1Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, BharatiVidyapeeth Deemed University, Pune-SataraRoad, Katraj, Pune 411043, Maharashtra, India, 2Department of Biotechnology, Modern College of Arts, Science and Commerce, Shivajinagar, Pune 411005, Maharashtra, India
Received: 24 Jun 2017 Revised and Accepted: 31 Aug 2017
Objective:To study 1) Optimization of nutritional and environmental parameters to enhance theyield of EPS by HalomonassmyrnensisSVD III isolated from seawater,West Coast of Maharashtra, India and 2) Purification and characterization of the EPS produced.
Methods:The isolate was grown in Sehgal and Gibbons (SG) medium broth supplemented with 3% glucose, at 37°C, 120 rpm for 7 d. Optimization of different parameters was carried out with one factor at a time approach. EPS was isolated from cell-free supernatantof the culture broth bycentrifugation and precipitation using chilled ethanol, after removal of proteins by trichloroacetic acid (TCA) treatment.Characterization of the purified EPS was carried out with respect to fourier-transform infrared (FTIR) spectrum, 1H nuclear magnetic resonance (NMR) spectrum and mass spectrometry (MS) analysis.
Results:Two-fold increase in the yield of EPS (23 g/l) by the selected isolate was obtained by using culture conditions as 10% inoculum size having cell density of 107 cells/ml, pH 6, incubation temperature 45°C, 3% carbohydrate, 0.5% yeast extract as nitrogen source, 20% salt concentration and 7 d of incubation period. Characterization of the purified EPS suggested thepresence of dominated glycosidic linkages and heptasaccharide nature of the molecule. As the present strain is halophilic, 20% NaCl was found to be optimum.
Conclusion:Optimization studies resulted in two-fold increase in the yield of EPS which is of heptasaccharide nature.
Keywords:HalomonassmyrnensisSVD III, Exopolysaccharide, Characterization of EPS by FTIR, MS, 1H NMR, Glycosidic linkages, Heptasaccharide, Moderately halophilic bacterium
© 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/)
Microbial exopolysaccharides are polysaccharides produced by microbes extracellularlyas capsule or slime. These microbial EPS generally are categorized into 2 broad classes namely homopolysaccharide composed of single units of monosaccharides and heteropolysaccharide composed of two or more units of monosaccharides.
Microbial EPS are non-toxic, biodegradable and renewable in nature . They play an important role in protection against desiccation  and also useful in forming biofilms [3,4]. Their various applications include use as gelling agents, biosurfactants, emulsifiers, viscosifiers[5-7],biosorbants[8,9], biologically active antimicrobials, anticancer agents and antioxidants[10-13].
Dextran is the first industrial EPS produced by Leuconostocmesenteroidesfollowed by the xanthan gum which is produced by Xanthomonascampestrisas another approved food additive . Many Bacillus species are reported for EPS production [16-20]. Production of EPS from alkaliphilicVagococcuscarniphilus was investigated.
EPS from extremophilic microorganisms especially halophiles are comparatively less reported. Production of EPS from halophilic bacteria within extreme marine habitat along with its biological activities was reviewed . Haloferaxmediterranei, a halophilicarchaeonwas reported for the production of EPS with excellent rheological properties and has application in oil recovery [22,6].EPS from Halomonasventosae and Halomonasanticariensis has psuedoplasticbehavior. Moderately halophilicHalomonascerinasp. nov.was isolated from saline soils in Spain which produced EPS .
The yield, composition and structure of the EPS vary with the microorganism and conditions of fermentation. There are different requirements of carbon, nitrogen sources, temperature, pH, minerals etc. for different microorganisms for EPS production. The nutritional and environmental conditions influence theyield of EPS . EPS yield was found to be increased when marine bacteria were grown on limited nutrients such as phosphorous, sulphur, nitrogen and potassium . During optimization of EPS, the selection of carbon source plays an important role. When growth medium was amended with sucrose, the highest EPS yield was obtained fromHahellachejuensis isolated from Cheju Island, Republic of Korea . The haloalkaliphilic microorganism, Halomonasalkaliantartica strain CRSS which was isolated from asaline lake in Antarctica produced maximum EPS when acetatewas used as a carbon source . EPS production requires high carbon content and less nitrogen quantity in the production medium.
EPS is characterized by analyzing the hydrolysate of EPS by High-performance liquid chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR) etc.
EPS from the microbial origin can be used as antiviral, antitumor and immunogenic agents and also for their functional properties like gel formation and rheology. Dextrans are explored as a plasma substitute and about 6% dextran with 50,000-100,000 relative molecular weight has equivalent viscosity and colloid-osmotic properties to blood plasma. Also, it can be used as non-irritant absorbent wound dressings . EPS curdlanhas antitumor activity. EPS is also used in encapsulated drugs and lotion because of its gel formation property. Although polysaccharides can be seen in thepreparation of vaccines, some practical issues pose difficulties in this regard such as poor immune response by polysaccharide antigens. This can be addressed by chemical modification . EPS from Halomonasmauraand H.eurihalina has immunomodulating activity [31-34]. H.stenophila produced EPS having antitumor activity .
Due to surfactant and bio emulsifier activity of EPS, they have importance in enhancing oil recovery. As most of the oil recovery fields are observed in saline environments, EPS from halophiles may have anadvantage. Biological activities of EPS produced from marine halophilic bacteria have been reviewed . Halomonas is the potential genus producing EPS having efficient emulsifying activity among halophilicbacteria.Halomonasalkaliantartica,H.ventose, H. anticariencis and H.maurawere found to produce EPS that are proposed to have arole as anemulsifying agent in oil recovery [36,28,23,32].
As compared to other extremophiles like alkaliphiles and thermophiles, less attention has been paid to halophiles. Very few researchers have carried out optimization and characterization studies on EPS of halophiles as can be seen from the literature. The present studies were aimed at exploitation of halophiles from unexplored saline environments from West Coast of Maharashtra, India;optimization of different parameters to enhance theyield of EPS and its characterization to understand its functional groups and linkages. An attempt has been made to reach to the rationale that EPS from halophilic microorganisms could be useful for recovery of oil from theocean where there is a saline environment. Likewise, EPS from halophiles are considered to have immunomodulatory activity. Such kinds of studies are yet to be expanded and hence halophilic bacteria were selected for EPS production.
Halomonassmyrnensis SVD III was isolated from ocean water of Deobaug, West Coast of Maharashtra, India . The organism was found to produce EPS. Present study was carried out to investigate the effect of various parameters on EPS production byHalomonassmyrnensis SVD III (GeneBank accession number-KX057990) and its characterization.
Chemicals and reagents
All the chemicals were of laboratory grade and purchased from Hi-media, Merck, SD Fine Chemicals Ltd. and Qualigens, India.
HalomonassmyrnensisSVD III isolated from ocean water collected in Deobaug, West Coast ofMaharashtra, India was used in the present study. The culture was stored at 4°Con Sehgal and Gibbons (SG) medium+15% NaCl concentration for further study.
Inoculum was prepared for theproduction of EPS by growing the culture in SG medium to have acell density of 107cells/ml as measured by total viable count (TVC) method. The SG medium containing 3% glucose was used for theproduction of EPS. The composition of SG medium was casamino acids-0.75%, yeast extract-1%, potassium chloride-0.2%, trisodiumcitrate-0.3%, magnesium sulphate-2%, sodium chloride-15%, pH-7.2.
Effect of various parameters on EPS production byHalomonassmyrnensis SVD III was studied. The strain SVD III was cultivated in SG medium and incubated at 37°C, 120 rpm for 7 d. The culture was used at 10% inoculum sizewith 107cells/ml for all experiments. Effect of one parameter was studied at a time keeping other parameters constant. All experiments were carried out in triplicate using 50 ml SG medium in 250 ml Erlenmeyer flask. At the end of each experiment, EPS was extracted and estimated gravimetrically.
Effect of temperature on EPS production was studied by varying the temperature as 25°C, 37°C and 45°C.The most suitable temperature was selected to determine effect of pH (5, 6, 7, 8 and 9), incubation period (1 to 8 d),inoculum size (1%, 5% and 10%), concentration of NaCl (10, 15, 20 and 25%), concentration of carbon source (3%, 4%, 5% and 6% glucose) and nitrogen source (0.5%, 1%, 1.5% and 2% yeast extract) on production of EPS.
Isolation and purification of EPS
The culture broth was centrifuged at 8000 rpm for 10 min. The cell-free culture broth (CFCB) was collected and 20%trichloroacetic acid (TCA) added to the same. CFCB was then kept on ice for 30 min to precipitate proteins followed by the centrifugation at 8000 rpm for 10 min. The supernatant was then collected in aglass tube and two volumes of chilled ethanol added. The solution was kept at 4°C overnight to precipitate EPS [6,30]. The amount of EPS was calculated based on dry weight estimation by gravimetric method.
Characterization of EPS
The purified EPS was characterized using fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) [6,12,18,31].
The purified EPS was analyzed for FTIRspectroscopy analysis. Thesample was analyzed between infrared spectrum 3500-400 cm-1 using Bruker-TENSOR 37 IR spectrophotometer (USA).The purified EPS was subjected to NMRspectroscopy analysis usingBruker-Ascend 500 MHz (USA).Dimethylsulfonate (DMSO) was used as asolvent system.Applications of NMR used were1Hi.e. hydrogen-1 NMR/proton NMR in which analysis is done with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules . The purified EPS was analyzed by MS using Bruker-HRMS Impact HD Q-TOF MS (USA). Parameters for this study were as follows;source type-ESI, Ion polarity-positive, scan begin-50 m/z, scan end-1200 m/z.
In the present study, EPS was isolatedand characterized from HalomonassmyrnensisSVD III.
Effect of different media components on the production of EPS was investigated. Effect of carbohydrate concentrationwas checked by using different glucose concentration as 3%, 4%, 5% and 6%. Maximum EPS yield of 10.5g/l was obtained at 3% glucose concentration, which decreased with increase in sugar concentration (fig.1).
Fig.1: Optimization of carbohydrate concentration showing 3% as the optimum concentration of glucose with 10.5 g/l yield of EPS. The number of experiments was 3 and data given in mean standard deviation (SD) and standard error (SE)
As regards effect of theincubation period, it was found that EPS yield gradually increased with incubation period up to 7th day and then remained stationary (fig.2). EPS yield obtained on 7th day was 10 g/l.
Fig.2: Optimization of incubation period illustrating 7 d as the optimum incubation period with EPS yield of 10 g/l. The number of experiments was 3 and data given in mean SD and SE
EPS yield gradually increased with increase in inoculum size. At 10% inoculum size, EPS yield was found to be 9.75 g/l (fig. 3). Effect of different pH on EPS yield was checked and maximum EPS yield was observed at pH-6 which is 12.68 g/l followed by agradual decrease with increase in pH value (fig. 4).
As regards effect of different salt concentrations, it was observed that maximum EPS was produced at 20% salt concentration as 10.65 g/l (Fig.5). EPS production checked at different incubation temperature indicated that at ahigher temperature, theyield of EPS is increased. At 45 °C EPS yield was observed as 23.95 g/l (fig. 6).
Fig.3: Optimization of inoculum size indicating 10% inoculum size as optimum with EPS yield of 9.75 g/l. The number of experiments was 3 and data given in mean SD and SE
Fig.4: Optimization of pH showing 6 as the optimum pH yielding 12.68 g/l EPS. The number of experiments was 3 and data given in SD and SE
Fig.5:Optimization of salt concentration–20% salt as the optimum with EPS yield of 10.65 g/l. The number of experiments was 3 and data given in mean SD and SE
Fig.6:Optimization of temperature indicating 45°Ctemperature as the optimum with EPS yield of 23.95 g/l. The number of experiments was 3 and data given in mean SD and SE
Fig.7: Optimization of yeast extract concentration showing 0.5% yeast extract as the optimum nitrogen source yielding 11.34 g/l EPS. The number of experiments was 3 and data given in mean SD and SE
Different concentrations of yeast extract were studied for their effects on EPS yield. EPS yield was found maximum 11.34 g/l at 0.5% concentration of yeast extract (fig. 7).
Thus the optimum conditions for production of EPS by H.smyrnensis were found to be 3% glucose, 20% NaCl, theincubation period of 7 d, pH 6, temperature 45°C, 0.5% yeast extract andinoculum size 10%. Under all optimum conditions, the EPS yield was 23 g/l indicating two-foldincrease.
In FTIR spectra, the bands of 1770.94 cm-1 and 1664.23 cm-1 indicate thepresence of the carbonyl group. Bands of 1122.54 cm-1 and 1076.14 cm-1 are dominated by glycosidic linkages (strong C-O bonds) of the polysaccharide. The band at 3320.47 cm-1 is due to broad presence of the OH group. The–CH2 wagging is observed at band 1334.03 cm-1. The band 647.08 cm-1 is due to the out of plane bending of–OH group (fig.8).
1H NMR spectra of the EPS sample showed the signals between the ranges of 3.659-4.932 ppm indicating protons on anomeric carbons which suggests the sample to be heptasaccharide. The signal at 3.361 ppm corresponds to thepresence of protonated carbon adjacent to theelectronegative group. The signal at 2.506 ppm is attributed to presence of protonated carbon adjacent to less electronegative groups (fig. 9).
Mass spectrometry suggested thepresence of an oligosaccharide. The sample showed the peak at 1132.1940 which could be because of about 6-7 monosaccharide units. There are some peaks seen with the loss of 60 units suggesting thepresence of sugar which has lost C2H4O2. Base peak is seen at 782.4424 (fig. 10).
Fig.8: Fourier transform infrared spectroscopy analysis showing glycosidic linkages between 1122 and 1076-1 cm, carbonyl groups between 1770 and 1664 in the EPS produced
Fig.9:1H nuclear magnetic resonance analysis:signals between 3.659 and 4.932 showing heptasaccharide nature of the EPS
Fig.10: Mass spectrometry analysis indicating presence of oligosaccharide in the EPS produced
EPS is known to be produced extracellular during stationary phase and hence incubation period of 7 d was found to be optimum for production of EPS. It was observed that higher temperature plays asupportive role in EPS production. Halophilic microorganisms are known to grow optimally in the temperature range of 35°C to 55°C.In the present study also H.smyrnensis was found to produce maximum EPS at 45°C.Since nitrogen is required for onlygrowth of the organism, 0.5% yeast extract was found to be adequate for theproduction of EPS. As the organism H.smyrnensis is moderately halophilic, 20%concentration of NaClwas found to be optimum for production of EPS. EPS is a biopolymer of carbohydrates and hence3% concentration ofglucose was found to enhance theyield of EPS. Halomonassp. AAD6isolated from soil samples from CamaltiSaltern area in Turkey, when grown in the presence of sucrose in defined media, produced highest EPS production levels of 1.073 g/l. This strain was further identified as Halomonassmyrnensissp. nov.. The present strain of H.smyrnensis SVD III produced maximum 23.95 g/l EPS at 45°C temperature (fig.6). Thus the EPS production was 24 times higher than the type strain of H.smyrnensissp. nov.Characterization data of the extracted EPS fromtype strain of H.smyrnensissp. nov. Suggested that it was a levan type of EPS while the EPS produced bythepresent strain of H.smyrnensis SVD III was of heptasaccharide nature.
Optimization of production of EPS by bacteria from extreme marine habitats has been reviewed . Optimization of production of EPS and its characterization from Ophiocordycepsdipterigenawas studied . Optimization of different nutrient media for the production of EPS by Bacllussubtilis has been described .Characterization of EPS produced by Bacillus cereus and Brachybacterium species was carried out .Production of EPS by alkalitolerant and halotolerantVagococcuscarniphilusisolated from alkaline soda lake of Lonar, India was described, production of EPS was optimized for different environmental conditions and characterized the EPS for total carbohydrate content and monosaccharides . In the present study, optimization of production of EPS from H.smyrnensis SVD III was studied.
Production of EPS by amoderatelyhalophilic bacterium isolated from asalt lake in Romania was described. The production of EPS was optimized for culture conditions and composition of the culture medium. The polymer was characterized by FTIR and spectrophotometrically by measuring absorption at 260 nm and fluorescence emission at 530 nm. The EPS was found to be thermostable . In the present study, the EPS produced by H.smyrnensis SVD IIIwas characterized by FTIR, 1H NMR and MS.
In the light of work carried out on theproduction of EPS and its characterization from different microorganisms including halophilicmicroorganisms by different researchers over the globe,the EPS produced by the present strain of Halomonassmyrnensis SVD III thus appears to be different from the reported halophilic micro-organisms.
HalomonassmyrnensisSVD III isolated from ocean water from West Coast of Maharashtra, India was found to produce 23 g/l EPS under all optimum conditions namely 3% glucose, 20% NaCl, incubation period of 7 d, pH 6, incubation temperature 45°C, 0.5% yeast extract and inoculum size of 10%. The optimization studies resulted in two fold increase in yield of EPS. The characterization of EPS produced revealed heptasaccharide nature and dominance of glycosidic linkages (strong C-O bonds) in the polysaccharide.
The authors thank Dr. Sujata Kale of Aabasaheb Garware College, Pune for interpretation of results of FTIR, 1H NMR and MS. The authors are thankful to Dr. Prabhakar Ranjekar, Ex-Director, Interactive Research School for Health Affairs, Pune, Bharati Vidyapeeth Deemed University for his encouragement and support.
Siddharth Deshmukh:Ph. D. thesis work, conducting all the experiments, interpretation of results, writing and revising the manuscript
Pradnya Kanekar (Research co-guide): Concept and designing of experiments, interpretation of results, editing the manuscript
Rama Bhadekar (Reseach guide): Overall supervision on the laboratory experimental work, execution of the experimental work in thelaboratory, editing the manuscript.
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How to cite this article
SiddharthDeshmukh, PradnyaKanekar, Rama Bhadekar. Production and characterization of anexopolysaccharide from marine moderately halophilic bacterium HalomonassmyrnensisSVD III.Int J Pharm PharmSci 2017;9(10):146-151.