Isolation, characterization and optimization of dye degrading bacteria from natural source

Table of contents

Table of figures

Table of tables

List of abbreviations

Isolation, characterization and optimization of dye degrading bacteria from natural source: an overview

Abstract

1. Introduction
1.1 Objectives

2. Review of literature
2.1 Reactive dyes
2.2 Removal methods
2.3 Physical methods
2.4 By adsorption
2.5 By ion exchange
2.6 By membrane filtration
2.7 Chemical methods
2.8 Advanced oxidation process (AOP)
2.9 Electrochemical Method
2.10 Biological methods
2.11 Anaerobic treatment
2.12 Enzymatic treatments

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Soil sample collection
4.3 Quantitative analysis of microorganisms present in the collected soil
4.4 Isolation of potential dye decolourizers
4.5 Pure culture preparation
4.6 Morphological and biochemical tests
4.7 Statistical analysis

5. Results and discussion
5.1 Dye degradation studies
5.2 Optimization studies

6. Conclusions

References

ACKNOWLEDGEMENTS

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully.

We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. Joseph V.J, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the co-operation of many farmers.

Lastly, we extend our indebt thanks to patents, friends, and well wishers for their love and support.

Prem Jose Vazhacharickal*, John Joseph, Jiby John Mathew, Sajeshkumar N.K and Delmy Abraham

*Address for correspondence

Assistant Professor

Department of Biotechnology

Mar Augusthinose College

Ramapuram-686576

Kerala, India

Table of figures

Figure 1. Map of Kerala showing the soil sample collection point. Authors own work

Figure 2. Details of a) soil sample collected, b) diluted soil samples containing bacterial isolate 1, c) diluted soil samples containing bacterial isolate 2, d) and e) gram staining of bacterial isolate 1 and 2, f) various biochemical tests for identification of the bacterial isolates. Authors own images

Figure 3. Details of a) and b) various biochemical tests of bacterial isolate 2, soil sample collected, c) and d) dye degrading capacity of the bacterial isolate 1), e) and f) dye degrading capacity of the bacterial isolate 2. Authors own images

Figure 4. Dye optimization of a) bacterial isolate 1 at pH 4, b) bacterial isolate 1 at pH 6, c) bacterial isolate 1 at pH 8, d) bacterial isolate 1 at 37°C, e) bacterial isolate 1 at 33.5°C, f) bacterial isolate 1 at 40°C. Authors own images

Table of tables

Table 1. Various biochemical tests of the isolated bacterial strains (isolate 1 and 2)

Table 2. Effect of pH and temperature at different concentrations using the isolated bacterial strains (isolate 1 and 2; n=3)

List of abbreviations

illustration not visible in this excerpt

Isolation, characterization and optimization of dye degrading bacteria from natural source: an overview

Prem Jose Vazhacharickal1, John Joseph2, Jiby John Mathew1, Sajeshkumar N.K1 , Delmy Abraham2, Bilu Kurian2, and Sreelakshmi V.P2

1 Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

2 Department of Bioscience, Indira Gandhi College of Arts and Science, Nellikuzhi, Kerala, India-686691

Abstract

In this study an attempt was made to evaluate the colour degradation capabilities by collecting the contaminated soil sample from Kalady area and serial dilution was done upto 10-6. From the dilution 10-5 was taken and spread plated on Nutrient agar. From the above plate, isolated colonies was obtained which was found to be Bacillus sp and Pseudomonas sp respectively by morphological, microscopical and biochemical method. The isolated colonies was taken for degradation studies with 1% dye and 1% inoculum in Nutrient broth and OD values and colour change was noted. It was found to be Bacillus sp has more degrading capacity in yellow colour than Pseudmonas sp. The optimization studies were done with Bacillus sp having different concentration of colour (2, 4, 6) with varying pH (4,6,8) and temperature (37°C, 40°C and room temperature). The result was found to be having the concentration of colour with 4% having pH 4 and temperature 37°C.

Keywords: Azo dyes; Dye degradation; Biochemical identification.

1. Introduction

India’s dye industry produces every type of dyes and pigments. Production of dye stuff and pigments in India is close to 80,000 tones. India is the second largest exporter of dyestuffs and intermediates (developing countries) after China. The textile industry accounts for the largest consumption of dyestuffs, at nearly 80% (Carliell et al., 1995). Industrialization is vital to nation’s economy because it serves as a vehicle for development. However, there are associated problems resulting from the introduction of industrial waste products into the environment. Many of these products are problematic because of persistence (low biodegradability) and/or toxicity.

Environmental pollution has been identified as a major problem in the modern world. The increasing demand for drinkable water, and its dwindling supply, has made the treatment and reuse of industrial effluents an attractive option. One of the most important environmental pollution problems is the colour in water courses; although some of these colours are normally present and of “natural” origins (colour originates from the activity of microorganisms in ponds), a considerable proportion, especially conurbation, originates from industrial effluents are associated with the production and use of dyes.

Azo dyes, the largest chemical class of dyes with the greatest variety of colours, have been used extensively for textile, dyeing, and paper painting (Carliell et al., 1995). These dyes cannot be easily degraded, and some are toxic to higher animals. Over 7 × 105 metric tonnes of synthetic dyes are produced worldwide every year for dyeing and printing, and out of this, about 5% - 10% are discharged with wastewater. The amount of dye lost depends on the class of dye applied: it varies from 2% loss with the use of basic dyes to about 50% loss in certain reactive sulfonated dyes (Dafale et al., 2008). The presence of dyes in aqueous ecosystem diminishes photosynthesis by impeding light penetration into deeper layers thereby deteriorating water quality and lowering the gas solubility. Furthermore, the dyes and their degraded by-products may be toxic to flora and fauna (Talarposhti, 2001). Azo dyes consist of diazotized amine coupled with an amine or phenol, and contain one or more azo linkages. At least 300 different varieties of azo dyes are extensively used in the textile, paper, food, cosmetics and pharmaceutical industries. The effect of pH, temperature, type and concentration of respiration substrates, and oxygen tension on the rate of biological reduction of a variety of azo dyes have previously been investigated (Wuhrmann et al., 1980; Deng et al., 2008). Several combinations of treatment methods have been developed so far in order to effectively process cotton- textile wastewater, with decolorization among the main goals for these processes. Chemical coagulation/flocculation techniques, usually combined with activated sludge treatment, have been among the most common processing methods mainly due to the ease of their application. However, the above methods require high amounts of raw materials [coagulants] and also yield large amount of waste solids, leading to the elevation of the total treatment cost. Advanced oxidation processes (Ozonation, UV/H2O2) are based on the generation of hydroxyl radicals, which are mainly highly reactive oxidants. They are environmental friendly techniques, since no solid wastes are produced. However, they are not cost effective due to the high consumption of both energy and raw material. Active carbon adsorption and nano-filtration techniques are able to remove dyes from wastewater. Although, the main disadvantage of these methods is the production of a secondary waste stream (or waste solid) that requires further treatment or disposal.

Microorganisms can play a very significant role in decomposition and ultimate mineralization of these dyes (Razo-Flores et al., 1996). Environmental biotechnology is based on ability of microorganism (both bacterial and fungal) to decompose larger chemical compounds, which are xenobiotics. Many researchers have studied in detail and have isolated several microbial strains having potential to decolorize a large number of dyes belonging to different classes have been isolated (Levine, 1991). Biodegradation of reactive azo dyes present in textile wastewater is a complicated procedure due to versatility in structure of dyes. The factors such as temperature, dissolved oxygen, redox mediators, type of microorganisms and amount of nutrients, type and chemical structure of dye are under study. Many research reports are available, which explain successful decolorization of dyes by using purified microbial cultures. But these findings do not find much application in practical treatment system due to complexity and heterogeneity of chemical compounds present in textile wastewater. Over the Past decades, Biological decolorization has been investigated as a method to transform, degrade or mineralize azo dyes. Moreover, such decolorization and degradation is an environmentally friendly and cost competitive alternative to chemical decomposition process. Unfortunately, most azo dyes are recalcitrant to aerobic degradation by bacterial cells. However, there are few known microorganisms that have the ability to reductively cleave azo bonds under aerobic conditions.

It was reported that cell extracts or membrane – permeabilized cells reduce azo dyes more efficiently than entire cells, especially in the case of sulphonatedazo dyes (Mezohegyi, et al., 2012). The flavin reductases are located in the cytoplasm of the cells (Alaton and Balcioglu, 2001), which suggests that anaerobic reduction of azo dyes is an intracellular process and the permeation of azo dyes across the cell membrane is a rate limiting factor. On the contrary, using different bacterial cultures, other authors found similar removal rates for polymeric azo dyes and other analogue compounds with much simpler structures (Olukanni et al., 2006) an extracellular mechanism for azo dye reduction being proposed.

1.1 Objectives

The objectives of this study to isolate and characterize dye degrading bacteria and its evaluation.

2. Review of literature

2.1 Reactive dyes

A dye is described as a coloured substance with affinity to substrate applied. Dyes are soluble at some stage of the application process, whereas pigments in general retain basically their particulate or crystalline form during application. These are used to impart colour to materials of which it becomes an integral part. Aromatic ring structure coupled with a side chain is usually required for resonance and in turn imparts colour. Based on the origin and complex molecular structure, dyes can be classified into three categories: (1) Anionic: acid, direct and reactive dyes; (2) Cationic: basic dyes; and (3) Non-ionic: disperse dyes (Gong et al., 1993; Mishra and Tripathy, 1993; Fu & Viraraghavan, 2001; Greluk and Hubicki, 2010). It has been estimated that over 10,000 different textile dyes and pigments were in common use (Easton, 1995; Robinson et al., 2001). Also it is reported that there are over 100,000 commercial dyes are available with a production of over 7 × 105 metric tons per year (Zollinger, 1987; Fu and Viraraghavan, 2001) Among the various classes of dyes, reactive dyes are one of the prominent and most widely used types of azo dyes and are too difficult to eliminate. They are extensively used in different industries, including rubber, textiles, cosmetics, paper, leather, pharmaceutical and food (Aksu & Donmez, 2005; Vijayaraghavan & Yun, 2008; Wang et al., 2009). Because these dyes have favourable characteristics, such as wide colour spectrum , bright colour and colour shades, high wet fastness profiles, ease of application, brilliant colours and minimum energy consumption (Lee & Pavlostathis, 2004; Aksu, 2005; Vijayaraghavan and Yun, 2008).

The most common group reactive dyes are azo, anthraquinone, phthalocyanine (Axelsson et al., 2006) and reactive group dyes (Lin and Peng, 1994; Sanghi et al., 2006; Daneshvar et al., 2007). Most of these dyes are toxic and carcinogenic (Acuner and Dilek, 2004). Disposal of these dyes into the environment causes serious damage, like they may significantly affect the photosynthetic activity of hydrophytes by reducing light penetration (Aksu et al., 2007) and also they may be toxic to some aquatic organisms due to their breakdown products (Hao et al., 2000; He et al., 2007). Once they are released, they not only produce toxic amines by reductive cleavage of azo linkages which causes severe effects on human beings through damaging the vital organs such as brain, liver, kidneys, central nervous and reproductive systems (Aksu, 2005; Iscen et al., 2007) and light penetration (Brown and De Vito, 1993; O’mahony et al., 2002; Yesilada et al., 2003; Forgacs et al., 2004; Kalyani et al., 2008) in aquatic environment. Therefore, their removal causes a big environmental concern in industrialized countries and is subjected to many scientific researches. It is estimated that 10–20% of reactive dyes remain in wastewater during the production and nearly 50% of reactive dyes are lost through hydrolysis during the dyeing process and their removal from effluent is difficult by conventional physical/chemical as well as biological treatment (Manu and Chaudhari, 2002; Li et al., 2009; Greluk and Hubicki, 2010). Therefore, a large quantity of the dyes appears in wastewater (Heinfling et al., 1997). These dyestuffs are designed to resist biodegradation.

Synthetic reactive dyes are considered as recalcitrant xenobiotic compounds, due to the presence of an N=N bond and groups such as aromatic rings that are not easily degraded. The discharge of these coloured compounds into the environment causes considerable non-aesthetic pollution and serious health risks (Martínez Huitle and Brillas, 2009).

2.2 Removal methods

Many processes were employed to remove dye molecules from industry effluents and the treatment methods can be divided into the following categories:

2.3 Physical methods

Physical methods such as Adsorption (Chatterjee et al., 2010a; Chatterjee et al., 2010b), Ion exchange (Labanda et al., 2009) and Membrane filtration (Ahmad and Puasa, 2007) were employed in the removal of dyes. The main disadvantages of these physical methods were they simply transfer the dye molecules to another phase rather than destroying them and they were effective only when the effluent volume is small (Robinson et al., 2001).

2.4 By adsorption

Adsorption is the transfer of solute dye molecule at the interface between two immiscible phases in contact with one another. The removal of colour from dye industrial effluents by the adsorption process using granular activated carbon has emerged as a practical and economical approach.

2.5 By ion exchange

Removal of anions and cations from dye industry effluent can be carried out by Ion exchange method by passing the waste water through the beds of ion exchange resins where some undesirable cations or anions of waste water get exchanged for sodium or hydrogen ions of the resin. Greluk and Hubicki (2010) recommended the adsorption/ion exchange as an alternative method for the removal of reactive dyes. Application of commercial anion exchange resins to water contaminated with a broad range of reactive dyes were studied by Karcher et al. (2001) and reported that anion exchangers possess excellent adsorption capacity (200–1200 μmol/g) as well as efficient regeneration property for their removal and recovery. The applicability of ion exchange resin containing acrylic matrix for removing other classes of dyes were well documented by Bayramoglu et al. (2009), Dulman et al. (2009), Wawrzkiewicz and Hubicki (2009) and Barsanescu et al. (2009). Thus Acrylic anion exchangers is more advantage than styrenics by exhibiting high efficiency of anion exchange capacities and polluting less.

2.6 By membrane filtration

Reverse osmosis (RO) and electro dialysis are the important examples of membrane filtration technology. Electrolyte is important in dyeing process for exhaustion of dye. The concentration of neutral electrolyte like sodium chloride (NaCl) in the dyeing bath is in the range of 25/30 g/L for deep tone, 41.5 g/L for light tone and extended to 50 g/L in some exceptional cases. The exhaustion stage in reactive dyeing on cotton also requires sufficient quantity of salt. The contribution of reverse osmosis in removing this high salt concentration is of great. This RO reject can be reused again in the process. For reactive dyeing on cotton, the presence of electrolytes in the waste water causes an increase in the hydrolysed dye affinity making it difficult to extract. The total dissolved solids from waste water were removed by reverse osmosis. Though it is suitable for removing ions and larger species from dye bath effluents with high efficiency, it possesses some disadvantages like clogging of the membrane by dyes after long usage and high capital cost. In electro dialysis, the dissolved salts (ionic in nature) can also be removed by impressing an electrical potential across the water, resulting in the migration of cations and anions to respective electrodes via anionic and cationic permeable membranes.

To avoid membrane fouling it is essential that turbidity, suspended solids, colloids and trace organics are to be removed prior to electro dialysis.

2.7 Chemical methods

Chemical methods such as chemical oxidation (Osugi et al., 2009), electrochemical degradation (Yi et al., 2008), and ozonation (Moussavi & Mahmoudi, 2009) were employed in dye removal effectively. The treatment of synthetic dye house effluent byozonation and hydrogen peroxide in combination with Ultraviolet light was vast in literature. A variety of oxidizing agents were used to decolorize wastes by oxidation techniques effectively. Among that sodium hypochlorite decolorizes dye bath efficiently. Even though it is a low cost technique, it forms absorbable toxic organic halides. Ozone on decomposition generates oxygen and free radicals. The later combines with coloring agents of effluent, resulting in the destruction of colors. The main disadvantage of this technique is that it requires an effective sludge producing pre-treatment. Also, these chemical methods with high cost were rarely used in the actual treatment process and the disposal of sludge containing chemicals at the end of treatment requires further use of chemicals (Crini, 2006; Forgacs et al., 2004).

2.8 Advanced oxidation process (AOP)

Philippe et al. (1998); Slokar & Le Marechal (1998) were reported that the conventional water treatment technologies such as solvent extraction, activated carbon adsorption and chemical treatment process such as oxidation by ozone (O3) often produce hazardous by-products and generate large amount of solid wastes, which require costly disposal or regeneration method. Due to these reasons, Mahadwad et al. (2011) considerable attention had been focused on complete oxidation of organic compounds to harmless products such as carbon dioxide (CO2) and water (H2O) by the AOP. El-Dein et al. (2003) supported the AOP and reported that it provides a promising alternative method to treat the textile wastewater. The UV-driven AOPs use UV light with an oxidizer such as H2O2 and/or ozone to generate hydroxyl radicals (OH-) that attack organic compounds non selectively with a high reaction rate. Based on the studies from Shu et al. (1994) and Galindo & Kalt (1998) it was observed that the decolorization of textile dyes using H2O2/UV had shown it to decolorize dilute aqueous solutions (20 mg/L) of azo dyes.

2.9 Electrochemical Method

The requirement of chemicals and the temperature to carry the electro chemical reaction is less than those of other equivalent non-electrochemical treatment. It can also prevent the production of unwanted side products. But, if suspended or colloidal solids were high in concentration in the waste water, they slow down the electrochemical reaction. Therefore, those materials need to be sufficiently removed before electrochemical oxidation. Ceron et al. (2004) reported that many of the commercially used dyes are resistant to biological and physico-chemical methods (Delee et al., 1998; (Vandevivere et al., 1998; Anbia et al., 2010). Also Ceron et al. (2004) suggested that coagulation (Vandevivere et al., 1998), coagulation – electro oxidation (Xiong et al., 2001), adsorption (Morais et al., 1999), electrolysis (Davila-Jimenez et al., 2000photolysis (Ince, 1999) and ozonation are promising in terms of performance. But in terms of economic aspect, these methods have become most challenging problem. Consequently, Gutierrez et al. (2001) discussed the interest in electrochemical methods to decolourise and degrade dye molecules.

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