Unveiling the Intricacies of Microbial Pigments as Sustainable Alternatives to Synthetic Colorants: Recent Trends and Advancements

Abstract

Bio-pigments are the colored primary and secondary metabolites released by microbes under stress conditions and are crucial for adaptation. Bio-pigments are being widely accepted for industrial utilization due to their natural form, organic source, and biodegradability. Also, the ease of cultivation, scalability and cost-effectiveness in terms of pigment extraction is bringing bio-pigments into the limelight. Chemical dyes are carcinogenic and pose a serious threat to human lives, which is another issue that environmentalists must address. However, bacterial pigments are safe to employ; therefore, the food, pharmaceutical, textile, and cosmetics sectors may all benefit from their applications. The therapeutic nature of bacterial pigments is revealed because of their antimicrobial, anticancer, cytotoxic, and remarkable antioxidant properties. Bio-pigments also have multifaceted properties and thus can be an attractive source for the next generation to live a sustainable life. The present review discusses the importance of bacterial pigments over synthetic dyes and their therapeutic and industrial potential. Extensive literature has been reviewed on the biomedical application of bacterial pigments, and further opportunities and future challenges have been discussed.

1. Introduction

The course of adaptation is vital for survival and evolution of microbes. Environmental stressors and cellular damage triggers adaptive mechanisms and the synthesis of pigments that are vivid secondary metabolites created by certain microorganisms in response to diverse environmental factors (Figure 1). These microorganisms possess multifunctional biotic properties and exhibit a range of activities [1]. For example, several bio-pigments have antioxidant properties and are compatible with optimizing functional foods or nutraceuticals [2,3].

Before the 19th century, natural bio-pigments from plants (Curcuma, Indigo) and animals were widely used in food, cosmetics and textiles due to their safety. The synthesis of mauve by W. Perkins in 1850 boosted the bio-pigment industry, impacting India’s traditional Indigo sector. By 1900, around 80 synthetic pigments were used in food despite limited knowledge of their toxicity and purity, leading to some reported toxic effects. According to a study, the majority of synthetic pigments cannot be used in food or cosmetics due to their lethal side effects [4]. The synthetic additives present two issues: first, several synthetic pigments have recently been removed due to concerns about their toxicity or allergenicity; second, even when synthetic additives have been proven safe at sufficient levels, they still appear as “artificial colorants” on the eatable’s packings; therefore, this makes it unclear to consumers. Natural color additives have begun to replace some artificial colorants due to rising concerns over their safety. For instance, artificial colorants, including tartrazin and azorubin, induce allergies [5]. The emerging need for natural pigments, both from natural and synthetic sources, can be seen in the food business. Developing new sources of natural pigments and improving the extraction methods establish new ways to easily launch the products into the market [6]. As the demand for the food industry grows, so does the availability of traditionally made pigments, necessitating the development of new alternatives, such as colorant synthesis through fermentation, as it is said to be the most prominent method for pigment production at a cheaper scale. Riboflavin and β-carotene are the two pigments that are currently generated commercially by fermentation [7].

The very first bio-pigment was derived from milk in 1879 and was named lactoflavin. Later, another variant of yellow dye was extracted from the same source, named riboflavin [8]. This breakthrough shed light on bio-pigment isolation from natural resources. Microbial pigments have been extensively researched and are commonly employed for adding color to clothing, textiles, cosmetics, and even food. These pigments, which are derived from microorganisms, undergo a thorough examination and are frequently utilized in various industries to enhance the visual appeal of their products. They play a vital role in providing vibrant hues and shades to fabrics, makeup and consumables, making them visually appealing to consumers. The utilization of microbial pigments not only adds aesthetic value but also opens up new possibilities for sustainable and eco-friendly coloring options in different applications. Studies have revealed that pigments isolated from microorganisms also have substantial pharmaceutical properties. Several microorganisms have been reported to secrete bio-pigments that comprise functional properties. For instance, the pigment isolated from Haematococcus pluvialis used as a food additive and pigment from Paracoccus denitrificans exhibit antiplasmic and anticancer properties [9]. In comparison with other plant-based or chemical-based pigments, microbial pigments are more stable, water-soluble, and rapidly produced. However, they have certain limitations when it comes to exposure to light, heat or an acidic pH [10].

The widespread use of synthetic dyes and the waste from this sector pose a substantial hazard to the atmosphere [9]. A survey indicated that in the textile industry, on an average investment of $2.3 billion every year, 1.3 million metric tons of dye are produced and only 15% is used; the rest leads to environmental pollution [11]. The ever-increasing expansion of the textile industry poses concerns regarding environmental preservation and seeks a substitute for synthetic dyes.

2. Microbial Pigments: Production and Market

Natural pigments are usually five to ten times as expensive as their synthetic equivalents. In 1987, the market for natural pigments was expected to be worth $35 million, but by 2000, it had grown to $250 million [12]. Natural pigments are expected to have a market value of between $350 and $600 million, with an expected increase in market value from $1.5 billion by the end of 2020 to $2.5 billion by 2025. The two biggest markets for food pigments are in Europe and the US. By the year 2031, the organic pigments market is anticipated to reach a value of $8.4 billion. From 2022 through 2031, this expansion is anticipated to occur at a compound annual growth rate (CAGR) of 4.2%. The market is highly fragmented, with major companies such as Clariant, DIC Corporation, BASF SE, Ferro Corporation, and Lanxess holding significant market shares. The market is influenced by various factors, including the increasing demand for high-performance pigments, growing awareness about the environmental impact of pigments, and the need for bio-based paints and coatings. The market for organic pigments is divided into different geographical regions, namely North America, Europe, Asia-Pacific, and LAMEA. Each region represents a distinct market segment in terms of organic pigments. The Asia-Pacific region is projected to be the dominant market, holding the largest market share among these regions. This is due to the region’s rapid industrialization and growing population. North America and Europe are important markets for organic pigments as well. These regions are propelled by the existence of major industry players and the need for top-notch pigments across various sectors.

The most common pigment to be used in food and pharma industries is carotenoids. According to worldwide market surveys, the market potential for carotenoids in foods, medications, beverages, cosmetics, animal feed and dietary supplements was estimated to be 26.1, 9.2, 6.5, 34.8, and 23.5%, respectively, between the years 2018 and 2024. At this point, the process of synthesizing carotenoids is primarily reliant on chemical methods, accounting for around 80–90% of the overall synthesis [13]. Researchers tend to shift to microbial carotenoid synthesis due to their affordability and sustainability [14]. There are only certain bacterial-derived pigments in the market that have received FDA approval, such as riboflavin extracted from Eremothecium gossypii, lycopene and alpha-carotene extracted from Bacillus trispora, astaxanthin extracted from Xanthophyllomyces dendrorhous and prodigiosin from Monascus sp. [15].

3. Habitats of Pigmented Microorganisms

Pigment-producing bacteria are found in both marine and terrestrial environments. These bacteria are varied and may be present deep in the water in tropical and arctic climates. According to research, pigment-producing microorganisms are more substantial and can withstand extreme environmental conditions. Pigment-producing bacteria fall into two categories: real marine species and adaptable bacteria (terrestrial species and evolved into marine species). It is believed that adaptable bacteria synthesize unique bioactive chemicals that help them adapt to aquatic environments [16]. Marine bacteria are more diverse in pigments due to their nutritional availability and geographical locations [17,18,19,20,21,22]. Bacterial sp. such as Micropeneta laotica (dimorphic), Penicillium purpurogenum (xerophilic), Thermus sp. (thermophilic), Salinibacter (extreme halophilic), Kocuria polaris (psychrophilic), Acidobacterium (acidophilic), Halorubrum sp. (polyextremophile), Diaphanoeca grandis (radioresistance) and Cellulophaga lytica (color mimic) show polyextremophilic characteristics, which help them to survive under stress conditions [23,24,25,26,27]. Sponge, mussels, sea cucumbers, sea grass, sediments and microbial and algal mats in cold region lakes, corals, lagoons, oil-contaminated soil and sea ice have all been stated to be devoid of bacterial colors up to this point [16]. However, a few studies have specifically stated that sponges and sea grass can be a significant source of microorganisms with pigments [28,29]. Few studies have been extensively done on extreme climatic conditions due to high UV radiation and low temperatures. It has been reported that high frequencies of pigmented microbes can be isolated from glacial areas, algal mats, sediments, snow and sea weeds. Pigments serve multiple functions, including photoprotection and UV absorption, which help microbes to survive in harsh conditions. High pigmentation rates were also noted in 79% of bacterial isolates from ice cores and 57% from Antarctic ice cores, with a similar trend in samples from Antarctic lakes and supraglacial systems. This microbial pigmentation diversity holds considerable biotechnological potential, offering novel compounds with applications in various fields [1]. Furthermore, there is another study on extremophile pigmented microbes isolated from central Europe. They require more extreme conditions to grow and develop. Microbes are basically isolated from caves and karst; besides their low nutrient value, they also have severe depletion in oxygen and extreme redox potential [1,2]. It is yet unknown what influences how these bacteria move about in various environments and geographical regions. As a result, new untapped sources need to be looked into to discover these natural pigment-producing bacteria.

4. Analytics of Bio-Pigments

To meet expanding desires for microbial pigments on the global market, genetic engineering and fermentation techniques are mostly used to mass-produce low-cost, exceptionally durable pigments (Figure 2). A variety of instrumental-based logistic approaches may be employed to purify and analyze these pigments for physical and chemical properties. There are several methods available to achieve the desired outcome. These methods include Fourier transform infrared spectroscopy, ultraviolet–visible spectroscopy, thin-layer chromatography, energy-dispersive X-ray spectroscopy, nuclear magnetic resonance, high-performance liquid chromatography, and gel filtration chromatography [6].

Microbial pigments are becoming more popular in the market as a result of their distinctive attributes as anticancer agents, mutagenesis inhibitors, antioxidants, and bio-indicators. These properties make them a promising source in textiles, cosmetics, food, and pharmaceutical industries [30,31]. Various strains of microorganisms, including Bacillus sp., Sarcina sp., Serratia sp., Rhodotorula sp., Penicillium sp., Monascus sp., Streptomyces sp. and Cryptococcus sp., are capable of synthesizing all kinds of pigments [32]. Other than bacteria, fungi and yeast are also reported to produce pigments, like Aspergillus nidulans, Fusarium verticillioides and Rhodotorula sp. An anti-tumor pigment known as dolastatin has been isolated from marine invertebrates like mollusks and sea hares [33]. Epibiotic bacteria present in various vertebrates and invertebrates like seagrass, corals, mollusks, sponges, bryozoans, nudibranchs, ascidians and tunicates are responsible for pigment production [34]. Epibiotic bacterial species such as Pseudoalteromonas, Serratia marcescens and Pseudoalteromonas tunicate produce a yellow pigment known as tambjamine [35,36,37]. According to a theory, the bryozoan uses tambjamine as a chemical defense mechanism to prevent the spotted kelpfish Gibbonsia elegans from eating on it [38,39]. These microbial pigments can use different kinds of substrates, as shown in

Table 1. Different microbes and types of pigments and substrates used for their enhanced production.

Pigment	Organism Involved	Substrate Used for Production	Production Rate (mg/L)	Reference
Melanin	Pseudomonas sp.	Vegetable waste	2.79	[40]
	Aspergillus carbonarius	Apple, black carrot, pomegranate, red beet pulps	61.84	[41]
Prodigiosin	Serratia marcescens	Peanut oil cake	40,000	[42]
		Brown sugar	8	[43]
		Wheat bran and sunflower oil	240	[44]
		Tannery fleshing	33,000	[45]
		Sesame seed	17	[46]
	Streptomyces sp.	Dairy processing wastewater	47,000	[47]
Violacein	Chromobacterium vilaceum	Liquid pineapple waste	16.25	[48]
		Sugarcane waste	820	[49]
		Rapeseed cake	12.93	[50]
Carotenoids	Sarcina sp.	Apple pomace	12.87	[51]
	Sporidiobolus pararoseus	Corn steep liquor	40 mg/L	[52]
	Rhodotorula glutinis	Chicken feathers	92	[53]
	Rhodotorula achenorium	Whey filtrate	262	[54]
	Rhodotorula rubra	Sugarcane juice	30.39 mg/g	[55]
	Rhodotorula mucilaginosa	Coffee pulp	16.36	[56]
Astaxanthin	Haematococcus pluvialis	Piggery wastewater	83.9	[57]
	Phaffia rhodozyma
		Juice of date	23.8	[58]
		molasses	15.3	[59]
		Grape juice	9.8 µg/mL	[60]
Riboflavin	Bacillus subtilis	Corn steep liquor	26.8	[61]
Pyocyanin	Pseudomonas aeruginosa	Grape seed	4 µg/mL	[62]
		Cotton seed meal	4 µg/mL	[62]

, to enhance their production, also making them cost-effective.

5. Genetically Modified Organisms in the Pigment Industry

5.1. Metabolic Engineering

Genetic engineering allows for the modification of bacteria to generate specific pigments. This method has led to a significant improvement in fermentation efficiency and an additional cost reduction. To synthesize these pigments, various strategies have been proposed to develop GMOs (genetically modified organisms). These strategies include over-expressing the key enzymes and the insertion or deletion of specific genes involved in the biosynthesis of pigments. These genetic modifications not only improve microbial strains but also help to overcome the toxicity arising from synthetic dyes [63]. For instance, a GM strain of Corynebacterium glutamicum has been developed by manipulating the transcriptional repressor gene crtR to enhance the production of a specific carotenoid called decaprenoxanthin [64]. Another GM strain, Rhodobacter sphaeroides, has been developed by using gene manipulation methods to enhance lycopene production [65]. Furthermore, scientists have successfully manipulated E. coli at the genetic level to produce various pigments, including β-carotene and zeaxanthin. One notable achievement in this field is the synthesis of a modified strain of Escherichia coli through the alteration of a gene involved in ATP synthesis, pentose phosphate, and the TCA (citric acid cycle). This modified strain of Escherichia coli has been specifically designed to synthesize β-carotene [66]. Hence, the application of GM microorganisms can be widely employed for the large-scale manufacturing of pigments that possess various medicinal and industrial uses.

5.2. Gene Cloning

Gene cloning in pigmented microorganisms is also a part of genetically modified organisms involving isolation and insertion of specific pigment-producing genes into the genome of host microorganisms to enhance or introduce pigment production. This process has significant implications for industrial biotechnology, environmental monitoring and medical applications. The first step in gene cloning involves identifying and isolating the gene responsible for pigmented production. This is typically achieved through techniques such as polymerase chain reaction or restriction enzyme digestion. The isolated gene is then inserted into a plasmid vector, which will carry the gene into the host microorganism [67,68,69]. Plasmids are circular DNA molecules that can be replicated independently within a bacterial cell. Vectors often contain selectable markers, such as antibiotic resistance genes, to facilitate the identification of successfully transformed cells. The recombinant plasmid is introduced into host microorganisms through a process known as transformation. Methods such as electroporation or chemical transformation are commonly used to induce the uptake of plasmid DNA by the host cells. Following transformation, the host cells are cultured on selective media containing antibiotics. Only those cells that have successfully taken up the plasmid will survive. These cells are then screened for pigment production using spectrophotometric or chromatographic methods. The cloned gene is expressed under controlled conditions to maximize pigment production. This may involve optimizing growth conditions such as pH, temperature, and nutrient availability, as well as using inducible promoters to control the timing and level of gene expression [69,70]. This involves Serratia marcescens producing prodigiosin pigment having antimicrobial and anticancer properties.

The biosynthetic pathway involves several genes, including pigC, pigD and pigE. These genes can be cloned into a suitable host to enhance pigment production [70,71,72,73,74]. The gene involved in biosynthesis can be isolated using PCR with specific primers and then cloned into expression vectors such as pET21a, which is suitable for high-level expression in E. coli. The recombinant plasmid is introduced into E. coli via electroporation. Transformed cells are selected on LB plates containing ampicillin. Positive clones are screened for prodigiosin production by measuring the absorbance at 535 nm. The expression conditions, including temperature, IPTG concentration (for induction) and media composition, are optimized to enhance violacein yield [70,72,73,74,75]. Experiments are conducted to determine the optimal conditions for maximum pigment production. Another scientist also studied melanin pigment isolated from Pseudomonas putida. This gene is cloned into an expression vector, such as pET21b, which is suitable for use in E. coli. Moreover, red carotenoid pigment termed lycopene is isolated from Pantoea ananatis. These genes are then cloned into an expression vector, such as pTrc99A, suitable for use in E. coli [70,74,75,76,77].

CRISPR-Cas9 (Clustered regularly interspaced palindromic repeats) has sparked a trend, with numerous laboratories utilizing this technology to explore novel approaches in biotechnology, particularly genome engineering. Therefore, the CRISPR-Cas9 system can effectively be utilized in bacteria, yeast, and fungi for metabolic engineering. This approach enables these organisms to function as cellular factories, resulting in the cost-effective production of natural food colors [78]. Nielsen et al. [79] have devised a system for Aspergillus nidulans that incorporates the CRISPR-Cas9 system to find potential applications in various fungal systems without requiring significant modifications. The researchers demonstrated its effectiveness in a wide range of filamentous fungi, highlighting its versatility and adaptability. The CRISPR-Cas9 system has also been implemented in Talaromyces atroroseus, which is used as a natural red food color in food industries. Furthermore, it has also been employed in recent studies involving Penicillium chrysogenum to improve filamentous properties [80].

6. Significance of Pigmented Bacteria

Pigmented microorganisms are significant due to their diverse applications in various fields, including pharmaceuticals, medicine and industry. These microorganisms produce pigments with unique properties, such as antimicrobial, antioxidant and anticancer activities, making them valuable for drug development and therapeutic purposes. In agriculture, microbial pigments can act as natural pesticides and growth promoters, contributing to sustainable farming practices. Additionally, these pigments are used in the food and textile industries as natural colorants, offering a safer alternative to synthetic dyes. The study and utilization of pigmented microorganisms are expanding, driven by their potential in biotechnological innovations and environmental sustainability. The following review discusses the applications of various pigmented microorganisms in detail.

6.1. Cosmetic Industries

Traditionally, herbal pastes, oils and mineral ingredients were used to protect the skin against infections and harsh conditions. People have used cosmetics for self-expression, to make temporary tattoos and to color their faces. In some cultures, cosmetics were even used for spiritual purposes; for example, in ancient Egypt, kohl was applied around the eyes to ward off evil spirits. With time, using advanced techniques, people began experimenting with new ingredients and products to create more effective beauty products [81]. The cosmetic industry consumes a global market nowadays, with 2000 manufacturers in the USA itself [82]. Globally, skin-whitening agents are the most desirable cosmetic products, as lighter skin tone is the prominent beauty factor among various Asian cultures. Investment in skin-whitening agents is increasing annually, especially in the markets of China, Japan and India [83]. Skin color is defined by various intrinsic and extrinsic factors like type of skin, genetic background, exposure to sun and pollution, melanin, pigment, etc. [84,85,86]. Though pigmentation is important for protecting skin against harmful UV injuries, excess pigment results in aesthetic problems, such as ephelides pigmentation, melasma and post-inflammatory hyperpigmentation [83,84,85,86,87].

Traditional drugs like hydroquinone, corticosteroids and amino mercuric chloride brighten skin by either preventing melanocyte maturation or interfering with the process termed melanogenesis. The majority, if not all, of the stated drugs are, however, intimately linked to side effects such as prickling, contact dermatitis, irritation, high toxicity and sensitivity [88,89,90,91]. Cosmetic R&D industries effortfully develop innovative whitening agents that limit tyrosinase activity (TYR) to reduce the adverse effect on melanocytes and minimize hyperpigmentation. At present, natural skin-whitening agents are thus gaining interest in the cosmetic and pharmaceutical industries [92]. Recent research has demonstrated that melanin pigment, which was isolated from Streptomyces bellus MSA1, can be used in the production of bio-lip cream when coupled with beeswax, coconut oil, and lanolin [93]. Melanin has a high UV absorption tendency and, therefore, is a crucial ingredient in sunscreens, plastic films, varnishes, and cosmetics [94]. Few studies have reported on the sunscreen action and antioxidant properties of bacterial strains, which can be a valuable tool for the cosmetics industry [95,96]. Melanin is an important skin pigment, as it creates an epidermal–melanin barrier that reduces UV transmission to underlying skin cells, and UV-induced DNA photoproducts are a major cause of skin darkening. Moreover, bacterial pigments are stable and, hence, can be used as coloring agents in other cosmetic products, such as eye shadows, nail paints, hair colors, etc. [97,98].

6.2. Food and Pharmaceutical Industries

Pigments having potential health and nutritional benefits are highly valued and in demand and are mostly preferred by food industries. Therefore, finding new sources for natural and safe food colorants is required, rather than using synthetic colorants like tartrazine, reactive blue, vat green, and sunset yellow [99]. Microbial-extracted pigment carotenoids show an expanding business because they enhance food organoleptic properties without affecting food quality, eliminate many diseases, and have anti-aging properties [100]. Despite several benefits, microbial pigments have several drawbacks, such as less shelf-life and colors fading over time, sensitivity towards light, heat, pH and temperature, etc. To overcome this situation, scientists have discovered the use of nano-formulation, encapsulation, and non-emulsion techniques to enhance the stability of the pigment [101,102]. Encapsulated violet pigment shows more effective coloration in yogurts and jellies [103]. A wide range of microbial pigments are being utilized in various food and beverage industries. These pigments consist of beta-carotene and lycopene obtained from Blakeslea trispora; astaxanthin derived from different bacteria, algae, and Xanthophyllomyces dendrorhous; pink-red pigment obtained from Penicillium oxalicum; pigments from Monascus sp.; and riboflavin obtained from Aphis gossypii, Dipterocarpus globosus, Candida guilliermondii and Eremothecium ashbyii [31,101,104]. Research has also emphasized the commercial potential of anthraquinone and azaphilone-producing strains, such as Penicillium sp. and Talaromyces sp., for food coloration [105,106,107]. Also, carotenoid pigments are highly used as food additives for animals or aquatic species [108,109]. Fungal species also produce colors, but extensive tests are required to check the virulence and toxicity [106,110,111].

Additionally, the expanding interest in adding microbe feed additions to livestock diets is a result of the rising cost of feed made from plants and animals. Over the past ten years, experts in the fields of nutrition and microbiology have extensively explored and revealed the various advantages associated with the utilization of microorganisms as feed additives, viz. (i) microbial cells contain a higher amount of protein in their biomass compared to grains, and the amino acid content is of high quality compared to eggs; (ii) certain beneficial microbes can produce vitamins, including vitamin B12, which cannot be performed by eukaryotic organisms; (iii) microbial production is not dependent on seasons and can be carried out in various climates without the need for specific planting zones; and (iv) microorganisms can be cultivated using waste materials, offering a potential solution to cost-related issues [112]. Photosynthetic bacteria (PSB) are acknowledged as a highly advantageous source of microbial feed due to their abundant content of protein, lipids, essential amino acids, fatty acids, ubiquinone-10 (CoQ10), vitamins and carotenoids [113,114,115,116]. Figure 3 depicts the ecological functions and other applications of important microbial pigments. The bacterium Enterococcus faecalis PA2, which undergoes photosynthesis, has been recently identified as a potential carotenoid producer. It exhibits a comparatively high level of biomass production [117]. In terms of photosynthesis, PSBs can produce various kinds of carotenoids, such as monocyclic, bicyclic and acyclic groups. Certain carotenoids have been exclusively found in PSB and can be added to animal feed, specifically aqua feeds, to enhance coloration and boost the immune response [25,118].

6.3. Therapeutic Significance

Microbial pigments come in a variation of colors and are recognized as defense systems against UV irradiation, enhancing the pigment’s stability and allowing microorganisms to better adapt to their environment compared to non-pigmented microorganisms [21,118,119]. Some bioactive pigmented bacteria show symbiotic or epibiotic relationships with their hosts and protect their hosts from other pathogenic microbes and predatory species [120]. Extremophilic bacteria, including Thermus filiformis, Halococcus morrhuae and Halobacterium salinarum, create C50-carotenoids, which are crucial for the integrity of cell membranes [121]. It has been proven that bacterial phenazines control the cellular gene terminologies that help the bacteria survive and synthesize biofilms [122]. Carotenoids can decrease the presence of oxygen radicals that are generated by reactive nitrogen and oxygen species, as well as other nonbiological radicals within the cells [123]. A recent study has shown that the violet pigment produced by Chromobacterium violaceum can safeguard the lipid membranes of rat liver microsomes, soya bean and egg phosphatidylcholine liposomes from peroxidation caused by reactive hydroxyl radicals [124].

Violacein pigments are also known to be effective against other bacteria as well. Pigments produced by Janthinobacterium lividum and Chromobacterium violaceum are seen to restrict the growth of a variety of Gram-positive bacteria, such as Staphylococcus aureus and Bacillus subtilis. Furthermore, it has been concluded that pigment produced by these two species also has antifungal properties against Candida albicans, suggesting that violacein may be useful in the treatment of fungal infections. The ability to produce violacein pigments is an evolutionary advantage for these bacteria, as it provides them with a means of self-defense against predators and other hostile microorganisms [125]. Anti-phagocytosis properties are thought to be one of the positive functions of bacterial pigments. Similar to indigoidine, some Roseobacter strains create blue quinine compounds that eliminate other possibly rival bacteria [126]. Pyoverdine pigment generated by Pseudomonas fluorescens aids iron transport in plant cells as well [127]. Prodigiosin pigment-producing Vibrio strains are a thousand times more stable than non-pigment-producing Vibrio sp. when exposed to UV radiation [128].

Black pigmentation is said to have developed as a result of living things adjusting to harsh environmental circumstances. Melanin pigments, also termed cellular protectors, not only neutralize toxins like antibiotics and other drugs but also act as a survival factor for tolerating stressful conditions like starvation, high temperature, and hyperosmotic stress in Vibro cholerae [129,130]. Research has shown that pigment-producing marine bacteria are more resistant to drugs and heavy metals [131]. For mammals, insects, and microbes, the functional significance of this kind of pigment has been thoroughly examined [132,133]. The function of pigment in plants is not fully uncovered, but the research suggests that the dark color may also benefit them in certain ways. Melanin-based color is crucial for camouflage in plants, just like it is in mammals. The hull color in the majority of wild grains is black. The ripe, black-hulled seeds that drop to the ground are thought to be undetectable to birds over the backdrop of soil [134]. In theory, seeds with a black grain color have the potential to germinate earlier compared to yellow seeds. This is because black-grained seeds can absorb a greater amount of solar energy from light surfaces, converting it into heat. If we distinguish between black and white barley races, it is revealed that the black variety tends to reach maturity earlier than the white variety [135]. Melanins give seed shells more tensile strength, shielding them from harm. Additionally, due to its toxicity, melanin confers resistance to insects and vermin [136]. Black seed coverings on sunflower seeds protect them against mole larvae harm better than white-colored seeds [137]. Because melanins are a potent source of antioxidants, they can provide seeds with more vigor and protect them from environmental stresses [138,139]. Fungal melanins also protect against oxidative stress and reactive oxygen species, as well as against antimicrobial agents. In addition, they are known to be involved in the signaling and regulation of gene expression in fungi. In addition, melanins are believed to have a role in the formation of biofilms by offering a protective barrier and assisting in the cohesion of cells [140].

Table 2. Various microbial pigments and their therapeutic applications.

Pigment Produced	Microbial Source	Therapeutic Application	Reference
Prodigiosin	Serratia marcescens	Human colon cancer cells	[141]
Violacein	C. violaceum	Colorectal cancer	[142]
Monascin	Monascus purpureus	Teratogenic effects on chicken embryos	[143]
Carotenoids	Haematococcus pluvialis	Food additives	[144]
Bromoalterochromide	Pseudoalteromonas maricaloris	Cytotoxic effect on sea urchins	[145]
Tambjamine	Atapozoa sp.	Ichthyodeterrent activities	[146]
Anthraquinones	Phoma multirostrata	Herbicidal activities	[147]
Astaxanthin	Phaffia rhodozyma	Anti-aging properties	[148]
Astaxanthin	Phaffia rhodozyma	Antiproliferative activity	[149]
Monascus	Red mold dioscorea	Anti-diabetic activity	[150]
Carotenoid	Streptomyces mediolani	Antioxidant activity	[151]
Astaxanthin	Haematococcus pluvialis	Anti-atherosclerotic activity	[152]
Anthraquinones	Phoma foveata	Inhibition of HIV	[147]
Prodigiosin	S. marcescens	Antitrypanosomal activity	[153]
Violacein	Chromobacterium violaceum	Anti-malarial activity	[154]
Flexirubin	Flavobacterium	Treatment of skin disease	[155]
Prodigiosin	S. marcescens	Antileishmanial activity	[156]
Violacein	Janthinobacterium lividum	Antiprotozoal activity	[125]
Violacein	Plasmodium falciparum	Antiparasitic activity	[154]
Prodigiosin	Hahella chejuensis	Anti-algicidal activity	[157]
Prodigiosin	S. marcescens	Antifouling activity	[158]
Flexirubin	Flavobacterium sp.	Anti-tuberculosis activity	[159]
Phycobilioproteins	Cyanobacterial sp.	Anti-alzhelmeric activity	[159]
Cycloprodigiosin hydrochloride	Pseudoalteromonas denitrificans	Immunosuppressive activity	[160]
Phenazine	Streptomyces sp.	Antiviral activities	[161]
Anthraquinone	Trichoderma harzianum	Antifungal activity	[161]
Phycocyanin	Spirulina	Phycofluoures for DNA probes	[159]

enlists various microbial pigments, source organisms and their therapeutic effects.

Moreover, bio-pigments protect their host against predators. The endophytic fungi’s anthraquinones protect its host plants from pests and other microbes [162]. Tambjamine shows natural defense mechanisms against predators in a variety of microorganisms [37]. Interestingly, the fungus called Candida purpurea uses a variety of colors, including orange, red, yellow, and black, to warn off predators [163]. Pigments produced by microbes play a significant role in various antibacterial activities [164], virulence [142], antioxidant activity [124], ultraviolet absorption [143] and membrane stability [144].

6.3.1. Antibacterial Properties

Around 700,000 people die globally each year, and if essential steps are not taken, this number is likely to reach 10 million by 2050. The World Health Organization (WHO) has found 12 microorganisms that pose the greatest threat to humans due to their antibiotic resistance [145]. Melanins, flavins, carotenoids, monascins, quinones and violacein are a few examples of antibacterial pigments [146]. The soil bacterium Massilia sp. generates a violet pigment with deoxy violacein and violacein that has potential antibacterial activity against Bacillus subtilis, Staphylococcus aureus and E. coli [147]. A different study found that the bioactive pigment of Streptomyces hygroscopicus has been active against Klebsiella sp. and E. coli cultures, as well as VRSA (vancomycin-resistant Staphylococcus aureus), MRSA (methicillin-resistant Staphylococcus aureus) and ESBL (extended-spectrum beta-lactamase) [148]. Red pigment derived from Neisseria meningitidis was found to inhibit Gram-negative bacteria like E. coli and Pseudomonas sp. [149]. E. coli and Salmonella typhi were both susceptible to the antibacterial effects of Bacillus megaterium SU15’s orange pigment [149]. Furthermore, there is a pigment with a green hue that is generated by a specific strain of Bacillus cereus, known as cerein. This particular pigment has bactericidal properties against other strains of Bacillus cereus [150]. Certain research provides clear evidence that a melanin pigment extracted from Streptomyces djakartensis displayed potent anti-bacterial effects against E. coli, Staphylococcus aureus, Klebsiella pneumoniae and P. aeruginosa, even at minimal concentrations [151].

6.3.2. Antifungal Properties

Fungal pigments are secondary metabolites that are synthesized by mycelium in specific circumstances, such as nutrient scarcity or adverse environmental conditions. These pigments serve as a response to stress conditions and are not essential for the primary functioning of organisms [152]. Over the past 20 years, numerous lethal fungal and fungal-like illnesses have been documented in both plants and animals, endangering the safety of our food sources [153]. Amphotericin B, which is traditionally used as an antifungal treatment, is expensive and hazardous. Natural resources (plants and microbes) are assessed for their antifungal abilities to solve these issues. Serratia marcescens and Neisseria sp. are several pigmented bacteria with antifungal capabilities [154]. Prodigiosin is a tripyrole pigment that was initially identified in Serratia marcescens and is an efficient biocontrol agent for cyclamen’s grey mold [155]. When prodigiosin and chitinolytic enzymes were used together, a synergistic inhibitory effect was seen [46]. The red color pigment that was extracted and purified from the soil bacterium Neisseria sp. had potent activity against a variety of fungal species, including Candida sp., Aspergillus sp. and Trichoderma sp. [156]. Additionally, Janthinobacterium lividum violacein’s phytopathogenic fungi that cause white root rot are susceptible to its fungicidal effects [157]. In addition, during the investigation of the antifungal properties of Janthinobacterium lividum against aspergillus unguis in both pure culture and in the presence of Pseudomonas chlororaphis, it was discovered that the Janthinobacterium lividum culture exhibited the highest effectiveness. It inhibited the growth of mold mycelium by 46.3% and the Pseudomonas strain by only 21%. However, in the presence of Janthinobacterium lividum, the degree of suppression increased to 32%. This suggests that there are competing interactions between the bacteria, resulting in changes to overall antagonistic activity [158].

6.3.3. Anticancer Properties

Various factors contribute to the development of malignancy in somatic cells, including carcinogenic pollutants, unhealthy dietary patterns, oncogenic bacteria and viruses, and other unidentified causes. These factors collectively contribute to the initiation and progression of abnormal cell growth in the body [159]. Oncology chemotherapy has a major drawback, i.e., toxicity and resistance. It has been reported by various scientists that bacterial pigments could be the solution to these problems. These limits are overcome by influencing autophagy pathway apoptosis in cancer cell lines. Out of all bacterial pigments, violacein induces apoptosis in HL60 cells. It is a type of cancer cell line that is used as an example to study myeloid leukemia disease but does not present in normal lymphocytes [160]. Streptomyces glaucescens NEAE-H produced black extracellular melanin that was harmful to a skin cancer cell line [94]. Human hepatoma and glioblastoma cells were strongly suppressed by the blue-green pigment pyocyanin [162,163]. With an IC50 gene, the yellow pigment from S. griseoaurantiacus was one of the types of carotenoids that significantly caused cytotoxicity against cervical cancer-causing cells [164]. Flexirubin, a type of carotenoid extracted from Chryseobacterium artocarpi, induced programmed cell death in MCF7 breast cancer cells and exhibited enhanced effects when used in conjunction with silver nanoparticles [165].

In human cervical and laryngeal cancer cells, prodigiosin (PG) demonstrated potent anticancer and apoptotic effects [166]. Doxorubicin (Dox) and PG together have a synergistic impact on oral squamous cell cancer [167]. The working of PG and its processing is yet to be defined. Scientists have noticed that the red-colored pigment can suppress Wnt/-catenin signaling and lower the levels of cyclin D1 [168]. It has been suggested that PG offers therapeutic potential for treating advanced breast malignancies.

6.3.4. Antiparasitic Properties

Parasitic infections are among the most lethal and rapidly spreading infections transmitted by vectors. Examples of these diseases include malaria, encephalitis, leishmaniasis, and helminthiasis. They invade the host’s body and hinder its growth by depriving it of vital nutrients. Roughly one billion individuals, which accounts for approximately one-sixth of the global population, have experienced at least one type of parasitic infection in their lifetime [169]. These statistics are cause for concern, but the ramifications are not limited to this. Parasitic infections affect not only humans but also animals and plants, resulting in economic losses. Developing treatments that harm parasites without harming the host is always a difficult task due to the complex nature of the disease and the advanced virulence of the parasites. A study conducted on bacteria capable of surviving in cold temperatures in Antarctica discovered that certain strains of Pseudomonas sp. produce pigments with bioactive properties that can combat parasites [170]. Studies like these demonstrate the true effectiveness of microbial pigments and uncover the untapped potential of psychrophiles. Microbial pigments, such as violacein and deoxy violacein, are widely recognized in the pharmaceutical industry for their antimicrobial properties. However, in recent years, their ability to combat parasites, particularly Plasmodium falciparum and Trypanosoma cruzi, has gained attention. Efforts are ongoing to scale up the production of violacein to harness its potential as a drug against malaria and trypanosomiasis [171]. Furthermore, the use of bacterial pigments has demonstrated potential in mitigating plant nematode infestations. As an example, the pigment derived from Serratia marcescens has been found to effectively stop the juvenile stages of Radopholus similis and Meloidogyne javanica when administered in a low dosage [172]. The findings from this research showcase the potential of bacterial pigments in fighting against harmful parasites, presenting new avenues for exploration.

6.3.5. Immunosuppressing Properties

A marine bacterium called Pseudoalteromonas sp. has been discovered to contain a non-toxic bioactive pigment known as cycloprodogiosin hydrochloride. This pigment has shown the capacity to selectively prohibit the production of T-cells. Other members of the prodigiosin family are believed to have an impact on murine splenocytes. These findings offer novel opportunities to harness the power of bioactive pigments in developing immunosuppressive treatment drugs [173]. Another strain, MS-02-063, which is genetically related to the γ-proteobacterium Hahella sp., has been found to produce a red pigment known as RP-063. This pigment has also demonstrated immunosuppressive activity, although it differs from other well-known compounds in its mechanism of action. RP-063 inhibits the proliferation of lymphocytes to reduce the release of superoxide from these cells [160]. In addition to cycloprodigiosin hydrochloride and RP-063, there is another prodigiosin called 2,2′-[3-methoxy-1′amyl-5′-methyl-4-(1”-pyrryl) dipyrryl-methene (MAMPDM). MAMPDM is well known to inhibit the proliferation of both B-cells and T-cells [174]. It has been shown to induce apoptosis in Con A-stimulated cells, further demonstrating its selective inhibition of T-cells. Overall, these findings highlight the potential of bioactive pigments, such as cycloprodogiosin hydrochloride, RP-063 and MAMPDM, as promising candidates for the development of immunosuppressive drugs. Further studies in this area could lead to the discovery of new therapeutic options for conditions that involve excessive immune responses [175].

7. Bio-Colorants as Bio-Indicators

The term bio-indicator is defined as an indication of certain biochemical activities. Pigments generated from bacteria show greater application for antibacterial activity, antioxidant activity and various textile industries. Pigments showing fluorescent colors indicate the working of a certain chemical reaction. Phycoerythrin is a pigment used in calculating the rate of peroxy radical scavenging in human plasma; darker pigments signify its reaction with radicals, otherwise, it is fluorescent [176]. Some pigments are used in the detection of heavy metals present in soil or water. Some of the major examples include Vogesella indigofera producing a blue color when associated with normal environmental conditions, but when exposed to heavy metals, no such coloration can be seen [177]. Also, cyanobacteria act as good bio-indicators that detect heavy metals present in water by reducing the carotenoid content in cyanobacteria. Some pigmented bacteria are also used in monitoring temperature changes; Pantoea agglomerans is one of the pigmented microbes that gives a deep blue color when affected by higher temperatures [178].

8. Limitations of Pigmented Microbes

Though microbial pigments are essential and have numerous applications, we cannot deny the pathogenicity of microbial pigments, too. This can be seen in aquaculture farms and humans [179]. Children and adults have both been documented to contract infections from Chromobacterium violaceum, which produces violaceum [180]. Another violacein-producing bacterium, Janthinobacterium lividum, caused massive mortality in the Korean hatchery of rainbow trout Oncorhynchus mykiss [181]. In addition to humans, Serratia marcescens produces prodigiosin that also affects insects, other invertebrates and animals [182]. It has been found that certain Serratia marcescens and Chromobacterium violaceum strains are devious human infections [183,184]. There is no proof that the violacein and prodigiosin pigments play a part in the virulence functions of the microbe [185].

Moreover, pigments, including bacterial melanins [186] and pyoverdines [187], also have antibacterial activity against pathogens. Mycotoxins, such as citrinin and 4,15-diacetoxyscirpenol, are produced by the red pigment-producing fungus Fusarium and Monascus [188]. If we could simply identify the causes of this pathogenicity, we would be able to overcome the symptoms and prevent more infections.

9. Conclusions

Pigment-producing microbes are more likely to give multifaceted applications in all kinds of industries, like textiles, food, medicine, etc. The exploration of pigment-producing bacteria, fungi and cyanobacteria isolated from different environments, like aerobic, terrestrial and marine, is essential. It is important to recognize the convergent and divergent evolutionary patterns of these microbes. Moreover, the prime focus should be on introducing alternative substrates, like wastes from agro-industries, fisheries and poultry farms, as nutritional media to enhance microbial growth and pigment efficacy. This practice would provide a cheap and economical source for cultivation and a great approach to waste management. The potential of bacterial pigments is immense, and the future seems to be even brighter.

The potential of bacterial pigments for additional uses, such as biodegradable dyes, natural food colorants or even as a source of bioactive substances, has also become more apparent in this field. Understanding the interactions between various substances and their effects on the human body should be a major area of research. Additionally, clinical lab facilities must evaluate the effectiveness and safety of these chemicals to determine any related adverse effects. These investigations will aid our understanding of the therapeutic value of bio-pigments. It is crucial to go further into comprehending how these pigments’ chemical markings affect their biological functions. Investigating the complex relationship between structure and function can help us better understand the many roles they play.