Compositions for microbially produced pre-dispersed colourant using microbially produced dispersant agents and methods thereon

Abstract

The invention relates to microbial colorant compositions and associated processes to produce the same. Microbial colorant compositions are produced by combining microbially derived pigment molecules and microbial biosurfactants. The pigment molecules are obtained either directly from a fermentation broth or by extracting a precursor and transforming it into a pigment using chemical or enzymatic processes. A separately produced biosurfactant, such as a rhamnolipid or surfactin, is added to the obtained pigment molecule to create a pre-dispersed formulation. The biosurfactant may be added at various steps of This approach improves pigment dispersion, solubility, and substrate compatibility, enabling stable aqueous formulations without synthetic additives. The resulting compositions are suitable for wide range of applications including in textiles, food, cosmetics, and polymer materials. Processes for producing and combining the pigment and biosurfactant are also disclosed, including variations in fermentation, extraction, and formulation stages.

Description

COMPOSITIONS FOR MICROBIALLY PRODUCED PRE-DISPERSED COLOURANT USING MICROBIALLY PRODUCED DISPERSANT AGENTS AND METHODS THEREONTECHNICAL FIELD

[0001] The present disclosure generally relates to processes for production of microbial colorants, and more particularly related to processes for producing predispersed colorant using microbially produced dispersant agents.BACKGROUND OF THE INVENTION

[0002] In recent times, the production of dyes from microorganisms has gained prominence as a sustainable alternative to traditional chemical processes. This method of microbial dye production is celebrated for its reduced environmental footprint, leveraging the natural processes of microorganisms to create colorants with less waste and pollution. The process not only aligns with the growing demand for eco-friendly products but also offers innovative solutions to longstanding environmental issues associated with the dye and pigment industry.

[0003] However, application and use of microbial colorants is not without challenges. In some cases, pigments extracted from microbial production lack suitable properties, such as surface properties, for use in an industrial application. For example, violacein is widely produced by microorganisms around the world and is an important bioactive compound that can also be used to create natural, sustainable dyes. Violacein and some other pigments are typically insoluble in water and may require a surfactant to yield stable and homogenous dispersion for various dye applications such as textile dye, food coloring, and plastic coloring.

[0004] Surfactants and dispersing agents are chemicals which are typically used to promote uniform and stable suspension of small particles in a matrix. Dispersing agents could be added to pigments to prevent agglomeration of individual dye particles and create a homogenous dye stuff or dye solution. In order to improve sustainability and environmental burden of microbially produced dye stuff, using plantbased or microbially produced surfactants is desired.

[0005] In this disclosure, procedures and composition for producing 100% ornear 100% microbial dyestuff by using microbially biosynthesized color compounds and microbially grown dispersing agents.SUMMARY

[0006] Provided herein is a colorant composition including a microbial pigment molecule produced by fermenting a microorganism culture to yield a fermented broth, and processing the fermented broth to obtain the microbial pigment molecule, and a microbial biosurfactant, wherein the microbial biosurfactant is added to the microbial pigment molecule to produce the colorant composition.

[0007] The microbial pigment molecule may be extracted by treating the fermented broth.

[0008] The microbial pigment molecule may be extracted by at least one of filtration, solvent-based extraction, and surfactant-based extraction. Extraction of the microbial pigment molecule may include at least one of agitation, bead milling, high pressure homogenization, freeze-thaw, ultrasonication, and centrifugation.

[0009] The microbial pigment molecule may be produced by extracting a pigment precursor molecule from the fermented broth and transforming the pigment precursor molecule to the microbial pigment molecule through chemical or enzymatic modification. The pigment precursor molecule may include at least one of tryptophan, glutamine, phenazines, tyrosine, or a catechol compound.

[0010] The microbial biosurfactant may be produced by fermenting a second microorganism culture to yield a second fermented broth and extracting the microbial biosurfactant from the second fermented broth.

[0011] The biosurfactant may be selected from the group including rhamnolipids, surfactins, sophorolipids, trehalolipids, and mannosylerythritol lipids.

[0012] Provided herein is a process for producing a colorant composition, the process including producing a microbial pigment molecule or pigment precursor molecule by fermenting a first microbial seed culture to yield a first fermented broth, and processing the first fermented broth to obtain the pigment molecule or pigment precursor molecule, and adding a microbial biosurfactant to the pigment molecule or pigment precursor molecule to produce the colorant composition.

[0013] Processing the first fermented both to obtain the pigment molecule orpigment precursor molecule may include extracting the pigment or pigment precursor molecule from the first fermented broth by purification, and dehydrating the purified pigment or pigment precursor molecule, wherein the microbial biosurfactant is combined with the dehydrated pigment or pigment precursor molecule to produce the colorant composition.

[0014] Extracting the pigment or pigment precursor molecule may include at least one of filtration, solvent-based extraction, and surfactant-based extraction.

[0015] Extracting the pigment or pigment precursor molecule may include at least one of agitation, bead milling, high pressure homogenization, freeze-thaw, ultrasonication, and centrifugation.

[0016] The process may further include producing the microbial biosurfactant by fermenting a second microbial seed culture to yield a second fermented broth, and processing the second fermented broth to obtain the microbial biosurfactant. Processing the second fermented broth to obtain the microbial surfactant may include extracting the microbial surfactant from the second fermented broth by purification, and dehydrating the microbial surfactant.

[0017] Extracting the microbial surfactant may include at least one of filtration, solvent-based extraction, and surfactant-based extraction.

[0018] Extracting the microbial surfactant may include at least one of agitation, bead milling, high pressure homogenization, freeze-thaw, ultrasonication, and centrifugation.

[0019] Provided herein is a process for producing a colorant, the process including fermenting a microbial seed culture to yield a fermented broth, adding a microbial biosurfactant to the fermented broth to form a broth composition, treating the broth composition to extract a microbial pigment or pigment precursor molecule, and producing the colorant from the extracted pigment or pigment precursor molecule.

[0020] Provided herein is a process for producing a colorant, the process including fermenting a microbial seed culture to yield a fermented broth, extracting a microbial pigment or pigment precursor molecule from the fermented broth by purification, adding a microbial biosurfactant to the purified pigment or pigment precursor molecule, and producing the colorant by dehydrating the combined microbial biosurfactant and purified pigment or pigment precursor molecule.

[0021] Other aspects and features will become apparent to those ordinarily skilled in the art upon review of the following description of specific disclosed embodiments in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In the following, embodiments of the present disclosure will be described with reference to the appended drawings. However, various embodiments of the present disclosure are not limited to the arrangements shown in the drawings.

[0023] Figure 1 A to 1 C are schematic flow diagrams for microbially produced, pre-dispersed colorants, according to an embodiment;

[0024] Figure 2 is a schematic diagram showing various elements pertaining to the colorant production process of Figure 1A, according to one embodiment.DETAILED DESCRIPTION

[0025] Various compositions or processes will be described below to provide an example of each claimed embodiment. No embodiment described below limits any claimed embodiment and any claimed embodiment may cover processes or compositions that differ from those described below. The claimed embodiments are not limited to compositions or processes having all the features of any one composition or process described below or to features common to multiple or all of the compositions described below.

[0026] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure.

[0027] Referring to Figure 1 A, a general process for producing a target colorant composition (as shown by 324 in Figure 2) from a microorganism is shown generally at 100, according to an embodiment. The process 100 comprises a pigment production process 110 and a biosurfactant production process 150.

[0028] At 112, the process 110 includes preparing a microbial colorant seed culture (shown as 310 in Figure 2). According to one embodiment, the colorant seed culture 310 includes one or more microbial agents, such as bacteria, yeast, or fungi, that include a gene pathway responsible for creating a pigment molecule, or a precursor molecule for a pigment molecule. The microbial agents may include bacterial strains such as bacteria from one or more of the genera Janthinobacterium,Chromobacter, Duganella, Collimonas, Massilia, Pseudoalteromonas, Escherichia, Citrobacter, Corynebacterium, and Streptomycese that may or may not be genetically modified. Various microbial agents along with their corresponding produced pigment molecules are studied and may be known or readily available to a person skilled in the art from commercially available resources or scientific publications. For example, bacteria from the genera Janthinobacterium and Chromobacter may produce violacein (C20H13N3O3) presenting a purple color, or bacteria from the genera Bacillus and Streptomyces may produce melanin (C18H10N2O4) presenting a dark brown or black color. In a further example, bacteria from the genera Pseudoalteromonas and Streptomycese may produce prodigiosin (C20H25N3O) presenting a red color. Other examples of pigment molecules produced by microorganisms include melanin, flexirubin, cartenoids, indigoidine, and riboflavin.

[0029] In another embodiment the seed culture 310 prepared at 110 includes one or more microbial agents, such as bacteria, yeast, or fungi, that have a gene pathway responsible for creating a pigment precursor molecule, for example tryptophan or tyrosinase. The microbial agents may include bacterial strains such as bacteria from one or more of the genera Janthinobacterium, Chromobacter, Duganella, Collimonas, Massilia, Pseudoalteromonas, Escherichia, Citrobacter, Corynebacterium, and Streptomyces that may or may not be genetically modified. Various microbial agents along with their corresponding produced colorant precursor molecules are studied and may be known or readily available to a person skilled in the art from commercially available resources or scientific publications. For example, bacteria from the genera Janthinobacterium or Chromobacter may produce tryptophan (C11H12N2O2) that, through an additional conditioning step, such as oxidative condensation, can produce violacein, presenting a purple color. As another example bacteria from the genera Serratia or Streptomyces may produce MAP (2-methyl-3-n- amyl-pyrrole) or MBC (4-methoxy-2,2'-bipyrrole-5-carbaldehyde), that through an additional conditioning step of condensation, will create prodigiosin(C2oH2sN30) presenting a pink or red color. In other embodiments, microbial agents could be used to produce other pigment precursor molecules including but not limited to glutamine, phenazines, or a catechol compound.

[0030] In other embodiments, the colorant seed culture 310 may be prepared from other microorganisms such as yeast and fungi, such as from the genera Yarrowia,Saccharomyces and Pichia, capable of producing or metabolizing precursor or pigment molecules.

[0031] The microbial agents may include natural or modified (i.e. , engineered) microorganisms. Natural microorganisms are naturally occurring and may be found or extracted from nature. Engineered microorganisms are created synthetically, for example, through genetic engineering. According to one example, a particular yeast such as Pichia pastoris may be engineered to include a gene pathway to produce prodigiosin. Preferably the microorganism is engineered to result in production of pigment or precursor molecules in high quality and high yield.

[0032] Preparing the colorant seed culture at 112 may include taking small amounts of microbial agents from a head sample and growing the small amounts in a suitable culture medium including complex organic and inorganic sources, for example, that provide optimal multiplication and reproduction to create healthy microbial agents.

[0033] At 114, the process 110 further includes transferring the microbial seed culture 310 to a fermenter or a bioreactor (shown as 314 in Figure 2), in a nutrient-rich fermentation medium (shown as 312 in Figure 2). In an embodiment, the medium includes a carbon source and different macro- and micro-nutrients such as salts and amino acids. The nutrient may be sourced from industrial by-products, such as glycerol, or waste resources, such as agricultural waste obtained as waste source from farms or produce refineries. In an embodiment, beet pulp is used as a fermentation nutrient including both nitrogen and carbon for the Pichia pastoris yeasts to metabolize during the fermentation at 114.

[0034] According to a further embodiment, J. lividum B65593 is used as the microbial agent in the colorant seed culture 310 to produce violacein. The microbial strain is a purple-pigmented rod-shaped Gram-negative bacterium of the Proteobacteria phylum and the Oxalobacterteriaceae family. Violacein is a purplecolored dye molecule. Violacein includes a dimeric structure including 5-hydroxyindol, oxindole, and 2-pyrolidone subunits developed by condensation of two modified tryptophan molecules. Violacein is a natural bio-colorant with high demand due to its properties such as biodegradability, non-toxicity, and specific differences in color tones. Sustainable and economic production of violacein through microbialfermentation is an attractive prospect to replace synthetic dyes originating from petroleum products. The carbon source included in the fermentation medium 312 for production of violacein may be sourced from crude glycerol, molasses, sugarcane bagasse, rice bran, wheat bran, and fruit waste. The nitrogen source may be sourced from organic sources such as tryptone, peptone, yeast extract, meat extract, soymeal, corn steep liquor, and chicken feather digestate, and inorganic sources such as ammonium chloride, ammonium sulphate, and diammonium hydrogen phosphate.

[0035] The colorant seed culture 310 and the fermentation medium 312 may be mixed and diluted, for example by adding water to the mixture. The colorant seed culture 310 and the fermentation medium 312 are placed in the fermenter 314. The internal environment of the fermenter 314 may be configured during the fermentation at 314 to yield optimal and efficient production of colorant output. In an embodiment, fermentation conditions such as temperature, pH, dissolved oxygen (DO) levels, aeration, agitation, and fermentation duration are controlled and monitored throughout the fermentation process at 314 for optimal production yield.

[0036] The selection of the fermentation medium 312 and the fermentation conditions may largely depend on the microbial strain used in the colorant seed culture 310. For example, some microbial strains are acid-fermenting while others are basefermenting, and some are anaerobic while others are aerobic. A person skilled in the art can appreciate that some of the processes, methods, and compositions disclosed herein are largely dependent on specific microbial agents used for bioproduction of colorants, and examples mentioned herein are not to be interpreted as a limitation of the claimed embodiments.

[0037] The harvested materials at the end of the fermentation step at 114 is a fermented broth (shown as 316 in Figure 2) that is a liquid, rich in microbial biomass and produced intracellular or extracellular pigment or precursor molecules. The fermentation at 114 may be a batch fermentation, a continuous fermentation, or a fed- batch fermentation process or sub-process. In an embodiment, the fermentation at 114 is a submerged fermentation process or sub-process or a solid substrate fermentation process or sub-process.

[0038] At 116, the process 110 further includes processing the liquid fermented broth 316 to extract and purify intracellular or extracellular pigment moleculesproduced from the fermented broth 316. The target molecule extraction and purification at 116 includes filtering and separating pigment or precursor molecules from residual biomass (i.e. , spent microbial biomass 322 in Figure 2) and unutilized nutrient medium in the fermented broth 316. Typically, the target molecule extraction and purification at 116 may depend on the intracellularity or extracellularity of the pigment or precursor molecules. At step 116, solvents and / or surfactants, may be used to help extract the target molecules from cells.

[0039] Non-aqueous colorant extraction methods may be preferred to aqueous methods so that minimal or no water is wasted during the extraction at 116. Accordingly, further wastewater treatment may not be necessary, for example, to comply with ever tightening environmental regulations. Accordingly, non-aqueous colorant extraction methods may result in simple and economic down streaming processes that can immensely reduce the overall cost of the colorant production.

[0040] In an embodiment, the colorant extraction and purification at 116 includes a solvent-based extraction method in which a solvent, particularly an organic solvent, is added to the fermented broth 316 to create a solution mixture for convenient and efficient separation and isolation of target molecules from undesired products of the fermented broth 316. The solution mixture may be further processed. The solution mixture may be agitated (e.g. sonicated) to facilitate cell disruption and dissolution of pigment molecules in the solution mixture while leaving insoluble residual biomass in a precipitated layer. The precipitated residual biomass may be separated after a gravity separation method, for example.

[0041] The precipitated residual biomass, i.e., spent microbial biomass 322 in Figure 2, may be reused in other processes.

[0042] In some embodiments, surfactant-based extraction methods may be used instead of or in conjunction with solvent-based extraction methods. The surfactant-based methods may have similar steps to the solvent-based extraction methods.

[0043] In other embodiments, one or more other cell disruption, extraction, and recovery methods such as bead milling, high pressure homogenization, freeze-thaw, ultrasonication, chemical-based extraction, centrifugation, and filtration may be used alone, with the solvent-based extraction, or surfactant-based extraction methods. Theoptimal extraction method may be determined based on intracellular or extracellular pigment production, among other factors.

[0044] In another embodiment, the fermented broth 316, or a combination of the fermented broth 316 and the solvent / surfactant, is passed through one or more filter systems, such microfiltration with 0.1 to 1 pm sized filter and nanofiltration with 1 to 10 nm sized filter, trapping particles larger than the pigment or precursor molecule size. The filtered liquid or slurry may be a mix of solvent, nutrient medium, biomass, and the target molecule which may be purified through silica gel column chromatography, for example, to obtain a crude paste.

[0045] At 118 (shown as 318 in Figure 2), in the case of a pigment molecule, the process 110 further includes dehydrating the crude pigment (or colorant) solution or medium, for example using lyophilization techniques (for example using a VirTis industrial lyophilizer), spray drying, drum drying, or oven drying to achieve a completely dry and crude pigment in powder form. In an embodiment, the crude pigment may not be dehydrated and may be used in liquid or paste format. The resulting produced colorant or pigment (shown as 320 in Figure 2) of the process 110 may be further processed or directly used for a variety of applications such as use as a colorant in textile dyeing, food and beverage production, pharmaceutical production, and cosmetic production. Throughout this document, the produced colorant 130 may be also referred to as primary colorant or primary pigment.

[0046] At 118 (shown as 318 in Figure 2), in the case of a precursor molecule, the process 110 may further include dehydrating the precursor molecule solution or medium, for example using lyophilization techniques (for example using a VirTis industrial lyophilizer), spray drying, drum drying, or oven drying to achieve a completely dry and precursor in powder form. In an embodiment, the crude precursor molecule may not be dehydrated and may be used in liquid or paste format. The precursor may undergo the conditioning step in an external reactor to transform into a colorant molecule. This step may or may not include the addition of other small molecules into the colorant. For example, tryptophan, as a precursor molecule, can undergo oxidation, non-enzymatic, and enzymatic transformations to produce violacein (generally blue-purple), indigogine (generally blue), deoxyviolacein (generally purple). By controlling the formation of colorants from tryptophan (e.g, controlling temperature, time, pH, added enzymes) and integration of additives suchas amino acids into the created molecule, a wide range of colorants can be produced, including unconventional green, blue, and purple. Similarly, Santarcangelo et. al., “Generation and structure elucidation of a red colorant formed by oxidative coupling of chlorogenic acid and tryptophan” Food Chemistry. 2023, showed combining tryptophan with chlorogenic acid can yield a red colorant for use on textile substrates, food products and more under eco-friendly conditions.

[0047] In an embodiment, further processing is conducted on the extracted colorant 320. For example, certain additives may be added to the dehydrated pigment or dye powder to increase surface tension, pigment solubility, or pigment bonding to a target substrate for longer lasting and more intense colorant application. The used additive may be obtained using proprietary and naturally driven formulations. More particularly, some pigments, such as melanin, bear limited solubility in typical colorant solvents and for creating uniform dispersion throughout a colorant composition. Surfactants are often used to help disperse pigments or dyes by reducing surface tension and facilitating their incorporation into a material. Surfactants can help stabilize the dispersion of pigments by preventing them from settling or agglomerating. They achieve this by forming a protective layer around the pigment particles, preventing them from clumping together. Surfactants can improve the wetting properties of pigments, allowing them to spread more easily over the surface of the material. This helps in achieving better coverage and adhesion of the pigment to the material. Surfactants can also enhance the color intensity of pigments by improving their dispersion and interaction with a material's surface. This can result in brighter and more vibrant colors. Surfactants can also control the surface tension of the material, which is important for various applications such as coating, painting, or printing. Controlled surface tension ensures proper spreading and adhesion of colorant composition onto a substrate.

[0048] Surfactants may be produced from various sources including naturally sourced (e.g. plants, bacterial, insects, minerals), chemically synthesized, and biosynthesized (e.g. bioproduction based on bacterial and yeast growth) production methods.

[0049] Biosynthesized surfactants can include non-ionic and ionic surfactants, depending on their chemical structure and the microorganism responsible for their production. The choice between non-ionic and ionic surfactants for aiding in the dyeingof fabric depends on various factors, including the type of dye, the substrate (e.g. the fabric substrate such as cotton, leather, and polymer), and the specific requirements of applying or depositing the dye on the substrate. In order to produce 100% (or in the range of 80-100%) microbial dyestuff, microbial biosurfactants can be produced and mixed with the microbially extracted pigments 320.

[0050] The process 100 further includes preparing a microbial biosurfactant (as shown by 350 in Figure 2) through a microbial biosurfactant production process 150. The biosurfactant production 150 may include similar general steps of gene engineering, downstreaming, and upstreaming used in the colorant production process 110. At 152 biosurfactant seed culture (shown as 340 in Figure 2) is prepared. The biosurfactant seed culture 340 generally includes one or more microbial agents, such as microbes or bacteria, that include a gene pathway responsible for creating surfactant molecules. The microbial agents may include bacterial strains such as bacteria from one or more of the genera Pseudomonas, Bacillus, and Acinetobacter. Various microbial agents along with their corresponding pathways and the produced biosurfactant are studied and may be known or readily available to a person skilled in the art from commercially available resources or scientific publications. For example, bacteria from the species Bacillus subtillis and Pseudomonas aeruginosa can produce surfactins (C53H93N7O13) and rhamnolipids (C32H58O13). Other examples of biosurfactants produced by microorganisms include sophorolipids, trehalolipids and mannosylerithritol lipids.

[0051] In other embodiments, the biosurfactant seed culture 340 may be prepared from other microorganisms such as fungi and yeast, such as from Candida bombicola and Pseudozyma rugulosa, capable of producing or metabolizing biosurfactants. Additionally, the microbial agents may include natural or modified (i.e., engineered) microorganisms.

[0052] Preparing the biosurfactant seed culture at 152 may include taking small amounts of microbial agents from a head sample and growing the small amounts in a suitable culture medium including complex organic and inorganic sources, for example, that provide optimal multiplication and reproduction to create healthy microbial agents.

[0053] At 154, the process 150 further includes transferring the biosurfactantseed culture 340 to a fermenter or a bioreactor (shown as 344 in Figure 2), in a nutrient-rich fermentation medium (shown as 342 in Figure 2). In an embodiment, the medium includes a carbon source and different macro- and micro-nutrients such as salts and amino acids.

[0054] The biosurfactant seed culture 340 and the fermentation medium 342 may be mixed and diluted, for example by adding water to the mixture. The biosurfactant seed culture 340 and the fermentation medium 342 are placed in the fermenter 344. The internal environment of the fermenter 344 may be configured during the fermentation at 154 to yield optimal and efficient production of biosurfactant output. In an embodiment, fermentation conditions such as temperature, pH, DO levels, aeration, agitation, and fermentation duration are controlled and monitored throughout the fermentation at 154 for optimal production yield.

[0055] The selection of the fermentation medium 342 and the fermentation conditions may largely depend on the microbial strain used in the biosurfactant seed culture 340. The harvested materials at the end of the fermentation step at 154 include a fermented broth (shown as 346 in Figure 2) that is a liquid, rich in microbial biomass and produced biosurfactants. At 156, the process 150 further includes processing the liquid fermented broth 346 to extract and purify biosurfactant. The biosurfactant extraction and purification at 156 may include filtering and separating biosurfactants from residual biomass and unutilized nutrient medium in the fermented broth 356. Similar to the pigment purification step 116, the biosurfactant purification step 156 may involve solvent extraction, surfactant extraction, and other cell disruption methods such as bead milling, high pressure homogenization, freeze-thaw, ultrasonication, chemical-based extraction, centrifugation, and filtration may be used alone or in conjunction with each other. The optimal extraction method may be determined based on the type of the biosurfactant and the biomass. The disrupted cells may further pass through one or more filter systems, such microfiltration and nanofiltration. The filtered liquid or slurry may be a mix of solvent, nutrient medium, biomass, and biosurfactant which may be purified through silica gel column chromatography, for example, to obtain a crude biosurfactant medium.

[0056] At 158 (shown as 348 in Figure 2), the process 150 further includes dehydrating the crude biosurfactant medium, for example using lyophilization techniques, spray drying, drum drying, or oven drying to achieve a completely drybiosurfactant 350 potentially in powder form.

[0057] In step 160a, the extracted pigment 320 and biosurfactant 350 are mixed and combined to produce the target colorant composition 324. For example, a colorant formula consists of the pigment or pigment precursors in a ratio between 1 :1 to 2:1 pigment or precursor to surfactant. Other additives (such as microbial darkening pigments, or other functional microbial agents) may be added to the composition at step 160a to produce desired properties for the target colorant composition 324. Other additives may also include a powdering agent, for example maltodextrin to assist the dye becoming a powder therefore enhancing its performance in dyestuff application to a final substrate. The additives may be a cross-linking molecule, such as citric acid or tannic acid, that would improve adhesion of the target colorant composition to the final substrate. The additives may be one or a combination of the above-mentioned additives to produce the target colorant composition 324. The pigment 320 and biosurfactant 350 may be mixed under conditions that promote stable dispersion, thereby forming a pre-dispersed colorant composition suitable for storage, transport, or immediate use in dyeing applications.

[0058] At step 170 the target colorant composition 324 is used as dyestuff and is applied to various materials and substrates for various purposes and industries. The target colorant composition 324 can be a pre-dispersed composition in which the pigment or pigment precursor is uniformly distributed within a biosurfactant-containing medium prior to final application. This pre-dispersed composition eliminates the need for end-user dispersion or solubilization steps, and ensures consistent color distribution during use in various applications. For example, a pre-dispersed cotton dye composition that has been formulated with a pigment to microbial surfactant ratio of 2:1 can be added to a dyeing bath in an amount between 1 % and 40% weight of fabric. The combination of the surfactant and pigment facilitates a uniform dispersion of the pigment or precursors and enhances its interaction with the substrate. Agitation is applied to ensure homogenous distribution and penetration of the microbial pigment or pigment precursors into the textile fibers. The dyeing process is carried out at temperatures between 20°C and 100°C and pH ranges from 3 to 9, for a period of 15 minutes to 12 hours.

[0059] Referring to Figure 1 B now, the process 100 may involve adding the produced biosurfactant 350 at step 116 (i.e. , before purifying the pigment) to improvesurface tension, wetting, solubility, and color intensity of the extracted pigments 320, and to help cell disruption and separating pigment or precursor molecules from cells. Improving the solubility of the target molecules in water, at this stage, also makes it easier to separate and purify the target molecules at the pigment or precursor purification step 116. That is, the surfactant is dehydrated at step 158 and added to the microbial pigment production 110 process between growth of the pigment 114 and purification of the pigment at 116b. Therefore, following dehydration at step 118b, a colorant formula is achieved at 160b. The colorant formula at 160b may be generally the same as the colorant at 160a, but achieved through a different process, or may be different from the colorant formula at 160a . In some embodiments, additional biosurfactant 350 may be added to the colorant formula at 160b to improve its dispersion properties.

[0060] Referring to Figure 1C, the process 100 may involve adding the produced biosurfactant 350 after purification of pigment or precursor molecules at step 116, but before the dehydration step 118c. That is, the surfactant is dehydrated at step 158 and added to the microbial pigment production 110 process between purification of the pigment 116 and dehydration of the pigment 118c. Therefore, following dehydration at step 118c, a colorant formula is achieved at 160c. The colorant formula at 160c may be generally the same as the colorant formula at 160a, but achieved through a different process, or may be different from the colorant formula at 160a.

[0061] Examples

[0062] Example 1 : Microbial Production of Melanin-Based Pre- Dispersed Colorant Using Rhamnolipids

[0063] A microbial seed culture of Cryptococcus neoformans is prepared in LB medium. The strain is then transferred to a 5 L bioreactor containing 3.5 L of a fermentation medium composed of beet pulp hydrolysate (as carbon and nitrogen source), trace minerals, and tyrosine. Fermentation is carried out at 28°C, pH 7.2, with aeration (1 vvm) and agitation (400 rpm) for 72 hours. Melanin was produced as an intracellular pigment. At the end of fermentation, the broth was centrifuged (10,000 rpm, 10 min) and the pellet was treated with ethanol and 1 % rhamnolipids in a sonication bath for 15 minutes. After filtration, the extracted melanin-rich ethanol phase was separated, concentrated through filtration and spray dried to obtain melaninpigment powder.

[0064] In a separate process, a biosurfactant-producing strain of Pseudomonas aeruginosa was fermented in a glucose-peptone-yeast extract medium for 48 hours at 30°C. Rhamnolipids were extracted using ethyl acetate, dried under vacuum, and redissolved in sterile deionized water at 20% w / v.

[0065] The dried melanin powder was reconstituted in 2% aqueous rhamnolipid solution to a final ratio of 2:1 (pigment: surfactant by weight), creating a pre-dispersed brown colorant paste with uniform distribution and no visible settling after 24 hours. The pre-dispersed colorant was diluted to 5% w / w and applied to cotton fabric by immersion at 60°C and pH 5.5 for 1 hour, followed by cold rinsing and air drying. The fabric showed even coloration and excellent wash fastness.

[0066] Example 2: Production of Violacein-Based Colorant via Precursor Fermentation and Biosurfactant Blending

[0067] A recombinant strain of Escherichia coii, which is engineered for high- level expression of a pigment precursor molecule (e.g., tryptophan), is selected. A seed culture of the selected strain is fermented in a 10 L stirred-tank bioreactor operated in fed-batch mode using glucose-limited feed. The medium was formulated using agricultural waste-derived substrates, including sugar beet pulp hydrolysate (as the carbon source) and corn steep liquor (as the nitrogen source). The fermentation is carried out in 37°C, pH of 6.8 ± 0.1 , while dissolved oxygen is maintained at >30% via cascade aeration. Glycerol is also fed every 8 hours, and total fermentation time is at 72 hours. The fermentation results in a fermented broth that is rich in precursor molecules secreted into the supernatant.

[0068] The supernatant was separated from cells via centrifugation at 5,000 rpm. The precursor molecule is recovered from the clarified broth using a filtration technique. The extract was then subjected to a chemical conversion step under mild oxidative and enzymatic conditions, resulting in the formation of a violacein-based purple pigment solution. The solution was concentrated to form a thick slurry.

[0069] In parallel, sophorolipids were produced using Candida bombicola cultured in a medium comprising glucose and soybean oil. Fermentation was carried out at 28°C for 96 hours. The biosurfactant-rich broth was extracted with ethyl acetate, concentrated under vacuum, and reconstituted in water at 20% w / v.

[0070] The pigment slurry was combined with the aqueous sophorolipid solution (pigment to biosurfactant ratio of 2:1). The mixture was spray-dried to yield a pre-dispersed purple pigment composition with aqueous re-dispersibility and longterm colloidal stability.

[0071] While specific embodiments have been described and illustrated, such embodiments should be considered illustrative only and not as limiting the disclosed embodiments as construed in accordance with the accompanying claims.

Claims

CLAIMS1 . A colorant composition comprising: a microbial pigment molecule produced by: fermenting a microorganism culture to yield a fermented broth; and processing the fermented broth to obtain the microbial pigment molecule; and a microbial biosurfactant; wherein the microbial biosurfactant is added to the microbial pigment molecule to produce the colorant composition.

2. The composition of claim 1 , wherein the microbial pigment molecule is extracted by treating the fermented broth.

3. The composition of claim 2, wherein the microbial pigment molecule is extracted by at least one of: filtration, solvent-based extraction, and surfactantbased extraction.

4. The composition of claim 3 wherein extraction of the microbial pigment molecule includes at least one of: agitation, bead milling, high pressure homogenization, freeze-thaw, ultrasonication, and centrifugation.

5. The composition of claim 1 , wherein the microbial pigment molecule is produced by extracting a pigment precursor molecule from the fermented broth and transforming the pigment precursor molecule to the microbial pigment molecule through chemical or enzymatic modification.

6. The composition of claim 5, wherein the pigment precursor molecule comprises tryptophan, glutamine, phenazines, tyrosine, or a catechol compound.

7. The composition of any of claims 1 to 6, wherein the microbial biosurfactant is produced by fermenting a second microorganism culture to yield a second fermented broth and extracting the microbial biosurfactant from the second fermented broth.

8. The composition of any of claims 1 to 7, wherein the biosurfactant is selected from the group consisting of: rhamnolipids, surfactins, sophorolipids, trehalolipids, and mannosylerythritol lipids.

9. A process for producing a colorant composition, the process comprising: producing a microbial pigment molecule or pigment precursor molecule by: fermenting a first microbial seed culture to yield a first fermented broth; and processing the first fermented broth to obtain the pigment molecule or pigment precursor molecule; and adding a microbial biosurfactant to the pigment molecule or pigment precursor molecule to produce the colorant composition.

10. The process of claim 9 wherein processing the first fermented both to obtain the pigment molecule or pigment precursor molecule includes: extracting the pigment or pigment precursor molecule from the first fermented broth by purification; and dehydrating the purified pigment or pigment precursor molecule; wherein the microbial biosurfactant is combined with the dehydrated pigment or pigment precursor molecule to produce the colorant composition.

11. The process of claim 10, wherein extracting the pigment or pigment precursor molecule includes at least one of: filtration, solvent-based extraction, and surfactant-based extraction.

12. The process of claim 10 wherein extracting the pigment or pigment precursor molecule includes at least one of: agitation, bead milling, high pressure homogenization, freeze-thaw, ultrasonication, and centrifugation.

13. The process of claim 9 further comprising: producing the microbial biosurfactant by: fermenting a second microbial seed culture to yield a second fermented broth; and processing the second fermented broth to obtain the microbial biosurfactant.

14. The process of claim 13 wherein processing the second fermented broth to obtain the microbial surfactant includes: extracting the microbial surfactant from the second fermented broth by purification; and dehydrating the microbial surfactant.

15. The process of claim 14, wherein extracting the microbial surfactant includes at least one of: filtration, solvent-based extraction, and surfactant-based extraction.

16. The process of claim 14 wherein extracting the microbial surfactant includes at least one of: agitation, bead milling, high pressure homogenization, freezethaw, ultrasonication, and centrifugation.

17. A process for producing a colorant, the process comprising: fermenting a microbial seed culture to yield a fermented broth; adding a microbial biosurfactant to the fermented broth to form a broth composition; treating the broth composition to extract a microbial pigment or pigment precursor molecule; and producing the colorant from the extracted pigment or pigment precursor molecule.

18. A process for producing a colorant, the process comprising: fermenting a microbial seed culture to yield a fermented broth;extracting a microbial pigment or pigment precursor molecule from the fermented broth by purification; adding a microbial biosurfactant to the purified pigment or pigment precursor molecule; and producing the colorant by dehydrating the combined microbial biosurfactant and purified pigment or pigment precursor molecule.

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