An Overview on Industrial and Medical Applications of Bio-Pigments Synthesized by Marine Bacteria

Marine bacterial species contribute to a significant part of the oceanic population, which substantially produces biologically effectual moieties having various medical and industrial applications. The use of marine-derived bacterial pigments displays a snowballing effect in recent times, being natural, environmentally safe, and health beneficial compounds. Although isolating marine bacteria is a strenuous task, these are still a compelling subject for researchers, due to their promising avenues for numerous applications. Marine-derived bacterial pigments serve as valuable products in the food, pharmaceutical, textile, and cosmetic industries due to their beneficial attributes, including anticancer, antimicrobial, antioxidant, and cytotoxic activities. Biodegradability and higher environmental compatibility further strengthen the use of marine bio-pigments over artificially acquired colored molecules. Besides that, hazardous effects associated with the consumption of synthetic colors further substantiated the use of marine dyes as color additives in industries as well. This review sheds light on marine bacterial sources of pigmented compounds along with their industrial applicability and therapeutic insights based on the data available in the literature. It also encompasses the need for introducing bacterial bio-pigments in global pigment industry, highlighting their future potential, aiming to contribute to the worldwide economy.


Microbial Pigments
The production of bio-pigments from bacterial species is being conducted globally with soaring interest under the research of microbial autecology. A massive array of these compounds, also referred to as "bioactive pigmented molecules", can be derived from both Gram-positive and Gram-negative bacterial species. Production of these pigments in the marine environment is mediated through the complex mechanism of "quorum sensing" [1] or also can be induced through exposure to different stress conditions in external environments. Quorum sensing is the mechanism whereby individual bacterial cells can coordinate with others in their colony to carry out constitutive functions especially involving the secretion of numerous specific chemical compounds. These compounds can help them with survival, competence, bioluminescence, biofilm formation, and even sporulation, etc. Bio-pigments can be produced by triggering regulatory quorum sensing mechanisms of these species and can be extensively used in various bio-medical and bio-industrial sectors, including textiles, food, pharmaceutical, and cosmetic industries, owing to their beneficial attributes and biological activities [2,3]. These are moreover convenient to harvest in large volumes through utilizing simple gene manipulating strategies. The rising

Marine Ecosystem as a Source of Pigment Producing Bacterial Species
The study of a likely natural ecosystem serves as the initial-most important research step needed to find an environment that can entertain the diversity of bio-pigment sources. The marine environment is a habitat for almost 80% of all life forms [14]. It serves as a rich source of aquatic microbial species that exhibit comparatively more augmented diversity than their telluric counterparts [15]. The marine environment is presently being considered as an attractive fount for bio-pigment sources [16]. Numerous bacterial isolates from such biotopes have already been tested for pigment production. At the same time, many of them are also being utilized for various industrial purposes as well [15]. The preference of pigments produced by marine microorganisms is based on their ability to persist in extremities such as highly acidic/alkaline environments (pH < 4 and >9), extreme temperatures (−2-15 • C and 60-110 • C), and under limited substrate availability [17,18]. Apart from bacterial isolates, halophilic archaea are extensively disseminated in the marine ecosystem. Pigmented compounds from marine archaea are also prioritized owing to their ability to tolerate hyper saline and basic pH environments, besides their potential to withstand osmolytes (such as 2-sulfotrehalose) or high ionic strength [19,20].
Concerns regarding environmental conservation and consumers' preferences have stimulated the interests of researchers and stake holders in exploring nontoxic, eco-friendly, and biodegradable commodities. Bacterially produced bio-pigments (bpBPs) have growing importance not only on account of their dyeing potential, but also due to their medicinal properties. Likewise, awareness regarding the carcinogenic and other pernicious effects of synthetic colorants has kindled a fresh enthusiasm towards the utilization of bacterial pigments in the food industry as safer alternatives to use as antioxidants, color intensifiers, flavor enhancers, and food additives.
Extraction of natural pigments from microorganisms populating environments exclusive of soil is a topic of current interest. Marine environment has become a captivating subject matter for microbiologists, pharmacologists, and biochemists in order to extract water based bacterial pigments. With the recent increase in awareness towards the benefits of natural over synthetic products, the bio-pigment industry is likely to increase its global market. The review aims at discussing the therapeutic and industrial significance of marine derived bacterial pigments helping to delineate the consequence of furthering the scope of these studies. It provides a comprehensive overview of potentiality and competence of marine bacteria as a source of bio-pigments by critically summarizing the scientific researches and accumulated data in the literature and the prominence of these bio-pigments in strengthening the overall pigment market by reviewing latest industry market research, reports, and statistics.

Marine Bacterial Species as Sources of Bio-Pigments
The marine environment has been investigated for almost 300,000 known species, which constitutes only a small fraction of the total number of explorable pigment producing bacterial species. Bacterial species isolated from marine sediments or seawater such as Streptomyces sp., Pontibacter korlensis sp., Pseudomonas sp., Bacillus sp., and Vibrio sp. produce an array of pigmented compounds including prodigiosin, astaxanthin, pyocyanin, melanin, and beta carotene, respectively (Table 1). These pigments belong to a range of compound classes, for instance, carotenes are a subclass of carotenoids that have unsaturated polyhydrocarbon structures, prodiginines have a pyrrolyldipyrromethene core structure, tambjamines are alkaloid molecules, while violacein compounds are indole derivatives derived from tryptophan metabolism ( Figure 1) [1,2,21]. These and other such pigments, despite their class diversity, share a functional likeness due to the presence of aromatic rings in their structures.

Biosynthesis of Bacterial Pigments
The potential of marine bacterial isolates as a leading source of bio-pigments demands an extensive understanding of bio-mechanisms responsible for yielding pigmented molecules. Different studies have reported the proposed biosynthetic pathways of pigment production by marine bacterial isolates along with biochemically characterized enzymatic transformations ( Figure 2). However, it is still unclear if the proposed pathways are distinct for marine or terrestrial bacterial species, or may be the same in both cases.    Pyrrole-2-carboxyl thioester is generated by the transfer of pyrrole-2carboxyl group of PigG to the cysteine active site at PigJ. Phosphopantetheinylated ACP domains of PigH provide binding sites for malonyl group of malonyl-CoA. Decarboxylation of bound malonyl results in condensation with pyrrole-2-carboxyl thioester and leads to the formation of pyrrolyl-β-ketothioester on PigH. Generation of 4-hydroxy-2,2 -bipyrrole-5-methanol (HBM) occurs by decarboxylation between serine and pyrrolyl-β-ketothioester, catalyzed by PigH [80,82]. 4-hydroxy-2,2 -bipyrrole-5-carbaldehyde (HBC) is formed when PigM oxidizes the alcohol group of HBM. Methyltransferase (PigF) and oxidoreductase (PigN) further carries out the methylation of HBC hydroxyl group to form MBC [81]. After the formation of MAP and MBC, PigC utilizes ATP to perform terminal condensation of these pyrroles, synthesizing prodigiosin.
Cycloprodigiosin (cPrG) biosynthesis: The cyclization of undecylprodiginine in order to form metacycloprodigiosin and butyl-meta-cycloheptylprodiginine is carried out by mcpG and redG, respectively [83]. Studies also revealed that a homologus gene (PRUB680) encodes an alkylglycerol monooxygenase-like protein away from pig biosynthetic gene cluster [84]. The respective enzyme demonstrates regiospecificity through C-H activation, resulting in cyclization of prodigiosin to form cPrG [78].

Biosynthesis of Carotenoids
Carotenoids are yellow, orange, and red colored pigmented compounds that are further subdivided into carotenes and xanthophylls. So far, 700 carotenoids have been reported, and among them beta-carotene, lutein, canthaxanthin, astaxanthin, lycopene, and zeaxanthin are the highly valued carotenoids [87]. Universal precursors for C40 and C50 carotenoid biosynthesis are two 5 C subunits: isopentenyl diphosphate (IPP) plus its isomeric form dimethylallyl diphosphate (DMAPP). IPP/DMAPP isomerase (IDI) carries out the isomerization of IPP into DMAPP. Geranylgeranyl diphosphate (GGPP) synthase further catalyzes the addition of one DMAPP molecule with three IPP molecules to generate an immediate precursor, i.e., C20 geranylgeranyl diphosphate (GGPP) [88]. Phytoene synthase carries out the first committed step of carotenoid biosynthesis i.e., condensing two GGPP molecules to form phytoene (C40), which is further desaturated by phytoene desaturase by the incorporation of four double bonds in its structure. This desaturated structure is a red-colored compound unlike its colorless parent molecule, and is called lycopene. Lycopene further undergoes several modifications to produce different carotenoids. Beta-carotene is generated by the cyclization of lycopene, carried out by lycopene betacyclase. It is then converted into canthaxanthin and zeaxanthin by catalytic activity of two protein classes: beta-carotene ketolase and beta-carotene hydroxylase, respectively, next to the formation of astaxanthin [30]. Beta-carotene ketolase represented by CrtW and CrtO types adds the ketone group to carbon 4/40 of the b-ionone ring. However, beta-carotene hydroxylase, encompassed by CrtR, CrtZ, and P450 types carries out the hydroxylation of carbon 3/30 of the b-ionone ring [89]. 2,2 -β-ionone ring hydroxylase introduces hydroxyl group to the β-ionone ring of astaxanthin and results in the formation of 2,2 -dihydroxy-astaxanthin ( Figure 2b) [33].

Biosynthesis of Scytonemin
Biosynthesis of scytonemin involves three scytonemin biosynthetic enzymes; ScyA, ScyB, and ScyC. ScyB carries out the conversion of L-tryptophan to 3-indole pyruvic acid. ScyA (thiamin-dependent enzyme) performs the coupling of 3-indole pyruvic acid with phydroxyphenylpyruvic acid and results in the formation of b-ketoacid, whose cyclization is further carried out by ScyC (enzyme annotated as hypothetical protein) [90]. The resulting tricyclic ketone resembles half of the skeleton of scytonemin (Figure 2c) [91].

Antibacterial Activity
Antibacterial properties of various bacterially produced bio-pigments of marine origin have been reported against an array of bacterial species, e.g., prodigiosin, cycloprodiogisin (from Z. rubidus sp. S1-1), and the yellow pigment (extracted from Micrococcus sp. strain MP76) have shown antibacterial activity against Staphylococcus aureus sp. and Escherichia coli sp. [98,99]. Other bacterial strains that are reportedly inhibited by prodigiosin and cycloprodigiosin are Bacillus subtilis sp. and Salmonella enterica serovar Typhimurium [98]. Likewise, the yellow pigment has shown activity against P. aeruginosa sp. as well [99]. Norprodigiosin synthesized by marine Serratia sp. has also been reported to exhibit inhibition activity against Vibrio paraheamolyticus sp. and B. subtilis sp. [32]. These studies strengthen the utilization of bpBPs as potential alternatives to synthetic medicinal compounds.
Furthermore, inhibition activities recorded against Citrobacter sp. by pyocyanin and pyorubin [58] and P. aeruginosa sp. by violacein pigment (purified from Antarctic Iodobacter sp.) [100], further stretches the range of marine-derived bpBP's potential against pathogenic bacterial species to opportunistic bacterial species. There are numerous correspondingly published studies. The pigment "melanin" from marine Streptomyces sp., for instance, demonstrated antibacterial activity against E. coli sp., S. typhi sp., S. paratyphi sp., Proteus mirabilis sp., Vibrio cholera sp., S. aureus sp., and Klebsiella oxytoca sp. [68]. A bright pinkorange colored pigment extracted from Salinicoccus sp. (isolated from Nellore sea coast) also showed antimicrobial potential against several bacterial strains including E.coli sp., Klebsiella pneumoniae sp., B. subtilis sp., Proteus vulgaris sp., P. aeruginosa sp., and S. aureus sp. [101]. Hence, these and similar other studies all indicate the exploration of marine bacterial species as a dynamic approach to derive antibacterial compounds.
A few studies also seemingly suggest that a single pigment from different species may exhibit activities against various target microorganisms. One example is violacein, a violet colored pigment extracted from Antarctic bacterium Janthinobacterium sp. SMN 33.6, which showed antibacterial activity against multi-resistant bacteria: S. aureus sp. ATCC 25923, E. coli sp. ATCC 25922, Kocuria rhyzophila sp. ATCC 9341, and S. typhimurium sp. ATCC 14028 [102], and that extracted from Collimonas sp. showed antibacterial activity against Micrococcus luteus sp. [67].

Antifungal Activity
Studies have also been carried out to determine the antifungal potential of natural pigmented compounds. Several studies have reported the antifungal activity of marinederived bacterial pigments, among which violacein from Chromobacterium sp. and prodiginine pigments (prodigiosin and cycloprodigiosin) extracted from Indonesian marine bacterium P. rubra sp. reported to exhibit antagonistic activity against Candida albicans sp. [23,103]. Violacein also inhibited several other fungal strains, including Penicillium expansum sp., Fusarium oxysporum sp., Rhizoctonia solani sp., and Aspergillus flavus sp. Studies have also reported that violacein (extracted from a pure Chromobacterium sp.) shows comparable antifungal activity to that of bavistin and amphotericin B, highlighting the potential of marine-derived bpBPs as effective antifungal agents over existing synthetic antifungal compounds [103].

Anticancer Activity
Exploring anticancer compounds from marine microbes has been considered a hot spot in natural product research. Several studies have been carried out in order to examine the antitumor ability of marine bacterial pigments. Anticancer activity of marinederived bpBPs has been explored against several cancerous cell lines. Astaxanthin and 2-(p-hydroxybenzyl) prodigiosin (HBPG) isolated from P. kolensis sp. and P. rubra sp. displayed significant cytotoxicity against human breast cancer cell line (MCF-7) and human ovarian adenocarcinoma cell line, respectively [38,104]. PCA (Phenazine -1-carboxylic acid) pigment extracted from marine P. aeruginosa sp. GS-33 correspondingly showed inhibition against SK-MEL-2 (human skin melanoma cell line) [105]. Another pigment violacein extracted from Antarctic bacterium isolate, identified as a member of the genus Janthinobacterium (named as Janthinobacterium sp. strain UV13), revealed its antiproliferative activity in HeLa cells. Studies further confirmed the potential of violacein as an anticancer agent to cisplatin drug (anticancer chemotherapy drug) in cervix cell carcinoma [106]. It has also been reported that a single pigment can express anticancer activity against multiple cancerous cell lines. Synthetically derived tambjamines isolated from the marine bacterium P. tunicata sp. have shown significant apoptosis inducing effects against various cancer cell lines including glioblastoma cell line (SF-295), M14 melanoma cell line (MDA-MB-435), ileocecal colorectal adenocarcinoma cell line (HCT-8), and promyelocytic leukemia cells (HL-60) [107]. Carotenoid pigments extracted from marine Arthrobacter sp. G2O (isolated from the Caspian Sea) exhibited antitumor activity on esophageal squamous cancerous cells [108]. Likewise, prodigiosin homolog extracted from marine bacterium Serratia proteamacula sp. was also found to exhibit high antitumor activity [109], indicating the potential of marine-derived bpBPs in antitumor therapy.

Antiviral Activity
The advancing viral pandemics have taken a toll over the limited pool of existing antiviral agents, which has led to a rigorous search for newer, natural compounds with better antiviral capacities. Various studies on marine bpBPs suggest them as potential candidates. Prodigiosin extracted from Serratia rubidaea sp. RAM_Alex showed antiviral activity against hepatitis C virus (HCV) upon injecting HepG2 (human liver cancer cell line) cells with 2% of HCV infected serum (Table 2) [116]. Other carotenoid pigments (from Natrialba sp. M6) have also displayed complete elimination of HCV and clearance of 89.42% of hepatitis B virus (HBV) [117], indicating the use of marine pigments as availing antiviral agents. Table 2. Therapeutic applications of bio-pigments extracted from marine bacterial isolates.

Bio-Pigments as Dyeing Agents
The worldwide demand for clothes is rising exponentially. Newly, there is an increase in the insistence of incorporating antimicrobial properties in fabrics. Lee et al. identified a novel marine bacterium Z. rubidus sp. S1-1 that produced two significant pigments, i.e., prodigiosin and cycloprodigiosin. These were used to dye cotton and silk fabrics. Results revealed that the application of red-pigmented extract solution on fabrics reduced the growth rate of S. aureus sp. KCTC 1916 by 96.62% to 99.98% and E. coli sp. KCTC 1924 by 91.37% to 96.98% [98]. Furthermore, Vibrio sp. isolated from marine sediment produced a bright red colored prodiginine pigment that was used to dye nylon 66, silk, wool, acrylic, and modacrylic fabrics to obtain a pretty deep-colored shade. The dyed silk and wool fabrics also showed antibacterial activity against E. coli sp. and S. aureus sp. [121]. Researchers at Ulsan National Institute have also reported the synthesis of antibacterial fabric by using violacein pigment extracted from C. violacea sp. [122,123]. Prodigiosin pigment extracted from Serratia sp. BTWJ8 effectively dyed paper, PMMA (Polymethyl methacrylate sheets), and rubber latex. Rubber is commonly used in day to day life either in houses or industries. PMMA have been widely utilized for the construction of lenses for exterior lights of automobiles. Different concentrations of prodigiosin produced variable color shades that revealed its affectivity as a coloring agent [124].

Use in Cosmetics
The cosmetic industry is an expeditiously emerging global business market. About 2000 companies in the United States of America are cosmetic manufacturers. It is estimated that American adults use seven different skincare products per day for everyday grooming [125]. The cosmetic industry has a worth of 10.4, 10.6, and 13.01 billion euros in the UK, France, and Germany, respectively [126]. Considering the cosmetic market value worldwide, researchers have also made efforts to explore the use of marine-derived bpBPs in skincare products. The addition of the pigment PCA in a solution of commercial sunscreen enhanced its UV-B (ultraviolet B-rays) protection and increased the SPF (sun protection factor) values up to 10% to 30% [105].
Similarly, melanin incorporated cream (named cream F3) was synthesized by concentrates of seaweed (Gelidium spinosum) and melanin pigment (extracted from marine bacterium Halomonas venusta sp.). Cream F3 showed high SPF values and photoprotective activity and demonstrated great effectivity in wound healing as well. Moreover, the formulated cream also exhibited antibacterial activity against skin pathogens; Streptococcus pyogenes sp. (MTCC 442), and S. aureus sp. (MTCC 96) [127]. Another research reported the effectivity of melanin (extracted from marine bacterium Vibrio natriegens sp.) in protecting mammalian cells from UV irradiation. Results revealed 90% survival rate of HeLa cells in melanized cell culture [128]. In another report, Bio lip balm made from crude pigment (extracted from S. bellus sp. MSA1) in a mixture of coconut oil, lanolin, and shredded bee wax [114] suggested the use of melanin pigment as a significant ingredient in several beauty care products as well.

Antifouling Agent
Billions of dollars have been spent each year to control fouling activities on different objects placed in the marine environment. Biofouling on ships such as dreadnoughts in-creased the roughness of the hull, which promotes frictional resistance, ultimately leading to an increase in fuel consumption and other corresponding environmental compliances. Heavy metal-based antifoulants cause severe environmental complications, which further mandate the need for "eco-friendly" antifouling agents. Researchers have also revealed the use of marine-derived bpBPs for their role as an antifouling agent, for instance, prodigiosin extracted from Serratia. sp. was reported to exhibit antifouling activity against marine fouling bacterial species such as Gallionella sp. and Alteromonas sp. It also inhibited the adhesion of Cyanobacterium sp. on the glass surface [129]. Likewise, another pigment, polymelanin synthesized by the marine bacterium P. lipolytica sp., prevented metamorphosis and decreased the invertebrate larval settlement [130], hence indicating the role of marine bacterial pigments as potential antifoulants.

Photosensitizers
The use of prodigiosin has also been reported as photosensitizers in solar cells. The high photostability of extracted prodigiosin demonstrated its use as a sensitizer in dye-sensitized solar cells (DSSC) ( Table 3) [131]. This study suggests the viability of bpBPs in addition to that of prodigiosin for the construction of cost-effective and low tech industrially produced DSSC. Table 3. Industrial applications of bio-pigments extracted from marine bacterial isolates.

Industrial Importance and Global Market Trends of Pigmented Compounds
Pigments are already utilized in various nutritional supplements, antibiotics, skin care, and other industrial products ( Table 4). The most valuable pigments in the global market are beta-carotene, lutein, and astaxanthin ( Figure 3). Astaxanthin has its wide use in nutraceutical industries owing to its antioxidant properties and numerous health benefits. It has also been in wide use in cosmetic industries due to its antiaging activity. Moreover, astaxanthin is being utilized in aquaculture industries to carry out the pigmentation of shrimps, trouts, and salmons. At the industrial scale, astaxanthin production is accomplished using Paracoccus sp. It was predicted that the sales volume of astaxanthin by the year 2020 would be 1.1 billion US dollars [132], and the astaxanthin market is estimated to reach up to 3.4 billion US dollars with CAGR (compound annual growth rate) of 16.2% in 2027 [133].  FDA accepted the use of beta-carotene as a color additive in food products in the year 1964. Additionally, in 1977, the use of beta-carotene got approved in cosmetics also. The E-Number allotted to beta-carotene is E160a. Over and above, canthaxanthin use in food and broiler chicken feed got authorized in 1969, and the E-Number assigned to it is E161g [159]. Lycopene is also being utilized for many industrial purposes, and it is reckoned that the lycopene market will grow at a rate of 5.3% CAGR, by the end of 2026 [160]. The global market potential of carotenoid pigments is estimated to reach up to 5.7% by 2022 [166]. Europe and USA are the key business markets for carotenoid pigments [118]. It is expected that the global carotenoid market will increase from 1397.59 million US$ in 2018 to 2124.68 million dollars by the end of 2025, at the CAGR of 6.16% [167]. For a long time, different classes of pigmented compounds have occupied the entire market due to their wide range of applications in different industries ( Figure 4). It is anticipated that by the year 2022, the global market of food colorants will reach up to 3.75 billion US$, along with farming colorant market, to touch 2.03 billion US$ by the year 2022 [166]. Europe holds the forefront for cutting synthetic colorants' economy by utilizing natural dyes, which make up 85% of total dyes produced. It is evaluated that growing interests towards ready to eat and pre-packaged food items in China, India, and Middle-East countries will also drive the market of food colorants in the Asia Pacific as well [168].

Conclusions
Marine bacterial pigments can be a potential substitute for synthetic products to fulfill market demand and to ensure the public well-being. Putting aside the fact that synthetic medicines combat bacterial infections, they also pose adverse effects in terms of health. Likewise, artificial colorants due to the presence of azo dyes and heavy metals can also ignite cancer and other allergies. Microbial pigments derived from marine bacteria can be a promising approach to tackle the detrimental effects of these synthetic compounds.
Furthermore, marine bacteria tolerate a vast range of environmental conditions. Due to their unique biological properties, natural pigments from the marine environment also have wide range of applications in pharmaceutical, food, cosmetics, paper, and textile industries [1]. Marine bacterial species can be cultured in vitro and are genetically modified to get the desired level of pigments. Anyhow, there are certain limitations in implementing these naturally derived marine bacterial pigments on an industrial scale significantly: less percentage annual production, technological imperfections, low stability, high need for cost investments, and health complications [11]. For replacing synthetic products, efforts are required to explore new microbial sources and finding better optimization techniques to enhance the production of pigmented compounds. Genetic engineering and other strain development techniques should also be further studied to harvest bioactive pigments from marine bacterial species. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.