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Microbial conversion of selected azo dyes and their breakdown products
N. Yemashova* and S. Kalyuzhnyi*
*Department of Enzymology, Chemistry Faculty, Moscow State University, 119992 Moscow, Russia (E-mail: nat_yem@mail.ru; svk@enz.chem.msu.ru) Abstract Four selected azo dyes (acid orange 6, acid orange 7, methyl orange and methyl red) were completely decolourised in the presence of anaerobic granular sludge, while only methyl red was degraded in aerobic conditions using a conventional activated sludge. Additional experiments with culture broth devoid of cells showed that anaerobic decolourisation of azo dyes was performed by extracellular reducing agents produced by anaerobic bacteria. This was further confirmed by abiotic experiments with sulphide and NADH. The presence of redox mediators such as riboflavin led to dramatic acceleration of the anaerobic biodecolourisation process. The azo dye reduction products were found to be sulphanilic acid and 4-aminoresorcinol for acid orange 6; sulphanilic acid and 1-amino-2-naphthol for acid orange 7; N,N-dimethyl-1,4-phenylenediamine and sulphanilic acid for methyl orange; and N,N-dimethyl-1, 4-phenylenediamine and anthranilic acid for methyl red. Anaerobic toxicity assays showed that the azo dyes were more toxic than their breakdown products (aromatic amines), except 1-amino-2-naphthol. In the presence of activated sludge, only anthranilic acid was completely mineralised while sulphanilic acid was persistent. 4-aminoresorcinol, 1-amino-2-naphthol and N,N-dimethyl-1,4-phenylenediamine underwent autooxidation in aerobic conditions yielding coloured polymeric products. On the contrary, in the presence of granular methanogenic sludge, 4-aminoresorcinol, 1-amino-2-naphthol and anthranilic acid were quantitatively methanised, sulphanilic acid was partially (70%) mineralised while N,N-dimethyl-1,4phenylenediamine was only demethylated producing 1,4-phenylenediamine as an end product. Keywords Aerobic; anaerobic; azo dye

Water Science & Technology Vol 53 No 11 pp 163­171 Q IWA Publishing 2006

Introduction

Azo dyes ­ aromatics substituted with azo groups (-N¼ N-) ­ represent a major class of all colourants used worldwide. The presence of such compounds in industrial wastewater may create serious environmental problems (Hildenbrand et al., 1999). Biological methods of organic pollution removal are commonly considered to be the most economically effective and environmentally sound. In spite of the fact that recalcitrance and toxicity of azo dyes may make biological treatment (especially aerobic ones) difficult (Razo-Flores et al., 1997), a number of researches demonstrated azo dyes' reduction to colourless aromatic amines by anaerobic microbial consortia (Kalyuzhnyi and Sklyar, 2000; O'Neill et al., 2000; Rajaguru et al., 2000; Tan, 2001; van der Zee et al., 2001; Yemashova et al., 2004). Meantime, a wide range of azo dye structures decolourised in various anaerobic environments allows the assumption that the decomposition of azo dyes by splitting their azo bonds is a common property of any anaerobic sludge. At the present time, there is no generic opinion concerning the mechanism of azo dye decolourisation under anaerobic conditions. Generally speaking, the reduction of azo dye may imply different mechanisms, such as enzymatic, mediated, pure chemical, intracellular and extracellular (Stolz, 2001; van der Zee et al., 2001, 2003). For example, it was demonstrated that ubiquitous sources of electrons, such as reduced forms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), were able to reduce azo dyes in the absence of any enzymes (Nam and Renganathan, 2000). Another extracellular reducing
doi: 10.2166/wst.2006.349
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agent ­ sulphide ­ produced via respiration by sulphate reducing bacteria also chemically decolourises azo dyes (Yoo, 2002; Yemashova et al., 2004). Azo dye reduction was also greatly accelerated by the addition of redox mediators such as anthraquinone-sulphonate (dos Santos et al., 2004). On the other hand, aromatic amines released from azo dye decomposition are known, or suspected, to be carcinogens for humans (Hildenbrand et al., 1999). Meanwhile, aromatic amines can be easily biodegraded aerobically through hydroxylation and ring opening of the aromatic moiety. Consequently, it is suggested to combine the anaerobic cleavage of the azo dyes with the aerobic biodegradation for the amines formed (Kalyuzhnyi and Sklyar, 2000; O'Neill et al., 2000; Rajaguru et al., 2000; Tan, 2001). However, such an approach has serious drawbacks. Many of the aminoaromatics formed during the anaerobic decolourisation of azo dyes (especially with hydroxyl groups) are unstable under aerobic conditions and are extremely prone to autooxidation yielding recalcitrant polymeric products (Stolz, 2001). To avoid this, special attention should be given to the possibility of degradation of such compounds by anaerobic microorganisms. Some researches showed that aromatic amines substituted with hydroxyl or carboxyl groups could be degraded under anaerobic conditions (Battersby and Wilson, 1989; Razo-Flores et al., 1997; Kalyuzhnyi et al., 2000). The objective of this paper was to obtain a deeper insight into mechanisms of anaerobic decolourisation of four selected azo dyes (acid orange 6, acid orange 7, methyl orange and methyl red), as well as on anaerobic biodegradation of their breakdown products (aromatic amines) and compare the results obtained with the corresponding aerobic biodegradation data.
Materials and methods
Biomass and basal medium

N. Yemashova and S. Kalyuzhnyi
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Methanogenic granular sludge (16 g VSS/L, aceticlastic activity ­ 0.19 g CH4-COD/g/ VSS/L) from the EGSB-reactor treating brewery wastewater (Efes-Moscow) and activated sludge (6 g VSS/L) from Kur'yanovskaya municipal aeration station (Moscow) were used for experiments. The basal medium in all batch experiments contained (mg L21): NaHCO3 (5,000), NH4Cl (280), CaCl2·2H2O (10), K2HPO4 (250), MgSO4·7H2O (100), yeast extract (100), H3BO3 (0.05), FeCl3·4H2O (2), ZnCl2 (0.05), MnCl2·4H2O (0.05), CuCl2·2H2O (0.03), (NH4)2SeO3·5H2O (0.05), AlCl3·6H2O (2), NiCl2·6H2O (0.05), Na2SeO3·5H2O (0.1), 36% HCl to adjust the pH value to 7.
Assays

The toxicity of the azo dyes at concentrations of 150 ­ 1,200 mg/L and aromatic amines at concentrations of 100 ­ 300 mg/L was determined using aceticlastic methanogenic activity assay (Razo-Flores et al., 1997). These assays were performed in 120 mL closed glass serum bottles containing 50 mL of basal medium. After flushing with argon, the aqueous solution of investigated dyes and granular sludge (1.0 g VSS L21) were added. Then, 0.5 mL of acetate was added from a solution containing 2.85 M sodium acetate. The assay bottles were incubated at 30 8C for several days. The concentration of CH4 and pressure in the headspace of the serum flasks were recorded throughout the incubation. Specific aceticlastic activities of the sludge were calculated from a linear segment of kinetic curves of methane production. The aceticlastic activity of the untreated sludge with a toxicant was taken as control. From the concentration dependence of the specific activities, 50% of the inhibiting concentration (IC50) values for the azo dyes and aromatic amines were estimated. The assays were performed in duplicate. The anaerobic biodegradation assays with azo dyes and aromatic amines were performed in 120 mL closed serum bottles filled with 50 mL of basal medium, granular sludge (10 vol. %) and 0.2 ­ 1 mM corresponding substrate. Since some biodegradable


organics were introduced with the sludge, no other primary electron donors were added. Moreover, in the preliminary experiments, it was shown that their addition (e.g. ethanol) did not enhance biodegradation rates (data not shown). The headspace of the bottles was flushed with argon. The assays were performed in duplicate at 30 8C. The concentrations of azo dyes, aromatic amines, methane, carbon dioxide, sulphide and ammonium were measured. To take into account a production of methane from the cells' autolysis, control experiments were carried out without aromatic substrates. Since the concentration of azo dyes may decrease due to adsorption onto the sludge particles, the experiments with autoclaved sludge (killed cells) were performed for checking this effect. In experiments with riboflavin, the latter was added to the standard biodegradation assays at concentrations of 5­15 mM (sludge concentration ­ 5 vol.%). The aerobic biodegradation assays with azo dyes and aromatic amines were carried out at 27 8C in 100 mL shake flasks filled with 40 mL of basal medium, activated sludge (10 vol.%) and 0.2 ­ 0.9 mM corresponding substrate. The assays were performed for 30 d using a thermostatic rotary shaker (New Brunswick Scientific, USA) with 180 rpm. The concentrations of azo dyes and aromatic amines were monitored. The chemical azo dyes reduction assays were performed identically to the anaerobic biological assays with the exception that sulphide was added to the final concentrations of 5, 0.5 or 0.05 mM instead of granular sludge (sterile conditions). Concentrations of dyes were 0.5, 0.25 or 0.025 mM, respectively. In the assays with reduction by NADH, concentration of azo dyes was 0.25 mM and concentration of NADH was 0.5 mM. For elucidation of azo dyes' decolourisation mechanisms, the assay with culture broth devoid of microorganisms was performed. After 10 days of anaerobic biodegradation tests, the culture broth was passed through a 0.20 mm syringe microfilter (Corning Inc, USA) used for microbial broth sterilisation, and was then placed into the sterile closed 15 mL flasks. In this way, anaerobiosis was not disturbed. The flasks were flushed with argon, cultivation was performed at 30 8C for 40 d and the concentrations of azo dyes tested were 0.17 ­ 0.4 mM.
Chemicals

N. Yemashova and S. Kalyuzhnyi

All azo dyes and 1-amino-2-naphthol were purchased from Acrus (USA), 4-aminoresorcinol, N,N-dimethyl-1,4-phenylenediamine, anthranilic acid, sulphanilic acid were purchased from Aldrich (USA). All chemicals were of the analytical grade and were not further purified. All aromatic substrates used are presented in Figure 1.
Analysis

The methane and carbon dioxide contents in the headspace of the serum flasks were determined on a gas chromatograph LHM-8MD (Yagat, Russia) equipped with a steel column (2 m) packed with Porapak QS. The temperature of the column, injector port and conductivity detector was 50 8C. Argon was used as a carrier gas with a flow rate of 30 mLàmin21. The volume of gas sample was 200 mL. The azo dyes' concentrations were measured spectrophotometrically with a UV-1202 spectrophotometer (Shimadzu, Japan) at a maximum absorbance wavelength (pH 7.0) of 428, 431, 465 and 490 nm for acid orange 6, methyl red, methyl orange and acid orange 7, respectively. Samples were centrifuged and aliquots of 100 mL were diluted in a 2 mL 0.1 M phosphate buffer (pH 7.0) solution and measured in a 1.0 cm quartz cuvette. The aromatic amines formed were analysed with high performance liquid chromatography (HPLC). The reverse phase column was packed with a Diasorb 130-C16T and had a film thickness of 6 mm. The chromatograph (Gilson, France) was equipped with a UV detector operated at 225 nm. Methanol with 1% acetic acid was used as a liquid phase with a flow rate of

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HO HO3S NN OH HO3S N N

HO

Acid orange 6
N. Yemashova and S. Kalyuzhnyi
166

Acid orange 7 COOH CH3 CH3 NN CH3 Methyl red NH3 N CH3

HO3S

N

N

N

Methyl orange NH
3

OH

OH 4-aminoresorcinol NH
2

H 3C

N

CH3

N,N-dimethyl-1,4-phenylenediamine NH3 NH2 OH

COOH

SO3H anthranilic acid sulphanilic acid 1-amino-2-naphthol

Figure 1 Structural formulas of N-substituted aromatic substrates used

1mLàmin21. The pressure was 14 MPa and the volume of aqueous sample was 10 mL. Ammonium and sulphide were measured spectrophotometrically as described by Fawcett and Scott (1960) and Truper and Schlegel (1964), respectively.
Results and discussion
Biodecolourisation of azo dyes

In the presence of activated sludge, only methyl red became colourless, whereas the other azo dyes were persistent during 25 days of aerobic incubation (Figure 2a). This fact may be explained by reduced electron density of the aromatic ring caused by electron-withdrawing azo group making difficult electrophilic oxygenase attack ­ characteristic for aerobic microorganisms. On the contrary, in the presence of granular methanogenic sludge, all the azo dyes tested were completely decolourised under anaerobic conditions (Figure 2b). Azo dyes become colourless as a result of reductive splitting of azo group with formation of aromatic amines: Ar12N ¼ N2Ar2 × 4½H ! Ar1 NH2 × H2 N 2 Ar2 : Ï1÷

The bioreduction of azo dyes proceeded without significant lag periods and followed the first-order kinetics (Yemashova et al., 2004) with respect to dye concentration (Figure 2b). The corresponding constants were calculated from the data of Figure 2b and their


(a) 1.5 Azo dye, mM
1.2 0.9 0.6 0.3 0 0

Acid orange 6 Methyl orange

Acid orange 7 Methyl red

(b) Azo dye, mM

1 0.8 0.6 0.4 0.2

Acid orange 6 Acid orange 7 Methyl orange Methyl red

N. Yemashova and S. Kalyuzhnyi

0 5 10 15 20 25 0 4 8 12 16

Time, d
Figure 2 Aerobic (a) and anaerobic (b) biodegradation of selected azo dyes

Time, d

values are presented in Table 1. It is seen that methyl red and acid orange 6 were the most prone and persistent dyes for anaerobic reduction, respectively. The addition of ethanol as a primary electron donor for reaction (1) did not enhance azo dye decolourisation rate (data not shown). It should also be noted that incubation of azo dyes with activated sludge in anaerobic conditions also led to their decolourisation with the first order constants comparable to those observed in the presence of methanogenic sludge (Table 1), witnessing that this ability is not an exclusive property of only anaerobic sludges. Azo dye reduction products identified by HPLC were found to be sulphanilic acid and 4-aminoresorcinol for acid orange 6; sulphanilic acid and 1-amino-2-naphthol for acid orange 7; N,N-dimethyl-1,4-phenylenediamine and sulphanilic acid for methyl orange; and N,N-dimethyl-1,4-phenylenediamine and anthranilic acid for methyl red (Table 2).
Mechanism of anaerobic azo dye reduction

For degradation of azo dyes, the vital functions of microorganisms are necessary because no azo dye reduction was found in the presence of autoclaved anaerobic sludge (data not shown). On the contrary, the culture broth obtained by anaerobic sludge liquor filtration through 0.2 mm microfilter (i.e. devoid of cells but containing the products of their vital functions) performed a complete decolourisation of all the azo dyes tested (Figure 3a). Thus, splitting of azo dye appears to be a result of action of reducing agents presented in anaerobic culture broth and followed the pseudo first-order kinetics without any lag period (Figure 3a). However, the observed constants (Table 1) were lower compared to those in the presence of anaerobic biomass, which is thus beneficial for maintaining the pools of the reducing agents. Since sulphide and NADH are usually encountered in anaerobic environments, azo dyes' decolourisation was studied without any sludge but in the presence of both compounds. It was found that these reducing agents completely decomposed all the azo dyes studied (data for acid orange 6 are shown in Figure 3b as an example). This abiotic azo dye's reduction also followed pseudo first-order kinetics with respect to the dye concentration though some lag-periods were observed (Figure 3b). The corresponding constants are presented in Table 1. For sulphide, it was found that the rate of reduction increased with an increase of its concentration (Table 1). However, the sulphide concentrations measured in the anaerobic culture broth were always below 0.05 mM. This implies that the observed rates of azo dyes' reduction by anaerobic sludge (Table 1) cannot be explained by the action of sulphide only; some other reducing agents such as NADH and other redox mediators can also contribute to this process. The importance of the presence of redox mediators in anaerobic media for bioreduction of azo dyes was demonstrated by the addition of riboflavin to the standard anaerobic biodegradability assay (Figure 4). It is seen (data for acid orange 6 are shown in Figure 4

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Table 1 The 1st order constants (day
Azo dye

21

) of azo dye reduction by sludges, broth, sulphide and NADH
Broth* Activated sludge# Sulphide, 5 mM Sulphide, 0.5 mM Sulphide, 0.05 mM NADH, 0.5 mM

Methanogenic sludge

Acid orange 6 Acid orange 7 Methyl orange Methyl red

0.20 0.30 0.49 2.0

^ ^ ^ ^

0.02 0.03 0.03 0.2

0.09 0.13 0.36 0.18

^ ^ ^ ^

0.01 0.02 0.03 0.01

0.14 0.32 0.60 0.48

^ ^ ^ ^

0.01 0.05 0.06 0.04

1.07 1.44 20 3.9

^ ^ ^ ^

0.06 0.09 1 0.2

0.10 0.40 0.88 0.41

*Anaerobic culture broth devoid of cells # Anaerobic conditions

N. Yemashova and S. Kalyuzhnyi
^ ^ ^ ^ 0.01 0.08 0.04 0.05 0.07 0.29 0.20 0.26 ^ ^ ^ ^ 0.01 0.04 0.02 0.04 0.10 0.62 0.13 0.11 ^ ^ ^ ^ 0.01 0.06 0.01 0.01

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Table 2 Anaerobic toxicity of azo dyes and corresponding aromatic amines
Azo dye IC50 of azo dye, mM IC50 of corresponding aromatic amines, mM

Acid orange 6 Acid orange 7 Methyl orange Methyl red

2.50 0.90 0.25 0.99

sulphanilic sulphanilic sulphanilic anthranilic

acid acid acid acid

non non non non

toxic* toxic* toxic* toxic*

4-aminoresorcinol non toxic* 1-amino-2-naphthol 0.54 N,N-dimethyl-1,4-phenylenediamine 1.26 N,N-dimethyl-1,4-phenylenediamine 1.26

N. Yemashova and S. Kalyuzhnyi

*Non toxic until 2 mM concentration

as an example) that even such a low riboflavin concentration as 5 mM led to the dramatic acceleration of azo dye reduction.
Anaerobic toxicity of azo dyes and corresponding aromatic amines

Analysis of the data presented in Table 2 showed that in all cases, except 1-amino-2naphthol, the azo dyes were more toxic than their breakdown products. This fact is in agreement with literature data when aromatic amines are generally regarded as being less toxic compounds for anaerobic microorganisms compared to azo dyes (Razo-Flores et al., 1997).
Biodegradation of aromatic amines

In the presence of activated sludge, only anthranilic acid was completely mineralised while sulphanilic acid was persistent (Figure 5). The latter fact is in contradiction to literature data (Tan, 2001) where aerobic biodegradation of sulphanilic acid was observed. Probably, the activated sludge used by us required an acclimation to this substrate. Meanwhile, 4-aminoresorcinol, 1-amino-2-naphthol and N,N-dimethyl-1,4-phenylenediamine, after exposure to aerobic conditions, underwent autoxidation and polymerisation yielding coloured products which was evident from a largely altered UV-VIS spectrum. Thus, aerobic treatment is not a solution for removal of these amines. On the contrary, in the presence of granular methanogenic sludge, 4-aminoresorcinol (Figure 6a), 1-amino-2naphthol (Figure 6b) and anthranilic acid (data not shown) were quantitatively mineralised to methane, carbon dioxide and ammonium according to the following equations: C6 H7 O2 N × 4H2 O ! 3CH4 × 3CO2 × NH
3 3

for 4-aminoresorcinol; for 1-amino-2-naphthol for anthranilic acid:

C10 H9 ON × 8H2 O ! 5:5CH4 × 4:5CO2 × NH C7 H7 O2 N × 5H2 O ! 3:5CH4 × 3:5CO2 × NH

3

Sulphanilic acid was also slowly degraded in anaerobic conditions producing methane, ammonium and sulphide; however, the initial substrate was not fully mineralised
(a) 0.4 Azo dye, mM
0.3 0.2 0.1 0 0 6 12 18 24 30 36

Acid orange 6, mM

Acid orange 6 Acid orange 7 Methyl orange Methyl red

(b) 0.25
0.2 0.15 0.1 0.05 0 0 7 14 21 28 35 Sulphide NADH

Time, d

Time, d

Figure 3 Decolourisation of azo dyes by anaerobic culture broth devoid of cells (a) as well as sulphide and NADH (initial concentration of both ­ 0.5 mM) (b)

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1.2

Acid orange 6, mM

0.9 0.6 0.3 0 0 4 8 12 16

0 5 10 15

Acid, mM

CH4, NH3, mM

CH4, NH3, mM

1.5 1

0.4 0.3 0.2

0.25 1.5 0.2 1 0.5 0.05 0 0 7 14 21 28 35 42 0 0.15 0.1

0.5 0 0 7 14 21 28 35 42

0.1 0

Time, d

Time, d

Figure 6 Anaerobic biodegradation of 4-aminoresorcinol (a) and 1-amino-2-naphthol (b)

(30% left) after 100 d of anaerobic incubation (data not shown). N,N-dimethyl-1,4-phenylenediamine was demethylated but phenylenediamine formed was not further degraded in anaerobic conditions during 100 d of incubation (data not shown). According to our knowledge, this is the first report demonstrating anaerobic biodegradation of 4-aminoresorcinol, 1-amino-2-naphthol and sulphanilic acid. Two latter findings are in contradiction to some researchers (Bras et al., 2001; Tan, 2001) who reported on the nonbiodegradability of these substances in anaerobic conditions.

Conclusions

170

1. All four selected azo dyes were completely decolourised by an anaerobic granular sludge, while only methyl red was aerobically biodegraded.

1-amino-2-naphthol, mM

4-aminoresorcinol, mM

N. Yemashova and S. Kalyuzhnyi

20

Time, d
Figure 4 Influence of riboflavin concentrations (numbers on graph in mM) on acid orange 6 and anaerobic biodecolourisation (5 vol.% sludge)

1.2 0.9 0.6 0.3 0 0 5 10 15 20 25

Anthranilic

Sulphanilic

Time, d
Figure 5 Aerobic biodegradation of anthranilic and sulphanilic acids

(a)

2

CH4 NH3 4-aminoresorcinol

0.5

(b) 2

CH4 NH3 1-amino-2-naphthol

0.3


2. The anaerobic decolourisation of azo dyes proceeds due to the action of extracellular reducing agents (sulphide, NADH etc) produced by anaerobic bacteria. The presence of redox mediators such as riboflavin led to a dramatic acceleration of this process. 3. Anaerobic toxicity assays showed that the azo dyes were more toxic than their breakdown products (aromatic amines), except 1-amino-2-naphthol. 4. Under aerobic conditions, only anthranilic acid was completely mineralised while sulphanilic acid was persistent. 4-aminoresorcinol, 1-amino-2-naphthol and N,N-dimethyl-1,4phenylenediamine underwent autoxidation in these conditions yielding coloured products. 5. Under anaerobic conditions, 4-aminoresorcinol, 1-amino-2-naphthol and anthranilic acid were quantitatively (sulphanilic acid partially) methanised, while N,N-dimethyl1,4-phenylenediamine was only demethylated producing 1,4-phenylenediamine as an end product.

N. Yemashova and S. Kalyuzhnyi

References
Battersby, N.S. and Wilson, V. (1989). Survey of anaerobic biodegradation potential of organic chemical in digesting sludge. Appl. Environ. Microbiol., 55, 433 ­ 439. Bras, R., Ferra, M.I.A., Pinheiro, H.M. and Goncalves, I.C. (2001). Batch tests for assessing decolourisation of azo dyes by methanogenic and mixed cultures. J. Biotechnol., 89, 155 ­ 162. dos Santos, A.B., Bisschops, I.A.E., Cervantes, F.J. and van Lier, J.B. (2004). Effect of different redox mediators during thermophilic azo dye reduction by anaerobic granular sludge and comparative study between mesophilic (308C) and thermophilic (558C) treatment for decolourisation of textile wastewaters. Chemosphere, 55, 1149 ­ 1157. Fawcett, J.K. and Scott, J.E. (1960). A rapid and precise method for the determination of urea. J. Clin. Path., 13, 156 ­ 159. Hildenbrand, S., Schmahl, F.W., Wodarz, R., Kimmel, R. and Dartsch, P.C. (1999). Azo dyes and carcinogenic aromatic amines in cell cultures. Int. Arch. Occup. Environ. Health, 72, 52 ­ 56. Kalyuzhnyi, S. and Sklyar, V. (2000). Biomineralization of azo dyes and their breakdown products in anaerobic-aerobic hybrid and UASB reactors. Wat. Sci. Technol., 41(12), 23 ­ 30. Kalyuzhnyi, S., Sklyar, V., Mosolova, T., Kucherenko, K., Russkova, J. and Degtyaryova, N. (2000). Methanogenic biodegradation of aromatic amines. Wat. Sci. Technol., 42(5 ­ 6), 363 ­ 370. Nam, S. and Renganathan, V. (2000). Non-enzymatic reduction of azo dyes by NADH. Chemosphere, 40, 351 ­ 357. O'Neill, C., Lopez, A., Esteves, S., Hawkes, F.R., Hawkes, D.L. and Wilcox, S.J. (2000). Azo-dye degradation in anaerobic-aerobic treatment system operating on simulated textile effluent. Appl. Microbiol. Biotechnol., 53, 249 ­ 254. Rajaguru, P., Kalaiselvi, K., Palanivel, M. and Subburam, V. (2000). Biodegradation of azo dyes in sequential anaerobic-aerobic system. Appl. Microbiol. Biotechnol., 54, 268 ­ 273. Razo-Flores, E., Donlon, B., Lettinga, G. and Field, J.A. (1997). Biotransformation and biodegradation of N-substituted aromatics in methanogenic granular sludge. FEMS Microb. Rev., 20, 525 ­ 538. Stolz, A. (2001). Basic and applied aspects in the microbial degradation of azo dyes. Appl. Microbiol. Biotechnol., 56, 69 ­ 80. Tan, N.C.G. (2001). Integrated and Sequential Anaerobic/aerobic Biodegradation of Azo Dyes. PhD thesis, Wageningen University, The Netherlands. Truper, H.G. and Schlegel, H.G. (1964). Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells of Chromatium okenii. Antonie van Leeuwenhoek, 30, 225 ­ 238. Yemashova, N., Telegina, A., Kotova, I., Netrusov, A. and Kalyuzhnyi, S. (2004). Decolourization and partial degradation of selected azo dyes by methanogenic sludge. Appl. Biochem. Biotechnol., 119, 31 ­ 40. Yoo, E.S. (2002). Kinetics of chemical decolorization of the azo dye C.I Reactive Orange 96 by sulfide. Chemosphere, 47, 925 ­ 931. van der Zee, F.P., Lettinga, G. and Field, J.A. (2001). Azo dye decolourisation by anaerobic granular sludge. Chemosphere, 44, 1169 ­ 1176. van der Zee, F.P., Bisschops, I.A.E., Blanchard, V.G., Bouwman, R.H.M., Lettinga, G. and Field, J.A. (2003). The contribution of biotic and abiotic processes during azo dye reduction in anaerobic sludge. Water Res., 37, 3098 ­ 3109.

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