Pigments from Microorganisms: A Sustainable Alternative for Synthetic Food Coloring

Abstract

Microbial pigments are gaining acceptance as a green, sustainable substitute for synthetic food pigments due to growing health issues and their adverse health impacts. This review provides an overview of the potential of microbial pigments as natural food colorants and the advantages of microbial pigments over synthetic pigments. Microbial pigments are a natural source of color with medicinal properties like anticancer, antimicrobial, and antioxidant activity. Important pigments covered are astaxanthin, phycocyanin, prodigiosin, riboflavin, β-carotene, violacein, melanin, and lycopene, and their microbial origins and characteristics. The review also covers commercial production of microbial pigments, i.e., strain development and fermentation processes. Microbial pigments also find extensive applications in food industries, including preservatives for food. Also covered are their pharmacological activity and other applications, such as in the formation of nanoparticles. Finally, the challenges and future directions of microbial pigment production are covered, including the need for cost-effective production, regulatory acceptability, and the potential of genetic engineering and fermentation-based technologies to enhance pigment yield and quality.

1. Introduction

Colors are utilized to enhance the appearance of different commercial products such as food, clothing, and drugs. Food color is both a visual and practical cue, as well as an indicator of freshness and safety [1]. Although color is an important sensory characteristic of food, it is not a texture of food or a nutrient, or a flavor indicator [2]. Most importantly, color can affect how consumers perceive and anticipate these characteristics.

Application of synthetic colors in foods is prevalent due to the fact that they give permanent effects and result in strong color without altering the taste [3]. Some of the most common synthetic food colors include Erythrosine, Sunset Yellow FCF (E110), Allura Red, Tartrazine, Blue No. 1, and Blue No. 2, as shown in Figure 1. These food colors enhance the visual appeal and acceptability of various food products, including candy, sodas, and meat [4,5].

Nonetheless, the use of synthetic food dyes has raised considerable health concerns. As noted in recent research, studies on the effects on individuals consuming excessive amounts of synthetic food additives have shown that excessive intake can result in cardiovascular disease, gastrointestinal complications, respiratory diseases, dermatologic problems, and neurologic adverse reactions [6]. There is significant evidence indicating an association between food additive intolerance and hypersensitivity, hyperactivity in children, physical and mental ailments, allergies, and cancer [7].

Synthetic food colorings, especially azo dyes such as Allura Red and Tartrazine, have been linked to oral problems, diseases of the cardiovascular system, and carcinogenic activity [3]. These dyes can stain dentin and increase bacterial growth in dental crowns, contributing to plaque formation. Additionally, certain bacterial pathogens can elicit inflammatory reactions in the body and enter the bloodstream, thus potentially contributing to plaque build-up along major arteries of the heart and a host of cardiovascular diseases, including ischemic heart disease, strokes, coronary artery disease, and heart attacks [6].

Gupta et al. reported that synthetic food colorants, such as Tartrazine, Amaranth, and Erythrosine, can indeed have toxic effects on human lymphocyte cells in vitro and can interact directly with DNA [8]. Significant cytotoxicity and genotoxicity were observed with an (8 mM) treatment of high-concentration Amaranth; the frequency of sister chromatid exchanges increased by 1.7-fold compared to the control. At 2–8 mM, Erythrosine showed potent cytotoxicity and cytostaticity, while Tartrazine was hazardous at 4–8 mM dosages. Remarkably, using spectroscopy and electrophoresis, these dyes were demonstrated to bind to calf thymus DNA, resulting in the degradation of linear double-stranded DNA. PCR was used for DNA amplification (treated with a colorant) and showed reduced and delayed electrophoretic mobility, demonstrating that dye molecules can directly bind to DNA [9].

Because they are unsafe for health, pigments from microorganisms are a good option and a fine substitute for food colorants [10,11]. Microbial sources are more advantageous than plant sources for natural food colorants because they can be mass-produced, are readily available, provide high quantities, and are easy to handle [12,13]. For example, genetically modified Yarrowia lipolytica strains yield β-carotene at concentrations of up to 400 mg/L within 72–96 h, while the extraction of β-carotene from marigold flowers provides only 15–20 mg/kg of petals and the flowers take weeks to grow [13,14]. Therefore, the production of β-carotene with microbes not only increases the yield by a significant amount but also decreases the cost of production by 25–35% compared to traditional plant extraction techniques using optimized bioreactor systems [15].

There are different sources of pigments from microorganisms such as unicellular fungi, bacteria, photosynthetic bacteria, microalgae, and cyanobacteria [16]. Photosynthetic microorganisms, particularly microalgae and cyanobacteria, are excellent sources for pigment production while simultaneously capturing CO₂, offering a dual benefit of valuable product generation and greenhouse gas mitigation [17,18,19]. Many microbial pigments can serve as colorants, as well as providing medicinal health benefits. Compared to synthetic colors, natural pigments can offer multiple physiological and biochemical properties, including antioxidant, antimicrobial, anticancer, and immunomodulatory properties, and can serve as substitutes in various industries [20]. The pigments produced by various fungi, such as azaphilones, carotenoids, quinones, and melanin, have various pharmacological activities and may help to treat infections, cancers, Alzheimer’s disease, and cardiovascular disorders [21]. Also, their behavior towards the host’s immune system can result in favorable characteristics in the control of fungal infections [22]. Because of their many advantages over synthetic colorants, microbial pigments have attracted considerable interest in the food industry. They offer a safe, eco-friendly, and sustainable alternative to synthetic dyes [13,23].

Microbial pigments have been reviewed in many reviews, but most of them have dealt primarily with their general advantages, biosynthetic processes, and drug properties [13,24,25,26,27,28]. To completely understand the industrial feasibility of microbial pigments, including their benefits over synthetic pigments and regulatory considerations, more research is necessary. This review integrates a comprehensive classification of microbial pigments based on their structure, industrial relevance, and various uses, such as in food industries and nanoparticle synthesis. It also explores commercialization challenges and recent advances in bioprocess optimization. This review offers a new perspective that enhances and extends previous studies.

2. Importance of Microbial Pigments

Microbial pigments are gaining consumer popularity because of their low environmental footprint and potential health benefits. One of the main benefits is that microbial pigments are fully biodegradable; therefore, the disposal of these products does not harm the environment [29]. In addition, few or no chemical processes are involved in the manufacturing of microbial pigments, thus minimizing adverse effects and environmental damage [14]. Moreover, in addition to their environmental benefits, microbial pigments provide a colorful range of muted, relaxing colors, making them popular among consumers looking for a more natural and harmonious aesthetic [30].

Synthetic colors are resistant to typical reduction and oxidation processes, making them highly challenging to remove from industrial effluents [31]. Their destruction produces byproducts that have been linked to health risks, while natural color degradation byproducts have not been found to include such toxic substances [32]. Synthetic color-based effluents can pose severe harm to water streams and ecosystems because of their synthetic origin and complicated molecular structures. For example, degradation of azo dyes, accounting for over half of all dyes manufactured annually, can result in the discharge of carcinogenic aromatic amines [33]. The aromatic amines, primarily azo dye degradation products, are recognized as primary carcinogenic compounds and pose serious risks to human health and the environment [34]. Microbial pigments are safer and more environmentally friendly because they originate from renewable sources, have a simple structure, and are biodegradable [29]. Unlike synthetic dyes, which may release harmful degradation byproducts, microbial pigments are increasingly in demand in a broad array of manufacturing industries because of their eco-friendliness and bioactivity [16,35]. They offer several advantages over synthetic colorants, including their low or non-toxicity in ecotoxicological and cytotoxicity tests [23]. This gives them the capacity to be a safer alternative when applied in food, pharmaceuticals, textiles, and other industries [20,36].

Even though microbial pigments are well known for being biodegradable and environmentally friendly, their full environmental evaluation requires strong life cycle assessment (LCA). LCA research indicates that microbial pigments, as much as they are currently constrained by high water and energy requirements during fermentation and extraction, have substantially smaller environmental impacts in terms of greenhouse gas emissions, toxicity, and fossil resource depletion compared to their synthetic equivalents [11,20]. The utilization of renewable substrates and the optimization of bioprocesses can further enhance the circular economy-based sustainability of microbial pigment production [37]. These findings have been confirmed by recent studies through comparative LCA of microbial and synthetic pigments [38,39].

Many hazardous and allergenic synthetic colors are currently prohibited. Upon reviewing the advantages and disadvantages of microbial food colorants in comparison with synthetic food colorants, it is clear that microbial alternatives provide environmentally sustainable and potentially beneficial health options (

Table 1. Advantages and disadvantages of microbial and synthetic food colorants.

	Synthetic Food Colorants	Microbial Food Colorants
Advantages	High stability against oxygen, light, and heat (>100 °C) High pH stability (stable across pH 2–9) High tinctorial strength Availability of all shades of color Synthetic food colorants are less expensive than natural food colorants Widely approved by FDA/EFSA, e.g., Tartrazine E102, Allura Red E129	Biodegradable and environmentally friendly Generally regarded as safe and non-toxic Exhibit bioactivities such as anticancer, antimicrobial, and antioxidant activities, among others Can be produced using agro-industrial waste as a substrate (support the circular economy)
Disadvantages	Often linked to adverse health effects (carcinogenicity, hyperactivity in children, allergies) Environmental persistence and non-biodegradability	Relatively low stability towards oxygen, light, and heat (degrade above 60–80 °C) Low pH stability (stable between pH 4 and 8) Relatively low color saturation Not all shades of color are available Higher production costs and regulatory constraints Regulatory approval and large-scale standardization remain challenges Limited approvals by FDA/EFSA (e.g., β-carotene, Monascus pigment)

) [40].

However, there have been numerous hindrances to their large-scale application in industries. The production cost is high and the yield is low compared to synthetic pigments. Large-scale industrial production of pigments based on microorganisms to meet industrial requirements remains a challenge, necessitating developments in biotechnology, genetic engineering, and new fermentation techniques to enhance viability and cost-effectiveness [41]. Food laws are also a hindrance to the use of new sources of natural pigments, as they require costly toxicology tests and production processes [42].

3. Classification and Some Important Food-Grade Pigments

Pigments are classified into synthetic and natural colorants. Natural pigments are classified based on their source, chemical structure, solubility, and color systems. Natural pigments are widely classified into three groups based on their sources: plant pigments, animal pigments, and microbial pigments [43]. In the current research, we specifically emphasize the pigments of microorganisms. Among the major microbial pigments used as food colorants, astaxanthin, riboflavin, phycocyanin, lycopene, prodigiosin, β-carotene, violacein, and melanin are some of the major ones (Figure 2). Table 2 provides a comprehensive list of pigments, including their sources and characteristics.

3.1. Lycopene

Lycopene is an all-trans molecule with 2 non-conjugated and 11 conjugated double bonds for its red color and bio-protective activity induced by plants and microorganisms [44]. Its biosynthesis in microorganisms occurs primarily through the MEP or MVA pathway, leading to the formation of GGPP, a primary precursor to carotenoids (Figure 3) [45,46]. Blakeslea trispora, a filamentous fungus, is a commercial producer of lycopene [47]. Lycopene production in microbes can be greatly enhanced by optimizing culture conditions as well as through metabolic engineering strategies [48]. Lycopene production in B. trispora can be enhanced by the addition of inhibitors like pyridine or imidazole that inhibit lycopene cyclase activity [49]. Escherichia coli was genetically engineered to produce lycopene effectively by overexpression of the carotenoid biosynthetic gene cluster. Strain LYC010, for instance, produced 3.52 g/L (50.6 mg/g DCW) of lycopene in fed-batch fermentation [50]. Microbial lycopene has gained attention for food, pharmaceutical, and cosmetic applications because of its natural origin, stability, and health-protective activity. It exhibits antimicrobial, antioxidant, anti-inflammatory, and anticancer activity. It was shown to have preventive activity against a wide array of diseases, including diabetes and cardiovascular diseases [51].

3.2. β-Carotene

This reddish-orange pigment is derived primarily from Flavibacterium multivorum, Dunaliella salina, Phycomyces, Phaffia rhodozyma, Rhodosporidium, Rhodotorula mucilagenosa, Sporobolomyces, Blakeslea trispora, and Sporidiobolus [52]. Microbial biosynthesis of β-carotene occurs through the isoprenoid pathway via the methylerythritol phosphate (MEP) or the mevalonate (MVA) pathway to give the essential precursor geranylgeranyl pyrophosphate (GGPP) [53]. They both merge to give isopentenyl diphosphate (IPP) and the latter’s isomer, dimethylallyl diphosphate (DMAPP), the universal precursors to all the isoprenoids, including β-carotene (Figure 3) [46]. β-carotene, a major provitamin A carotenoid, has received tremendous attention due to its antioxidant activity, coloring property, and widespread applications in food, pharmaceutical, and nutraceutical industries [54]. It is blessed with many health benefits such as immunity boosting, preventing cardiovascular disease and cancer, and causing anti-aging effects [55].

3.3. Canthaxanthin

Microbial biosynthesis of canthaxanthin includes the transformation of β-carotene to canthaxanthin through ketolation reactions catalyzed by β-carotene ketolase enzymes. The enzymes at the 4 and 4′ positions of the β-ionone rings of β-carotene introduce keto groups to give the red-orange color of canthaxanthin (Figure 3) [56,57]. Various microorganisms, including Saccharomyces cerevisiae, Mucor circinelloides, and Escherichia coli, can naturally produce canthaxanthin [56,57,58]. The chemistry of canthaxanthin is such that its antioxidant activity is due to its singlet oxygen quenching ability and free radical scavenging activity [59]. Its antioxidant activity is due to its conjugated double-bond chemistry, which allows it to effectively inactivate reactive oxygen species [60]. Amazingly, canthaxanthin has been shown to protect LDL cholesterol from oxidation and induce antioxidant enzymes like catalase and superoxide dismutase [59].

3.4. Astaxanthin

Astaxanthin is a lipophilic diketo carotenoid pigment [61]. It is a red-orange xanthophyll carotenoid pigment, primarily synthesized by microalgae and red unicellular fungi. Microorganisms commercially used to manufacture astaxanthin include Haematococcus pluvialis, a green microalga, and the heterobasidiomycetous unicellular fungus Xanthophyllomyces dendrorhous (Phaffia rhodozyma teleomorph) [62,63]. It is a highly effective antioxidant carotenoid and has attracted significant attention due to its health potential. Its lipophilic character and poor water solubility limit its bioavailability and application in industries [64,65]. Its bioavailability has been improved by employing various strategies in recent research, i.e., glycosylation. Glycosylation was found to enhance the water solubility, bioavailability, photostability, and biological activities of astaxanthin to a significant level [66]. Heterologous glycosylated astaxanthin production was first reported in Yarrowia lipolytica with a yield of 1.47 mg/L. The process can be improved to increase water solubility, photostability, and biological activity [66].

3.5. Phycocyanin

Phycocyanin is a natural blue pigment mainly isolated from microalgae, particularly cyanobacteria such as Spirulina platensis and Aphanizomenon flosaquae [67]. Phycocyanin is an important accessory light-harvesting pigment extending the light absorption spectrum of photosynthesis. It consists of α and β protein subunits that are covalently linked to phycocyanobilin chromophores, which are responsible for its blue color and fluorescence [68]. It has attracted a lot of attention in the food sector because of its positive impact on health, including anti-inflammatory, antitumor, and antioxidant properties [69]. It is used in dietary supplements, foods, and drinks that are high in protein.

3.6. Prodigiosin

Prodigiosin is a red tripyrrole pigment that is biosynthesized mainly by Serratia marcescens and other bacterial species like Vibrio psychoeerytrus, Streptomyces sp., Pseudomonas sp., Rugamonas rubra, Pseudoalteromonas rubra, Actinomycetes sp., and Streptoverticillium rubrireticuli [70,71,72]. Microbial biosynthesis of prodigiosin is a bipartite process involving independent biosynthesis of two major precursors, 2-methyl-3-n-amyl-pyrrole (MAP) and 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC), which are enzymatically coupled to form the final pigment [73]. It is characterized by its unique pyrrolylpyrromethane skeleton and has a molecular weight of 323–324 Da [74]. It exhibits antineoplastic, antibacterial, antibiotic, antimalarial, anti-inflammatory, anticancer, and antitumor properties [75,76,77].

3.7. Riboflavin

The yellow water-soluble bacterial pigment or vitamin B2 is produced by the ascomycete Ashbya gossypii; Candida famata, a filamentous fungus; and the bacterium Bacillus subtilis [78]. It plays a crucial role as a precursor for the essential cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both of which are essential in numerous redox reactions in all of the kingdoms of life [79]. The isoalloxazine ring structure of flavins is responsible for their characteristic yellow fluorescence and redox properties [80]. They are used in many products, including fruit drinks, energy drinks, baby foods, dairy products, cereals, sauces, and vitamin-fortified milk [81].

3.8. Violacein

Violacein is a purple pigment that is biosynthesized by a number of bacteria through the condensation of two tryptophan units [82,83]. The pigment, which was initially isolated from Chromobacterium violaceum, has been a focus of extensive research interest owing to its variability in biological activities and potential applications in the future [84,85]. This indole-derived pigment possesses a typical UV-visible spectrum with maximum light absorbance at 575 nm, which remains unchanged in a range of bacterial sources [86]. Different bacteria of different genera, including Pseudoalteromonas, Iodobacter, Duganella, Janthinobacterium, Massilia, and Collimonas, are able to produce violacein [87].

3.9. Melanin

Melanins are heterogeneous pigments found in all types of life which are synthesized via different biochemical pathways. Two of the most common pathways for melanin synthesis are the DOPA pathway, which is initiated through conversion of L-tyrosine, and the DHN pathway, which is initiated through malonyl-coenzyme A (Figure 4) [88,89]. Melanin is a common and heterogeneous pigment found in all biological kingdoms, i.e., animals, plants, and microbes [90,91]. Melanin is a high-molecular weight pigment and usually dark brown to black in color, but reddish-yellow in color under some conditions [92]. Melanin is classified into five classes on the basis of the chemical structure of the precursor: neuromelanin (not typically produced by microorganisms), eumelanin, allomelanin, pheomelanin, and pyomelanin [93]. Melanin is a multifunctional molecule in a diverse range of microbial species, like Burkholderia cenocepacia, Rubrivivax benzoatilyticus JA2, Streptomyces cyaneus, Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum, and Paracoccidioides brasiliensis [94,95,96,97,98]. Melanin possesses certain physicochemical characteristics, is biocompatible and biostable, and is a candidate compound for various applications [99]. Melanin and melanin nanoparticles (MNPs) have been reported to have desirable applications in cancer theranostics due to their unique properties. MNPs have high photothermal conversion efficiency as photothermal agents under near-infrared (NIR) light, which can destroy tumors under photothermal therapy (PTT) efficiently [100]. The photothermal ability of MNPs can also trigger immunogenic cell death and tumor-associated antigen release, which may activate antitumor immune responses [101].

Table 2. Microbial pigments, sources, characteristics, and applications.

Pigment	Source Organism(s)	Major Industrial Producer (Biotechnological Source)	Mol. Formula and Solubility	Application	Stability Profile (pH/Temp. Light)	Refs.
Astaxanthin (orange-pink-red)	Serratia marcescens, Halobacterium salinarium, Agrobacterium aurantiacum, Paracoccus carotinifaciens, Pseudoalteromonas rubra	Haematococcus pluvialis [GRAS GRN 294/580], Xanthophyllomyces dendrorhous [No GRAS], and engineered E. coli (introducing genes from H. pluvialis, like β-carotenoid hydroxylase and ketolase) [no GRAS]	C40H52O4 596.8 g/mol lipid soluble	Animal and fish food, food colorants, anti-aging, and memory improvement	pH 5–8, stable up to 70 °C, moderately light-sensitive	[102,103,104,105,106,107,108,109,110]
Phycocyanin (blue)	Pseudomonas aeruginosa, Aphanizomenon flos-aquae, Spirulina sp.	Spirulina platensis [GRAS GRN 424]	C33H38N4O6 water-soluble	Ice creams and sweets	pH 6.5–7.5, unstable > 45 °C, very light-sensitive	[111,112,113,114,115,116,117]
Prodigiosin (red)	Pseudoalteromonas rubra, Serratia marcescens	Recombinant E. coli (Sau3A fragments of S. marcescens DNA were introduced into E. coli K-12), Pseudomonas putida KT2440	C20H25N3O 323.4 g/mol water-insoluble	Colorants in yogurt, milk, and carbonated beverages	pH 4–6, degrades > 60 °C, light-sensitive	[70,118,119,120,121,122]
Riboflavin (yellow)	Debaryomyces subglobosus, Ashbya gossypii, Clostridium acetobutylicum	Ashbya gossypii, Bacillus subtilis [GRAS under 21 CFR 184.1695]	C17H20N4O6 376.4 g/mol water-soluble	Food industries and dietary supplements	pH 5–7, stable up to 100 °C, extremely light-sensitive (photo-labile)	[78,123,124,125,126,127]
β-carotene (yellow)	Rhodotorula gracilis, Rhodotorula rubra, Blakeslea trispora	Yarrowia lipolytica [GRAS GRN632 and BSL1 status]	C40H56 536.9 g/mol water-insoluble	Vitamin A sources, food industries, and boosts immunity	pH 4–8, degrades > 60 °C, highly light-sensitive	[128,129,130,131,132]
Violacein (purple)	Chromobacterium violaceum, Janthinobacterium lividum, Pseudoalteromonas spp., Pseudoalteromonas tunicata	E. coli strain (B8/pTRPH1-pVio-VioE), Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica	C20H13N3O3 343.3 g/mol	Cosmetics, textiles, medicine, and food industries	pH 5–7, stable < 60 °C, light-sensitive	[84,124,133,134]
Melanin (black)	Burkholderia cenocepacia, Rubrivivax benzoatilyticus JA2	Gliocephalotrichum simplex, MEL1 mutant of Aspergillus nidulans, Streptomyces kathirae strain SC-1	C18H10N2O4 318.3 g/mol	Cosmetic creams, food industries, and anti-HIV activity	pH 2–10, highly heat-stable, light-stable	[135,136,137,138,139]
Lycopene (red)	Fusarium sp., Blakeslea trispora	Blakeslea trispora [no GRAS but approved in EU (2006/721/EC)]	C40H56 536.9 g/mol	Meat colorants	pH 4–7, degrades > 60 °C, highly light-sensitive	[140,141,142]
Canthaxanthin (orange-pink)	Halobacterium sp., Bradyrhizobium sp., Fusarium Sporotrichioides	Haematococcus pluvialis [GRAS GRN 294], genetically engineered Mucor circinelloides, and Saccharomyces cerevisiae [no GRAS]	C40H52O2 564.8 g/mol	Food colorant, salmon food and poultry feed	pH 4–9, stable up to 80 °C, relatively light-stable	[143,144,145]

GRAS: U.S. FDA Generally Recognized as Safe. GRN: GRAS Notification Number. “No GRAS” = no official FDA recognition, but the organism or product may be accepted elsewhere (e.g., EU Novel Food Catalogue).] [Note: Some listed pigment-producing microorganisms, e.g., Serratia marcescens, Burkholderia cenocepacia, Pseudomonas aeruginosa, Citrobacter freundii, and Chromobacterium violaceum, are Risk Group 2 (RG2) organisms and opportunistic pathogens [146,147]. Their use in food, pharmaceutical, or cosmetic industries should be preceded by strict biosafety screening and regulatory approval. Biosafety Level 1 and GRAS-listed strains should be used in large-scale applications.

Table 2. Microbial pigments, sources, characteristics, and applications.

Pigment	Source Organism(s)	Major Industrial Producer (Biotechnological Source)	Mol. Formula and Solubility	Application	Stability Profile (pH/Temp. Light)	Refs.
Astaxanthin (orange-pink-red)	Serratia marcescens, Halobacterium salinarium, Agrobacterium aurantiacum, Paracoccus carotinifaciens, Pseudoalteromonas rubra	Haematococcus pluvialis [GRAS GRN 294/580], Xanthophyllomyces dendrorhous [No GRAS], and engineered E. coli (introducing genes from H. pluvialis, like β-carotenoid hydroxylase and ketolase) [no GRAS]	C40H52O4 596.8 g/mol lipid soluble	Animal and fish food, food colorants, anti-aging, and memory improvement	pH 5–8, stable up to 70 °C, moderately light-sensitive	[102,103,104,105,106,107,108,109,110]
Phycocyanin (blue)	Pseudomonas aeruginosa, Aphanizomenon flos-aquae, Spirulina sp.	Spirulina platensis [GRAS GRN 424]	C33H38N4O6 water-soluble	Ice creams and sweets	pH 6.5–7.5, unstable > 45 °C, very light-sensitive	[111,112,113,114,115,116,117]
Prodigiosin (red)	Pseudoalteromonas rubra, Serratia marcescens	Recombinant E. coli (Sau3A fragments of S. marcescens DNA were introduced into E. coli K-12), Pseudomonas putida KT2440	C20H25N3O 323.4 g/mol water-insoluble	Colorants in yogurt, milk, and carbonated beverages	pH 4–6, degrades > 60 °C, light-sensitive	[70,118,119,120,121,122]
Riboflavin (yellow)	Debaryomyces subglobosus, Ashbya gossypii, Clostridium acetobutylicum	Ashbya gossypii, Bacillus subtilis [GRAS under 21 CFR 184.1695]	C17H20N4O6 376.4 g/mol water-soluble	Food industries and dietary supplements	pH 5–7, stable up to 100 °C, extremely light-sensitive (photo-labile)	[78,123,124,125,126,127]
β-carotene (yellow)	Rhodotorula gracilis, Rhodotorula rubra, Blakeslea trispora	Yarrowia lipolytica [GRAS GRN632 and BSL1 status]	C40H56 536.9 g/mol water-insoluble	Vitamin A sources, food industries, and boosts immunity	pH 4–8, degrades > 60 °C, highly light-sensitive	[128,129,130,131,132]
Violacein (purple)	Chromobacterium violaceum, Janthinobacterium lividum, Pseudoalteromonas spp., Pseudoalteromonas tunicata	E. coli strain (B8/pTRPH1-pVio-VioE), Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica	C20H13N3O3 343.3 g/mol	Cosmetics, textiles, medicine, and food industries	pH 5–7, stable < 60 °C, light-sensitive	[84,124,133,134]
Melanin (black)	Burkholderia cenocepacia, Rubrivivax benzoatilyticus JA2	Gliocephalotrichum simplex, MEL1 mutant of Aspergillus nidulans, Streptomyces kathirae strain SC-1	C18H10N2O4 318.3 g/mol	Cosmetic creams, food industries, and anti-HIV activity	pH 2–10, highly heat-stable, light-stable	[135,136,137,138,139]
Lycopene (red)	Fusarium sp., Blakeslea trispora	Blakeslea trispora [no GRAS but approved in EU (2006/721/EC)]	C40H56 536.9 g/mol	Meat colorants	pH 4–7, degrades > 60 °C, highly light-sensitive	[140,141,142]
Canthaxanthin (orange-pink)	Halobacterium sp., Bradyrhizobium sp., Fusarium Sporotrichioides	Haematococcus pluvialis [GRAS GRN 294], genetically engineered Mucor circinelloides, and Saccharomyces cerevisiae [no GRAS]	C40H52O2 564.8 g/mol	Food colorant, salmon food and poultry feed	pH 4–9, stable up to 80 °C, relatively light-stable	[143,144,145]

GRAS: U.S. FDA Generally Recognized as Safe. GRN: GRAS Notification Number. “No GRAS” = no official FDA recognition, but the organism or product may be accepted elsewhere (e.g., EU Novel Food Catalogue).] [Note: Some listed pigment-producing microorganisms, e.g., Serratia marcescens, Burkholderia cenocepacia, Pseudomonas aeruginosa, Citrobacter freundii, and Chromobacterium violaceum, are Risk Group 2 (RG2) organisms and opportunistic pathogens [146,147]. Their use in food, pharmaceutical, or cosmetic industries should be preceded by strict biosafety screening and regulatory approval. Biosafety Level 1 and GRAS-listed strains should be used in large-scale applications.

4. Economic Evaluation of Microbial Pigments

Microbial pigment production measurement through bioprocesses is intricate due to low-technology goods, insufficient statistical information at a regional level, and the scattered nature of production among many small enterprises globally. There is greater interest in the use of microbial pigments, however. Natural pigments are sometimes more costly than synthetic pigments [148]. Typically, synthetic colors have a lower price range of USD 700–800 for each 100 g. In the food sectors, the benefits of microbial pigments are longer-term considerations, other than just direct cost considerations [149]. The production cost of bacterial β-carotene is around USD 1000 per kilogram and that of chemically synthesized β-carotene is around USD 500 per kilogram [150]. The current market prices place microbial (natural) β-carotene between USD 350 and USD 7500 per kilogram and synthetic β-carotene between USD 200 and USD 2000 per kilogram depending upon purity, formulation, and production scale [151]. The β-carotene market worldwide was worth approximately USD 1.03 billion in 2023, and the natural β-carotene market alone was estimated to be worth around USD 1.28 billion in 2024 [152]. The synthetic β-carotene market was worth around USD 256 million in 2024 [153]. Advanced fermentation techniques and genetically modified strains can be used to reduce the production cost of substrates. Some microbes can utilize agro-industrial waste as a substrate, considerably reducing costs for raw materials as well as promoting sustainability by reducing waste.

Cortes et al. (2017) forecasted that the market for natural food pigments would expand at a compound annual growth rate (CAGR) of 6.22% during 2015–2019 [154]. Gürses et al. (2016) projected that the overall size and amount of consumption of pigments and dyes in the global market would be 9.9 million tons and amount to USD 26.53 billion in 2017, boosted by expansion in key end-use markets [43]. The β-carotene market was expected to grow from USD 247 million by the end of 2007 to USD 285 million by the end of 2015, with a CAGR of 1.8%. The moderate growth rate indicates a steady demand for β-carotene across industries [154]. The astaxanthin market’s projected growth rate of 16.2% CAGR and the anticipated value of USD 3.4 billion by 2027 align with the rapid expansion observed in several related industries [155]. The industrial production of astaxanthin is carried out utilizing Paracoccus sp. It was anticipated that, by 2021, the volume of astaxanthin sales would amount to USD 1.1 billion. With a CAGR of 16.2%, the astaxanthin market is predicted to attain a value of USD 3.4 billion by 2027 [156]. The color market is not only growing but evolving. The increasing demand for natural colorants, particularly those derived from microorganisms, is reshaping the industry.

5. Industrial Considerations in the Use of Bacterial and Fungal Pigment Producers

Bacteria and fungi are widely exploited industrially for pigment production primarily due to their faster growth rates, resilience to different substrates, and high yields of micronutrients and pigments [16]. Interestingly, while the two have industrial advantages in terms of providing numerous sources of pigments, they also present certain challenges that require special considerations.

Fungi offer bioavailability, cost, and scalability advantages in terms of cell culture and downstream processing. They can produce a variety of pigments, such as carotenoids, melanin, polyketides, and azaphilones [157]. Pigment biosynthesis in microorganisms can be increased under various stress conditions. Monascus ruber CGMCC 10910 led to a significant increase in yellow pigment production after being subjected to high-glucose stress conditions. The intracellular and extracellular pigment yield increased by 26.31% and 94.86%, respectively [158]. Also, high salt stress resulted in a 40% increment in pigment yield [159]. Rhodotorula mucilaginosa AJB01 showed an increase in carotenoid production under osmotic stress and UV light exposure [160].

Additional pigment and secondary metabolite yields could be obtained using genera such as Talaromyces, Monascus, and Fusarium. They are eco-sustainable and can be grown on low-budget agro-industrial wastes (solid-state fermentation), and thus represent a low-cost form of production. Certain fungi strains present biosafety concerns, e.g., the co-production of mycotoxins by Monascus purpureus citrinin [161]. This can be addressed by screening of the strains, mutagenesis, or genome editing. Filamentous growth also imposes mixing and oxygen transfer issues in bioreactors, which necessitate special reactor designs or agitation regimes [162].

Bacterial pigments are generally used because of their easy growth, rapid production, and simplicity of pigment extraction. In particular, Serratia marcescens and Streptomyces spp. are more or less flexible than fungi that grow rapidly and have low nutrient requirements, which makes them ideal for continuous fermentation systems [36]. These processes facilitate the production of pigments due to their simplified metabolic pathways, relatively short life cycles, and tractability to genetic manipulation. However, they are relatively vulnerable to contamination and may have stringent aseptic requirements, particularly in open systems. High pigment yields, in addition to considerations such as high biosafety (GRAS status) requirements, strain genetic stability, growth patterns, and the aerobic fermentation systems exploited, are knowledge perspectives that are relatively helpful for industrial management based on the criteria of strain selection [16,41].

Contamination management must be of concern at scale and across industries. Solid-state fermentation (SSF) systems, which use fungi, are less likely to be contaminated since they work under low-water activity conditions, in contrast to submerged fermentation (SmF) systems, primarily for bacteria, which need rigorous sterilization and closed systems. Automated monitoring and online analytics minimize variability and batch inconsistency [163].

6. Advances in Large-Scale Production and Genetic Engineering of Microbial Pigments

6.1. Industrial Production of Microbial Pigments

Before producing large quantities of pigments, it is critical to characterize the pigment producer, pigment, and pigment properties. Screening methods have also been developed for this purpose. One of the fastest and simplest methods for detecting pigments is the use of a portable handheld Raman spectrometer [27]. Moreover, it is important to have prior knowledge of the metabolic pathways of pigment producers to identify and eliminate toxic and pathogenic pigment producers. For example, this information has been useful in the manufacture of food products such as QuornTM, as it can also identify cytotoxic compounds (e.g., 4,15-diacetoxyscirpenol) produced by Fusarium venenatum [164].

Monascus purpureus produces red pigments for food coloring, but also produces citrinin, a nephrotoxic mycotoxin that can harm humans and animals. Furthermore, researchers have used several genetic engineering techniques in an attempt to reduce or eliminate citrinin production in order not to impact pigment generation (essentially increasing the amount of pigment) [165]. The CRISPR/Cas9 system was developed to delete large genomic regions such as biosynthetic gene clusters of polykaryotic fungi such as M. purpureus. The researchers successfully deleted the citrinin biosynthetic gene cluster (15 kb) in a commercial strain and characterized stable homokaryotic mutants that no longer produce citrinin. Moreover, their genetic engineering increased the production of pigment by 2–5% [166].

Robust analytical techniques are critical in the industrial production of microbial pigments to achieve product quality, safety, and regulatory acceptance. High-Performance Liquid Chromatography (HPLC) in combination with Mass Spectrometry (MS), and techniques like UV-Visible Spectroscopy, LC-MS, FTIR, and NMR enable precise structural identification and quality control of microbial pigments. Utilizing such analytical methods, industries can maintain routine pigment quality, provide batch-to-batch analysis, and maintain high levels of purity for mass application and regulatory approvals. Such comprehensive validation is essential for full utilization of microbial pigments in food, pharmaceutical, and cosmetic industries [167].

The pigment manufacturing industry must use a large-scale production method that aligns with emerging fermentation techniques. For instance, SSF and SmF use fermentation principles to produce pigments, but they differ depending on the situation or the preset variables. Each of these two techniques uses different variables to determine which substrates are available, the microorganism type, and desired pigment attributes. SSF offers advantages, such as lower energy requirements and reduced wastewater production, making it an environmentally friendly option [168].

SmF can use more energy with energy systems; however, it is often preferred for many large-scale industrial applications owing to the control over the processes and technologies available for later scale-up and automation of larger production [169]. Additionally, SmF allows for the growth of microorganisms in nutrient-rich liquid media at an optimal pH, and aeration facilitates higher yields. SmF can easily be scaled; however, it incurs higher capital and operational costs. Alternatively, SSF can use solid substrates based on agro-industrial byproducts, making them eco-friendly and more cost-effective by leveraging low-cost materials [170].

The Response Surface Methodology (RSM) has been found to be an effective tool for optimizing the conditions of media to maximize pigment yield in a variety of fermentation processes [171]. Several studies have demonstrated that RSM can be applied to determine optimal operating conditions to enhance production. For instance, in the production of red and orange pigments, RSM was applied to optimize the conditions for submerged fermentation of Talaromyces albobiverticillius 30548. Optimum pigment and biomass yields were established at a pH of 6.4, with an agitation rate of 164 rpm, a temperature of 24 °C, and a fermentation period of 149 h [172].

Wild-type strains generally require more time to produce pigments and often do not yield sufficient quantities to make the process commercially viable, highlighting the importance of strain development [173]. To improve a strain, it is treated with common mutagens such as ethyl methyl sulfonate (EMS), 1-methyl-3-nitro-1-nitrosoguanidine (NTG), and ultraviolet (UV) to cause an increase in pigment production. After being treated with EMS, UV, NTG, and microwave radiation, there was an enhancement in pigment production by Serratia marcescens and Haematococcus pluvialis [174].

To achieve industrial feasibility, a bioreactor needs to be designed with respect to the microbial strain, type of pigment produced, and yield. Bubble column reactors, stirred-tank bioreactors (STRs), and airlift bioreactors are usually used based on the type of pigment and microbial strain. STRs are the most popular, as oxygen transfer is efficient, while airlift bioreactors are appropriate for shear-sensitive microorganisms that require enhanced oxygen transfer. Bubble column reactors have high mass transfer rates and are frequently used in aerobic microbial fermentation [175]. Bioreactors used at an industrial level can range in volume from 10 L to 100,000 L, and working parameters, such as pH, agitation, temperature, feed of nutrients, and dissolved oxygen, must be monitored continuously [176].

Pigment synthesis is significantly impacted by nutritional enrichment or shortage. Pigment synthesis may be impeded by organic acids generated during Monascus ruber culture under oxygenated conditions [177]. Introducing rice and wheat feed (either mixed grains or broken leftovers) has demonstrated the enrichment of carotenoid production in fungi and unicellular fungi [178]. It was demonstrated that Acinetobacter wofii produced more pink pigment when given methanol as its only carbon source [179]. Carotenoids are produced by Mycobacterium tuberculosis in response to acidic stress (pH 5.0–6.0) and prolonged anaerobic growth [180]. When succinate was supplied as the only carbon source, Pseudomonas fluorescens produced a significant amount of water-soluble, yellow-green, fluorescent pigment; however, when citric or malic acids were employed as substrates, no pigment was produced [112]. Several substrates have been found to be strong stimulators for different pigment molecules, including phenylalanine, tryptophan, and, most importantly, tyrosine. However, further research is needed to determine whether other substrates are also effective in improving pigmentation [181].

Pigment extraction is essential for producing high-quality products after fermentation. Traditionally, extraction is carried out with a solvent and has historically been laborious and complicated. Except for steam distillation, other methodologies use ethanol to extract pigments, and when traditional solvents are used, this process is not economical. However, in modern extraction techniques, non-toxic solvents, including supercritical CO₂ and ethyl acetate, have been increasingly utilized in alignment with green chemistry principles [14,182]. Some techniques include membrane filtration, a non-solvent process; ultrasound-assisted extraction, which enhances the extraction of the pigment and reduces the amount of solvent; and supercritical fluid extraction techniques, which utilize CO₂ as a solvent in the extraction process [183].

Safety and stability are also paramount in the scaling of the production of commercial applications in food, cosmetics, and pharmaceuticals [184]. Stabilizing processes for pigments include encapsulating treatments, for example, biopolymer coatings and nano-emulsions, which are utilized to improve the stability of pigments throughout transportation, handling, and storage [185]. On the other hand, regulatory approval is also required for industrial-scale production. Organizations such as the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA) require rigorous quality control procedures, including toxicity tests, stability tests, and compliance with Generally Recognized as Safe (GRAS) regulations [186]. These tests ensure that microbial pigments are safe for consumption.

6.2. Genetically Engineered Microorganisms for Pigment Production

Recent advances in genetic engineering have enabled the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology to enhance pigment production in microbes. The approach facilitates easy microorganism engineering to produce natural colors by expressing color-producing genes in their genomes using the CRISPR-Cas9 system [187]. For instance, in Escherichia coli recombinant cultures, the MEP pathway has been enhanced through co-expression of enzymes such as isopentenyl pyrophosphate isomerase (FNI) and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and has resulted in significantly elevated β-carotene production [46]. At the same time, overexpression of dxs encoding the very first committed step in the MEP pathway enabled production of lycopene in Corynebacterium glutamicum [188].

Microorganism-based synthesis of anthocyanins has proven to be an effective alternative to conventional plant extraction techniques. This method has a number of advantages, including reduced production time, purer yield, reduced amount of waste, and greater batch-to-batch consistency in the yields. Different microbial systems have been engineered to produce anthocyanins, both prokaryotic and eukaryotic organisms [189]. For instance, Escherichia coli has been genetically engineered to produce cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside efficiently [190]. Anthocyanins have also been produced with other microorganisms such as Saccharomyces cerevisiae and Corynebacterium glutamicum [191]. Although issues are still present, including enhancing enzyme efficiency and stabilizing the products, the process of using microorganisms to make anthocyanins is evolving, showing potential for large-scale production of these pigments of interest [192]. Some genetically engineered microorganisms and processes for pigment manufacture are listed in Table 3.

One notable success was the engineering of Yarrowia lipolytica using CRISPR-Cas9 to enhance β-carotene production by optimizing the MVA pathway. In one study, researchers achieved a remarkable 24-fold (408 mg/L) increase in β-carotene production. This was accomplished by overexpressing several genes related to the MVA pathway, including GGS1, ERG13, and HMG, as well as multiple copies of carB and carp [193]. Future experiments utilizing CRISPR-Cas9 and genetic engineering should focus on achieving cost-effective and high-yield pigment production at a commercial scale.

Though CRISPR-Cas9 technology is applied in genetically modified microorganisms (GEMs) within the food sector, it is encountering severe challenges regarding regulation, biosafety, and acceptability for consumers. Most countries have regulatory systems that categorize CRISPR-edited strains applied for food purposes in the form of genetically modified organism (GMO) regulations. This categorization calls for elaborate risk assessments, traceability, and labeling procedures, which may prove to be cumbersome and contentious [194]. Moreover, consumer attitudes have been conservative due to poor awareness and ethical objections to gene editing technologies.

Successful commercialization of genetically modified microbes (GEMs) in the pigment industry relies on the convergence of socio-technical factors that take into account biosafety, regulatory matters, and public engagement. These are critical to the usability and acceptability of GEMs in the pigment industry.

There has to be an appropriate regulatory system. It should include safety, environmental, and ethical evaluations to secure public confidence and government approval [195]. This will guarantee that GEMs comply with existing environmental and health and safety policies. Open and science-based policies help reduce GEM risks and give the public and stakeholders confidence in the safety of these technologies.

Public acceptance is also an important socio-technical factor. Activists and consumers are stakeholders who must be addressed and persuaded in a way that will promote their acceptance of GEMs. Public campaigns, education, and debate on the benefits and safety of GEMs will minimize fear and resistance [196]. Effective communication campaigns that address public ethical and safety concerns can create a good image and build a more positive climate for commercialization.

Finally, considering socio-economic impacts, such as job creation and enabling a circular bioeconomy, can be beneficial for the commercialization of GEMs. Focusing on the overall economic impact, these innovations can be supported even further by policymakers and investors [197].

Table 3. Some genetically modified microorganisms.

Pigment	Microorganism	Genetic Engineering Strategy	Yield Before Modification	Reported Yield After Modification	Refs.
Astaxanthin	E. coli	Optimization of the astaxanthin biosynthetic pathway with multivariate modular methods	Not reported	Not reported	[198]
β-carotene	E. coli	Overexpression of dxs (1-deoxy-D-xylulose-5-phosphate synthase) and idi (isopentenyl diphosphate isomerase), CrtE (GGPP synthase), CrtB (phytoene synthase), and CrtI (phytoene desaturase)	~20 mg/L	~450 mg/L	[199]
Violacein	E. coli	Violacein biosynthesis genes’ heterologous expression	Not reported	Not reported	[200]
Astaxanthin	Y. lipolytica	Combination of HpCrtZ and HpCrtW from H. pluvialis	Not reported	3.3 g/L or 41.3 mg/g DCW under fed-batch fer mentation conditions	[201]
β-carotene	Y. lipolytica	Optimized promoter gene pairs based on Golden Gate DNA assembly	17 mg/L	408 mg/L	[131,193]
Violacein	Y. lipolytica	Golden Gate Assembly method	Not reported	70.04 mg/L	[202]
Astaxanthin	S. cerevisiae	Recombination of CrtW and CrtZ genes in vitro and in vivo	Not reported	4.7 mg/g DCW	[203]
β-carotene	S. cerevisiae	Integration of CrtI, CrtE, and CrtYB (bifunctional phytoene synthase and lycopene cyclase) and additional copies of tHMG1 from X. dendrorhous using CRISPR-Cas9	Not reported	5.9 mg/g dry weight	[204]
Lycopene	S. cerevisiae	Assembly of IDI, CrtE, and CrtB	Not reported	41.8 mg/g DCW	[205,206]
β-carotene	S. cerevisiae	Lipase expression and introduction of the β-carotene synthetic pathway from X. dendrorhous	Not reported	772.8 mg/L	[207,208]

Dry cell weight (DCW). Note: Yields are reported either as volumetric titer (mg/L or g/L) or specific productivity (mg/g DCW), depending on the source. These units reflect different aspects of production performance and are not directly comparable.

Table 3. Some genetically modified microorganisms.

Pigment	Microorganism	Genetic Engineering Strategy	Yield Before Modification	Reported Yield After Modification	Refs.
Astaxanthin	E. coli	Optimization of the astaxanthin biosynthetic pathway with multivariate modular methods	Not reported	Not reported	[198]
β-carotene	E. coli	Overexpression of dxs (1-deoxy-D-xylulose-5-phosphate synthase) and idi (isopentenyl diphosphate isomerase), CrtE (GGPP synthase), CrtB (phytoene synthase), and CrtI (phytoene desaturase)	~20 mg/L	~450 mg/L	[199]
Violacein	E. coli	Violacein biosynthesis genes’ heterologous expression	Not reported	Not reported	[200]
Astaxanthin	Y. lipolytica	Combination of HpCrtZ and HpCrtW from H. pluvialis	Not reported	3.3 g/L or 41.3 mg/g DCW under fed-batch fer mentation conditions	[201]
β-carotene	Y. lipolytica	Optimized promoter gene pairs based on Golden Gate DNA assembly	17 mg/L	408 mg/L	[131,193]
Violacein	Y. lipolytica	Golden Gate Assembly method	Not reported	70.04 mg/L	[202]
Astaxanthin	S. cerevisiae	Recombination of CrtW and CrtZ genes in vitro and in vivo	Not reported	4.7 mg/g DCW	[203]
β-carotene	S. cerevisiae	Integration of CrtI, CrtE, and CrtYB (bifunctional phytoene synthase and lycopene cyclase) and additional copies of tHMG1 from X. dendrorhous using CRISPR-Cas9	Not reported	5.9 mg/g dry weight	[204]
Lycopene	S. cerevisiae	Assembly of IDI, CrtE, and CrtB	Not reported	41.8 mg/g DCW	[205,206]
β-carotene	S. cerevisiae	Lipase expression and introduction of the β-carotene synthetic pathway from X. dendrorhous	Not reported	772.8 mg/L	[207,208]

Dry cell weight (DCW). Note: Yields are reported either as volumetric titer (mg/L or g/L) or specific productivity (mg/g DCW), depending on the source. These units reflect different aspects of production performance and are not directly comparable.

6.3. Issues and Implications of Different Pigment-Producing Microorganisms

Selection of microbial hosts for pigment manufacture must be performed carefully in light of their divergent biosafety profiles, metabolic characteristics, and downstream processing considerations. Microbial systems offer scalable, environmentally benign substitutes to synthetic pigments, but their use in industry must be compatible with safety, reproducibility, and acceptability to regulators. These considerations must be given special emphasis when industries request natural pigment replacements, where microbial producers must be selected and regulated with careful regard to their intrinsic risks, limitations, and practicability.

Bacterial pigment producers such as Serratia marcescens, Pseudomonas aeruginosa, and Chromobacterium violaceum are severe biosafety concerns. Serratia marcescens, a widely reported prodigiosin producer, is an opportunistic pathogen with nosocomial infection and antibiotic resistance linkages [209]. Its quorum-sensing system regulates virulence factors and biofilm formation, which is a severe biosafety risk factor [210]. Although photodynamic inactivation (PDI) and gene manipulations such as ptrA gene disruption have been fruitful in curtailing virulence at the expense of pigment production, these approaches emphasize the imperative need for stringent safety evaluations [211,212]. S. marcescens is economical to culture on inexpensive substrates such as oilseed cakes and waste from industry, providing a window of opportunity for sustainable pigment production if biosafety concerns are addressed. Pseudomonas aeruginosa is a Biosafety Level 2 (BSL-2) pathogen, primarily due to its production of pyocyanin (a blue-green pigment), a virulence factor that plays a widespread role in its contribution to pathogenicity [213]. Pyocyanin is a redox-active phenazine compound that not only contributes to the bacterium’s virulence but also the danger it poses to host tissues, particularly in immunocompromised hosts or cystic fibrosis patients [214,215]. Chromobacterium violaceum, the natural violacein producer, is also a safety concern due to its established virulence in humans, primarily through its type III secretion systems (T3SSs), implicated in cytotoxicity and tissue destruction. These risks restrict its industrial applications to non-ingestible or topical products under stringent regulatory control [216]. Janthinobacterium lividum, which produces violacein and deoxyviolacein, also possesses pathogenicity, and hence safety screening is necessary [217]. Bacterial pigment synthesis is generally strain-specific, the differences in yield, stability, and regulation of biosynthesis complicating standardization. Overcoming these challenges depends on developments in strain enhancement and metabolic engineering. Through manipulation of gene pathways and fermentation conditions, improved yields and more stable pigment biosynthesis are possible [184]. Utilization of biotechnological innovations, e.g., heterologous hosts or purified enzymes, can improve the reliability and efficiency of pigment biosynthesis. These methods not only enhance yields but also offer improved control of pigment character, i.e., color and chemical composition [184]. The fast growth of bacteria also poses the risk of contamination, which requires strict aseptic control. Use of closed systems and advanced filtration processes can ensure sterility at the time of production, especially in continuous or large-scale fermentative systems [218]. These issues are major hurdles to regulatory approval, scalability, and commercial feasibility.

In contrast, fungal pigment producers are known for producing a wide range of natural pigments, but their industrial use presents several safety, scalability, and regulatory concerns. Most critical among these are the high co-production rates of mycotoxins with pigments. For example, Monascus purpureus yields citrinin, a nephrotoxic metabolite, restricting its application for food and pharmaceutical applications [219]. Even after attempts at removal of citrinin by means of strain improvement, mutagenesis, or gene editing, residual mycotoxins need ongoing safety assessment, despite genetic or screening remediation [166,220]. Similarly, Penicillium oxalicum and Talaromyces atroroseus are bioactive pigment-producing species but phylogenetically closely related to mycotoxin-producing strains like Penicillium citrinum and Aspergillus flavus and need extensive screening [221]. Fungi like Fusarium fujikuroi that can also produce bikaverin and fusarubin belong to genera which have phytopathogenic or toxic strains and hence are industrially problematic [222,223].

Filamentous fungi present engineering challenges because of their mycelial morphology, resulting in high broth viscosity, inadequate mixing, and oxygen transfer problems [224,225]. These factors hinder scalability and necessitate specially designed bioreactors. Pigment yield and composition are extremely sensitive to cultural conditions of pH, temperature, and nutrient ratio, resulting in batch-to-batch variability and variable product quality [226]. Moreover, the intracellular site of the pigment necessitates mechanical or enzymatic cell breakage and solvent extraction, which results in increased downstream costs and also raises the problem of solvent residues and environmental toxicity.

Microalgae are also promising sources of high-value pigments like astaxanthin, β-carotene, lutein, and phycocyanin [227]. Their large-scale use is, however, restricted by some technical and economic limitations. Cultivation demands costly photobioreactors or well-regulated open ponds with strict control of light, CO₂, temperature, and supply of nutrients to achieve maximum pigment yield. Environmental fluctuation, contamination with competing microorganisms, and culture crashes often undermine yield and operational efficiency [228]. Open ponds, although less expensive, are plagued by contamination, low yields, and high water losses [229].

The majority of microalgal pigments are intracellular, requiring energy-prone harvesting, drying, cell disruption, and solvent extraction, all contributing to processing expense and environmental footprint [230]. Though some species such as Haematococcus pluvialis and Dunaliella salina possess GRAS status, numerous other strains lack defined safety profiles and need extensive toxicological evaluation. Spirulina platensis, a phycocyanin source, has the propensity to accumulate heavy metals when grown in uncontrolled conditions, raising further safety issues [231]. Microalgae’s biomass and pigment composition are highly stress-sensitive to extrinsic conditions, which is a cause of batch-to-batch variation and quality control problems [12]. Furthermore, genetically engineered microalgal strains also face additional regulatory scrutiny and limited public acceptability in some markets [232]. All of these factors compromise the cost-effectiveness, scale-up, and regulatory acceptability of microalgal pigments in the context of commercial interest. Therefore, organism-specific issues must be addressed by targeted strain selection, process optimization, biosafety validation, and regulatory compliance to realize the full industrial potential of microbial pigment production.

7. Applications in the Food Industry

Most bacteria and fungi have been thoroughly studied for their ability to produce food colors.

Table 4. Commercially utilized microbial pigments in industries [233].

Pigment Name	Color	Industry Name	Producing Microorganism
Phycocyanin	Blue	Fermentalg	Arthrospira sp. (formerly Spirulina sp.) and Galdieria sulphuraria
Lycopene	Red	DSM Nutritional Products	Blakeslea trispora
β-Carotene	Yellow-orange	DSM Nutritional Products	Blakeslea trispora
β-Carotene	Yellow-orange	Henkel-Cognis Australia	Dunaliella salina
Astaxanthin	Pink-red	JX Nippon Oil & Energy	Paracoccus carotinifaciens
Astaxanthin	Pink-red	AstaReal	Haematococcus pluvialis
Anthraquinones	Red	Natural Red	Penicillium oxalicum and many others

provides a comprehensive collection of commercialized microbial pigments, together with information about their source organisms. Many microbial pigments are already used at the industrial level in the food industry.

7.1. Pigment-Producing Microorganisms and Applications in the Food Industry

Monascus purpureus has been shown to produce a diverse range of pigments, such as red, orange, and yellow compounds. The primary pigments are dimeric yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin), and red (rubropunctamine and monascorubramine) molecules [234]. The pigments are commonly applied in food, especially in Asian countries, and are also referred to as “red yeast rice”. These pigments are also found in processed meats and sausage [235]. Talaromyces atroroseus is another non-toxigenic fungal species that can produce azaphilone biosynthetic families of colors, such as mitorubrins and Monascus pigments, while not producing mycotoxins, which are suitable for beverages and confectionery products and for coloring in other applications.

Microbial pigments also have a great impact on the dairy industry regarding the color and quality of countless products. One example is strawberry milk, which is colored by β-carotene, a natural pigment synthesized by Blakeslea trispora [236]. Brevibacterium linens produces carotenoid pigments that provide washed-rind cheeses with their characteristic orange-red color. Penicillium purpurogenum and Talaromyces spp. are sources of reddish and orange azaphilones, which are incorporated into a variety of baked goods [21].

Actinorhodin, a blue-pigmented secondary metabolite with antibiotic qualities, is produced by Streptomyces coelicolor and may represent a novel food coloring [237]. Carotenoids are biopigments that are synthesized by various microorganisms, such as bacteria like Micrococcus luteus. These pigments are extensively used in food as food colorants of natural origin, especially in meat [235].

Microbial pigments employed in the food and beverage industry are of particular interest as natural colorants because of their biodegradability and possible health benefits. Talaromyces sp. has been found to be capable of synthesizing water-soluble red pigments, which find applications in fruit juices [238]. Astaxanthin, used in health-oriented foods, nutraceuticals, sports nutrition drinks, and beverage products, is synthesized by Paracoccus carotinifaciens and Xanthophyllomyces dendrorhous.

7.2. Food Preservatives

Microbial pigments are also favored in the food industry as preservatives because of their antibacterial and antioxidant properties, as well as consumer demand, safety, and potential health benefits [174]. Prodigiosin, produced by Serratia marcescens, exhibits strong antioxidant and antimicrobial activities. It is a potential scavenger of DPPH and ABTS radicals at low levels (10 mg/L) and also a growth inhibitor of foodborne microorganisms, increasing food product shelf life [239].

Several pigments, especially anthocyanins, flavonoid water-soluble pigments, have also been reported to exhibit considerable antibacterial activity against a variety of pathogens. These natural constituents have been known to demonstrate potential in increasing shelf life and preservation of food against spoilage. Anthocyanins are pH-sensitive, and this allows them to sense chemical and microbial alterations in foods. They have high antioxidant activities and antimicrobial activities, thereby increasing food product shelf life [240].

Carotenoids have numerous activities, such as harvesting light energy, neutralizing oxidants, and functioning as virulence agents and efficient antioxidants capable of scavenging singlet oxygen and peroxyl radicals generated during photooxidation [241]. Because of these activities, they help to preserve food quality by preventing light-induced oxidative damage.

7.3. Pharmacological Activities

Microbial pigments show a wide range of pharmacological activities, including antioxidant, antibacterial, anti-inflammatory, antiviral, immunomodulatory, and anticancer activity; hence, they are regarded as future drug candidates [36].

Prodigiosin, isolated from Serratia marcescens JSSCPM1, has been reported to exhibit antibacterial activity against Gram-negative bacteria [242]. Key research has validated that prodigiosin causes bactericidal activity through induction of programmed cell death (PCD) in bacterial cells. It involves DNA fragmentation, production of reactive oxygen species (ROS), and induction of proteases with caspase-like substrate specificity. Prodigiosin is internalized by bacterial cells, and the majority of it is present in the membrane and nuclear fractions, thus ensuring that it comes into contact with bacterial DNA and allowing intracellular mobility. Furthermore, it greatly inhibits bacterial motility [243].

Monascus ruber pigments were also found to be active against Staphylococcus aureus and Escherichia coli (MIC: 10–20 mg/mL) [244]. Their mode of action operates mainly through membrane disruption. The orange pigment of Monascus ruber inhibits the cytoplasmic membranes of bacteria, enhancing the electric conductivity of bacterial suspensions and leading to cell death. The pigment can be incorporated into phospholipid bilayers, which also suggests that it can disrupt bacterial membranes if the pigment is bound to cellular membranes [245].

The antioxidant activity of pigments such as prodigiosin from Streptomyces sp. WMA-LM31 reached 62.5% against protein and lipid oxidative stress [246]. Carotenoids such as β-carotene, astaxanthin from Xanthophyllomyces dendrorhous, and torularhodin from Rhodotorula spp. neutralize ROS, prevent lipid peroxidation, and increase cellular antioxidant defense [247,248,249]. Melanin from Aspergillus niger and Cryptococcus neoformans possesses redox buffering activity and UV protection [250]. Melanin is also involved in photoprotection and immunomodulation by absorption of UV and degradation of ROS in UV-induced oxidative stress, and therefore a potential sun screen ingredient found in nature [251,252,253].

Violacein, derived from Chromobacterium violaceum, has received significant attention because of its broad range of biological activity, including antiviral activity. It activates the human Toll-like Receptor 8 (TLR8) signaling pathway and thus enhances innate immunity. The receptor-dependent process indicates that violacein can enhance the body’s own antiviral defense; however, the exact interactions of this process with particular viruses like HSV or DENV are not yet fully known [254].

Torularhodin and naphthoquinone derivatives have proven to be very promising as anti-inflammatory drugs through different mechanisms. One of the significant mechanisms through which torularhodin exerts its anti-inflammatory effect is by activating the Nrf2/NF-κB signaling pathways. Oxidative stress and neuroinflammation are key factors in a very large number of degenerative diseases, and torularhodin reverses the issues by facilitating the translocation of Nrf2, thereby upregulating antioxidant enzymes such as HO-1. Concurrently, it inhibits the NF-κB pathway, which is proven to mediate inflammatory cytokine expression [255].

Talaromyces sp. SK-S009 produces naphthoquinone analogs, which have been reported to inhibit cyclooxygenase and lipoxygenase pathways. These pathways are crucial for the biosynthesis of pro-inflammatory mediators, and therefore such analogs are promising for the treatment of inflammatory disorders [256].

Certain microbial pigments demonstrated anticancer activity through apoptosis induction, metastasis inhibition, and the targeting of crucial cellular pathways. Indole-type pigments were active against inhibiting CDC25 phosphatases, SIRT1, ferriprotoporphyrin IX, and DNA binding [253]. Prodigiosin and analogs were cytotoxic to 60 human cancer cell lines and blocked Wnt/β-catenin signaling through the inhibition of LRP6, DVL, and GSK3β [75,257]. Prodigiosin was also described as an in vivo tumor growth and metastasis inhibitor, an MMP-2 inhibitor, and a RhoA pathway modulator [258]. Rubropunctatin, a Monascus pigment, demonstrated anticancer activity against human cell lines HepG2, BGC-823, MKN45, SH-SY5Y, and AGS [259]. A summary or table of some microbial pigments and their bioactivity is illustrated in

Table 5. Pigment-producing microorganisms and their bioactivity.

Microorganisms	Pigment	Color	Notable Functional Benefit	Ref.
Agrobacterium aurantiacum	Astaxanthin	Pink-red	Antioxidant, anticancer, anti-inflammatory	[260]
Ashbya gossypi	Riboflavin	Yellow	Anticancer, antioxidant	[81]
Blakeslea trispora	B-carotene, Lycopene	Cream/red	Anticancer, antioxidant	[261]
Chlorococcum, Chlorella	Lutein	Yellow	Antioxidant	[262]
Cordyceps unilateralis	Naphtoquinone	Deep blood-red	Anticancer, antibacterial, trypanocidal	[263]
Dunaliella salina	B-Carotene	Red, orange	Suppression of cholesterol synthesis, antioxidant, anticancer	[264]
Erythrobacter sp. SDW2	Xanthophyll	Yellow	Antioxidant activity and potential for foods, cosmetics, and pharmaceuticals	[265]
Flavobacterium sp., Paracoccus zeaxanthinifaciens	Zeaxanthin	Yellow	Antioxidant, photoprotectant	[266]
Fusarium sporotrichioides	Lycopene	Red	Anticancer, antioxidant	[261]
Haloferax volcanii	Carotenoids	Yellow	Anticancer activity (human liver carcinoma cell HepG2)	[267]
Halomonas sp.	Carotenoid	Yellow	Antimicrobial activity	[268]
Kocuria marina, Meiothermus	Carotenoids	Yellow	Antioxidant activity	[269]
Kocuria sp. QWT-12	Carotenoids	Yellow	Anticancer (breast cancer cell lines MCF-7)	[269]
Micrococcus terreus	--------	Yellow	Anticancer activity against cervical and liver cancer	[270]
Monascus purpureus	Monascin Ankaflavin	Red-yellow	Antitumor, anti-inflammatory	[271]
Pacilomyces farinosus	Anthraquinone	Red	Antifungal, virucidal	[272]
Paracoccus carotinifaciens	Astaxanthin	Pink-red	Antioxidant, anticancer, anti-inflammatory	[273]
Pedobacter	Carotenoids	Yellow	Antioxidant activity	[274]
Phaffia rhodozyma	Astaxanthin	Pink-red	Anti-inflammatory, antioxidant, anticancer	[275]
Porphyridium cruentum	Phycoerythrin	Red	Antioxidant, antitumor, immunoregulatory	[276]
Pseudoalteromonas rubra	Prodigiosin	Red	Immunosuppressant, anticancer	[277]
Pseudoalteromonas sp.	Prodigiosin	Red	Cytotoxicity against U937 leukemia cells	[278]
Pseudoalteromonas	Violoacein	Purple	Antioxidant activity	[279,280]
Pseudomonas aeruginosa	Phyocyanin	Blue-green	Ciliary dysmotility, cytotoxicity	[281]
Pseudomonas Aeruginosa P1.S9	--------	Blue-green	Cytotoxic activities, antibacterial, antioxidant	[282]
Pseudomonas balearica	Melanin	Black	Antimicrobial activity against phytopathogenic strains	[283]
Rhodococcus maris	--------	Yellow	The risk of breast cancer was shown to be reduced	[284]
Rhodosporidium toruloides	Carotenoid	Yellow–orange-red	Antioxidant activity	[285]
Saccharomyces neoformans var. nigricans	Melanin	Black melanin	Antimicrobial, antibiofilm, antioxidant	[286]
Pseudomoalteromonas rubra	Prodigiosin	Red	Anticancer activity against human cervix carcinoma	[287]
Staphylococcus aureus	Staphyloxanthin Zeaxanthin	Golden yellow	Antioxidant	[288]
Streptomyces hygroscopicus	--------	Yellow	Antibacterial activity against MRSA, VRSA, and ESBL cultures	[289]
Streptoverticillium rubrireticuli	Prodigiosin	Red	Antibacterial, antimalarial, antineoplastic	[290]
Vibrio owensii TNKJ.CR.24-7 (MH488980.1)	B Carotene	Yellow	Antibacterial activity	[291]
Xanthophyllomyces dendrorhous	Astaxanthin	Pink-red	Antioxidant, anticancer, anti-inflammatory	[275]

.

7.4. Alternative Applications

Various applications, apart from bioactivity, such as numerous microbes, display a range of pigment synthesis with varied applications. For example, Rhodotorula paludigena produces a carotenoid pigment that is used to enhance the skin pigmentation of ornamental fish [292].

Talaromyces ruber generates an extracellular fluorescent pigment that is used for wood coloring [293]. Vibrio sp., a marine bacterium, produces a red pigment that can facilitate wound healing and exhibits antibacterial properties [294]. Nigrospora aurantiaca produces a red pigment called bostrycin, which is well known for its remarkable staining resistance in cotton fabric [295]. Isaria spp. and Fusarium spp. produce purple and ruby-red pigments, respectively, which are specifically used in the leather dyeing process [296]. Yarrowia lipolytica W29 produces pyomelanin, a brown pigment that functions as an antioxidant and boosts the sun protection factor (SPF) in sunscreens [297]. Serratia rubidaea can produce prodigiosin, which is used to dye fabrics and is also antibacterial [298].

Various microbes, such as Candida lipolytica, Trichosporon cutaneum, Lipomyces starkeyi, Cryptococcus curvatus, and Yarrowia lipolytica, can be used for pigment production when they are grown on agro-industrial residues, especially lignocellulosic residues. Colors derived from these microorganisms possess outstanding photoprotective, antimicrobial, and antioxidant properties. These properties make them extremely suitable for skincare products, limiting the occurrence of wrinkles and acting as a defense against sunburn [299].

Furthermore, using agro-industrial waste, Actinomucor elegans and Umbelopsis isabellina synthesized carotenoids that are recognized for their antioxidant properties [300]. Talaromyces albobiverticillius utilizes pineapple peels as a substrate to generate a red pigment. This pigment has various functions, like dyeing cotton fabric, and it also has antibacterial and antioxidant properties [301].

8. Emerging Application: Microbial Pigment-Mediated Nanoparticle Synthesis

Different microbial pigments have been used to prepare nanoparticles with diverse chemical and physical characteristics. Bacterial pigment nanoparticles of different sizes ranged between 10 and 100 nm, but in a single case, Au particles were 2000 nm in size. Algal and fungal pigment-mediated metal nanoparticles have size ranges of 5–25 nm and 1–60 nm, respectively [302]. Fungal pigments have significant potential for producing smaller nanoparticles.

However, the process is not yet understood. El-Baz et al. have postulated a likely process for the increase in redox potential with an increase in concentration of red pigment of Monascus extract. Redox potential has been found to play a critical role in the stabilization and reduction of AgNPs [303]. Microbial pigments have several negatively charged functional groups like hydroxyl, ketone, and carboxyl groups, which allow Ag+ ions to adsorb [304]. These pigment-metal complexes initiate the formation of superoxide free radicals (O₂⁻) from dissolved oxygen during photolytic activity, leading to the reduction of Ag+ to Ag⁰ (Figure 5) [305].

Rubropunctatin, a water-insoluble pigment, was purified and utilized as a blocking and reducing agent [306]. The rubropunctatin AgNPs (R-AgNPs) synthesized were well dispersed in solution, spherical in morphology, and had an average size of 13.54 nm. The R-AgNPs are highly active against bacteria, and they have even reduced in vitro toxicity. This indicates that bioactive rubropunctatin functionalized on AgNPs enhances bioavailability and synergistically boosts their activity [307].

Table 6. Microbial pigments used in the synthesis of nanoparticles.

Microorganism	Pigment	NPs	Biomedical Applications	Ref.
Talaromyces purpurogenus	-----	AgNPs	Antimicrobial and anticancer	[308]
Talaromyces australis	-----	AgNPs	Antibacterial	[309]
Serratia nematodophila (NMCC 76), C. violaceum (KM226331)	-----	AgNPs, AuNPs	Antiparasitic, antiplasmodial	[310]
Streptomyces coelicolor	Actinorhodin	AgNPs	Antibacterial	[311]
Xanthomonas sp.	Xanthomonadin	AgNPs	Photoprotecting, antioxidant	[312]
Monascus purpureus NRRL 1992	-----	AgNPs	Antibacterial, antifungal	[313]
Monascus ruber	-----	AgNPs	Antibacterial, antioxidant, catalytic degradation of toxic dyes	[314]
Thermomyces sp.	Yellow	AgNPs	Textile application	[315]
Yarrowia lipolytica NCYC789	Melanin	AuNPs	Antibacterial	[316]
Penicillium chrysogenum	Melanin	MgONPs	Antibacterial, antifungal	[317]
Gordonia amicalis HS-11	Carotenoids	AuNPs	Free radical scavenging activity	[318]
Streptomycetes coelicolor	Actinorhodin	AgNPs	Enhancement of antibacterial activity	[311]

shows the numerous microbes and pigments used in the production of nanoparticles, as well as their uses.

9. Challenges and Future Perspectives

Microorganisms can produce colors both externally and internally. Although the industrial synthesis of food pigments from microorganisms has several benefits, the species employed must be culturable and productive within a restricted region and time [13]. They should be non-pathogenic, non-toxic, capable of growth on a variety of low-cost raw materials, and stable in extreme physical and chemical conditions [112]. Despite the availability of many varieties, the industrial use of natural pigments from microbiological sources is challenging. Some microorganisms are easy to screen but difficult to scale-up commercially. Microbial pigment purification and isolation from fermentation broth is a time-consuming, low-yielding, and costly process [174]. Additionally, these pigments may interact with other food components, leading to undesirable odors and flavors [27]. Temperature, pH, light, UV, oxygen, and heat all have an impact on microbial color, which can cause fading and shorten shelf life. Also, environmental factors such as metal ions, proteins, and organic compounds might influence their natural color [27]. Ending the present lag in microbial pigment production is a priority in an effort to unleash their full potential as bioactive, environmentally friendly alternatives to synthetic dyes. Solving the issues of low yields, high expense, and low scalability through the advancements in metabolic engineering, strain improvement, and process design will assist in making production commercially viable.

In summary, microbial pigments are an extremely promising group of natural pigments with applications ranging from the food, pharmaceutical, and cosmetic industries to the environment. Microbial pigments are bioactive, biodegradable, and safe for manufacture on renewable substrates, and hence are suitable alternatives to synthetic dyes. But full industrial realization will involve a coordinated effort in strain performance optimization, yield improvement through metabolic and process engineering, and solving stability and regulatory problems. Future research will be required to promote life cycle analyses, multi-omics approaches to pathway elucidation, and scalable downstream processing technology. With ongoing inter-disciplinary innovation and policy support, microbial pigments have the potential to play a central part in the transition to cleaner and greener industrial applications.