by

Arka Mukherjee, Ruchira Mitra, Dr.Debjani Dutta*

Biological decolorization of Biological decolorization of Malachite Green by a Malachite Green by a carotenoid producing carotenoid producing

bacteriumbacterium

Department of BiotechnologyNational Institute of Technology

Durgapur India

Structure :

Malachite Green (MG)

MG is a triphenyl methane dye, used extensively in textile industry.MG alone accounts for two-third of the total production of dye containing wastewater.It has been banned worldwide due to its carcinogenicity and toxicity.

Molar mass : 364.911 g/mol

Bioremediation of MGTraditional wastewater treatment methods (like coagulation, ozonation, ion-

exchange, reverse osmosis, etc. )

less effective

expensive

produces high amount of sludge

Biological processes low cost,

effectiveness produce less sludge

environmental sustainability

A wide variety of microorganisms capable of removing wide range of dyes have been reported… Examples …

Bacteria : Escherichia coli NO3, Pseudomonas luteola, Aeromonas hydrophila; fungi: Aspergillus niger, Aspergillus terricola, yeasts: Saccharomyces cerevisiae, Candidatropicalis, C. lipolytica; algae: Spirogyra species, Chlorella vulgaris

Kocuria marina DAGII Kingdom : BacteriaPhylum : Actinobacteria

Order : ActinomycetalesFamily : Micrococcaceae

Genus : KocuriaSPECIES : marina

Isolated from soil in Department of

Biotechnology, NIT-Durgapur

Gram Positive

Aerobic

Yellow coloured

Non-motile

Carotenoid producing

•Carotenoid produced by the bacterium is known as β-cryptoxanthin (β-crx).•β-CRX belongs to the class of xanthophylls.

Fig.1:-β-CRX

•Distribution - found in many vegetables and fruits, mainly papaya, mango, peaches, oranges,corn and watermelon.•Strong antioxidant - prevents free radical damage to cells and DNA.•Pro-vitamin A - Precursor of vitamin A, which is an essential nutrient needed for eyesight, growth, development and immune response.•Anticancer - Recent studies suggest that it reduces the risk of lung cancer & colon cancer.

β-cryptoxanthin (β-CRX)

*Recently it has been found that Kocuria marina DAG II is decolorizing Malachite Green…

A : Bacterial Culture

B : Media supplemented with MG and inoculated with the bacterium

C : Bacterial Culture after MG decolorization by the bacterium

A CB

•To study the effect of MG concentration on decolorization by Kocuria marina DAGII.

•To study the effect of inoculum size on MG decolorization by the bacterium.

•To analyze the obtained results by statistical methods.

Cultivation of Kocuria marina DAGII

• Glucose(7.5 g/l), maltose(10g/l), yeast extract(10 g/l), peptone(5 g/l) and NaCl(4 g/l), pH - 7.9

• Autoclaved at 15psi for 20 mins at 121°C, Inoculum – 1%, Incubated at 25°C and 150 rpm. Dye solution

1000mg/L dye solution by dissolving MG in distilled water. It was further diluted to obtain

the desired dye concentration during experiments. Decolorization experiment

• Desired concentration of dye was added from the stock solution to the freshly prepared

culture flasks.

• Sample was collected centrifuged at 10000rpm for 15 mins.

• The supernatant was collected and it was analyzed under UV-vis spectrophotometer at

617nm (λmax).

• Decolorization% was calculated by using the following formula:-

Effect of initial dye concentration on decolorization.

Effect of increasing inoculum size on decolorization.

*Effect of initial dye concentration on decolorization

Decolorization of MG was studied at various increasing concentrations of dye i.e., 10, 20, 30, 40, 50, 60 ppm.

Rate of decolorization decreased with increasing dye concentrations. 99% decolorization of MG was observed at 10, 20ppm within 2 and 7h, respectively

After 20ppm, the rate of decolorization decreased indicating reduction in decolorization with increase in dye concentration.

Only 36, 29, 21, 14% decolorization was observed at 30, 40, 50 and 60 ppm, respectively after 8h

Inoculum percentage was increased from 2 and 3% to study the effect of increased cell

concentration on MG decolorization.

98% decolorization took place at 40 and 50ppm when the inoculum was increased from 2 and

3%.

At 60ppm, 50 and 60% decolorization was achieved for 2 and 3% inoculum.

It could be inferred that decolorization could be increased by increasing cell concentration.

Two-way ANOVA was done by using Graphpad Prism 5 to determine the significant parameter

for MG decolorization.

Effect of initial dye concentration on MG decolorization was more significant (p

value=0.0076) than that of inoculum percentage (p value=0.0219).

With increasing MG concentration,the decolorization% also decreases.

The decolorization% for a particular concentration of MG increases when the amount of

inoculum was increased.

By statistical analysis,it was shown that the effect of initial dye concentration was more

significant than inoculum percentage for MG decolorization.

To study the effect of 1% inoculum on higher MG concentration such as 70ppm,80ppm.

To check the same effect by using higher inoculum concentration such as 2%,3%.

To determine the reason behind MG decolorization.

This research work was financially supported by the grant from the National Institute of Technology, Durgapur, India.

[1] Daneshvar, N., Ayazloo, M., Khataee, A.R., and Pourhassan , M., “Biological decolorization of dye solution containing Malachite Green by

microalgae Cosmarium sp.”, Bioresource Technology, 98, 6, April 2007, pp. 1176-1182.

[2] Chen, K.C, Wu, J.Y., Liou, D.J., and Hwang S. C. J., “Decolorization of the textile dyes by newly isolated bacterial strains”, Journal of

Biotechnology, 101, 1, February 2003, pp. 57-68.

[3] Daneshvar, N., Khataee, A.R., Rasoulifard, M.H., and Pourhassan, M., “Biodegradation of dye solution containing Malachite Green:

Optimization of effective parameters using Taguchi method”, Journal of Hazardous Materials, 143, 1, May 2007, pp. 214-219.

[4] Nath, J., and Ray, L., “Biosorption of Malachite green from aqueous solution by dry cells of Bacillus cereus M116 (MTCC 5521)” Journal of

Environmental Chemical Engineering, 3,1, March 2015, pp. 386-394.

[5] Shedbalkar, U., and Jadhav, J. P., “Detoxification of malachite green and textile industrial effluent by Penicillium ochrochloron”,

Biotechnology and Bioprocess Engineering, 16, February 2011, pp. 196-204.

[6] Lv, G.Y., Cheng, J.H., Chen, X.Y., Zhang, Z.F., and Fan, L. F., “Biological decolorization of malachite green by Deinococcus radiodurans

R1”, Bioresource Technology, 144, September 2013, pp. 275–280.

[7] Parshetti, G., Kalme, S., Saratale, G., and Govindwar, S., “Biodegradation of Malachite Green by Kocuria rosea MTCC 1532”, Acta Chimica

Slovenica, 53, 2006, pp. 492–498.

[8] Mitra, R., Samanta, A.K., Chaudhuri, S., and Dutta, D., “Growth kinetics and carotenoid production of a newly isolated bacterium on glucose

as a carbon source during batch fermentation”, in 5th Annual International Conference on Advances in Biotechnology (BioTech 2015), IIT

Kanpur, India, March 13-15, 2015, DOI: 10.5176/2251-2489_BioTech15.90.

 

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Wavelength (nm)

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