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Biotechnology & Applied Microbiology
Synthetic dyes and colourants have been the mainstay of the pigment industry. Researchers are eager to find a more environment friendly and non-toxic substitute because these synthetic dyes have a negative impact on the environment and people’s health. Microbial pigments might be an alternative to synthetic pigments. Microbial pigments are categorized as secondary metabolites and are mainly produced due to impaired metabolism under stressful conditions. These pigments have vibrant shades and possess nutritional and therapeutic properties compared to synthetic pigment. Microbial pigments are now widely used within the pharmaceuticals, food, paints, and textile industries. The pharmaceutical industries currently use bacterial pigments as a medicine alternative for cancer and many other bacterial infections. Their growing popularity is a result of their low cost, biodegradable, non-carcinogenic, and environmentally beneficial attributes.
- biocolorant
- biotechnology
- pigments
- pharmaceutical applications
1. Introduction
Natural dyes have gained more concern for their application because they are more compatible with the environment due to their better biodegradability. Natural dyes have been utilised for a very long time; they served as colouring agents in the earliest known human civilizations. However, as synthetic colours were introduced to the market, they started to lose their charm. These synthetic dyes were much cheaper, user-friendly, came in a variety of colours, and were reproducible compared to their natural alternatives. The plant-based dye industries collapsed at the beginning of the twentieth century, whereas the synthetic dye business flourished as a result of recent advances in the field. The industries using synthetic dyes ended up dumping the toxic chemical wastes into freshwater sources, such as rivers and ponds, which led to algal blooms, ultimately contributing to greenhouse gases [1]. These industrial pollutants were hazardous and carcinogenic for consumers as well. It was not until recently that the synthetic dyes ill effects started to surface, and industries were forced to look in a new direction. Because of these demerits of synthetic dyes, their application nowadays is considered “toxic contaminants” and are, thus, less oftenly used [2]. Natural dyes made from plants are already in the market, but there are disadvantages that must be taken into consideration. It might be expensive to grow plants specifically for dyeing purpose. Furthermore, plant-based pigments are rapidly denatured in the presence of changed pH and might not provide batch reprodubility. Consequently, researchers used a different strategy involving microbes [3].
Bacteria, yeasts, fungi, and algae are a few of the microbes that typically generate natural colours. Several categories that have been documented to be synthesised by these microorganisms, including carotenoids, flavins, phenazines, violaceins, melanins, and many others that are mentioned in the paper later. These pigments are essential for the microorganisms’ innate ability to adapt to harsh environmental circumstances and perform specific cellular functions (for example, photosynthesis in photosynthetic micro-organisms) [4]. Such naturally synthesized pigments are now used in various industries, such as textile, cosmetics, pharmaceuticals, and food, because of their exotic properties and advantageous characteristics, which are useful for both human health and the environment [5].
2. Overview of Natural Pigments, Plant Pigments and Microbial Pigments
A pigment can be defined as a chemical compound capable of absorbing a fraction of photons falling on it and transmitting the rest, which falls in the detection range of human eyes [10]. However, these compounds are not the same as phosphorescent, fluorescent and other luminescent materials, with inherent, light-emitting properties. Pigments selectively absorb a particular wavelength, which imparts them with their distinctive colours, and since they possess this property, they are used to colour plastics, fabrics, cosmetics, foods, and ink/paints. The pigment is any coloured material produced by a living organism (Figure 1). Physical organs, such as nails, hair, skin, iris, and fur, also bear unique pigments known as melanin [11]. Additionally, other organisms, such as reptiles (involving chameleon, garden lizards), amphibians (such as frogs), teleost (involving fishes like kill fish, eel, catfish, minnow), and insects (involving stick insect, grasshoppers, lake flies), have specialized pigment-producing cells called chromatophores that enable them to change the colour of their skin under specific conditions. Thus, pigmentation in biological systems aids the survival of organisms by enabling them to mimic and camouflage with their immediate environment [12].
Figure 1. Classification of natural pigments on the basis of organisms. The natural pigments can be obtained from different organisms involving plants (photosynthetic and protective pigments), animals (Chromatophores and carotenoids), and micro-organisms (prodigines, phycobiliproteins, oxyindoles), which have various pharmaceutical and industrial applications.
In some cases, pigments might also serve as the basis for sexual selection, signalling, and aposematism [13]. For example, in guppies, the orange spots are determined by carotenoid uptake in their diet. Males with a low-carotenoid diet have dull orange spots and are less preferred by females over the males with high-carotenoid uptake [14]. Pigments (chlorophyll) aid in complex chemical cascades, such as photosynthesis in plants. Pigment colours should not be confused with structural colours; structural colours are iridescent, whereas pigment colour remains the same from different angles.
Prokaryotes are known to synthesize pigments, just like animals and plants do. Some of these pigments aid in the synthesis of complex carbohydrates by photosynthetic bacteria, while others protect them from ultraviolet (UV) damage [15]. Prokaryotes can be divided into two major categories: autotrophic prokaryotes and heterotrophic prokaryotes. Autotrophic prokaryotes bear pigments that help them participate in photosynthesis; chlorophyll, carotene, and xanthenes are the most commonly found pigments in autotrophic prokaryotes. The heterotrophic prokaryotes produce some accessory pigments, which helps in the survival of these organisms in extreme niches. For example, the membrane bound, yellow-coloured pigment xanthomodin is secreted naturally by the bacteria Xanthomonas oryzae pv. oryzae. In addition, xanthomodin has been shown to possess photodamage protection properties to the bacteria [16]. The biosynthesis of these accessory pigments is crucial for the taxonomic characterization and identification of novel bacteria and helps establish genetic relatedness amongst new species and the pre-existing ones.
3. Ecological Distribution of Microbial Pigments
Pigmented micro-organisms, also known as chromogenic microorganism, belonging to, e.g., bacteria, microalgae, archaea (mainly haloarchaea), have been isolated from diverse environmental and geographical conditions, including terrestrial, aerial, and marine locations [32]. Research is being conducted extensively on coloured microorganisms isolated from marine habitats, including seawater, marine sediment, sponge [33], sea ice (including Algoriphagus), sun saltern, microbial mats [34] and many other locations due to the variety of colours and novelty. Several pigment-producing microorganisms have also been found in a variety of stress environments, such as lava caves [35], hot springs [36], etc., in addition to normal environments. There are also specific geographic locations where the frequency of the pigmented microorganisms is higher than other occurrences. For example, Hermansson et al. (1987), discovered that the frequency and occurrence of pigmented bacteria were higher at the air–water interface than bulk water [37]. Additionally, glaciers, ice cores, salt lakes, and deep-sea hydrothermal vents are a few locations with large concentrations of coloured microorganisms [38]. Thus, it can be shown that the pigment-producing microorganisms are extensively dispersed around the world, providing enormous opportunity for scientists to learn about their medicinal and industrial uses.
4. Advantages of Microbial Pigments over Synthetic Pigments
Applications of bacterial pigments are more favoured than synthetic pigments because of a number of advantages. Bacterial pigments are simple to grow and secure for use by humans. Furthermore, their extraction methods and scaling up processes are more economical. Additionally, pigments are secondary metabolites created by a living creature that support the cell in numerous ways, including photosynthesis, UV protection, defence against competing species, and even energy-storing molecules [39]. They are a good contender for the current pigment industry because of their ease of growing, resistance to temperature and pH changes, variety, and non-toxic/eco-friendliness. Additionally, bacterial pigments possess great medicinal qualities, making them even more deserving of replacement for synthetic dyes. For instance, bacterial pigments, such as zeaxanthin, astaxanthin, yellow and orange carotenoids, prodigiosin, violacein, pyocyanin, and actinorhodin are the subject of intense research for their possible use in modern medicine [39]. The ability to biosynthesize any natural pigment using a bacterial host cell is another advancement in recombinant DNA technology that has further cemented bacteria’s place in the pigment industry. Now, rather than growing quintals of plants for their pigments, bacterial culture can achieve the same results [9]. This simplistic beauty of working with prokaryotes attracts more research into the topic.
5. Pigment Production from Bacteria and Genes Involved in Pigment Production
Bacterial pigments fall into one of two categories: soluble pigments that quickly permeate into the surrounding medium and are referred to as extracellular pigments; and insoluble pigments are confined to the interior of the cell and are referred to as intracellular pigments. In extracellular pigments, the liquid culture can be directly processed through chromatography to isolate the pigment. On the other hand, intracellular pigments first require the culture to be sonicated; this breaks the cell open to release the pigment into the surrounding media (Figure 2).
Figure 2. Extraction and purification of microbial pigments. The process of production, extraction, and purification of microbial pigments is explained in the figure and is numbered serially from 1 to 6 respectively.
Bacterial pigments are secondary metabolites synthesized only when the bacterial species are grown in appropriate growth conditions. The pigments, such as orange carotenoids, protect the cell from the damage caused by harmful ultraviolet light when the bacterial culture is exposed to direct sunlight [19]. Some pigments are made in the lag phase where the cell is experiencing resource exhaustion and thus, pigmentation is also sensitive to the kind of medium utilized for production. Different bacterial species have special growth requirements under which they produce specific pigments. The percentage yield can be calculated on a laboratory scale by closely monitoring the purified pigment’s absorbance; the more the absorbance, the better the pigment production efficiency will be. The maximum yield and quality of the pigments can be simply attained via media optimization and standardization of incubation conditions [40,41]. Bacterial culture will be unable to surpass the lag until the right conditions are provided; otherwise, the quality and quantity of the pigment produced will be of an inferior kind.
Molecular cloning can enhance pigment production by developing high yielding strains that will reduce the cost of maintaining stringent fermentation conditions and produce more pigment per unit mass. However, the type of pigment being purified depends greatly on the downstream purification of the pigment from the fermentation media. Therefore, a proper purification scheme is required for pigment purification. Since most bacterial pigments are insoluble in water, they are selectively solubilized in organic solvents, such as methanol, ethanol, acetone, ethyl acetate, or hexane. Once the pigment is obtained in the organic solvent, the organic solvent is allowed to evaporate. The powdered residue left behind is our pure pigment and can be used for functional analysis [42]. There are different types of pigments, such as canthaxanthin, β-carotene, violacein, phenazines, etc. Each pigment is produced by different kinds of micro-organisms via a tightly regulated biochemical pathway catalysed by a number of enzymes that are encoded by other genes, respectively.
6. The Utilization of Genome Engineering Techniques to Enhance Bacterial Pigment Production
Biosynthesis of several industrially important pigments, such as carotenoids, lutein, zeaxanthin, etc., can be hosted in microorganisms using modern genetic engineering techniques that do not naturally produce them, or if they do, they are made in minute quantities [9]. Therefore, various strategies have been postulated to develop genetically engineered microorganisms to synthesize the desired pigments, such as over-expression of the key enzymes involved in the pigment biosynthesis, insertion/deletion of specific genes, etc. Such genetic modifications not only help in strain improvement, but also help in mitigating the toxic issues, which are caused by synthetic dyes.
Corynebacterium glutamicum has been metabolically engineered for the co-production of a secreted amino acid (L-lysine) along with a cell-bound carotenoid (β-carotene) using two feedstocks, namely, xylose and arabinose. Furthermore, a genetically engineered strain of C. glutamicum for the transcriptional repressor gene crtR has also been developed to overproduce the carotenoid decaprenoxanthin [50]. In addition, Escherichia coli has been genetically engineered to obtain several pigments, such as β-carotene, zeaxanthin, etc. For example, via modification of a single gene of ATP synthesis, pentose phosphate, and TCA cycle, a modified E. coli has been constructed to synthesize β-carotene [39].
Recent advances in genetic engineering have helped scientists engineer certain microorganisms, such as E.coli and yeast, for large-scale carotenoid production. For example, an engineered E.coli has been constructed to synthesize zeaxanthin from lycopene using two fusion protein-mediated substrate channels and introduce tunable intergenic regions to express crtY, and crtZ genes encode for the enzyme lycopene β-cyclase and a β-carotene hydroxylase, respectively [51]. Rhodobacter sphaeroides was genetically engineered for the production of lycopene by many modifications; firstly, its crtI3 gene was replaced by crtI4 gene from Rhodospirillum rubrum. Secondly, the crtC gene was deleted and, finally, the zwf gene was knocked out along with the integration of dxs gene, which blocked the competitive pentose phosphate pathway and reinforced the methyl erythritol phosphate pathway [52]. Thus, genetically engineered micro-organisms can be extensively employed for the large-scale production of pigments with many therapeutic and industrial uses.
7. Application of the Bacterial Pigments in the Pharmaceutical Industry
Since the discovery of antibiotics, the fatality due to bacterial infections has drastically reduced. Over the past decades, not only has the average lifespan of human beings increased, but we have also learned how to tackle health care emergencies. Thanks to our ever-expanding pharma industry, which invest in the research and development of new drugs and manage their production and circulation amongst the human population. However, as years pass by, our knowledge of modern medicine still stands incompetent in front of the present-day challenges. Due to the injudicious circulation of heavy antibiotics, multidrug-resistant (MDR) strains of pathogenic microorganisms have developed. This is one of the challenges that have rendered us helpless. Another one is the unsolved puzzles of diseases, such as cancer, which lay an irreversible impact on patients and their families. To tackle the serious issues of antimicrobial resistance and expensive healthcare, the pharma industry was compelled to look into a new direction—‘bacterial pigments’. Bacterial pigments in recent years of research have revalued their importance; they might be the next big breakthrough in the field of modern medicine.
8. Application of Microbial Pigments in Food Industries
Vibrant and colourful food items appeal to the senses of people of all age groups. There are pieces of evidence that prove the early Egyptian and Roman empires used natural food colours in their food to make their product more appealing [99]. Food industries have also begun to rely on food colourants, and to keep costs down, they encourage the use of synthetically manufactured food colourants due to their stability and low price. However, synthetic food colours are made out of the by-products of petroleum wastes and are not health-benefiting. In some cases, the long term use of artificial colouring can ill impact the health of the consumers [100,101]. Plant-based colouring agents are often costly and unstable; hence, researchers are shifting their focus towards bacterial pigments since they are more stable and easier to produce than plant-based pigments [102]. However, when compared to synthetic pigments, microbial pigments exhibit reduced stability in specific environmental circumstances (light, pH, oxygen, UV, temperature), which causes them to reduce their shelf-life and lose their colour over time [102,103]. Thus, certain techniques have been developed, such as micro-encapsulation, preparations of nanoemulsion, or nanoformulations, which increase the market value of microbial pigments making them more durable for industrial applications [104,105,106]. With the knowledge of recombinant DNA technology, bacterial pigment production can be increased multifold. Microorganisms produce pigments, such as flavin, melanins, monascins, violaceins, carotenoids, quinies, and many others [32], which can be employed in the modern food industry, not only as colourants, but also as pro stabilizers due to their free radical scavenging activity [107]. For example, a pinkish-red pigment, astaxanthin, produced by the bacteria Agrobacterium aurantiacum and Paracoccus carotinifaciens, is an excellent antioxidant, which, when used as a colourant in food items, not only imparts an attractive colour, but also increases the shelf life of the product, thereby acting as a preservative as well [108]. Similar to astaxanthin is canthaxanthin, produced by Bradyrhizobium Sepp. This orange-coloured pigment is also a potent antioxidant currently being used in the food industry [109].
As discussed earlier, bacterial pigments also have many therapeutic properties associated with them; thus, the inclusion of such pigments in food products will enhance the visual appeal of the foods and fortify them with essential medicinal properties. For example, Heptyl prodigiosinis, a pink anti-plasmodial pigment (isolated from α-Proteobacteria), prodigiosin, a red anticancer pigment (isolated from Serratia marcescenes), and pyocyanin, a proinflammatory green pigment (isolated from Pseudomonas spp.) [27], are a few examples of the pigments already being used by the food industry, which are also having therapeutic applications. In addition, many pigments, such as staphyloxanthin (isolated from Staphylococcus aureus), trypanthirin (isolated from Cytophaga/Flexibacteria AM13,1 Strain) [110], and undecylprodigiosin (isolated from Serratia marcescenes), are under laboratory analysis and soon might be used in the food industry as a non-toxic, therapeutic food colourant. With more research investigations directed towards the search for new bacterial pigments and soon, a new class of immune-fortified foods might gain popularity, as these foods would be not only appealing, but also impart therapeutic immunity to the consumer, especially in the prevailing times where humanity is more than ever prone to infectious diseases and lifestyle disorders.
9. Application of Microbial Pigments in the Cosmetic Industry
Since synthetic dyes have toxicity and carcinogenicity issues, cosmetic industries also switch to safer alternatives. One of these is low cost yet highly efficient bacterial pigments. Most of these pigments are isolated from marine bacteria or extremophiles [111]. The degradation of the dermal and epidermal layer’s extracellular matrix is the primary factor behind the skin’s ageing, which is mainly controlled by the intrinsic factor (genetics and personal diet). However, the external environment (smoke, pollution, UV exposure, weather, etc.) contributes to the premature ageing of the skin. Amongst all the known pigments, β-carotenoids are the active ingredients used in anti-ageing creams. Carotenoid is a lipid-soluble pigment with a characteristic carrot-like orange colour produced by Deinococcus radiodurans [112], having an excellent capacity to prevent the production of ROS, which causes extensive damage to cells. Therefore, it is used in anti-ageing formulations, such as provitamin A. The anti-ageing property of cosmetics is due to high percentages of antioxidants. One of the pigments produced by Haematococcus pluvialis is astaxanthin, which is known to have excellent antioxidant properties. These antioxidants scavenge upon the free radicals produced by the cells [113]. The other two most commonly used antioxidants in the cosmetics industry are; myxol and saproxanthin. These pigments are members of the carotenoid family and are isolated from strains of marine bacteria belonging to the Flavobacteriaceae family [114].
Prolonged exposure to UV radiations causes dermatoheliosis, also known as photo-ageing [115]. In the long run, UV exposure can also cause DNA damage and can cause skin cancer. Hence, nowadays, moisturizersizers and sunscreens add pigments that reprimand the UV damage. Most of the extremophiles produce pigments that assist them in avoiding DNA damage. Scytonemin is one such pigment, a UV-A inducible pigment made by cyanobacteria that may aid in UV radiation protection due to its potential for absorption in the UV-A and UV-B range [116,117].
The modern-day cosmetic industry relies on bacterial pigments as effective preservatives since chemical preservatives can be harmful or toxic to the user and might decompose into undesirable compounds, rendering the product ineffective. A polyacetylene pigment, falcarindiol, obtained from the chloroform extract of Crithmum maritimum [118], has antimicrobial effects against bacteria Micrococcus luteus and Bacillus cereus, thereby imparting long shelf life to the product. However, bacterial pigments have still not been profoundly studied for their skin whitening, but astaxanthin, a carotenoid, is known to have depigmentation properties that aid in the lightening of the skin spots developed due to skin ageing by reducing melanin production [18]. Therefore, companies in cosmetic industries are investing more in researching marine bacterial pigments and utilization in preparing cosmetic concoctions, which are safe and more efficient in function.
10. Application of Microbial Pigments in Textile Industry
Since they are non-carcinogenic and safe for the environment, the textile industry now favours microbial pigments. Synthetic colours can have a harmful impact on your health in a number of ways, including skin responses and the emission of potentially dangerous compounds during synthesis. Prodigiosins, which are bright red in colour isolated from Vibrio sp., can be used for dyeing silk, wool, nylon and acrylics [119]. Additionally, prodigiosin, produced by Serratia marcescens SB08, has found its potential use as a natural dye for various fabrics, such as acrylics, silk, polyesters and cotton [120]. The fabrics dyed with such microbial-derived pigments also retained anti-microbial activities against microbes, such as P. aeruginosa, E.coli, and Bacillus subtilis, making the fabrics much safer for human use.
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms11030614
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