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Ramesh, C.;  Prasastha, V.R.;  Venkatachalam, M.;  Dufossé, L. Pigments Production from Potential Microbes in Submerged Fermentation. Encyclopedia. Available online: https://encyclopedia.pub/entry/28932 (accessed on 09 July 2025).
Ramesh C,  Prasastha VR,  Venkatachalam M,  Dufossé L. Pigments Production from Potential Microbes in Submerged Fermentation. Encyclopedia. Available at: https://encyclopedia.pub/entry/28932. Accessed July 09, 2025.
Ramesh, Chatragadda, V. R. Prasastha, Mekala Venkatachalam, Laurent Dufossé. "Pigments Production from Potential Microbes in Submerged Fermentation" Encyclopedia, https://encyclopedia.pub/entry/28932 (accessed July 09, 2025).
Ramesh, C.,  Prasastha, V.R.,  Venkatachalam, M., & Dufossé, L. (2022, October 11). Pigments Production from Potential Microbes in Submerged Fermentation. In Encyclopedia. https://encyclopedia.pub/entry/28932
Ramesh, Chatragadda, et al. "Pigments Production from Potential Microbes in Submerged Fermentation." Encyclopedia. Web. 11 October, 2022.
Pigments Production from Potential Microbes in Submerged Fermentation
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This entry is adapted from the peer-reviewed paper 10.3390/fermentation8090460

Pigments from bacteria, fungi, yeast, cyanobacteria, and microalgae have been gaining more demand in the food, leather, and textile industries due to their natural origin and effective bioactive functions. Mass production of microbial pigments using inexpensive and ecofriendly agro-industrial residues is gaining more demand in the current research due to their low cost, natural origin, waste utilization, and high pigment stimulating characteristics. A wide range of natural substrates has been employed in submerged fermentation as carbon and nitrogen sources to enhance the pigment production from these microorganisms to obtain the required quantity of pigments. Submerged fermentation is proven to yield more pigment when added with agro-waste residues.

microbial pigments submerged fermentation natural substrates antimicrobial and anticancer activities

1. Introduction

The invisible microbial world comprises both non-pigmented and an array of pigmented microbes. They are visible to the human eye when cultured on agar plates. In the microbial world, numerous pigmented bacteria [1][2], fungi [3][4][5][6], yeast [7][8][9][10], cyanobacteria [11], and microalgae [12][13] have been identified. Pigment production in these organisms is mostly taxa or species specific, and their intracellular or extracellular pigments are extracted using traditional and green methodologies [14][15]. In general, these pigments are produced by microbes to tolerate environmental stress, for survival and competition [16]. Various microbial pigments of different chemical origins have demonstrated a wide range of applications [1][17][18]. Among the numerous known microbial pigments, prodigiosin [19][20][21], violacein [22], carotenoids [10][23][24][25][26][27], melanins [28][29], azaphilones [30][31], phycocyanin [32], phycoerythrobilin [33][34], and riboflavin [35] pigments have gained more demand in the global context as value-added products and medicine [23].
Although vast literature is available on plant pigments [36][37][38], microbial pigments are gaining more demand due to easy cultivation, large-scale production throughout the year, and biodegradability. Another reason for microbial pigment demand is the side effects posed from synthetic colorants, such as hyperactivity disorder, cancer, allergies, and teratogenicity [39][40][41]. More significantly, synthetic dye effluents released from textile dyeing have been found to cause plant growth inhibition, air pollution, water contamination, and human illnesses [42]. Hence, many microbial pigments are used as food colorants [43]. The increasing demand for microbial pigments [2][44][45], mostly carotenoids, by various companies [10][23], is a sign of replacing the overuse of synthetic colorants in a wide range of applications.

2. High Pigment Yielding Natural Substrates

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 [46].
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 [47]. However, only some substrates are demonstrated to yield more pigment [10][45][47][48][49]. Several studies have investigated a large number of agro-industrial substrates in solid-state fermentation compared to submerged fermentation [50]. 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 [51]. 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 [52] and peanut oil cake [53].
Monascus purpureus culture produced more pigment yield when tested with corncob hydrolysate [54], bakery waste hydrolysate [55], brewer’s spent grain [56], and glucose fermentation medium added with rice straw hydrolysate [57]. Monascus ruber produced significantly low pigment yield when supplemented with sugarcane bagasse hydrolysate [58]. 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 [59]. Many agro-industrial residues tested were found to enhance carotenoid production from several yeast species [10][45]. Loquat kernel extract [60], sugar beet molasses [61], and sugar cane extracts [62] were the two substrates reported to enhance yeast carotenoids greatly.

3. Submerged Culture Conditions for Pigment Production

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 research 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 [51]. 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 [63], bakery waste hydrolysate [55], and corncob hydrolysate [54], 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 [64] and 26 °C [65], respectively. The pigment yield levels from microbes depends on the type of substrate used in submerged fermentation.

4. Rapid Identification of Microbial Pigments

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 [66]. 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 (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.

5. Need for Targeted Drug Research on Microbial Pigments

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 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][67]. 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 [68]. 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 [69][70][71]. 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 [72][73]. 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 [74]. 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.

6. Role of Pigmented Microbes in Climate Change

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 [75]. 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][76]. 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 [77] as well as aquatic microbes, especially marine microbes [78][79][80], 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 [81][82] and biocontrol agents of phytopathogens [81][82][83] and insects [84]. Prodiginine obtained via mutasynthesis in Pseudomonas putida was reported to enhance the root growth of Arabidopsis thaliana at low concentrations [82]. Serratia marcescens isolated from cattle manure vermicompost [81] and halotolerant bacteria Bacillus and Halobacillus isolated from groundnut plants’ rhizosphere [84] showed growth-promoting abilities [81][85] and inhibited phytopathogenic fungi [81]. The prodigiosin pigment of S. marcescens isolated from Digitaria decumbens grass compost [83] and the rhizosphere of Bacopa monnieri acted as a biocontrol agent to phytopathogens [86]. 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 [87]. Liquid extracts from Spirulina platensis showed a seed germination effect on the groundnut Arachis hypogaea [88]. 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 [89]. 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 [90][91][92]. 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 [93][94]. Similarly, AMF of the phylum Glomeromycota boost water and nutrient exchange in plant roots through their hyphae [95][96]. Many Rhizobium spp., colonize the plant root cells of some plants of the legume family, which helps in nitrogen fixation [97].
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 [98][99]. 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 [100]. 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 [101]. 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 [102]. 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 [103]. 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 [104], the ability of pigmented yeast to promote plant growth has not been studied, whereas, similar to macroalgal culture beds [105], several investigations found that large-scale culture of microalgae in open systems, bioreactors [106][107][108][109][110][111][112][113], and integrated culture systems [114] 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

The numerous applications of microbial pigments in food, textile, leather, cosmetic, and drug industries have been reviewed very often in the last five years by various researchers [1][2][68][115][116][117][118][119]. Here, researchers detail the selected and recent applications of microbial pigments in various areas. Undecylprodigiosin, a prodigiosin pigment derivative, was reported to have dyeing, food colorant, and antimicrobial properties [66][120]. Particularly, undecylprodigiosin and other unidentified pigment molecules have been demonstrated to show a high affinity to staining transverse sections of Tridax procumbens [66], indicating the application of these pigments as natural stains in laboratory studies. Recently, the prodigiosin pigment extracted from Serratia plymuthica has been used to develop an antibacterial (against S. aureus and P. aeruginosa) food packaging system in combination with bacterial cellulose and a chitosan composite [121]. Similarly, the development of antimicrobial textiles for hospital-acquired infections has been demonstrated using prodigiosin extracted from Serratia rubidaea [122]. The flexirubin pigment extracted from Chryseobacterium artocarpi was used to make soaps [123]. Tyrian purple indigoid (originated from Murex) synthesized from E. coli has potential dye applications [124]. Indigoid pigments are reported to have chemosensory and semiconductor properties [125]. Phycocyanin extracted from Arthrospira platensis is used as a fluorescent probe in medical, food safety, and environmental research [126][127]. Microalgae is one of the major sources of nutraceuticals, pharmaceuticals, and biogases [128], especially having anticancer properties [27][129][130]. Thus, microalgae-based pigments have also been gaining more attraction in the industry compared to bacterial and fungal pigments.

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Subjects: Biotechnology & Applied Microbiology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Chatragadda Ramesh
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V. R. Prasastha
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Mekala Venkatachalam
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Laurent Dufossé
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