Fungal Underground Q'n'A

1. Introduction

Considering the harmful effects of synthetic dyes on human health and to the environment, developmental process for obtaining pigments from natural sources has become significant worldwide. Microbial pigments have gained attention owing to a growing interest of the industry in safer products, easily degradable, eco-friendly and do not cause harmful effects. The pigment production from microorganisms is considered more advantageous because it is a more efficient and cost-effective process than chemical synthesis of pigments. Microorganisms are also more feasible sources of pigments in comparison to pigments extracted from plants and animals because they do not have seasonal constraints, do not compete for limited farming land with actual foods, and can be produced easily in the cheap culture medium with high yields [1–6]. Besides, the microorganisms produce an extraordinary range of pigments that include several chemical classes such as carotenoids, melanins, flavins, phenazines, quinones, monascins, violacein, or indigo, as shown in Table 1.

Among microbial species, fungi represent an economically significant source of these compounds because they can act as microbial cell factories producing high yields of metabolites with great diverse chemical structures combined with ease of large-scale cultivation [7–9].

As shown in Table 1, some fungal species produce a dark-brown pigment, known as melanin. In general, this pigment is located in the outermost layer of the cell wall associated with chitin (referred as cell wall-bound melanin), but in some fungi, melanin can also be found outside the fungal cell, usually in the form of granules in culture fluids [10].

Fungal melanins are negatively charged, hydrophobic pigments of high molecular weight formed by oxidative polymerization from phenolic and/or indolic compounds, such as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol or 1,8-dihydroxynaphthalene (DHN) or 3,4-dihydroxyphenylalanine (DOPA). Most Ascomycota fungi synthesize DHN-melanin from the polyketide synthase pathway, whereas few species are able to produce melanin through L-DOPA, in a pathway that resembles mammalian melanin biosynthesis [11–13].

The melanin pigment is not essential for fungal development, but it has been reported to act as “fungal armor” due to its ability to protect the microorganisms from harmful environmental conditions. In vitro studies have shown that melanized fungi are more resistant to UV light-induced and oxidant-mediated damages, temperature extremes, hydrolytic enzymes, heavy metal toxicity, and antimicrobial drugs than those nonmelanized [10, 14–17]. Recent studies have shown that in industrial and roadside areas, there is an increase in the proportion of dark melanin-containing fungi, as Cladosporium and Alternaria, which were more resistant to contamination by heavy metals and unsaturated hydrocarbons. Radionuclide contamination also led to a change in fungal communities, with an increased proportion of melanized fungi. For example, melanized fungal species as Cladosporium spp., Alternaria alternata, Aureobasidium pullulans, and Hormoconis resinae were found to colonize the walls of the damaged reactor at Chernobyl where they are exposed to a high constant radiation field [18, 19].

The presence of melanin in the cell wall is also correlated with enhanced virulence of parasitic fungi, as Paracoccidioides brasiliensis, Sporothrix schenckii, and Exophiala (Wangiella) dermatitidis [17, 20, 21]. This pigment protects the conidia against digestion by proteases and hydrolases secreted by competitive microorganisms or against bactericidal and fungicidal proteins of animal origin, such as defensins, magainins, or protegrins [22]. This effect was observed for Cryptococcus neoformans, whose in vitro melanization has been associated with resistance against host effector cells, oxidants, microbicidal peptides, and amphotericin B [23–25], and in Wangiella (Exophiala) dermatitidis, when the polyketide synthase gene WdPKS1 associated on melanin production was disrupted, this strain has become more susceptible to voriconazole and amphotericin B [26]. Others studies suggest that melanin contributes to fungal pathogenesis because this pigment alters the host defense response mechanisms, decreases phagocytosis, and reduces the toxicity of microbicidal peptides, reactive oxygen species, and antifungal drugs as well as to play a significant role in fungal cell wall mechanical strength [27, 28].

Although the molecular structure of fungal melanin remains enigmatic, significant progress has been made in understanding particular aspects of its macro- and microstructure. These advances allow to elucidate the molecular mechanisms of the various biological functions of melanin [22]. Studies have shown that the effect of melanin enhancing the survival of fungi under adverse conditions can be mainly due to its powerful free radical scavenger properties, acting as a “sponge” for other free radicals generated by the fungus in response to environmental stress [20, 29, 30]. Apart from this scavenging ability, melanin exhibits other biological activities, including thermoregulatory, photoprotective, antimicrobial, antiviral, cytotoxic, anti-inflammatory, radioprotective, and immunomodulatory [13, 17, 18, 31–34].

Since melanin has characteristics of functional materials and bioorganic, a growing number of researchers see this pigment with great interest, taking advantage of their properties for numerous biotechnological applications in cosmetics, pharmaceutical, electronic, and food processing industries [12, 19, 35].

The purpose of this chapter involves a comprehensive discussion of the research on fungal melanin, including the recently discovered biological activities and the potential use of this pigment for several biotechnological applications. Additionally, we discussed the ways to explore the metabolic potential of the pigment-producing fungi by manipulation of cultivation conditions to improve performance of the process, increasing yields, and reducing cost, for large-scale production.

2. Factors influencing the melanin production

Microbial pigment production is now one of the emerging fields of research due to its potential for various industrial applications, as foodstuff, cosmetics, pharmaceutical, and textile manufacturing processes. However, it is known that for the success of microbial fermentation processes, it is necessary to choose the correct productive culture strain and to determine the appropriate cultivation conditions [4, 8, 36].

An ideal pigment-producing microorganism should be capable of using a wide range of C and N sources; must be tolerant to pH, temperature, and minerals concentration; and must give reasonable pigments yield. The nontoxic and nonpathogenic natures, coupled with easy separation from cell biomass, are also preferred qualities. The potential of using filamentous fungi as pigment sources is due to their extraordinary metabolic versatility because they can be cultivated over a wide range of temperatures (10–50°C), pH (2–11), salinity (0–34%), and water activity (0.6–1) and under oligotrophic or nutrient-rich conditions. They can grow in different culture systems (submerged and solid), and fermentation protocols have been established for large-scale industrial processes. In addition, these organisms can be genetically modified to increase productivity and quality of the produced pigments [37, 38].

In order to improve performance and reduce the cost of pigments produced by microbial fermentation, it is essential to identify the nutritional and physical factors that have a greater influence on the cell growth and metabolite biosynthesis [4, 6, 39, 40].

Several studies have shown that the composition of the growth medium, nature and concentration of carbon and nitrogen sources, minerals, vitamins, temperature, pH, the presence of oxygen and aeration, light, stress, and irradiation, among others, affect the growth and pigment production in fungi and that the manipulation of the culture conditions can result in enhanced pigment production [41–47].

Experimental evidences indicate that the growth temperature influences the performance of the pigment production process, but this effect depends on the type of organism. Pseudomonas requires 35–36°C for its growth and pigment production, while in Monascus purpureus, maximum pigment production was observed at 30°C with a reduction of the yield at 37°C [48]. Another study in Monascus sp. J101 reported that the yield of pigment at 25°C was ten times higher than at 30°C, probably due to long growing (120 hours) and lower viscosity of the broth at 25°C compared to 30°C [49]. Studies developed in our laboratory, using a melanin-overproducing mutant (MEL1) from Aspergillus nidulans fungus, showed that the higher production of pigment occurred at incubation temperature of 28°C compared to 37°C [50].

Researches support that the pH of the medium also affects the growth of fungi and type of pigment produced. In species of Monascus, the pH influences the yield and quality of the produced pigment, with the highest red pigment excretion and production at alkaline pH [51, 52]. Studies on wood-inhabiting fungi indicate that pH of the substrate potentially plays an important role in fungal melanin formation. Fungi Trametes versicolor and Xylaria polymorpha tested on wood substrates produced maximum pigmentation at the pH range 4.5–5.0, except for Scytalidium cuboideum, which produce maximum intensity of red pigment at pH 6 and blue pigment at pH 8 [53]. In our study with the hypermelanized mutant (MEL1) from A. nidulans, we observed an increase in the production of pigment when the initial pH of the culture was at 6.8 compared to pH 8.0 [50]. Metabolically, the effects of pH and temperature on fungal pigment production is associated with changes in protein activity, so that the culture conditions may control certain activities such as cell growth, production of primary and secondary metabolites, fermentation, and oxidation processes of the cell [54].

The influence of light on intra- and extracellular pigment production was studied in five pigment-producing fungi: M. purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides, and Penicillium purpurogenum [55]. These authors concluded that the cultivation in the total absence of light increased biomass and production of extracellular and intracellular pigments in all fungi. The fungi grown under red light have no effect, and green or yellow light resulted in worsening effect in all the fungi, thus postulating the existence of photoreceptors responsive to dark and light in all the fungi. In a similar study, [56] noted that the production of pigment by Monascus species also was favored when the fungus was grown in the dark.

Some studies report that the pigment synthesis requires proper aeration probably related to the oxygen dependency of some enzymatic reactions responsible for the production of pigment. In Monascus ruber, it was observed that the highest levels of pigments production were obtained at an aeration rate of 0.05 L min⁻¹, which appeared to be clearly sufficient for providing the fungus with oxygen and removing carbon dioxide [57]. In our studies, it was noted that no melanin pigment production takes place during stationary cultivation of hypermelanized mutant (MEL1) from A. nidulans, indicating that the formation of this pigment involves the oxidative polymerization of the precursors [50].

Carbon and nitrogen are necessary for cellular metabolism, and these sources are related to the formation of biomass, the type produced pigment, and the yield of the desired substance. These nutrients may regulate the expression of genes of interest and activate important metabolic pathways for the production of pigments [45, 58, 59]. In general, glucose, an excellent carbon source for growth, interferes with the formation of many secondary metabolites, including pigments. For example, the pigment production by Penicillium sp. was evaluated in the presence of 10 different carbon sources, and the maximum mycelial growth was obtained with fructose, whereas the maximum pigment production was obtained with soluble starch [60]. This result shows that the increased biomass does not necessarily result in increased pigment production because pigments produced by fungi are secondary metabolites whose production usually occurs at the late growth phase (idiophase) of these microorganisms [61]. The pigment production capability of fungal species belonging to the genera Penicillium, Aspergillus, Epicoccum, Lecanicillium, and Fusarium was evaluated in different culture media, and the results showed that the complex media, as potato dextrose (PD) and malt extract (ME), favored increased pigment production [47]. According to the authors, these media contain nutrients that can regulate the expression of genes of interest and activate metabolic pathways important for the production of pigments.

Studies have demonstrated that the promoting or repressing effect of a nitrogen source on pigment production is strain dependent. It has been reported that various types of peptone, used as a nitrogen source, are able to promote an increase in the production of pigments in many species of fungi [55, 59, 62, 63]. However, M. purpureus was not able to grow in media containing peptone, and a maximum yield of the pigment was achieved when the media were supplemented with yeast extract (1%) and monosodium glutamate (5%) as nitrogen source [41]. In M. ruber, the use of glutamic acid as a nitrogen source showed promising results, either as stimulating the accumulation of extracellular pigments or contributing to increase the efficiency of the pigment production process [45]. The production of high amounts of extracellular melanin by the fungus Gliocephalotrichum simplex was obtained in cultures supplemented with tyrosine (2.5%) and peptone (1%) [64].

The optimization of medium composition is an important strategy to increase pigment production because some sources of carbon and nitrogen can be more easily assimilated and promote higher yields of the desired product. During the optimization experiments to enhance the production of melanin by Auricularia auricula, it was observed that soluble starch, tyrosine, peptone, CaCO₃, and K₂HPO₄ had positive effects, while glucose, (NH₄)₂SO₄, MgSO₄, CuSO₄, and FeSO₄ negatively impacted melanin production [46]. In other study with A. auricula, it was observed that yeast extract, tyrosine, and lactose have significant effects on pigment production and the optimization of medium resulted in 2.14-fold higher melanin concentration than that of the unoptimized medium [65].

Since the substrates for the production of pigment strongly influence the cost of the bioprocess, there is a need to select cheap and efficient substrates to make the process economically viable on the industrial scale. Large amounts of agro-industrial residues generated from diverse economic activities have attracted strong industry interest on the utilization of these residues as inexpensive substrates to support the growth of microorganisms in bioprocesses. This strategy may represent an added value to the industry and also helps in solving pollution problems, reducing or preventing their disposal in the environment [1, 66, 67].

Various studies have reported the successful utilization of agro-industrial residues for the production of fungal pigments. The use of corn cob powder as a substrate for production of pigments by M. purpureus resulted in greater pigment production [68] than other substrates, as jackfruit seed [69], corn steep liquor [70], and grape waste [71]. In the black yeast Hortaea werneckii, it was observed that rice bran acts as the cheapest source for increased production of melanin by than wheat bran and coconut cake [72]. Wheat bran extract, L-tyrosine, and CuSO₄ represent the best combination of medium components to obtain the maximum melanin yield from the fungus A. auricula in submerged culture [73]. A study conducted in our laboratory evaluated the use of corn steep liquor, sugarcane bagasse, and molasses as nutritional source on pigment production by melanin-overproducing mutant (MEL1) from A. nidulans. We observed that, in the presence of 0.2% corn steep liquor, an increase in the pigment production occurred, while a high yield of biomass was obtained at a concentration of 2%. The supplementation of medium with molasses and sugar cane bagasse hydrolysate did not have a positive effect on pigment production but promoted an increase in the fungal growth. These results indicate that corn steep liquor contains substances that stimulate the synthesis of pigment and it represents a low-cost fermentation medium for large-scale production of the pigment melanin by MEL1 mutant for future industrial applications [74].

3. Pathways of melanin biosynthesis

Various techniques, including electron paramagnetic resonance [75], X-ray diffraction [76], infrared, ultraviolet and visible spectroscopy [77], and nuclear magnetic resonance [78], have been used to elucidate the melanin structure from different organisms. These studies have shown that fungi can produce different types of melanins by oxidative polymerization of phenolic or indolic compounds [11, 27].

Melanin in cell walls of Basidiomycotina is derived from phenolic precursors, as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol. In the parasitic fungus Ustilago maydis, polymerization of catechol dimers with the formation of fibrils of melanin was shown [79]. The precursor of melanin in Agaricus bisporus and other Basidiomycetes is a metabolite of the shikimic acid pathway-γ-glutaminyl-4-hydroxybenzene oxidized under the action of peroxidase and/or phenolase into γ-glutaminyl-3,4-benzoquinone, followed by its polymerization [80]. C. neoformans, a pathogenic basidiomycetous yeast, is known to synthesize DOPA-melanin when o-diphenolic compounds, such as 3,4-dihydroxyphenylalanine, are present in the culture medium. This fungus may use a wide array of substrates, such as D- and L-dopamine [81], homogentisic acid [82], catecholamines, and other phenolic compounds [83], maximizing its ability to produce melanin. Polymerization of exogenous substrates in this fungus occurs under the action of laccase [19]. However, it is important to emphasize that different properties are observed for melanins derived from different substrates. Comparison of the catecholamines L-dopa, methyldopa, epinephrine, and norepinephrine shows differences in term of color, yield, and thickness of the cell wall melanin layer. It was also observed that the pigments vary in the strength of the stable free radical signal detectable by EPR [13, 83].

In the Ascomycota fungi, melanin pigment is generally synthesized from the pentaketide pathway in which 1,8-dihydroxynaphthalene (DHN) is the immediate precursor of the polymer, as described by Bell and Wheeler [11] based on genetic and biochemical evidence obtained from Verticillium dahliae and W. dermatitidis [84, 85]. Figure 1 shows a general model for fungal dihydroxynaphthalene (DHN)-melanin biosynthesis. In this pathway, the polyketide synthase (PKS) converts malonyl-CoA to 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which undergoes several reduction and dehydration reactions to produce scytalone, 1,3,8-trihydroxynaphthalene (THN), and vermelone. A further dehydration step leads to the intermediate 1,8-dihydroxynaphthalene (DHN), which is polymerized to DHN-melanin, possibly by a laccase enzyme [10, 13, 27].

However, some species of this class, including Cladosporium resinae, Epicoccum nigrum, Hendersonula toruloidea, Eurotium echinulatum, Humicola grisea, and Hypoxylon archeri, do not produce this type of pigment [11, 28, 86–88]. In the genus Aspergillus, DHN-melanin has not been identified in some members, as A. nidulans and A. niger. Bull [89] identified dopachrome (indole 5,6-quinone 2-carboxylic acid) and melanochrome (indole 5,6-quinone), which are intermediates in the DOPA-melanin pathway, in A. nidulans mutants defective in the production of melanin. Other studies confirmed the indolic nature of the melanin produced by A. nidulans [11, 90]. In A. nidulans strains, one tyrosinase was identified as the enzyme responsible for the production of melanin pigment, based on its substrate specificity (DOPA substrate) and susceptibility to inhibitors [91, 92]. In a recent study, our group characterized the pigment produced by A. nidulans mutants as DOPA-melanin according to the results obtained with specific inhibitors of DHN- and DOPA-melanin pathways [93].

The production of DOPA-melanin has also been investigated in other fungi such as Neurospora crassa [94], Podospora anserina [95], A. nidulans [91], A. oryzae, and C. neoformans [96]. A biosynthesis pathway for fungal DOPA-melanin, proposed by [11], is shown in Figure 2, which strongly resembles the pathway found in mammalian cells, though some of the details may differ.

In this pathway, there are two possible starting molecules, L-dopa and tyrosine. If L-dopa is the precursor molecule, it is oxidized to dopaquinone by laccase. If tyrosine is the precursor, it is first converted to L-dopa and then dopaquinone. The same enzyme, tyrosinase, carries out both steps. Dopaquinone, a highly reactive intermediate, forms leucodopachrome, which is then oxidized to dopachrome. Hydroxylation (and decarboxylation) yields dihydroxyindoles, which can polymerize spontaneously to form DOPA-melanin [10, 27, 97].

Some fungi have more than one biosynthetic pathway of melanins. For example, Aspergillus fumigatus synthesizes DHN-melanin [98] and also produces a second type of melanin, piomelanins, from homogentisic acid by the tyrosine degradation pathway that protects the cell wall of hyphae from ROS, and gray-green DHN-melanins determine the structural integrity of the cell wall of conidia and their adhesive properties [99]. In Agaricus bisporus, melanins are formed from DOPA by tyrosinase and from γ-glutaminyl-4-hydroxybenzene by peroxidase and phenolase [100].

The extracellular fungal melanin, which is found in culture fluids usually in the form of granules, can be formed from some culture components, which are autoxidized or are oxidized by phenoloxidases released from the fungus during autolysis [10, 11, 27].

4. Biological activities of melanin

Despite the difference in their origins, melanin pigments have a number of common characteristics that allow them to fulfill their protective function. Several biological functions of melanins are closely associated to their chemical composition and structure. The presence of unpaired electrons in the melanin structure is responsible for various properties, including antioxidant, semiconductor, optical, electronic, and radio- and photoprotective [19].

The effect of melanin enhancing the survival of fungi under adverse conditions is mainly due to its function as an extracellular redox buffer, which can neutralize oxidants generated by the fungus in response to environmental stress [19]. It has been reported that melanin contributes for virulence of C. neoformans, protecting the pathogen against free radicals generated immunologically [29]. In W. dermatitidis and A. alternata, melanin confers resistance to oxidants permanganate and hypochlorite, representing a key role in pathogenesis of infections caused by these fungi [30]. Studies have shown that melanin of zoopathogenic and phytopathogenic fungi is essential for their parasitizing, due to its antioxidant properties [101].

Melanin pigment extracted from several fungal species has shown the ability to scavenge free radicals (reactive nitrogen and oxygen species), becoming a potential natural antioxidant. Melanins produced by Exophiala pisciphila and Aspergillus bridgeri ICTF-201 exhibited a significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity comparable with that of synthetic melanin, indicating its antioxidant potential [102, 103]. Melanin produced by Schizophyllum commune showed high free radical scavenging activity in a dose-dependent manner, when the melanin concentration was increased from 10 to 50 μg, the scavenging activity was also increased from 87% to 96%, similar to those obtained using ascorbic acid (standard compound used to measure free radical scavenging activity) [34]. Melanin pigment of Fonsecaea pedrosoi has antioxidant potential by reducing Fe(III) to Fe(II), ensuring the balance of its redox chemical microenvironment and minimizing the effect of oxidation of fundamental structures on fungal growth [104]. Similar results were also observed for melanin from Ophiocordyceps sinensis, which proved to be an effective DPPH radical scavenger and a strong ferrous iron chelator [105]. The chelating power of fungal melanin can be explained by various functional groups present in the structure of this pigment, which provide an array of multiple nonequivalent binding sites for metal ions [14, 22].

It has been reported that substances acting as antioxidants protect cells from ROS-mediated DNA damage, which can result in mutation and subsequent carcinogenesis. The excess free radicals may attack cellular constituents, as the cell membrane, nucleic acid, protein, enzymes, and other biomolecules, by peroxidation, resulting in the severe damage of cell functions and subsequent serious deleterious effects on the organism [106]. It has been reported that melanin protects melanocytes and keratinocytes from the induction of DNA strand broken by hydrogen peroxide, indicating that this pigment also has an important antioxidant role in the skin [107]. Studies in our laboratory showed that melanin extracted from hyperpigment-productive mutant (MEL1) of A. nidulans has the ability to scavenge the biological oxidants, as HOCl, and may be a promising material in cosmetic formulations to protect the skin against possible oxidative damage [31].

There is experimental evidence that fungal melanin may also act as an anti-aging drug, due to its action in reducing the generation of free radicals, clearing away the free radicals produced in excess, and enhancing the activities of antioxidant enzymes. Studies have shown that one of the major causes of aging is the surplus free radicals produced during the oxidative metabolism in the human body [108]. It was demonstrated that the melanin produced by fungus Lachnum singerianum YM296 significantly inhibited the formation of lipid peroxidation products and slowed down the aging process, elevating the levels of superoxide dismutase, glutathione peroxidase, and catalase and decreasing the level of malondialdehyde in mice liver and brain homogenate and serum, suggesting that this pigment could be used as a new anti-aging drug [109].

Researches have also shown that some fungal melanin exhibits immunomodulatory activity through the inhibition of pro-inflammatory cytokine production in T lymphocytes and monocytes, as well as fibroblasts and endothelial cells [12, 110, 111]. During an inflammatory response, cells of the innate and acquired immune systems release a variety of mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukins (IL), and the reactive nitrogen and oxygen species, which are implicated in the pathogenesis of a number of inflammatory diseases [112]. [113] reported that treatment of macrophages activated in vitro with melanin from the fungus F. pedrosoi inhibited the production of nitric oxide and Th1 cytokines. The study performed by [114] showed that the expression of inducible nitric oxide synthase gene decreased and lower levels of cytokines, such as IL-12 and TNF-α, were observed when activated macrophages were incubated with melanized cells of the Fonsecaea monophora fungus. Our studies demonstrated that melanin extracted from a highly melanized mutant (MEL1) of A. nidulans inhibited NO production in LPS-stimulated macrophages, with a maximum response of 82% inhibition, and also showed a dose-dependent inhibitory effect on TNF-α production, reaching an inhibition of 51.86% at a melanin concentration of 100 μg/mL. These results suggest that melanin from A. nidulans has potential as an anti-inflammatory agent and may be used in the future for development of new drugs with therapeutic utility [32].

Some studies have proposed that fungal melanin exhibits anti-radiation activity in vivo and in vivo and then could be explored as a probable radioprotector [16, 115]. Since melanin has a stable free radical population, it is thought that the radioprotective properties of this pigment result from a combination of physical shielding and quenching of cytotoxic free radicals generated by radiation [18]. [116] showed that Lachnum extracellular melanin (LEM404) had strong anti-ultraviolet radiation activity because the survival rates of Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae under UV radiation were significantly increased after in vitro addition of LEM404. Compared with the control groups, the antioxidant defense systems, such as superoxide dismutase and glutathione peroxidase activities, were improved significantly in mice of experiment groups, and the reactive oxygen species detected by malondialdehyde content were decreased significantly. These results confirmed that fungal melanin could be used as component of photoprotective creams mainly for its free radical scavenging rather than its light absorption properties. The probable mechanisms of radioprotection by melanin appear to be modulated in pro-survival pathways, immune system, and prevention of oxidative stress. It was reported that melanin isolated from the fungus G. simplex reduced the radiation-induced overproduction of pro-inflammatory cytokines (IL-6 and TNF-α), which might help in the recovery from radiation injury by preventing the aggravation of inflammation and oxidative stress [33]. This study confirmed the possible use of melanin-coated nanoparticles for protecting against radiotoxicity during radioimmunotherapy [117].

Recent studies have demonstrated that, in addition to the ability of transferring electrons arising under the action of radiation, melanin also possesses ionic conductivity due to its ability to transform any type of radiation energy not only into heat but also use it for the maintenance of redox processes in cells [118]. It was assumed that melanin pigments, participating in redox reactions, are able to perceive the energy of radiation (UV, visible light, and radiation) and convert it into useful reducing power for metabolic processes. This hypothesis is supported by the discovery of melanized fungi in soils contaminated by radioactive nuclides and areas around the damaged Chernobyl nuclear reactors, which not only survive high radiation levels but also have enhanced growth upon exposure [16, 19, 119, 120]. Owing to its semiconductor property, melanin becomes a promising material for organic bioelectronic devices like transistors, sensors, and batteries [121].

Fungal melanins also exhibit growth inhibitory effect against various microorganisms. The extracellular melanin isolated from S. commune showed significant antibacterial activity against E. coli, Proteus sp., Klebsiella pneumonia, and Pseudomonas fluorescens and antifungal activity against dermatophytic fungi, Trichophyton simii, and T. rubrum [34]. The A. auricula melanin displayed inhibitory activity on biofilm formation of the three bacterial strains, E. coli K-12, Pseudomonas aeruginosa PAO1, and P. fluorescens P-3, and there was a proportional reduction in biofilm biomass with the increase in pigment concentration. Confocal laser scanning microscopy (CLSM) analyses showed that the three strains formed thick and compact biofilms when grown in the absence of pigment, but the presence of A. auricula melanin resulted in thinner and looser cell aggregations on surfaces instead of normal biofilm architecture. This study suggested that A. auricular melanin inhibits quorum-sensing (QS)-regulated biofilm formation in all strains tested without interfering with their growth [122]. Silver nanoparticles incorporated Yarrowia lipolytica melanin exhibited antimicrobial activity against the pathogen Salmonella paratyphi, and they were also effective at disrupting biofilms on polystyrene as well as glass surfaces [123]. These nanoparticles displayed excellent antifungal properties toward an Aspergillus sp. isolated from a wall surface, suggesting the application of these nanoparticles as effective paint additives. The melanin-silver nanostructures with broad-spectrum antimicrobial activity against food pathogens also have potential applicability in food processing and food packaging industries [124].

The anti-cell proliferation effect of fungal melanin in tumoral cell lines has already been demonstrated. [34] reported that the extracellular melanin produced by the fungus S. commune was effective against human epidermoid larynx carcinoma cell line (HEP-2) in a concentration-dependent manner, indicating its potential application in cancer chemoprevention and chemotherapy.

The evaluation of the effect of fungal melanin on non-tumor cells is also interesting because it may serve as alternative to acute in vivo toxicity testing, avoiding the indiscriminate use of animals. The melanin produced by A. bridgeri was evaluated in vitro cytotoxicity assay using cell lines TE 355.Sk derived from normal human skin fibroblasts and HEK-293 derived from human embryonic kidney cells, and no cytotoxicity was observed against the two cell lines [103]. In our studies, the toxicity of the melanin from A. nidulans was also evaluated due to its potential practical application as antioxidant and anti-inflammatory agent. The results showed that the viability of mouse macrophages remained greater than 90% when these cells were treated with a high melanin concentration (100 μg/mL), indicating that this pigment has low cytotoxicity [32]. We also showed that the toxicity of A. nidulans melanin on mouse fibroblast McCoy cell line, after metabolic activation with hepatic S9 microsomal fraction, was much lesser (CI₅₀ = 413.4 ± 3.1 μg/mL) than known cytotoxic agents such as cyclophosphamide (CI₅₀ = 15 ± 1.2 μg/mL). In this study, we demonstrated that this melanin pigment did not induce gene mutations in different strains of Salmonella typhimurium used in the Ames assay. Based on these results, we suggest that the melanin produced by A. nidulans does not cause significant damage to the cellular components and might be used in the future for development of new therapeutic drugs [32].

5. Biotechnological applications of melanin

With the current knowledge about physical and chemical properties and the broad spectrum of biological activities, fungal melanins have attracted growing interest for their potential use in the fields of biomedicine, dermocosmetics, nanotechnology, and materials science.

6. Conclusion

Melanin possesses physicochemical properties and biological activities that make it a suitable biomaterial for a wide range of applications in cosmetic, pharmaceutical, electronic, and food processing industries. In addition, this pigment has a considerable interest biotechnological because it can be produced on a large scale with low cost, making its use for future practical applications economically advantageous. However, it is necessary to expand the knowledge about the structure-property-function relationships for the development of melanin-based technology. In the context, we hope that the information in this book will be useful and will encourage a greater number of researches on fungal melanin, which might be useful to deploy innovative and sustainable solutions for human health and the environment.