Bacterial pigments: A colorful palette reservoir for biotechnological applications

Abstract Synthetic derivatives are currently used instead of pigments in many applicative fields, from food to feed, from pharmaceutical to diagnostic, from agronomy to industry. Progress in organic chemistry allowed to obtain rather cheap compounds covering the whole color spectrum. However, several concerns arise from this chemical approach, as it is mainly based on nonrenewable resources such as fossil oil, and the toxicity or carcinogenic properties of products and/or precursors may be harmful for personnel involved in the productive processes. In this scenario, microorganisms and their pigments represent a colorful world to discover and reconsider. Each living bacterial strain may be a source of secondary metabolites with peculiar functions. The aim of this review is to link the physiological role of bacterial pigments with their potential use in different biotechnological fields. This enormous potential supports the big challenge for the development of strategies useful to identify, produce, and purify the right pigment for the desired application. At the end of this ideal journey through the world of bacterial pigments, the attention will be focused on melanin compounds, whose production relies upon different techniques ranging from natural producers, heterologous hosts, or isolated enzymes. In a green workflow, the microorganisms represent the starting and final point of pigment production.


INTRODUCTION
The term Phoenicians origins from the Greek "phoenix," the red purple pigment extracted from mollusc shells to dye textiles 1 . Currently, microbiologists and biotechnologists, behaving as "modern Phoenicians," focus their attention on the microbial world as a plentiful source of pigments, which are exploited by different industrial fields, including the alimentary, cosmetic, pharmaceutical, and environmental industry. Indeed, most prokaryotic microorganisms produce chemically different colored compounds, carrying out diverse physiological functions. Bacteria and Archaea are widespread and may inhabit extreme environments, including oceans, volcanos, saline, which constitute an endless reservoir of microorganisms producing bioactive colored compounds. In this scenario, the availability of plentiful sources for microbial pigments has a great potential for the discovery of new bacterial-derived dyes 2 . As much as the attention for the environment sustainability is increasing, this natural and unlimited colorful palette represents an environmentally friendly source that can substitute synthetic analogue compounds. Bacteria as a source for biocolorants are preferred to other natural sources, including plants and fungi, because of their stability and availability for cultivation throughout the year 3 . As an added value, bacterial cultivation may follow the principles of the circular economy aiming at waste elimination. Exploitation of agricultural wastes, such as molasses, seeds, and peels, not only allows to address disposal and environment pollution issues, but also to grow biocolor producing microorganisms 4 . The United States Food and Drug Administration (US FDA) and European Food Safety Authority (EFSA) have approved few bacterial pigments for the nutraceutical industry, such as astaxanthin and β-carotene from phototrophic bacteria, thus far 5,6 . Indigo, one of the oldest pigments for dyeing textiles, especially denim, is currently obtained by chemical synthesis. As this strategy is based on fossil feedstocks, scientists propose a more environmentally sustainable biotechnological process that exploits microbial biocatalytic systems 7 . In this context, it is reasonable to predict that microorganisms will be even more investigated in the future with the intent to solve problems related to human and animal health.
The aim of this review is to focus the attention on the most investigated pigments produced by bacteria and highlight the link between their physiological role and the possible biotechnological applications. In this last regard, a more clear-cut knowledge of the biological functions of pigment producers has, at least, two important implications: (1) it allows the discovery of potential applications in different biotechnological fields, and (2) it favors the development of appropriate strategies to implement their production. Moreover, the discovery of the genetic determinants as well as of the regulatory circuits involved in the biochemical synthesis allow to choose the more appropriate biotechnological process with maximum yield of pigment production in the same producer, either in a heterologous host or in an in vitro system.

BACTERIAL PIGMENTS AND VISIBLE LIGHT IRRADIATION
In the world of bacterial pigments, the sunlight plays a dual role by both permitting to perceive each pigment specific color and by taking part in their biological and ecological functions (Fig. 1).
1. The electromagnetic spectrum is the range of all possible wavelengths of radiation emitted by the sun, and visible light constitutes only a part of this spectrum. Bacterial pigments absorb only certain wavelengths of the visible light and reflect the color of each unabsorbed wavelength. In plants, algae and cyanobacteria, chlorophyll a (Chl a) has a unique and crucial role in converting light energy into chemical energy by absorbing wavelengths from both ends of the visible spectrum (blue and red), but not from green. As green is reflected, Chl appears green. Some pigments, such as melanin, absorb a broad range of the electromagnetic spectrum, conferring a brownish or black appearance to bacterial colonies on solid medium 8 . 2. Photosynthetic pigments, such as Chls (Chls) and bacteriochlorophylls (Bchls), carotenoids, ficobiliproteins, are fundamental for "phototrophic" bacteria, where visible light represents the energy source, alternative to organic or inorganic chemicals 9 . Pigments from light harvesting (LH) complexes capture photon energy and transfer it to reaction centers (RCs) pigments, where photochemical reactions promote an electron flow. Phototrophic or facultative phototrophic bacteria convert light energy into chemical energy either in the form of ATP, and/or of the reducing molecule, NADPH. Among these microorganisms, those defined photoautotrophic satisfy their carbon requirements exploiting ATP and NADPH to fix atmospheric carbon dioxide, whereas those defined as photoheterotrophic use environmental organic compounds. Bacteria often optimize their fitness by producing a mixture of pigments to absorb energy from a wider range of visible-light wavelengths. Full access to sunlight is not achievable by all microorganisms since some bacteria grow underwater, where light intensity decreases with depth and the water absorbs certain wavelengths. The competition in the same ecological niche, among phototrophic F I G U R E 1 Schematic representation of the main physiological roles of bacterial pigments. The cited pigments produced by phototrophic and nonphototrophic microorganisms are involved in both light-dependent and light-independent mechanisms. Each pigment family involved in capturing sunlight radiation absorbs visible light in the specified range. In the dark, pigments increase the microbial fitness acting as virulence factors, antioxidants, antimicrobials organisms, drives their ability to capture light of specific wavelengths as a function of their own pigment reservoir as well as of light intensity 10,11 . 3. In phototrophic bacteria, the activation of Chls and Bchls causes the development of photo-oxidative stress that may be counteracted by accessory pigments. Almost 60 years ago, Griffith highlighted the antioxidant property of the carotenoid family. He reported damage in the carotenoid-deficient facultative phototrophic Rhodobacter sphaeroides during photosynthesis in an aerobic atmosphere 12 . In this bacterial species, Bchl a reaches a triplet excited state (3 Bchl a*) during solar energy capture 13 and carotenoids play an important role in protecting cells from photo-oxidative stress induced by photodynamic reactions. In the so-called "type I" photodynamic reactions, electrons flow from the excited photosensitizer (3 Bchl a*) to surrounding molecules and/or oxygen to produce radicals and/or reactive oxygen species (ROS), that is, O 2 -, H 2 O 2 , OH . . In "type II" photodynamic reactions, energy flows from 3 Bchl a* to oxygen ( 3 O 2 ), which is subsequently converted to singlet oxygen ( 1 O 2 ), a high reactive species that destroys biomolecules such as lipids, proteins, and DNA, causing cellular impairment and death 14 . In nonphototrophic bacteria, sunlight radiation may promote photo-oxidative stress, and pigments, such as melanins and phenazines, may counteract this photodegradation 15 . In spore of Bacillus subtilis, melanin protects from UV irradiation 16 19 focused the attention on interesting questions on the use of light in nonphotosynthetic biological systems. Indeed, light can also be used to activate a number of photosensory compounds and proteins designed to carry out tasks other than energy production 19 . Blue light has also been found to regulate several physiological processes such as metabolic pathways, motility, and virulence 20 . In both photosynthetic and nonphotosynthetic bacteria, the lateral gene transfer is suggested to be responsible for the production of pigments allowing to exploit new environmental and metabolic mechanisms 21 .

WIDESPREAD PHYSIOLOGICAL FUNCTIONS OF BACTERIAL PIGMENTS
As previously mentioned, pigments related to phototrophic metabolism, exploit sunlight radiation as a positive energy donor and may also counteract the subsequent induction of negative photo-oxidative stress. Pigments that are not involved in phototrophic metabolism play different roles, which may be either linked, or not, to sunlight radiation. In this scenario, it is functionally important to distinguish between phototrophic and nonphototrophic pigments. In the following sections, the structure and function of the more widely investigated bacterial pigments are shown.

Photosynthetic pigments
In phototrophic bacteria, pigments are pivotal elements in the context of refined mechanisms underlying exploitation of solar radiation. Chls and BChls, carotenoids, ficobiliproteins, form the LH antenna and the RC of photosynthetic complexes in bacteria 22,23 . The term "chlorophototrophs" indicate organisms that have photochemical RCs and perform Chl-based phototrophy. Among these, green sulfur bacteria, green nonsulfur bacteria, purple bacteria, and heliobacteria are non-oxygen evolving, whereas cyanobacteria release oxygen 24 . The last one played an essential role in the evolution of complex life, by contributing to transform a reducing early Earth environment into an oxygenated world 25 . In facultative phototrophic bacteria (i.e., Rhodobacter spp.), microorganisms can switch between energy sources, from light to chemicals, for survival. The formation of photosynthetic complexes is influenced by the light intensity in such a way that pigments are produced and are essential only under certain environmental conditions 26 . The biodiversity of phototrophic bacteria, in terms of metabolism, habitat, environmental niche, growth conditions, depends also on the richness and variety of their pigments. Phototrophs exploit Chls and hundreds of carotenoids to utilize most of the solar radiation that reaches the earth, from the near-UV (∼350 nm) to the near infrared (∼1050 nm) 23 . Each bacterial pigment allows the absorption of narrow ranges of the light spectrum. This diversity permits to improve bacterial health and overcomes the competition in the same environmental niche. The spatial arrangement of photosynthetic pigments is fundamental for producing energy, and bacteria have evolved diverse supramolecular antenna structures, for example, phycobilisomes in cyanobacteria, chlorosomes in green bacteria, or carotenoids-Bchls complexes as well as photochemical RCs containing various Bchls or Chls 23 .

Chlorophylls and bacteriochlorophylls
Chlorophylls are the more widespread photosynthetic bacterial pigments, known also as "pigments of life" involved both in LH and photochemistry 27 . Chls are a group of macrocyclic tetrapyrrole containing a central Mg ++ . A phytol moiety, consisting in a long chain of esterifying alcohols, influences the aggregation of Chls and, therefore, the interaction with the environment 28 . Many anoxygenic phototrophs possess Bchls that may contain a central Zn ++ in the tetrapyrrole macrocycle. Different types of Chls (Chl a, b, d; divinyl-Chl a and b; 81-hydroxy-Chl a) and/or Bchls (Bchl a, b, c, d, e, and g) collect and convert solar energy into chemical energy. The range of light that can be absorbed by (bacterio) chlorins for photosynthesis ranges from ∼350 to ∼1050 nm in the UV and near infrared. Their spectral absorption properties change depending on the substituents on the macrocycle and on the interaction with other Chls, carotenoids, proteins, and lipids. Although (B)Chls are different in their structure and spectral properties, their synthesis is based on a common precursor, protoporphyrin IX, and phototrophs share highly conserved enzymes responsible for most of the biosynthetic pathway.
The metabolic precursor of all (B)Chls is protoporphyrin IX (Proto-IX), which is also the precursor of heme and heme derivatives such as the bilin chromophores in phycobiliproteins (PBPs). Proto-IX is synthesized from eight molecules of 5-aminolevulinic acid (5-ALA) 23 . Most Chls and Bchls are often embedded in Chl-binding proteins, forming the antenna structures 29 . The spectral properties of the photosynthetic apparatus are determined by the arrangements of Chls and Bchls, depending on whether they are self-assembled or organized within a protein scaffold, as well as by the chemical and spectroscopic properties of these pigments 30 . The chemical structures of Chl a and Bchl a are represented in Figure 2.

Primary carotenoids
Primary carotenoid is the term used to specifically indicate carotenoids involved in photoprotection and LH in phototrophs. Carotenoids are polyisoprenoid, and according to their chemical composition, are classified as carotenes (C, H) or xanthophylls (C, H, and O) and absorb in the blue-green range of the UV-visible spectrum, 31 . Among different, carotenoids, β-carotene is orange, lutein is yellow, astaxanthin red-pink, fucoxanthin green-brown, β-cryptoxanthin orange-red 32 . Hundreds of carotenoids have been identified and their biosynthetic pathways involve the transformation of acetil-CoA in C5 isoprenoid precursors that condense to C10, C15 and C20, C30 derivatives, to form C40 polyene chains of 8-13 alternate double bonds with benzene ring at one or both ends of the molecules 33,34 . More recently, C50 carotenoids have been isolated from Antarctica heterotrophic microorganisms 35 . Aerobic anoxygenic phototrophic bacteria, such as heliobacteria, green sulfur and not sulfur bacteria, purple bacteria, use carotenes or xan-thophylls to capture 400-500 nm light, which is then transferred to Chl, thus protecting from photo-oxidative stress. Carotenoids neutralize ROS through an electron transfer that causes the allylic hydrogen abstraction and addition reactions 31 . Furthermore, carotenoids counteract 1 O 2 , by forming a reactive carotenoid molecule and the triplet nonreactive 3 O 2 . In these conditions, the excitation energy dissipates through rotational and vibrational interactions, that is, release of heat, regenerating the original carotenoid molecule. The primary function of carotenoid pigments in phototrophs is to act as chemical buffers against photo-oxidation of other cell constituents by Chl, thus conferring a high degree of immunity to endogenous photosensitization 36 . The chemical structure of β-carotene is represented in Figure 2.

Phycobiliproteins
PBPs represent a family of accessory pigments. Phycocyanin, phycoerythrin, phycoerythrocyanin, and allophycocyanin are the most investigated PBP. Each protein is formed by two different polypeptides (α, β), which probably derive from ancient gene duplication events and assemble as trimers (αβ) 3 or hexamers (αβ) 6 . Each PBP contains multiple chromophoric bilin prosthetic groups, which confer extremely high absorbance coefficients to PBP. Phycobilins such as phycocyanobilin (A max = 640 nm) (represented in Fig. 2), phycoerythrobilin (A max = 550 nm), phycourobilin (A max = 490 nm), and phycoviolobilin (A max = 590 nm) are linked by carbon bridges, noncyclic tetrapyrroles lacking metal atoms in their structure 37 . Phycobilins are enzymatically synthetized from the precursor 5-ALA, derived from glutamic acid, in a process requiring tRNA Glu. The condensation of two 5-ALA to porphobilinogen is the first step of a pathway leading to hydroxymethylbilane, and then to uroporphyrinogen III. From this latter molecule, cyanobacteria produce protoporphyrin, the phycobilins precursor and Chls. PBPs are responsible for LH and transfer into photosystems with high efficiency 37 . In cyanobacteria, PBP absorb blue-green light, in a wavelength range of the visible spectrum that cannot be absorbed by Chls. Indeed, cyanobacteria are able to colonize environments such as sea depths, which are rich in blue-green light. Furthermore, PBPs are antioxidant agents like cyanobacterial carotenoids 38 .

Nonphototrophic bacterial pigments
Nonphototrophic bacteria produce a variety of pigments, whose role in the ecological environment has only be partially characterized thus far. The following paragraphs aim F I G U R E 2 Chemical structures of photosynthetic and nonphotosynthetic pigments cited in the text. Chlorophyll a, bacteriochlorophyll a, β-carotene, and phycocyanobilin were chosen as representative photosynthetic pigments. Among bacterial pigments not involved in photosynthetic processes, staphyloxanthin, isolalloxazine, melanins (shown as a polymer of homogentisic acid), pyocyanin, prodigiosin, indigo, and violacein were included to describe the main features of a selected list of the most investigated nonphototrophic bacterial pigments.

Secondary carotenoids
Secondary carotenoids refer to carotenoids which are not involved in phototrophic metabolism. Among nonphotosynthetic carotenogenic bacteria, S. aureus strains produce a membrane-bound "secondary" carotenoid, known as staphyloxanthin (C 51 H 78 O 8 ) (Fig. 2). Staphyloxanthin is a secondary metabolite, which is not necessary for Staphy-lococcus aureus growth, instead it is used by the pathogen to survive in infected hosts and to elude the immune system 39 . Liu et al. 40 showed that a S. aureus mutant with disrupted carotenoid biosynthesis was more susceptible to oxidant toxicity, indicating that carotenoids may represent virulence factors. Furthermore, carotenoids can convert prooxidants metals, such as iron and copper derivatives, into harmless molecules, acting as metal chelators 33 .
Recently, the zeaxanthin diglucoside was extracted from an endophytic Pseudomonas spp. strain. Fidan and Zhan 41 suggest the potential use of this strain as a plant-promoting strain for agricultural applications. The production of surface-active compounds and carotenoid pigments by Gordonia spp. allows this group of microorganisms to grow under different conditions 42 .

Flavins
Flavins are yellow pigments derived from isoalloxazine (Fig. 2), a tricyclic heterocycle containing oxygen and nitrogen. Among flavins, the main microbial pigment is riboflavin, known also as vitamin B2. Riboflavin biosynthesis occurs through seven steps catalyzed by enzymes encoded from five genes, organized in the rib operon (ribGBAHT) 43 . In a successive step, flavokinase RibC catalyzes the transformation of riboflavin and ATP into flavin mononucleotide (FMN), which is then transformed into various molecules by the enzymes RosB, RosC, and RosA, ultimately producing roseoflavin 44 . Riboflavin is involved in cellular metabolism as a structural component of the coenzymes, FMN and flavin adenine dinucleotide, playing key roles in redox homeostasis, protein folding, DNA repair, fatty acid β-oxidation, amino acid oxidation 45 . Roseoflavin and toxoflavin are structural riboflavin-analogs isolated from Streptomyces spp. and Burkholderia spp., respectively, showing antimicrobial activity 44,46 .

Melanins
Melanins are a heterogeneous group of pigments with an undefined structure, composed of polymeric compounds formed by oxidation and polymerization of phenolic, indolic, or homogentisic acid monomeric units ( Fig. 2) 47 . Melanins absorb light from all the electromagnetic spectrum and are brown/black, but yellow-reddish melanins have also been described 48 . The genetic background of melanogenesis in prokaryotic microorganisms has been investigated in several producers. In Streptomyces antibioticus, l-methionine induces the expression of the melC operon that controls melanin production through the activity of an apotyrosinase 49 . Among melanin producer microorganisms, pyomelanin synthesis in Pseudomonas aeruginosa has been well illustrated and involves the conversion of l-tyrosine in 4-hydroxyphenylpyruvate through the action of the aromatic aminotransferase TyrB. Successively, 4-hydroxyphenylpyruvate dioxygenase (HPPD) converts 4-hydroxyphenylpyruvate into homogentisic acid, which undergoes oxidation to form acetic benzoquinone. This latter compound self-polymerizes to produce pyomelanin 50 . Homologue key enzymes have been isolated from Shewanella colwelliana, Vibrio cholera, Hypomonas sp 51 . Recently, the production of pyomelanin in Klebsiella pneumoniae, Alcaligenes faecalis, Enterobacter spp., and Vibrio splendidus was shown to be linked to the activity of HPPD 52,53 . Marinomonas mediterranea contains a laccase (ppoA gene) involved in melanogenesis through a two-component system of signal transduction 54 . Melanin is an energy transporter and virulence factor with remarkable protective properties from UV irradiation and environmental stress 55 . Some melanin derivates have been isolated from endospore coats to protect from UV radiation 48 .

Phenazine compounds
Many bacterial species produce phenazines, a group of nitrogen containing heterocyclic compounds with different properties depending on the type and position of substituent groups 56 . Even if Pseudomonads are the most investigated phenazine producers, other Gram-negative and Gram-positive bacteria produce phenazines such as Sorangium, Brevibacterium, Burkholderia, Erwinia, Pantoea agglomerans, and Streptomyces. In silico analyses highlighted that a biosynthetic operon core is required for the synthesis of the three-ringed phenazine structure, which is probably shared among different genera by horizontal gene transfer 57 . Indeed, the pigmentation of P. aeruginosa is the result of the production of different phenazines such as pyocyanin, besides pyoverdine, pyomelanin, and pyorubin [58][59][60][61] . The most studied phenazine is pyocyanin (5-N-methyl-1-hydroxyphenazine) (Fig. 2). Pyocyanin is a water-soluble and nonfluorescent phenazine that changes color according to its oxidation state: it is blue in the completely oxidized state, and colorless in the reduced state 56 . Pyocyanin acts as an electron shuttle and can modify cellular redox state by altering electron flow patterns. In P. aeruginosa PAO1 strain, the presence of two quorum sensing regulated operons (phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2) leads to the synthesis of phenazine-1-carboxylic acid (PCA), which is subsequently converted to pyocyanin by a methyltransferase and a monooxygenase, encoded by phzM and a phzS, respectively 62 . Pyoverdine, a fluorescent yellow green siderophore, plays an important role in iron uptake 59 ; pyorubin is a nonfluorescent red pigment helping in protecting microorganism from oxidative stress 61 . Pyocyanin and the other phenazines are virulence factors showing antimicrobial properties and conferring a selective advantage over other microbes in the natural environment. These pigments regulate cellular gene expression that trigger bacterial survival and biofilm formation 56 .

Heterocyclic pigments
Heterocyclic compounds possess a cyclic structure with two or more different kinds of atoms. Among heterocyclic pigments, the so-called prodigiosins (PGs) are red, whereas indigo and indigoidine are blue, and violacein is purple 3 . Three members of the PG family, PG (Fig. 2), undecylprodigiosin, and cycloprodigiosin hydrochloride, are characterized by a common pyrrolylpyrromethene skeleton 63 . Serratia spp., Zooshikella spp., and actinobacteria are the main producers of PGs. Serratia marcescens contributes to the biocontrol of plant diseases by inhibiting the growth of several phytopathogens. PGs confer the typical blood-like appearance to starchy foods contaminated by S. marcescens 64 . Indigo, represented in Figure 2, is a dark blue heterocyclic pigment deriving from the degradation of indole, which represents a versatile bacterial messenger influencing bacterial physiology and virulence. Indole compounds are considered as interkingdom signaling molecule involved in the pathogenesis of human diseases as well as in the control of animal behavior 65 . Many bacterial oxidoreductases, such as naphthalene dioxygenase, biphenyl dioxygenase, phenol hydroxylase and cytochrome P450 hydroxylase, oxidize indole to indoxyl, which is further dimerized into indigoids. Indole is not the inherent substrate for these enzymes, which are crucial to catabolize energetic substrates. However, most of these enzymes are responsible for the conversion of indole into the nontoxic dye indigo, through an oxygenation reaction depending on the presence of electron-donating cofactors. Indigo seems to be a cellular waste, rather than playing a physiological role in the bacterium life 66 .
Violacein, represented in Figure 2, is a bisindole produced by several Gram-negative nonphylogenetically related genera and may be isolated from very different environmental niches, including marine, freshwater, and soil environments 67 . Its biosynthesis begins with ltryptophan and it is catalyzed by enzymes encoded by the vioABCDE operon 68 . This violet pigment is a secondary metabolite often associated to biofilm formation and its production is regulated by quorum sensing signaling. Violacein shows antibacterial properties against Gram-positive bacteria, such as S. aureus, and displays toxicity toward bacteriovorus predators, such as Caenorhabditis elegans 67 . The antioxidant activity of violacein has been described as involved in membrane defense against oxidative stress 69 , eukaryotic predation, and fungal diseases 70,71 .

BIOTECHNOLOGICAL APPLICATIONS OF BACTERIAL PIGMENTS
A multidisciplinary scientific approach is fundamental for the development of modern biotechnology, whose progress is greatly influencing the economic world and the lifestyle of human beings. The United Nation Convention on Biological Diversity defines biotechnology as "any technolog-ical application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." 72 Bacterial pigments fit very well within this definition.
An increasing number of studies and reviews report the great potential of bacterial pigments in different applicative biotechnological fields. An overview of the possible uses of pigments can be illustrated with the biotechnology "rainbow code," which symbolizes different biotechnology branches with different colors. Thus, "red biotechnology" is for human health applications, "yellow biotechnology" is for food and nutrition, "white" indicates industrial biotechnology, and "green biotechnology" refers to agriculture, plant, and environment 72 . A synopsis of the potential applications of bacterial pigments in the different fields is summarized in Figure 3.

Bacterial pigments and red biotechnology
Red biotechnology aims to discover new drugs 72 . In these times more than ever, the discovery and emergence of new viruses, microbial superbugs and multidrug-resistant infections and cancers, represent human life-threatening issues. A study estimated that 10 million deaths due to antimicrobial resistance would occur every year after 2050. In addition, in the same year, cancer incidence will double as a consequence of population growth and ageing 73 . Furthermore, the current pandemic spread of SARS-CoV-2 is leading toward unpredictable and alarming scenarios. While biomedical research is principally aimed at obtaining an efficient vaccine, old and new drugs are administered to counteract the clinical manifestations of viral infection in different host tissues, comprising the respiratory, urinary, and cardiovascular systems 74 . Among putative antiviral drugs, bacterial pigments represent an unlimited reservoir of natural compounds to face new and old pathogens as well as cancer.

Antibacterial pigments
The antimicrobial activity is often associated with pigmented secondary metabolites that control the growth of bacterial competitors and, thus, increase the fitness of bacteria in each environment. Although it is well established that pigments help the producer to eliminate other species of bacteria colonizing the same niche, a definite mechanism of action was hypothesized for few compounds 79 . Among phenazines, pyocyanin, which is responsible for Pseudomonas spp. blue-green color, was formerly defined as colicin owing to its ability to inhibit Escherichia coli growth 80 . Pyocyanin conferred antimicrobial activity against other P. aeruginosa physiological competitors, such as S. aureus, Staphylococcus saprophyticus, and Enterococcus faecalis 81 . Pyocyanin has been suggested to interfere with the cell membrane respiratory chain, thus impairing energy-requiring, membrane-bound metabolic processes, such as active transport into the cell 82 .
Melanin from Pseudomonas balearica has been proposed as a biocontrol agent of the phytopathogenic Erwinia 83 . Streptomyces davaonensis and Streptomyces cinnabarinus produce roseoflavin, a promising broadspectrum antibiotic, inhibiting the growth of S. aureus, E. faecalis, Streptococcus pyogenes, Listeria monocytogenes 84 .
Violacein producers are generally sessile bacteria, which are constitutively more vulnerable to predation. This last observation lends support to the hypothesis that the purple pigment serves to accomplish a defensive mechanism 67 . Violacein extracted from Chromobacterium violaceum showed strong antibacterial activity against S. aureus, possibly via the disruption of the membrane integrity, as recently suggested 85 . A recent review reported the renewed interest in anti-infective activities of the red pigment PG, against both Gram-negative and Grampositive pathogens 86 . PG drastically alters the cell membrane integrity of B. subtilis and E. coli 87 . Both heterocyclic pigments, violacein and PG, altered the permeability of the cytoplasmic membrane and, consequently, the physiology of the cell. Bacteria cell membrane is the cellular compartment mediating most of the functions that in eukaryotic cells take place in organelles. Alteration of membrane integrity impairs the proton gradient and ATP synthesis; consequently, some essential functions, such as solute transport into the bacterial cell, DNA, and peptidoglycan synthesis, are compromised. Pigments behave more as disinfectants than antibiotics, as the former have a broader spectrum of activity than the latter.
Recent investigations highlight the efficacy of the most promising pigments in killing multidrug resistant strains and inhibiting/eradicating pathogen biofilms. Violacein was active against S. aureus methicillin-resistant (MRSA) strains 86 and inhibited the biofilm formation of Staphylococcus epidermidis, an opportunistic pathogen that forms adherent communities on catheters causing chronic infections and sepsis in hospitalized patients 88 . PG inhibited the biofilm formation of P. aeruginosa by ROS production 89 . The combination of natural pigments with traditional antibiotics may represent a strategy to counteract the spread of superbugs. In this context, PG in combination with β-lactamic antibiotics showed a synergistic effect against a MRSA strain 90 .

4.1.3
Antifungal and antiparasitic activities Among phenazines, 5-methylphenazine-1-carboxylic acid (5MPCA), was efficacious against pathogenic fungi, such as C. albicans 94 . At concentrations higher than those of pentamidine, violacein showed antileishmanial activity without remarkable side effects 95 . Antimalarial activity was observed for PG and violacein. The latter was active against chloroquine resistant strains of Plasmodium falciparum 96 .

Anticancer
Toxicity, adverse events, and resistance represent major issues for oncology chemotherapy. Several reports in literature are available to demonstrate the possible efficacy of bacterial pigments in overcoming these limitations, by influencing apoptosis or autophagy pathways in cancer cell lines. Interestingly, violacein selectively induced apoptosis in HL60 cells, a cancer cell line used as a model to study myeloid leukemia, but not in normal lymphocytes 97 . The black extracellular melanin, from Streptomyces glaucescens NEAE-H, was cytotoxic against a skin cancer cell line 98 . The blue green pigment pyocyanin significantly inhibited human hepatoma cells and glioblastoma cells 99,100 . Among carotenoids, the yellow pigment from Streptomyces griseoaurantiacus induced a significant cytotoxicity against cervical cancer cells with low IC 50 101 . Flexirubin, a carotenoid from Chryseobacterium artocarpi, induced apoptosis in breast cancer cells MCF-7 and the combination with silver nanoparticles showed synergistic effects 102 . PG showed strong anticancer and apoptosis effects on human cervical and laryngeal cancer cells 103 . Indeed, prodiginines are also attractive options because several multidrug resistance pumps, which can confer resistance to anticancer chemotherapy drugs, do not interact with them 104,105 . The combination of PG with doxorubicin determined a synergistic effect in oral squamous cell carcinoma 106 . The mechanism of action of PG is actually under investigation. Wang et al. 107 observed the ability of the red pigment to inhibit Wnt/β-catenin signaling and reduce cyclin D1 levels. PG has been proposed to have therapeutic activity against advanced breast cancers.

Diagnostic approaches
In the red biotechnology, bacterial pigments, behaving as fluorophores, may be exploited in diagnostic applications. Since 1989, the use of phycoerythrin has been suggested to evaluate the rate of peroxyl radical scavenging in human plasma 108 . Upon UV irradiation, PBPs may be used in flow cytometry and histochemistry 109 . In the last twenty years, the photodynamic therapy (PDT) emerged as a promising treatment for both cancer and antimicrobial clinical settings. PDT is a technique based on the irradiation of dyes (photosensitizers or PSs) to induce, in the presence of oxygen, the production of ROS and 1 O 2 , which are toxic for eukaryotic and prokaryotic cells, respectively (Fig. 4). In this context, Staron et al. 110 suggested to search for possible PSs among different bacterial pigments, including Chls, carotenoids, flavonoids, indigoids, phenothiazines, and porphyrins. The photosynthetic pigments, Chls and Bchls, have been proposed as excellent natural photosensitizers. In particular, Bchl a was modified to photodynamically treat a preclinical model of colon cancer 111 , conjugates of Bchl a and Chl a were found to be effective against bacteria, such as E. coli 112 . Yoshii et al. 113 observed that human skin melanoma cells were damaged after neoxanthin, fucoxanthin, and siphonaxanthin irradiation. These carotenoids share energy states above 1 O 2 , and this feature seems to confer photosensitizing properties in contrast to other carotenoids, such as β-carotene, which share energy state below 1 O 2 113 . Indeed, most pigments possess many conjugated double bonds that can absorb visible light, behaving as ideal PSs. On these bases, the exploitation of bacterial pigments in PDT for antitumor and antibacterial applications deserves much attention. It must be said, however, that many pigments have constitutive antimicrobial and/or antitumor activities regardless of light irradiation, as previously described.

Bacterial pigments and yellow biotechnology
The use of colorants in food and alimentary industry mainly relies on synthetic compounds, which are characterized by average low costs of production and high chemical stability. The occurrence of possible adverse events, such as allergenicity, toxicity, and carcinogenicity, makes the production of natural alternatives a main goal in the so-called "yellow biotechnology." 114 Although, the US FDA, the EFSA, and the World Health Organization have imposed safe dosages for the use of food colors 115 , some confusion in the distinction between natural and synthetic additives still persists, and a legal definition for "natural compound" has not been adopted yet 116 . Many colored substances deriving from living organisms, such as plants, fungi, and bacteria, are devoid of the negative drawbacks of the chemical counterparts and display antioxidant, antibacterial, or anticancer activities. Several bacterial pigments show a potential use as food grade additives. For example, the interest in β-carotene is increasing due to its use as a colorant in food industry in concentrations between 2 and 50 ppm, in juices, drinks, butter, margarine, and cheese. It is also used as a nutritional supplement, being the precursor of provitamin A, and the involvement of vitamin A in the vision process is well known. Furthermore, hypovitaminosis A represents one of the major nutritional problems in least developed regions of the world 117 . The extraction of β-carotene from microbial feedstocks could meet the increasing request of the world food market that is based on synthetic derivatives. However, the production process of this pigment is hampered by solubility, stability, melting point, and low bioavailability issues. The delivery of carotenoids through polymeric nanocapsules may overcome such drawbacks 118 . The yellow zeaxanthin, from Flavobacterium spp., was suggested as an antioxidant colorant in food 119 and as an additive in poultry feeds 120 . Canthaxanthin from Bradyrhizobium spp., an orange/deep pink pigment, displayed potent antioxidant properties 121 . Aphanizomenon spp. belonging to cyanobacteria produce the blue pigment phycocyanin, which is used in food and beverage industry 116 . Most studies have been done in Spirulina spp. cyanobacteria with a high phycocyanin content reaching the 20% of its dry weight. However, the sensitivity of phycocyanin to heat treatment results in precipitation and fading of the blue color and limits its use in food. The addition of sugars and polyhydric alcohols, safe for consumption, could overcome this issue and stabilize proteins 122 .
The red pigment PG, produced by S. marcescens, represents a further source replacing synthetic dies used in food industry. Although PG offers several limitations including solubility and short stability upon exposure to pH, light and high temperatures, an alternative delivery system was suggested. Kappa-carrageenan and maltodextrin were used to encapsulate the red pigment that was proposed in a spray-dried formulation, as a coloring agent in yogurt, milk, and carbonated drinks. The moisture content, particle size, and color intensity of PG encapsulated were optimized for food consumption 123 .
Violacein, produced by C. violaceum, is a powerful antioxidant stimulating mucosal defense mechanisms. There are interesting data on the ability of violacein to interfere with the composition of the rat gut microbiota. The oral administration of the violet pigment to rats in low (50 μg/ml) and high (500 μg/ml) doses for a month influenced the composition of the microflora: Bacilli and Clostridia were abundant in the low violacein dose, while Bacilli followed by Clostridia and Actinobacteria were present as the major components for the high violacein dose 68 . If the observed changes will be evaluated as beneficial to the host, the use of bacterial pigments as prebiotics could represent an important add value in the food industry. Melanin pigments are used as food colorants and nutritional supplements, and soil bacteria grown on fruit waste have been used in food industry, as proposed by Tarangini and Mishra 124 . Kiran et al. 125 proposed a very interesting use of melanin from actinobacterium Nocardiopsis alba MSA10. The synthesis of silver nanoparticles mediated by melanin was developed for their potential incorporation in food packaging materials and antimicrobials for stored fruits and foods. However, the efficacy of melanin-silver nanoconjugates on the shelf life of packed food products needs to be investigated 125 .
Riboflavin is accepted in dairy products, drinks, and baby foods 126 . Recently, the EFSA Panel on Additives and Products or Substances used in Animal Feed concluded that the use of riboflavin produced using B. subtilis KCCM-10445 as a feed additive for all animal species poses a risk for the spread of viable cells and antibiotic resistance determinants 45 . A valid alternative is the use of lactic acid bacteria (LAB) that can be used in the food industry owing to the acknowledged status of qualified presumption of safety 127 . As Lactobacilli produce riboflavin, the development of vitamin B2 enriched probiotic food was investigated. Riboflavin-hyperproducer LAB were obtained by the selection of variants carrying few mutations in rib operon that codifies enzymes involved in vitamin B2 biosynthesis. Among the obtained mutants, Lactobacillus fermentum PBCC11.5 was used to fortify bread. Bread produced using the inoculum of yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of the final vitamin B2 content 127 . This strategy permits to overcome the spread of resistance determinants.
Though much promising in perspective, the use of natural colors in "yellow biotechnology" is challenging, both in term of cost production and product stability. When used in confectionary manufacturing, for example, natural pigments are as much as 20 times more expensive than synthetic compounds 128 . Another drawback of natural pigments is due to their sensitivity to environmental conditions such as light, temperature, pH, and oxygen concentration 129 . In general, the low bioavailability, solubility, and stability of bacterial pigments such as carotenoids can be overcome with the help of nanotechnology formulations. Micro-encapsulation or production of nanoemulsions with surfactants, allow a more effective food coloring, with consequent cost reduction 130 . Given the great potential in health benefits displayed by natural pigments, many challenges are waiting to be addressed at the biotechnological level for the identification of new producing species and the construction of hyper-producing strains. The possible identification of low-cost fermentation substrates together with the increase of natural color shelf life will ensure the availability of substances which shouldn't be considered any longer as mere food additives.

Bacterial pigments and white biotechnology
Different bacterial pigments find applications in industrial production.
The phototrophs possess molecular complexes with defined optical response regions that pave the way for photonic materials based on biopigment assemblies. Pale suggested the use of bacterial Chls in nanophotonics, for instance, in organic solar cells, light emission diodes, and lasers 131 . For example, a solar cell manufactured from this material would be able to function even in cloudy days 131 . Similarly, the PBPs involved in bacterial photosystems LH, could be exploited in optics to assembly efficient light-trapping devices for capturing solar energy under low light 37 . Carotenoid pigments isolated from the UV-resistant Antarctic (red) bacterium Hymenobacter spp. and (yellow) Chryseobacterium spp. belong to the xanthophyllin family and represent a promising tool as photosensitizers in the production of dye-sensitized solar cells technology. Sunlight photoactivation of purified carotenoids form the photoanode and deliver electrons to a titaniumcoated conductive glass. A redox electrolyte fills the space between electrodes and fulfils the role of regenerating the oxidized dye. This technology is still at the first stage of research and development and more efforts should be addressed to the identification of new biomolecules producing microorganisms 132 .
Furthermore, various carotenoids such as astaxanthin, lycopene, β-carotene, and canthaxanthin are being commercialized to some extent and may find applications in cosmetics, thanks to their antioxidant properties 133 .
The bright red pigment PG from Vibrio spp. and S. marcescens was used to dye fibers such as nylon, acrylics, cotton, and silk. Though dyeing performances mostly varied with the fiber nature, colors were maintained upon variation of external conditions such as washing, perspiration, and rubbing 134 . PG could be a good candidate for coloration of polyolefins, such as polyethylene ultrathene. The pigment suspension from S. marcescens strain 9986 was introduced gradually up to rolled polymer sheet for the equilibrium coloration of the dyed stuff without spraying in the air 135 . In the cosmetic industry, PG showed to increase by 20-65 % the sunscreen protection factor of dermatological creams. The addition of PG to extracts of Aloe vera leaf, and Cucumis sativus fruit increased of one order of magnitude the protection factor 136 .
In a similar way, violacein, from C. violaceum, was used to color pure cotton, pure silk, rayon, and polyester. Notably, fiber dyeing was obtained either by simple fabric dipping into the dye solution or by boiling with bacterial cells; the color intensity varied with the dipping time and temperature 137 . Interestingly, an important aspect of marine bacteria belonging to the genus Pseudoalteromonas producing violacein in the crude extract, is their intolerance, and death, at human body temperature, suggesting their safety for industrial purposes 138 . Violacein is a component of several cosmetic products, displaying both rapid and prolonged contact with the skin, airways, or mucous membranes, such as antiperspirants, lipsticks, eye makeup 139 . Violacein from Pseudoalteromonas sp. (DSM 13623) was proposed for economical use in large amounts for consumer and environmental-friendly products, especially in textile and toy industries 140 .
Indigo is one of the oldest textile dyes for popular blue denim, originally prepared from plant material and nowadays chemically synthesized from fossil feedstocks. In the perspective to develop a more sustainable and environmental-friendly biotechnological productive process for this popular dye, a recent review reported an overview of the various microbial enzymes, which can produce indigo, highlighting the advantages and disadvantages of each biocatalytic system 141 . Besides naturally occurring enzymes, development of protein engineering for indigo production represents a promising and important resource. However, a large-scale industrial biotechnological indigo production is not available, yet 141 . Besides textile industry, indigo was used for the construction of sustainable electronic devices. Indigo is an unusual organic semiconductor, featuring a highly planar, but relatively small cross-conjugated, π-electron system. This pigment undergoes rapid and reversible oxidoreduction processes, thus favoring the charge flow for biotransistors and inverters setup 142 . Similarly, melanins display a semiconductor like behavior for efficient dissipation of electromagnetic energy, such as heat 143 . In general, melanins are attractive natural polyphenolic compounds with broadband absorption in the UV-visible spectrum and, for this reason, can find many biotechnological applications 144 . Fresh melanin seems to be the product of proto-molecules organization within onionlike nanostructures. Much interest is devoted to the proposed porphyrin-like tetramer structure, where the exposed nitrogen atoms in the core allow metal ion binding and the creation of larger molecules with high potential energy storage, usable in alternative energy batteries [145][146][147] . The presence of catechol residues and basic aminoacids inspired the production of adhesive films and/or nanoparticles via oxidation in moderate basic aqueous solutions or hydroalcoholic solutions. In the initial phases of the oxidative reaction, monomers or small aggregates can coat surfaces, whereas, when the reaction proceeds, the inherent size increases owing to polymerization, giving rise to the precipitation of nanoparticles, resembling natural melanin granules 148,149 . The presence of hydroxyl-, carboxyl-, and quinone functional groups renders melanin able to bind and retain metal ions, although with variable binding affinity. This has been applied in the industrial treatment of waste waters contaminated with Pb(II), Cu(II)m, Cd(II), and Zn(II) 144 .
Phenazines are a class of soluble pigments produced, among others, by P. aeruginosa and are characterized by electron transfer properties 150 . This attitude seems to be exploited not only by producers, but also by other bacterial species, making these compounds a "collective good" to be used as an electron shuttle 151 . Phenazines are a promising tool in biofuel cells, where electrons generated by microbial consortia are not directly transferred to cognate receptors but, instead, are diverted to an electrode with production of electrical energy 152 . Interestingly, though redox conditions in microbial fuel cells are disadvantageous for aerobic bacterial species, P. aeruginosa becomes dominant in bacterial communities. On the other hand, constant oxidation of the electron shuttle phenazine causes overproduction of the dye concomitant with the enhancement of the bacterial concentration 153 .

Bacterial pigments and green biotechnology
Bacterial pigments and/or pigmented bacteria may also be used for "green biotechnology" applications in the agricultural bioremediation fields.
In agriculture, bioagents are preferred to chemical pesticides as they can be more selective and safer than chemical insecticides. Bacterial pigments represent an arsenal of compounds potentially useful for this aim. An insecticide containing violacein was effective in preventing plant mycosis, such as grass pythium blight, sclerotinia stem rot, bean sprout seedling blight, and plant parasitic nematode diseases such as watermelon Meloidogyne spp. diseases 140 . The potency of PG was proved against Drosophila larvae 154 , Aedes aegypti, and Anopheles stephensi 155 . The previously described antiviral activity of PG against B. mori nucleopolyhedrovirus 77 could be exploited to inhibit virus propagation and counteract deleterious effect on sericulture. A possible oxidative stress caused by PG seems to be involved in the toxic effects against Microcystis aeruginosa cells. Downregulation of gene transcription and cell lysis may represent a promising tool for combating Microcystis blooms and consequent environmental pollution 156 . Another interesting agronomic use of bacterial pigments concerns the insecticidal crystal proteins produced by Bacillus thuringiensis. As a melanin producer mutant of this bacterium protected the protein from UV radiation damage, the authors indicated that it could be useful for the industrial production of light-stable, environmental-friendly insecticides 157 .
Phenazines produced by Pseudomonas chlororaphis display beneficial properties on plant roots, as the redox potential enables phenazines to cope with ROS generated by drought stress 158 . Importantly, P. chlororaphis represents a model suitable for whole-cell application, as it displays low toxicity in humans. The growth of the plant pathogen R. solani is inhibited by phenazine derivatives produced by Burkholderia cepacia, whereas PCA showed activity against the hyphae of the pathogen Botrytis cinerea 159 . As this latter microorganism causes great losses in the whole production chain of strawberries, grapes, and tomatoes, PCA may represent a promising natural remedy for post-harvest control 160 . Pyoverdine produced by Pseudomonas putida strain B2017 also displayed antifungal activity. As this bacterial species does not produce HCN, pyocyanin, biosurfactants, or toxic metabolites, it may represent a promising biocontrol agent, without hazardous effects on nontarget organisms 161 .
Bacteria producing pigments can also be used as biofertilizer. Among phototrophic microorganisms, purple nonsulfur bacteria produce pigments such as carotenoids (e.g., spirilloxanthin, rhodopin, okenone, rhodopinal) that, together with vitamins and other plant growth-promoting substances, counteract environmental stress and contribute to plant benefits 162 .
In bioremediation field, metals persisting and accumulating in the environment pose a threat to human health and ecosystems because they cannot be degraded or destroyed. As the methods commonly used for their removal are expensive and resource intensive, new green technologies are necessary 163 . Due to the metal affinity and high adsorption capacity of melanins, melanogenic bacteria can be used for bioremediation purposes. Nanoparticles obtained from the humic compound pyomelanin, purified from Pseudomonas stutzeri or Azotobacter chroococcum could bind Hg(II), Cu(II), Cr(VI), and Pb(II), showing promising potential in ion sequestration from polluted aqueous environments 164,165 . Iron-and copperfunctionalized pyomelanin was used for trivalent and pentavalent arsenic removal from contaminated wastewaters. Interestingly, the system could be re-used after ion reloading upon arsenic removal from melanin 166 . The release of radionuclides poses risks to ecosystems and requires innovative technologies for disposal of these substances and mitigation their detrimental effects. Turick et al. 167 showed that, in uranium contaminated soils, tyrosine addition enhanced melanin production by indigenous microorganisms, resulting in uranium stable sequestration for up to 13 months. The fluorescent siderophore pyoverdine is commonly produced by Pseudomonas sp. strains and it is involved in iron homeostasis. Its ability to bind divalent ions, such as Cu(II) in soil matrices suggested a possible use in regulating copper photo-availability in vineyard topsoil. Copper containing pesticides are not completely banned and are still widely used as fungicidal compounds with possible drawbacks on plant photosynthesis inhibition. The possible use of pyoverdine producing bacteria could represent a promising strategy to promote copper phyto-extraction 168 .
Gu and Cheung 169 observed a phenotypic change in the bacterium Vogesella indigofera upon exposure to environmental Cr 6+ ions, which inhibited the routine production of the blue dye indigoidine. In this view, the microorganism could be used as a biosensor for specific metal detection in polluted areas, with a putative detection sensitivity between 200 and 300 μg/ml. The same pigment seemed to protect the organism during growth in cold water (below 15 • C) and could be exploited as a cryopreservative additive 170 .
The treatment of waste waters represents a further green application of bacterial pigments. In the study of Gustavsson, the production of melanin was obtained in E. coli expressing a recombinant tyrosinase on external cell surface. This system proved to be very efficient in pharmaceutical contaminants removal from waste waters and a rapid regeneration of melanin matrix was obtained by simple pH adjustment 171 .
In this context and as previously reported, it is essential to highlight that the use of bacterial pigments is better than synthetic dyes that are not environmental friendly. The latter offer some limitations such as the requirement of hazardous chemicals and the disposal of hazardous wastes in the ecosystem. Furthermore, the process to obtain bacterial pigments can be managed in order to limit the impact on the environment. To this end, various agricultural products and byproducts such as corncob, sugarcane bagasse, grape waste, jackfruit seed, corn steep liquor, wheat substrates, and cassava were successfully used as growth medium for the cultivation of bacteria to produce pigments 172 . Thus, the bioprocess to obtain bacterial pigments and their exploitation in different application fields are part of green technology.

BIOTECHNOLOGICAL STRATEGIES TO PRODUCE BACTERIAL PIGMENTS
Different strategies can be developed to exploit bacterial pigments for biotechnological purposes. Once the pigment is identified, the best candidate among bacterial producers must be chosen. Indeed, the knowledge of the genetic background can drive the choice for the best producer. Microorganisms become biological machineries for pigment production, and it is essential to get a complete picture of the genetic determinants codifying the enzymes involved in pigment biosynthesis and the related regulatory network. Biotechnologists can choose between in vivo or in vitro strategies to obtain bacterial pigments. The first techniques use whole cells as factories of pigments synthesized in the cytoplasm and in some cases released in the extracellular environment. The in vitro approach resorts to one or more isolated enzymes.
A tentative roadmap can be outlined as follows: a. In the physiological context, the yield of metabolite production by the producer is highly dependent on environmental conditions. Wild strains are prone to produce pigments at low concentrations. The optimization of culture conditions in laboratory settings can increase the yield of pigment production, both in batch and in fermentation processes. b. Genetic engineering approaches may be used for the improvement of natural producer strains. Site directed or random mutagenesis, focusing on genetic determinants or regulators, lead to the selection of hyperproducer strains. c. The choice of a heterologous host carrying selected genes or operons can overcome the issues related to those natural producers that are unsafe and/or difficult to cultivate. d. The preparation of one or more isolated enzymes involved in the synthesis of pigments represents an alternative choice for in vitro production of microbial pigments.

Melanin pigments as an example of biotechnological resource
To outline different strategies that exploit microbial cells, it is useful to focus the attention on one family of pigments. Among pigments, melanins are the most versatile for the high variety of potential applications. Melanin polymers represent a family of pigments widely distributed among prokaryotic and eukaryotic domains, even if with different chemical structures. As previously described, bacterial melanins may be used in different biotechnological branches, such as "red" (antimicrobial activity), "yellow" (antioxidant in food/feed), "green" (control of phytopathogen and bioremediation), and "white biotechnology" (bioplastics, optical lenses). Biotechnological strategies, both in vitro and in vivo, may be chosen to produce pyomelanin, (Fig. 5) as described herein.

Optimization of culture conditions for native producers
A sample of soil, seawater, and vegetable can be the source of unknown and promising melanin producers. Melanin production in vitro is easily noticeable by medium darkening. Batch cultivation in appropriate environmental conditions can improve the bioproduction of melanin. A P. balearica strain was isolated from the marine green alga Ulva lactuca, and grown in a semisynthetic medium containing l-tyrosine, as sole carbon source, producing F I G U R E 5 Schematic representation of four different biotechnological strategies used to produce pyomelanin. The Pseudomonas balearica strain was grown under different cultivation parameters to optimize the production of pyomelanin (panel A). A pyomelanin hyperproducer strain was obtained in Pseudomonas aeruginosa PAO1 upon knock out of hmgA gene codifying homogentisate-1,2-dioxygenase and involved in tyrosine catabolism, as represented in panel B. In panel C, Escherichia coli cells overexpress hpd gene codifying 4-hydroxyphenylpiruvate dioxygenase from PAO1 strain. This approach represents the use of a heterologous host to produce large amounts of pyomelanin for biotechnological purposes. The chance to produce pyomelanin can be pursued through tyrosinase as isolated enzyme. Tyrosine is transformed into L-DOPA that, in turn, can lead to the synthesis of melanin-like polymers 29 mg/L of melanin (Fig. 5A). The addition of yeast extract enhanced the production of melanin up to 110 mg/L. Different temperatures (20-45 • C) and the medium pH (pH 5.0-9.0) influenced the yields of melanin production, with the temperature range of 30-37 • C, and a pH ∼8 representing the optimal conditions 83 . A more recent study reported the production of pyomelanin by marine P. stutzeri BTCZ10 after 180 h of growth in tyrosine basal broth (∼50 mg/L), yielding lower concentrations with respect to the former study 173 . The importance of this topic emerged in a recent review by Pavan et al. 174 who sorted different melanin producers by melanin yield (g/L) and considered the parameters affecting pigment production, such as the addition of tyrosine and metal ions, the substrate chosen as source of carbon and energy, and the biotransformation time. The review outlines the add-value of applying statistical analyses to increase pigment production. Optimization of pigment production results from the combination of analyses, performed before and after biotransformation, with the adjustment of critical growth parameters, such as pH, temperature, and agitation 174 . Tarangini and Mishra 124 inoculated a nutrient agar with a garden soil sample to isolate different melanin-producing colonies. Among these, the 16S rDNA sequencing identified a new species, Bacillus safensis ZJHD1-43, which was cultivated in fruit waste and produced a high yield of melanin (∼7 mg/ml). Interestingly, the combination of two statistical approaches, central composite design and response surface methodology, optimized the production of melanin considering two critical parameters (pH and temperature) 124 .

5.3
Natural or artificial selection of pigment hyperproducer strains P. aeruginosa produces different type of pigments, such as pyomelanin, pyocyanin, pyoverdin, and pyorubin. In their environmental niches, mutant isolates, that are able to produce high amounts of pigments, fit better than wild-type strains. For example, melanogenic clinical isolates of P. aeruginosa regularly isolated from cystic fibrosis patients were more competitive compared to wild-type parents 175 . In chronic infection isolates, the pigmentation originates from the accumulation of homogentisic acid, due to large chromosomal deletions that cause the lack of hmgA gene encoding homogentisate-1,2-dioxygenase. The inactivation of hmgA results in the secretion of homogentisic acid, which autoxidizes and self-polymerizes to form pyomelanin 60 . Single point mutations occurring in this gene impair the function of the dioxygenase causing the accumulation of pyomelanin 176 .
The inactivation of hmgA gene can be pursued by in vitro molecular approaches, through random or site-directed mutagenesis. A transposon bank was constructed in the model microorganism P. aeruginosa PAO1, and, among ∼2000 mutants screened on LB agar, one showed redbrown colonies and a gentamycin cassette interrupted the hmgA gene (Fig. 2B). The FT-IR spectroscopy analysis of the supernatant from the mutant culture minimal medium, supplemented with 5 mM tyrosine, was compatible with pyomelanin 15 .

Pigment production in heterologous hosts
In different natural contexts, melanin produced by opportunistic pathogens plays the role of virulence factor. Thus, the exploitation of melanogenic bacteria for biotechnological applications poses some issues related to safety. Furthermore, wild strains grown in vitro do not reach high amount of biomass, necessary for scaling up processes. Genetic engineering offers different strategies to overcome these limits and it improves the yield of pigment production. In particular, the expression of key enzymes in a heterologous host is a good strategy. E. coli represents an optimal host because it does not produce melanin. E. coli W3110 expressing the tyrosinase coding gene (melA) from the nitrogen-fixing Rhizobium etli CFN42 was used to transform tyrosine into melanin 177 . Chavez-Bejar developed an engineering process by cloning a mutated gene form of melA from R. etli in E. coli in order to direct the carbon flow from central metabolism into the L-tyrosine biosynthetic pathways. To this end, an E. coli strain lacking the sugar phosphotransferase system and tyr R repressor was obtained. However, the ability to grow on glucose was recovered by replacing the native promoter region of galP in the chromosome with the strong trc (trp-lac) promoter. Furthermore, key enzymes of the l-tyrosine pathway were overexpressed as feedback inhibition versions. The result was an engineered process of melanin production, relaying on glucose as carbon source, which greatly reduced the production cost with respect to l-tyrosine as raw material 178 .
The cloning of hpd coding for 4-hydroxyphenyl pyruvate dyoxigenase from P. aeruginosa PAO1, conferred E. coli JM109 the ability to produce pyomelanin 179 . Furthermore, the cloning of the genetic determinant under the control of an inducible promoter permits to turn on the enzyme expression, and, consequently, the pigment production at the chosen time. The administration of arabinose 0.1% increased threefold the production of pyomelanin in E. coli, as the transcription of the hpd gene was controlled by the pBAD promoter which, in turn, is regulated by araC gene product. Upon addition of arabinose, repression is relieved and AraC regulator contributes to maintain the promoter in a transcription-proficient conformation, thereby permitting gene expression (Fig. 5C) 179 .

Isolated enzymes
An auspicious technology to produce the bioactive pigment melanin is represented by the use of purified bacterial tyrosinases. These copper containing monooxygenases catalyze the o-hydroxylation of tyrosine (monophenol) to 3,4-dihydroxyphenylalanine or DOPA (o-diphenol) and its subsequent oxidation to dopaquinone. The o-quinone can be transformed into melanin through a series of nonenzymatic reactions in which molecular oxygen acts as the oxidant species 180 . Five classes of prokaryotic tyrosinases have been identified, thus far, which differ in domain organization and in the necessity of auxiliary proteins (caddie proteins) for correct folding and protein activity 180 . Ren et al. 181 describe the expression in E. coli of the tyrosinase gene from Verrucomicrobium spinosum and the identification of optimal cultural conditions for soluble and active protein production both in batch and fed-batch systems 181 . Protein engineering led to expression and purification of a C-terminal deletion of the enzyme with inherent increase in catalytic activity (from 30 to 100-fold) 182 . A C-terminal domain is reported in about 98% of mushroom tyrosinases, where enzymes are synthesized in a latent isoform activated by proteolytic cleavage. Notably, expression of the engineered tyrosinase isoform leads to a fully active enzyme, which, like Rhizobium etli tyrosinase, does not require copper chaperone for proper folding. Verrucomicrobium spinosum tyrosinase shows specific activity toward L-tyrosine and L-DOPA, but Molloy reported that genetically engineered isoforms of the enzyme from Ralstonia solanacearum show improved catalytic efficiency on d-isomers 183 . Enzymatic immobilization of melanin on plastic surfaces allowed to apply tyrosinases catalytic properties for nerve growth. Moreover, melanin shows bacteriostatic activity and may be useful in preventing microbial contamination 180 .
As reported above, l-DOPA is an intermediate in melanin production deriving from monophenolase activity (EC 1.14.18.1) of tyrosinases on l-tyrosine, which is of high therapeutic value as a first line drug for Parkinson's disease treatment. The possibility to exploit the activity of immobilized tyrosinases in the presence of reducing agents, such as ascorbate, which prevent production of dopaquinone and melanin, may represent a challenging industrial approach for l-DOPA production. In this perspective, isolated tyrosinase shows a double edge activity in the production of both bioactive pigments and pharmacologically relevant intermediates.

CONCLUSIONS
The increasing demand for ecofriendly and biodegradable supplies urges to the production of natural colorants. Most of living bacteria produce different pigments that increase their fitness and survival under physiological and/or stress conditions. Thus, prokaryotic biodiversity represents a colorful natural palette to exploit. Furthermore, it is desirable to increase the number of cultivable species, also from extreme environments, with the dual advantage to ameliorate our knowledge on the microbial world and to expand the availability of pigment sources. Bacteria are the natural source of many pigments and represent the biotechnological tool to produce them. Indeed, the use of microorganisms to produce colorants is commercially and economically promising because of the ability to control growth conditions and to ensure renewability. Microorganisms are easily cultivated and propagated, and unlike vegetables, microbial growth is not influenced by seasonality. Furthermore, according to the modern circular economy, waste substrates can be used for bulk production. Pigment extraction from high quantity of biomass continuously growing in bioreactors is usually a simple process. The boost in biotechnological techniques offers many strategies to optimize pigment production, from natural producers and heterologous hosts to isolated enzymes in vitro.
The review highlighted several issues related to the biotechnological exploitation of bacterial pigments. For example, the use of pigments in the pharmacological, food, and feed fields may be difficult owing to their low solubility and the enormous progress in nanotechnology science is helping to discover new delivery systems to overcome this limit. Moreover, several pigment producers and/or the heterologous hosts pose a threat because of their virulence arsenal or antibiotic resistance, respectively. The efforts of researchers should be aimed at selecting microorganisms lacking any "dangerous" determinant. The engineering of safe probiotics could give a boost to develop the bacterial pigment world.
Furthermore, nowadays, there is a gap between the potential market and the patents available for bacterial pigments. It could be beneficial to create a world in silico-platform collecting information on microorganism species and their pigments (chemical structure, spectral absorbance, putative physiological role, production pathways, genetic determinants, and control). The final user, that is, a biotechnological company, could be allowed to find the compound fitting with the specific commercial demand as well as the researchers with the related expertise. The optimization of bioprocesses, even if protected by corresponding patents, could be shared in the same platform, thus enriching the entire scientific community. In conclusion, the best applications for each bacterial pigment could be found to improve the life quality of human being.

C O N F L I C T O F I N T E R E S T
The authors report no potential conflict of interest.