The concentration (intensity) of microbial pigments usually varies according to species and strain. In the laboratory conditions, some pigmented microbes produce high-quantity and high-concentration pigments, while some produce low-quantity and low-intensity pigments. The use of a variety of natural/synthetic substrates to stimulate and enhance the yield of microbial pigments has been reviewed, and indicated natural substrates as a potential nutrient element in microbial pigment production [45]. Thus, the use of genetic engineering modification techniques, which are costly and time consuming, have limitations but remain an option to improve strains. However, it is not an essential step to improve pigment production unless the strain has proven to have specific bioactivity or coloring applications. In exceptional cases, mutagenesis and genetic engineering techniques are implemented for strain improvement as well as to enhance pigment production from a low pigment yielding microbe with potential application [96].

The use of natural substrates and adsorbents [45] in fermentation plays an important role in enhancing cell volume during frothing. Several studies have demonstrated the application of natural substrates on the yield of various microbial pigments due to the presence of rich carbon–nitrogen residues [48]. However, only some substrates are demonstrated to yield more pigment [10,45,46,47,48]. Several studies have investigated a large number of agro-industrial substrates in solid-state fermentation compared to submerged fermentation [97]. Here, substrates with high pigment yielding ability used in submerged fermentation are alone detailed briefly for further implications.

Higher prodigiosin production from S. marcescens was achieved using peanut broth (38.75 mg mL⁻¹) than other substrates [61]. For more substrates with a good yield of prodigiosin pigment, refer to Han et al. (2021) [20]. Among the several tested substrates, prodigiosin pigment production was enhanced greatly with cassava wastewater [98] and peanut oil cake [99].

Monascus purpureus culture produced more pigment yield when tested with corncob hydrolysate [100], bakery waste hydrolysate [101], brewer’s spent grain [102], and glucose fermentation medium added with rice straw hydrolysate [103]. Monascus ruber produced significantly low pigment yield when supplemented with sugarcane bagasse hydrolysate [104]. The waste extract medium made up of various peels of inedible fruit matter was reported to enhance carotenoid production from several species of Rhodosporidium, especially from Rhodosporidium toruloides [105]. Many agro-industrial residues tested were found to enhance carotenoid production from several yeast species [10,45]. Loquat kernel extract [66], sugar beet molasses [106], and sugar cane extracts [107] were the two substrates reported to enhance yeast carotenoids greatly.

Usually, pigmented bacteria, fungi, and yeast are highly sensitive to physicochemical parameters. Thus, these microbes require a variety of in vitro culture conditions to yield more pigments in either solid-state or submerged fermentation. It is necessary to investigate the optimized culture conditions for each species or strain. Therefore, optimization of experimental design studies using artificial neural networks, Box–Behnken design, central composite design, Plackett–Burman design, and response surface modeling have been used to identify the key physicochemical factors that trigger high pigment production in microbes. Regardless of species, a review of the literature suggests that most pigmented microbes, except few cases, produce pigments at temperatures ranging between 22–28 °C, pH 5–6, and agitation at 100–150 rpm [1,10].

The maximum prodigiosin pigment production from S. marcescens was observed at 28 °C (38.75 mg/mL) compared to 30 °C (25.98 mg/mL) when cultured in peanut seed broth but not with other substrates tested [61]. Many species of fungi and yeast were observed to produce carotenoid pigments under various parameters such as temperature, pH, agitation, and light availability [10]. Monascus purpureus, when cultured with whey powder [128], bakery waste hydrolysate [101], and corncob hydrolysate [100], was able to produce more pigments at 30 °C. Numerous yeast species have been observed to yield more carotenoid pigments at pH 5 and temperature below 30 °C [10]. On the other hand, carotenoid production from microalgae Haematococcus pluvialis and Phormidium autumnale have demonstrated the maximum yield at 23 °C [79] and 26 °C [158], respectively. The pigment yield levels from microbes depends on the type of substrate used in submerged fermentation.

The identification of pigments from microbes is easier compared to non-pigmented microbial compounds. Identification of non-pigmented compounds on thin-layer chromatography (TLC) requires additional tests and UV visualization. However, rapid extraction, purification, and identification of pigments has become easy due to color appearance. For instance, the TLC technique, a simple and cost-effective method, was quick and effective to purify and identify red pigments [162]. TLC is a cheaper technique compared to other chromatographic techniques such as high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC), which are basically costly instruments that require more maintenance and costly consumables to process samples. Hence, TLC outstands as the cheapest and most efficient method to purify pigments. It is not clear whether or not pigmented microbes display cellular vitiligo (a condition in which the bacterial cell wall may display patchy loss of pigmentation) condition. However, it is easier to purify extracted pigments (intra- and extracellular) of any microbe using TLC. Some wild fungal species and some cultured species on agar plates release droplets of concentrated pigmented molecules on their filaments’ surface. These compounds are collected using a syringe and mixed (authors’ unpublished data) in methanol (because methanol has high polarity and better extractive yield) to test their bioactivity and colorant properties. It is not possible to obtain enough quantity of pigment droplets to test a large number of cytotoxicity and antimicrobial assays using this approach. Thus, this approach serves as a simple and rapid technique to determine the bioactive nature of pigment droplets using fewer bioassays. Thereby, this rapid method allows researchers to decide whether or not to choose a pigmented microbe that releases pigment droplets for submerged fermentation. Upon confirming the biological properties, pigment droplets can be purified easily using TLC. After obtaining clear, distinct pigment bands with TLC;TLC plates are allowed to dry at room temperature to evaporate the solvents on the silica gel. Then those bands are scraped using sterile pointed blades, and the eluted pigments are collected in a micro-vial to identify the pigments using HPLC, Fourier-transform infrared spectroscopy (FT-IR), liquid chromatography–mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) analyses.

Numerous studies have widely studied the potential biological properties of microbial pigments as antimicrobial and anticancer agents. The antimicrobial activities of microbial pigments against common pathogens and/or using strains that are available in their laboratory have been reported very often. However, the current research need is to find the molecules that combat multidrug-resistant microbes (MDRM) and a variety of cancer cells. Therefore, a routine antimicrobial investigation using pathogens (which are not of current interest) may be useful only for documentation but not in drug development research if considering the following reasons: (1) to find an effective pigment molecule against targeted MDRM and currently emerging pathogens, and (2) to develop a potential anticancer pigment molecule. Research work merely focusing on routine antibacterial properties for documentation and publications may no longer support the rapid development of drugs and help public health. Therefore, it is urgent to realize that the targeted research on the above two points using microbial pigments is very important to save time, research budgets, and hard work. In addition, one of the important notes is finding effective pigment molecules for rapid development of food colorant drug applications without repeating or duplicating the works performed before. The literature review has indicated the photodynamic photopigment therapy (i.e., the activation of photosensitizing pigments by light energy to treat a variety of diseases and infections) as an effective method to treat cancer and several microbial infections [45,163]. Therefore, studies that deal with microbial pigments need to perform photopigment therapy based on antimicrobial and other biological properties to understand the bioactive nature of microbial pigments in the presence and absence of light treatments.

Furthermore, the use of animal models in in vivo studies has constraints such as finance and ethics. In this regard, Galleria mellonella has been identified as a widely used, cheaper, and alternative model to study the cytotoxicity effect of a candidate drug. This invertebrate model requires no ethical approvals, is significantly cheaper, and its short lifespan enables it to be an ideal invertebrate model for high-throughput research [164]. The response of G. mellonella, which shares some similarities with the mammalian innate immune system, is the most crucial feature that makes it a useful preclinical in vivo model [165,166,167]. In comparison to mammals, this mini-host has economic and ethical benefits, and its short lifespan makes it an ideal model for high-throughput investigations of a variety of compounds [168,169]. They can readily be cultivated at 37°C in an incubator, giving researchers more control over the experimental situation and allowing them to examine clinically relevant human pathogens at a temperature similar to the human host, resulting in precise and reliable data [170]. Therefore, alternative invertebrate models such as G. mellonella larva may be used as an effective and rapid preclinical in vivo model to determine the cytotoxicity of pigments.

The global temperature has been increasing in recent years due to anthropogenic gases released from industrialization, automobiles, and the enormous use of greenhouse-gas-releasing systems [171]. Therefore, the current research trend has turned towards green energy, green chemistry, and green earth concepts. The toxic gases and water discharges released from the synthetic colorant manufacturing industries and textile industries using synthetic colorants are entering the atmosphere [39,42,172]. Therefore, the use of microbial pigments over synthetic colorants would eliminate toxic gases and other pollutants emitted from parties manufacturing and utilizing synthetic colorants. Therefore, efforts in this direction to implement natural pigments in every industry that uses pigments are needed urgently to arrest industrial emissions and to overcome environmental pollution, global warming, and climate change. The combination of green-energy-based industries and pigmented microbes could pave the way to reducing the industrial-based atmospheric and liquid chemical effluents. It is the need of the hour to understand the importance of the ecosystem rather than showing interest in color-appealing things (originated from industries) without knowing their (toxic emissions released from an attractive product that uses synthetic colorants) negative impacts on the environment and health. In addition, utilizing natural substrates over synthetic chemical substrates in any fermentation system may indirectly reduce the industrial emissions (by reducing synthetic chemical demand and emissions released from chemical manufacturing industries) into the atmosphere.

Light-harvesting primary pigments are known to capture CO₂ from the atmosphere. It is evident that many bacterial and fungal species found in agroecosystems [173] as well as aquatic microbes, especially marine microbes [174,175,176], are directly involved in carbon sequestration. However, little is known about the role of pigmented bacteria, fungi, and yeast in CO₂ sequestration, indicating the research gap to be studied. Nevertheless, pigments originating from microbes, especially bacteria (pigmented fungi and yeast are the least studied in this context), could indirectly help CO₂ capture by acting as potential growth promoters of plants [177,178] and biocontrol agents of phytopathogens [177,178,179] and insects [180]. Prodiginine obtained via mutasynthesis in Pseudomonas putida was reported to enhance the root growth of Arabidopsis thaliana at low concentrations [178]. Serratia marcescens isolated from cattle manure vermicompost [177] and halotolerant bacteria Bacillus and Halobacillus isolated from groundnut plants’ rhizosphere [180] showed growth-promoting abilities [177,181] and inhibited phytopathogenic fungi [177]. The prodigiosin pigment of S. marcescens isolated from Digitaria decumbens grass compost [179] and the rhizosphere of Bacopa monnieri acted as a biocontrol agent to phytopathogens [182]. Cell-free culture filtrates of pink pigmented Methylobacterium strains when added with 1.09 to 9.89 µg·mL⁻¹ of cytokinins showed a seed germination effect on wheat Triticum aestivum [183]. Liquid extracts from Spirulina platensis showed a seed germination effect on the groundnut Arachis hypogaea [184]. These studies indicate that microbial pigments could protect plants from phytopathogens, promote plant growth, and indirectly facilitate CO₂ capture by protecting plants from chloroplast damage and photosynthesis arrest.

Fungal species have also been shown to be involved in CO₂ sequestration, in particular, soil fungi dramatically benefit the environment and ecosystem in a positive way [185]. Numerous research studies have shown that arbuscular mycorrhizal fungi (AMF) play a role as climate change warriors. The mycorrhizal fungi have a symbiotic relationship with plants by colonizing the root cells, where they form a large hyphal network and exert major control on transporting carbon [186,187,188]. In addition, the symbiotic association of fungi with plants has fundamental effects on the plant physiology and growth, and especially helps to utilize phosphorus and nitrogen, thus aiding in stimulating plant growth. For example, hyphae of AMF produce a glycoprotein called glomalin, which protects hyphae against nutrient or water losses, glues together soil aggregates, and improves nutrient cycling as well as nutrient uptake in plants [189,190]. Similarly, AMF of the phylum Glomeromycota boost water and nutrient exchange in plant roots through their hyphae [191,192]. Many Rhizobium spp., colonize the plant root cells of some plants of the legume family, which helps in nitrogen fixation [193].

Ectomycorrhizal fungal (EMF) species of Suillus, Piloderma, and Cortinarius are predominant in boreal forests and are likely to play a crucial role in storing soil carbon in mycorrhizal forests [194,195]. Furthermore, species of Suillus and Cortinarius are involved in forest restoration and are linked to rapid turnover of microbial biomass and efficient nitrogen utilization in the forest plants [196]. In addition to these benefits, it was reported that several species of Trichoderma have been used as successful biocontrol agents owing to this potential action against phytopathogens. In a study by Lombardi et al., it was found that Trichoderma spp. stimulated strawberry plant growth, improved fruit yield, and improved the accumulation of anthocyanins and antioxidants in red ripened fruits [197]. Piriformospora indica also exhibited a multifunctional role in diverse plant species mainly by regulating plant metabolism and improving plant tolerance to various biotic and abiotic stresses [198]. Considering the role of fungal pigments in carbon sequestration and their influence on ecosystem function, little is known. However, it was demonstrated that the highly melanized fungus Cenococcum geophilum is drought-tolerant in water-stressed habitats. It is suggested that melanin is an important functional trait that allowed the hyphae to penetrate deeper into the soil to access water, and it is considered that melanin production helps with this function [199]. Hence, by closely work together with plants, fungal communities can potentially strengthen their defense mechanisms, improve resilience to plant diseases, enhance nutrient uptake through well-developed roots, store soil carbon, and so on.

Although several species of yeast have demonstrated plant-growth-promoting ability [200], the ability of pigmented yeast to promote plant growth has not been studied, whereas, similar to macroalgal culture beds [201], several investigations found that large-scale culture of microalgae in open systems, bioreactors [202,203,204,205,206,207,208,209], and integrated culture systems [210] play an important role in the mitigation of atmospheric CO₂. In this way, bacteria, fungi, and microalgae offer multifaceted applications to society and protect the environment by regulating CO₂ levels.

7. Current Applications of Microbial Pigments