Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University, Katraj, Pune 411046, India
Received: 17 Jun 2017 Revised and Accepted: 31 Aug 2017
Objective:To study the production of pigments by Kocuria sp. BRI 36, their characteristics and influence of heavy metals on pigments.
Methods:The effects of various physical and chemical parameters on pigments production by Kocuria sp. BRI 36 were examined. Pigments were extracted and partially characterised by Thin Layer Chromatography (TLC) and Fourier Transform Infrared spectroscopy (FTIR). The effects of heavy metals such aspb2+,Cd 2+,Ni2+and Cr3+were studied on pigment production. Antimicrobial activity and stability studies of crude pigment were also conducted.
Results:Kocuria sp. BRI 36 isolated from cold oceanic region maximally produced red-orange pigment in presence of glucose (5% w/v)and protease peptone (0.2% w/v)at pH 7.5, 10±1°C. Thin layer chromatography (TLC) analysis revealed the occurrence of three different compounds in the crude pigment belonging to carotenoid and xanthophyll group. Metals like Ni2+and Cr3+adversely affected pigment production while Pb2+and Cd2+enhanced the yield. The significant features of Kocuria sp. BRI 36 pigment are i) antimicrobial activity against Gram-positive and Gram-negative bacteria, ii) maximum stability at pH 7.5and 10±1°Cand iii)~38% color lossat 50±1°Cin 5 h.
Conclusion:Our results suggest application potential of Kocuria sp. BRI 36 pigments in various biotechnological fields.
Keywords:Antimicrobial activity, Carotenoid, Halotolerant,Metals, Pigment
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Pigments are colourful compounds that are produced naturally or synthetically. Natural pigments produced by bacteria, fungi, plants, insects etc. have better bio-degradability and environment acceptability over synthetic pigments. Bacterial pigments could play a key role as additives incolorful beverages, textile industries as natural colorant .Biopigments are also known to posses antimicrobial and antitumor activity.Among various types of pigments reported from bacteria, acarotenoid group of pigments are more widely studied with respect to their applications. Kulkarni et al. reported theapplication of yellow pigment in thedyeing of fabric produced by Kocuria flava sp. HO-9041 similarly application of bright red pigment prodigiosin for dying of wool, nylon, acrylic and silk had been suggested by Alihosseini et al. and Ahmed et al. . While Alcantara et al.  have demonstrated the application of zeaxanthin from Flavobacterium sp in food as an additive in poultry feeds. Bradyrhizobium sp. strain was described as a canthaxanthin (4,4’-diketo-b-carotene) producer which has been usedas aqua feed to impart the desired flesh colour in farmed salmonids .
Considering the demands of bio-pigments in various applications, better quality natural colorant with higher stability is the needof the hour. Among the genus Kocuria, seventeen species have been described so far  and are found to produce pigments like ethinenone, echinenone,beta-carotene, lycopene, canthaxanthin, alfa carotene etc . However, Kocuria sp. from extreme habitats has not been studied in depth. Also, effects of heavy metals on pigment production and/or therole of Kocuria pigment in thedetection of metals has not been examined yet. In view of this, the present paper deals with production and partial characterization of carotenoid pigment produced by Kocuria sp. BRI 36 , an isolate from thecold oceanic region. The present paper also discusses an effect of heavy metals on pigment production and its potential in heavy metal detection.
MATERIALS AND METHODS
The halotolerant (15% NaCl tolerance) Kocuria sp. BRI 36 was used in this work. The organism was grown in Mineral Salt Medium (MSM) at 25±2°C for 48 h with shaking at 120 rpm . It was further used for inoculation in all the experiments at 10% concentration.
Chemicals and reagents
All chemicals used were of analytical grade. The media components were purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India).The stock solutionsof cadmium, nickel, lead and chromium at a concentration of 1000 ppm each were purchased fromSigma-Aldrich.
Extraction and estimation
The culture of Kocuria sp. BRI 36 grown for 48 h was centrifuged at 8000 rpm for 15 min. The harvested cells were washed with sterile distilled H2O and suspended in 1 ml chloroform. The pigment was extracted using themethod described by Ahmad et al. 2012 . The pigment was concentrated by rotary evaporator at 40±2°C (IKA RV 10) and dried at 37±2°C for 24h. The powder was used as crude pigment for further experiments. Its λmaxwas determined by using UV visible spectrophotometer (Thermo Fisher scientific 10 UV scanning) in the range of 200 nm to 700 nm.
The effect of various physical and chemical parameters on pigment production was evaluated by varying one parameter at a time and keeping the other parameters constant.The one giving best result was used in further experiments. At the end of each experiment, a pigment was extracted and its absorbance was measured at its λmax.
Effect of heavy metals
Kocuria sp. BRI 36 exhibits very high tolerance to heavy metals viz. lead, cadmium, chromium and nickel . Immobilized cells of BRI 36 were used to determine the effect of metals on pigment colour.Immobilization was achieved using 2.5% sodium alginate and 50 × 10-3 mole calcium chloride.The beads formed were exposed to 10-40 ppm concentration of each metal for 24 h.
To check the effect of heavy metals on pigment production,BRI 36 was cultivated in a previously standardized medium supplemented with different concentrations (1 to 5ppm) of Pb2+and Cd 2+whereas it was 5 to15 ppm for Ni2+and Cr3+. At the end of incubation, cells were separated by centrifugation at 8000 rpm for 15min, thepigment was extracted and its absorbance was measured at λmax.
The experiments for characterization of crude pigment were performed using the sample dissolved in phosphate buffered saline (PBS) at 5 mg/mlconcentration.
The stability of crude pigment was determined in terms of its absorbance at λmax.The effect of various conditions of pH (5.0, 7.0, 9.0) on stability was examined at room temperature. The pH showing maximum stability was selected to investigate effect of temperature (10 to 50°C). These conditions were further used to analyse an effect of dark and light conditions for different time intervals (24, 48, 72 h) on stability. Percent color loss was determined by using the following equation.
Where ODi = Initial OD;and ODt= OD at time (t)
Antibacterial activity of the crude pigment (5-0.013mg/ml) against several microbial strains was determined by the 96-well plate microdilution method . Different clinical isolates used were E. coli, Bacillus, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella and Salmonella paratyphi. 125 µl double strength growth medium was added to thefirst column of the 96-well microplate. After 48 h of incubation at 37°C, the optical density was measured at 600 nm. The growth percentages at different pigment concentrations for each microorganism were calculated as:
Where ODi = Initial OD;and ODt= OD at time (t)
Thin layer chromatography (TLC) and fouriertransform infrared spectroscopy (FTIR)
Kocuria sp. BRI 36 was grown under optimized conditions and the pigment was extracted as described above. TLC analysis of the crude pigment was carried out as described by Vora et al. . One mg of crude red-orange pigment was directly used for FTIR (Brucker, tensor 37) analysis. The conditions used were 16 scans at a resolution of 4cm-1 measured between 400 and 4000cm-1.
The experiments were performed in triplicates and thestandard deviation was calculated. One-way ANOVA was applied to determine thesignificant value (p<0.05).
RESULTS AND DISCUSSION
Estimation of pigment
The extracted pigment from Kocuria sp. BRI 36 was dissolved in PBS at aconcentration of 5mg/ml. spectrophotometric analysis showed 475 nm as its λmax. Previous reports on different species of Kocuria have also shown maximum absorbance of carotenoid pigment in the range of 471-477nm .
Different parameters influencing the pigment production were studied individually by varying one parameter at a time. Taking one parameter at a time represented an efficient way to optimise production of microbial metabolites and/or biological processes [16,17].We observed maximum pigment production at 10°C (0.29±0.01) (fig. 1a) and pH 7.5 (0.23±0.005) (fig. 1b). It decreased with increase in temperature. The response of microorganism to low temperature in terms of increasing proportion of unsaturated fatty acids is well documented . It helps in increasing membrane fluidity. Medicharla et al. have suggested arole of carotenoid in maintaining rigidity of membrane at low temperature. Kocuria carniphila MY and Kocuria polaris MO were also found to produce carotenoid optimally at 10°C and at neutral pH . As shown in fig. 1c, pigment absorption was highest at 5% glucose (0.22±0.005). Kocuria sp. K70 showed better pigment production at 1% lactose while for Arthrobacter sp., Serratia marcescens, Brevibacterium marisglucose proved to be the better source . We have used different organic and inorganic nitrogen compounds to examine their effect and among all, protease peptone (0.73±0.0005)was found to be the best (fig. 1d). Optimal effect of organic nitrogen on pigment production had been also observed by Kim and Park  in Kocuria sp.SmilarlyEl-Sharouny  and Subhasree et al. have reported higher pigment production in presence of peptone and yeast extract from Kocuria carniphilaMY and Kocuria Polaris MO, respectively. Kocuria sp. BRI36 (this work) is a halotolerant isolate from cold region and can grow upto 15% w/v NaClconcentration .However, increase in NaCl concentration above 5% decreased the pigment yield (fig. 1e). We have come across a few studies which focus on biotechnological production of pigments at NaCl concentrations of ≥ 15% w/v NaCl. For example, pigment production by photosynthetic halophiles at saturated NaCl (35% w/v NaCl), conferring diverse ecological advantages to the halophile which helps them to dominate their habitat [24, 25].Another species of Kocuria sp. K70, have exhibited pigment production at 2% NaCl . Similarly,Pseudomonas sp. isolated from marine environment had also displayed production at 2% w/v NaCl .NaCl both reduces water activity and osmotic stress in microbial cells;the cellular stress imposed triggers increase in various types of metabolites [27, 28]. The reduction in water activity is pertinent to the current study as this is the parameter which stimulates anincrease in secondary metabolites.
Fig.1: Effect of various parameters on pigment production by Kocuria sp. BRI 36 by varying one factor at a time, a) temperature, n=6,[0.29±0.01]b) pH, n= 8, [0.23±0.005]c) glucose concentration, n=5, [0.23±0.005]d) nitrogen source, n=7, [0.73±0.0005]and e) NaClconcentration, n=7, [0.23±0.0005]. Data were analyzed by two–way ANOVA (p<0.05) and vertical bars represent standard error. Values in the square brackets indicate mean±SD
Effect of heavy metals
Experiments were carried out to evaluate theeffect of Pb2+, Cd2+, Ni2+and Cr3+at various concentrations on pigment colour using immobilized pigmented biomass of Kocuria sp. BRI 36.Increase in metal concentration caused loss of pigment colour at the end of 24 h (fig. 2).These observations suggesta possible application of Kocuria pigment in thedetection of metal contamination, although further experimentation is necessary. Application of immobilized cells of CynobacteriaAnabaena cylindrical for detection of Cu and Pb had been shown by Wong and Teo . On similar lines, immobilized cells of Kocuria sp. BRI 36may prove helpfulin developing biosensor. With regards to theeffect of metals on pigment production, apositive effect was observed when themedium was emended with Pb2+and Cd2+(fig. 3a,c). Pigment production increased with increase in metal concentration upto 5 ppm on the contrary, increase in concentration of Ni2+and Cr3+adversely affected pigment production (fig. 3b,d).Enhancing theeffect of cadmium on pigment production by Bacillus safensis and Pseudomonasaerogenosahad been also reported by Priyalaxmi et al.  and Abdul-sada  respectively.Whereas, chromium was found to augment yellow pigmentation in S. aureus upto 100µg/ml .It may be attributed to increased pigment synthase (s) action as it was observed in case of red pigment producing Monascus sp. When cultivated inFe, Zn and Mn .To our knowledge, this is the first report analysing theeffect of heavy metals on pigment production in Kocuria sp.
Fig.2: Effect of different heavy metals on pigment colour produced by Kocuria sp. BRI 36 when exposed to different concentrations (10 ppm - 40 ppm) ofa) Cd2+b) Ni2+c) Pb2+and d) Cr3+
Fig.3: Effect of different heavy metals on pigment production. Kocuria sp. BRI 36 was cultivated in previously standardized medium supplemented with different concentrations ofa) Cd2+, n=7,[0.6±0.05] b) Ni2+,n=4,[1.85±0.005]c) Pb2+n=7,[0.34±0.005]and d) Cr3+, n=6,[1.2±0.05].Data were analyzed by two–way ANOVA (p<0.05) and vertical bars represent standard error. Values in the square brackets indicate mean±SD
The absorbance of pigment at 475 nm at room temperature and pH 7.5 was considered 100% and colour loss was determined with respect to that. Crude pigment showed ~77% stability at 10°C at the end of 5 h, while theincrease in temperature caused aloss in stability with ~38% colour loss at 50°C after 5 h (fig. 4a). Exposure of the pigment to various pH at room temperature indicated ~90% stability at pH 7.0 (fig. 4b). Studies on theeffect of dark and light conditions on pigment stability showed ~ 80% and 50% stability respectively at the end of 72 h (fig. 4c). Similar studies were carried out by Shatila et al. and they have shown 80% stability in orange colour pigment producing Exiguobacterium aurantiacum FH after exposure to light for 24 h. Thus our results indicated application potential of Kocuria sp. BRI36 pigment in textile, food, decorative articles as acolouring agent. Fig. 8 shows its use in preparation of colourful candle using paraffin wax.
Fig.4: Effect of a) temperature (10 to 50°C), b) pH (5, 7 and 9), c) dark and light conditions on stability of crude pigment extracted from Kocuria sp. BRI 36
Antimicrobial activity of the crude pigment was studied against E. Coli, Pseudomonas aerugenosa and Bacillus subtilis. The activity increased with increasing concentration of pigment (0.015 to 5 mg/ml). We observed 83% growth inhibition in Pseudomonas aeruginosa and Bacillus subtilis while it was 75% in case of E. coli at 0.015 mg/ml concentration. The maximum effect was recorded in Pseudomonas aeruginosa with more than 95% inhibition at 5 mg/ml concentration (fig. 5).
Similarly, Kushwaha et al.  reported antimicrobial activity of the pigment produced by psychrotrophicKocuria sp. against Bacillus cereus, Staphylococcus aureus, Vibrio choler,Shigella dysenteriae at 40 to 80 μg/ml pigment concentration. Carotenoid pigment isolated from Micrococcus sp. had also showed inhibitory effect on Gram-positive organism when tested in the concentration range of 0.25 to 2.0 mg/ml . Thus, our results demonstrate thepotential of BRI 36 pigment for application in pharmaceutical and cosmetic products.
Fig.5: Anti microbial activity of crude pigment (5-0.013mg/ml) against different clinical isolates viz. E. coli,Bacillus subtilis', Staphylococcus aureus, Pseudomonasaeruginosa, Shigella dysenteriae', and Salmonella paratyphi
TLC and FTIR
The crude pigment was found to be a mixture of three different compounds corresponding to the Rf values of 0.177, 0.387, 0.9182 (fig. 7) as observed in TLC experiments. The Rf values (0.387 and 0.9182) are in accordance to reported Rf values of carotenoid pigments . Rf value of 0.177 indicates thepresence of xanthophyll which is an oxygenated derivative of carotenoid . FTIR spectra of crude pigment gave the prominent peaks at 2921.69, 2852.76 and 1066.72 cm-1 as depicted in fig. 6. Other peaks were observed at 1737.15, 1627.53, 1460.14, 1220.65, 518.85 cm-1. Comparatively very less literature is available on psychrotrophic bacterial carotenoid pigment using FTIR spectroscopy. The bands at 2921.69 cm-1 are due to asymmetrical stretching vibration of aliphatic CH group while at 2852.76 cm-1 are due to asymmetrical stretching vibration of thesame group as is interpreted by Latha and Jeevaratnam .The peak at 1460 cm-1may be due to asymmetrical deformation vibration of CH3 groups. Bands at 1657 cm-1may be due to thepresence of theolefinic functional group. Peak at 1734.42 cm-1is due to>C1=O group probably ester. However thecomplete structure of compounds cannot be determined based on IR data. Vibrational peaks are most likely due to oxidation and/or deformation in polyene chain .
Fig.6: Thin layer chromatogram of the crude pigment,the sample was resolved using butanol:ethanol:water (9:1:1) system
Fig.7: FTIR analysis of the crude pigment. One mg of crude red-orange pigment was directly used for FTIR analysis. The conditions used were 16 scans at a resolution of 4 cm-1 measured between 400 and 4000cm-1
Fig.8: Preparation of colour candle using crude pigment
Crude pigment produced by Kocuria sp. BRI 36 is a mixture of three different compounds with significant stability at high temperature and pH. Glucose and protease peptone found to affect pigment production positively while heavy metals like Cr3+and Ni2+had negative effect on pigment production. Other recent studies found that the ecophysiology of pigments produced by bacteria can be exploited for biotechnological purposes . The findings of the current study suggest that phylogenetically diverse types of microbes may potentially yield biotechnologically valuable pigments, and further bio-prospecting efforts are needed to determine how much-untapped potential there is in hitherto uncharacterised microbial pigments.
We gratefully acknowledge financial support from Bharati Vidyapeeth Deemed University, Pune to undertake this work.
All the experiments were performed by Anuradha Mulik. Priyanka Kumbhar assisted her for optimization and characterization experiments. Planning of experiments, result analysis, manuscript writing and reviewing werecarried out by Dr. Rama Bhadekar.
CONFLICT OF INTERESTS
No conflict of interest was reported by the authors
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How to cite this article
Anuradha Mulik, Priyanka Kumbhar, Rama Bhadekar. Production and partial characterization of pigments produced by Kocuria spbri 36: influence of heavy metals.Int J Pharm Pharm Sci 2017;9(10):137-145.