TECHNICAL FIELD

[0001] The present disclosure generally relates to processes for scalable production of microbial colorants, and more particularly related to processes for fermentation, extraction, and recovery of microbial pigments.

BACKGROUND OF THE INVENTION

[0002] The typical textile dyeing processes use an abundance of chemicals (estimated at 70,000 tonnes annually) resulting in a large volume of wastewater and serious toxicity caused due to release of the toxic chemicals into the environment and main water streams. Moreover, this massive industry uses toxic chemicals from non-renewable resources, which is unsustainable.

[0003] Many of the chemicals used for dyeing, once in water, are hard to separate, resulting in permanent harm and pollution of the environment and water sources for downstream usages. At least 72 chemicals, which are called “forever chemicals”, have made their way into human/plant/animal tissue and can never be removed.

[0004] The pigments may be used as colorants in a variety of industries and applications including textile dying, food and beverage production, pharmaceutical production, and cosmetic production.

[0005] Accordingly, there is a need for developing high performance, clean (e.g., nontoxic), sustainable textile dye as a desirable goal for the textile industry. There is particularly a need for dyes that incorporate circular production systems for producing clean microbial dye for the most effective and non-pollutive process.

SUMMARY

[0006] Provided is a process to obtain a target colorant from a microorganism, the process including fermenting a culture including the microorganism and a nutrition-rich fermentation medium in a first fermentation batch to harvest a fermented broth, treating the fermented broth to produce a target colorant-rich medium and a spent microbial biomass, and utilizing the spent microbial biomass as a partial nutrition-rich medium in a second fermentation batch. Treating the fermented broth may include a solvent-based extraction method to extract the colorant from the fermented broth.

[0007] The solvent-based extraction method may include a) adding a solvent to the fermented broth to form a slurry, b) sonicating the slurry, c) subjecting the sonicated slurry to gravity separation under continuous agitation for about 12 to 36 hours to separate an insoluble biomass from the slurry thereby resulting in the target colorant-rich medium, d) adding the solvent to the insoluble biomass to form a second slurry and repeating elements (b) and (c), and e) repeating element (d) for 1 to 3 times to form the spent microbial biomass.

[0008] The solvent may be selected from an alcohol, an ether, a ketone, and a combination thereof.

[0009] The solvent may be selected from an oxygenated solvent selected from an alcohol, an ether, a ketone, water and a combination thereof.

[0010] The solvent may not include water.

[0011] The solvent may be added to the fermented broth in about 100% v/v.

[0012] The spent microbial biomass may be sterilized prior to utilization in the second fermentation batch.

[0013] The culture may include a microbial strain selected from bacteria, yeasts, fungi, and a combination thereof.

[0014] The microbial strain may be selected from a natural microorganism, an engineered microorganism, and a combination thereof.

[0015] The culture may include a microbial strain selected from a bacterium from a genus of Janthinobacterium, Chromobacter , Duganella, Collimonas, Massilia, Pseudoalteromonas, Escherichia, Citrobacter, Corynebacterium, and Streptomycese.

[0016] The seed culture may be inoculated in the range of 1 to 15% v/v of the fermentation medium.

[0017] The process may further include preparing the culture.

[0018] The process may further include dehydrating the extracted colorant or the target colorant-rich medium.

[0019] Provided is a fermentation medium for fermenting a microbial seed culture to produce a colorant, the fermentation medium including a spent microbial biomass. The spent microbial biomass is produced in a process including, in a prior fermentation batch, fermenting the seed culture and an initial nutrition-rich fermentation medium to harvest a fermented broth, treating the fermented broth to extract a colorant-rich medium, thereby providing the spent microbial biomass, and sterilizing the spent microbial biomass.

[0020] 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

[0021] 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.

[0022] Figure 1 is a schematic flow diagram of a general microorganism-based production of colorant, according to an embodiment;

[0023] Figure 2 is a process flow diagram showing elements pertaining to the colorant extraction and recovery process of Figure 1, according to an embodiment;

[0024] Figure 3 is a schematic diagram showing various elements pertaining to the colorant production process of Figure 1, according to one embodiment; and

[0025] Figure 4 is a graph comparing the concentration of violacein in the fermented broth over time, in a first fermentation batch vs. a subsequent fermentation batch with spent microbial biomass reuse, according to the current disclosure.

DETAILED DESCRIPTION

[0026] 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 of the features of any one composition or process described below or to features common to multiple or all of the compositions described below.

[0027] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure.

[0028] The following abbreviations are used throughout this disclosure: Abbreviation Meaning

SMB Spent Microbial Biomass

OD Optical Density

DO Dissolved Oxygen

[0029] According to various aspects, the disclosed process pertains to the production, extraction, and recovery of colorants using microorganisms and using methods of disclosed biomass recirculation. Key benefits of the disclosed process include efficiency gains from recirculating the spent microbial biomass and further include providing a comparatively non- pollutive process. SMB may be used as a partial carbon and nitrogen source as the biomass itself is rich in complex carbohydrates and the cell materials are rich in proteins, thus making the colorant production process circular, economic, and environmentally friendly.

[0030] Referring to Figure 1, a general process for producing a colorant from a microorganism is shown generally at 100, according to an embodiment. At 102, the process 100 includes preparing a microbial seed culture (shown as 110 in Figure 3). The microbial seed culture 110 generally includes one or more microbial agents, such as microbes or bacteria, that include a gene pathway responsible for creating 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. Various microbial agents along with their corresponding produced pigment molecules are well 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. 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.

[0031] In other embodiments, the seed culture 110 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 pigment molecules.

[0032] 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 artificially, 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 molecules in high quality and high yield.

[0033] Preparing the microbial seed culture at 102 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.

[0034] At 104, the process 100 further includes transferring the microbial seed culture 110 to a fermenter or a bioreactor (shown as 114 in Figure 3), in a nutrient-rich fermentation medium (shown as 112 in Figure 3). In an embodiment, the medium includes proteins and different macro- and micro-nutrients. 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 104.

[0035] According to a further embodiment, J.lividum B65593 is used as the microbial agent in the seed culture 110 to produce violacein. The microbial strain is a purple-pigmented rod-shaped Gram-negative bacterium of the Proteobacteria phylum and the Oxalobacterteriacecie family. Violacein is a purple-colored pigment. 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-pigment 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 microbial fermentation is an attractive prospect to replace synthetic dyes originating from petroleum products. The carbon source included in the fermentation medium 112 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, com steep liquor, and chicken feather digestate, and inorganic sources such as ammonium chloride, ammonium sulphate, and diammonium hydrogen phosphate. [0036] The seed culture 110 and the fermentation medium 112 may be mixed and diluted, for example by adding water to the mixture. The seed culture 110 and the fermentation medium 112 are placed in the fermenter 114. The internal environment of the fermenter 114 may be configured during the fermentation at 104 to yield optimal and efficient production of colorant 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 104 for optimal production yield.

[0037] The selection of the fermentation medium 112 and the fermentation conditions may largely depend on the microbial strain used in the seed culture 110. For example, some microbial strains are acid-fermenting while others are base-fermenting. 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.

[0038] The harvested materials at the end of the fermentation at 104 is a fermented broth (shown as 116 in Figure 2 and Figure 3) that is a liquid, rich in microbial biomass and produced intracellular or extracellular pigment molecules. The fermentation at 104 may be a batch fermentation or a fed-batch fermentation process or sub-process. In an embodiment, the fermentation at 104 is a submerged fermentation process or sub-process or a solid substrate fermentation process or sub-process.

[0039] At 106, the process 100 further includes processing the liquid fermented broth 116 to extract and purify intracellular or extracellular pigment molecules produced from the fermented broth 116. The colorant extraction and purification at 106 includes filtering and separating pigment molecules from residual biomass and unutilized nutrient medium in the fermented broth 116.

[0040] Non-aqueous colorant extraction methods may be preferred to aqueous methods so that minimal or no water is wasted during the extraction at 106. 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.

[0041] In an embodiment, the colorant extraction and purification at 106 includes a solvent-based extraction method (shown as 120 in Figure 2 and Figure 3) in which a solvent (shown as 126 in Figure 3), particularly an organic solvent, is added to the fermented broth 116 to create a solution mixture for convenient and efficient separation and isolation of pigment molecules from undesired products of the fermented broth 116. The solution mixture may be further processed. The solution mixture may be 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.

[0042] 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 or with the solvent-based extraction. The optimal extraction method may be determined based on intracellular or extracellular pigment production, among other factors.

[0043] In another embodiment, the fermented broth 116, or a combination of the fermented broth 116 and the solvent 126, is passed through a gravity filter, such as a 4 to 6 pm sized filter, trapping particles larger than the pigment molecule size. The filtered liquid may be a mix of solvent, nutrient medium, biomass, and pigment which may be purified through silica gel column chromatography, for example, to obtain a purified pigment paste.

[0044] Referring to Figure 2 now, a solvent-based process flow diagram pertaining to the colorant extraction and purification sub-process at 106 is shown at 120 according to one embodiment. At 162, the sub-process 106 includes adding a solvent 126 to the fermented broth 116 to create a solution mixture. In an embodiment, the solvent 126 includes a solvent selected from an alcohol, an ether, a ketone, and a combination thereof.

[0045] In a further embodiment, the solvent 126 includes an oxygenated solvent with a low boiling point selected from an alcohol (e.g., ethanol and methanol), carboxylate ethers (e.g., ethyl acetate), ketones (e.g., acetone), and a combination thereof.

[0046] In a further embodiment, water is used as the solvent 126 alone or in combination with one or more of the exemplary solvents previously mentioned. However as mentioned hereinabove, non-aqueous extraction methods may be preferred to aqueous methods due to reduced wastewater production.

[0047] In an embodiment, the volume of solvent 126 added to the fermented broth 116 is equal to the volume of the fermented broth 116. [0048] At 164, the sub-process 106 further includes sonicating the solution mixture, for example, in a bath sonicator at room temperature, to maximize cell disruption and create a homogenous solution mixture.

[0049] At 166, the sub-process 106 further includes applying gravity fdtration to the solution mixture. The homogenous solution mixture may be left under continuous agitation, over a period of time such as 18 to 36 hours and at a temperature between 20° to 50° C before being gravity separated. As result of the gravity separation, extracted pigment (shown as 130 in Figure 3), dissolved in the solvent medium, is separated from the insoluble biomass.

[0050] At 168, the sub-process 106 further includes collecting the extracted pigment 130, for example in a pigment solution tank.

[0051] At 170, the sub-process 106 further includes collecting the residual insoluble biomass. The residual insoluble biomass may further include a significant amount of pigment molecules.

[0052] At 172, the sub-process 106 further includes subjecting the residual insoluble biomass to another round of the sub-process from 162 to 170 in case further pigment extraction is desirable, while additional extracted colorant or pigment is collected at 168. Subjecting the residual insoluble biomass to several rounds of solvent extraction may advantageously result in higher pigment yields and greater overall productivity and efficiency of the pigment production process.

[0053] In an embodiment, the residual insoluble biomass is mixed with 50% (v/v) of the solvent used in the previous round of solvent extraction.

[0054] At 172, if further pigment extraction from the residual biomass is not desirable, the residual insoluble biomass is collected at 174 as spent microbial biomass (SMB)(shown as 140 in Figure 3). Generally, SMB 140 is substantially void of pigment molecules while being rich in proteins and carbohydrate sources due to the content of non-pigment cell materials. Thus, SMB 140 may be recycled and recirculated as a partial carbon and nitrogen source as part of the fermentation medium 112 of the next batch of the fermentation process, for example. SMB 140 may be particularly suitable as a nutrition source for the next batch of fermentation process because SMB 140 has a predictable composition, is highly repeatable, and is well- defined since it is essentially produced and collected in highly controlled processes and conditions. Recirculation of SMB 140 results in significant waste reduction from the pigment production process 100 and less use of externally sourced fermentation medium. Recycling SMB 140 as a partial nutrient source of the fermentation medium further supports circular bioeconomy and may reduce the overall cost for microbial production of colorant.

[0055] Referring to Figure 3, shown therein is a schematic diagram showing various elements 300 pertaining to the disclosed microbial colorant production process 100. The SMB 140 collected as a waste biomass from one fermentation batch fermented at 104 and is recirculated and reused as a partial nutrient source in the fermentation medium 112 of a subsequent fermentation batch fermented at 104.

[0056] In an embodiment, the SMB 140 includes toxic compounds with potential inhibitory effects that may negatively impact metabolic behavior of the microbial agents during fermentation at 104. To ensure maximum sterility and avoid contamination in the subsequent fermentation at 104, the SMB 140 along with the other components used in the fermentation medium 112, are treated at 121° C for 30 minutes at 1 atmospheric pressure.

[0057] Referring back to Figure 1, at 108 the process 100 further includes dehydrating the purified pigment (or colorant) solution or medium, for example using lyophilization techniques (for example using a VirTis industrial lyophilizer) to achieve a completely dry pure pigment in powder form.

[0058] In an embodiment, the purified pigment may not be dehydrated and may be used in liquid or paste format.

[0059] The resulting produced colorant of the process 100 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.

[0060] In an embodiment, further processing is conducted on the extracted and recovered pigment. For example, certain additives may be added to the dehydrated pigment powder to increase 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.

[0061] The processes and compositions of this disclosure are further explained by the following examples in more detail. It should be understood that the examples do not restrict the scope of the claimed embodiments.

[0062] Examples

[0063] Example 1- Seed Culture Preparation [0064] A seed culture was prepared by taking 1 ml of J.lividum B65593 strain from a master culture including the strain preserved at 80°C in 20% (v/v) of glycerol. The 1 ml sample was inoculated in 200 ml of preculture medium in an Erlenmeyer flask with baffle and incubated at a shaker for agitation at room temperature for almost a full day. The preculture medium was 3-5 g/L tryptone; 3-5 g/L of yeast extract; 1-3 g/L ammonium sulphate; 1-3 g/L of K2HPO4; and 1-3 g/L of glucose. The flasks were maintained at pH between 6 to 8 and incubated at 20 to 25°C at shaker with 150 to 200 rpm agitation for 16 to 24 hours of incubation.

[0065] Example 2- Fermentation medium and fermenter conditions for first fermentation batch

[0066] 5-10% (v/v) of inoculum volume of the prepared seed culture was used for inoculation in a 5L fermenter.

[0067] The fermentation medium was 3-6 g/L tryptone; 3-6 g/L of yeast extract; 1-3 g/L of ammonium sulphate; 1-3 g/L of K2HPO4; and 10-15 g/L of crude glycerol. The pH of the fermenter was maintained between 6 to 8 and incubated at 20 to 26°C with 30 to 40% dissolved oxygen. The fermentation was of a batch type and ran for 48 hours of fermentation.

[0068] To analyze the trend of biomass and violacein formation during the fermentation process, samples were taken from the fermenter at intervals of every 12 hours of fermentation time (0, 12, 24, 36, 48 hours). For each sample, dried cell weight (g/L), optical density and violacein content were determined. The dried cell weight was measured using gravimetric method, OD was analyzed using a spectrophotometer at 600 nm wavelength, and violacein content was determined using high pressure liquid chromatography (HPLC).

[0069] A similar measurement was conducted for a subsequent fermentation batch with SMB used as a partial substrate for the fermentation process, as detailed under example 4. The comparative results of the first fermentation batch without SMB versus the subsequent batch with SMB are demonstrated in Figure 4.

[0070] Example 3 - Solvent-based extraction of colorant

[0071] A multi-phase solvent extraction method was used to recover violacein from the fermented broth harvested from the fermentation process of Example 2. At the first phase, a combination of ethyl acetate and methanol in the ratio of 4:1 was added to the fermented broth with equal volume to that of the volume of the fermented broth. The resulting solution mixture was sonicated for about 20 minutes at ambient temperature. The sonicated solution medium was then kept for gravity separation for 24 hours at 37°C with continuous agitation. The extracted violacein in solvent was collected in a liquid tank. The residual biomass was also collected for a second phase of solvent-based extraction.

[0072] At the second extraction phase, solvent including only ethyl acetate was used with a 50% volume of the solvent volume that was used for the first extraction phase. The resulting solution mixture was subjected to a similar sonication, rest, and gravity separation as the first extraction phase. Similarly, the extracted violacein pigment in solvent was collected and added to the liquid tank. Also, similarly, the residual biomass was collected for use in a subsequent solvent-based extraction in a third phase.

[0073] At the third extraction phase, the same solvent as in the second phase of extraction, with a 50% volume of the solvent volume used for the second extraction phase, was mixed with the residual biomass. Similar sonication, rest, and gravity separation was repeated on the solution mixture and the separated violacein pigments in the solvent were collected and added to the liquid tank. The SMB, resulting from the residual biomass of the third phase, was also collected and reused in a subsequent fermentation batch.

[0074] All of the extracted violacein pigments, collected in the liquid tank, were further dried in a rotary evaporator and freeze-dried to obtain violacein powder with 85% (v/v) of recovery.

[0075] Example 4- SMB reuse in a subsequent fermentation batch

[0076] The SMB left from the solvent-based extraction, along with other components of a modified fermentation medium (as noted below), were sterilized by being autoclaved at 121°C for 30 minutes at 1 atmospheric pressure. The sterilized SMB was used as a substrate for the subsequent fermentation batch along with a modified fermentation medium comprising: 3-5 g/L tryptone; 3-5 g/L of yeast extract; 1-3 ammonium sulphate; 1-3 g/L of K2HPO4; and 10-13.5 g/L of crude glycerol. The same seed culture as the first fermentation batch with a 5- 10% (v/v) of inoculum volume was used. The pH of the fermenter was maintained between 6 to 8 and incubated at 20 to 25°C with 30-40% DO. The fermentation was of a batch type and ran for 48 hours of fermentation.

[0077] The reuse of the SMB reduced the use of newly sourced carbon (e.g., glycerol) and nitrogen (e.g., tryptone and yeast extract) sources by nearly 10% (w/v) and 16% (w/v) respectively, compared to the carbon and nitrogen sources used for the first batch of fermentation that did not utilize SMB. As shown in Figure 4 in a graph 400, the use of SMB as a partial substrate resulted in almost a similar final violacein content of 252 mg/L.

[0078] 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.