Melanin Production by Xanthomonas campestris pv. vesicatoria under the Effect of Aromatic Oil’s Vapour Phase - A New Report

Introduction

We are all familiar with the role of melanin pigment in imparting colour to human skin and hair. They are ubiquitous in nature, found in all living organisms from microbes to plants and animals. These pigments are produced by the oxidative process known as melanogenesis by polymerization of phenolic compounds, where L-tyrosine or L-DOPA [1] or catechol [2] or HGA [3, 4] are used as the precursors. Melanin obtained from microbes varies in colour viz. yellow, red, brown and black, and based on these they are classified as eumelanin, pheomelanin, and allomelanin [5, 6]. Nowadays, production and extraction of melanin from microorganisms is of great interest, owing to its versatile applications. Melanin offers multiple antibiotic resistance [7], UV protection [8, 9], exhibit significant antibacterial [10, 11], antifungal [10], antioxidant activity [10, 11] along with anticancer potential [10, 12]. It enhances the survival and competitive abilities of organisms in harsh conditions [13, 14]. The organism producing melanin can be used for heavy metal remediation, where melanin is involved in absorption and accumulation of the heavy metals within the tissue [15]. It has been used as a reducing and stabilizing agent for the synthesis of silver and gold nanostructures [16]. It has the metal binding capacity [9], and is used in the fabrication of radio-protective materials, food packaging, cosmetics, and in medicine [6]. Apart from this, melanin nanoparticles also have strong antibacterial activity [11] and are used in heavy metal absorption [17]. Owing to the increasing demand for melanin in the market and its versatile applications, economically feasible methods of melanin production from microorganisms should be identified.

Aromatic oils are extracted from plants, they have the characteristic fragrance of the plant and contain specific volatile compounds. These aromatic/essential oils are extensively used in perfumes, incense, cosmetics, fresheners and cleaning products like soaps and detergents, and to add flavor in food and beverages. They are reported to have antifungal [18], antibacterial [19, 20] and insect repellent ability [21], and thus have been proved to be a potential alternative to synthetic chemicals used presently. Apart from this, they are also reported to be used in aromatherapy in human beings, either by inhalation or by applying it to the affected body part after dilution of the oil [22]. However, use of aromatic oils in the production of pigments in microbes is unknown, and therefore studied in the present experimentation by using Xanthomonas campestris pv. v_esicatoria_ (Xcv), plant pathogenic bacteria causing bacterial blight disease in tomato (Lycopersicon esculentum Mill.) and pepper (Capsicum annum L.). Species of the genus Xanthomonas typically produce yellow pigment xanthomonadin except Xanthomonas campestris pv. phaseoli which produce melanin-like pigment [4]. In the present study, the effect of aromatic oils vapour phase (aroma) was observed in the production of melanin pigment in the plant pathogenic bacteria Xcv.

Discussion

The colonies of Xanthomonas spp. usually produces yellow pigmentation in the growth medium, which is a typical characteristic of the genus. However, due to the effect of vapour phase (aroma) of aromatic oils viz. of dhavana, henna, kasturi, firdous and rose, Xcv produced extracellular dark brown pigmentation in growth media, similar to melanin. Generally, melanin’s are insoluble in water and organic solvent, but they are soluble in alkaline solution. They are oxidized by H2O2, and are readily precipitated by HCl. Similar results were obtained for the extracellular pigment produced by Xcv, and the chemical characteristics of the pigment are in agreement with the previous reports [1, 10, 16]. In the UV-Vis spectrum at 200 nm to 900 nm wavelength range, the extracellular pigment produced by Xcv (Figure 3), shown higher absorption in the ultraviolet region (between 250 nm to 350 nm), and it progressively declined towards visible light, which is a peculiar character of melanin pigment. The UV-Vis spectrum of Xcv pigment was similar to the previous reports of UV-Vis spectrum of melanin pigment [1, 10, 16]. In FTIR spectroscopy, the melanin isolated from Pseudomonas sp. showed similar absorption peaks at 3273 cm-1, 2929 cm-1, and 1625 cm−1 [11]. In addition to these peaks, some other peaks were observed at 1539.25 cm-1 corresponding to N-H bending; 1423.51 cm-1 showing characteristic CH2 -CH3 bending, and another peak at 1240.27 cm-1 which was attributed to phenolic compounds in melanin produced by Streptomyces glaucescens NEAE-H [12]. The FTIR spectroscopy results of the extracellular pigment isolated from Xcv (Figure 4) are in agreement with the other previous reports of melanin isolated from microorganisms [1, 10, 24, 25].

Dark brown pigment has been earlier reported in few bacteria and fungus, mostly growing in diverse and extreme environmental conditions v_iz. from saline water of Lonar lake [26], water samples of saline belt [27], cold, dry and intense ultraviolet radiated area of Antarctic [8], sewage water sample [5], Arabian sea sediments [7]. Melanin pigment can protect microorganisms, such as bacteria and fungi, against thermal as well as chemical and biochemical stresses through the generation of reactive oxygen species [26]. Melanin enhances the survival and competitive abilities of organisms in harsh conditions [13, 14]. Here in the present study melanin was produced by _Xcv when exposed to vapour phase (aroma) of aromatic oils, it may be an adaptive response of the bacterium Xcv to its surrounding environment.

Melanin is produced predominantly via tyrosinase in many microorganisms where L-tyrosine or L-DOPA is the precursors [12, 16, 26, 27]. Some microorganisms produce melanin without supplementation of tyrosine in media [24], whereas for some microorganism tyrosine has to be incorporated in media [5, 26, 27, 28]. In Xanthomonas campestris pv. phaseoli, it is formed due to secretion and subsequent oxidation of homogentisic acid [4]. Melanin production is also reported via the activity of laccases, catecholases, poltketide synthase and 4-hydroxyphenylacetic acid hydroxylase enzyme pathways [1, 2, 3, 4, 6]. In Azotobacter chroococcum active polyphenol oxidase can produce melanin from catechol [2]. In Klebsiella pneumoniae, melanin is produced from

4-hydroxyphenylacetic acid hydroxylase and not tyrosinase [3]. Shrishailnath, et al. [1] also found biosynthesis of melanin from L-tyrosine via 4-hydroxyphenylacetic acid hydroxylase, an unusual pathway of melanogenesis. 4-hydroxyphenylacetic acid hydroxylase catalyzes the hydroxylation of other aromatic compounds, which leads to the formation of dibenzoquinone and other o-quinone derivatives, which then polymerize spontaneously to melanin-like polymers [1, 3]. Here in the present study, one of the melanogenesis enzymes in Xcv may have produced melanin by oxidation of the aromatic compounds of aromatic oil. Due to the versatile properties of melanin viz. photoprotective, antioxidant (radical scavenging activity), anticancer, antimicrobial, heavy metal scavenger [9, 10, 11, 12, 17], it has been used in a wide range of applications, from cosmetics, pharmaceutical, clinical, agricultural to food industry [9, 29]. The antimicrobial and antioxidant properties of melanin were demonstrated by Arun, et al. [10], where they reported the antibacterial activity of the extracellular melanin produced by Schizophyllum commune against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas fluorescens and antifungal activity against Trichophyton simii and T. rubrum at 100 μg/ ml. They also found that melanin showed high free radical scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) at 50 μg/ml concentration. Another interesting application of melanin is its photoprotective effect on bioinsecticides. The bioinsecticide made of Bacillus thuringiensis var. israelensis (BTI) is used to controls the growth of mosquito larvae, but its effectivity is greatly affected by UV radiation. The melanin produced from the bacteria Aeromonas media strain WS showed a photoprotective effect on BTI against UV and Sunlight radiation [30]. One more important application of melanin is its use for bioremediation purposes [29], melanin nanoparticles produced by Pseudomonas stutzeri HMGM- 7 were found to have −54.9 mV zeta potential, and could efficiently adsorb different heavy metals like lead, chromium, and selenium from drinking water due to their metal affinity and high adsorption ability [17]. Some other applications of melanin include its use in the production/synthesis of nanoparticles, packaging material, microbial fuel cells and sensitized solar cells [29].

The extensive use of melanin has created a huge demand for melanin in the market. The cost of 1 gm of melanin is around 444 $ which means it is costlier than the most expensive metal gold [31]. This initiated the need for exploring an environmentally friendly and economically feasible process for melanin production. Scientists have mentioned the possibility of melanin pigment synthesis from microorganisms as an alternative option for commercial production of melanin for industrial use [1, 9, 25, 29]. Here in the present investigation, 4.61 ± 0.07 and 5.13 ± 0.08 mg/mL melanin was produced by Xcv under the effect of firdous and rose aromatic oils, respectively_. This is better when compared to earlier reports, for example, Kurian [7] reported the melanin produce of 11 different bacteria isolated from 96m depth Arabian sea sediments and it ranged between 27 μg/ ml to 504 μg/ml in tyrosine basal broth. While _Schizophyllum commune produced 250 μg/l [10], Yarrowia lipolytica NCIM 3590 produced 160 mg/l [16], Streptomyces glaucescens strain NEAE-H produced 350 mg/l [12] and Streptomyces sp. produced 2.2 g/l [28].

The amount of melanin produced by bacteria is remarkable, but it depends upon various factors. An increase in melanin yield from 163 to 513 mg/l was reported by Thaira, et al. [17] in Pseudomonas stutzeri strain HMGM-7 after optimization of different parameters like media, carbon and nitrogen source in media, pH of media, temperature, inoculum load, incubation time. Whereas, Klebsiella sp. GSK produced 130 mg/l melanin in minimal medium with L -tyrosine, but then the melanin yield increased 4 times to 540mg/l when supplemented with 50 mg/l L-DOPA along with L -tyrosine [1]. For high production of melanin use of expensive media like Peptone yeast extract iron broth [12], Tryptone soya broth [17], minimal media broth [1], Luria Bertani broth [11] and so on [10, 16, 28] are used, and most of the time they are supplemented with L- tyrosine [1, 11] or L- DOPA [1, 16] or metal ion [12] to enhance the yield. In order to reduce the production cost Tarangini, et al. [25] used an economical fruit waste extract for melanin production, he successfully optimized the melanin production and obtained ~ 6.9 mg/ml melanin yield (at optimum conditions) from a garden soil microbe Bacillus safensis. Here, Nutrient broth was used for melanin production without supplementation of L- tyrosine or L-DOPA which is economical than the other specific media used for melanin production in previous reports. Also, the aromatic oils used here are economic, as they are commercially available synthetic aromatic oils. Thus, microorganisms from different environmental conditions can be tested under the effect of the vapour phase of aromatic oils to identify high melanin yielding microbe.

Conclusion

To conclude, here we have come across a very different phenomenon i.e. when Xcv was exposed to vapour phase of specific aromatic oil viz. firdous (Combination of Jasminum officinale, Cinnamomum verum and Nelumbo nucifera) and rose (Rosa damascena), the bacteria produced extracellular dark brown melanin pigment. It is the first report of melanin production by plant pathogenic bacteria Xcv and the use of aromatic oils vapour phase (aroma) to produce melanin pigment in bacteria is also novel, thus this technique of melanin production can be further explored for obtaining this costly pigment with minimal cost.

Acknowledgment

The author would like to thank Department of Plant Pathology and Agricultural Microbiology, Mahatma Phule Krishi Vidyapeeth, Rahuri for providing the required facilities. The first author would like to thank Dr. Mohinder Kadian, International Potato Center (CIP), SWCA, New Delhi to permit the author to do in-service PhD.