Biological ink, coating, and related method

Abstract

To provide a method for producing pigment / coloring agent suitable for industrial requirements from sustainable resources.SOLUTION: Provided is a method for producing pigment from microbial biomass. Pigment may be designed black pigment. The method may include a heat treatment process of carbonizing microbial biomass. Biomass in a carbonized state and a state before carbonization can be chemically and / or mechanically cleaned. In another step, biomass is pulverized via pulverization of powdering process. Pulverization / powdering may be performed at any various points of the process. In some embodiments, biomass has a particle size of 0. to 100 microns.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on November 7, 2018, the entire contents of which are incorporated herein by reference. This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 756 / 968, entitled "BIOLOGICAL INKS AND COATINGS AND ASSOCIATED METHODS, SYSTEMS AND DEVICES," filed on Oct. 23, 2013.

[0002] The disclosed technology relates generally to the production of pigments and colorants from microbial biomass. [Background technology]

[0003] Pigments and colorants are an industry worth over $30 billion per year. However, the production of these compositions involves the production 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 their ability to produce particle sizes small enough to be suitable for most industrial applications. Thus, there is a need in the art for methods to produce pigments / colorants from sustainable resources that are suitable for industrial requirements. Summary of the Invention

[0004] A method is disclosed for producing engineered carbon black pigments from microbial biomass by heat treating the microbial biomass to form a carbonized biomass; washing the carbonized biomass; and grinding the carbonized biomass to a particle size of about 0.01 microns to about 100 microns to form powdered microbechar. In certain embodiments, the microbial biomass comprises a plurality of prokaryotic cells. In exemplary embodiments, the plurality of prokaryotic cells has an average size of less than 20 μm. According to certain implementations, the microbial biomass comprises bleached prokaryotic cells. In exemplary embodiments, the bleached prokaryotic cells are of the genus Arthrospira.

[0005] In certain embodiments, the method further comprises washing the microbial biomass prior to the heat treatment step. In an implementation, the washing step is with water. In a further implementation, the washing is an acid wash, e.g., the microbial biomass is incubated in a solution with an acidic pH, such as 1.5. In an exemplary implementation, the acid wash may be followed by one or more water washes. In a further implementation, the acid wash may be followed by or instead of the acid wash, a wash with a basic solution.

[0006] According to further aspects, the washing of the carbonized biomass is an acid wash. In an exemplary embodiment, the acid wash comprises immersing the carbonized biomass in a solution with a stabilized pH of less than about 2. In an exemplary embodiment of these implementations, the carbonized biomass is at the reduced pH for a time length of about 1 minute to about 1 hour. In a further implementation, the step following the acid wash is washing the carbonized biomass with water after the acid wash. In an exemplary aspect of these implementations, the acid wash and subsequent water wash produce porous microbial charcoal.

[0007] According to a particular embodiment, the grinding step is performed by an apparatus selected from the group consisting of a ball / media mill, a long gap mill, an air classifier, a jet mill, a three-roll mill, a basket mill, a cryomill, and a cyclone mill. In a particular implementation, the grinding step is performed by an apparatus selected from the group consisting of a jaw crusher, a hammer mill, a saw mill, an impact dryer mill, a granulator, a guillotine, an impact dryer mill, a lump breaker, a knife mill, a pin mill, a roller crusher, a roller mill ... The grinding process is carried out without the use of shears, rotary mills, vibratory tumblers, or magnetic tumblers, thus reducing the cost and complexity of the process. In certain alternative embodiments, the grinding step is carried out by a method selected from the group consisting of ammonia freeze explosion, steam hydrolysis, and wet oxidation.

[0008] In an exemplary embodiment, the grinding process is carried out until the powdered microbial charcoal has an average particle size diameter of less than about 10 microns. Further disclosed herein is an engineered carbon black pigment comprising carbonized biomass derived from microbial biomass and having a particle size of from about 0.01 microns to about 100 microns.

[0009] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes exemplary embodiments of the disclosed devices, systems, and methods. As will be recognized, the disclosed devices, systems, and methods are capable of modification in various obvious aspects, all without departing from the spirit and scope of the present disclosure. Accordingly, the drawings and detailed description should be regarded as illustrative in nature and not as restrictive. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a flow chart illustrating a method according to one embodiment. [Diagram 2] FIG. 2 is a flow chart illustrating a method according to one embodiment. [Diagram 3] FIG. 3 is a flow chart illustrating a method according to one embodiment. [Figure 4] FIG. 4 is a flow chart illustrating a method according to one embodiment. [Diagram 5] FIG. 5 is a flow chart illustrating a method according to one embodiment. [Figure 6] FIG. 6 is a flow chart illustrating a method according to one embodiment. [Figure 7] FIG. 7 shows pigments from a baseline TGA of decolorized spirulina subjected to heat treatment at different temperatures, and pigments from a baseline TGA of decolorized spirulina subjected to heat treatment at different temperatures and acid washed prior to making the pigment dispersion. [Figure 8] FIG. 8 shows magnified images of decolorized Spirulina including: unsieved Earthrise Spirulina; unsieved, pre-carbonized, unwashed LinaX; sieved, pre-carbonized, unwashed LinaX; unwashed, LinaX with TGA at 200° C.; unwashed, LinaX with TGA at 300° C.; and unwashed, LinaX with TGA at 400° C. [Figure 9] FIG. 9 shows defatted Nannochloropsis pigment subjected to heat treatment at different temperatures, and defatted Nannochloropsis pigment subjected to heat treatment at different temperatures and acid washed prior to making the pigment dispersion. [Figure 10] FIG. 10 shows a close-up image of the degreased Nannochloropsis including sieved, pre-carbonized degreased Qualitas; degreased, 200° C. TGA Qualitas; degreased, 300° C. TGA Qualitas; degreased, 400° C. TGA Qualitas; degreased, 500° C. TGA Qualitas; degreased, 600° C. TGA Qualitas; and degreased, 700° C. TGA Qualitas. [Figure 11] FIG. 11 shows images of unwashed, acid washed, and base washed pigments from bamboo charcoal, Wakefield biochar, Cool planet, Biochar Now, and LinaX. [Figure 12] FIG. 12 shows images of pigments from unwashed and acid washed commercial biochars from Biochar Now, Take, Cool planet, Wakefield, and LinaX. [Figure 13]Figure 13 shows a close-up image of unwashed Cool Plant ground in a herb grinder for 5 minutes; unwashed SEEK Bamboo ground in a herb grinder for 4 minutes; unwashed Wakefield Biochar ground in a mortar and pestle for 2 minutes; acid washed Wakefield Biochar; unwashed Biochar Now; and acid washed Biochar Now. [Figure 14] FIG. 14 shows pigments from E. coli, yeast, Spirulina, Nannochloropsis, and Chlorella after treatment at 200° C. and 500° C. [Figure 15] FIG. 15 shows the pigment from pine sawdust after treatment at 200° C. and 500° C. [Figure 16] FIG. 16 shows magnified images of E. coli before carbonization, after treatment at 200° C., and after treatment at 500° C., as well as magnified images of yeast before carbonization, after treatment at 200° C., and after treatment at 500° C. [Figure 17] FIG. 17 shows enlarged images of Nannochloropsis before carbonization, after treatment at 200° C., and after treatment at 500° C., and enlarged images of Chlorella before carbonization, after treatment at 200° C., and after treatment at 500° C. [Figure 18] FIG. 18 shows magnified images of Spirulina before carbonization, after treatment at 200° C., and after treatment at 500° C., as well as magnified images of pine sawdust before carbonization, after treatment at 200° C., and after treatment at 500° C. [Figure 19] FIG. 19 shows pigments from residual biomass of Nannochloropsis treated at 200° C. in WC Nannochloropsis, degreased Qualitas, and degreased Qualitas deproteinized; from residual biomass of Nannochloropsis treated at 500° C. in WC Nannochloropsis, degreased Qualitas, and degreased Qualitas deproteinized; from residual biomass of Spirulina treated at 200° C. in WC Spirulina prewashed with water, acid, and acid and water; from residual biomass of Spirulina treated at 500° C. in WC Spirulina prewashed with water, acid, and acid and water prewashed. [Figure 20]FIG. 20 shows enlarged images of degreased Qualitas before carbonization; degreased Qualitas at 200° C.; degreased Qualitas at 500° C.; degreased Qualitas before carbonization; deproteinized Qualitas at 200° C.; and deproteinized Qualitas at 500° C. [Figure 21] FIG. 21 shows images of pigment from decolorized Spirulina pre-washed with water and treated at 200° C., 300° C., and 500° C.; from decolorized Spirulina pre-washed with acid and treated at 200° C., 300° C., and 500° C.; and from decolorized Spirulina pre-washed with acid and water and treated at 200° C., 300° C., and 500° C. [Figure 22] FIG. 22 shows images of pigments from decolorized Spirulina pre-washed with acid and treated at 500° C.; from decolorized Spirulina pre-washed with acid and water and treated at 500° C.; from decolorized Spirulina pre-washed with acid, treated at 500° C. and post-washed with acid; from decolorized Spirulina pre-washed with water and treated at 500° C.; and from decolorized Spirulina pre-washed with water, treated at 500° C. and post-washed with acid. [Figure 23] FIG. 23 shows images of pigments from WC Spirulina, water pre-washed Spirulina, acid pre-washed Spirulina, and acid and water pre-washed Spirulina treated at 200° C., and pigments from WC Spirulina, water pre-washed Spirulina, acid pre-washed Spirulina, and acid and water pre-washed Spirulina treated at 500° C. [Figure 24] FIG. 24 shows enlarged images of the following decolorized Spirulina: LinaX before carbonization, pre-washed with water; LinaX pre-washed with water, TGA at 200° C.; LinaX pre-washed with water, TGA at 300° C.; and LinaX pre-washed with water, TGA at 500° C. [Diagram 25] FIG. 25 shows magnified images of the following decolorized Spirulina: LinaX before carbonization, pre-washed with acid; LinaX pre-washed with acid, TGA at 200° C.; LinaX pre-washed with acid, TGA at 300° C.; and LinaX pre-washed with acid, crushed, TGA at 500° C. [Figure 26]FIG. 26 shows magnified images of the following decolorized Spirulina: LinaX before carbonization, pre-washed with water and acid; LinaX pre-washed with water and acid, TGA at 200° C.; LinaX pre-washed with water and acid, TGA at 300° C.; and LinaX pre-washed with water and acid, crushed, TGA at 500° C. [Figure 27] FIG. 27 shows the pigments from Printex 300 carbon black, acid washed ETIA, and unwashed ETIA. [Figure 28] FIG. 28 shows the unwashed pigment (12 μm), acid washed pigment (10 μm), and acid washed pigment (7.5-5 μm) from Q. Degreasing after 500° C. treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, a further aspect includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms a further aspect. It is further understood that the endpoints of each range are meaningful both in relation to the other endpoint, and independently of the other endpoint. It is also understood that a number of values ​​are disclosed herein, and each such value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, when a value of "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0012] Reference herein and in the concluding claims to parts by weight of a particular element or component in a composition indicates the weight relationship between that element or component and any other element or component in the composition or article for which the parts by weight are expressed. Thus, 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 are present in a weight ratio of 2:5, and are present in that ratio regardless of whether additional components are included in the formulation.

[0013] Weight percentages (wt %) of ingredients are based on the total weight of the formulation or composition in which the ingredient is included, unless otherwise specified. As used herein, the terms "desirably" or "desirably" mean that the subsequently described event or circumstance may or may not occur, and that the description includes cases where said event or circumstance occurs and cases where it does not occur.

[0014] A method is disclosed for producing an engineered carbon black pigment from a microbial biomass by heat treating the microbial biomass to form a carbonized biomass; washing the carbonized biomass; and grinding the carbonized biomass to a particle size of about 0.01 microns to about 100 microns to form a powdered microbial char. In certain aspects, the microbial biomass comprises a plurality of prokaryotic cells. In an exemplary aspect, the plurality of prokaryotic cells has an average size of less than 20 μm. According to certain implementations, the microbial biomass comprises decolorized prokaryotic cells. In an exemplary embodiment, the decolorized prokaryotic cells are of the genus Arthrospira.

[0015] In certain embodiments, the method further comprises washing the microbial biomass prior to the heat treatment step. In an implementation, the washing step is with water. In a further implementation, the washing is an acid wash, e.g., the microbial biomass is incubated in a solution with an acidic pH, such as 1.5. In an exemplary implementation, the acid wash may be followed by one or more water washes. In a further implementation, the acid wash may be followed by or instead of the acid wash, a wash with a basic solution.

[0016] In certain aspects, the microbial biomass is dried prior to the heat treatment step. In an exemplary implementation, the microbial biomass is dried at a temperature of about ambient temperature to about 300° C. prior to the heat treatment step. In certain embodiments, the heat treatment step is carried out for a time length of about 1 second to about 24 hours. In an exemplary implementation, the time length is about 5 minutes to about 40 minutes. In each run, the duration is approximately 10 minutes.

[0017] In certain implementations, the heat treatment step is carried out until a predetermined endpoint is reached. In exemplary embodiments, the heat treatment step is carried out until the carbonized biomass contains between about 20% and about 75% fixed carbon. In further embodiments, the heat treatment step is carried out until the approximate volatiles level in the carbonized biomass is less than about 25%. In still further embodiments, the heat treatment step is carried out until the oxygen concentration in the carbonized biomass is less than about 20%. In still further embodiments, the heat treatment step is carried out until the oxygen concentration is less than about 10%. In still further embodiments, the heat treatment step is carried out until the ash concentration in the carbonized biomass is less than about 20%. According to further implementations, the endpoint of the heat treatment step is reached when the carbonized biomass has a predetermined fixed carbon to oxygen ratio. In exemplary implementations 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 about 0.30 oxygen to carbon (e.g., 3 parts final oxygen to 10 parts final carbon).

[0018] According to further aspects, the washing of the carbonized biomass is an acid wash. In an exemplary embodiment, the acid wash comprises immersing the carbonized biomass in a solution with a stabilized pH of less than about 2. In an exemplary embodiment of these implementations, the carbonized biomass is at the reduced pH for a time length of about 1 minute to about 1 hour. In a further implementation, the step following the acid wash is washing the carbonized biomass with water after the acid wash. In an exemplary aspect of these implementations, the acid wash and subsequent water wash produce porous microbial charcoal.

[0019] According to certain aspects, the grinding step is performed by an apparatus selected from the group consisting of ball / media mills, long gap mills, air classifiers, jet mills, three-roll mills, basket mills, cryomills, and cyclone mills. In certain implementations, the grinding step is performed without the use of jaw crushers, hammer mills, sawmills, impact dryer mills, granulators, guillotines, impact dryer mills, lump breakers, knife mills, pin mills, roller crushers, rotary mills, vibratory tumblers, or magnetic tumblers, thus reducing the cost and complexity of the process. In certain alternative embodiments, the grinding step is performed by a method selected from the group consisting of ammonia freeze explosion, steam hydrolysis, and wet oxidation.

[0020] In an exemplary embodiment, the grinding process is carried out until the powdered microbial charcoal has an average particle size diameter of less than about 10 microns. Powdered microbial charcoal may be further processed or modified for a variety of downstream applications according to the methods disclosed herein.

[0021] Microbial biomass Various embodiments disclosed or contemplated herein relate to the use of autotrophic and / or heterotrophic biomass, including plant cells, bacteria, and photosynthetic microorganisms, as inks, coatings, and / or colorants. Naturally derived, engineered, and / or processed cells may be used to obtain certain colored / colored cells / cultures. Certain implementations relate to the use of partial or whole cells to act as alternative colorants in inks and other formulations, whereby extraction of coloring molecules from the cells is not required.

[0022] In certain implementations, the microbial biomass comprises a plurality of prokaryotic cells. In an exemplary embodiment, the plurality of prokaryotic cells has an average single cell size of less than 20 μm. In certain implementations, the microbial biomass comprises bleached prokaryotic cells. In an exemplary embodiment, the bleached prokaryotic cells are of the genus Arthrospira.

[0023] Various implementations use colony-forming types of bacteria, algae, and blue-green algae. In the ink formulation, the aggregate size is less than 100 microns. One embodiment of the present disclosure relates to implementations where the pigment portion is about 0.01-100 microns. It is understood that this size allows for an increased amount of pigment particles to be dispersed to an acceptable density so that a strong color can be obtained. In various implementations, the size of 0.01-100 microns can be achieved in several ways. In certain implementations, the size can be achieved by growing biological cells of the appropriate size. In alternative implementations, the size can be achieved by grinding cells or cell aggregates to the appropriate size (0.01-100 microns). In yet other implementations, both 0.01-100 micron diameter cells as well as grinding of aggregates may be used.

[0024] According to certain implementations, the microbial biomass comprises a plurality of microbial cells. Microbial cells suitable for the disclosed microbial methods include heterotrophic, autotrophic, mixotrophic, or extremophilic microorganisms, including microalgae, algae, macroalgae, cyanobacteria, fungi, and bacteria. In certain implementations, the plurality of cells is a mixture of the aforementioned cells. According to certain embodiments, the microorganism comprising the plurality of 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 elongates PCC 7942, Nostoc sp., Synechococcus 6911. , Synechococcus leopoliensis, plankthorax rubescens, Synechococcus sp. PCC 7002, Arthrococcus Arthospira platensis PCC 7345, Haematococcus pluvailis, Navicula pelliculosa, Cryptomonas erosa, Rhodomonas minuta, Porphyridium purpureum, Phaeodactylum tricomutum, Nannochloropsis spp., Synechocystis spp., Synechococcus spp., Nostoc spp., Planktothorax spp., Arthospira spp., Haematococcus spp., Navicula spp., Cryptomonas spp., Rhodomonas spp., Porphyridium spp., Phaeodactylum spp., Nannochloropsis spp., Volvox spp., Anabena spp., Chlorella spp., Euglena spp., Achnantes spp. 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 sp., Janthinobacterium sp. sp., Streptomyces sp., Xanthomonas sp., Sarcina sp., Serratia sp., Rhizobium sp., Prevotela sp., Actinomyces sp., Staphylococcus sp., Proteus sp., Micrococus sp., Rugamonas sp. ), Pseudomonas sp., Helicobacter sp. ), Saccharomyces sp., Candida sp., Leucosporidium sp., Rhodotorula sp., Schizosporidium sp., Schizosaccharomyces sp., Dekker sp., Brettanomyces sp., Synechocystis sp., Synechococcus spp., Nostoc spp., Planktothorax spp., Arthospira spp., Haematococcus spp., Navicula spp., Cryptomonas spp., Rhodomonas spp., Porphyridium spp., Fusobacterium spp., Geodactylum spp., Nannochloropsis spp., Volvox spp., Anabaena spp., Chlorella spp., Euglena spp., Achnanthes spp., Botryococcus spp., Chaetoceros spp., Chroococcus spp., Cosmarium spp., Microcystis spp., Microspora spp., Pediastrum spp., Scenedesmus spp., Spirogyra spp., Spirulina spp., Zygnema spp., Chlorobium spp., Escherichia spp., Spirillum spp., Chromobacterium spp., Janssen spp. Synobacterium spp., Streptomyces spp., Xanthomonas spp., Sarcina spp., Serratia spp., Rhizobium spp., Prevotella spp., Actinomyces spp., Staphylococcus spp., Proteus spp., Micrococcus spp., Rugamonas spp., Pseudomonas spp., Helicobacter spp., Saccharomyces spp., Candida spp., Leucosporidium spp., Rhodotorula spp., Schizosaccharomyces spp., Deckel spp., and Brettanomyces spp. It will be appreciated by those skilled in the art that other microorganisms are possible.

[0025] According to certain embodiments, each of the intact microbial cells has a diameter of less than about 100 microns. According to certain implementations of these embodiments, the microorganism is a Haematococcus, Euglena, and / or Odontella species.

[0026] According to further implementations, each of the intact microbial cells has a diameter of less than about 10 microns. According to certain implementations of these embodiments, the microorganism may be one or more of the following: Planktothorax spp., Arthospira spp., Synechocystis spp., Synechococcus spp., Nostoc spp., Planktothorax spp., Arthospira spp., Haematococcus spp., Navicula spp., Cryptomonas spp., Rhodomonas spp., Porphyridium spp., Phaeodactylum spp., Nannochloropsis spp., Achnanthes spp., Botryococcus spp., Chaetoceros spp., Chroococcus spp., Cosmarium spp., Microcystis spp., Microspora spp., Pediastrum spp., Scenedesmus spp., Spirogyra ... Lurinus spp., Zygnema spp., Chlorobium spp., Escherichia spp., Spirillum spp., Chromobacterium spp., Jansinobacterium spp., Streptomyces spp., Xanthomonas spp., Sarcina spp., Serratia spp., Rhizobium spp., Prevotella spp., Actinomyces spp., Staphylococcus spp., Proteus spp., Micrococcus spp., Rugamonas spp., Pseudomonas spp., Helicobacter spp., Saccharomyces spp., Candida spp., Leucosporidium spp., Rhodotorula spp., Schizosaccharomyces spp., Deckel spp., Brettanomyces spp., Lactobacillus sp., P. Pyrococcus sp., Corynebacterium sp. , Aspergillus sp., Bacillus sp., Streptococcus Streptococcus sp., Acetobacter sp., Clostridium sp., Trichoderma sp., Penicillium sp., Prochlorococcus sp., Anabaena sp., Chlorella sp., Thermosynechococcus sp., Cladophora sp., Chlamydomonas sp., Gloeocapsa sp., Ana Anabaenopsis sp., Calothrix sp., Osira Oscillatoria sp., Gloebacter sp., Cyanidioschyzon sp., Crypthecodinium sp., and / or Galdieria sp.

[0027] In certain embodiments, the plurality of microbial cells comprising the microbial biomass comprises intact whole-cell microorganisms. In alternative embodiments, the microbial biomass comprises disrupted microbial cells (e.g., where the integrity of the cell wall and / or cell membrane has been disrupted). In certain aspects of these embodiments, the microbial biomass comprises disrupted microbial cell components. According to certain implementations of these embodiments, one or more microbial components are removed from the microbial biomass. In exemplary implementations, lipids, amino acids, minerals, and / or colorant molecules are removed from the microbial biomass.

[0028] In certain embodiments, the plurality of microbial cells has had the staining agent removed, while the cells remain otherwise intact. Purification of microbial biomass In one aspect, the microbial biomass is subjected to a purification process. In certain implementations, the purification process removes growth medium from the microbial biomass. According to further implementations, the purification process further includes removing intracellular components. According to certain embodiments, the intracellular components are one or more of the following components: lipids, amino acids, minerals, and colorant molecules. In certain embodiments, the removed cellular components are combinations of the aforementioned components.

[0029] According to certain aspects, intracellular components are removed by mechanical washing, either alone or in combination with a chemical washing process. In certain alternative embodiments, chemical washing may be performed without mechanical washing. In certain aspects, the purification step removes salts and other components of the growth medium and / or solutions resulting from any of the processing steps disclosed herein. In exemplary embodiments, contaminants or unwanted materials found in the cell growth environment, cell concentrate, cell solution, and / or cell powder are removed through the purification step.

[0030] In certain embodiments, the chemical washing step involves washing the microbial biomass with an acid solution (eg, hydrochloric acid, formic acid, acetic acid, phosphoric acid), water, and / or a phosphate buffered saline solution. In certain embodiments, the purification step further comprises rinsing the biomass with a liquid and incubating the biomass for about 1 minute to about 24 hours. In certain implementations, the wash liquid is an aqueous, organic, or ionic liquid.

[0031] In exemplary aspects of these embodiments, the biomass is incubated at a temperature of about -30°C to about 300°C. In certain exemplary aspects of these embodiments, the microbial biomass is treated with a phosphoric acid wash solution (and made into a dispersion in 1 M phosphoric acid and digested (dissolved) with stirring at 70° C. for 1 hour. The heat is then removed and the suspension is mixed at room temperature for an additional period of time. In an exemplary embodiment, the period of time is about 24 hours. According to these embodiments, the extended digestion at room temperature solubilizes mineral impurities, followed by filtration and washing with deionized (DI) water).

[0032] According to yet a further embodiment, the purification process further comprises rinsing the biomass with a detergent solution. According to yet a further embodiment, the purification step further comprises sonication of the microbial biomass.

[0033] Cleaning before heat treatment In certain implementations, the microbial biomass is washed prior to heat treatment. This washing step may be performed in addition to or in place of the purification step in certain implementations. In certain implementations, no pre-heat treatment washing step is performed. According to certain embodiments, the washing is an acid wash (e.g., pH 1.5 with HCl). In further embodiments, the acid wash is followed by one or more water washes. In certain implementations, the acid wash may be performed according to the following procedure: the carbonized biomass may be incubated with acid (e.g., 1% hydrochloric acid (15.9 mL in 500 mL DI water)) followed by two or more washes with DI water. According to these exemplary embodiments, each wash is performed for about 15 minutes at ambient temperature in a shaking incubator. After washing, the carbonized biomass may be centrifuged and then resuspended for subsequent washing or further processing.

[0034] In certain embodiments, the purification and / or washing steps result in substantial changes to the structural characteristics of the individual cells, while in certain alternative embodiments, the purification and / or washing steps have no or minimal effect on the structural characteristics of the individual cells.

[0035] Drying In certain aspects, the microbial biomass is dried to reduce the moisture content and concentrate the cells. In certain implementations, the microbial biomass is dried until the moisture content is about 10%-20%. In further embodiments, the microbial biomass is dried until the moisture content reaches about 5% or less. The drying step may be carried out according to various techniques known in the art. In exemplary embodiments, the 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, electrofloation, roller press, etc. belt harvesters, capillary extraction, simple heating / evaporation, high Drain cyclones, crossflow, assisted separation (magnetic, electric, dielectric, acoustic), granular bed filters, precoat filters, disc stack centrifugation, clos The mixture is dried by flow filtration, decanter centrifugation, spray drying, or organic flocculation. Drying may be performed by the 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.

[0036] In certain exemplary embodiments, the microbial biomass is dried by flocculation. According to these embodiments, polyvalent metal salts may be used. In certain embodiments, self-flocculation is used, for example at pH 8-13, and is induced by the addition of polyelectrolytes, polymers, chitosan, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and / or sodium carbonate.

[0037] Heat Treatment According to certain embodiments, following drying of the microbial biomass, the microbial biomass is subjected to heat treatment to produce microbial charcoal. In certain aspects, the heat treatment is carried out in a reaction vessel. In an exemplary implementation, the reaction vessel can be sealed to exclude any additional gases from being introduced during the production process. In one embodiment, an inert gas can be added to the vessel to push out any unwanted gases, such as carbon dioxide, oxygen, and any other reactive gas species. In certain alternative embodiments, air and other reactive gases are added to the combustion chamber to increase the overall combustion temperature and promote chemical reactions within the chamber. In other embodiments, different types of inert and reactive gases can be introduced into the reaction chamber in subsequent steps to obtain different types of reactions at different times during the heating process. Suitable reaction vessels include various reaction vessels known in the art. Exemplary reaction vessels include, but are not limited to: batch reactors, rotary kilns (vertical or horizontal), shaft furnaces, fluidized beds, spouted beds, entrained beds, screw reactors, herreshoff over / multiple hearth furnaces, Torbed reactors, microwave reactors, compact moving beds, belt reactors, and the like. These include dryer / reactors and fixed bed reactors.

[0038] In certain aspects, the thermal treatment of the cells is carried out by a process selected from the group consisting of pyrolysis, gasification, combustion, thermo-oxidative decomposition, torrefaction, and hydrothermal carbonization, hi certain embodiments, the thermal treatment step comprises using a combination of the above processes.

[0039] According to one particular implementation, the heat treatment step is at a temperature range of about 100° C. to about 2000° C. In a further particular implementation, the temperature range is about 100° C. to about 1000° C. In one embodiment, the heat treatment temperature range is 200° C. to about 800° C. In a further embodiment, the heat treatment temperature range is 250° C. to about 750° C. In a further embodiment, the heat treatment temperature range is 300° C. to about 700° C. In a further embodiment, the heat treatment temperature range is 350° C. to about 750° C. In yet a further embodiment, the heat treatment temperature range is 400° C. to about 700° C. In yet a further implementation, the heat treatment step is about 400° C. In certain exemplary implementations, the temperature is increased in stepwise intervals. In certain alternative implementations, the temperature is increased at a constant rate over a predetermined interval.

[0040] In certain embodiments, the heat treatment step is carried out for a time length of about 1 second to about 24 hours. In further embodiments, the time length is from about and the heat treatment step is carried out at about 600° C. for a time length of about 5 to 7 minutes.

[0041] In certain implementations, the heat treatment process is carried out until a predetermined endpoint is reached. In exemplary implementations, the endpoint is reached when the carbonized biomass contains between about 20% and about 75% fixed carbon. In further embodiments, the endpoint is reached when the carbonized biomass contains between about 20% and about 50% fixed carbon. In yet further embodiments, the endpoint is reached when the carbonized biomass contains between about 20% and about 30% fixed carbon.

[0042] In a further embodiment, the heat treatment step is carried out until the approximate volatile content level in the carbonized biomass is less than about 25%. In a further embodiment, the heat treatment step is carried out until the approximate volatile content level in the carbonized biomass is less than about 20%. In yet a further embodiment, the heat treatment step is carried out until the approximate volatile content level in the carbonized biomass is between about 15% and about 25%.

[0043] In yet a further embodiment, the heat treatment step is carried out until the oxygen concentration in the carbonized biomass is less than about 20%. In yet a further embodiment, the heat treatment step is carried out until the oxygen concentration is about 10-15%.

[0044] According to still further embodiments, the heat treatment step is carried out until the ash concentration in the carbonized biomass is less than about 20%. According to certain further embodiments, the heat treatment step is carried out until the ash concentration in the carbonized biomass is about 10%-20%. In still further embodiments, the heat treatment step is carried out until the ash concentration in the carbonized biomass is less than about 10%.

[0045] In certain aspects, the end point of the heat treatment step is determined by a predetermined ratio of oxygen to fixed carbon. In an exemplary implementation of these embodiments, the end point of the heat treatment is reached when the final oxygen to final carbon ratio of the carbonized biomass is less than about 0.30 oxygen to carbon (e.g., 3 parts final oxygen to 10 parts final carbon).

[0046] In certain embodiments, the endpoint is reached when two or more of the above parameters are achieved. Crushing In certain implementations, milling of cell culture components is required to obtain acceptable pigment particle sizes or cell aggregate sizes with particle size values ​​between 0.01 microns and 100 microns. In certain embodiments, the milling step is performed by an apparatus selected from the group consisting of a mortar / pestle, rotary tumbler, vibratory tumbler, magnetic tumbler, roll mill, bead mill agitator, disk mill, basket mill, jet mill, ball mill, jaw crusher, rotor mill, cutting mill knife mill, cryomill, hammer mill, pin mill, cyclone mill, and classifier mill.

[0047] According to a further embodiment, the grinding process is carried out by ammonia freeze explosion, steam hydrolysis, etc. and wet oxidation methods. According to yet a further embodiment, the grinding step is carried out by ultrasonication.

[0048] In one particular embodiment, the grinding process is carried out until the powdered microbial charcoal has an average particle size diameter of less than about 10 microns. In certain implementations, the grinding step includes adding one or more mechanical grinding additives to the carbonized biomass during grinding. According to further embodiments, the one or more mechanical grinding additives are selected from the list consisting of steel, chrome, stainless steel, ceramic, rubber, stoneware, aluminum, magnesium, zirconia, porcelain, silica, and glass. According to certain further embodiments, the mechanical grinding additive has a particle size in the range of about 1 / 32 inch to about 5 inches in diameter.

[0049] In certain aspects, the grinding step includes adding one or more chemical grinding additives to the carbonized biomass during grinding. In certain implementations of these embodiments, the one or more chemical grinding additives are selected from the list consisting of dispersants, surfactants, wetting agents, abrasives, detergent soaps, hyperdispersants, nonionic high HLB polyalkoxylated surfactants, nonionic polymers, antifoam agents, water, resins, surface tension modifiers, hydrophobic anionic polymers, acetylenic diols, and acetylenic diols.

[0050] Post-milling modification of powdered microbial charcoal. According to certain embodiments, the disclosed method further comprises modifying the powdered microbial charcoal after the grinding step. In certain aspects, these post-grinding modification steps aim to reduce the individual particle size. In further aspects, these steps are carried out to achieve desired particle surface properties to make the microbial charcoal suitable for a particular application. According to certain embodiments, the post-grinding modification aims to reduce the heavy metal content of the microbial charcoal. In further embodiments, the post-grinding treatment aims to remove soluble inorganic salts and / or reduce the ash content. In yet further embodiments, the post-grinding modification reduces the total dissolved solids concentration. In further implementations, the post-grinding modification includes adjusting the pH and / or increasing the surface area of ​​the particles. In further aspects, the post-grinding treatment aims to further reduce the moisture content of the microbial charcoal.

[0051] In certain aspects, the modification of the microbial char is by adding chemical additives to the microbial char. According to certain implementations 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.

[0052] In one particular implementation, surface modification of the powdered microbial charcoal is performed through hydrogen peroxide treatment. In an exemplary embodiment, after powderization, the microbial charcoal is isolated by freeze-drying and a purity analysis is performed. After isolation, the microbial charcoal is further functionalized with a 30% (w / w) hydrogen peroxide solution and refluxed. In one particular embodiment, refluxing is performed at 60° C. for about 24 hours. After refluxing, excess hydrogen peroxide is removed. In an exemplary embodiment, hydrogen peroxide is removed by dialysis against DI water in tubing until no residual peroxide is detected. The final functionalized powder is then freeze-dried again and analyzed by SEM EDS to help measure the degree of surface modification.

[0053] According to further aspects, modifying the powdered microbial charcoal includes drying the microbial charcoal. According to these embodiments, the drying step can be performed by drum filtration, dead-end filtration, microfiltration, ultrafiltration, pressure filtration, vacuum filtration, tangential flow filtration, diatomaceous earth filtration, membrane filtration, magnetic filtration, or the like. The drying step is carried out by a method selected from the list consisting of: air separation, forward osmosis, electroflotation, roller press, belt harvester, capillary extraction, simple heating / evaporation, hydrocyclone, cross-flow, assisted separation (magnetic, electric, dielectric, acoustic), grain bed filter, precoat filter, disc stack centrifuge, cross-flow filtration, decanter centrifuge, and organic flocculation. In certain embodiments, the drying step is carried out by a combination of the aforementioned methods.

[0054] In certain aspects, the post-pulverization drying step is carried out until a predetermined moisture reduction threshold is met. In certain exemplary embodiments, the drying step is carried out until the moisture content of the microbial charcoal is reduced to less than about 8%.

[0055] 1, in some embodiments, method 100 includes various optional steps and substeps that may be performed in any order. As will be appreciated, in various embodiments, the method may begin at any step and proceed accordingly.

[0056] In one embodiment, the method 100 includes selecting or providing cells intended for carbonization (Box 102). In another step, various components of the cells are removed by chemical and / or mechanical processes (Box 104). In another step, the cells are dried (Box 106).

[0057] In various of these embodiments, the dried cells are heat treated in another step (box 108). In a further step, the heat treated cells are washed by chemical and / or mechanical washing (box 110). The washed cells may then be dried in a subsequent step (box 112). In another step, the washed and dried cells are then ground and / or powdered (box 114). In a subsequent step, a dispersion is made (box 116). In a final step, the final formulation for a particular market is manufactured (box 118).

[0058] In alternative embodiments, method 100 begins with heat treating the cells (108). In these and other embodiments, heat treatment (108) is followed by chemical and / or mechanical cleaning (box 110) and drying (112). Further steps include grinding and / or pulverization (box 14). Another step includes creating a dispersion. Yet further steps include the final formulation or end product for a particular market (box 118).

[0059] In various alternative embodiments, as shown in FIG. 2, the method 100 has a first step of obtaining cells intended for carbonization (box 102). In a second step, the obtained cells are chemically and / or mechanically washed (box 132). In another step, the washed cells are dried (box 134). In a further step, the dried cells are crushed and / or powdered (box 136). In a still further step, the cells are dry and heat treated (box 138). In a next step, the heat treated cells are crushed and / or powdered (box 140). A dispersion is then made (box 142). In a subsequent step, the final product is manufactured according to the final formulation for a particular market (box 144).

[0060] FIG. 3 illustrates a further embodiment of method 100. In a first step, cells intended for carbonization are selected and / or obtained (box 150). In a further step, various cellular components are removed from the cells via chemical and / or physical treatment (box 152). In a further step, the cells are physically and / or mechanically washed (box 154). In some implementations, the steps of removal and washing may be repeated periodically. The next step, in some embodiments, is to create a dry product by drying (box 156). Furthermore, the dry product may be ground and / or pulverized via a grinding and / or powdering process. In one embodiment, the cells are dried and / or powdered (Box 158). In another step, the cells are heat treated (Box 160). In various embodiments, the heat treatment (Box 160) is a dry heat treatment. In a further step, the heat treated product is ground and / or powdered via a grinding and / or powdering process (Box 162). In another step, a dispersion is made (Box 164). In a final step, the final formulation is manufactured for the desired market (Box 166).

[0061] Another alternative implementation of method 100 is shown in FIG. 4. In this embodiment, method 100 starts with cells intended for carbonization (box 170). The cells are then chemically and / or mechanically treated to remove cellular components (box 172). The cells are then chemically and / or mechanically washed (box 174). In some embodiments, the removal and washing steps may be repeated and / or cyclically performed until the desired intermediate product is obtained. In another step, the cells are dried (box 176). In a next step, the cells are subjected to a dry heat treatment (box 178). In another step, the cells are crushed and / or powdered (box 180). In another step, a dispersion is made (box 182). In a next step, the cells are made into a final formulation (box 184).

[0062] 5 shows another exemplary embodiment of the method 100. First, the cells to be used in the process are provided (box 200). Second, the cells are chemically and / or mechanically treated to remove various components of the cells (box 202). Next, the cells are chemically and / or mechanically washed (box 204). In a fourth step, the cells are subjected to a wet heat treatment (box 206). In another step, the heat-treated cells are ground and / or powdered through a grinding and / or powdering process (box 208). In the next step, a dispersion is made with the cells (box 210). In a final step, the cells are made into a final formulation (box 212).

[0063] Another alternative embodiment is shown in Figure 6. In one step, cells for method 100 are provided (box 220). In another step, the cells are subjected to a chemical and / or mechanical treatment to remove cellular components (box 222). In another step, the cells are subjected to a moist heat treatment (box 224). Another step includes grinding and / or powdering the cells (box 226). In another step, a dispersion is made (box 228). In a final step, an ink or final product is manufactured according to the final formulation (box 230).

[0064] Those skilled in the art will understand that the various steps disclosed herein may be included or excluded depending on the nature of the microbial biomass used and the desired end product characteristics. As a non-limiting example, in certain embodiments, the method is a heat treatment step, followed by a washing step, followed by a grinding step. In a further embodiment, the method is a heat treatment step, followed by a grinding step. In yet a further embodiment, the method is a washing step, followed by a heat treatment step, followed by a grinding step. In yet a further embodiment, the method is a washing step, followed by a heat treatment step, followed by a grinding step, followed by a washing step. In yet a further embodiment, the method is a washing step, followed by a heat treatment step, followed by a grinding step, followed by a washing step. In yet a further embodiment, the method is a washing step, followed by a heat treatment step, followed by a washing step, followed by a grinding step.

[0065] Ink formulation The concentration of treated microbial biomass may range from 0.1-100% (w / w) of the total composition of the ink and / or colorant formulation. In addition to replacing the pigment portion of known formulations, the microbial cells may also partially or completely replace the addition of other formulation components (resins, oils / carriers, additives) commonly found in formulations developed for specific industries in which this colorant technology may be utilized. This is because changes in the overall cellular components (concentrations and / or types of fatty acids, proteins, carbohydrates, minerals, etc.) within a particular cell type may have a significant effect on the overall composition of the ink and / or colorant formulation. The pigments can be used in organic solvent-based colorant formulations as well as in water-based or radiation-curable colorant formulations, as they can modify the need for certain formulation ingredients that traditionally need to be added to the formulation.

[0066] Alternatively, certain methods may be applied to enhance the integrity of biological cells when they are utilized as colorants in various industries. Strategies may include, but are not limited to, the addition of preservatives to cell cultures prior to colorant formulation. These preservatives act to enhance the integrity of individual cells when various treatments are applied to prepare the cells for the colorant. These preservatives may include, but are not limited to, mono- and polysaccharides, mono- and polysaccharide alcohols, glucosylglycerol, glycerol, dextrose, saccharose, trehalose, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, methanol, dextrin, dimethyl sulfoxide (DMSO), sodium benzoate, potassium metabisulfite, sodium citrate, sodium chloride, cellulose sulfate, Locke's solution, ectoine, Ringer's solution, gelatin, plasma, amino acids, peptides, or proteins or combinations thereof, such as late embryonic accumulation (LEA) proteins, either undiluted or at any concentration.

[0067] Additional strategies to enhance the integrity of cells used in ink and / or colorant formulations include encapsulation of biological cells. Encapsulation vehicles include, but are not limited to, alginate-polylysine-alginate (APA) microcapsules, sodium alginate, cellulose sulfate, calcium algenate, bovine serum albumin, collagen, chitosan, gelatin, either neat or at any concentration. It may include agarose and formalin.

[0068] In certain implementations, the ink and / or colorant compositions incorporating the above discussed technology may or may not be combined with any mixture of molecular components commonly used in the particular target industry, including, but not limited to: pigments, resins, oils / carriers, additives, binders and / or resins, acrylic resins, oligomers, copolymers, UV / EB resins, styrene resins, xylene, toluene, and urethane resins, anti-wear additives, adhesion promoters, air-release additives, anti-blocking additives, and the like. Additives, anti-denting additives, anti-floating additives, anti-agglomeration Additives, anti-flooding additives, anti-foaming additives, anti-gas additives (anti gassing additives), anti-sag additives, anti-caking additives, anti-static additives, fungicides, barrier additives, carrier bases, catalysts, chelating additives, chemical resistance additives, coalescing aids, color locking additives, conductive additives, corrosion prevention additives agents, coupling agents, crosslinkers / chain extenders, hardeners, deaeration / gassing agents, drying agents / drying accelerators, dispersing additives, emulsifying additives, film foaming additives, flame retardants, flexibility agents, flow and leveling additives, optical brighteners, free flowing additives, gloss enhancers, hardeners, heat resistance agents, impact modifiers, initiators, lubricants, mar / scratch resistance additives, matting additives, moisture scavengers, opacifiers, pH adjusters, plasticizers, strengthening additives, release aids, wear resistance additives, skid / skid resistance, slip additives, solvent resistance additives, special effect additives, and wetting additives.

[0069] The pigments and colorants made according to the disclosed methods are suitable for use in a variety of industrial applications, including, but not limited to: paint manufacturing industry, ink manufacturing industry, pigment manufacturing industry, coatings industry, colorant industry, inkjet industry, printing industry, textile industry, food color industry, functional food industry, agricultural industry, soap and grooming industry, cosmetics industry, and any of the above.

[0070] Printing types that may be used with the present invention include, but are not limited to: silk and rotary screen coating, screen printing, offset lithography, flexography, forward roll coating, reverse roll coating, spray coating, air knife coating, anilox coating, flexo coating, dip coating, metering rod coating, roller coating, kiss coating, extrusion coating, curtain coating, dip coating, spin coating, digital printing, inkjet and xerography, gravure, 3D printing, dye sublimation, pad printing, relief printing, intaglio printing, radiation curable, thermal dye sublimation, and any subset of the printing types described herein.

[0071] Ink types that may utilize the present technology include, but are not limited to: water-based inks, solvent inks, vegetable-based inks, latex-based inks, radiation curable inks, phase change inks, and any subset of the printing types described herein.

[0072] In one particular implementation, the preparation of the ink and / or colorant formulation involves several steps. In one step, 25 ml of a high density culture of the microorganism (measured by the optical density of the culture, which is about OD730 of 15) is centrifuged in a suitable centrifuge device at a range of 3000-10000×g. In a further step, the supernatant is decanted, leaving only the cells and a small amount of growth medium in the tube. The concentration of the remaining cells should be 5%-25% of the total volume. To this solution, 200 mg of cellulose (to give a concentration of 40 g / l) is added to the culture, and 200 uL of gum arabic is added to give a concentration of gum arabic of 4% of the total solution volume. This results in 5 ml of a final ink solution suitable for use in a screen printer.

[0073] In certain implementations, grinding of cell culture components is required to obtain acceptable pigment particle sizes or cell aggregate sizes with particle size values ​​between 0.01 microns and 100 microns. Particle size reduction methods that may be used may include, but are not limited to: mortar / pestle, rotary tumbler, vibratory tumbler, magnetic tumbler, roll mill, bead mill agitator, disk mill, basket mill, jet mill, ball mill, jaw crusher, rotor mill, cutting mill, and knife mill. In addition to the method of grinding, various physical grinding media, as well as chemical additives, may be introduced into any of the grinding methods mentioned above to further increase the grinding efficiency. Physical additives such as grinding media may include, but are not limited to: steel, chrome, stainless steel, ceramic, rubber, stoneware, aluminum, magnesium, zirconia, porcelain, silica, and glass. The grinding media may be obtained in various sizes ranging from 1 / 32 inch to 5 inch diameter. Chemical additives may include dispersants, surfactants, wetting agents, abrasives, detergent soaps, hyperdispersants, nonionic high HLB polyalkoxylated surfactants, nonionic polymers, defoamers, water, resins, surface tension modifiers, hydrophobic anionic polymers, acetylenic diols, and acetylenic diols.

[0074] Production of biological cell-derived black colorant via thermal process Biogenic black biochar pigments used as colorants in various formulations and in various industries: In one particular implementation, the black biogenic pigments can be used in various possible ink and / or colorant embodiments. As previously described in this document, various biological sources can be utilized as "starting" materials for producing the black biogenic pigments. Several mechanisms can be used to achieve the black biogenic pigments. One embodiment of the production mechanism includes a processing step that is performed after the growth, isolation, and dehydration of the biomass. This processing step uses a thermal treatment process that can be applied to microbial cultures that include one or more of the biomass sources previously described in this document. Pyrolysis, gasification, combustion, and / or a combination of these processes can be used to produce the final microbial black pigment. In this process, the microbial cultures Growth medium is removed from the cell culture so that the moisture content of the algae culture is in the range of 0%-75% (volume / volume). This pre-drying may include, but is not limited to, the following processes: ambient air drying, forced air drying, steam drying, centrifugation, spray drying, and jet milling. This dried algae culture is then placed in a suitable container that can be sealed to exclude any additional gases from being introduced during the production process. In one embodiment, an inert gas may be added to the container to push out any unwanted gases such as carbon dioxide, oxygen, and any other reactive gas species. In a separate embodiment, air and other reactive gases are added to the combustion chamber to increase the overall combustion temperature and promote chemical reactions within the chamber. In another embodiment, different types of inert and reactive gases may be introduced into the reaction chamber in subsequent steps to obtain different types of reactions at different points during the heating process. Once the microbial biomass is added to the appropriate chamber, the chamber is heated to a constant temperature that may be in the range of 100 degrees Celsius to 1000 degrees Celsius or any temperature therebetween. Alternatively, multiple temperature steps may be applied to the biomass chamber over a certain time frame. Once the appropriate temperature setting is reached, the temperature is held for a certain length of time, which may range from 1 minute to 24 hours. This is repeated for any temperature setting that may be required throughout the heating process. The length of time it takes to reach a stable holding temperature may be a fast ramp (moving between two treatment temperatures in as little as a few seconds), or a relatively slow ramp, such as 6 hours, or anywhere in between these indicated ramp rates. Once the desired heating schedule is complete, cooling of the microbial biomass may be done following a gradual or stepwise process along the lines of what was described in the heating process. Further processing of the algae biomass may or may not be required. In one embodiment, a rinse is applied to the biomass.The rinsing process involves adding liquid to the biomass and heating the biomass to a temperature between 30 degrees Celsius and 300 degrees Celsius for a set length of time that may be comprised within the range of 5 minutes to 12 hours. These processes may be performed once or may be performed multiple times on the same batch of microbial biomass. An additional drying step may or may not be included in the process. Certain equipment and / or processes may be used and / or performed to reduce the overall amount of particle agglomeration that may occur during the pigment treatment process. A grinding process may or may not be performed after the final drying process is completed. Additionally, surface treatment for increased surface area and smaller particle size may be achieved by gas treatment, chemical treatment, or other surface treatments known to those skilled in the art of pigment manufacturing.

[0075] Working Example The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the articles, devices, and / or methods claimed herein are made and evaluated, are intended to be merely exemplary of the invention, and are not intended to limit the scope of what the inventors regard as their invention. However, it should be understood by those of ordinary skill in the art in light of the present disclosure that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0076] procedure All data was collected according to the following procedures unless otherwise noted. TGA Procedure (Carbonization Process): Thermogravimetric analysis (TGA) is a thermal analysis method in which the mass of a sample is measured over time as the temperature is changed. A maximum amount of powdered material sieved to <50 μm was loaded into a 5 mL TGA cup (typically 2-3 g). The analytical instrument was purged with nitrogen gas for 15 min before the start of the analysis and during the course of the analysis to create an inert atmosphere around the cup. The analysis was performed at 30 °C / min. The carbonization was performed by heating at 500°C / min to 150°C, followed by a 50°C / min ramp to the set temperature. The test duration (hold at set temperature) was 15 minutes, followed by passive cooling after completion of the test. Yield measurements were performed by measuring the mass of each sample before and after the test. All samples were carbonized at a set temperature of 500°C, unless otherwise noted in the individual tables.

[0077] PSD Procedure (Median and Mean Size Measurement): The particle size distribution of the samples was measured using a Horiba LA-950 V2 particle size distribution analyzer. For samples containing larger particles (>100 μm), a manual dry screening method using sieves of various sizes was used in combination with the Horiba instrument.

[0078] SEM Procedure (SEM Images): Scanning electron microscope images were taken of all samples before and after TGA. Images were taken using a Jeol / EQ InTouchscope. 5-10 mg of sample was sprinkled onto a double-sided carbon fixed to the SEM stage. To reduce any charging during imaging, samples were pre-coated with gold and palladium by sputtering. Images were taken at magnifications of 150x, 500x, and 1500x.

[0079] Preparation of Pigment Dispersions for Color Analysis: (Average L, Average A, and Average B Measurements, and Drawdown Images) A dispersion master mix was prepared:

[0080] [Table 1]

[0081] 2g of zirconium beads were mixed with 1.1ml of dispersion master mix. 0.2g of TGA sample was added to this dispersion mix. Pigment dispersions were prepared by bead beating method using Biospec 3110Bx Mini beadbeater at a speed of 42R for 3 minutes (30 seconds duration with 1 minute off). The tubes were incubated at room temperature for 15 minutes and cooled. 150L was used for particle size characterization using grind gauge. 250uL was used for drawdown on Leneta ink test sheet. The drawdown was allowed to dry for 24 hours, after which L,a,b measurements were taken using a 3nH YD5050 spectrodensitometer.

[0082] Acid wash for color analysis: (avg L, avg A, avg B - Post carbon wash data) Post-TGA acid washes were performed on baseline, baseline reanalysis, and pre-wash samples. 1 g of post-TGA sample was washed with 25 ml of 1% hydrochloric acid (1:25 biomass ratio). Washing was completed with 15 min of shaking incubation. Acid-washed char was recovered by centrifugation at 5000 g for 5 min. A water wash was performed with 25 ml of DI water to remove residual acid and salts dissolved in the water. The washed char was dried overnight at 55° C. Dispersions from the acid-washed char were prepared as described above. The color difference between unwashed and acid-washed was recorded.

[0083] Dispersion Drawdown: 250 μl of the pigment dispersion was drawn down at a constant speed using a #7 bar onto a vellum opaque Leneta ink test sheet. The ink was allowed to dry for 24 hours and then measured using a spectrodensitometer. L,a,b measurements were taken using a 3D LCMS spectroscopy. Three L,a,b values ​​were taken at three different areas on the drawdown and averaged.

[0084] Example 1 The data in the following tables (Tables 1 and 2) and Figures 7 and 8 specifically show data collected for several different forms of the prokaryotic genus Spirulina. Experiment Name: Baseline Study All starting biomass materials were hand sieved to less than 50 μm prior to carbonization.

[0085] List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - WC=whole cell - NO CHAR = data collected on pre-char sample. - Unwashed = data collected after carbonization and before washing. - Post carbonization wash = data collected after an acid wash was performed on the carbonized material. - Spirulina decolorization = Prior to sampling, colored molecules were removed via solvent stripping. - Temperature indicates the temperature at which the sample was carbonized for the specified time. - Biomass source / alternative name structure: -- Spirulina: Earthrise Functional Food

[0086] [Table 2]

[0087] [Table 3]

[0088] Example 2: The data in the following tables (Tables 3 and 4) and Figures 9 and 10 specifically show data collected for several different forms of the eukaryotic genus Nannochloropsis. Experiment Name: Baseline Reanalysis List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - WC=whole cell - NO CHAR = data collected on pre-char sample. - Unwashed = data collected after carbonization and before washing. - Post carbonization wash = data collected after an acid wash was performed on the carbonized material. - Delipidated Nannochloropsis = lipid molecules were removed via solvent stripping prior to sampling. - Temperature indicates the temperature at which the sample was carbonized for the specified time. - Biomass source / alternative name structure: -- Defatted Nannochloropsis: Qualitas Health

[0089] [Table 4]

[0090] [Table 5]

[0091] Example 3: The data in the following tables (Tables 5 and 6) and Figures 10-20 specifically show data collected for several prokaryotic and eukaryotic species in several different forms. Experiment Name: Species Differences / Residual Biomass List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - WC=whole cell - NO CHAR = data collected on pre-char sample. - Unwashed = data collected after carbonization and before washing. - Delipidated Nannochloropsis = lipid molecules were removed via solvent stripping prior to sampling. - Delipidated / Deproteinized Nannochloropsis = Both lipids and proteins have been removed via separate processes. - Temperature indicates the temperature at which the sample was carbonized for the specified time. - Biomass source / alternative name structure: -- E. coli - Intracellular strain -- Yeast-endostrain waste biomass -- Whole Cell Spirulina - Earthrise Brand, purchased from retail stores -- Whole Cell Nannochloropsis - Qualitas Health -- Whole Cell Chlorella - Manna brand, purchased from retail stores -- Pine sawdust - purchased commercially -- Defatted Nannochloropsis - Qualitas Health -- Defatted / Deproteinized Nannochloropsis - Qualitas Health

[0092] [Table 6-1]

[0093] [Table 6-2]

[0094] [Table 7-1]

[0095] [Table 7-2]

[0096] Example 4: The data in the following tables (Tables 7 and 8) and Figures 21-26 specifically show data collected for several different forms of Spirulina spp. Biomass Cleaning Details: - 1:25 biomass ratio (20 g LinaX per 500 mL wash volume) in a 1 L Erlenmeyer flask - Cleaning solution: -- Acid: 1% hydrochloric acid (15.9 mL in 500 mL DI water) -- Water: DI water (washed twice to ensure removal of as much acid, salt, etc. as possible) - For each wash, incubate for 15 minutes at room temperature in an incubator with shaking at 150 rpm - Each flask was centrifuged at 5000 x g in 10 x 50 mL Falcon tubes and the supernatant was poured off and either dried or the pellet was resuspended in an equal volume of water and returned to the flask for the wash incubation. - The washed samples as pellets in Falcon tubes were dried overnight in an incubator at 55°C.

[0097] List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - WC=whole cell - NO CHAR = data collected on pre-char sample. - Unwashed = data collected after carbonization and before washing. - Post carbonization wash = data collected after an acid wash was performed on the carbonized material. - Acid.Water = Before carbonization, the samples were washed with an acid solution and also with an aqueous solution. - Acid = samples were washed with an acid solution before carbonization. - Water = samples were washed with an aqueous solution prior to carbonization. - Temperature indicates the temperature at which the sample was carbonized for the specified time. - Biomass source / alternative name structure: -- Spirulina: Earthrise Functional Food

[0098] [Table 8]

[0099] [Table 9]

[0100] Example 5: The data in the following tables (Tables 9 and 10) and figures specifically show data collected for several different forms of Spirulina spp. Experiment Name: Ash Test Biomass Cleaning Details: - 1:25 biomass ratio (20 g LinaX per 500 mL wash volume), 1 L In Erlenmeyer flask - Cleaning solution: -- Acid: 1% hydrochloric acid (15.9 mL in 500 mL DI water) -- Water: DI water (washed twice to ensure removal of as much acid, salt, etc. as possible) - For each wash, incubate for 15 minutes at room temperature in an incubator with shaking at 150 rpm - Each flask was centrifuged at 5000 x g in 10 x 50 mL Falcon tubes and the supernatant was poured off and either dried or the pellet was resuspended in an equal volume of water and returned to the flask for the wash incubation. - The washed samples as pellets in Falcon tubes were dried overnight in an incubator at 55°C.

[0101] List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - Uncarbonized / Unwashed = The material was not subjected to any form of pre-cleaning and was not carbonized. - Carbonized / Unwashed = The material was carbonized at 500°C but the product was not washed either before or after the carbonization process. - Carbonization / washing = the material was carbonized and then after the carbonization process it was washed with acid and rinsed with water. - Unwashed = data collected after carbonization and before washing. - Temperature indicates the temperature at which the sample was carbonized for the specified time. - Biomass source / alternative name structure: -- Spirulina: Earthrise Functional Food

[0102] [Table 10]

[0103] [Table 11]

[0104] Example 6: The data in the following tables (Tables 11 and 12) specifically represent data collected on commercially available biomass samples that were pre-carbonized by an entity other than Living Ink prior to any testing or data collection. Experiment Name: Commercial Biomass Washing Details: - Acid Wash - 1:25 biomass ratio (200g in 5L of 1% acid) with 1% hydrochloric acid, performed in a 5 gallon bucket. Washing time was 20 minutes. The washed char was collected on a filter plate. This was followed by a 5L water wash. The cake was collected from the filter plate and dried at room temperature. - Base wash - 1:25 biomass ratio with 2% sodium hydroxide (2g in 50mL of 2% NaOH solution). Washed for 20 minutes and collected the washed char using centrifugation. Followed by washing with 50ml water. The washed char was dried at 55°C overnight.

[0105] List of terms: - Approximate - Analysis for moisture, ash, volatile matter, and fixed carbon as specified in accordance with ASTM D3172. - FINAL - For coal and coke, the determination of the carbon and hydrogen in the material as found in the gaseous products of its complete combustion, the determination of the sulfur, nitrogen, and ash in the material as a whole, and the calculation of the oxygen by difference. This data was determined in accordance with ASTM D3176. - Unwashed = the material did not undergo any form of post-carbonization cleaning. - Acid / water = charred samples were washed with an acid solution after carbonization and washed with an aqueous solution. - Base / Water = Charred sample was washed with a base solution after charring and washed with an aqueous solution. - Unwashed = data collected after carbonization and before washing. - Post carbonization wash = data collected after an acid wash was performed on the carbonized material. - Biomass source / alternative name structure: -- Wood-based biochar: purchased from Wakefield Biochar -- Coconut charcoal: Purchased from Cool Planet -- Bamboo charcoal: Purchased from SEEK -- Mixed biomass: Purchase from Biochar Now

[0106] [Table 12]

[0107] [Table 13]

[0108] Although the present disclosure has been described with reference to preferred embodiments, those skilled in the art will recognize that changes can be made in form and detail without departing from the spirit and scope of the disclosed devices, systems, and methods.

Claims

1. 1. A method for producing carbon black pigment from microbial biomass, comprising: a. removing intracellular components from the microbial biomass; b. heat treating the microbial biomass to form a carbonized biomass; c. washing the carbonized biomass; and d. grinding the carbonized biomass to a particle size of 0.01 μm to 100 μm to form a powdered microbial charcoal; A method comprising:

2. The method of claim 1 , wherein the microbial biomass comprises a plurality of prokaryotic cells.

3. The method of claim 2 , wherein the microbial biomass comprises decolorized prokaryotic cells.

4. The method of claim 3 , wherein the bleached prokaryotic cells are of the genus Arthrospira.

5. 3. The method of claim 2, wherein the prokaryotic cells have an average cell size of less than 20 μm.

6. 10. The method of claim 1, further comprising washing the microbial biomass prior to the heat treatment step.

7. The method of claim 6 , wherein the microbial biomass is washed with an acid.

8. 10. The method of claim 1, wherein the microbial biomass is dried at a temperature between 30°C and 300°C prior to the heat treatment step.

9. 7. The method of claim 6, wherein the microbial biomass is dried until the moisture content is reduced to less than 10% by weight.

10. 10. The method of claim 1, wherein the thermal treatment of the microbial biomass is carried out by a process selected from the group consisting of pyrolysis, gasification, combustion, thermo-oxidative decomposition, torrefaction, hydrothermal liquefaction, and hydrothermal carbonization.

11. The method of claim 1 , wherein the heat treatment step is carried out at a temperature in the range of 300° C. to 700° C.

12. 10. The method of claim 1, wherein the heat treating step is carried out for a length of time between 1 second and 24 hours.

13. 13. The method of claim 12, wherein the period of time is between 5 minutes and 40 minutes.

14. 14. The method of claim 13, wherein the period of time is 10 minutes.

15. 13. The method of claim 12, wherein the heat treating step is carried out until the carbonized biomass contains between 20% and 30% fixed carbon by weight.

16. 13. The method of claim 12, wherein the heat treating step is carried out until the approximate volatile content level in the carbonized biomass is less than 20% by weight.

17. 13. The method of claim 12, wherein the heat treatment step is carried out until the oxygen concentration in the carbonized biomass is less than 20% by weight.

18. 13. The method of claim 12, wherein the heat treating step is carried out until the oxygen concentration is between 10% and 15% by weight.

19. 20. The method of claim 18, wherein the heat treatment step is carried out until the ash concentration in the carbonized biomass is less than 20% by weight.

20. 2. The method of claim 1, wherein the washing of the carbonized biomass is an acid wash, the acid wash comprising lowering the pH of the carbonized biomass to less than 2.

21. 21. The method of claim 20, comprising washing the carbonized biomass at a reduced pH of less than 2 for a period of time between 1 minute and 1 hour.

22. 22. The method of claim 21, further comprising washing the carbonized biomass with water following the acid wash.

23. 10. The method of claim 1, wherein the grinding step is performed by an apparatus selected from the group consisting of a ball / media mill, a long gap mill, an air classifier, a jet mill, a three-roll mill, a basket mill, a cryomill, and a cyclone mill.

24. 2. The method of claim 1, wherein the grinding step is carried out without the use of a jaw crusher, a hammer mill, a saw mill, an impact dryer mill, a granulator, a guillotine, an impact dryer mill, a lump breaker, a knife mill, a pin mill, a roller crusher, a rotary mill, a vibratory tumbler, or a magnetic tumbler.

25. The method of claim 1, wherein the grinding step is carried out until the average particle size diameter of the powdered microbial charcoal is less than 10 μm.

26. 2. The method of claim 1, wherein the grinding step comprises adding one or more mechanical grinding additives to the carbonized biomass during grinding, the mechanical grinding additives having a particle size in the range of 0.79 mm to 127 mm diameter.

27. 10. The method of claim 1, wherein the grinding step comprises adding one or more chemical grinding additives to the carbonized biomass during grinding.

28. 28. The method of claim 27, wherein the one or more chemical grinding additives are selected from the list consisting of dispersants, surfactants, wetting agents, abrasives, detergent soaps, hyperdispersants, nonionic high HLB polyalkoxylated surfactants, nonionic polymers, defoamers, water, resins, surface tension modifiers, hydrophobic anionic polymers, acetylenic diols, and acetylenic diols.

29. 10. The method of claim 1, further comprising modifying the powdered microbial charcoal after the grinding step.

30. 30. The method of claim 29, wherein the modification of the carbonized biomass is by adding a chemical additive to the carbonized biomass.

31. 31. The method of claim 30, wherein the chemical additive is selected from the list consisting of aromatic compounds, alcohols, surfactants, oils / fats / fatty acids / lipids, water, ionic liquids, hydrogenation, chemical hydrolysis, enzymatic hydrolysis, alkaline solvents, carbon dioxide, chlorine gas, sulfur gas, nitrogen gas, and oxygen gas.

32. 30. The method of claim 29, wherein the modifying of the powdered microbial charcoal comprises drying the microbial charcoal.

33. 33. The method of claim 32, wherein the drying step is by coalescence.

34. 2. The method of claim 1, wherein the drying step is carried out until the moisture content of the microbial charcoal is reduced to less than 8% by weight.

35. 1. A method for producing carbon black pigment from microbial biomass, comprising: a. removing intracellular components from the microbial biomass; b. heat treating the microbial biomass comprising a plurality of decolorized Arthrospira cells to form a carbonized biomass; and c. grinding the carbonized biomass to a particle size of 0.01 μm to 100 μm to form a powdered microbial charcoal; A method comprising:

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