Na2SO4, which are released in the gas phase. Si, K, and Ca are the main causative elements of aggregation, forming CaO, SiO2, and K2O, due to their tendency to melt easily. Minerals can catalyze secondary reactions by reacting with certain sugar byproducts, such as levoglucosane, to form other volatile substances such as levoglucocenon, furan derivatives, and light oxygenates. The presence of high mineral content in biomass affects the distribution of products by reducing oil yield and increasing char and gaseous products. The catalytic activity of ash alters the dynamics of combustion and gasification. It has been shown that reducing the ash content of biomass by washing increases the peak combustion temperature but decreases the peak temperature of the gasification mass loss rate.

[0055] Further implementations include the addition of species to biomass that convert simple carbohydrates into gaseous species. Further implementations include the addition of binders that can sequester simple carbohydrates and / or proteins.

[0056] Drying of microbial biomass In certain embodiments, the microbial biomass is dried to reduce its moisture content and concentrate the cells. In certain executions, the microbial biomass is dried until it reaches a moisture content of about 10% to about 20%, with 15% being an example. In further embodiments, the microbial biomass is dried until its moisture content reaches about 5% or less.

[0057] The drying process may be carried out according to various techniques known in the art. In exemplary embodiments, cells are dried by drum filtration, filtration / drying, dead-end filtration, microfiltration, ultrafiltration, pressure filtration, vacuum filtration, tangential flow filtration, diatomaceous earth filtration, membrane filtration, magnetic separation, forward osmosis, electrofloat, roller press, belt harvesters, capillary extraction, simple heating / evaporation, hydrocyclone, cross-flow, assisted separation (magnetic, electrical, dielectric, acoustic), granular bed filtration, pre-coat filtration, disc stack centrifugation, cross-flow filtration, decanter centrifugation, spray drying, or organic flocculation. Drying may be carried out by techniques described in Advancement and Challenges in Harvesting Techniques for Recovery of Microalgae Biomass, Difusa et al., the entire contents of which are incorporated herein by reference.

[0058] According to a particular embodiment, the method further includes a step of drying the biomass. This drying step may be carried out by one of several techniques known in the art. Exemplary drying methods include, but are not limited to, drum drying, spray drying, tray drying, pulse combustion drying, evaporative drying, convection drying, freeze drying, spiral plate drying, surface tension dryers, vacuum tray dryers, solar conduction dryers, osmotic dryers, membrane separation dryers, sedimentation dryers, froth floatation separation, centrifugation, and / or filtration.

[0059] In a particular execution, the drying step removes approximately 30-99% of the total solution from the biomass. In a particular alternative execution, the drying step is omitted from the disclosed method.

[0060] heat treatment According to a particular embodiment, following drying of microbial biomass, the microbial biomass is subjected to heat treatment to produce microbial char. In a particular embodiment, the heat treatment is carried out in a reaction vessel. In an exemplary execution, the reaction vessel can be sealed to prevent the introduction of any additional gases during the production process. In one embodiment, an inert gas may be added to the vessel to push out any unwanted gases, such as carbon dioxide, oxygen, and any other reactive gas species. In a particular alternative embodiment, air and other reactive gases are added to the combustion chamber to increase the overall combustion temperature and to facilitate the chemical reaction within the chamber. In another embodiment, various types of inert and reactive gases may be introduced into the reaction chamber in subsequent steps to obtain various types of reactions at different points in the course of the heating process. Suitable reaction vessels include a variety of reaction vessels known in the art. Examples of reaction vessels include, but are not limited to, batch reactors, rotary kilns (vertical or horizontal), shaft furnaces, fluidized beds, spouted beds, co-beds, screw reactors, Herreshoff over / multiple hearth furnaces, Torbed reactors, microwave reactors, compact moving beds, belt dryers / reactors, and fixed-bed reactors.

[0061] In certain embodiments, the heat treatment of cells is carried out by a process selected from the group consisting of pyrolysis, gasification, combustion, thermal oxidative decomposition, roasting, and hydrothermal carbonization. In certain embodiments, the heat treatment step includes using a combination of the above processes.

[0062] In one particular execution, the heat treatment process is in a temperature range of approximately 100°C to approximately 2000°C. In one particular further execution, the temperature range is approximately 100°C to approximately 1000°C. In a further embodiment, the heat treatment temperature range is 200°C to approximately 800°C. In a further embodiment, the heat treatment temperature range is 250°C to approximately 750°C. In a further embodiment, the heat treatment temperature range is 300°C to approximately 700°C. In a further embodiment, the heat treatment temperature range is 350°C to approximately 750°C. In a further embodiment, the heat treatment temperature range is 400°C to approximately 700°C. In a further execution, the heat treatment process is approximately 550°C. In one particular exemplary execution, the temperature is increased in a stepwise manner. In one particular alternative execution, the temperature is increased at a constant rate over a predetermined interval.

[0063] In certain embodiments, the heat treatment process is carried out over a time duration of approximately 1 second to approximately 24 hours. In further embodiments, the time duration is approximately 5 to 7 minutes, and the heat treatment process is carried out at approximately 600°C.

[0064] In a particular execution, the heat treatment process is carried out until a predetermined endpoint is reached. In an exemplary execution, the endpoint is reached when the carbonized biomass contains approximately 20% to 75% fixed carbon. In a further embodiment, the endpoint is reached when the carbonized biomass contains approximately 20% to 50% fixed carbon. In yet another embodiment, the endpoint is reached when the carbonized biomass contains approximately 20% to 30% fixed carbon.

[0065] In further embodiments, the heat treatment process is carried out until the carbonized biomass is reduced to approximately 15% to 60% of the initial starting mass. In further embodiments, the heat treatment process is carried out until the approximate volatile content level in the carbonized biomass is less than approximately 25%. In even further embodiments, the heat treatment process is carried out until the approximate volatile content level in the carbonized biomass is less than approximately 20%. In even further embodiments, the heat treatment process is carried out until the approximate volatile content level in the carbonized biomass is between approximately 15% and approximately 25%.

[0066] In further embodiments, the heat treatment process is carried out until the oxygen concentration in the carbonized biomass is less than approximately 20%. In even further embodiments, the heat treatment process is carried out until the oxygen concentration is approximately 10-15%.

[0067] In a further embodiment, the heat treatment process is carried out until the ash concentration in the carbonized biomass is less than approximately 20%. In a specific further embodiment, the heat treatment process is carried out until the ash concentration in the carbonized biomass is about 10% to 20%. In a further embodiment, the heat treatment process is carried out until the ash concentration in the carbonized biomass is less than approximately 10%.

[0068] In certain embodiments, the endpoint of the heat treatment process is determined by a predetermined ratio of oxygen to fixed carbon. In exemplary executions of these embodiments, the endpoint of the heat treatment is reached when the final oxygen-to-final carbon ratio of the carbonized biomass is less than approximately 0.30 oxygen-to-carbon (e.g., 3 parts final oxygen to 10 parts final carbon).

[0069] In a particular embodiment, the endpoint is reached when two or more of the above parameters are met.

[0070] Cleaning after heat treatment In further implementations, the disclosed method also includes a post-thermal treatment cleaning / activation step for biomass. In exemplary implementations, cleaning may be carried out by cleaning the carbonized biomass with a substance that makes the cleaning agent acidic or basic. In exemplary embodiments, one or more of the following may be used: hydrochloric acid, hydrochloric acid, bleach, phosphoric acid, potassium hydroxide, sodium hydroxide, calcium chloride, and zinc chloride. In certain implementations, the concentration of the acid or base is about 0.2% to about 20% of the cleaning agent. In certain implementations in which an acidic cleaning agent is used, the cleaning agent has a pH of about -1.0 to about 6. In exemplary implementations, the cleaning agent has a pH of about 0.1 to about 3.0.

[0071] According to a particular embodiment, the post-heat treatment washing is repeated 1 to 10 or more times. In a particular run, each washing may have a duration of about 30 seconds to about 12 hours. Such washing may act to remove about 60% to about 100% of the minerals present in / around / on the carbonized biomass.

[0072] In certain executions, water washing is also included in the post-heat treatment washing process. In certain embodiments, water washing is performed before, after, or both before and after acid / base washing. While not bound by any specific theory, the post-carbonization washing process is thought to perform one or more of the following functions: i) breaking down larger particles into smaller ones (e.g., by dissolving biomass substances that can cause cell adhesion and cell bonding, thereby changing the surface area of ​​the particles and the electromagnetic charge associated with the particles themselves); ii) removing mineral species, resulting in a mineral content (ash content) of 1% to 30% of the total mass of the material. In exemplary embodiments, the ash content is less than about 10%. This results in a higher concentration of carbon in the sample and, therefore, a blacker material, since mineral species are usually grayish-white; iii) removing surface minerals that, when removed, allow for a higher surface area while maintaining the particle volume without changing it; iv) breaking down cellular components that make the biomass stick together more "sticky"; acid washing can change the charge of the biomass.

[0073] Crushing In certain embodiments, grinding is not required after the heat treatment process, and the microbial char can be used as a pigment without further treatment or with chemical treatment alone. However, in certain alternative embodiments, grinding of the microbial char is required to obtain the desired pigment particle size or cell aggregate diameter, which is a particle size value of 0.01 microns to 100 microns. In certain embodiments, the grinding process is carried out by an apparatus selected from the group consisting of mortars and pestles, rotary tumblers, vibrating tumblers, magnetic tumblers, roll mills, bead mill agitators, disc mills, basket mills, jet mills, ball mills, jaw crushers, rotor mills, cutting mills, knife mills, cryo mills, hammer mills, pin mills, cyclone mills, and classification mills.

[0074] In further embodiments, the grinding step is carried out by a method selected from the group consisting of ammonia freeze-explosion, steam hydrolysis, and wet oxidation. In a further embodiment, the grinding process is carried out by ultrasonic treatment.

[0075] According to one particular embodiment, the grinding process is carried out until the average particle size diameter of the powdered microbial charcoal is less than approximately 10 microns. In a particular execution, the grinding step includes adding one or more mechanical grinding additives to the carbonized biomass during the grinding process. According to a further embodiment, one or more mechanical grinding additives are selected from a list consisting of steel, chromium, stainless steel, ceramics, rubber, stoneware, aluminum, magnesium, zirconia, porcelain, silica, and glass. According to a particular further embodiment, the mechanical grinding additives have particle sizes ranging from about 1 / 32 inch to about 5 inches in diameter.

[0076] In certain embodiments, the grinding step includes adding one or more chemical grinding additives to the carbonized biomass during the grinding process. In certain executions of these embodiments, one or more chemical grinding additives are selected from a list consisting of dispersants, surfactants, wetting agents, abrasives, soap detergents, superdispersants, nonionic high HLB polyalkoxylated surfactants, nonionic polymers, defoamers, water, resins, surface tension modifiers, hydrophobic anionic polymers, acetylene diols, and acetylenediols.

[0077] Modification of powdered microbial charcoal after crushing According to certain embodiments, the disclosed method further includes modifying the powdered microbial charcoal after the grinding step. In certain embodiments, these post-grinding modification steps are intended to reduce the size of individual particles. In further embodiments, these steps are performed to achieve desired properties of the particle surface in order to make the microbial charcoal suitable for a particular application. According to certain embodiments, the post-grinding modification is intended to reduce the heavy metal content of the microbial charcoal. In further embodiments, the post-grinding treatment is intended to remove soluble inorganic salts and / or reduce the ash content. In further embodiments, the post-grinding modification is intended to reduce the total dissolved solid concentration. In further implementations, the post-grinding modification includes adjusting the pH and / or increasing the surface area of ​​the particles. In further embodiments, the post-grinding treatment is intended to further reduce the moisture content of the microbial charcoal.

[0078] In certain embodiments, the modification of microbial charcoal is performed by adding chemical additives to the microbial charcoal. According to certain executions of these embodiments, the chemical additives may be aromatic compounds, alcohols, salts (e.g., ammonium persulfate), surfactants (e.g., Avenel), oils / fats / fatty acids / lipids, water (e.g., steam), ionic liquids, hydrogenation, chemical hydrolysis, enzymatic hydrolysis, alkaline solvents (e.g., sodium hydroxide, ammonia, carbon dioxide), carbon dioxide, chlorine gas, sulfur gas, nitrogen gas, and oxygen gas.

[0079] In a particular execution, surface modification of pulverized microbial charcoal is carried out through hydrogen peroxide treatment. In an exemplary embodiment, after pulverization, the microbial charcoal is separated by freeze-drying and purity analysis is performed. After separation, the microbial charcoal is further functionalized with a 30% (mW / mW) hydrogen peroxide solution and refluxed. In a particular embodiment, refluxing is carried out at approximately 60°C for approximately 24 hours. After refluxing, excess hydrogen peroxide is removed. In an exemplary embodiment, hydrogen peroxide is removed by dialysis against DI water in a tube until no residual peroxide is detected. The final functionalized powder is then freeze-dried again and analyzed by SEM EDS to aid in measuring the degree of surface modification.

[0080] In a further embodiment, the modification of microbial charcoal includes drying the microbial charcoal. According to these embodiments, this drying step is carried out by a method selected from the list consisting of drum filtration, dead-end filtration, microfiltration, ultrafiltration, pressure filtration, vacuum filtration, tangential flow filtration, diatomaceous earth filtration, membrane filtration, magnetic separation, forward osmosis, electroflotation, roller press, belt harvester, capillary extraction, simple heating / evaporation, hydrocyclone, cross-flow, assisted separation (magnetic, electrical, dielectric, acoustic), granular bed filtration, pre-coat filtration, disc stack centrifugation, cross-flow filtration, decanter centrifugation, and organic flocculation. In a particular embodiment, the drying step is carried out by a combination of the aforementioned methods.

[0081] In certain embodiments, the post-pulverization drying process is carried out until a predetermined threshold for moisture reduction is met. In certain exemplary embodiments, the drying process is carried out until the moisture content of the microbial charcoal is reduced to less than about 8%.

[0082] This drying process removes between 30% and 99% of the total solution from the biomass. This drying may or may not be included in this process.

[0083] Any / all of the above, or any combination thereof. In a particular embodiment, the method further includes a step of washing the microbial biomass before the heat treatment step.

[0084] In a particular embodiment, the disclosed method is carried out according to the following steps: In the first step, biomass, which may be wet or dry, is added to an aqueous solution of a calcium salt, which is a first ion source or precipitate precursor. In various executions, calcium chloride is the calcium source. The concentration of the calcium salt in the aqueous suspension is about 0.75–30% by weight, about 1–20% by weight, or about 1.5–10% by weight.

[0085] In a further step, the biomass is suspended in a solution. The concentration of the biomass suspended in the aqueous solution of calcium salt is in the range of approximately 0.1–40% by weight as solids, or alternatively, in the range of approximately 2–20% by weight. The suspension is prepared at a temperature in the range of approximately 0–40°C or 10–35°C.

[0086] In a further optional step, the mixture may be sheared or sonicated to disrupt the cell membranes. This step may also accelerate the absorption of calcium salts by the cells.

[0087] In another step, the mixture is stirred at 0–40°C for 0–20 hours, 1–20 hours, or 0.5–2 hours. In another step, an aqueous solution of phosphate, which is a second ion source or precipitate precursor counterion, is added to the mixture. The second ion source or precipitate precursor counterion, phosphate, may be in solid form. In certain runs, trisodium phosphate is used. The phosphate concentration may be about 1.25–50% by weight, about 2–30%, or about 2.5–20%, calculated based on the final volume of the biomass mixture.

[0088] In the following optional steps, the mixture is stirred at 0–40°C for 1–20 hours. In a particular run, the mixture is stirred at 0–35°C for 0–20 hours, or in some runs, for about 0.5–2 hours.

[0089] In the next step, the precipitate and biomass are filtered out. The precipitate and biomass may be filtered out of the liquid using various filtration methods, such as gravity sedimentation, centrifugal sedimentation, and filter press. In various implementations, a filter press is used.

[0090] In another step, the solid is subsequently dried and carbonized. In a further step, the charcoal is acid-washed to remove ash. In certain runs, the acid is used to dissolve various mineral components. In various runs, the acid is hydrochloric acid. In certain runs, the acid has a pH of approximately -1.0 to 6, or approximately 0.1 to 3.0.

[0091] Examples The following examples are provided to give a complete disclosure and description of the methods by which the articles, apparatus, and / or methods claimed herein are made and evaluated, and are intended to be merely illustrative of the invention and not intended to limit the scope of what the inventors consider to be their invention. However, it should be understood that in light of this disclosure, many modifications can be made to the particular embodiments disclosed, and similar or analogous results can still be obtained without departing from the spirit and scope of the invention.

[0092] Example 1 In this example, 3.46 g of calcium chloride was added to 100.97 g of DI water. Next, 8.28 g of yeast was added to the calcium chloride solution to obtain a biomass suspension. This mixture was sheared for 3 minutes at 2000 RPM using a shear mixer equipped with 1.5-inch blades, and then stirred at 100°F using a magnetic stirrer plate.

[0093] Next, 7.38 g of trisodium phosphate was added to 149.81 g of DI water. Then, the entire trisodium phosphate aqueous solution was added to the biomass mixture at once. The resulting final mixture was stirred at RT for 1.5 hours and centrifuged at 2500 RPM for 5 minutes. The supernatant was discarded and the solid was dried at 160°F. All samples were treated in a heated screw-type pyrolysis apparatus SPIRAJOULE® SPJ HT in a nitrogen gas environment at a temperature of 565°C for 20 minutes.

[0094] Example 2 In this example, 2.99 g of calcium chloride was added to 99.50 g of DI water. Next, 8.11 g of Spirulina species was added to the calcium chloride aqueous solution to obtain a biomass suspension. This mixture was sheared for 2 minutes at 2000 RPM using a shear mixer equipped with 1.5-inch blades, and then stirred at 150°F using a magnetic stirrer plate.

[0095] Next, 7.49 g of trisodium phosphate was added to 149.52 g of DI water. The entire trisodium phosphate aqueous solution was added to the biomass mixture at once. The resulting final mixture was stirred at RT for 2 hours and centrifuged at 2500 RPM for 5 minutes. The supernatant was discarded and the solid was dried at 160°F. All samples were treated in a heated screw-type pyrolysis apparatus SPIRAJOULE® SPJ HT in a nitrogen gas environment at a temperature of 565°C for 20 minutes.

[0096] Example 3 In one example, salt was removed from the biomass before carbonization so that the biomass would melt together. The presence of salt on the biomass before carbonization (i) keeps the particles separate, and (ii) creates pores in the biomass.

[0097] Figure 1A shows biomass treated with a soluble anion / cation mixture and subsequently carbonized. Figure 1B shows biomass that was salt-removed by acid washing before carbonization and then carbonized. In Figure 1B, the biomass had amorphous characteristics. Figure 1C shows the "rescue" biomass from Figure 1B, i.e., the biomass from Figure 1B was retreated with a soluble anion / cation mixture and then this retreated material was carbonized. In Figure 1C, many pores are visible.

[0098] Example 4 In this example, the biomass used was photosynthetic microorganisms sold in stores as whole-cell cyanobacteria with all salts washed off (Earthrise, Spirulina (photosynthetic prokaryotes), Spirulina species found in grocery stores). Figure 2A shows the untreated carbonized biomass. Figure 2B shows the biomass treated with a soluble anion / cation mixture and subsequently carbonized. In Figure 2B, separate particles with a highly porous surface are shown.

[0099] Example 5 In this example, the biomass used was whole-cell yeast from a fermenter (researcher from UC-Anschutz, yeast (heterotrophic eukaryote)). Figure 3A shows the untreated carbonized biomass. Figure 3B shows the biomass treated with a soluble anion / cation mixture and then carbonized.

[0100] Example 6 In this example, adding salt to the biomass before carbonization produced char with high fluidity / pouring properties. A summary of the results for Example 4 is shown in Table 1. Figure 4A shows Spirulina biomass after all salt has been washed off and then carbonized. In Figure 4A, the biomass spread out into a mushroom shape and became a solid, non-flowing substance, different from its original state. Figure 4B shows Spirulina biomass after washing, followed by the addition of salt using a soluble anion / cation mixture and then carbonization. In the example in Figure 4B, the carbonized biomass remained a fluid substance without changing its form.

[0101] Figure 5A shows untreated yeast biomass that has been carbonized and formed a hard substance. As can be seen from Figure 5A, the carbonized biomass solidified into a pack-like structure in the center of the container, changing its form from its original state. Figure 5B shows yeast biomass treated with a soluble anion / cation mixture before carbonization. The salt-treated carbonized biomass filled the container after carbonization and maintained its fluidity without carbonizing into a hard substance.

[0102] Example 7 As shown in Tables 2 to 4, adding salt to the biomass before carbonization and acid washing after carbonization significantly increased the blackness value. Furthermore, adding salt to the biomass before carbonization also significantly increased the fluidity of the carbonized biomass, as evidenced by the fact that the carbonized biomass did not stick together and flowed freely.

[0103] Example 8 Figure 6A shows an example of salt crystals formed on salt-added carbonized biomass. In Figure 6B, the salt is removed from the salt-added carbonized biomass via acid treatment, thus revealing the highly porous nature of the resulting carbonized biomass.

[0104] Imaging Figures 1-3: Scanning electron microscope images were acquired for all samples after TGA. Images were acquired using a Jeol / EQ InTouchscope. 5-10 mg of sample was sprinkled onto double-sided carbon fixed to the SEM stage. To reduce any charging during imaging, the samples were pre-coated with gold and palladium using a sputtering rig. Images were acquired at magnifications of 150×, 500×, and 1500×.

[0105] Figures 4 and 5: Optical images of the sample were acquired after TGA and photographed in a 5 ml crucible used to carbonize the material within the TGA. The images were taken with a mobile phone. Figure 6: Scanning electron microscope images were acquired for all samples after TGA. Image acquisition was performed using a Phenom Prox Desktop scanning electron microscope equipped with an energy-dispersive X-ray spectrometer. 5–10 mg of sample was sprinkled onto double-sided carbon fixed to the SEM stage. To reduce any charging during imaging, the samples were pre-coated with gold and palladium using a sputtering rig. Images are at 100,000× magnification.

[0106] While this disclosure has been described with reference to preferred embodiments, those skilled in the art will recognize that modifications can be made, both formally and in detail, without departing from the spirit and scope of the disclosed apparatus, systems, and methods.

Claims

1. A method for producing carbon black pigment from microbial biomass, To provide a microbial biomass solution containing multiple microbial cells in an aqueous solvent, By adding the first soluble ion to the microbial biomass solution, the plurality of microbial cells are nucleated. The method involves adding a second soluble ion to the microbial biomass solution to initiate crystal formation in and / or on the plurality of microbial cells, thereby forming a plurality of crystalline outer shell-coated microbial cells, wherein the charge of the first soluble ion is opposite to the charge of the second soluble ion, and the crystal is formed from the precipitation of the first and second ions, and The process involves heat-treating the aforementioned plurality of crystalline shell-coated microbial cells to form carbonized biomass. Washing the aforementioned carbonized biomass to form microbial charcoal, Methods that include...

2. The method according to claim 1, wherein the first ion is an anion and the second ion is a cation.

3. The method according to claim 1, wherein the first ion is a cation and the second ion is an anion.

4. The method according to claim 3, wherein the cation is calcium and the anion is a phosphate ion.

5. The method according to claim 1, wherein the first ion and the second ion are present in stoichiometric ratios.

6. The method according to claim 1, further comprising a nucleation step of performing a nucleation incubation by incubating the first ion with a plurality of microbial cells for a duration of 5 minutes to 2 hours.

7. The method according to claim 6, wherein the incubation step further comprises heating the microbial biomass solution to a temperature of 32°C to 65°C.

8. The method according to claim 6, wherein the incubation step further comprises stirring the microbial biomass solution.

9. The method according to claim 8, wherein the stirring step is performed by shear mixing the microbial biomass solution at 2000 RPM for 2 minutes.

10. The method according to claim 1, wherein the crystal formation step further comprises a crystallization incubation by incubating the nucleated microbial biomass solution with the second ion for a duration of 5 minutes to 2 hours.

11. The method according to claim 1, wherein the plurality of crystalline shell-coated microorganisms have crystal formation on their cell surface and / or the plurality of crystalline shell-coated microorganisms have crystal formation within their cells.

12. The method according to claim 1, wherein the microbial biomass includes a plurality of prokaryotic cells, and the average cell size of the prokaryotic cells is less than 50 μm.

13. The method according to claim 1, wherein the microbial biomass is dried at a temperature of 30°C to 300°C prior to the heat treatment step, until the moisture content of the microbial biomass is reduced to less than 15%.

14. The method according to claim 1, wherein the heat treatment step is carried out until the carbonized biomass contains 20% to 70% fixed carbon.

15. The method according to claim 1, wherein the heat treatment step is carried out until the oxygen concentration becomes 10 to 15%.

16. The method according to claim 1, wherein the washing of the carbonized biomass is acid washing, and the acid washing includes reducing the pH of the carbonized biomass to less than 2 over a period of time from 1 minute to 1 hour.

17. The method according to claim 16, further comprising washing the carbonized biomass with water following the acid washing, wherein the acid washing and subsequent water washing produce porous microbial charcoal.

18. The method according to claim 1, further comprising a grinding step following the washing of the microbial charcoal, wherein the grinding step is carried out until the average particle size diameter of the powdered microbial charcoal is less than 10 microns.

19. The method according to claim 1, wherein the carbon black pigment produced has increased fluidity compared to microbial biomass produced by equivalent heat treatment without crystal formation.

20. The method according to claim 1, wherein the manufactured carbon black pigment has increased porosity compared to microbial biomass obtained by equivalent heat treatment without crystal formation, and / or the manufactured carbon black pigment has increased blackness compared to microbial biomass obtained by equivalent heat treatment without crystal formation.

21. A method for producing carbon black pigment designed from microbial biomass, The microbial biomass containing multiple crystalline outer shell-coated microbial cells is subjected to heat treatment to form carbonized biomass, and The washing of the carbonized biomass is acid washing. A method comprising the washing step of lowering the pH of the carbonized biomass to less than 2 over a period of time from 1 minute to 1 hour to form microbial charcoal.

Images