Journal of Research in Biology
Peanut Oil Cake: A Novel Substrate for Enhanced Cell Growth and
Prodigiosin Production from Serratia marcescens CF-53
Keywords:
Serratia marcescens, red pigment, prodigiosin, pea nut oil cake, fermentation.
ABSTRACT:
Different agro-wastes such as peanut oil cake (POC), coconut oil cake (COC), sesame oil cake (SOC), safflower seed oil cake (SFOC) and cotton seed oil cake (CSOC) were screened for prodigiosin production through fermentation employing Serratia marcescens-CF-53. POC was highly beneficial for the pigment production. Fermentation parameters have been standardized; maximum amount of pigment was obtained (~40 mg ml-1) in POC extract at 30oC for 42 h using 8% inoculum density (1X107cell/ml) compared to PG broth (14.2 mg ml-1). The pigment yield was almost three fold higher than that of the PG broth. Use of POC extract as a raw material for pigment production could be of great commercial significance. This report appears to be the first one on prodigiosin production using POC as a substrate.
549-557 | JRB | 2012 | Vol 2 | No 6
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Authors:
Chandrashekhar Naik1, Srisevita JM1,
Shushma KN1, Farah Noorin1, Shilpa AC1,
Muttanna CD1, Darshan N, Sannadurgappa D2.
Institution:
1. Microbiology and
Downstream Processing Laboratory, Department
of Biotechnology,
Sir M Visvesvaraya Institute of Technology, Yalahanka, Bangalore- 562 157.
2. Centre for Sustainable Technologies, Indian Institute of Science (IISc),
Bangalore-560 012.
Corresponding author:
Chandrashekhar Naik.
Email:
cknaikg@gmail.com
Phone No:
+91-080- 2846 7248.
Fax:
+91-080- 2846 7081.
Web Address:
http://jresearchbiology.com/documents/RA0251.pdf.
Dates:
Received: 02 Jun 2012 Accepted: 16 Jul 2012 Published: 03 Aug 2012
Article Citation:
Chandrashekhar Naik, Srisevita JM, Shushma KN, Farah Noorin, Shilpa AC,
Muttanna CD, Darshan N, Sannadurgappa D.
Peanut Oil Cake: A Novel Substrate for Enhanced Cell Growth and Prodigiosin Production from Serratia marcescens CF-53.
Journal of Research in Biology (2012) 2(6): 549-557
Journal of Research in Biology
Original Research
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
INTRODUCTION:
The interest in prodigiosin and prodigiosin like pigments has been on the increase due to the biological activities of these compounds. The pigment prodigiosin, an alkaloid compound, has antibacterial, antifungal, antimalarial, antiproliferative and immunosuppressive properties (Montaner and Tomas, 2001; Soto et al., 2004). It also induces apoptosis in certain cancer cells (Furstner, 2003; Perez et al., 2003). This secondary metabolite is produced by several microorganisms like Serratia, Pseudomonas, Streptomyces and certain marine bacteria (Gerber, 1975; Giri et al., 2004) through quorum sensing (Fuqua et al., 1996; Wei et al., 2006). The naturally occurring prodigiosin and prodigiosin like pigments exist in cyclic and acyclic forms and contain 4 methoxy-2, 2’ bipyrole ring (Manderville, 2001). Prodigiosin, itself an acyclic form, contains a 2 mehtyl-3-pentyl-pyrrole ring. The cyclic forms include metacycloprodigiosin, nonylprodigiosin, streptorubin B and cycloprodigiosin (Manderville, 2001).
The gram negative Serratia marcescens are prolific producers of red pigment (Rustom et al., 1990). From an industrial point of view and because of its biological potentialities, there is a necessity to develop a high throughput and cost-effective bioprocess for large scale prodigiosin production. Earlier workers have reported many differential, selective and synthetic media such as, nutrient broth, peptone glycerol broth (PGB), LB broth etc, (Montaner et al., 2000; Haddix and Werner, 2000) for prodigiosin and prodigiosin like pigment production. The yield of the pigment is varying: 3 g L-1 (Cang et al., 2000) incorporating ethanol in the medium, 152 mg l-1 (Wei and Chen, 2005) in LB broth and oil supplements, and 0.08 g-4.28 gL-1 employing different carbon and nitrogen sources (Min-Jung-Song et al., 2005). Giri et al, (2004) obtained 17 to 39 g L-1 yield and opined pea nut and sesame powder and their oils to be good substrates for prodigiosin biosynthesis. Our earlier works on citric acid production had revealed that the some agro wastes could be beneficially employed to enhance citric acid production (Lingappa et al., 2002). It was, hence, considered worthwhile to attempt enhancing prodigiosin production using such agro-wastes.
The pea nut cakes obtained after extraction of oil are at best used as a cattle feed supplementation. Our earlier studies had revealed the presence of sufficient sugar and fat content in these cakes. However, no attempts appear to have been made to use these agro-wastes for prodigiosin production. Hence, the present work was undertaken to evaluate production of prodigiosin and prodigiosin like pigments employing S. marcescens CF-53.
MATERIALS AND METHODS
Microorganism and cultural conditions
A red color pigment producing bacterial strain was isolated from soils near dairy industry waste disposal points in and around Bangalore and identified as Serratia marcescens CF-53 and deposited at Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India (Accession No. MTCC7643). Sub culturing and maintenance was on peptone glycerol agar (PGA) slants. Seed culture of S. marcescens CF-53 to be used as inocula for the experimental work was maintained in PG broth.
Screening of substrates and culture medium
Different agro-based substrates, the peanut oil cake (POC), the coconut oil cake (COC), the sesame oil cake (SOC), the safflower seed oil cake (SFOC) and the cotton seed oil cake (CSOC) were obtained from local market in Bangalore and powdered. To 100 ml water, 5 g of each cake powder was separately added and boiled for 30 min and allowed to cool. The supernatant was further centrifuged at 7000 rpm for 15 min to obtain a debris-free solution. To the clarified solution, agar was added (15-20 gL-1) after due adjustment of pH to 7.0 and autoclave sterilized at 121°C for 20 min. The agar plates
then prepared were employed in preliminary screening studies to evaluate as to which substrate was most suitable for prodigiosin and prodigiosin like pigment by S. marcescens CF-53. After the preliminary screening the peanut cake was similarly processed and the media without agar served as the substrate and PG broth as the control for the pigment production. The flasks were inoculated with 0.5 ml of (1 X 107 cells/ml) 24 h cell suspension of S. marcescens CF-53 and incubated at 30°C in an orbital shaker at 200 rpm for 48 h. The initial inoculum was 2% to avoid coloration of the starter culture in the beginning only.
Effect of biotic and abiotic parameters
To standardize the culture parameters for biomass yield and prodigiosin production, the effect of varying pH, temperature, incubation period, inoculum size, substrate concentration as well as supplementation of different carbohydrates, nitrogen sources, salts and metal ions were studied.
The production of prodigiosin by Serratia marcescens CF-53 is very sensitive; therefore, the selected parameters were optimized to achieve higher yield of prodigiosin. Once a fermentation parameter was optimized, the optimum level of that parameter was employed in the subsequent studies wherein another parameter was to be optimized.
Quantification of Prodigiosin
Culture broth (1 ml) was mixed vigorously with 3 ml of methanol in a vortex mixer and then centrifuged at 5000 rpm for 5 minutes. An amount of 0.8 ml supernatant was further mixed with 0.2 ml of acidified methanol. The optical density of the resulting solution was measured as per Wei and Chen (2005).
Dry cell weight determination
Cell concentration was determined by measuring the dry cell weight (DCW). Culture broth was filtered through a washed and preweighed micropore filter (0.2µm). The cell-loaded filter was dried at 105°C and reweighed to determine cell biomass (Wei and Chen, 2005).
Purification of pigment for Mass Spectroscopic analysis
S. marcescens-CF53 grown in POC extract was centrifuged at 10000 rpm for 20 minutes and the cell biomass was collected. The pigment from the biomass was extracted with acidified methanol (1N HCl 1 ml: methanol 24 ml) and the extract was centrifuged at 5000 rpm for 15 min. The supernatant was concentrated in vacuum evaporator at 60°C to obtain crude pigment. The crude concentrated pigment was dissolved in methanol and the solution was passed through hexane balanced silica gel (mesh 60-120) column. The trapped pigment within the column was eluted with 10 M ethyl acetate to liberate the adsorbed pigment. Further, elute was harvested and dried in a vacuum drier at 60oC to obtain the purified product. Small fractions of the pigment were collected and silica gel thin layer chromatography (TLC) was performed using the chloroform/methanol/ water (80:15:05 v/v) as solvent system. Fractions having single red spot with Rf value of 0.72 were pooled and the solvent was evaporated. Pooled samples were dissolved in methanol for molecular mass determination by ESI-MS (Min-Jung-Song, et al., 2005).
Scale up studies in Bioreactor
The fermentations were carried out in a 2 L lab scale bioreactor (Sartorius B-Lite, India) equipped with a pH, temperature and DO electrode. The fermentation was started after inoculating 24 h seed culture of the Serratia marcescens CF53 (8% v/v) into the POC extract broth. Fermentations were conducted under the condition of temperature 30oC, air flow rate of 2 vvm with an agitation speed of 200 rpm and initial pH 7.0, unless mentioned otherwise. The working volume was 1 L with foam level controlled by automatic addition of 1% antifoam (mineral oil). Culture pH was controlled by using 2.5 N HCL and 2.5 N NaOH as and when required. Samples were drawn periodically to estimate the yield of
prodigiosin.
RESULTS AND DISCUSSION
The results of the studies regarding prodigiosin production by S. marcescens CF-53 employing different substrates are presented in Figure 1. The richness of the pigment and its production was observed to be maximum in POC medium. Variation in the color of the pigment produced in different substrates could be due to the compositions of the extracts of different substrates. Therefore, POC extract medium was the preferred choice-medium to carry out systematic investigations on medium improvement and process optimization for the higher yield of prodigiosin by Serratia marcescens CF-53.
Effect of pH
Maximum yield of the pigment was obtained at pH 7 in POC and the control PG broth growth media (Fig. 2A), 14.2 mg/ml and 8.5 mg /ml respectively. Thus the pigment yield in POC was almost 1.6 times that in PG both. It has been reported that the secondary metabolites are generally produced in the pH range of 6-8 (Sole et al., 1997). Differences in the yield would depend on medium composition. PG broth contains peptone and yeast extract as nitrogen source and glycerol as carbon source and thus lacks a direct source of carbon (Wei et al., 2005). On the other hand, POC extract naturally inherits oil as its carbon source and basically contains saturated and unsaturated fatty acids, carbohydrates, minerals, vitamins etc.
Effect of temperature
The results of the influence on growth and pigment production by S. marcescens CF-53 are presented in Fig. 2B. Production of prodigiosin and cell dry weight was maximum at 30°C in both the POC extract broth (18.2 mg ml-1 and cell dry weight 35.2 mg ml-1, respectively) and the PG broth (10.2 mg ml-1 and cell dry weight 23.5 mg ml-1, respectively). The increase is almost 1.7 times in pigment yield and 1.4 times in cell growth in POC broth when compared to those in the PG broth. In case of PG broth, the viscosity of glycerol decreases at this temperature leading to increased accessibility of the
carbon source to the microorganism (Giri et al., 2004). Pigment production was totally blocked at 40°C in PG broth (on par with the report of Giri et al., 2004) and at 42oC in the POC broth. The impact of physiological role of temperature in blocking prodigiosin synthesis seems to vary with mediums of different substrate composition (Giri et al., 2004).
Effect of incubation period
Incubation period plays a very important role in the production of bacterial secondary metabolites. Incubation period varied in the range of 6 to 60 h. Pigment production commenced from 8th h in PG broth, while the same started from 10th h of growth in POC medium (Fig. 2C) and it was linear from 18th to 42nd h in case of POC broth and from 12th to 36th h in case of PG broth, the maximum yields being 28.6mg ml-1 and dry cell weight 45.8 mg ml-1 in the POC broth and the same being ~13 mg ml-1 and 25.6mg ml-1 respectively in PG broth. This is almost a 2.2 fold increase in pigment yield in POC when compared to its yield in the PG broth. The early onset of pigment production in PG broth appears to be due to its rich nitrogen sources. PG broth contains peptone, meat and beef extracts which are digests of plant or animal proteins and glycerol is the carbon source (Giri et al., 2004). The POC broth on the other hand is a complex and undefined nutrient source of saturated and unsaturated fatty acids yielding carbon, proteins, minerals, vitamins and hence, the delay in pigment production onset might have been caused.
Effect of inoculum density
Lower initial inocula in both the broths led to lower prodigiosin production when compared with higher initial inocula (Fig. 3A). Pigment production was maximum with 8% initial inoculum density. It was 36.2 mg ml-1 in POC broth and only 14.2 mg ml-1 in PG broth, almost 2.6 fold pigment production being observed in POC broth. Pigment expression mainly depends upon quorum sensing phenomenon (Fuqua et al., 1996). Growth in liquid culture at low cell density allows low level expression of a membrane
permeable positive regulator of prodigiosin expression. The intracellular concentration of the regulator remains low at low cell density due to its diffusion across the cell membrane after synthesis. However, as the cell density increases, the intracellular concentration of regulator increases to a threshold needed for quicker activation of prodigiosin expression (Haddix and Werner, 2000).
Effect of substrate concentration
Substrate concentration too influences the production of pigment and biomass (Fig. 3B). Maximum pigment production was observed at 8% (w/v) of POC extract. But with higher substrate concentrations, biomass production alone was benefited, not pigment production. Probably higher substrate concentrations suppress prodigiosin production. The prodigiosin production was 39.8 mg ml-1 in POC broth compared to PG broth (14.2 mg ml-1) in 42 h, almost 2.8 times in POC medium than in PG broth.
Effect of carbohydrates and Nitrogenous sources
Varying productions of pigment and biomass could be obtained in PG broth amended with different carbon sources. Yet, maximum pigment and biomass production were obtained in POC medium alone, without any supplementary additions of carbon sources (Table 1). Supplementing different carbon sources to PG broth yielded interesting results; in many instances, there is no correlation between biomass yield and pigment production. Glucose appeared to inhibit pigment production, probably by catalytic repression or lowering the media pH as also observed by Sole et al., (1997); hence the use of glycerol as glucose alternative in PG broth to support prodigiosin like pigments
(Montaner et al., 2000, Bae et al., 2001). Though tryptone, yeast extract and soybean meal among the various nitrogen sources induced better pigment and biomass production than the PG broth, they were not even half of those observed with POC medium alone (Table 2). These results support similar observations of Wei and Chen (2005). Various salts (NaCl, KCl, NaNO3, Na2SO4 , CH3COONa) at 1% (w/v) as well as metal ions (CuSO4, CoCl2, FeSO4, MnSO4, ZnSO4, MgSO4 and CaCl2) at 0.1% (w/v) inhibited pigment production (data not shown), thus supporting the observations of Melvin and Elaine (1973).
Determination of molecular mass by ESI-MS
The purified pigment from the culture broth was analyzed by ESI-MS (Figure 4). One major peak was observed; its signal at 325.20 matches the molecular weight of prodigiosin (Montaner and Tomas, 2001). Therefore, the pigment obtained in this present study is ascertained to be prodigiosin, the same red pigment produced by Serratia spp. and Streptomyces spp. (Gerber, 1975).
Scale up studies in bioreactor
Conditions for cell growth and prodigiosin production in Ehrlenmeyer flasks become limited due to substrate consumption as well as metabolite accumulation. Therefore, studies were carried out in the bioreactor employing the biotic and abiotic parameters already standardized hitherto (Fig. 5) The maximum amount of prodigiosin recovered was ~ 40 mg ml l-1 and dry cell weight was 65.2 mg ml-1 (data not shown). This yield appears to be ~3 fold higher than that reported by Bae et al., (2001) who employed novel integrated bioreactor with internal absorbent resins. However, Min-jung-Song et al., (2005) reported that resins exhibit inhibitory effects on prodigiosin production. In the present study, neither internal nor external adsorbents were employed; yet high levels of prodigiosin productions could be achieved using conventional batch fermentation process.
CONCLUSION
The present study has revealed the feasibility of a cost-effective fermentation strategy that can lead to enhanced production of prodigiosin pigment employing Serratia marcescens CF-53. Further, the significance of the present study is that an economically cheap and easily available byproduct can be used, without any external addition of nutrients (sugars, proteins, minerals etc.) for prodigiosin production using peanut oil cake as substrate for fermentation. Studies are in progress for large scale pigment production employing Serratia marcescens CF-53 using pea nut oil cake as the substrate.
ACKNOWLEDGEMENT
The authors wish to thank the Sri Krishnadevaraya Education Trust (KET) for financial support in executing this project. We would also like to
express our sincere thanks to Dr. H.G. Nagendra and Dr. P.M. Nimbargi for their advice during manuscript preparation.
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Cang S, Sananda M, Johdo O, Ohta, S, Nagamatsu Y, Yoshimoto A. 2000. High production of prodigiosin by Serratia marscecens grown on ethanol. Biotechnol. Lett. 22:1761-1765.
D’Aoust JY, Gerber NN. 1974. Isolation and purification of prodigiosin from Vibrio psychroerythrus. J. Bacteriol., 118:756-757.
Furstner A. 2003. Chemistry and biology of roseophilin and the prodigiosin alkaloids: a survey of the last 2500 years. Angew. Chem. Int. Ed., 42:3582-3603.
Fuqua C, Winans SC, Greenberg EP, 1996. Census and consensus in bacterial ecosystem: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol., 50:727-751.
Gerber NN. 1975. Prodigiosin like pigments. CRC Crit. Rev. Microbiol., 3:469-485.
Giri AV, Anandkumar N, Muthukumaran G, Pennathur G. 2004. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil. BMC Microbiol., 4:1-10.
Haddix PL and Werner TF. 2000. Spectrophotometric assay of gene expression; Serratia marcescens pigmentation. Bioscene, 26(4):3-12
Lingappa K, Chandrashekhar Naik, Vivek Babu CS, Ramakrishna D, Venkata reddy M. 2002. A Novel substrate for citric acid production under solid state fermentation. Indian Journal of Microbiology 42:347-349.
Manderville RA. 2001. Synthesis, proton affinity and anticancer properties of the prodigiosin-group natural products. Curr. Medicinal Chem., 1(2):195-218.
Melvin PS, Elaine FM. 1973. Effect of iron and salt on prodigiosin synthesis in Serratia marcescens. J. Bacteriol., 114(3):99-1006
Min-Jung Song, Jungdon Bae, Dae Sil Lee, Chang-Ho Kim, Seung-Wook Kim, Suk-In Hong. 2006. Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. J. Biosc. Bioeng., 101:157-161.
Montaner B, Navarro S, Pique M, Vilasca M, Martinell M, Giralt E, Gil J, Perez-Tomas R. 2000. Prodigiosin from the supernatant of Serratia marcescens induce apoptasis in haematopoietic cancer cell lines. Br. J. Pharmocol., 131:585-593.
Montaner B, Perez-Thomas R. 2001. Prodigiosin induced apoptosis in human colon cancer cells. Life Sci., 68:2025-2036.
Montserrat Sole, Nuria Rius, Alicia Francia, Loren JG. 1994. Letters in Applied Microbiology. 19:341-344.
Perez-Tomas R, Montaner B, Lalgostera E, Soto-cerrato V. 2003. The prodigiosins proapoptatic drugs with anti cancer properties. Biochem. Pharmocol., 66:1447-1452.
Pryce L Haddix, Terry F, Werner. 2000. Spectrophotometric assay of gene expression: Serratia marcescens Pigmentation. 26(4):3-13.
Rustom SM, Valiollah heidarynejad, Alka MP, Prafulla JD. 1990. Isolation and characterization of Serratia marcescens mutants defective in prodigiosin biosynthesis. Curr. Microbiol., 20:95-103.
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Sole M, Francia A, Rius N, Loren JG. 1997. The role of pH in the ‘glucose effect’ on prodigiosin production by non proliferating cells of Serratia marcescens. Lett. Appl. Microbio., 25:81-84.
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Yu-Hong Wei, Wei-Chuan Chen. 2005. Enhanced production of Prodigiosin like pigment from Serratia marcescens SMAR by medium improvement and oil-supplementation strategies. J. Biosc. Bio eng., 99(6):616-622.
Naik et al.,2012
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Fig 1. Growth and pigment production profile of Serratia marcescens CF-53 on different substrates. (a) COC extract agar; (b) SOC extract agar; (c) POC extract agar; (d) PGA; (e) SFOC extract agar and (f)CSOC extract agar
Fig 2. Effect of pH, temperature and incubation time on prodigiosin production by Serratia marcescens CF-53; (○) Pigment in POC extract; (●) pigment in PG broth; (Δ) Cell growth in POC extract; (▼) Cell growth in PG broth
Naik et al.,2012
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Fig 3. Effect of inoculum size and substrate (POC) concentration on growth and prodigiosin production by
Serratia marcescens CF-53; A, (○) Pigment in POC extract; (●) pigment in PG broth; (▼) Cell growth in POC extract; (Δ) Cell growth in PG broth: B, (●) pigment in PG broth; (○) Cell growth in POC extract.
Fig 4. Electro spray Ionization Mass spectrometry (ESI-MS) shows the prodigiosin isolated from Serratia marcescens CF-53 has molecular weight of 325.20 Da.
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Table 1. Effect of carbon source on cell growth and pigment production by S. marcescens CF-53
Carbon source |
Cell dry weight (mg ml-1) |
Pigment (mg ml-1) |
No carbon |
20.00 |
1.00 |
Glucose |
21.00 |
1.58 |
Galactose |
22.98 |
3.10 |
Fructose |
23.00 |
6.15 |
Sucrose |
25.00 |
3.16 |
Lactose |
21.30 |
6.14 |
Maltose |
23.10 |
5.15 |
Manitol |
23.33 |
3.60 |
Malibiose |
19.80 |
0.09 |
Rafinose |
15.60 |
0.04 |
Xylose |
16.60 |
0.04 |
Cellobiose |
14.20 |
0.10 |
Arbinose |
10.98 |
0.10 |
Rhamnnose |
19.86 |
1.13 |
Soluble starch |
29.08 |
11.50 |
Corn starch |
35.50 |
10.00 |
Potato starch |
37.60 |
13.08 |
Rice starch |
37.08 |
12.00 |
POC extract |
62.80 |
39.80 |
Medium PG broth: 10 g carbon source, 10 g Bactopeptone, 10 g Beef extract, 2 g K2HPO4l-1, 2 g NaCl, 10 ml glycerol in 1 l distilled water adjusted to pH 7 and incubated in incubator shaker at 200 rpm at 28oC for 48 h. Two batches of growth were analyzed in replicates.
Fig 5. Scale up studies in bioreactor with POC medium by Serratia marcescens CF-53
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Table 2. Effect of nitrogen source on cell growth and pigment production by S. marcescens CF-53
Nitrogen source |
Cell dry weight (mg ml-1) |
Pigment (mg ml-1) |
No nitrogen |
0.00 |
0.00 |
Yeast extract (YE) |
24.56 |
8.12 |
Soybean meal |
35.41 |
14.12 |
Cas-aminoacids (CAS) |
26.22 |
10.88 |
Tryptone |
24.52 |
9.87 |
Urea |
13.23 |
4.01 |
Dried yeast |
23.29 |
6.05 |
NH4NO3 |
14.70 |
2.03 |
NH4Cl |
13.15 |
0.01 |
NH4SO4 |
14.20 |
0.02 |
POC extract |
62.82 |
39.80 |
Medium PG broth: (see Table-1) was used which contained an indicated nitrogen source (20 g l-1) in place of Bactopeptone and Beef extract. Two batches of growth were analyzed in replicates.
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Naik et al.,2012
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