Fuel Processing Technology 92 (2011) 401–406

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Fuel Processing Technology

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Bioethanol production from renewable polymer lichenan using lichenase from analkalothermophilic Thermomonospora sp. and thermotolerant yeast

Vishnu Menon, Rupesh Divate, Mala Rao ⁎Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008. India

⁎ Corresponding author. Tel.: +91 20 25902228; fax:E-mail address: mb.rao@ncl.res.in (M. Rao).

0378-3820/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.fuproc.2010.10.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 June 2010Received in revised form 14 October 2010Accepted 20 October 2010Available online 16 November 2010

Keywords:LichenanLichenaseHydrolysisSynergismEthanolThermotolerant yeast

Biomass feedstocks available decentrally will be more commodious for localized biorefinery approach thanthe exhaustive large scale and centralized plants driven by cost intensive technology. Lichen is present in awide range of habitats in a distributed manner. A maximum hydrolysis of 73%–76% for lichenan from Cetrariaislandica, Usnea barbata and Parmelia sp. were obtained in 24 h using lichenase from an alkalothermophilicThermomonospora sp. wherein the hydrolysis was 100% with commercial enzyme Accellerase™1000. Thesynergistic role of β-glucosidase in lichenan hydrolysis was demonstrated by the exogenous addition ofβ-glucosidase to Thermomonospora lichenase which resulted in complete hydrolysis. The hydrolysates oflichenan obtained using Accellerase or a cocktail of Thermomonospora lichenase and β-glucosidase whenfermented with free cells of Saccharomyces at 40 °C produced an ethanol yield of 0.45 g/g–0.48 g/g withtheoretical conversion efficiencies of 93%–96%. The Ca-alginate immobilized yeast cells were reused eighttimes at 40 °C with 100% fermentation efficiency.

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1. Introduction

The increasing energy demands have evoked a worldwideattention on the utilization of renewable resources, particularlyagricultural and industrial byproducts for the production of biofuels.The development of second generation biofuel is technically a costintensive process and serves as a key enabler to the billion-ton biofuelvision. The exploitation of feedstocks containing polysaccharidesother than cellulose is vital for exploring the feasibility of biofuelproduction. Biodegradation of these polymers such as lichenan,xyloglucan, glucomannan and β-glucan and their processing tovalue added products could be turned to an industrial opportunity.Despite their obvious potential as renewable polymers, lichenan hasnot yet been evaluated for ethanol production.

Lichen is present in a wide range of habitats throughout the worldand dominates terrestrial ecosystems accounting for 20,000 speciesglobally. The Indian subcontinent has 2450 species of lichens, of whichIndia alone has about 2040 species [1]. Parmelia is a large genus oflichenized fungus with a global distribution [2], extending from theArctic to the Antarctic continent but concentrated in temperateregions [3,4]. Lichen consists of a major polysaccharide lichenan,accounting for 62% of the total carbohydrate content and is found inspecies such as Cetraria islandica (Icelandic moss), Usnea barbata,Alectoria ochroleuca etc. [5]. Lichenan is a linear homopolysaccharide

of β-D-glucopyranose residues (Glcp) linked by a mixture of β-(1→3)and β-(1→4) linkages [6].

Structurally lichenan from lichen consists of 82% of 1,3-linkedcellotriosyl and -cellotetraosyl units and longer cellulose like (18%)segments [7]. Despite their close chemical similarity to cellulose, 1,3-1,4-β-D-glucans are soluble, a property that appears to be related to the evendistribution of β-(1→3) bonds along the chains, with a resultingdisruption of linear orientation effects [8] and is easily amenable toenzymatic hydrolysis. 1,3-1,4-β-D-glucanase or lichenase (E.C.3.2.1.73) isan inducible endoglucanase that specifically cleaves the β-1,4 glycosidicbonds in 3-O-substituted glucopyranose units to yield 3-O-β-D-cellobio-syl-D-glucose and 3-O-β-D-cellotriosyl-D-glucose [9]. They exhibit strictsubstrate specificity for the cleavage of β-1,4 linkages adjacent to β-1,3-glycosidic bonds. We have earlier reported the characterization of alichenase from an alkalothermophilic Thermomonospora sp. [10].

Ethanol production from renewable feedstocks is a technically costexhaustive process and centralized pilot plants demand expensiveframework and an adept manpower. In developing countries, farmingis widely distributed and advancement of decentralized plants usinglichens as a feedstock may be feasible. Different technologiesdeveloped for a particular feedstock may not be haulable to otherresources as they differ depending on the raw material pegged.Therefore a logical approach is to develop processes for producingethanol from alternative feedstocks while waiting for the lignocellu-losic biomass ethanol technology to be ready for commercialization.Hence visualizing a biorefinery for the production of bioethanol fromlichen at localized regions for which cultivation is regional and climatespecific may be beckoning.

402 V. Menon et al. / Fuel Processing Technology 92 (2011) 401–406

The current report explicates for the first time the possibility oflichenan as a potential polysaccharide for the production of biofuelusing lichenase produced from an alkalothermophilic Thermomonos-pora sp. and a newly isolated thermotolerant yeast. The enzymatichydrolysis of lichenan from three different lichens, C. islandica (CL),U. barbata (UL), and Parmelia sp. (PL) were carried out with lichenasefrom Thermomonospora sp. and a commercial enzyme Acceller-ase™1000. The cooperative action of lichenase and β-glucosidasewere investigated using Thermomonospora lichenase and β-glucosi-dase. The study was also focused to gauge the potency of thermo-tolerant yeast for ethanol production in SHF models at 40 °C.

2. Materials and methods

2.1. Raw materials

Lichenan from C. islandica and U. barbata was purchased fromSigma-Aldrich Co. St. Louis, MO, USA and Parmelia sp. was obtainedlocally. Parmelia sp. thalli was washed with distilled water, andextracted with ethanol at 80 °C for 2 h. The residue obtained wasfurther extracted with water at 100 °C for 2 h. The aqueous extractwas repeatedly freeze and thawed five times and the residue wascentrifuged at 10,000 rpm for 45 min. The residue obtained was airdried andwas powdered [11]. The total glucan (lichenan) content wasdetermined by the orcinol-sulphuric acid method [12]. For C. islandicaand U. barbata a value of 99% and for Parmelia sp. a value of 98% wasobtained.

2.2. Chemicals

All chemicals were of analytical grade. 3,5-Dinitrosalysilic acid(DNS) and β-glucosidase from almond were obtained from Sigma-Aldrich Co. St. Louis, MO, USA. Ethanol was purchased from Les AlcoolsDe Commerce Inc, Brampton, Ontario.

2.3. Thermotolerant yeast

Thermotolerant yeast was isolated from rotten strawberries byenrichment in a media containing (g/l): glucose, 50.0; malt extract,3.0; yeast extract, 3.0; peptone, 5.0; agar, 20.0; at pH 6.0±0.2 at 40 °C.After enrichment for 48 h, the sample was used to isolate the yeast.The yeast was further purified by single colony plating. The yeast wasidentified as Saccharomyces sp. by physiological, biochemical and 18srRNA. Inoculum was prepared by growing the organism on a rotaryshaker at 180 rpm for 48 h at 40 °C, in a growth medium similar toenrichment medium except that the glucose concentration was 40 g/l.After 48 h the inoculum was centrifuged at 5000 rpm for 20 min andthe supernatant was discarded. The cells were washed twice withsterile 0.9% NaCl solution and centrifuged again. The supernatant wasremoved and the cells were weighed and used for fermentationstudies.

2.4. Microorganism, cultivation condition and enzyme production

Alkalothermophilic Thermomonospora sp. was maintained on LuriaBertani wheat bran slants at pH 10 and 50 °C [13]. Thermomonosporawas grown in 500 ml Erlenmeyer flask containing 100 ml of modifiedReese medium at 50 °C on a rotary shaker maintained at 200 rpm.Sterile 10% sodium carbonate was used to adjust the pH of themedium to 9. A 10% inoculum was added from the inoculum flaskgrown for 48 h at 50 °C. At the end of 96 h the fermentation broth wascentrifuged at 10,000 rpm for 15 min at 4 °C [10]. The supernatantwas concentrated using ammonium sulphate (0–90%) and was thesource of enzyme. The preparation contained 7200 IU/ml and 7 IU/mlof lichenase and β-glucosidase respectively.

The Commercial enzyme Accellerase™1000was a kind gift fromDrRaj Lad and Dr Surendra Bade, Danisco US Inc. Genencor Division, USA.The preparation showed 6500 IU/ml of lichenase and 450 IU/ml ofβ-glucosidase.

2.5. Enzyme assays

The activity of lichenase was measured by incubating 1 ml of assaymixture containing 0.5 ml of 1% lichenan and 0.5 ml of suitably dilutedThermomonospora lichenase/commercial enzyme in 50 mM phos-phate buffer, pH 7/50 mM acetate buffer, pH 4.5 respectively for30 min at 50 °C. Enzyme and reagent blanks were also simultaneouslyincubated with the test samples. The reducing sugar formed wasestimated by DNS [14]. One unit of enzyme activity is defined as theamount of enzyme required to liberate 1 μmol of reducing sugar perminute under the assay conditions.

β-glucosidase was measured according to the standard procedurerecommended by the Commission on Biotechnology, IUPAC [15]. Oneunit of enzyme activity is defined as the amount of enzyme requiredto liberate 1 μmol of p-nitrophenol per minute from PNP-β-D-glucoside under the assay conditions. Protein concentration wasdetermined according to the method of Bradford, using bovine serumalbumin as standard [16].

2.6. Immobilization of thermotolerant Saccharomyces in Ca-alginatebeads

Saccharomyces cells were harvested after 48 h of growth at 40 °Cby centrifugation at 5000 rpm for 20 min, washed and 1 g (wet cellweight) was added to 10 ml of 2% (w/v) Na-alginate [17]. Thesuspension was then extruded dropwise through a 5 ml syringe into agently stirred cold solution of CaCl2 and hardened at 4 °C for 1 h in thissolution. Particle integrity and absence of microbial contaminationwere ensured by means of optical microscopy. The beads (meandiameter of 2 mm) were washed with distilled water and used forfermentation.

2.7. Enzymatic hydrolysis

The hydrolysis of lichenan from three genera of lichens was carriedout at different substrate concentrations (25 g/l and 50 g/l) and enzymeconcentrations of 200 and 400 U/g for Thermomonospora lichenase and100, 200 and 400 U/g for Accellerase™1000. The synergistic effect ofThermomonospora lichenase and β-glucosidase in overall hydrolysis oflichenan was elucidated using 400 U/g of Thermomonospora lichenaseand twodifferent concentrations (25 U/mland50 U/ml)ofβ-glucosidaseat a substrate loading of 25 g/l and 50 g/l. The hydrolysis was carried outin a stoppered flask in 25 ml reaction volume containing appropriateconcentrations of substrates in 50 mM phosphate buffer, pH 7.0 forThermomonospora lichenase and 50 mM acetate buffer, pH 4.8 forAccellerase under static condition. Hydrolysis was terminated by boilingat 100 °C for 5 min at the end of stipulated time intervals and filtered andthe filtrate was assayed for reducing sugar by DNS method [14]. Theenzymatic hydrolysis was carried out for 24 h. Appropriate substrate andenzyme controls were performed.

2.8. Separate hydrolysis and fermentation

Ethanol production from hydrolysates obtained after saccharificationof lichenan using Accellerase™1000 and a cocktail of Thermomonosporalichenase and β-glucosidase were carried out. Lichenan at 50 g/l wasincubated separately with 200 U/g of commercial enzyme and a cocktailof 400 U/g Thermomonospora lichenase and 50 U/ml of β-glucosidase at50 °C for 24 h. Ethanol productionwas also performedwith hydrolysatesobtained after saccharification of lichenan at 100 g/l level using 400 U/gof Accellerase™1000. SHF experiments were performed at shake flask

0 10 20 30 40 500

10

20

30

40

50

60

70

80

90

100

Hyd

roly

sis

(%)

Time (h)

Citreria islandica

Usnea barbata

Parmelia sp

Fig. 1. Saccharification of lichenan with Accellerase™1000 (400U/g) at a substrateloading of 100 g/l.

403V. Menon et al. / Fuel Processing Technology 92 (2011) 401–406

level with 50 ml hydrolysate. The hydrolysate was filtered andsupplemented with nutrients (g/l); urea; 5, MgSO4 7H2O; 5, K2HPO4; 2and inoculated with 10% free and immobilized yeast and fermentationwas carriedout at 40 °C.Aliquotswerewithdrawn,andsugar andethanolwere determined. An attempt was also made to test the efficacy of theyeast to ferment synthetic medium containing 100 g/l glucose at 40 °Cand 45 °C.

2.9. Reuse of immobilized yeast

The hydrolysates obtained in separate hydrolysis and fermentation(SHF) were used to investigate the ability of thermotolerantSaccharomyces for reprocessing. The Ca-alginate immobilized cells(10%) were added to hydrolysates supplemented with nutrients andfermentation was performed at 40 °C for 48 h. Afterward, the Ca-alginate beads were retrieved and reused repeatedly with freshcharge of hydrolysates and the ethanol formedwas assayed at the endof each cycle of operation.

2.10. Analytical methods

The hydrolysates were analyzed by high performance liquidchromatography (HPLC) (Waters) for the presence of carbohydratesusingWaters Sugar Pack Column 6.5×300mm. Themobile phase usedwas Milli Q water with 100 μM EDTA and 200 μM CaCl2. The flow ratewas maintained at 0.4 ml/min keeping the oven temperature at 70 °C.The sugars were detected by Waters 2410 refractive index detector.Ethanol was estimated by gas chromatography (GC) (Master DANI)with a BP1 (fused silica bonded phase) column (30.0 m×0.32 mm) atoven temperature of 85 °C and flame ionization detector (FID) at200 °C. The ethanol standards were prepared using commercialethanol. Nitrogen with a flow rate of 0.5 ml/min was used as carriergas.

3. Results and discussion

3.1. Enzymatic hydrolysis

The saccharification of lichenan from Citreria, Usnea and Parmeliasp. using different substrate loadings was carried out with varyingconcentrations of Thermomonospora lichenase and Accellerase. Amaximum hydrolysis of 76% and 68% was obtained for lichenan at asubstrate loading of 25 g/l and 50 g/l respectively with 400 U/g ofThermomonospora lichenase in 24 h. Lichenan from three differentlichens was completely converted into soluble sugars forming atransparent solution within 24 h at a substrate loading of 25 g/l and50 g/l by Accellerase at 200 U/g (Table 1). At higher substrate

Table 1Saccharification of lichenan from three different lichens at different substrate and enzyme

Substrates SBC (g/l) 25 50

Percent hydrolysis

Thermomonospora lichenase (U/g)

Time (h) 200 400 200

Citreria islandica 4 44.59 56.43 39.3212 58.31 67.89 48.0724 68.08 74.41 61.89

Usnea barbata 4 46.29 58.16 40.6812 54.61 62.38 51.2124 69.20 75.84 64.88

Parmelia sp. 4 49.88 53.28 42.3812 55.98 60.21 54.0224 67.20 72.50 59.20

SBC—substrate concentration.

concentration of 100 g/l and double the enzyme dosage (400 U/g),the hydrolysis was complete in 48 h (Fig. 1). Accellerase consistentlyshowed increase in saccharification as compared to Thermomonosporalichenase.

The end product analysis of the hydrolysates obtained aftersaccharification of lichenan with Accellerase™1000 shows glucoseas the end product wherein with Thermomonospora lichenase showscellobiose as the major end product with traces of glucose and higheroligosaccharides.

The different hydrolysis patterns exhibited by similar dosage ofThermomonospora and commercial enzyme may be due to the varyinglevels of β-glucosidase activities. Accellerase™1000 had higher ratioof lichenase to β-glucosidase as opposed to Thermomonospora(Section 2.4). The complete hydrolysis of lichenan requires anaccessory enzyme, β-glucosidase which breaks down the cellobioseto glucose preventing the end product inhibition.

An experiment was designed to determine the synergistic effect ofβ-glucosidase and lichenasewhich is crucial for the overall hydrolysis oflichenan. Thiswas confirmed by the exogenous addition ofβ-glucosidaseto Thermomonospora lichenase thereby increasing the hydrolysis from75% to 100% in 24 h at a substrate loading of 25 g/l (Fig. 2). At a highersubstrate concentration (50 g/l) complete hydrolysis was achieved in48 h (data not shown). The end product analysis confirmed the pre-sence of glucose as themajor end product obtained from the hydrolysisof lichenan with Thermomonospora lichenase supplemented with

concentrations of Thermomonospora lichenase and Accellerase™1000.

25 50

Accellerase™1000 (U/g)

400 100 200 100 200

41.69 65.28 83.21 60.54 69.9858.35 76.81 90.25 72.18 87.3165.82 89.57 100 82.98 10052.68 67.27 89.58 58.93 80.2860.34 74.31 95.68 69.32 91.0768.18 87.34 100 81.23 10047.83 63.71 88.65 57.62 79.6458.21 75.98 94.67 70.28 88.7567.98 85.34 100 81.36 100

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 25

Citreria

Hyd

roly

sis

(%)

Time (h)

Control 25 U/ml β-glucosidase 50 U/ml β-glucosidase

Usnea Parmelia

Fig. 2. Hydrolysis of lichenan using a cocktail of Thermomonospora lichenase (400U/g) and β-glucosidase at 25 g/l level.

404 V. Menon et al. / Fuel Processing Technology 92 (2011) 401–406

exogenous β-glucosidase. Similar synergistic effects of β-glucosidaseand β-xylosidase are also reported in the overall hydrolysis of celluloseand hemicellulose [18,19].

3.2. Separate hydrolysis and fermentation

Separate hydrolysis and subsequent fermentation were carried outusing free cells of newly isolated Saccharomyces sp. at 40 °C. Thehydrolysates of lichenan from C. islandica at a substrate loading of50 g/l and 100 g/l obtained using Accellerase, when fermented withyeast produced a maximum of 23 g/l and 41 g/l of ethanol with a yieldof 0.46 g/g and 0.41 g/g respectively (Table 2). The hydrolysates oflichenan containing 50 g/l of reducing sugar obtained using a cocktailof Thermomonospora lichenase and β-glucosidase after fermentationwith thermotolerant yeast produced 22 g/l of ethanol with yield of

Table 2Ethanol production from enzymatic (Accellerase™) hydrolysates of lichen using free and im

SBC 50 g/l SBC

Substrate Time(h)

Sugar(g/l)

Ethanol (g/l) Ethanol yiel

Free Immobilized Free I

Citreria islandica 0 48.81 – –

12 32.85 8.38 9.84 0.17 024 18.28 15.48 17.61 0.32 036 1.13 22.64 23.65 0.46 048 0.54 13.46 16.88 0.28 0

Usnea barbata 0 49.6 – –

12 31.09 8.08 8.95 0.16 024 18.39 12.48 13.94 0.25 036 1.08 22.14 23.59 0.45 048 0.49 16.46 18.25 0.33 0

Parmelia sp 0 49.6 – –

12 32.97 9.68 11.38 0.2 024 17.68 16.87 19.67 0.34 036 2.04 21.17 23.72 0.43 048 0.88 18.48 20.69 0.37 0

0.48 g/g and a theoretical conversion efficiency of 88% (Fig. 3). As acontrol, when Saccharomyces was grown in a synthetic mediumcontaining 100 g/l glucose, 48 g/l ethanol with a yield of 0.48 g/g,productivity of 1.33 g/l/h and a theoretical conversion efficiency of 96%was achieved at 40 °C. The study was also focused to determine theefficacy of yeast to produce ethanol at higher temperatures. The ethanolconcentrations of 50 and 40 g/l with a yield of 0.48 and 0.41 g/g areobtained from 100 g/l glucose at 40 °C and 45 °C respectively (Fig. 4).

Ethanol production at high temperature has received muchattention because fermentation processes conducted at elevatedtemperatures will significantly reduce cooling costs [20]. Otheradvantages of elevated temperatures include more-efficient simulta-neous saccharification and fermentation, a continuous shift fromfermentation to distillation, reduced risk of contamination, andsuitability for use in tropical countries [21,22]. Attention has focused

mobilized Saccharomyces at 40 °C.

100 g/l SBC

d (g/g) Sugar(g/l)

Ethanol (g/l) Ethanol yield (g/g)

mmobilized Free Immobilized Free Immobilized

99.98 – –

.2 59.75 15.97 20.87 0.16 0.21

.36 31.28 26.47 34.35 0.26 0.34

.48 5.04 41.08 46.47 0.41 0.46

.35 1.08 37.68 33.07 0.38 0.3399.97 – –

.18 60.28 14.79 19.25 0.15 0.19

.28 29.66 25.48 32.09 0.25 0.32

.48 4.08 41.37 48.08 0.41 0.48

.37 1.25 35.88 41.36 0.36 0.4199.95 – –

.23 58.27 16.94 21.08 0.17 0.21

.4 30.11 25.07 35.22 0.25 0.35

.48 4.59 40.89 47.07 0.41 0.47

.42 1.33 34.78 40.69 0.35 0.41

0

10

20

30

40

50

Sugar (g/L)

Ethanol (g/L)

Ethanol yield(g/g)

Eth

ano

l (g

/L)

Citreria Usnea

Time (h)

0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50

Parmelia

0.0

0.1

0.2

0.3

0.4

0.5

Eth

ano

l Yield

(g/g

)

0

10

20

30

40

50S

ug

ar (

g/L

)

Fig. 3. Ethanol production from lichenan hydrolysates obtained with a cocktail of Thermomonospora lichenase and β-glucosidase using Saccharomyces at 40 °C.

405V. Menon et al. / Fuel Processing Technology 92 (2011) 401–406

on thermotolerant yeast species capable of producing ethanol atelevated temperatures. The isolated thermotolerant Saccharomycessp. was able to efficiently ferment the lichenan hydrolysates at 40 °Cwith conversion efficiencies of 82–92%.

3.3. Immobilization and reuse

Saccharomyces cells were immobilized in Ca-alginate matrix asdescribed in Section 2.9. The hydrolysate of lichenan from U. barbataat 50 g/l and 100 g/l level obtained using Accellerase when fermentedwith immobilized cells produced a maximum of 24 g/l and 48 g/l of

12 24 36 480

10

20

30

40

50

60

Ethanol (g/L) Productivity (g/L/h)

Time (h)

Eth

ano

l (g

/L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Pro

du

ctivity (g/L

/h)

Fig. 4. Time course for ethanol production using Saccharomyces at 40 °C and 45 °C filledobjects—40 °C, empty objects—45 °C.

ethanol with a yield of 0.48 g/g respectively (Table 2). Immobilizedcells showed a significant increase in ethanol yield as compared tofree cells. The Ca-alginate immobilized yeast was reprocessed for 10cycles using a batch mode at 40 °C. The immobilized yeast was reusedeight times without any apparent loss in capacity to ferment. Theimmobilized yeast produced ethanol with yield of 0.48 g/g, produc-tivity of 1.33 g/l/h and conversion efficiency of 96% for 8 successivebatches. Then the productivity declined with final conversionefficiencies of 62% in the 10th cycle (Fig. 5).

4. Conclusion

The present article introduces the concept to develop lichen as afeedstock for decentralized cultivation and production of biofuel. The

0 2 4 6 8 10

20

30

40

50

Eth

ano

l (g

/L)

Number of cycles

Citreria islandica Parmelia spUsnea barbata

Fig. 5. Reuse of Ca-alginate immobilized Saccharomyces for ethanol production fromlichenan hydrolysates at 40 °C.

406 V. Menon et al. / Fuel Processing Technology 92 (2011) 401–406

article enunciates for the first time the hydrolysis and ethanolproduction from a novel potential substrate lichenan using lichenaseand thermotolerant yeast. The experimental results demonstratedthe synergistic role of lichenase and β-glucosidase in the overallhydrolysis of lichenan thereby improving the saccharification andfermentation efficiency. The thermotolerant yeast was able to fermentefficiently the enzymatic hydrolysate of lichenan to ethanol in the SHFsystem at 40 °C. The immobilized yeast was reused for eight cycleswith 100% fermentation efficiency.

Acknowledgements

M.R. acknowledges the financial support from CSIR EmeritusScheme. V.M. acknowledges the junior research fellowship from CSIREmeritus Scheme, Govt. of India.

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