Red HE7B degradation using desulfonation by Pseudomonas ... - ENVIS

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Red HE7B (RHE7B, 100 mg lА1), a sulfonated azo dye, was decolorized at static condition by ... Extracellular lignin peroxidase (LiP) has played a crucial role in.


International Biodeterioration & Biodegradation 60 (2007) 327–333

Red HE7B degradation using desulfonation by Pseudomonas desmolyticum NCIM 2112 Satish Kalme, Gajanan Ghodake, Sanjay Govindwar Department of Biochemistry, Shivaji University, Kolhapur 416004, India Received 4 April 2007; received in revised form 24 May 2007; accepted 24 May 2007 Available online 10 July 2007

Abstract Red HE7B (RHE7B, 100 mg l1), a sulfonated azo dye, was decolorized at static condition by Pseudomonas desmolyticum NCIM 2112 in 72 h with 71% reduction in chemical oxygen demand (COD). Extracellular lignin peroxidase (LiP) has played a crucial role in breakdown of the dye by asymmetric cleavage and reductases in the initial 24 h incubation to break azo bonds of some dye molecules. Dye also induced the activity of aminopyrine N-demethylase, one of the enzymes of mixed function oxidase system. Decolorization and degradation were analyzed by using UV–vis and high-pressure liquid chromatography (HPLC). The Fourier transform infrared spectroscopy (FTIR) analysis revealed that P. desmolyticum preferred C–N and S¼O bonds to break down the RHE7B. GC–MS identification of 8-amino-naphthalene-1,3,6,7-tetraol and 2-hydroxyl-6-oxalyl-benzoic acid as final metabolites supports the degradation of RHE7B by desulfonation before and after ring cleavage. Aerobic degradation of amines and reduced phytotoxicity increased the applicability of this microorganism for dye removal. Scientific relevance of the paper: This is the first report on degradation of Red HE7B by oxidative enzymes and on further degradation by desulfonation before and after ring cleavage. r 2007 Elsevier Ltd. All rights reserved. Keywords: Anoxic; Biodegradation; Desulfonation; Pseudomonas desmolyticum; Red HE7B

1. Introduction Reactive sulfonated dyes are very soluble by design. As a result, not all are used up by textile fibers during the dyeing process and, therefore, discharge from dye houses results. The sulfonic acid groups that are introduced to increase the water solubility of the dye and the azo group (chromophoric group) confer resistance to microbial attack, making them recalcitrant to oxidative decolorization (Coughlin et al., 1999). There are some reports on aerobic desulfonation of sulfonated aromatic compounds (Kertesz et al., 1994; Kneifel et al., 1997) but most of the sulfonated azo dyes are hardly biodegraded under aerobic conditions. There are worm in the literature in decolorization of sulfonated dyes by chemicals, e.g., Reactive Black 5 by Candida oleophila Corresponding author. Tel.: +91 231 2609152; fax: +91 231 2691533.

E-mail address: [email protected] (S. Govindwar). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.05.006

(Lucas et al., 2005), Remazol dyes by Trichophyton rubrum LSK-27 (Yesiladali et al., 2006), Acid Red 151 and Acid Orange 12 by Kerstersia sp. (Vijaykumar et al., 2007) and Direct Red 81 by bacterial consortium (Junnarkar et al., 2006) but they report only on physico-chemical parameters involved. The studies reporting anaerobic degradation of Red HE7B (RHE7B) did not explain the metabolic pathway followed by the microorganism during degradation (Carliell et al., 1995; O’Neill et al., 2000). As initial breakdown of azo bond requires anaerobic/anoxic conditions, there is a need to study the fate of such sulfonated azo dyes in anoxic conditions. It has been reported that complete mineralization of dyes is possible only if anaerobic reduction is followed by aerobic oxidation of the amines formed in the reductive steps (Rajaguru et al., 2000). So, in the present study we have described the degradation of sulfonated dye RHE7B by Pseudomonas desmolyticum NCIM 2112 at static anoxic condition. Aerobic degradation of aromatic amines which are


S. Kalme et al. / International Biodeterioration & Biodegradation 60 (2007) 327–333

produced after anoxic digestion, enzymes involved in the degradation and the degradation mechanism has been explained. 2. Material and methods 2.1. Dyestuff and chemicals 2,20 -Azinobis (3-ethylbezthiazoline-6-sulfonate) (ABTS) and aminopyrine were purchased from Sigma-Aldrich, USA. Catechol, tartaric acid and n-propanol were purchased from Sisco Research Laboratories, India. The textile dye RHE7B was a generous gift from local textile industry, Solapur, India. All the chemicals used were of the highest purity (99%) available and of analytical grade.

2.2. Microorganism and culture conditions P. desmolyticum NCIM 2112 was obtained from National Center for Industrial Microorganisms (NCL, Pune, India). Pure culture was maintained on nutrient agar slants. The nutrient medium used for decolorization studies was composed of (g l1) NaCl 5, and peptone 5 and beef extract 3. The P. desmolyticum was gradually exposed to the increasing concentration of dye (0.1–0.3 g l1) to acclimatize as reported earlier (Kalme et al., 2007). This acclimatized microorganism was used for all the studies.

2.3. Decolorization experiments P. desmolyticum was grown for 24 h at 30 1C in 250 ml Erlenmeyer flasks containing 100 ml nutrient broth (pH: 6.8). After 24 h, 100 mg l1 dye was added to each flask and incubated at static, as well as shaking, condition at 30 1C for 120 rpm on orbital shaker. An aliquot (3 ml) was withdrawn (5000 g for 15 min) at different time intervals and decolorization was monitored by measuring absorbance at 552 nm. Change in pH and reduction in chemical oxygen demand (COD) (American Public Health Association, 1995) were also studied in the same sample. Growth of microorganism in dye-containing medium was determined by the gravimetric method after drying at 80 1C until constant weight. All decolorization experiments were performed in three sets. Abiotic controls (without microorganism) were always included. The percentage decolorization was calculated as reported earlier (Saratale et al., 2006).

2.4. Determination of enzyme activities P. desmolyticum was grown in nutrient broth at 30 1C for 24 h and centrifuged at 6000 g for 20 min. These cells were suspended in 50 mM potassium phosphate buffer and a cell-free extract was prepared as reported earlier (Kalme et al., 2007). In the cell-free extract and culture supernatant, laccase activity was determined in a reaction mixture of 2 ml containing 10% ABTS in 0.1 M acetate buffer (pH 4.9) and we measured the increase in optical density at 420 nm (Hatvani and Mecs, 2001). Tyrosinase activity was determined by the method reported earlier (Jadhav et al., 2007). Lignin peroxidase (LiP) activity was determined by monitoring the formation of propanaldehyde as reported by Parshetti et al. (2007). All enzyme assays were carried out at 30 1C where reference blanks contained all components except the enzyme. All enzyme assays were run in triplicate and average rates calculated and one unit of enzyme activity was defined as a change in absorbance unit min1 (mg protein)1. NADPH-dichlorophenol indophenol (DCIP) reductase and aminopyrine N-demethylase (AND) activities were determined using a procedure reported earlier by Salokhe and Govindwar (1999). In malachite green reductase (MGR) assay, malachite green reduction was calculated using the extinction coefficient of 8.4  103 mM1 cm1 (Jadhav and Govindwar, 2006).

2.5. Decolorization and biodegradation analysis Decolorization was monitored by UV–vis spectroscopic analysis (Hitachi U-2800) at different time intervals. For biodegradation analysis, 100 ml culture broth was taken at 24, 48 and 72 h intervals, centrifuged at 10,000 g and extraction of metabolites was carried out from supernatant using equal volumes of ethyl acetate. The extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. Highpressure liquid chromatography (HPLC) conditions applied were the same as reported earlier (Kalme et al., 2007) except the UV detector at 314 nm. The Fourier transform infrared spectroscopy (FTIR) analysis was done in the mid-IR region of 400–4000 cm1 with 16-scan speed. The samples were mixed with spectroscopically pure KBr in the ratio 7:93, pellets were fixed in sample holder, and the analyses were carried out. Rotary vacuum evaporated sample (extracted after 72 h decolorization period) was dissolved in methanol and GC–MS analysis of metabolites was carried out using a Hewlett-Packard 989 B MS Engine, equipped with integrated gas chromatograph with an HP1 column (30 m long, 0.25 mm i.d., nonpolar). Helium was used as carrier gas at a flow rate of 1.1 ml min1. The injector temperature was maintained at 300 1C and too oven conditions were the following: 100 1C kept constant for 2 min; raised upto 250 1C at a rate of 10 1C min1; raised up to 280 1C at a rate of 30 1C min1. The compounds were identified on the basis of mass spectra, using the NIST library.

2.6. Aerobic degradation of aromatic amines Five percent inoculum of P. desmolyticum was added (absorbance at 530 nm: 0.1) in 100 ml synthetic medium containing 0.1 g l1 individual aromatic amine and incubated at 30 1C at 150 rpm. At different time intervals, culture supernatant was collected (5000 g, 5 min) and diluted in 0.10 M sodium phosphate buffer solution (pH 7.0). The aerobic degradation of aromatic amines was analyzed spectrophotometrically (Hitachi U-2800) by measuring decrease in their concentration at their absorbance maxima (Tan et al., 2005): p-aminobenzene sulfonamide (p-ABSA, 260 nm), 4-aminobenzene sulfonic acid (4-ABS, 248 nm), 4amino-6-naphthol-2-sulfonic acid (4-A-6-NOH-2-S, 317 nm) and RHE7B metabolites produced at static anoxic condition (315 nm). Growth of P. desmolyticum on aromatic amines was measured at 530 nm periodically.

2.7. Phytotoxicity study This test was performed in order to assess the toxicity of the untreated dye in the concentration range 2000–10,000 ppm. As 10,000 ppm concentration of RHE7B dye showed maximum germination inhibition, the effect of dye metabolites was recorded at this concentration. Tests were carried out with two kinds of seeds commonly used in the Indian agriculture: Sorghum bicolor, Triticum aestivum as reported by Parshetti et al. (2006). Thirty-five individual seeds were used for the germination in each set and watered separately with 10 ml solution of RHE7B and its degradation product (10,000 ppm) per day for 7 days.

2.8. Statistical analysis For analysis of the data one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparison test was used. Readings were considered significant when P was p0.05.

3. Results 3.1. Effect of static and shaking conditions Acclimatization of the P. desmolyticum had reduced the time required for complete decolorization of 100 mg l1 dye from 168 to 72 h. Decolorization of RHE7B was 95% at

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40 35


30 60

25 20


15 10





36 48 Time (h)



0.006 0.004





0 0





Time (h)


Fig. 1. Effect of static (’) and shaking (m) condition on RHE7B decolorization and growth of P. desmolyticum NCIM 2112 at static (&) and shaking (n) condition of the nutrient broth.



0 0



5 0



Laccase/Tyrosinaseactivity (Units mg-1 ml-1)


45 Dry cell weight (mg)

Decolorization (%)


50 LiP activity (units mg-1 ml-1)



Fig. 2. Time course of extracellular (&) and intracellular (’) LiP, laccase (m) and tyrosinase (  ) in P. desmolyticum NCIM 2112 during RHE7B decolorization.


3.2. Enzyme activities during decolorization of RHE7B In order to gain additional insight into the decolorization mechanism, screening of oxidative enzyme activities were also monitored over time. At 48 h, induction in extracellular LiP activity was observed by 143% (as compared to 0 h activity). The activity of intracellular LiP remained unchanged throughout the decolorization process. Induction in laccase and tyrosinase activities was observed upto 72 h incubation (270% and 94%, respectively). Even after complete decolorization (at 96 h), a noticeable induction in LiP (intracellular 40% and extracellular 86%), laccase (550%) and tyrosinase (150%) have been observed (Fig. 2). There were no activities of laccase and tyrosinase in culture supernatant. AND, MGR and DCIP reductase activities were induced in the time course of 96 h by 187%, 101% and 4.5%, respectively. Especially, MGR activity was highly induced (214%) up to 24 h incubation during the decolorization process (data not shown). 3.3. Biodecolorization and biodegradation analysis UV–vis scan (400–800 nm) of supernatants at different time intervals showed decolorization and decrease in dye


0h 24 h 72 h

0.5 Optical density

static condition and 53% at shaking condition with growth of microorganism. Growth was observed to be more at shaking (0.46 g l1) as compared to static condition (0.32 g l1) (Fig. 1). To confirm whether this decolorization was due to microbial action or change in pH, the change in pH was recorded, which was in the range of 6.8–7.8 at shaking and 6.8–8.0 at static condition. UV–vis spectra of RHE7B did not show any change at this pH range. Reduction in COD was observed by 71% at static and 43% at shaking condition (data not shown). There was no decolorization in abiotic control.

0.4 0.3 0.2 0.1 0 400 440 480 520 560 600 640 680 720 760 800 Wavelength (nm)

Fig. 3. UV–vis spectral analysis of culture supernatant extracted during decolorization of RHE7B at different time intervals.

concentration from batch culture. Peaks observed at 552 and 500 nm (0 h) were decreased with shift in lmax showing a new peak at 440 nm with complete decolorization of dye (72 h) (Fig. 3). HPLC analysis of RHE7B dye showed a peak at retention time 1.82 min and the sample extracted after 72 h showed three major metabolites at retention times 1.94, 2.16 and 2.54 min (data not shown). The FTIR spectral comparison between control dye and samples extracted at different time intervals showed degradation of RHE7B in different metabolites by P. desmolyticum. FTIR spectra of RHE7B showed the presence of different peaks at 3421 cm1 for N–H stretching. It also showed vibration of N–H deformation+C–N stretching as a peak at 1541 cm1 and, C–H stretching at 2924 cm1. The presence of sulfonic acid was confirmed by two peaks at 1206

ARTICLE IN PRESS S. Kalme et al. / International Biodeterioration & Biodegradation 60 (2007) 327–333


(symmetric S¼O stretching) and 1041 cm1 (asymmetric S¼O stretching). In 24, 48 and 72 h extracted samples, the appearance of new peaks at 1670, 3413, 1667 and 3402 cm1 expressed the breakdown of RHE7B in various compounds showing C¼O stretching in amides, free N–H stretching in pyrroles and amines, respectively (Fig. 4). The GC–MS analysis of 72 h extracted sample showed 13 peaks. The compound at retention time 6.25 min was identified as N,N0 -bis-[1,3,5] triazin-2-yl-benzene-1,4-diamine, one of the metabolites generated after asymmetric cleavage of RHE7B by LiP (Table 1). Two more possible metabolites of asymmetric cleavage, 2-hydroxynaphthalene 1,5-disulfonic acid (2HN-1,5SA) and 6-diazenyl-5-hydroxy-4-imino-3,4-dihydronaphthalene 2,7-disulfonic acid (6Az-5H-4I-3,4HN-2,7SA) and 2-aminonaphthalene-1,5-

disulfonic acid (2AN-1,5SA), generated after azo bond cleavage by reductase were not detected in the sample (data not shown). But their presence was anticipated on the basis of the metabolites detected after desulfonation, before and after ring cleavage. 3.4. Biodegradation mechanism Desulfonation of 2HN-1,5SA before ring cleavage by dioxygenase enzyme produced naphthalene 1, 2, 3, 6, 7 pentaol (Table 1). 6Az-5H-4I-3,4HN-2,7SA was further degraded by desulfonation before ring cleavage. The desulfonation in hydrolysis product of 6Az-5H-4I-3,4HN2,7SA, due to monooxygenase reaction, was identified as 5,7-dihydroxy-4-imino-6-nitroso-3,4-dihydronaphthalene-2-

85.0 48 h extracted metabolites

80 75 3413.98



Control RHE7B

65 60

1411.34 1470.53

2924.23 2926.67


617.57 976.91



72 hextracted metabolites 55

768.97 796.86





1206.77 1454.74

50 1667.34

45 40 35

24 h extracted metabolites

3235.35 2928.27











cm-1 Fig. 4. FTIR comparison of control dye RHE7B and metabolites formed at different time intervals during degradation. Table 1 Mass spectral data, retention times, and proposed identities of metabolites formed after degradation of Red HE7B by P. desmolyticum NCIM 2112 Sl. no.a

Rt (min) and Mw (m/z)

Relative abundances in mass spectrum: m/z (% relative intensity)

Proposed compound


4.12, 283

50 (5), 66 (30), 74 (6), 94 (100), 283 (8)


8.99, 343 [MH]


13.88, 281

60 (8), 73 (100), 97 (8), 147 (13), 207 (6), 325 (10), 341 (14) 55 (8), 70 (81), 98 (6), 125 (36), 154 (100), 281 (3)

4 5

14.30, 208 [M+1] 15.08, 209 [MH]

5,7-Dihydroxy-4-imino-6-nitroso-3, 4dihydronaphthalene-2-sulfonic acid 1-Hydroxy-8-imino-3,6-disulfo-7,8dihydronaphthalene-2-diazonium 1,3-Dihydroxy-8-imino-6-sulfo-7,8dihydronaphthalene-2-diazonium 8-Aminonaphthalene-1,3,6,7-tetraol 2-Hydroxy-6-oxal-yl-benzoic acid

6 7

6.25, 267 [M+1] 13.60, 208 a

55 (6), 70 (84), 125 (46), 154 (100), 208 (3) 55 (9), 70 (77), 86 (18), 125 (12), 154 (100), 167 (4), 209 (2) 55 (23), 71 (16), 99 (100), 267 (2) 57 (15), 72 (31), 86 (15), 99 (21), 113 (68), 128 (100), 156 (40), 191 (3), 208 (6)

Structures of serial number 1–5 are in Fig. 5(a) and (b).

N,N0 -Bis-[1,3,5] triazin-2-yl-benzene-1,4-diamine Naphthalene-1,2,3,6,7-pentaol

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sulfonic acid (Fig. 5[1]), whereas oxidation product of 6Az5H-4I-3,4HN-2,7SA was identified as 1-hydroxy-8-imino3,6-disulfo-7,8-dihydronaphthalene-2-diazonium (Fig. 5[2]). The desulfonation of later intermediate due to monooxygenase enzyme was identified as 1,3-dihydroxy-8-imino-6-




3.5. Aerobic degradation of amines SO3H








[2] SO3H





monooxygenase O N






[3] SO3H





[1] N





3.6. Phytotoxicity analysis




[A ]




O2 S O3H















H 2O

Aerobic degradation of aromatic amines by P. desmolyticum was confirmed by decrease in absorbance maxima of respective amines with respect to incubation time (30 days) and growth of microorganism in synthetic medium containing amines. P. desmolyticum took 15 days to reach stationary growth phase during growth on RHE7B metabolites and 4-A-6-NOH-2-S. The degradations of these amines were, 95% and 94%, respectively. In the presence of p-ABSA and 4-ABS, the stationary growth phase occurred after 21 and 27 days (91% and 99% degradation), respectively (data not shown).




S O3H N H2

sulfo-7,8-dihydronaphthalene 2-diazonium (Fig. 5[3]). Further dioxygenase action and reduction in N2 had given a compound loosing both sulfonic groups, identified as 8amino-naphthalene-1, 3, 6, 7-tetraol (Fig. 5[4]). The desulfonation after ring cleavage in 2AN-1,5SA (azo bond cleavage metabolite) was identified as 2-hydroxy-6-oxalylbenzoic acid (Fig. 5[5]).


N hydrolysis



[E ]

monooxygenase O COOH

COOH OH [5 ]

Fig. 5. Proposed pathway for degradation by (a) desulfonation before ring cleavage in 6-diazenyl-5-hydroxy-4-imino-3,4-dihydro-naphthalene2,7-disulfonic acid. (b) Desulfonation after ring cleavage in 2-aminonaphthalene-1,5-disulfonic acid. The compounds represented by alphabets have not been found, but their existence is rationalized as necessary intermediates for the final products found. The compounds in Arabic numbers have been found in reaction mixture. Details of their GC–MS analysis are in Table 1.

After 7 days of incubation, untreated RHE7B (10,000 ppm) showed 99% and 98% germination inhibition in S. bicolor and T. aestivum, respectively. When metabolites formed after complete decolorization were applied at the same concentration, there was no germination inhibition in both the seeds. But the growth observed in presence of metabolites was not normal as compared to the growth in distilled water. The shoot and root lengths were decreased by 19% and 11%, respectively, in S. bicolor, whereas in T. aestivum, the shoot and root growths were affected by 14% and 57%, respectively (Table 2). 4. Discussion In this study, the observations suggest that the decolorization performance of P. desmolyticum was better at static anoxic condition where depletion in oxygen content is followed. The physiology of the possible reactions that result in a reductive cleavage of azo compounds under anoxic conditions differs significantly from the situation in the presence of oxygen, because several redox active compounds (e.g. reduced flavins or hydroquinones) rapidly react either with oxygen or with azo dyes. Therefore, decreased decolorization at shaking condition is due to competition between oxygen and the azo compounds for the reduced electron carriers. The co-metabolic activity by P. desmolyticum during decolorization was biomass and supplemented source dependent. The azo dye reduction in anaerobic incubation is a nonspecific and extracellular process in which reducing equivalents from either biological or chemical source are transferred to the dye (Stolz, 1999). The involvement of fungal peroxidases and laccases for the oxidation of


S. Kalme et al. / International Biodeterioration & Biodegradation 60 (2007) 327–333

Table 2 Phytotoxicity study of Red HE7B and its metabolites produced after complete decolorization (72 h) Dye concentration (ppm)

Plants studied Sorghum bicolor

Distilled water 10,000 Metabolites (RHE7B) (10,000)

Triticum aestivum

Germination inhibition (%)

Shoot length (cm)

Root length (cm)

Germination inhibition (%)

Shoot length (cm)

Root length (cm)

0.0 9970.5 0.0

8.88a71.30 0.570.1 7.2a70.90

6.94a71.02 0.4570.15 6.20a70.99

0.0 9870.5 0.0

14.16a72.31 0.670.1 12.26a71.80

11.88a71.20 0.470.15 5.12a,b71.67

Values are mean of two experiments. a Significantly different from sample grown in untreated Red HE7B at Po0.001. b Significantly different from sample grown in distilled water at Po0.05.

sulfonated and unsulfonated azo dyes has been reported (Kandelbauer et al., 2004; Zille et al., 2005). In this study, there was induction in extracellular LiP during decolorization, and this is opposite of that in the earlier study of Kalme et al., (2007). Extracellular LiP has played a crucial role in breaking down the dye by asymmetric cleavage and reductases in the initial 24 h incubation to break azo bonds of some dye molecules. Even after complete decolorization, upto 96 h, enzyme activities (laccase and tyrosinase) remained increasing in the batch culture. The induction in AND activity showed the involvement of mixed function oxidase system. The induction in oxidative enzymes (LiP, laccase) and tyrosinase upto complete decolorization period (72 h) are presumably responsible for the degradation of RHE7B. The elucidation of degradation pathways is of special interest considering health and environmental priorities. It was confirmed by Carliell et al. (1995) that 2-aminonapthalene-1,5-disulfonic acid was present after anaerobic digestion of RHE7B, thus showing that azo bond has been cleaved. In our study, we have observed the desulfonated product of 2-aminonapthalene-1,5-disulfonic acid (Fig. 5b). Desulfonation of sulfonated aromatic compounds after/before ring cleavage (Kertesz et al., 1994; Kneifel et al., 1997; Stolz, 1999) and symmetrical splitting of azo linkage or asymmetrical cleavage of sulfonated azo dyes (Lopez et al., 2004) has been studied at aerobic condition. In this study at static condition, the oxidation–reduction reactions made possible the degradation of RHE7B by desulfonation before and after ring cleavage by P. desmolyticum. The FTIR analysis revealed that P. desmolyticum preferred C–N and S¼O bonds to break down the RHE7B. This data was supported by GC–MS identification of 8-amino-naphthalene-1,3,6,7tetraol and 2-hydroxy-6-oxalyl-benzoic acid as final metabolites of RHE7B. Use of untreated and treated dyeing effluents in agriculture has direct impact on fertility of soil. Hence we considered assessing the phytotoxicity of the undecolorized and decolorized dyes important. The aromatic amines produced during static digestion of RHE7B were degraded

aerobically and reduced phytotoxicity of metabolites produced by the action of P. desmolyticum enables this microorganism to be used in biological treatment of industrial effluents containing the sulfonated azo dye RHE7B.

Acknowledgments The authors thank SAIF, IIT, Mumbai, for availing GC–MS facility and Common Facility Center, Shivaji University, Kolhapur, for FTIR facility.

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