Bacteriological Removal of Azo Dyes: An Eco-Friendly Approach




'Department of Microbiology and Biotechnology, NMSS Vellaichamy Nadar College, Madurai-625019, Tamil Nadu, India

  • -Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai-625021, Tamil Nadu, India
  • 1Department of Microbiology, Allagappa University, Karaikudi-630003, Tamil Nadu, India


Colors and dyes are commonly used in textile, paper, food, cosmetics, and pharmaceutical industries. There are above 1,00,000 different human- made synthetic dyes available on the market, and worldwide its production has around 7,00,000 tons/year (Hao et al., 2000). Most of the synthetic dyes are lethal to living organisms due to their toxic and carcinogenic properties. Most of the commonly used dyes in textile industries belong to a class of compounds called azo dyes, having the functional group R-N=N-R’ (where R and R’ can be either aiyl or alkyl). Wastewater from textile industries carries 10% of the dyestuffs, which has been a significant cause of environmental pollution and has been released into different water bodies. Azo dyes in the effluent give a strong color, high pH, higher chemical oxygen demand (COD), and lower dissolved oxygen (DO). The removal of dyes from industrial wastewaters could be very important due to their toxicity and carcinogenicity. The structural complexity of azo dyes makes effluent treatment difficult by conventional physicochemical methods due to then high cost and low effectiveness (Latif, 2010). Among the various effluent treatment practices, the biological method of remediation has significantly removed the dyes. Both fungal and bacterial bioremediation process is effective, which removes the dyes by either anaerobic or aerobic metabolism. Microbial consortia become the best alternative for the single-microbial treatment process. This chapter describes a brief review on methods of azo dye bioremediation.

8.2 DYES

Dye is a natural or synthetic colored substance that is used to impart color the substrate to which it binds and becomes an integral part. It is composed of chromophore and auxochromes groups. Chromophore group (-N=N, -C=0, -NO,, CH4, and quinoid group) is responsible for the color of dye while auxochromes group (-COOH, -NH,, -SO,H and -OH) intensify the color of the chromophore (Sapna Kochher and Sandeep Kumar, 2012). Dye is used for the coloring of paper, cotton, polyester, nylon, silk, leather, plastics, and hair. Pigments are also used for coloring the substances. Both dyes and pigments can absorb a particular wavelength of light. Most of the pigments are insoluble in water, so they do not have an affinity to the substrate. Dyes must have the characters such as (1) color the substrate, (2) soluble in water/solvents, (3) ability to absorb and retained by fiber (or) chemically combined with the substrate, and (4) ability to withstand washing or diy cleaning and resist to exposure to light.


Each class of dye has a very unique structure, chemistry, and bonding. For example, some can make strong bonds when it reacts chemically with the substrates, but others can be held by physical forces. Dyes are classified in numerous ways. Based on its origin, dyes are classified into (i) natural and (ii) synthetic dyes. Natural dyes are mainly obtained from plant resources such as roots, bark, leaves, berries, fruits or flower, wood, algae, and lichens. At the Neolithic period, people used these natural dye materials for coloring the textiles. Human inspired to search alternative coloring materials due to the scarcity and the limited color availabilities.

Human-made synthetic dyes were discovered in the late 19th century, which are prepared from petroleum by-products and earth minerals.

Besides, dyes are broadly classified based on their chemical structure particularly the presence of chromophore group and method of application. According to their chromophore groups, dyes are classified into six groups, such as (1) Nitro and Nitroso, (2) Azo, (3) Triaryl methane, (4) Anthraquinone, (5) Indigo, and (6) Sulfur. According to their application dyes are classified into (1) Acid, (2) Basic, (3) Direct, (4) Reactive, (5) Disperse, (6) Vat, (7) Mordant, and (8) Sulfur (Corbmann, 1983; Hao et al., 2000). Among the various dyestuffs, azo compound is effectively used for dyeing the textile products like fibers and fabrics.

8.2.2 AZO DYES

Azo dye is a class of synthetic organic dyes contains the functional group R-N=N-R,’ R and R’ can be either alkyl or aiyl group (Figure 8.1). The term azo comes from ‘azote’ (Latin name meaning nitrogen). The azo group is linked with naphthalene or benzene groups that may contain different functional groups such as methyl (-CH}), nitro (-NO,), chloro (-C1), hydroxyl (-OH), amino (-NH,), S03H, NR, and carboxyl (-COOH) leads to formation of different types of azo dyes (Zollinger, 1991). Azobenzene (Figure 8.2) is the chromophore group of azo dyes, the intensity and color of the molecule can be altered by changing the auxochromes group (Figure 8.2). The polar auxochromes make the dye either water-soluble or insoluble, and dye binds with fabric by forming a chemical bond.

Structure of azobenzene

FIGURE 8.1 Structure of azobenzene.

Based on the number of azo groups, azo dyes are classified into mono azo (examples, orange-II, acid orange-12, aniline yellow), diazo (examples, oil red, congo red, direct blue-1, sudanblack-B), triazole (example, direct blue-71), tetra-azo, and polyazole dyes (Figure 8.3).

Auxochrome group (green color) influences the color of dye

FIGURE 8.2 Auxochrome group (green color) influences the color of dye.

Azo dyes absorb particular wavelength of light in the visible spectrum because of their chemical structure (Chang et al., 2000), give bright, high-intensity colors and have fair to good fastness properties. Aryl azo compound has vivid color includes orange, yellow, and red as a consequence of electron delocalization. Azo dyes are used in dyeing of cloth, natural, and synthetic materials, ink, food, medicine, cosmetics, and paints (Telke et al., 2008). Azo dyes account for approximately 60-70% of all dyes used in food and textile industries because of economic feasibility, ease of the synthesis, stability, and the availability of different colors of azo dyes compared to other types of dyes (Chang et al., 2004).


In the market, there are several thousand numbers of azo dyes are used for various purposes, of these 500, and more azo dyes contain carcinogenic aromatic amines such as benzidine, 4-aminodiphenyl, 3,3’-dichlo- robenzidine and 2-naphthylamine in their chemical formulations. It has been estimated that textile industries consumed about two-third of the total dye produced annually (Stolz, 2001). The textile industry effluents are complex, having a variety of synthetic dyes and other products, such as acids, bases, dispersants, salts, detergents, humectants, oxidants, and so on. Textile dye in water can absorb and reflect the sunlight entering in aquatic ecosystem thereby reduce photosynthetic activity of algae and oxygenation ability of water. It can also affect germination rates and biomass of different plant species that provide habitat for living organisms and organic matter essential for soil fertility (Ghodake et al., 2009a).

Chemical structure of azo dyes

FIGURE 8.3 Chemical structure of azo dyes.

Toxic compounds from textile effluent enter into aquatic life pass via the food chain and finally reach the human being and cause physiological disorders such as sporadic fever, renal damage, hypertension, and cramps. Azo dyes and its degradation products are toxic, carcinogenic, and mutagenic. It causes human bladder cancer, hepatocarcinoma, and splenic sarcoma and induces chromosomal aberrations in experimental animal cells (Puvaneswari et al., 2006). Genotoxicity potential of the azo dye is based on its binding capability on double-stranded DNA, while mutagenicity has been associated with the formation of free radicals (Wang et al., 2012).

Synthetic azo compounds and its derivatives 3,3-dichlorobenzidine,

2-amino-4-nitrotoluene and phthalocyanine have been used in tattoo colorants (Baumler et al., 2000). Normal microflora of human skin does not produce the effect by their presence under nonnal circumstances. When azo dye exposed on the skin by tattoo ink, the topical use of skin colorants, and wearing colored textiles, the nonnal microflora of the skin react with the dye and produces potentially toxic aromatic amines (Levine, 1991; Chung et al., 1992; Platzek et al., 1999). If azo dyes entering into the human body, they are metabolized by azoreductase enzyme present in the gastrointestinal tract and liver that produces aromatic amines, and it create adverse effects (Platzek et al., 1999). For example, anaerobic metabolism of l-Amino-2-Naphthol based azo dyes (Sudan dyes) by human intestinal microflora leads to the fonnation of aromatic amines, l-Amino-2-iiaphthol, 1,4-phenylene-diamine, and 2,5-diaminotoluene, which cause harmful effects (Figure 8.4) (Xu et al., 2008).

When effluent containing azo dyes are entering into the water bodies, environmental microorganisms can readily reduce the dyes to produce carcinogenic amines under anaerobic conditions. The following three mechanisms convert azo dyes to the carcinogenic aromatic amine, such as (1) Aromatic amines are produced by reduction and cleavage of the azo bonds, (2) Oxidation of azo dye with a structure containing free aromatic amine group without reduction of azo linkage, and (3) Direct oxidation of azo linkage leads to activation of azo dyes to highly reactive electrophilic diazonium salts (Brown and Devito, 1993). Therefore, there is a need to remove/treat the textile effluents before releasing into the environments. Hence the government legislature is imposing textile industries to treat the effluent before discharging to the environment. Currently, several physical, chemical, and biological methods have been used to treat the effluents discharged from the dye industries (Figure 8.5).

Anaerobic metabolism of l-amino-2-naphthol-based azo dyes (Sudan dyes)

FIGURE 8.4 Anaerobic metabolism of l-amino-2-naphthol-based azo dyes (Sudan dyes).



Many different physical methods including membrane filtration, reverse osmosis, electrolysis, nanofiltration, and various absorption techniques are used to remove the dyes. Among the different methods, coagulation, and flocculation of dyes using Fe2+, Al2+, and Mg2+ salts is effective, but this method mainly remove disperse and sulfur dyes, and it showed very low capacity for other dyes (Khamb, 2012). Adsorption methods have been considered to the superior of other physical methods because of their efficacy for the removal of a wide variety of dyes, the simplicity of design, flexibility, and intensive to toxic pollutants. Adsorptions of dye rely on many physicochemical factors such as dye-adsorbents interaction, the surface area of adsorbent, temperature, contact time and particle size (Guptha and Suhas, 2009). Activated carbon is a very effective amongst all adsorbent, but it is not used due to its higher cost (Robinson et al., 2001). Peat fly ash, bentonite clay, polymeric resins, com/maize cobs, maize, and wheat bran and straw are also used for the removal of the color of dye in wastewater (Ramakrishna and Viraraghavan, 1997; Kobya, 2004; Sulak, 2007; Khamb, 2012). In addition, filtration methods such as ultrafiltration and nanofiltration have been used to clarify and concentrate dyes from the wastewater. The subsequent process of filtration after coagulation- flocculation revealed good color removal. However, membranes used for filtration have some limitations including high cost, periodic replacement due to clogging of pores and the production of secondary waste (Robinson et al., 2001; Dos Santos et al., 2007) (Figure 8.6).

Textile effluent treatment techniques

FIGURE 8.5 Textile effluent treatment techniques.

Methyl red degradation by Staphylococcus aureus

FIGURE 8.6 Methyl red degradation by Staphylococcus aureus.


Chemical oxidation is the common method used for the removal of colors from the effluents. These techniques use different oxidizing agents, such as hydrogen peroxide (H,0,), ozone (03), and permanganate (Mn04) to destmct or decomposition of dye molecules. In the presence of oxidizing agent, transformation of a group or modification of the chemical composition of a compound could takes place, which makes the dye susceptible to degradation (Metcalf, 2003). In this view, Ozone has been used to remove colors effectively from textile wastewater containing several azo dyes (Alaton et al., 2002). But, ozone shows ineffectiveness on removal of disperse dyes and also incompetent to remove COD because of its short half-life period (20 minutes) and the higher cost practical application of ozonation method has been restricted (Anjaneyulu et al., 2005). However, advanced oxidation processes have been successfully used for the removal of recalcitrant dyes present in the textile effluents. The effectiveness of this process is based on the formation of veiy powerful oxidation molecule like hydroxyl radical (OH-) that destmcts the hazardous dye molecules. Most commonly, the Fenton reaction method has been used to remove both soluble as well as insoluble dyes. Although it is a cheap and efficient color removal method, which generates high sludge that limits the usage of this process (Robinson et al., 2001). Electrochemical oxidation is a new technique to effectively destroy organic pollutant and produce nontoxic end products. However, little or no consumption of chemicals and the high cost of the electricity requirement limit its application. Physical/chemical methods of dye removal have several drawbacks, such as (i) cost, (ii) unable to remove xenobiotic azo dyes and their degradative products, (iii) chemical and photolytic stability of dyes and (iv) generation of a large amount of sludge (Anjaneyulu et al., 2005).


In the present scenario, biological decolorization methods are gaining importance to remove toxic waste from textile effluents. In this approach, microorganisms are widely studied because they acclimatize to a wide range of toxic compounds and develop new strains as expected, which can transform toxic compounds into a harmless form. Commonly, microbial decolorization of dyes may take place in two ways: (i) degradation of the chemical structure of dyes by microbial cells, and (ii) adsorption of dyes on living and dead cell biomass. Microbial degradation has been attracted in several ways because of (i) environmentally friendly, (ii) economically feasible, (iii) complete mineralization or nontoxic end product, (iv) less sludge production, and (v) less water consumption. However, the imperfection of bioremediation process limits its use at the field level (Bafana et al., 2011).

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