Biotechnology Report: Microorganism Degradation of Textile Dyes Study

This report focuses on the use of microorganisms for the biodegradation of toxic chemical textile dyes, specifically Mordant Black 17 (Calcon), found in textile industry effluents. The study details the isolation and characterization of dye-degrading bacteria from contaminated sites using biochemical and molecular methods, including 16s RNA sequencing. It further analyzes the decolourization activity of these microorganisms and characterizes the resulting metabolites through TLC, FTIR, and GC-MS analyses. The proposal outlines a plan for isolating and characterizing dye-degrading microorganisms, optimizing culture conditions, conducting decolourization assays, and identifying degradation metabolites, providing a cost-effective and environmentally friendly approach to treating industrial effluents. The budget and timeline for the experiments are also detailed, along with a list of references.

Running head: BIOTECHNOLOGY
BIOTECHNOLOGY
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Summary
This study highlights the use of microorganisms for the degradation of toxic chemical textile
dyes such as Mordant Black 17 (Calcon) that is present in the effluents of the textile
industries (Sunkar & Renugadevi, 2015). The paper illustrates the isolation of the dye
degrading bacteria from the site contaminated by the textile effluents. These are further
characterized using biochemical and molecular methods like 16s RNA sequencing method.
Additionally the paper analyses the decolourization activity of the microorganisms along with
the characterization of the metabolites that are produced after the decolourisation using
several analytical methods such as TLC analysis, FTIR analysis and GC–MS analysis.
Background
Treatment and reuse of industrial effluents is quite necessary for the provision of clean water
supply. With the increasing amount of application of chemical dyes in the textile industries,
which are toxic in nature, there is an immediate need to find ways for the degradation of these
chemical textile dyes (Chequer et al, 2013). These dyes are classified into acidic, basic,
disperse, azo, diazo, anthraquinone and metal complex based on their structure. Based on the
dyeing process, textile dyes are classified as reactive, direct, disperse, acid, basic and vat dyes
(Gupta et al., 2015). Earlier many traditional methods have been followed like chemical
degradation but with the increase on the focus of green technology, the method of
biodegradation is being implemented for the removal of the chemical dyes form the effluents
of the textile industries (Khan, Bhawana & Fulekar, 2013). Here the focus is on the use of
microorganisms for the degradation since this method is cost-effective as well as an
environmental friendly approach.

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Proposal plan
The study proposes that dye degrading microorganisms will be isolated from the sites that are
contaminated by chemical effluents from the textile industries. The organisms collected will
be further sampled using serial dilution and pour plated in nutrient agar plates. After an
incubation period of 24 hours, the colonies will be picked from these plates depending on the
proper colour, texture, and other morphological characteristics. This will be inoculated
separately in order to maintain a pure culture. For further use, glycerol stock solution of the
pure culture will be maintained at -20 C. For the 16s RNA sequencing of the culture, PCR
amplification followed by DNA sequencing will be one in order to isolate the genomic DNA
of the pure culture. The PCR product was sequenced bi-directionally using the forward,
reverse and internal primer. To identify the bacterium, the sequence data was aligned and
analysed to its closest neighbours (Karunya, Rose & Nachiyar, 2014).
To provide optimum growth conditions, the culture medium will be optimized using a variety
of mineral salts in varying compositions. An important factor is pH. Optimum pH conditions
needs to be provided for promoting effective growth of the organism. The carbon source will
also be varied such as glucose, which may be replaced by other sources such as lactose,
sucrose, fructose, maltose and dextrose, while optimising the culture medium. In order to
carry out the decolourization experiment, a stock solution of the dye will be needed to be
made of a particular concentration (Selvakumar, Manivasagan & Chinnappan, 2013).
The degradation experiments will be conducted by adding the culture in specific amounts
such as 100 mg l-1 into the culture medium and will be incubated overnight at 32 C at 150
rpm. Butanol extraction method will be carried out next for determination of the degradation.
The amount of decolourisation of the dye due to degradation will be measured by the change
in absorbance of culture supernatants at the maximum absorption wavelength (λmax) of 520
nm using a spectrophotometer. The rate of decolourization can will be calculated by

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considering the difference between the initial and the final absorption values of the
supernatant at λmax of the dye. This can be followed by a protein content and analysis and
the glucose utilization analysis with the help of Lowry method and DNS method respectively.
A triplicate set of experiments will be conducted in each case (Karunya, Rose & Nachiyar,
2014).
After the degradation assay, experiments will be conducted in order to identify the
metabolites that are formed after degradation. Characterizations include the TLC analysis,
FTIR analysis and GC–MS analysis. Thin layer chromatography (TLC) analysis for the
breakdown products will performed on fluorescent silica plates. Identification of
naphthoquinone is done by using the solvent system of isopropanol: acetic acid: water in the
ratio of 19:9:1. The compound will be identified by comparing the Rf values with that of
standard. Fourier Transform Infra-Red (FTIR) spectroscopic analysis of the spot from TLC
plate, (eluted using ethyl acetate) was carried out in a FTIR spectrophotometer.
Centrifugation will be carried out of the culture medium containing the degradation products
after the incubation period and the supernatant will be extracted thrice with equal volume of
ethyl acetate, dried over anhydrous Na2SO4. Next the solvent will be made to evaporate in a
rotary evaporator. Gas chromatography–mass spectroscopy (GC–MS) analysis of the ethyl
acetate extract will be performed by using GC–MS spectrometer. The column used was VF-5
ms, 30 m × 0.250 mm diameter with the film thickness of 0.25 m and the column oven was
programmed between 70 and 300 ◦C at the rate of 10 ◦C per minute with the injection
temperature of 240 ◦C. Mass spectra were recorded under scan mode in the range of 40–1000
m/z. Compounds were identified using WILEY8.LIB (Sunkar & Renugadevi, 2015).

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Budget
Experiments Budget in Canadian dollar (CAD)
Isolation and characterization of the
microorganism
767.40
Optimization of the culture medium 575.55
Decolourisation assay 959.25
Identification of the metabolites 1151.10
Total 3453.6
Timeline
Experiments Time required
Isolation and characterization of the
microorganism
2-3 months
Optimization of the culture medium 1 month
Decolourisation assay 2-3 weeks
Identification of the metabolites 1 – 2 months

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References
Chequer, F. M. D., de Oliveira, G. A. R., Ferraz, E. R. A., Cardoso, J. C., Zanoni, M. V. B.,
& de Oliveira, D. P. (2013). Textile dyes: dyeing process and environmental impact.
In Eco-friendly textile dyeing and finishing. InTech.
Gupta, V. K., Khamparia, S., Tyagi, I., Jaspal, D., & Malviya, A. (2015). Decolorization of
mixture of dyes: a critical review. Global Journal of Environmental Science and
Management, 1(1), 71-94.
Hassan, M. M., Alam, M. Z., & Anwar, M. N. (2013). Biodegradation of textile azo dyes by
bacteria isolated from dyeing industry effluent. Int Res J Biol Sci, 2(8), 27-31.
Karunya, A., Rose, C., & Nachiyar, C. V. (2014). Biodegradation of the textile dye Mordant
Black 17 (Calcon) by Moraxella osloensis isolated from textile effluent-contaminated
site. World Journal of Microbiology and Biotechnology, 30(3), 915-924.
Khan, R., Bhawana, P., & Fulekar, M. H. (2013). Microbial decolorization and degradation
of synthetic dyes: a review. Reviews in Environmental Science and
Bio/Technology, 12(1), 75-97.
Selvakumar, S., Manivasagan, R., & Chinnappan, K. (2013). Biodegradation and
decolourization of textile dye wastewater using Ganoderma lucidum. 3 Biotech, 3(1),
71-79.
Sriram, N., Reetha, D., & Saranraj, P. (2013). Biological degradation of Reactive dyes by
using bacteria isolated from dye effluent contaminated soil. Middle–East Journal of
Scientific Research, 17(12), 1695-1700.
Sunkar, S., & Renugadevi, K. (2015). Citrobacter freundii mediated degradation of textile
dye Mordant Black 17. Journal of Water Process Engineering, 8, 28-34.