Fungal Melanin

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This entry is adapted from the peer-reviewed paper 10.3390/jof9090891

Synthetic dyes are generally unsafe for human health or the environment, leading to the continuous search and growing demand for natural pigments that are considered safer, biodegrade more easily, and are environmentally beneficial. Among microorganisms, fungi represent an emerging source of pigments due to their many benefits; therefore, they are readily viable on an industrial scale. Among all the bioactive pigments produced by fungi, melanin is an enigmatic, multifunctional pigment that has been studied for more than 150 years. This dark pigment, which is produced via the oxidative polymerization of phenolic compounds, has been investigated for its potential to protect life from all kingdoms, including fungi, from biotic and abiotic stresses.

melanin bioactive pigment biotechnological potential fungi

1. Introduction

The negative consequences that synthetic dyes have on the environment and human health are well known. Therefore, the toxic effects caused by these synthetic chemicals and their industrial byproducts on human health and the environment represent a developing concern ^[1]^[2]^[3]. As a result, a critical demand for the extraction of pigments from natural sources, mostly micro-organisms, is expanding globally. Among micro-organisms, fungi comprise a group that is significant for the production of pigments because when grown on a large scale, they produce high yields of metabolites, serving as “microbial cell factories” and making the bioprocess commercially viable ^[4]. Amid the wide range of pigments produced by fungi there is a significant class of pigments called melanins, the production of which has been known about for more than 150 years ^[5]^[6]^[7]^[8]. A melanin is a secondary metabolite produced by fungi which contributes to the survival mechanism of fungi in unfavorable environments ^[9].

The term “melanin” is derived from the Greek word “melanos”, meaning “black”, and it was first used by the Swedish chemist Berzelius in 1840 ^[10]^[11]. Melanins are usually dark-brown- or black-colored pigments of high molecular weights which are negatively charged, hydrophobic in nature, and formed via the oxidative polymerization of phenolic or indolic compounds ^[12]. These pigments are widely synthesized by plants, animals, fungi, protists, protozoans, pathogenic bacteria, and helminths ^[13]. However, melanin is known for its distinctively unique and exceptional physicochemical and biological properties, such as its black color, insolubility in organic solvents, resistance to acid degradation, reduction of silver nitrate ammonia solutions, and provision of positive reactions to polyphenols ^[14]. It is the stability, insolubility, and resistant nature of melanin that make fungi capable of surviving in unfavorable environments, but these qualities also make it difficult to work with, limiting the analytical approaches that successfully identify and characterize the pigment.

Fungal melanin is generally located either extracellularly (secreted outside, in the environment) or intercellularly (in the cell wall). However, according to Agustinho and Nosanchuk ^[15], the distribution of melanin in the cell structure varies between species.

While melanin is sometimes found to be associated with the matrix, fibrillar in nature, and extending out of the cell wall, in other fungi, it is generated as a discrete and strongly delimited layer ^[16].

This pigment, on the other hand, demonstrates a variety of biological and functional actions, including defending the fungi from oxidative damage, UV radiation, enzymatic lysis, and extremely hot and cold temperatures. It serves as a physiological redox buffer, providing structural rigidity to cell walls and protection against antimicrobial drugs ^[17]. Melanin has been referred to as “fungal armour”, conferring advantages to fungi so they may survive in harsh conditions and increasing the virulence of a variety of fungi that are pathogenic to plants and humans ^[18].

2. Types of Melanin

With specificity to the fungal kingdom, types of melanin are classified as eumelanins, pheomelanins, allomelanins (DHN-melanins), and pyomelanins. These classifications are based on the chemical composition of the monomer subunit structure of the pigment and are widely accepted ^[19]. Both eumelanins and pheomelanins are derived from the common precursor dopaquinone, which is obtained via the oxidation of tyrosine or L-Dopa.

Eumelanins are dark brown or black pigments containing 6–9% nitrogen and 0–1% sulfur. Their precursor, dopaquinone, undergoes cyclization, resulting in cyclodopa, which is rapidly oxidized into dopachrome. Dopachrome is then converted into units of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to form eumelanins.

Pheomelanins are yellow-red pigments containing 8–11% nitrogen and 9–12% sulfur and are composed of benzothiazine monomer units. In the presence of cysteines, dopaquinones connect with cysteines to form 5-s cysteinyl dopa and 2-s cysteinyl dopa, which provide benzothiazin and benzothiazole intermediates that polymerize to produce pheomelanins.

Allomelanins are a heterogenous group of nitrogen-free polymers produced through the polymerization of di-DHN (1,8-dihydroxynaphthalene). Polyketide synthase (PKS) catalyzes the initial step of the pathway in which it converts the precursor malonyl CoA into 1,3,6,8-tetrahydroxynaphthalene (THN).

Pyomelanins involve tyrosine transaminase forming 4-hydroxyphenylpyruvate, which is converted into homogentisic acid by dioxygenase. The dioxygenase is spontaneously oxidized into benzoquinone acetate and polymerized to form a pyomelanin.

Glutaminyl-4-hydroxybenzene melanin (GHB) is another type of melanin that has been observed in the basidiospore wall of Agaricus bisporus, in which “browning” has been reported to be a common phenomenon wherein the melanogenic phenols are enzymatically converted into quinones and then evolve into melanins ^[20].

3. Biosynthesis

Specific to fungal melanin, based on precursors and the pathway taken, the two most significant types of melanin are DHN-melanin (1,8-dihydroxy naphthalene) and DOPA-melanin (L-3,4-dihydroxyphenylalanine) (Figure 1). Pathogenesis has been linked to both forms of melanin ^[21].

Figure 1. Biosynthetic pathways for various types of fungal melanins.

3.1. DHN Melanins

Most ascomycetes and deuteromycetes synthesize the 1,8-dihydroxy naphthalene (DHN) type of melanins through the polyketide pathway, using 1,8-DHN as a precursor. A general model for the biosynthesis of DHN-melanins has been explained below; however, it varies in different fungi ^[22].

As the initial step in the process of biosynthesis, malonyl-CoA/Acetyl CoA is converted into 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) by the enzyme polyketide synthase (PKS). The enzymatic dehydration of scytalone results in 1,3,8-trihydroxy naphthalene (1,3,8-THN), which is then further reduced to vermelone by a second reductase enzyme. The scytalone dehydratase catalyzes a further dehydration phase that results in the intermediate 1,8-dihydroxy naphthalene (DHN). A laccase subsequently catalyzes the dimerization and finally polymerizes the molecules to create DHN-melanins ^[23].

3.2. DOPA Melanins

Conversely, basidiomycetes and other imperfect fungi produce DOPA melanins, typically using L-DOPA as a precursor, similar to the mammalian pathway. The melanins formed from DOPA are eumelanins and pheomelanins ^[24].

With respect to the biosynthesis of DOPA melanins, phenol oxidases form a second major group of enzymes that produce pigments, and they fall into two subgroups—laccases and catechol oxidases (commonly called tyrosinases)—on the basis of the differences in their substrate specificities. However, both have copper ligands and require bound copper ions for their activity ^[25].

The synthesis of DOPA-melanin in fungi has been of scientific interest for a long time. Numerous fungi such as Neurospora crassa, Podospora anserina, Aspergillus nidulans, and Aspergillus oryzae, as well as pathogenic fungi like C. neoformans, have been studied in this regard ^[26].

Depending on the precursor molecule, either L-dopa or tyrosine is converted to produce dopaquinone. The process is catalyzed by tyrosinase, which acts as the primary rate-limiting enzyme of the reaction. Then, dopaquinone undergoes a series of reactions to form a eumelanin or pheomelanin, respectively ^[27].

Dopaquinone is a highly reactive intermediate. In the absence of thiols, dopaquinone forms leucodopachrome, which is then oxidized into dopachrome. Hydroxylation (and decarboxylation) yields dihydroxy-indoles, which can polymerize to form DOPA-melanins ^[28].

4. Optimization for Enhancing Pigment Production

The selection of appropriate fungal strains and the optimal culture conditions (suitable media, temperature, pH, and aeration) for pigment production are essential for effective fermentation. In this regard, filamentous fungi are favored sources of pigments as they may be grown under a wide range of cultivation conditions. They can grow in solid and submerged culture systems, and they have the potential to be used in large-scale industrial fermentations to produce a rational yield of pigment ^[29].

Microdochium bolleyi, first known as Gleosporium bolleyi and later as Aureobasidium bolleyi, is a common soil fungus, and it was observed that media composition and light are the two elements that influence the formation of the pigment melanin in this fungus. Pigment production was found to be at a maximum under alternating light and dark rhythms in the case of light, while among all the media investigated, PDA was able to significantly change its colony differentiation (i.e., from hyaline cells to melanin-pigmented chlamydospores) and create intense pigmentation ^[30].

Since pigments are secondary metabolites that are generally produced during a late growth phase (idiophase), an increase in mycelial growth does not necessarily imply an increase in pigment production.

The pigment output generated during fermentation is also associated with proper aeration relating to O₂ dependency. For example, the maximum pigment production was achieved in M. ruber at an aeration rate of 0.05 L/min; thus, this aeration rate was found to be suitable for oxygenating the fungus ^[31]. This suggests that the formation of this pigment requires the oxidative polymerization of the precursors.

Apart from the nutritional and physical factors involved in fermentation, the use of low-cost media has always been believed to be a convenient and cost-effective method for the production of large amounts of melanin. Agro-industrial residues have been studied and found to be successfully utilized as substitutes for expensive substrates that support fungal growth and pigmentation ^[32].

5. The Functional Role of Fungal Melanin

5.1. The Role of Melanin in Anti-Microbial Effects

The role of melanin as an anti-microbial agent is also interesting as it exhibits significant anti-bacterial and anti-fungal activities. For example, Arun et al. ^[33] reported that the mushroom fungus Schizophyllum commune produces melanin with an effective anti-bacterial activity against E. coli, Proteus sp., Klebsiella pneumonia, and Pseudomonas fluorescens and anti-fungal activity against Trichophyton simii and T. rubrum.

Similarly, the production of melanin from the endophytic fungus Phoma sp. RDSE17 was also studied to exhibit anti-bacterial potential against Bacillus subtilis, Staphylococcus aureus, E. coli, and Salmonella typhi and anti-fungal potential against A. flavus, A. niger, Rosellinia sp., and Ustilaginoidea virens ^[34]. Auricularia auricula melanin also has a high rate of inhibiting biofilm formation, acting against E. coli K-12, Pseudomonas aeruginosa PAO1, and P. fluorescens P-3 ^[35].

5.2. The Role of Melanin in Photoprotection

Prolonged exposure to the sun may lead to skin burns and the suppression of the immune system. The synthetic chemicals used in sunscreen products, such as benzophenone-3, cinnamate, and octocrylene, produce free radicals that cause damage to the skin ^[36]. Among all the biological pigments known, melanin has the ability to absorb all visible wavelengths. Hence, these pigments are found to act as UV light shields, and it has been confirmed via human experience that human DOPA-melanin protects the skin from sunlight. This suggests that melanin has anti-radiation properties, a significant application of the pigment ^[37].

Cladosporium sp., Alternaria alternata, Aureobasidium pullulans, and Hormoconis resinae are some of the melanized fungi that have been observed to colonize the damaged Chernobyl nuclear reactor, which has a strong radiation field ^[38].

The microscopic melanized fungi Cryomyces antarcticus and Cryomyces minteri also exhibit high levels of UV resistance for a few hours compared to their non-melanized strains ^[39].

5.3. The Role of Melanin in Thermoregulation

Studies suggest that the melanin-producing ability of fungi has been closely linked to their ability to survive under extreme conditions. These darkly pigmented polymers protect the cells against various stresses imposed by the environment and play an important role in thermoregulation, e.g., in C. neoformans, melanin protects the fungi against temperature extremes, making them thermostable; therefore, the fungi are able to survive in extreme heat (42–47 °C) and in cold (−20 °C) temperatures.

5.4. The Role of Melanin in Protection from Radiation

Due to its chemical structure, the presence of stable free radicals, and its spatial arrangement, melanin has also been observed to protect against extremely high levels of ionizing radiation. One of the classic examples of fungal melanins surviving in an extreme environment are the microbiota at the site of the Chernobyl nuclear reactor accident. More than 37 different kinds of fungi were found within the damaged reactor, and a significant number of black fungi were discovered in the polluted soil near the Chernobyl Nuclear Power Plant. Even though the radiation levels were increased manifold and were lethal in nature, fungi like Alternaria alternata and Cladosporium sphaerospermum were found on site.

5.5. The Role of Melanin in Protecting against Oxidative Damage

Melanin enhances the survival of fungi by neutralizing oxidants caused due to stress in the surrounding environment. There have been many reports suggesting that the melanins produced by ascomycetous fungi such as C. neoformans, Aspergillus nidulans, Aspergillus bridgeri, and A. alternata, as well as basidiomycetes like Exophiala pisciphila, Wangiella dermatitidis, Schizophyllum commune, and Inonotus hispidus, exhibit potent antioxidant activity ^[40].

Fungal melanin is a powerful antioxidant due to its unique electrochemical properties, which allow it to act as an electron donor and electron acceptor ^[41]. The antioxidant properties of fungal melanin are attributed to the addition of free radicals, probably because melanin has unpaired electrons that cause it to interact with peroxyl radicals ^[42].

Hypoxylon archeri melanin has a better ability to protect 80.95% 5-thio-2-nitrobenzoic acid (TNB) from oxidative damage by H₂O₂ than synthetic melanin. It also has a better effect when scavenging hydrogen peroxide oxygen radicals and promotes the production of other fungal polyphenol oxidases ^[43].

5.6. The Role of Melanin in Metal Chelation

There are various functional groups present in the structure of melanin which provide an array of multiple nonequivalent binding sites for metal ions. Due to this property, the pigment is able to bind to heavy metals that are potentially toxic to cells. In addition, if useful metals that are important to the cell physiology of a fungus but are present in fewer amounts in the environment, melanin can concentrate them in a manner that makes them available to the cell in sufficient amounts, thus conferring an important advantage to the survival of melanized fungi ^[44].

Melanin also possesses the ability to chelate metal, which enhances its biosorption capacity, and they remove rare earth elements from wastewater via metal complexing. Hence, in this manner, they provide an alternative means of cleaning industrial effluents from wastewater ^[45].

6. The Extraction and Purification of Melanin

6.1. Ultrasound-Assisted Extraction (UAE)

This technique utilizes ultrasound pressure waves to increase the efficacy of melanin extraction. In this process, a cavitation-induced mass transfer is generated in the fungal cell wall. As the cavitation bubbles are formed and collapsed, the generated energy induces an increase in the penetration of the solvent into the cell wall, due to which the cell wall is easily disrupted and the cellular matrix may be separated out ^[46].

6.2. Negative Pressure Cavitation (NPC)

In the case of NPC, the creation of negative pressure governs cavitation. This extraction proved to be more effective in the extraction of heat-sensitive compounds like polyphenols and polysaccharides ^[47].

6.3. Microwave-Assisted Extraction (MWE)

Microwave-assisted extraction is one of the most practical methods utilized in industrial purposes due to the availability of equipment, its convenient handling, and its high extraction efficiency. This is another advanced technique that was discovered recently. In this method, microwave energy is generated that heats the solvent in contact with the fungal biomass and releases the bioactive compounds out of the fungal cell ^[47]. This method was first used in the extraction of melanin from Lachnum singeranum YM296. The quantity of melanin produced was significantly increased in Lachnum singerianum YM296 with the aid of this technique and other optimized parameters ^[48].

The difficulty with this method is that the extraction process ends early because the solvent boils and eventually increases the temperature of the reaction mixture. As a result, the extraction yield is decreased because the targeted chemicals are not sufficiently dispersed from the sample into the solvent ^[49].

6.4. Hydrodynamic Cavitation Extraction (HCE)

Cavitation occurs due to pressure variations in the flowing liquid with respect to the change in the geometry of the constriction ^[47].

However, every extraction method has its own pros and cons, and therefore, no standard protocols for the extraction of melanin have been described until now. Hence, the extraction methods, fermentation conditions, and optimization parameters can be coordinated accordingly to obtain increased yields of melanin from different fungal sources ^[50].

7. The Characterization of Melanin

7.1. UV–Visible Spectroscopy

One of the characteristic methods for the identification of melanin in its preliminary stage is its maximum absorption in the UV-visible spectra (i.e., 200–400 nm). The complex conjugated molecules in the structure of melanin absorb and scatter UV light photons; for this reason, alkaline melanin solutions show significant optical absorption in the UV region (200–400 nm) which gradually declines toward longer wavelengths (400–800). This subsequent decline in absorption is almost linear. As a result, straight lines with negative slopes are formed when the logarithm of the absorbance of an alkaline melanin solution is plotted versus the wavelength. This linear connection forms a distinguishing characteristic for the qualitative identification of melanin in an extract ^[51].

7.2. Fourier Transform Infra-Red (FTIR) Spectroscopy

FTIR spectroscopy is another method for the detection of melanin in which functional groups are known. Through studying the structure of melanin, it is has already been established that eumelanins comprise polymeric units of indole-5,6-quinone and 5,6-dihydroxy-indole-2-carboxylic acid, and pheomelanins have benzothiazine and benzothiazole units in their structures. Therefore, the different functional groups in the heterocyclic rings of eumelanins and pheomelanins produce characteristic peaks in the spectra for the detection of the type of melanin ^[52].

7.3. Elemental Analysis

Elemental analysis studies of melanin help us understand the type of melanin on the basis of the composition of elements and the elemental ratio. Eumelanins contain high C, H, and N contents and lack sulfur because they are synthesized via L-dopa or L-tyrosine oxidation. On the other hand, pheomelanins have high sulfur contents (9–12%) as they are products of cysteinyl conjugates of DOPA.

7.4. Physicochemical Properties

Conventional physicochemical tests are typically used for the initial detection and characterization of melanin due to its distinctive solubility and reactivity characteristics. Melanin has a number of distinguishing characteristics, including a low level of solubility in distilled water, low levels of solubility in most organic and inorganic solvents (except for aqueous alkali), a resistance to degradation by concentrated acids, bleaching upon exposure to potassium permanganate, potassium dichromate, sodium hypochlorite, hydrogen peroxide, or other oxidizing agents, a positive reaction for polyphenols (via the FeCl₃ test), reacting with sodium dithionite and potassium solutions, and reacting with ammoniacal silver nitrate solutions ^[53].

7.5. Thermal Characterization

In comparison to other natural polymers, fungal melanin has greater heat stability. According to Gomez-Marin and Sanchez (2010), the graphite-like structure of the polymer is responsible for its thermal stability. Therefore, a thermogravimetric analysis (TGA) can be used to examine the heat stability of melanin. To assess the thermal behaviors of fungal melanins, TGA thermograms curves were utilized. Typically, a characteristic pattern of peaks is visible in the two stages of the heat deterioration of fungal melanin. First, an endothermal peak is formed due to the evaporation of bound water at approximately 250 °C. Following the endothermal peak, the second is an exothermal peak caused by the loss of CO₂ that occurs at 350 °C ^[54].

7.6. Nuclear Magnetic Resonance (NMR)

In order to further confirm the molecular structure of extracted melanin, a proton and carbon nuclear magnetic resonance (¹H-NMR, ¹³C-NMR) analysis can be carried out. The sample is subjected to an NMR analysis using various resonant frequencies and is usually referenced to tetramethylsilane. There are several chemical shifts in the ¹H-NMR spectra of melanin which can support the correct identification of the melanin’s molecular structure ^[55].

7.7. Electron Paramagnetic Resonance (EPR)

The EPR signal can be used to determine the existence of free radical centers in the structure of melanin, which are features of melanin biopolymers. EPR tests performed on pigmented soil fungi such as Cladosporium cladosporioides and mushroom fungi Schizophyllum commune have demonstrated the existence of melanin ^[56].

O-semiquinone radicals are produced by eumelanins, whereas o-semi quinone imine radicals are produced by pheomelanins. Even at higher microwave powers, the EPR spectrum of eumelanin displays a single line with a hyperfine form, whereas the EPR spectrum of pheomelanin exhibits a complicated shape with an unresolved structure, primarily due to the interactions between free radical electrons and the nearby nitrogen nuclei ^[57].

8. Applications of Melanins

8.1. Applications in Bio-Electronic Industries

Many interesting optoelectronic properties of melanin have been studied, such as its high optical absorption in the UV-Vis range, good electronic transmission, and ionic conductivity, which makes it a potential biomaterial for use in organic electronic devices with a less negative impact on the environment. Studies suggest that the electrical conductivity properties of melanin highly resemble those of amorphous semiconductors; therefore, melanins are considered organic semiconductors ^[58]. These are cheaper and easier to process relative to inorganic semiconductors like silicon and germanium and could therefore be used as a promising materials in sensors and photovoltaic devices (Figure 2) ^[59].

Figure 2. Applications of fungal melanin in various industries.

Owing to the optical and electronic properties of melanin, thin films have been produced from these pigments that can be used in solar cells, chemo-sensors, and many other detectors. Melanins are also employed as sensitizing pigments in DSSCs (dye-sensitized solar cells), which transform photons into their electrically excited state ^[60]. The ultraviolet absorption property of melanin pigment has also been extremely utilized in the manufacture of ophthalmic devices, protective eyewear packaging material, umbrellas, and other materials that protect from harmful radiation.

8.2. Applications in the Dyeing Industries

The biodegradable nature of fungal melanin as an eco-friendly dye demonstrates its potential uses in natural hair colors and cosmetics. On a similar note, the durability of fungal melanin for dyeing purposes was demonstrated via the melanin released by Lasiodiplodia theobromae, which was used to tint bleached poplar veneers.

8.3. Applications in Pharmaceutical Industries

Due to its promising antioxidant and anti-inflammatory effects, fungal melanin has been explored and widely utilized in drug development, therefore offering a fresh approach to boosting antioxidant therapy ^[61]. It has been reported to treat various types of malignant tumors in cancer, disorders of the immune system, diseases of blood origin, disorders due to disturbances in cell homeostasis, mental disorders such as schizophrenia, epilepsy, and other disorders involving the nervous and other regulatory systems ^[62]^[63]. The effective free radical scavenging ability of melanin enhances immunological function, triggers apoptosis, and prevents blood angiogenesis; for these reasons, fungal melanin has been developed as a powerful anticancer therapeutic component ^[64]. Owing to its ability to increase the permeability of the blood–brain barrier, melanin is also useful as a carrier for other therapeutic agents which must reach the brain tissue to produce their therapeutic responses. Melanin nanoparticles have been explored as potential biocompatible drug carriers, especially in relationd to pH targeting as it responds strongly to pH ^[65].

8.4. Applications in the Dermocosmetic Industry

In the dermocosmetic industry, melanin has especially been a subject of keen interest as it was found to have great potential when incorporated into skin photoprotection formulations, especially sunscreens that protect against the noxious effects of UV radiation ^[66]. The emerging demand for safer and chemically inert dermocosmetic products has encouraged the use of melanin as an active ingredient in sunscreens. This property of melanin is attributed to the high efficiency of the pigment in absorbing and scattering photons, simultaneously quenching the radicals in their excited state and scavenging reactive oxygen species ^[67].

In two separate studies using two different melanins, Kurian and Bhat compared the SPF values of commercially available sunscreens with the effect of melanin on the enhancement of the SPF value, demonstrating that melanin increased the SPF value by factors of 3.42 and 2.6 ^[68]^[69]. An allomelanin extracted from the black knot fungus also proved to be a great choice for cosmetics and as an anti-UV radiator ^[70].

8.5. Applications in Packaging Materials

The packaging industry has also seen an increase in the manufacturing of composite chemical materials made from fungal melanin. This potential of fungal melanin has been attributed to its antibacterial and antioxidant properties. Thin films developed from carvacrol and fungal melanin are effectively sold in the market, providing a future alternative to plastic films for green packaging and food packaging ^[71].

9. Conclusions

Due to its multifarious properties, melanin has been widely recognized for its potential in diverse applications. Among micro-organisms, fungi have emerged as a great source of this ubiquitous pigment as they are easier to upscale on the industrial level while maintaining diversity. However, knowing the potential applications of fungal melanins, they are still executed successfully very often. There lies a complexity in the structure of these biopolymers which is mainly due to the enzymatic imbalances in the biosynthetic pathways that alter their metabolic routes. This heterogenous nature of melanin is responsible for making the extraction and chemical characterization of the pigment difficult. With concerted efforts, a massive scope of work has been completed to circumvent challenges in the isolation of melanin and techniques for elucidating its structure. The development of new extraction methods that do not involve harsh treatments has enabled efficient extraction, and many analytical techniques have also been explored to successfully determine the structure of melanin. The optimization of the media used in fermentation also plays an important role in increasing the bioproduction of melanin.

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