Optimization and Characterization of Polyhydroxyalkanoates and its Copolymers Synthesized by Isolated Yeasts

 

Nupur Ojha, Nilanjana Das*

Bioremediation Laboratory, School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India

 

ABSTRACT:

The present study investigates the role of yeasts, isolated from different environments for its abilities to synthesize biodegradable polymer, Polyhydroxyalkanoates (PHA) and its copolymers. Serial dilution method and spread plate technique had been used for the isolation of yeasts from different samples. Screening had been done by crotonic assay followed by UV Spectrophotometric analysis. Growth parameters such as effect of carbon sources, incubation period, inoculum percentage, incubation temperature and pH of the production medium were optimized for each of the screened isolates. Characterization of PHA was done by FTIR and GC-MS analysis. Maximum PHA produced was observed after 96 hours of incubation period inoculated with 2% (v/v) of inoculum in minimal media of pH 8 containing various carbon sources viz. 5% sucrose for Isolate1; 5% glucose for Isolate 2; 3% starch for Isolate 3 and 5% Lactose for Isolate 4. Incubation temperature was maintained at 37˚C. Maximum PHA production was noted as 40 % in Isolate 1, 33% in Isolate 2, 29% in Isolate 3and 25% in Isolate 4 respectively on the basis of  dry cell weight under optimized condition. Prominent peaks obtained through FTIR analysis at wavelength 3273, 2922, 1739 and 1462 cm ^-1 showed the presence of stretching bands of –OH-,-CH-, -C=O- and –CH₃- group which are the characteristic  peaks of PHA polymer. GC-MS chromatogram detected  the peaks at retention time 3.063 and 19.25 min  were corresponding to the derivatized products of butanoic acid (polyhydroxybutyrate) and octadecanoic acid (polyhydroxyoctadecanoate).The present study demonstrated that yeast Isolate 1 could produce high amount of  PHA (40% on dry cell weight basis ) with hydroxybutyrate (HB) and hydroxyoctadecanoate (HOD) as its main constituents.

 

 

 

INTRODUCTION:

The widespread use of petrochemical based polyethylenes has been increasingly scrutinized as a source of environment and waste management problems. Recalcitrant nature of plastics has been quashing the fertility as well as microbiome of the soil environment. In response to these problems associated with petrochemical based polyethylenes and its effect on the environment, there has been a considerable interest towards the development and production of biodegradable plastics.

 

Based on the monomer units, biodegradable polymers have been classified into seven classes which are distinguished as polynucleotides, polyamides, polysaccharides, polyisoprenes, lignin, polyphosphate, and Polyesters¹. PHA is a family of polyesters composed primarily of R-3-hydroxyalkanoic acid monomers of molecular weight ranges from 2 x 10⁵ to 3 x 10⁶ Dalton. Based on the number of carbon atoms present in the monomer units, PHA have been classified into three groups, namely Short Chain Length (SCL) polymers, consisting of 3-hydroxy acids from 3 to 5 carbons, Medium Chain Length (MCL) polymers, consisting of 3-hydroxy acids from 6 to 16 carbons and Long Chain Length (LCL) polymers, consisting of 3-hydroxy acids more than 16 carbons in the monomer unit². PHAs are the most promising polyesters which can be used as bio plastics, because of its inherent properties such as biodegradability, water insolubility, thermoplasticity and physicochemical properties ³. It has high molecular weight with properties similar to conventional polyethylenes and have a wide range of applications, in the manufacturing of bottles, plastic utensils, fibers, packaging of food stuff and pharmaceutical drugs. It has remarkable applications in the medical industry listed from the drug delivery, bio-implantation as well as in the manufacturing of bone plating, surgical tools, adhesive stitches, and sutures ⁴. Over the past few decades, many PHA and its copolymers have been synthesized by a wide variety of microorganisms as intracellular inclusions when there is an excess of carbon sources with limiting nutrients present in the medium ⁵. Around 90 genera of bacterial species including Escherichia coli (Genetically engineered), recombinant Escherichia coli, Bacillus megaterium JK4h, Bacillus cereus 64-INS, Cupriavidus necator, Bacillus megaterium uyuni S29, Halomonas TD01 and Ralstonia eutropha, have been reported for its abilities to produce PHA and its copolymers ^6-7.

 

Some of the Cyanobacterial strains have also been reported which include Synechococcus MA19, Spirulina maxima, Gloeothece sp., Spirulina platensis, and Oscillatoria limosa. Moreover, some of the extremophiles such as Halorubrum litoreum, Halobiforma nitratireducens CGMCC, Haloferax mediterranei, and Halogeometricum borinquense E3 have been reported to be a good producer of PHA and its copolymers ^{6, 8}. But compared to these microorganisms, reports are scanty in case of yeast species. The major commercial drawback of the PHA producing bacterial strains is its high production cost of carbon sources, which is making them substantially more expensive comparative to the petroleum-based plastics ^9-10. Evidently, yeasts are an inexpensive, readily available source of biomass, physiologically more flexible and larger than bacteria ¹¹.

 

Moreover, the large-scale fermentation technology and separation technology is very well known for this organisms. Consequently, looking for eukaryotic cell systems like yeast which can able to produce PHA seems to be a beneficial alternative to the production of PHA. Furthermore, yeast cells retain their ability to accumulate a broad range of PHA to varying degrees under a wide range of external conditions. Though some concerned effects have already been reported using genetically modified yeast strains but no exploration work has been done for enhancing the PHA production in a cost effective way. Therefore, the purpose of the present study was to optimize different growth parameters for the enhancement of PHA production using cheap carbon sources and its characterization by FTIR and GC-MS analysis.

 

MATERIALS AND METHODS:

Isolation and Enrichment of Yeast Isolates:

Soil and domestic food waste samples were collected from plastic dumped ground, activated sludge, agricultural ground, contaminated food stuff, vegetable wastes, dairy products, and fruit juices aseptically and further processed for conducting experiments. Solvents used while conducting the experiments were purchased from Merck Pvt. Ltd. The other chemicals used for the preparation of reagents, solutions, and microbiological growth media were purchased from HiMedia laboratories Pvt. Ltd. and SISCO Research laboratory, Mumbai, India. Isolation of the yeast colonies was done by serial dilution method followed by spread plating technique on YEPD medium containing yeast extract (10 g/L); peptone (20 g/L); dextrose (20g/L) and agar (20 g/L). Plates were incubated at 30˚C for 48 h in shaking condition of 150 rpm ¹². The distinct yeast colonies were selected and sub-cultured for further characterization.

 

Culture Condition:

Yeast strains (stationary phase) grown in YEPD medium were harvested by centrifugation and  cells were washed once with autoclaved distilled water and resuspended in mineral salts medium (MSM) containing (g L^-1) K₂HPO₄ – 3g, Na₂HPO₄ – 6g, NH₄Cl – 2g, NaCl – 5g, MgSO₄ – 1g, CaCl₂ – 0.1g and FeCl₃ – 0.06g supplemented with 0.1% glucose, 0.5% of the detergent Tween 60 (Sigma, St. Louis, Mo.), and 0.1% fatty acid (oleic acid). Yeast cells were grown for 6 days at 32°C, pH 6 in shaking condition of 120 rpm ¹³.

 

Extraction of PHA:

The fermented yeast strains were centrifuged at 5000 rpm for 25 minutes at 4 °C. Cell pellets were washed twice with distilled water and dried in Hot air oven at 55˚C. Dried biomass of each of the yeast strains were scrapped out and its dry cell weight values were recorded. Dried biomass of yeast cells was agitated vigorously by adding 10 ml solution of 3 ml of sodium hypochlorite (4%) and 7ml of chloroform. The mixture of each yeast strains was centrifuged at 2500 rpm for 5 minutes to get three separate layers. The dissolved polymer can be obtained from the bottom layer and precipitated by adding methanol drop-wise-drop in 9:1 ratio. Precipitates were collected separately for each of the strains and evaporated by the addition of 1ml chloroform to get purified polymer¹⁴. Residual biomass of each of the strains was estimated as the difference between dry cell weight and dry weight of the extracted PHA. The percentage of the PHA production extracted from the potential isolates was estimated as the percentage composition of PHA present in the dry cell weight ^15-16.

 

Residual biomass (g/l) =

Dry Cell Weight (g/l) - Dry weight of extracted PHA (g/l)

 

Percentage of PHA on Dry Cell Weight Basis (%) =

 

Dry weight of the extracted PHA(g/L) X 100

            Dry Cell Weight (g/L)

 

Quantification of extracted PHA by Crotonic Acid Assay:

The amount of PHA present in the extracted samples can be determined by Ultraviolet Spectrophotometric analysis.  Polymer extracted from yeast isolate was dissolved in boiling chloroform and evaporated. Eventually, the polymers were mixed with 10ml of concentrated sulphuric acid in capped glass tube and heated for 10 min at 100˚C in a water bath separately. By adding sulphuric acid, the polymers extracted from each of the yeasts isolates converted into crotonic acid and the color inside the glass tube changes to brown color. Crotonic acid standard graph had been prepared with different increasing concentrations of 10-40 µg. The solutions were diluted using sterile distilled water to the dilution factor of 10 before the determination of PHA¹⁶. The PHA concentration of each of the yeast isolate was determined from an estimated standard graph in which the absorbance was plotted against the concentration of crotonic as a standard (235 nm).The absorbance of each of the diluted samples was measured by UV spectrophotometer between the range of 200-400 nm, where peak obtained between 230-240 nm confirmed the presence of PHA and its copolymers¹⁷.

 

Optimization of the growth Parameters:

The production of PHA in microorganisms is mainly influenced by the types of carbon sources and growth parameters used during culturing PHA-producing microorganisms. Effect of carbon sources was optimized using different carbon source such as glucose, sucrose, cellulose, starch, glycerol, lactose and maltose with increasing concentration ranging from1-7 %. The effect of other growth parameters such as Incubation period (h), inoculum percentage (v/v), pH and temperature were also optimized for each isolate. Potential yeast isolates were grown under the optimized growth conditions for estimating total increase in the PHA production.

 

Characterization of extracted PHA:

Fourier Transform-Infrared Spectroscopic (FT-IR) analysis:

The polymer extracted from best PHA producing yeast isolate was analyzed by FT-IR Spectroscopy. Infrared spectra (IR) from the range of 4000 to 400 cm ^-1were recorded on polymer films cast from a chloroform solution onto KBr plates using FT-IR (Shimadzu, DR-800) at 27°C and the functional groups were detected. Different conformations bands in the extracted polymer from the yeast isolate were exposed by FT-IR analysis ¹⁸.

 

Gas Chromatography-Mass Spectrophotometric (GC-MS) analysis:

GC-MS analysis was performed after methanolysis of extracted polymer. Sample was suspended in 1ml of chloroform and 1 ml of methanol containing 2.8 M H₂SO₄ in a screw capped tube, and then incubated at 100°C for 2 h. After cooling, 2.5 ml of demineralized water was added and the organic phase containing the resulting methyl ester group was analyzed by GC-MS spectrophotometer (JEOL GC MATEII). Helium (1 ml/min) was used as carrier gas. The injector and detector are at 250°C and 200°C respectively ¹⁹.

 

RESULTS AND DISCUSSION:

Isolation of Yeasts from Different Sources:

A total of fifteen yeast strains were isolated from the dilution plates of 10^-4 and 10^-5 dilution factor and further sub cultured separately on YEPD medium. Morphological characterization was done by simple staining followed by compound microscopic view magnified at 100 X using immersion oil. Results have been tabulated in Table 1. Isolated yeasts were further screened for its abilities to produce PHA and its copolymers.

 

Table 1: Morphological characterization of the isolated yeasts

Yeast Isolates	Pigmentation	Morphology	Mode of reproduction
Isolate1	Creamy; white	Raised, circular	Ovoidal
Isolate 2	Creamy; white	Raised, circular	Spheroidal
Isolate 3	Creamy; white	Flat, circular	Spheroidal
Isolate 4	Creamy; white	Raised, circular	Ogival
Isolate 5	Creamy; white	Raised, circular	Ovoidal
Isolate 6	Yellowish; cream	Raised, circular	Ogival
Isolate 7	Dusty ; white	Raised, irregular	Apiculate
Isolate 8	Creamy; white	Raised, clustered oval	Ovoidal
Isolate 9	Creamy; white	Raised, irregular	Ovoidal
Isolate 10	Creamy; white	Raised, circular	Cylindroidal
Isolate 11	Creamy; yellowish	Ovoid Flat, irregular	Cylindroidal
Isolate 12	Creamy; white	Raised smooth, irregular	Ovoidal
Isolate 13	Creamy; white	Raised , irregular	Cylindroidal
Isolate 14	Creamy; white	Raised, circular	Ovoidal
Isolate 15	Creamy; white	Raised, circular	Flask shaped

 

Extracted PHA:

Among fifteen yeast isolates, only four strains named as Isolate 1, Isolate 2, Isolate 3 and Isolate 4 were capable of producing PHA and its copolymers. Percentage of PHA produced of Isolate 1, Isolate 2, Isolate 3 and Isolate 4 were found to be 13 %, 7.2 %, 4.8 % and 3.2 % with respect to their biomass.

 

Quantification of extracted PHA by Crotonic Acid Assay:

The amount of PHA present in the extracted samples was determined by UV Spectrophotometric analysis. The UV absorbance spectrum shows distinct absorbance peak at 235 nm by all the four Isolates represented in figure 1. This confirmed that the retention time of absorbance peak obtained by the Isolate 1, Isolate 2, Isolate 3 and Isolate 4 was identical to that of crotonic acid. A similar trend was reported by Bhakti in 2015 and Santhanam in 2010 ^16-17.

 

Figure 1: Concentration of the extracted PHA was determined by UV Spectrophotometric analysis synthesized by yeast Isolate 1, Isolate 2, Isolate 3 and Isolate 4.

 

Optimization of different carbon sources and growth parameters:

The effect of different carbon sources and growth parameters such as Incubation period (h), Inoculums percentage (v/v), temperature and pH of the production medium were optimized. Results have been represented in figure 2 and figure 3.

 

Effect of different carbon sources:

Four yeast isolates showed different responses towards the concentration ranges of different carbon sources. Maximum PHA production presented in figure 2 was found to be 25%, 20%, 18% and 15% of its total biomass, in case of Isolate 1, Isolate 2, Isolate 3 and Isolate 4, grown in minimal media containing sucrose (5%), glucose (5%), starch (3%) and lactose (5%) respectively under optimized condition. In this study, the amount of carbon source used in the media influenced the dry cell weight of each of the yeast isolates. Similar results were reported by Chengium Zhu in 2009²⁰.

 

 

Figure 2: Representing the effect of different carbon sources with variable concentration range (1 to 7%) on PHA production in (a) Isolate 1, (b) Isolate 2, (c) Isolate 3 and (d) Isolate 4

 

Effect of Incubation Period:

Different incubation period viz. 24, 48, 72, 96, 120, 144, 168 and 192 h were studied for the production of PHA.  A gradual increase in the production of PHA by yeast isolate 1, Isolate 2, Isolate 3 and Isolate 4 were observed from 24 h (3%, 2%, 1% and 0.9% based on dry cell weight) which had increased to 30%, 26%, 21% and 18% at 96 h and then gradually decreased to 3%, 2%, 0.8% and 0.5 at 192 h. Results have been represented in figure 3(a). This clearly indicated that the maximum PHA production was favored at 96 h by all yeasts isolates. After this stage, the concentration of accumulated PHA was started decreasing that may be because of the degradation of polymer intracellularly by endogenous PHA depolymerase. A similar trend was reported by Desuoky in 2007 ²¹.

Effect of Inoculum:

The effect of initial inoculum density on growth and PHA production by Isolate 1, Isolate 2, Isolate 3 and Isolate 4 were studied by inoculating five different percentages of inoculum (1%, 2%, 3%, 4% and 5%) in the production medium. At 2% of inoculum, a significant increase had been observed in the yield of PHA by isolate 1, Isolate 2, Isolate 3 and Isolate 4 from 13, 7.2, 4.8 and 3.2 % to 30, 26, 21 and 19% respectively. Results have been represented in figure 3(b). This indicated that inoculum density of the culture significantly affects the production of PHA. A similar trend was observed by Kulkarni in 2010²².

 

Effect of Temperature:

Effect of incubation temperature on growth of yeast isolates and PHA production was studied over a range of temperature from 27 to 67°C (27, 32, 37, 47, 52, 57, 62 and 67°C) in the production medium. From the present study, the optimum temperature for growth as well as PHA production was observed at 37°C by isolate 1, Isolate 2, Isolate 3 and Isolate 4 respectively. However, at the extremes of the temperatures studied, i.e., 27 and 67°C, growth of the culture as well as PHA production was observed very low by all the yeast isolates. Results have been represented in figure 3(c). This indicated that incubation temperature of the culture significantly affects the production of PHA. Similar results were obtained for H. campisalis where maximum PHA production was observed at 37°C²³.

 

Effect of pH:

Effect of initial pH of production medium on growth of the culture and PHA production was studied ranging from 3 to 11.  A significant increase in the PHA production was observed at pH 8 with 37, 32, 28 and 25% of PHA produced on dry cell weight basis by isolate 1, Isolate 2, Isolate 3 and Isolate 4 respectively comparable at and decreased sharply at the extremes of acidic and alkaline pH, i.e., pH 3 and 11. Results have been represented in figure 3(d). This indicated that a wide range of pH of the production media significantly affects the production of PHA and the growth of the cultures. Similar results were obtained for H. boliviensis LC1 where pH of the PHA production medium was 8.5 ^23-25.

 

Figure 3: Representing different growth parameters viz. (a) effect of incubation period, (b) effect of inoculum, (c) effect of temperature (c) and (d) effect of pH of the medium. Highest PHA produced in Isolate 1, Isolate 2, Isolate 3 and Isolate 4 were observed after 96 hours of incubation period inoculated with 2% (v/v) of inoculum, incubated at 37˚C in minimal media of pH 8 supplemented by the optimized carbon sources

 

PHA Production under Optimized Condition:

Optimal growth parameters were optimized and the maximum PHA production was determined for each of the yeast isolates. PHA production in Isolate 1, Isolate 2, Isolate 3 and Isolate 4 was increased from 13 to 40%, 7 to 33%, 4.8 to 29% and 3.2 to 25% based on dry cell weight respectively after optimization. Results have been tabulated in table 2. A significant increase in the production of PHA was observed in isolate 1 with 12 g/L PHA content, whereas, in Isolate 2, Isolate 3 and Isolate 4 comparable difference were observed with 9, 7 and 5 g/L of PHA content. Results have been represented in figure 4. So far maximum PHA production reported by the wild-type yeast Rhodotorula minuta was found to be 2% of the total biomass ²⁶. However to increase the PHA production, some recombinant/transgenic strain such as Kloeckera sp. (KY1) and saccharomyces cerevisiae,²⁶ were also reported but the maximum PHA production were found to be 7.06% and 2.68%  only which was lower than the  results of the present studied yeast Isolate 1 (40%), Isolate 2 (33%), Isolate 3 (29%) and Isolate 4 (25%). Thus, based on the results of the present work, it can be concluded that the yeast isolate 1, Isolate 2, Isolate 3 and Isolate 4 have the capability to produce PHA.

 

Figure 4: Representing the maximum PHA production which had been increased from 13 to 40% in Isolate 1, 7 to 33% in Isolate 2, 4.8 to 29% in Isolate 3 and 3.2 to 25% in Isolate 4 after optimization

 

Table 2: Maximum PHA production in yeast Isolate 1, Isolate 2, Isolate 3 and Isolate 4 before and after optimization

Yeast Isolates	Growth Parameters	Carbon Source (wt./v)	Incubation Period (h)	Inoculum (v/v)	Temperature (˚C)	pH values	Dry Cell Weight (g/L)	PHA production (g/L)	PHA production (%)
Isolate 1	Before Optimization	Glucose (2%)	144	2%	32	7 ± 0.2	15 ± 0.5	2 ± 0.3	13 ± 0.2
	After Optimization	Sucrose (5%)	96	2%	37	8 ± 0.4	30 ± 0.3	12 ± 0.6	40 ± 0.3
Isolate 2	Before Optimization	Glucose (2%)	144	2%	32	7 ±  0.8	13 ± 0.8	1 ± 0.4	7 ± 0.8
	After Optimization	Glucose (5%)	96	2%	37	8 ± 0.3	27 ± 0.5	9 ± 0.4	33 ± 0.4
Isolate 3	Before Optimization	Glucose (2%)	144	2%	32	7 ± 0.4	12 ± 0.6	0.7 ± 0.7	4.8 ± 0.3
	After Optimization	Starch (3%)	96	2%	37	8 ± 0.6	24 ± 0.8	7 ± 0.5	29 ± 0.4
Isolate 4	Before Optimization	Glucose (2%)	144	2%	32	7 ± 0.6	10 ± 0.7	0.4 ± 0.4	3.2 ± 0.5
	After Optimization	Lactose (5%)	96	2%	37	8 ± 0.3	20 ± 0.4	5 ± 0.5	25 ± 0.4

 

Characterization of extracted PHA:

Fourier Transform Infrared Spectroscopy (FTIR) Analysis:

FT-IR spectroscopic analysis was done cm⁻¹ was performed to analyze the functional groups involved in PHA. The extracted polymers showed the intense absorption at 3273 cm^-1 for the O-bending group and at 2922 cm^-1 for C-H stretching group. Other strong prominent peaks at wave numbers 1724 and 1739 cm^-1 represented the presence of ester carbonyl (C=O) stretching groups in comparison with the standard polyhydroxybutyrate reported in the referred article ²⁷. Other adsorption bands at 1375 and 1462.04 cm^-1 were observed for -CH₃ group. Some of the sugar derivatives such as guluronic acid, manuronic acid, and uronic acid as well as β-glycosidic linkages between the sugar monomers were confirmed by the presence of a prominent peak at 1055 cm^-1 and 856 cm^-1. Similar trends were reported by Peter in 1999 ²⁸.

Gas Chromatography Mass Spectrophotometric (GC-MS) Analysis:

The gas chromatography-mass Spectrophotometric analysis of PHA for the Isolate 1 was done to determine the constituent acids present in the polymer following the method of Braunegg in 1978 ^29-30. Peaks obtained at retention time 3.063 and 19.925 min represented the corresponding derivatized product of butanoic acid and octadecanoic acid which showed the presence of polyhydroxybutyrate and polyhydroxyoctadecanoate respectively. Similar results were reported in case of Bacillus cereus and Halomonas campisalis ²⁶. According to the mass spectral library from NIST database, the compounds obtained at retention time 3.063 and 19.925 min were identified as Methyl-ester 2-hydroxybutyrate (HB) and Methyl ester-2-hydroxyoctadecanoate (HOD) respectively. Similar trend was reported by Wang Q in 2010 and Kim-Kang in 1991 ^31-32.

 

Figure. 5. FTIR analysis of the PHA produced by Isolate 1

 

Figure 6: (a) Chromatogram obtained by GC-MS analysis of the PHA produced by Isolate 1; Spectrum at (b) retention time 3.063 min and at (c) retention time 19.925 min

 

Table 3: The m/z signals corresponding to each RT values and the compounds identified by GC-MS analysis

Yeast Isolates	Retention Time (min)	Mass-to-charge ratio signals (m/z)	Compound Name	Molecular Weight	Formula
Isolate 1	3.063	83.0427, 47.0254, 48.0757, 49.0559, 50.0361, 82.0646, 70.1141, 84.9986, 87.0939, 91.1443 120.1017, 135.2289,  207.2209	Butanoic acid	146	C6H10O4
	19.925	44.0141, 98.1955, 55.1457, 207.2209, 133.2775, 177.2961, 208.1254, 209.1691, 281.3033, 256.4719 283.3204 342.1041 417.8946 493.4229 519.7276, 560.6905	Octadecanoic acid	624	C39H76O5

 

CONCLUSION:

From the present study, it can be concluded that yeast Isolate 1 was found to be the potential strain producing high amount of PHA (40%) and its copolymers compared to the recently reported wild-type yeasts like Saccharomyces cerevisiae (0.45%) and Kloeckera species (7%). FTIR spectrum of Isolate 1 shows the presence of stretching bands of –OH-,-CH-, -C=O- and –CH₃- groups confirming the presence of functional group of PHA polymer. GC-MS analysis detected the peaks at retention time 3.063 and 19.925 min which were corresponding to polyhydroxybutyrate and polyhydroxyoctadecanoate polymer. These monomer units may add new properties to the PHA polymer. Yeast Isolate 1 has been sent for molecular identification. Further study will be carried out to elucidate the metabolic pathway for the synthesis of PHA polymer, so that it can be implemented on a commercial scale.

 

ACKNOWLEDGEMENT:

The authors are grateful to the School of Advance Sciences, VIT University, Tamil Nadu, India for extending their support and providing laboratory facilities to complete this work.

 

CONFLICT OF INTERESTS:

There is no conflict of interests among the authors.

 

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Accepted on 20.02.2017           © RJPT All right reserved