Accepted Manuscript

Short Communication

Decolorization and biodegradation of congo red dye by a novel white rot fungus

Alternaria alternata CMERI F6

Samayita Chakraborty, Bikram Basak, Subhasish Dutta, Biswanath Bhunia,

Apurba Dey

PII: S0960-8524(13)01368-0

DOI: http://dx.doi.org/10.1016/j.biortech.2013.08.117

Reference: BITE 12316

To appear in: Bioresource Technology

Please cite this article as: Chakraborty, S., Basak, B., Dutta, S., Bhunia, B., Dey, A., Decolorization and

biodegradation of congo red dye by a novel white rot fungus Alternaria alternata CMERI F6, Bioresource

Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.08.117

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Decolorization and biodegradation of congo red dye by a novel white rot fungus 1

Alternaria alternata CMERI F6 2

Samayita Chakrabortya,1

, Bikram Basaka,1

, Subhasish Duttaa, Biswanath Bhunia

b,* 3

and Apurba Deya,* 4

aDepartment of Biotechnology, National Institute of Technology Durgapur, Mahatma Gandhi 5

Avenue, Durgapur-713209, India. 6

bDepartment of Bio Engineering, National Institute of Technology Agartala, Barjala, Tripura-7

799055, India 8

*Corresponding author 9

Tel No: +91-343-2754027, Fax No: +91-343-2547375, 10

Email: apurbadey.bt@gmail.com, apurba.dey@bt.nitdgp.ac.in, bbhunia@gmail.com 11

1Equal contribution. 12

Abstract 13

A novel white rot fungus Alternaria alternata CMERI F6 decolorized 99.99% of 600 14

mg/L congo red within 48 h in yeast extract-glucose medium at 25 oC, pH 5 and 150 rpm. 15

Physicochemical parameters like carbon and nitrogen sources, temperature, pH and aeration 16

were optimized to develop faster decolorization process. Dye decolorization rate was maximal 17

(20.21 mg/L h) at 25 oC, pH 5, 150 rpm and 800 mg/L dye, giving 78% final decolorization 18

efficiency. Scanning electron microscopy and X-Ray Diffraction analysis revealed that the 19

fungus become amorphous after dye adsorption. HPLC and FTIR analysis of the extracted 20

metabolites suggested that the decolorization occurred through biosorption and biodegradation. 21

Thermogravimetric analysis (TGA), differential thermal analysis (DTA) and acid-alkali and 70% 22

ethanol treatment revealed the efficient dye retention capability of the fungus. The foregoing 23

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results justify the applicability of the strain in removal of congo red from textile wastewaters and 1

their safe disposal. 2

Keywords: Decolorization; azo dyes; congo red; Alternaria alternata; biodegradation. 3

1. Introduction 4

With the growth of the worldwide textile industries in the recent years, a commensurate 5

increase in the use of synthetic dyes and the consequent environmental pollution due to discharge 6

of wastewater contaminated with dyestuffs remains a critical issue (Saratale et al., 2011). Azo 7

dyes are regularly used in various applications in food, pharmaceutical, paper, cosmetic, textile 8

and leather industries (Saratale et al., 2013). These dyes belong to the class of aromatic and 9

heterocyclic compounds having the azo bond (-N=N-) which are recalcitrant and even possess 10

carcinogenic property (Saratale et al., 2011). As they are introduced into the environment due to 11

the inefficiency during the dyeing process and subsequent improper discharge, they are 12

accumulated mainly in water bodies and have adverse effects in terms of dissolved oxygen (DO), 13

Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), color, etc (Saratale et 14

al., 2013; Solís et al., 2012). Therefore, their removal from wastewater is of utmost practical 15

importance before discharge of the wastewater into the environment (Ayed et al., 2011). 16

Several white rot fungi like Phanerochaete chrysosporium, Trametes versicolor, due to 17

their efficient lignolytic system, have been reported to degrade or sequester azo, heterocyclic, 18

reactive or polymeric dyes (Solís et al., 2012). However, Congo Red (CR) dye removal by a 19

white rot fungus Alternaria alternata has not been described previously. 20

This study was undertaken to investigate the potential of a novel white rot fungus 21

Alternaria alternata for decolorizing CR. Relationships between the dye decolorization process 22

and different physicochemical parameters were characterized to achieve maximum dye removal. 23

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The decolorization process was characterized using Microscopy, High performance liquid 1

chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and X-Ray diffraction 2

analysis (XRD). Thermal gravimetric analysis and differential thermal analysis (TG-DTA) was 3

employed to evaluate the dye retention property of the fungus at the higher temperature. 4

2. Methods: 5

2.1 Microorganism 6

The white rot fungus Alternaria alternata CMERI F6 (NCBI GenBank Accession 7

number: JQ781696) used in this study was procured from CSIR-Central Mechanical Engineering 8

Research Institute, Durgapur, India. The fungus was maintained by bimonthly subculture on 9

potato dextrose agar (PDA) and stored at 4 oC after incubation at 30

oC. 10

2.2 Culture condition of the microorganism 11

Different media (Table 1) were employed to examine the decolorization activity of the 12

strain. Apart from the inorganic media (IM) (Basak et al., 2013), a combination of yeast extract 13

(5g/L) and glucose (5g/L), yeast extract (5g/L) and peptone (5g/L), Peptone (5g/L) and glucose 14

(5g/L) were used to observe CR decolorization. 15

2.3 Decolorization experiments 16

In order to examine the decolorization performance, acclimatization was performed by 17

gradually exposing the fungus to the increasing concentration of CR 25 mg/L to 1000 mg/L. All 18

the experiments were carried out in triplicate in 250ml Erlenmeyer flasks with 50 ml media at 25 19

oC and pH 5 with constant shaking at 120 rpm. The decolorization performance was examined 20

spectrophotometrically (Rayleigh 2601, China) at the maximum absorption wavelength 498nm. 21

2.4 Effects of different physicochemical parameters on CR decolorization 22

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For the best set of decolorization observed, the experiments were conducted at different 1

temperatures (20-30 oC) at pH 5 and 120 rpm, at various pH (3-8) at 25

oC and 120 rpm, and at 2

different rpm (90,120, 150 and 180) at 25 oC and pH 5, with highest concentration of the dye 3

decolorized. The effects of heavy metal toxicity on the decolorization of CR by the fungus were 4

investigated at concentrations 100-200 mg/L in the yeast extract-glucose medium. The salts of 5

these metals used were HgCl2, Pb(NO3)2, and CdCl2. The CR decolorization percentage was 6

checked at regular interval. 7

2.5 Analytical procedures 8

Changes in morphological characteristics were examined using scanning electron 9

microscopy (SEM) (Zeiss, USA). The fungal sample were taken before and after CR 10

decolorization and prepared for SEM according to Yu et al (Yu et al., 2007). Further 11

characterization of macromolecular structure of the fungus before and after dye decolorization 12

was done by X-ray diffraction (XRD) analysis. Dried fungal mycelium (incubated without CR 13

and the mycelium that absorbed the dye) was analyzed using X´Pert Pro MRD (XL) X-ray 14

diffractometer (PANalytical Ltd, Cambridge, UK) equipped with radiation source used at 40 kV 15

and 30 mA (diffraction angle varied from 60o

to 10o). The samples were scanned at the rate of 16

3o/min with a Ni-filtered beam (wavelength 1.5406 Å). 17

The metabolites produced during CR degradation were extracted according to methods 18

described by Saratale et al. (2013). The extracted metabolites were dissolved in small amount of 19

HPLC grade methanol, filtered through 0.22µ membrane filter and then analyzed for 20

determination of metabolites by HPLC (WatersTM

600, USA, equipped with WatersTM

2489, 21

UV-visible detector and C18 hypersil column (4.6 mm x 250 mm; particle size 5 μm)) with a 22

mobile phase of methanol (80%) and deionized water (20%) at a flow rate of 1ml/min. An 23

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aliquot of 20µl was injected and analyzed by UV-visible detector at wavelength of 498nm. The 1

extracted metabolites as well as the dried fungal mycelium (before and after CR decolorization) 2

were also analyzed by FTIR (Jasco FT/IR 6300, Japan) and compared with the control dye. The 3

analysis was done in the mid-IR region of 400-4000 cm-1

with scan speed of 16. The samples 4

were prepared according to Ayed et al. (Ayed et al., 2011) 5

After CR decolorization, the fungal mycelium was separated from the culture medium, 6

suspended in distilled water and autoclaved to check whether the fungus is releasing the dye at 7

121 oC and 15 psi pressure. In order to examine the chemical stability, after CR decolorization, 8

the dried fungal mycelia was subjected to 1M HCL, 1M NaOH and 70% ethanol treatment for 1 9

h. Same experiment was repeated with hydrated mycelium. The absorption intensity of CR in 10

medium was measured spectroscopically at λ 498nm. 11

In addition, the Thermogravimetric-Differential Thermal Analysis (TG-DTA) was done 12

to check the thermometric stability of the fungus after CR decolorization with respect to weight 13

in an inert atmosphere. The instrument (Pyris Diamond TG-DTA, Perkin Elmer, USA) used 14

Helium gas to provide an inert atmosphere and the dye-absorbed fungal mycelium was made to 15

undergo identical thermal cycle for the change in percentage weight of the dye-adsorbed with 16

increasing temperature (at a constant rate of 10 oC/min, ambient to 350

oC). 10 mg sample of the 17

dye-absorbed fungal mass was loaded into an alumina crucible for each run under non-18

isothermal conditions and in a atmosphere of Helium supplied at 10 ml/min. 19

3. Results and discussion 20

3.1 Effect of supplementation of carbon and nitrogen sources on CR decolorization 21

The decolorization performance of CR in IM supplemented with various carbon sources 22

(Table 1) was studied in 250 ml Erlenmeyer flasks containing 50 ml media at 25 oC, pH 5 and 23

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120 rpm. IM supplemented with glucose and yeast extract showed 99.99% and 90% 1

decolorization of CR, respectively, indicating that glucose is the efficient carbon source for CR 2

decolorization by the fungus amongst the carbon sources taken under investigation, followed by 3

yeast extract (Table 1). Since, azo dyes generally do not act as carbon source as they are electron 4

deficient owing to presence of their azo linkage (-N=N-) (Solís et al., 2012) microbial 5

decolorization and degradation of azo dyes generally depends on the presence and type of a 6

carbon source used, because they act as electron donor for azo-dye reduction (Hou et al., 2012). 7

Following this result, a set of different media comprising combinations of yeast extract, glucose 8

and peptone were designed (Section 2.2). It was found that medium consisting of yeast extract 9

and glucose yielded better CR decolorization rate compared to any other media. Therefore, this 10

medium was selected for further decolorization experiment. 11

3.2 Effect of dye concentration on decolorization 12

Decolorization experiments were conducted with dye concentrations of 25-1000 mg/L 13

CR in yeast extract-glucose medium. As the sequence of experiments were performed for 14

acclimitization, it was found that with increasing concentrations of CR beyond 600 mg/L, the 15

amount of absorbed dye remained constant. However, the fungus was only able to retain its 16

decolorization capacity to 99.99% up to 600 mg/L CR resulting in 0.28-18.01 mg/L h in 17

decolorization rate (Fig. 1a-1d). Fig. 1b & 1d show that at 800 and 1000 mg/L concentration, CR 18

removal resulted in 78.37% and 60% final decolorization efficiency with removal rate of 10.86-19

20.21 and 10.52-16.59 mg/L h, respectively. This finding is also in accordance with previous 20

reports (Ayed et al., 2011; Saratale et al., 2013) and attributed to the fact that biosorption of dyes 21

is due to heteropolysaccharide and lipid components of the cell wall, which contain different 22

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charged functional groups like carboxyl, hydroxyl, phosphate and other charged groups, causing 1

strong attractive forces between the azo dye and the cell wall (Vitor & Corso, 2008). 2

3.3 Determination of optimum temperature, pH and agitation speed for decolorization of CR 3

Adaptation of the fungus to a broader range of pH and temperature can make it more 4

suitable for dye bioremediation. Experiments showed that 99.99% decolorization of 600 mg/L 5

CR was observed at 20, 25 and 30 oC within 44, 40, and 48 h, respectively. Fig. 2a shows that 6

CR reomoval rate was maximal (6 mg/L h) at 25 oC, followed by CR removal rate at 30 (5.28 7

mg/L h) and 20 oC (4.61 mg/L h), respectively. This is attributed to the fact that (a) in adsorptive 8

decolorization, optimum temperature plays a crucial role in increased surface activity and kinetic 9

energy of the dye molecules and (b) in degradative decolorization, optimum temperature is 10

necessary for optimal activity of dye degradative enzymes (Saratale et al., 2011). 11

A. alternata CMERI F6 showed the decolorization up to nearly 99.99% in the broad 12

range of pH (3-7). Although, the fungus could effectively decolorize (89%) CR at relatively 13

higher pH (8), it showed maximal rate (6.32 mg/L h) of CR decolorization at pH 5 as can be 14

observed from Fig. 2b. It is an established fact that by controlling the [H+] concentration, the pH 15

of the medium influences degradation of a pollutant (Basak et al., 2013). Hence, the dye removal 16

efficiency of the fungus at broad range of pH makes it potent candidate for removal of CR from 17

textile watewaters that differs significantly in pH (Saratale et al., 2011). 18

At agitation speed of 150 and 120 rpm, the fungus was able to decolorize 99.99% of 600 19

mg/L CR. The CR removal rate was also maximal (5.59 mg/L h) at 150 rpm showing that 20

optimum aeration is necessary for maximal CR removal (Fig 2c). In this case, agitation increased 21

mass and oxygen transfer between cells and the medium. In addition, enzyme activity also could 22

have depended on the presence of oxygen (Saratale et al., 2011). At agitation speed of 90 and 23

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180 rpm, the fungus showed relatively less decolorization performance, giving 93 and 89% 1

decolorization after 48 h of incubation, respectively, similar to Ali et al. (Ali et al., 2009). 2

3.4 Effect of heavy metals on decolorization performance 3

The presence of heavy metals in the media affected CR removal rate significantly. In 4

presence of 100 mg/L Pb(NO3)2 and CdCl2, 45 and 37% decolorization was observed within 48 5

h. However, when concentrations of both the salt were increased to 200 mg/L, CR removal rate 6

decreased drastically to 0.75 mg/L h and 1.57 mg/L h (Fig. 2d and 2e), resulting in only 16 and 7

30% CR decolorization, respectively. Presence of Hg was observed to have more severe impact 8

on CR decolorization rate. Fig. 3f shows at HgCl2 concentration of 100 and 200 mg/L, the 9

fungus showed significantly less decolorization rate of 1.32 and 0.37 mg/L h, giving only 13 and 10

6% removal of CR, respectively, similar to Pointing et al. (Pointing et al., 2000). 11

3.5 Biodecolorization and biodegradation analysis 12

Both light microscopy and scanning electron microscopy revealed the surface 13

morphology of the fungus after CR decolorization to be more amorphous in nature (Fig 1S, 14

supplementary data). This result is also in close agreement with the X-ray diffraction analysis. 15

The XRD analyses of the fungal mycelia before CR decolorization revealed that the value of 2θ 16

is 280

when the maximum count is observed, which is indicative of the amorphous in nature of 17

mycelia. The XRD of the fungal mycelia after the dye adsorption showed that the 2θ value 18

further decreased, indicating increase in amorphous nature as evident from the less sharper X-ray 19

diffraction peaks noted in Fig. 1S a and 1S b (supplementary data). 20

To investigate the possible mechanism of CR decolorization, HPLC and FTIR analyses 21

were carried out. HPLC elution profile showed three prominent peaks at retention time of 2.141, 22

2.609 and 3.235 min when metabolites separated from the sample obtained after decolorization, 23

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compared to peak obtained for control dye at retention time 1.861 min. The analysis has showed 1

that the peak of control dye has shifted to 2.141 min and there is a sharp decrease in the 2

concentration of the dye (Fig. 2S c and d, supplementary data). The new peaks formed indicated 3

the production of certain metabolites, confirming the degradation of CR by A. alternata CMERI 4

F6. Similar observations have also been reported in the literature (Saratale et al., 2013). In the 5

present study, generation of three peaks in HPLC chromatogram may be consistent with 6

formation of three metabolites of CR degradation described by Xu et al. (Xu et al., 2013). 7

The FTIR spectrum of the control dye (Fig. 3S, supplementary data) showed a broad peak 8

at 3340.1 cm-1

due to stretching of bonded O-H group, a peak at 1641.13 cm-1

due to N=N 9

stretching of azo group, along with other peaks at 2158.92 cm-1

and 2340.19 cm-1

. In addition, 10

peak at 1228.43 cm-1

represented primary aromatic amines with C-N vibration. The FTIR 11

spectrum of the extracted metabolites from the decolorized sample (Fig. 4S) shows peak at 12

3292.86 cm-1

and 1641.13 cm-1

. Other peaks at 2361 cm-1

and 2152 cm-1

indicate stretch due to C13

C band. As compared to the control dye, the disappearance of peak at 1228.43 cm-1

indicates 14

the possible breakage of C-N bond and degradation of CR by the fungus. Decolorization of 15

various dyes has also been reported to have occurred through both adsorption and degradation by 16

filamentous fungi (Jadhav et al., 2010). However, the toxicity of the end product of the 17

decolorization of CR has yet to be found out. 18

3.6 Thermal and chemical stability of the bioabsorbed dye 19

When the hydrated biomass was autoclaved at 121 oC at 15psi, no cell disruption, 20

desorption and dye liberation was observed visually. Moreover, on Spectrophotometric analysis 21

negligible change in the adsorption spectrum was noticed. 22

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The Thermogravimetric analysis (TG) and Differential Thermal Analysis (DTA) 1

provided information about desorption of the dye and other physical changes of the fungal 2

mycelium. Most of the weight loss of the mycelium due to pyrolysis of the fungus (after CR 3

adsorption) in inert atmosphere of Helium takes place at temperature up to 150 oC where most of 4

the moisture and volatiles are released. Beyond 150 oC there is only decomposition of 5

carbohydrates, protein and lipids and weight loss occurred mainly due to gasification (Agrawal 6

& Chakraborty, 2013). The DTA curve (Fig. 5S, supplementary data) showed that at about 80 7

oC, an exothermic effect appears resulting in the peak where the degradation of mycelium and 8

subsequent liberation of the dye has occurred completely. The strong exothermic effect signifies 9

that disintegration of the fungal mycelium and liberation of the dye requires relatively low 10

temperature in inert atmosphere. The low degradation temperature of the fungal mycelium that 11

adsorbed the dye, compared to the incineration temperature may help in removal of the dye from 12

the biomass in inert conditions after biosorption. 13

Upon treatment of dried fungal biomass with 1M HCl and 1M NaOH, the fungal 14

structure disintegrated but the dye was not released freely in the media and remained adsorbed to 15

the disintegrated fungal structures, except for 70% ethanol treatment. This can be attributed to 16

the dissolution of lipid content and dehydration of protein by ethanol which consequently 17

released the dye into the environment. However, relatively high resistance of the fungus to acid 18

or alkali treatment makes it an efficient and promising candidate for retaining adsorbed CR. 19

4. Conclusion 20

A. alternata CMERI F6 could decolorize 99.99% of 600 mg/L and 78% of 800 mg/L CR 21

within 48 h, at maximal decolorization rate of 18.01 mg/L h and 20.21 mg/L h, respectively. The 22

fungal strain proved to be an excellent candidate for removal of azo dyes within broad pH, 23

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temperature and aeration range. HPLC and FTIR study suggest that dye removal by the fungus 1

can be attributed to adsorption to biomass and degradation. The thermal and chemical stability 2

analyses disclosed that the fungus can retain the adsorbed dye despite different harsh treatments 3

like high temperature and acid-alkali treatment. 4

References 5

1. Agrawal, A., Chakraborty, S. 2013. A kinetic study of pyrolysis and combustion of microalgae 6

Chlorella vulgaris using thermo-gravimetric analysis. Bioresour. Technol., 128, 72-80. 7

2. Ali, N., Hameed, A., Siddiqui, M., Ghumro, P., Ahmed, S. 2009. Application of Aspergillus 8

niger SA1 for the enhanced bioremoval of azo dyes in simulated textile effluent. Afr. J. 9

Biotechnol., 8, 3839–3845. 10

3. Ayed, L., Mahdhi, A., Cheref, A., Bakhrouf, A. 2011. Decolorization and degradation of azo 11

dye Methyl Red by an isolated Sphingomonas paucimobilis: Biotoxicity and metabolites 12

characterization. Desalination, 274, 272-277. 13

4. Basak, B., Bhunia, B., Mukherjee, S., Dey, A. 2013. Optimization of physicochemical 14

parameters for phenol biodegradation by Candida tropicalis PHB5 using Taguchi 15

Methodology. Desalin. Water Treat., DOI:10.1080/19443994.2013.770638 16

5. Hou, B., Hu, Y., Sun, J. 2012. Performance and microbial diversity of microbial fuel cells 17

coupled with different cathode types during simultaneous azo dye decolorization and 18

electricity generation. Bioresour. Technol., 111, 105-10. 19

6. Jadhav, J.P., Kalyani, D.C., Telke, A.A., Phugare, S.S., Govindwar, S.P. 2010. Evaluation of 20

the efficacy of a bacterial consortium for the removal of color, reduction of heavy metals, 21

and toxicity from textile dye effluent. Bioresour. Technol., 101(1), 165-73. 22

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7. Pointing, S.B., Bucher, V.V.C., Vrijmoed, L.L.P. 2000. Dye decolorization by sub-tropical 1

basidiomycetous fungi and the effect of metals on decolorizing ability. World J. 2

Microbiol. Biotechnol., 16, 199-205. 3

8. Saratale, R.G., Gandhi, S.S., Purankar, M.V., Kurade, M.B., Govindwar, S.P., Oh, S.E., 4

Saratale, G.D. 2013. Decolorization and detoxification of sulfonated azo dye C.I. 5

Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS. J. Biosci. Bioeng., 6

115(6), 658-67. 7

9. Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P. 2011. Bacterial decolorization and 8

degradation of azo dyes: A review. J Taiwan Inst. Chem. Eng., 42, 138–157. 9

10. Solís, M., Solís, A., Pérez, H.I., Manjarrez, N., Flores, M. 2012. Microbial decolouration of 10

azo dyes: A review. Process Biochem., 47, 1723–1748. 11

11. Vitor, V., Corso, C.R. 2008. Decolorization of textile dye by Candida albicans isolated from 12

industrial effluents. J. Ind. Microbiol. Biotechnol., 35(11), 1353-7. 13

12. Xu, Q., Sun, J., Hu, Y.Y., Chen, J., Li, W.J. 2013. Characterization and interactions of anodic 14

isolates in microbial fuel cells explored for simultaneous electricity generation and 15

Congo red decolorization. Bioresour. Technol., 142, 101-8. 16

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Figure Legends 20

Figure 1: Effect of different CR concentrations on decolorization performance of the fungus. 21

Figure 2: Effect of temperature (a), pH (b), agitation speed (c) and different heavy metals (d, e, f) 22

on removal of CR by the fungus. 23

Highlights:

Optimum physicochemical parameters for congo red decolorization were

determined.

Effect of different heavy metals on congo red decolorization was evaluated.

Characterization of fungal morphology after dye removal was done using SEM and

XRD.

Congo red biodegradation analysis was done using HPLC and FTIR.

Evaluation of dye retention properties of the fungus by TG-DTA analysis.

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Table 1

Decolorization performance of Alternaria alternata CMERI F6 using different carbon sources.

acarbon sources added in a concentration of 0.5% w/v each.

bCongo red concentration 25 mg/L.

# Inorganic media composition adopted from Basak et al. (2013)

Mediaa % removal

b Time taken (h)

Inorganic media# (IM) 0 48

Inorganic media + Glucose 99.99 32

Inorganic media + Beef extract 50.00 40

Inorganic media + Peptone 79.80 44

Inorganic media + Yeast extract 90.00 36

Inorganic media + Sugarcane Bagasse 56.90 48