BIOREMEDIATION AND BIODEGRADATIONLecture Week 4Biodegradation of Azo Dyes

By Puan Norhayati Abdullah

Biodegradation of Azo Dyes

AbstractAn aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida(MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (910.5) and salinity (0.93.68 g/l) at ambient temperature (28 2 C). The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied. The presence of a H2O2 independent oxidase laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2. The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products. The enzymes present in the crude supernatant was found to be reusable for the dye degradation.

History of dyesSince prehistoric period, people have been using colourants to dye furs, textiles and other objects. These natural substances were mainly obtained from plants, vegetables or animals. Cave-drawing such as those in the Chauvet Cave in France and Altamira Cave in Spain showed the evidences of dyes application.

Yarn drying after being dyed in early American tradition, at Conner Prairie living history museum in Fishers, Indiana.

Some 35,000 years ago

Horses

Fighting Rhinos

Archaeological evidence shows that, particularly in India and the Middle East, dyeing has been carried out for over 5000 years. The dyes were obtained from animal, vegetable or mineral origin, with no or very little processing.

By far the greatest source of dyes has been from the plant kingdom, notably roots, berries, bark, leaves and wood, but only a few have ever been used on a commercial scale.

In 1856, synthetic dyes manufacturing started. Mauveine, also known as aniline purple and Perkins mauve, was the first synthetic dye produced. It was originally manufactured under the name Tyrian Purple.

Tyrian purple was discovered by an 18-year old William Henry Perkin, a talented English chemist, who worked at Royal College of Chemistry in London. Perkin patented his discovery and commercialized it.

Later on, a lot of synthetic new dyes started to appear in the market. Until now, almost all of the dyes appear in the market are synthetic dyes.

Perkins letter with an Example of Tyrian Purple dye on silk

Introduction to DyeA dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and may require a mordant to improve the fastness of the dye on the fiber. Chemical affinity is defined as electronic properties by which dissimilar chemical species are capable of forming chemical compounds. It can also refer to the tendency of an atom or compound to combine by chemical reaction with atoms or compounds of unlike composition

Both dyes and pigments appear to be colored because they absorb some wavelengths of light preferentially. In contrast with a dye, a pigment generally is insoluble, and has no affinity for the substrate.

Classification of dyes Dyes are characterized by their ability to absorb visible light (400-700 nm). Solids, liquids or gases can absorb light completely, partially or not at all. The part that is not absorbed can be reflected at the surface of solids, liquids or transmitted through gases. The reflected light reaches the retina in human eye and goes through a series of photochemical reaction.

Dyes contain chromophores and auxochromes Chromophores refer to functional groups which are unsaturated and they cause a compound to become coloured. Examples of chromophores are N=N-, -C=C-, -C=N-and -C=O Auxochromes are saturated functional groups and the presence of these groups in a compound greatly increases the color-yielding power of the compound. Such as -NH3, -COOH, -OH and NHR.

The huge number of commercial dyes is classified in terms of structure, colour and application according to Colour Index (C. I).

Society of Dyers and Colourists and the American Association of Textile Chemists and Colourists are societies which are responsible to revise Colour Index every three months.

Acid dyes are anionic compounds that are used for dyeing nitrogen-containing fibers such as silk (contains about 250 mmol/kg of ammonium groups), wool (contains about 850 mmol/kg of ammonium groups) and synthetic polyamides (contains about 30-50 mmol/kg of amino groups).

The name of acid dyes refers to the dyeing process, which means these dyes are applied to wool, silk and polyamides using from neutral to acidic solutions (pH 2-6). Most synthetic food colours falls in this category.

Direct dyes are compounds that have a high affinity for cellulose fibers.

Direct or substantive dyeing is normally carried out in a neutral or slightly alkaline dyebath, at or near boiling point, with the addition of either sodium chloride (NaCl) or sodium sulfate (Na2SO4). Direct dyes are used on cotton, paper, leather, wool, silk and nylon. They are also used as pH indicators and as biological stains.

During the dyeing process, van der Waals forces are involved - cause the sorption of dyes to cellulose fibers.

Reactive dyes refer to the coloured compounds with reactive group that form a covalent bonds with OH-, NH- or SH-groups in fibers. The reactive group is usually with two labile chloride or fluoride.

Metal complex dyes are dye compounds with metal atom. Usually metal atom that involved is chromium, copper or cobalt.

They are normally 1:1 complexes (1 metal atom with 1 dye molecule) or 1:2 complexes (1 metal atom with 2 dye molecules).

The addition of metal atom improves the fastness properties of the dye compounds.

Basic dye is a class of dyes that contain basic groups in their chemical structure. They are used for dyeing acid-containing fibers.

Basic dyes are mainly used in paper industry, printing industry, cosmetics industry and food industry.

Mordant dyes are dyes that naturally have no affinity to the fibers. Thus, mordant, a chemical compound, is needed to bind with fibers during the dyeing process.

Most of these dyes contain sulphonic acid groups and metal atom such as chromium, cobalt or nickel.

Dyes attachment to the fibers depends on the formation of metal-fiber bonds, and thus increases the strength of fastness properties.

Mordant dyes are used for wool, silk, nylon and leather.

Most of the disperse dyes are insoluble in water. They can be applied to cellulose acetate fibers and polyester fibers. The dyeing process for disperse dyes needs the help of chemical softeners or high temperature (>120oC) and thus allows dyes to penetrate the fibers.

Sulfur dyes are dyes that have S-containing and water-insoluble compounds.

They are produced by treating aromatics, phenol and amino phenols with sodium polysulfide or sulfur or both.

These dyes are mainly used for cellulose fibers (Abrahart, 1977).

Fixation of Dyes On Its Fiber

Azo DyesThere are more than 100,000 commercially available dyes whilst over 7 x 105 metric tons of dyestuffs are produced annually. Amongst the synthetic dyes, azo dyes are the largest group and it is estimated more than half of the annually produced amounts of dyes (for the year 1994 worldwide azo dyes production as 1 million tons) are azo dyes.

Azo dyes are basically characterized by the presence of one or more azo groups (-N = N-).

They are usually complex aromatic compounds which are chemically stable and more difficult to biodegrade in nature.

Hence, during the synthesis of azo dyes and dyeing process in industries, dyes that are lost to the industries wastewater remain recalcitrant.

Consequently affect photosynthetic activity in aquatic life due to the reduced light penetration.

Aryl azo compounds have vivid colours, especially reds, oranges, and yellows. Therefore, they are used as dyes, azo dyes for example Disperse Orange 1.

Some azo compounds, e.g. methyl orange, are used as acid-base indicators due to the different colours of their acid and salt forms. The development of azo dyes was an important step in the development of the chemical industry.

Disperse Orange 1 (4-anilino-4'-nitroazobenzene) is a dye of the azobenzene class. Disperse Orange 1 contains approximately 25% dye by weight, with the remaining mass consisting of NaCl and other salts.

Several of the azo dyes are mutagenic and carcinogenic including 4-phenylazoaniline and N-methyl and N,N-dimetyhl-4-phenylazoaniline (Sparado, 1992).

Their chemical structures are based on azobenzene and the azo naphthol derivatives.

e.g. Amaranth = Acid Red 27 = FD&C Red 2

Hazard of Azo DyesAzo dyes can be reduced by azoreductases in liver cells, skin surface microflora, kidney cells and intestinal bacteria and thus aromatic amines (aryl amines) are released. Walker (1970) pointed out that the metabolism of azo dyes in mammal (dog) occurred when the result showed the product of azo dye (sulphanilic acid) was found in the urine of dogs after they consumed Orange I.

A study carried out by Hildenbrand et al. (1999) on reduction of azo dyes in cell cultures showed that the addition of a dye, Resacor Blue 2F, to kidney cells and liver cells produced a carcinogenic aromatic amine 3,3-Dimethoxybenzidine.

In an international conference held in Wurzburg, Germany in October 1992, an incident was reported where 15 workers were working in distilling 2-naphthylamine plant. Long term of exposure to 2-naphthylamine caused all of these 15 workers to develop bladder cancer.

Over the years epidemiological studies have shown the evidence that long-term occupational exposure to certain aromatic amines such as benzidine, 4-aminobiphenyl and 2-naphthylamine, that are used in dye industries increases the risk of developing cancer.

Dye-degrading microorganismsThe efforts of isolating dye-degrading bacteria started in the 1970s Bacillus subtilis (1977) Aeromonas hydrophila (1978) Bacillus cereus (1980)

Dye-degrading bacterial cultures

Azo Dyes Removal Techniques A wide range of dye removal techniques have been studied and developed to remove dyes from the wastewater before discharging to the environment. These techniques involved adsorption, coagulation-flocculation, oxidation, membrane filtration and microbial degradation.

Physical and/or chemical methods Advantages Disadvantages Adsorption Good removal of a Absorbent requires regeneration wide variety of dyes or disposal

Membrane Removes all dye types Concentrated sludge technologies production Coagulation/ Economically feasible High sludge production flocculation

Oxidation Rapid process High energy costsTable: Current physical and chemical technologies for colour removal

Biological treatment is advantageous over the physical-chemical techniques as it is inexpensive and more environmental friendly.

However, conventional biological treatment techniques involving the use of activated sludge proved to be ineffective in terms of removing colour in the dye-containing wastewater.

Thus, since the 1990s, numerous of research papers have been published on combined anaerobic-aerobic treatment of dye-containing wastewater.

The anaerobic-aerobic treatment studies showed that a generally high colour removal can be achieved and some of the studies showed the evidence of mineralization of the dye compounds.

Biodegradation of azo dye occurs in two stages.first stage --reduction of azo linkage resulting in the formation of aromatic aminessecond stage --degradation of aromatic amines

First stage: reduction of azo bond

Second stage: degradation of aromatic amines

Degradation Potential Aerobic ConditionAzo dyes seldom degrade under aerobic conditions. The only aerobic degradation has been attributed to the fungus Phanerochaete chrysosporium and the action of Pseudomonas strains adapted to growth on carboxylated azo dyes (Pasti-Grigsby, 1992).

Phanerochaete chrysosporium is reported to degrade three azo dyes, congo red, orange II and tropaeolin O (Cripps, 1990).

Degradation Potential Anaerobic ConditionMany bacteria can reduce azo compounds to amines through cometabolism.

These microorganisms include Clostridium spp., Bacillus subtilis, Streptococcus spp., etc.

These aromatic amines however, can be highly toxic and carcinogenic.

Combined MetabolismIt was first reported that mineralization of azo dye was carried out through a combined mode of anaerobic-aerobic process. Example:Sulfonated azo dye mordant yellow 3 (MY 3) has been mineralized by this dual modeConsists of facultative organisms since the alternating presence and absence of oxygen cause no toxic effects.

Reduction of azo linkage under anaerobic conditions formed 6-aminonaphthalene-2-sulfonic acid (6A2NS).

Two organisms under aerobic metabolism use 6A2NS as source for carbon and energy.

The addition of 1-butanol caused a threefold enhancement in the degradation of MY3.