melanin from marine streptomyces sp. (mvcs13) with potential effect against ornamental fish...

Original Research Paper

Melanin from marine Streptomyces sp. (MVCS13) with potentialeffect against ornamental fish pathogens of Carassius auratus(Linnaeus, 1758)

P. Sivaperumal a,b,n, K. Kamala a, R. Rajaram b, Saurabh S. Mishra a

a Central Institute of Fisheries Education, ICAR-Deemed University, Mumbai 400061, Indiab Department of Marine Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India

a r t i c l e i n f o

Article history:Received 16 August 2014Received in revised form3 September 2014Accepted 15 September 2014

Keywords:MelaninFT-IROrnamental fish pathogensAntibacterial activity

a b s t r a c t

Melanin pigments were extracted from a wide variety of microorganisms including bacteria and fungi. Inthe present study isolation, identification and characterization of melanin from marine actinobacterium(Streptomyces sp. MVCS13) and its potential activities against fish pathogens were investigated. Cultureconditions and medium composition for the melanin production were optimized. Further, pigment wascharacterized by UV–vis absorption spectroscopy and FT-IR infrared spectrometry. It has potentialantibacterial activity against ornamental fish pathogens Vibrio sp. FPO5 (1570.01 mm) followed byAeromonas sp. FPO6 (1270.02 mm) which was isolated from Carassius auratus infected fish. Theminimum inhibitory concentration (MIC) ranges were observed between 1870.01 and 2770.03 μg/ml.The present study suggested that, on the basis of anti-bacterial activity and MIC test of melanin pigmentfrom Streptomyces sp., MVCS13 can be selected as an effective anti-bacterial agent for ornamental fishculture.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

With increasing intensification in commercial aquaculture, manyproducts are being made availabe for aquaculture practice withvarying success rate. Use of antibiotics is a common practice inaquaculture to control fish diseases. The fish diseases are mainlycaused by bacteria, fungus, virus and protozoa. Particularly, bacterialdiseases are liable for heavy mortality rates in fishes. Severalchemotherapeutic agents are used in the fishery sector for thetreatment of fish diseases caused by bacterial pathogens. However,the fish bacterial pathogens are becoming more resistant to currentlyused drugs (Kayis et al., 2009). Hence, the fishermen are facing lotsof difficulties to control the diseases caused by these bacteria, andthe fishing sector is more susceptible in connection with bacterialpathogens. The associated problem of antibiotic resistance in aqua-cluture has grown to be more complex (GESAMP, 1997) whichnecessitate the search for alternative drugs, preferably of naturalsources with no side effects to the host. There is worldwide interestin the development of processes for the production of antibioticsfrom primary metabolites like pigments kind of natural sources due

to the serious safety problemwith many artificial synthetic colorants,which have been widely used in agriculture field as feed and coloragents. However, over the decades, efforts for antibiotics and pharm-aceutically relevant compounds discovery have been shifted toprimary metabolites which have been produced by microbes. Theimportant primary metabolites like melanin frommicrobes and theirbiological role have received attention recently. Melanin is a com-mon term used for dark brown to black pigments of high molecularmass formed by oxidative polymerization of phenolic compoundsusually complexed with protein and carbohydrates. Actinobacteriahave long been described as capable of producing dark brown blackcolored metabolic polymers which are important not only as a usefulcriterion in taxonomic studies but also because of their resemblanceto soil humic substances. Melanins are not important for the growthand development in the organism's biology but play a significant rolein improving their survival and competitiveness. They are prolificsource of pigments and the vast majority of these compounds arederived from the single genus Streptomyces (Pathom-Aree et al.,2006). They showed a broad spectrum of biological roles, includingantioxidant (Goncalves and Pombeiro-Sponchiado, 2005), antimicro-bial activity (Casadevall et al., 2000), antitumor activity (El-Obeidet al., 2006), etc. Inspired by these facts, the aim of present researchwork was to evaluate the isolation and characterization of melanin

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Biocatalysis and Agricultural Biotechnology

http://dx.doi.org/10.1016/j.bcab.2014.09.0071878-8181/& 2014 Elsevier Ltd. All rights reserved.

n Corresponding author. Mobile: þ91 9892723141.E-mail address: [email protected] (P. Sivaperumal).

Please cite this article as: Sivaperumal, P., et al., Melanin from marine Streptomyces sp. (MVCS13) with potential effect againstornamental fish pathogens of Carassius auratus.... Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.007i

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from Streptomyces sp. MVCS13 and its antibacterial potential againstornamental fish pathogens isolated from Carassius auratus.

2. Materials and methods

2.1. Isolation and identification of marine actinobacteria

A marine actinobacterium Streptomyces sp. MVCS13 was isolatedfrom the marine sediments of Versova coast (Lat 19108026.12″N;Long 72148007.41″E), India and screened for melanin productionon Kuster's agar incubated for 7 days at 28 1C. After incubation,production of melanoid pigments was observed on the medium.Development of dark brown to black color around the media wasconsidered positive for melanin pigments.

Actinobacterial strains were identified based on their morpho-logical and biochemical characteristics as described in the Bergey'sManual of Determinative Bacteriology (Buchanan and Gibbons,1974). Micromorphology and sporulation were observed under alight microscope using the cover slip method with a well grownsporulated culture. The genus was identified based on their cellwall composition taking into account the presence or absence ofGlycine with Diaminopimelicacid isomers (DAP), LL-Diaminopi-melicacid, and meso-Diaminopimelicacid with sugar components.Comparing the chemical composition and micromorphology, thespecific genus was identified using the key of Nonomura (1974).

Molecular identification of the isolate was achieved by 16S rRNAgene sequencing. The DNA was isolated by the phenol chloroformmethod (Marmur, 1961). The universal primer was chosen for theactinobacterial 16S rRNA gene. Sequencing was done using forwardprimer 27F and reverse primer 1492R. PCRs were performed with thefollowing conditions: 35 cycles consisting of 95 1C for 1 min and72 1C for 5 min, followed by a final extension of 5 min at 72 1C. The16S rRNA gene sequence was analyzed by an automated DNAsequencer (Applied Biosystems) and sequences were edited withDNA Baser ver. 3.2.13., and initially aligned using ClustalW algorithm(Thompson et al., 1997) and then optimized visually. Phylogeneticanalysis of actinobacterial strain MVCS13 (16S rDNA) was carried outin tandem with representative actinobacterial sequences obtainedfrom GenBank data. A selection of representative species was mainlyfocused to know the evolutionary relationship of actinobacteriabased on nblast results with high similarity values and query cover-age. Neighbor-joining (NJ) method was employed using MEGA5 software.

2.2. Melanin production

Melanin production was carried out by following Manivasaganet al. (2013) with slight modification. Composition of the productionmedium used was Tyrosine agar (Glycerol 10 mL; L-Tyrosine 0.5 g;L-Asparagine 1 g; K2HPO4 0.5 g; MgSO4 �7H2O 0.5 g; NaCl 0.5 g;FeSO4 �7H2O 0.01 g; Trace Salt solution 1ml; pH 7.0 and 100%seawater). All the experiments were carried out in 500 ml Erlenmeyerflasks containing 100ml of Tyrosine agar medium. Sterile mediumwas inoculated with 5% of inoculums (3.1�104–5.4�104 CFU/ml),incubated at 28 1C and cultivated under agitation at 200 rpm for7 days. Growth and melanin production were determined from thesamples collected at 12 h intervals for which samples (5 ml) weretaken from each of three replicate tubes. The cells were harvested bycentrifugation at 12,000 rpm for 15 min, at 4 1C.

2.3. Melanin formation

Melanin pigment was estimated by taking 2 ml of the cultureand 1 ml of 0.4% substrate solution (L-Tyrosine or L-DOPA). Thereaction mixture was incubated at 37 1C for 30 min for L-Tyrosine

or 5 min for L-DOPA and black coloration resulting from dopa-chrome formation which was observed and read spectrophotome-trically at 300 nm (UV-1800-Shimadzu Scientific Instruments,USA). When there was no coloration within these periods, thereaction mixture was further incubated for as long as 2 h. Afterincubation, melanin was found to be formed within 30 min(Scribners et al., 1973). Three replicates were maintained for eachtreatment.

2.4. Melanin optimization

Melanin optimization was carried out by using different physicalparameters such as pH, temperature and nutritional parameters, i.e.carbon and nitrogen sources and trace salt components. The differentconcentrations were used to optimize medium composition formaximum melanin production. The kinetics of melanin productionwas followed in batch cultures under optimum conditions. Theexperiment was planned for 7 days starting from the log phase tostationary phase under submerged culture conditions. The resultingcell-free supernatant was removed by filtration followed by coldcentrifugation at 10,000 rpm at 4 1C for 20 min. The supernatant wasanalyzed for melanin production.

2.5. Purification of melanin

The culture broth was harvested by centrifugation at 10,000 rpmfor 15 min at 4 1C. The culture medium was centrifuged to removethe cells, and melanin was precipitated from the supernatant bychanging the pH to 3.0 with 5 N HCl. The precipitated melanin wasre-dissolved in distilled water at pH 8.0, centrifuged again anddialyzed against distilled water. The dialysis was carryout for at least24 h and stopped when the dialyzed solution reached the pH 4.5. Thedialyzed preparation of melanin was lyophilized. Further purificationwas carried out using ion exchange chromatography (SephadexG-50). The melanin powder was applied to a Sephadex G-50 column,pre-equilibrated with 20 mM potassium phosphate buffer (pH 7.0).After washing the column with 3 vol. of equilibration buffer, boundmelanins were eluted stepwise using phosphate buffers of increasingmolarity and decreasing pH values at room temperature. The flowrate was adjusted to 24 ml/h and fractions (1 ml each) were collected.The fractions showing high melanin content were pooled togetherand used as a purified melanin for further characterization study.

2.6. Characterization Studies

To observe the UV–vis absorption spectra of melanin, 0.1 mg ofmelanin was dissolved in alkaline distilled water, at pH 8.0 (Zhanget al., 2007), and the resulting solution was scanned with aspectrophotometer (UV-1800-Shimadzu Scientific Instruments,USA) to obtain its absorption spectra at wavelengths ranging from200 to 380 nm. For IR spectrum, the pigment was further purifiedby acid hydrolysis with 0.5 ml of 5 N HCl in a sealed glass vial andkept for 2 h at 100 1C. The IR spectrum of purified pigment wasrecorded with a Perkin-Elmer model 297IR spectrophotometer.One part of the purified pigment was mixed with 99 parts of driedpotassium bromide and it was scanned between 600 and 4000wave number (cm�1) at a speed of 1 μm/ min and with aprogrammed slit opening and air as reference.

2.7. Isolation and identification of fish pathogens from ornamentalfish C. auratus

Bacteria were isolated from fresh water ornamental fish ofC. auratus at Central Institute of Fisheries Education, Mumbai.Infected part of the fish was serially diluted and plated on Zobellmarine agar and incubated at 37 1C for 24 h. After incubation

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colonies were pure cultured and subcultured on Zobell marineagar slants. The morphological and biochemical tests were per-formed according to Bergey's manual of systematic bacteriology(Buchanan and Gibbons, 1974) and the molecular identification ofthe bacterial isolate was achieved by 16S rRNA gene sequencing asmentioned above in detail.

2.8. Antibacterial assay

Antibacterial activity of purified melanin pigment against 6 fishbacterial strains viz. Bacillus sp. FPO1, Aeromonas sp. FPO2, Citro-bacter sp. FPO3, Edwardsiella sp. FPO4, Vibrio sp. FPO5 andAeromonas sp. FPO6 was carried out using a disc diffusion method(Mercan et al., 2006). Filter paper (Whatmann No. 1) discs with5 mm diameter were impregnated with known amount testsamples of the lyophilized pigment and positive control containeda standard (streptomycin) antibiotic disc. The impregnated discsalong with control (incorporated with sterile water) were kept atthe center of Nutrient Agar Plates, seeded with test fish bacterialcultures. After incubation at room temperature (37 1C) for 24 hantibacterial activity expressed in terms of diameter of zone ofinhibition was measured and recorded. The minimum inhibitoryconcentrations (MICs) of the purified melanin were determined bya serial dilution technique (Noble and Sykes, 1977) in the presenceof standard streptomycin.

3. Results and discussion

3.1. Isolation and identification of marine actinobacteria

A marine actinobacterium (MVCS13) strain was isolated fromthe marine sediment sample collected from Versova coast, Mum-bai and it produced enormous melanoid pigments (Fig.1A). Thestrain produced aerial and substrate mycelia and vegetativehyphae produced an extensively branched mycelium and sporechain was spiral (Fig.1B). Examinations of whole cell hydrolysatesshowed the presence of LL-DAP with glycine, indicating that thestrain belongs to the cell wall chemo type I. This isolate wastentatively identified as Streptomyces sp. MVCS13 based on themorphology and it was confirmed by the 16S rDNA sequencing.The sequence was submitted to Gene Bank in NCBI (http://www.ncbi.nlm.nih.gov/nuccore/KC292199) with the accession number(KC292199). In this strain MVCS13 is phylogenetically close to thegenus Streptomyces (Fig. 1C).

3.2. Melanin production and optimization

In the present study, production and optimization of melaninwere carried out by using different concentrations of mediumcomposition with different variables. Among them L-Tyrosine, Aspar-agine, MgSo4, NaCl, FeSo4, Trace Salt solution, pH and temperature

Fig. 1. (A) Screening of melanin on Kuster's agar medium. (B) Spore morphology – spiral. (C) Neighbor-joining tree based on 16Sr DNA sequences.

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http://www.ncbi.nlm.nih.gov/nuccore/KC292199

http://www.ncbi.nlm.nih.gov/nuccore/KC292199




Fig. 2. (A–J) Production and optimization of melanin from Streptomyces sp., MVCS13.






were identified as most significant variables influencing melaninproduction (Fig. 2A–J). Optimum temperature (50 1C (Fig. 2J)) and pH(7.4 (Fig. 2I)) were found for highest melanin production from

Streptomyces sp. MVCS13. It seems that the melanin from Streptomycessp. MVCS13 was considerably similar to the Osmanthus fragransmelanin reported to date (Wang et al., 2006) because most of themelanin was stable only up to 25–50 1C. Previously a few researchershave reported melanin which is stable only in the pH ranges of 4–11.0(Wang et al., 2006). Among the different parameters, pH of the growthmedium is said to play an important role by inducing morphologicalchanges in microbes and their melanin secretion and the pH changesduring the microbial growth can also affect product stability in themedium (Gupta et al., 2003). The broad ranges of NaCl concentrations(0.25–1.25 g/L) were used and the moderate production was observedat 0.75 g/L (Fig. 2F), but the melanin production decreased when theNaCl concentration increased from 0.75 g to 1 g/L. Similarly Dharmarajet al. (2009) have also reported that in Streptomyces sp. highest

Fig. 3. Differentiation of melanin production between normal andoptimized media.

Fig. 4. UV spectrum of melanin from 200 nm to 380 nm.

Fig. 5. FT-IR spectrum of melanin.

Table 1Morphological and biochemical characteristics of bacterial strains isolated frominfected ornamental fish.

FPO1 FPO2 FPO3 FPO4 FPO5 FPO6

Gram staining þ � � � � �Shape Rod Rod Rod Rod Rod RodMotility M M M M M MIndole test � þ þ þ þ þMethyl red test þ þ � þ þVoges Proskeur test þ � � � � �Citrate utilization test � � þ � þ -Urease test � � þ � � �H2S � � þ þ � �Gas � � � � � �Nitrate reduction test � þ � � þ þCatalase tests þ þ þ þ þ þOxidase test þ þ � � þ þCarbohydrate testGlucose þ þ þ þ þ þMaltose þ þ þ þ þ þSucrose þ � þ � þ �

FPO1 (Bacillus sp.); FPO2 (Aeromonas sp.); FPO3 (Citrobacter sp.); FPO4 (Edwardsiellasp.); FPO5 (Vibrio sp.) FPO6 (Aeromonas sp.); þ , Positive; � , Negative; and M,motile.






production of melanin occurred in 2.5% of sodium chloride. It is worthmentioning here that NaCl-sensitive actinobacteria can increase theirtolerance to higher concentrations during successive cultivations witha significantly different absorbance in the visible spectrum, suggestingthe production of new metabolites. Comparatively highest melaninproduction (238.57 mg/ml) was observed from optimized mediumcomposition (Glycerol-10 g; L-Tyrosine 0.75 g/L, Asparagine1.5 g/L,K2HPO4 0.5 g/L; MgSo4 0.25 g/L, NaCl 0.75 g/L, FeSo4 0.015 g/L, TraceSalt solution 1.5 ml, pH 7.4 and temperature 50 1C) (Fig. 3). A fewreports are available concerning the optimization of growth para-meters for microbes, such as Escherichia coli, Bacillus thuringiensis(Vilas-Boas et al., 2005), Bacillus cereus (Zhang et al., 2007), Strepto-myces (Dastager et al., 2006) and Actinoalloteichus Sp., (Manivasaganet al., 2013) with melanin production. Melanin synthesis inhibitorscan be used to identify the different types of melanin (Rizner andWheeler, 2003), such as tricyclazole, pyroquilon, thalide and chlo-benthiazone which inhibit the synthesis of 1,8-dihydroxy naphtha-lene (DHN) melanin. However, DOPA melanin synthesis was inhibited

by tropolone, Kojic acid, 2- mercaptobenzimidazole and diethyldithio-carbamate (Elliott, 1995). In the present study, inhibition of melaninproduction was observed by the test of strain MVCS13 grown in Kojicacid (DOPA melanin inhibitor). Moreover, the marine actinobacteriumStreptomyces sp. MVCS13 showed growth in the presence of tricycla-zole (DHN melanin inhibitor), appeared normal and was similar tocontrol. These results showed that Streptomyces sp. MVCS13 strainsynthesized DOPA melanin.

3.3. Characterization studies

The ultraviolet (UV)–visible light absorption spectrum of thepurified melanin of the strain MVCS13 showed absorption peak at300 nm (Fig. 4). The absorption of wavelength is almost 300 nm inthe case of melanins. Hence, the strong UV absorption at 300 nm isoften used to identify melanins (Ravishankar et al., 1995). The FTIRspectrum of the melanin also characterized a broad absorptionband at 3422 cm�1 which revealed the presence of OH stretching

Fig. 6. (A) C. auratus ornamental fish. (B) Infected area. (C) Amplified DNA. (D) Neighbor-joining tree based on 16Sr DNA sequences for isolated fish pathogens.






and hydroxyl group. The peaks at 1096 cm�1 and 1035 cm�1 wereassigned to CN stretch bond related to aliphatic amines and thepeaks at 2959 and 2924 cm�1 were assigned to alkanes group CHstretch. The absorption bands at 2343 and 2361 cm�1 revealed thepresence of N–H and CQO groups, respectively. The peak at1625 cm�1 was CQC stretch and peaks at 1420, 1388 cm�1 wereof aromatic groups and are followed by the peaks at 874 and801 cm�1 due to CQO stretching (Fig. 5). This is due to thepresence of many complex conjugated structures in the melaninmolecule (Cockell and Knowland, 1999). Suryanarayanan et al.(2004) have described the IR spectrum of melanin with peaks near3352.5 cm�1 ascribed to OH and NH bonds.

3.4. Isolation and identification of fish pathogens

Bacterial disease is the most common infectious problem ofornamental fishes and most bacterial infections are caused byeither gram-positive or negative organisms. The common bacterialfish pathogens are natural inhabitants of the aquatic environment,whether it is freshwater or marine. Extreme stress conditions,including shipping, crowding, poor water quality and insufficientnutrition, may expose an ornamental fish to bacterial disease. Inthe present study a total of six bacterial isolates were obtainedfrom infected region of C. auratus through plating methods andthey were identified through morphological and biochemicalcharacteristics. These 6 isolates belongs to 5 genera such asBacillus sp. FPO1, Aeromonas sp. FPO2 and FPO6, Citrobacter sp.FPO3, Edwardsiella sp. FPO4 and Vibrio sp. FPO5 (Table 1). Accord-ing to the 16S rDNA analysis, all the isolates showed 100%similarity with respective genus (Fig. 6D). The dominant genusAeromonas is a complex group of gram negative ubiquitousbacteria commonly isolated from clinical, environmental, anddrinking water samples (Kuhn et al., 1997a). Aeromonas veroniihas been reported as a food borne pathogen causing infection infish, food producing animals, and humans (Austin and Austin,1993; Isonhood and Drake, 2002; Janda and Abbott, 1996). Aero-monas sp. has previously been isolated from ulcerative diseasedfish in the Indo-Pakistan region by Iqbal et al. (1998). Ornamentalfish bacterial diseases are still unknown; nevertheless, organismsbelonging to the potentially fish-pathogenic genera Aeromonas,Vibrio, and Bacillus were often isolated from the infected fish(Rahman et al., 2002). Previously McGarey et al. (1991) reportedthat representatives of Aeromonas sp. were recovered most repeat-edly, followed by Vibrio and Plesiomonas sp. Therefore, this study

conveys important information to the C. auratus ornamental fishbacterial disease in Indian aquaculture field.

3.5. Determination of antibacterial activity and MIC

Natural products are considered as an important source of newantibacterial agents. Drugs derived from natural products or drugssemi-synthetically obtained from natural sources corresponded to78% of the new drugs (Cragg et al., 1997). Marine forms naturalproducts comprise approximately a half of the total global biodi-versity; large-scale screening will continue to play an importantrole in the development of new drugs (Xu et al., 2004). Marinemicroorganisms are a rich source of structurally novel and biolo-gically active metabolites. Many chemically distinctive compoundsof marine origin with different biological activities have beenisolated and a number of them are under investigation and arebeing developed as new pharmaceuticals (Faulkner, 2000a, 2000b;Da Rocha et al., 2001; Schwartsmann et al., 2001). In the presentstudy, purified melanin at a concentration of 30 μg/disc showedantibacterial activity against 6 fish bacterial pathogens with respectto the standard 30 μg/disc streptomycin. The results are presentedin Table 2. The maximum zone of inhibition was found to be1570.01 mm (30 μg/disc) for Vibrio sp. FPO5 and 1270.02 mm(30 μg/disc) for Aeromonas sp. FPO6; whereas, 2270.01 and1670.03 mm for the standard streptomycin. Similarly Lin et al.(2005) have reported the effect of bacterial melanin on the activityof antibiotics against E. coli. The minimum inhibitory concentration(MIC) of the extract is shown in Table 2. It was found that thepurified melanin showed good activity against fish bacterial patho-gens. The minimum inhibitory concentration values of purifiedmelanin against the test organisms have indicated that the purifiedmelanin has shown less antibacterial activity, when compared tothe standard antibiotics tested streptomycin, as revealed by mini-mum inhibitory concentrations.

4. Conclusion

In the international market the production of pigments isincreasing every year due to the commercial use. According tothe results shown above, we found that the production andcharacterization of melanin from Streptomyces sp. MVCS13 areconsidered as most profitable. Another important finding is thatthe isolation and identification of six fish pathogens from affectedCarassius auratus ornamental fresh water fish are carried out andthe melanin pigments have potential antibacterial activity againstthese fish pathogens at in-vitro condition. It concluded that thepurified melanin pigment from Streptomyces sp. MVCS13 can beselected as effective anti-bacterial agent for ornamental fishculture.

Acknowledgment

The authors are grateful to CIFE, ICAR-Deemed University,Mumbai for providing facilities to carry out this study and thankfulto Dr. B.B. Nayak, Principal Scientist, PHT Division, CIFE, Mumbaifor giving valuable suggestion.

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Table 2Antibacterial activity and minimum inhibitory concentration (MIC) of the purifiedmelanin against ornamental fish pathogenic bacteria. The data is presented as themean7value standard deviation of three replicates.

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melanin from marine streptomyces sp. (mvcs13) with potential effect against ornamental fish...

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