Biochemical Engineering Journal 19 (2004) 75–79

Decolorization of azo-reactive dyes and cotton-textile wastewater usinganaerobic digestion and acetate-consuming bacteria

D. Georgiou∗, C. Metallinou, A. Aivasidis, E. Voudrias, K. GimouhopoulosDepartment of Environmental Engineering, Demokritos University of Thrace, 67100 Xanthi, Greece

Received 16 January 2003; accepted after revision 17 November 2003

Abstract

An anaerobic digestion technique was applied to azo-reactive dye aqueous solutions and cotton textile wastewater aiming at the colorelimination. A batch-mode water-jacketed anaerobic reactor and acclimatized acetate-consuming bacteria initially derived from the anaer-obic digester sludge of a municipal wastewater treatment plant were used for this study. Acetic acid solution and a pH-controller wereutilized to maintain the pH at the desired level (6.6–7.2) while the temperature was kept constant at 37◦C using an external water-bath.Acetic acid also served as an external substrate (electron-donor) supply for the bacteria metabolism. Complete decolorization of all dyesolutions was succeeded in 4–5 days of experimental run. The biodegradation ability of cotton textile wastewater was also examinedwithout the addition of external substrate supply (acetic acid) resulting to poor decolorization results. However, anaerobic digestion of thesame wastewater using the acetate-consuming bacteria and acetic acid as an external substrate supply lead to the complete decolorizationof the wastewater in 4 days of experiment.© 2003 Elsevier B.V. All rights reserved.

Keywords: Acetobacter; Acetic acid; Azo-reactive dyes; Anaerobic processes; Decolorization; Wastewater treatment

1. Introduction

Cotton textile wastewater has a high content of pollutingcompounds the sources of which are the natural impuritiesextracted from the cotton fiber, the processing chemicals andthe dyes. The discharge of this wastewater to the environ-ment causes aesthetic problems due to the remaining color(from the dyestuff used) and also damages the quality of thereceiving water. The color impedes light penetration and thedyes and/or their degradation derivatives can prove toxic toaquatic life[1–5].

Azo-reactive dyes are among the most common onesto dye cotton nowadays. They are non-biodegradable byconventional activated sludge treatment methods and theyrequire intense physical/chemical methods in order tobe removed from the wastewater. Chemical coagulation/flocculation and activated carbon adsorption techniqueshave been developed in order to remove the color. However,the latter methods can only transfer the contaminants (dyes)from one phase to the other leaving the problem essentiallyunsolved (solid waste is created)[6–10]. Advanced oxida-tion processes (UV/H2O2 irradiation, ozonation) have also

∗ Corresponding author. Tel.:+30-2541-28865; fax:+30-2541-62955.E-mail address: dgeorgio@env.duth.gr (D. Georgiou).

been proposed; however, they are not cost-effective due tothe high consumption of energy[11–14].

Anaerobic digestion of textile wastewater is a verypromising technique since it is cost-effective and envi-ronmentally safe. Azo-reactive dyes decompose underanaerobic (reductive) conditions due to the cleavage of theazo-bond (reaction 1) eliminating consequently the color ofthe wastewater. The reduction products (aromatic amines)should then be further treated using aerobic biologicaltreatment methods[5,15–20].

Ar-N = N-Ar ′ + 4e− + 4H+ → ArNH2 + H2NAr ′ (1)

The objective of this paper is to study the decolorizationof six different and most representative azo-reactive dyesutilized by a nearby cotton textile industry using an anaero-bic digestion technique and acetate-consuming bacteria. Fur-thermore, the same technique is to be applied to wastewaterobtained from the latter textile industry.

2. Materials and methods

2.1. Reagents

Azo-reactive dyes were obtained from DyStar (Germany).A total of six of the most representative and commonly

1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.bej.2003.11.003

76 D. Georgiou et al. / Biochemical Engineering Journal 19 (2004) 75–79

Table 1Dyes and their properties (DyStar)

Name Azo type Reactive group

Levafix yellow E-3GA Monoazo Difluorchlorpyrimidine (FCP)Levafix red ERN Monoazo Monohalogentriazine (MHT)

Levafix blue EBNA Diazo Difluorchlorpyrimidine (FCP)Vinylsulphonyl (VS)

Remazol yellow RR Monoazo Vinylsulphonyl (VS)

Remazol red RR Monoazo Vinylsulphonyl (VS)Monohalogentriazine (MHT)

Remazol black B Diazo Vinylsulphonyl× 2 (VS)

used dyes from both the Levafix and Remazol types weretested. The characteristics of these dyes were provided bythe manufacturing company (DyStar) and are summarizedin Table 1. The structural formulae of the reactive groups(Table 1) and also that of the Remazol black B dye are givenin Fig. 1. All dyes have a similar to the Remazol black Bdye structure; the only difference is the kind and number ofreactive groups (Table 1).

Textile wastewater was obtained from Fanco S.A. (Ko-motini, Greece), the characteristics of which are presentedin Table 2. The sample (alkaline with a reddish color) waswithdrawn from the equalization basin of the wastewaterprocessing plant.

Acetic acid, sodium acetate and glucose from Fischer Sci-entific were utilized. Moreover, a liquid fertilizer the com-position of which is shown inTable 3was used.

Fig. 1. The molecular structures of the Remazol black B dye and thereactive groups.

Table 2Textile wastewater characteristics (Fanco S.A.)

pH/Redox(mV)

BOD5

(mg/l)COD(mg/l)

Absorbance (m−1)

436 nm 525 nm 620 nm

9.0/+108 180 430 30.8 43.2 38.8

Table 3Composition of liquid fertilizer

Components %

N 12P2O5 4K2O 6MgO 0.2Fe, Cu, Mn, B, Zn, Mo (sulfate salts) 0.5

2.2. Apparatus and methods

All experiments were carried out in a 2 l batch-modewater-jacketed reactor that was slowly stirred using a mag-netic stirrer (Fig. 2). The pH was maintained in the rangeof 6.6–7.2 using acetic acid solution (10% v/v) and apH-controller, while the temperature was kept constant at37◦C using an external water bath. Acetic acid also servedas a substrate (electron-donor) for the bacteria growth;the liquid fertilizer (Table 3) was added (20 ml/l) in theacetic acid solution. Anaerobic acetate-consuming bacteria(a mixture of methanogens and sulfate reducers) that wereacclimatized for 2 months under the above conditions inan identical to the above-mentioned reactor (Fig. 2) wereused as the inoculum for our study. The initial biomasswas obtained from the sludge of the anaerobic digester of amunicipal wastewater treatment plant.

The dye-solutions were prepared as follows: 20 mg/l of thedye, 1 g/l sodium acetate and 10 ml/l fertilizer (Table 3) weredissolved in tap water and brought to the desired temperature(37◦C). The pH was also adjusted at the above-mentionedrange and then nitrogen gas was utilized in order to purge theair out of the reactor. Finally, 100 ml of the inoculum were

pH

Biogas

Redox

water, 37oC

water, 37oC

CH3COOH and Fertilizer

Fig. 2. The anaerobic batch-mode reactor.

D. Georgiou et al. / Biochemical Engineering Journal 19 (2004) 75–79 77

introduced into the reactor in order to start the anaerobicdigestion.

Samples were withdrawn from the reactor, filtered using0.2 �m pore filters and analyzed for color using a spec-trophotometer (WTW, Photolab Spektral). The yellow, redand blue colors were measured at 436, 525 and 620 nmrespectively. The biomass was measured by filtration of thesamples using 0.45 �m pore filters that were then dried inan oven at 105 ◦C for 2 h. Chemical oxygen demand (COD)measurements were also taken after centrifugation of thesamples.

3. Results and discussion

Anaerobic digestion of the dye-solutions took place suc-cessfully in all cases. Highly reductive conditions weredeveloped (redox = −270 to −360 mV) after 1 day of exper-iment indicating a highly active anaerobic biomass. The con-sumption of acetic acid from the bacteria lead to the increaseof the pH in the anaerobic reactor which in turn, lead tofurther addition of acetic acid (pH-controller signal). Thus,a regular addition of acetic acid in the reactor maintained ahigh COD (substrate) content (2000–2500 mg/l) preservingat the same time a highly active biomass. The production ofbiogas was also obvious at all times due to the rotten-eggcharacteristic smell (presence of H2S traces). Almost com-plete decolorization of all dye-solutions took place in 4–5

time (d)

0 1 2 3 4 5 6

Con

vers

ion

[(C

/Co)x

100]

0

20

40

60

80

10 0 Levafix yellow E-3GA

Levafix red ERN

Levafix blue EBNA

Remazol yellow RR

Remazol red RRRemazol black B

time (d)

0 1 2 3 4 5 6

Bio

mas

s (m

g/L)

0

50

100

150

200

250

300

350

400

Fig. 3. Decolorization of the azo-reactive dyes with time during theanaerobic digestion.

days of experimental run as shown in Fig. 3. This result cor-relates very well with previous findings on decolorizationof azo-reactive dyes during anaerobic digestion [5,15–18].

The next experiment comprised the study of the biodegra-dation ability of the textile wastewater (Table 2) without theaddition of an external substrate (acetic acid). Hydrochloricacid solution (0.5 M) was used instead in order to regulatethe pH at the desired level (6.6–7.2). The liquid fertilizer(Table 3) was also added in the wastewater (10 ml/l) and thehydrochloric acid solution (20 ml/l) in order to assist micro-bial growth. Finally, sludge from the anaerobic digester ofa municipal wastewater treatment plant was utilized as theinoculum for this experiment.

The results from the anaerobic digestion of the textilewastewater are shown in Fig. 4. Anaerobic digestion was in-deed developed in the first 2 days—due to the wastewater’sbiodegradable organic content—leading to a COD reductionof 40% while decolorization of around 30% for the yellowand red colors and 70% for the blue color took place. How-ever, the microbial activity and consequently the rate of theanaerobic digestion were decreased substantially after thesecond day. As shown in Fig. 4, the rate of COD degradationwas decreased to one fifth of the initial one while almost nofurther decolorization took place after the second day of theexperimental run. Moreover, the redox potential clearly in-dicates the low anaerobic biomass activity. For this reason,a small amount of a mixture of glucose and acetic acid was

time (d)

0 2 4 6 8 10 12 14 16 18

Con

vers

ion

[(C

/Co)

x100

]

0

20

40

60

80

100

120

140

160

180

yellow

red

blue

CODsubstrate addition

time (d)

0 2 4 6 8 10 12 14 16 18

Red

ox (

mV

)

-400

-300

-200

-100

0

100

200

Fig. 4. Anaerobic digestion of textile wastewater utilizing HCl solution.

78 D. Georgiou et al. / Biochemical Engineering Journal 19 (2004) 75–79

time (d)

0 1 2 3 4 5 6

Con

vers

ion

[(C

/Co)

x100

]

0

20

40

60

80

100 yellow

red

blue

time (d)

0 1 2 3 4 5 6

Red

ox (

mV

)

-400

-300

-200

-100

0

100

COD = 1200-1400 mg/L

Fig. 5. Anaerobic digestion of textile wastewater utilizing acetic acid andthe acetate-consuming bacteria.

introduced in the reactor increasing the COD to 660 mg/l.As depicted from the redox potential and the COD degra-dation rate (Fig. 4), the anaerobic bacteria were reactivatedby the introduction of new substrate—after an acclimatiza-tion period of 4 days—leading to the complete decoloriza-tion of the textile wastewater 8 days following substrateaddition. The wastewater’s initial red color vanished andonly 30% of the initial yellow color (absorbance at 436 nm)remained.

The above results clearly indicate that the organic contentof the textile wastewater is very low to act as a sufficientsubstrate for the growth of anaerobic bacteria and the com-plete decolorization of the wastewater. It is evident that anexternal substrate (electron-donor) supply is necessary forthe development of sufficient anaerobic digestion conditions.This result also comes in accordance with previous findingsrelated to anaerobic digestion of textile wastewater [21,22].

Following the above conclusions, anaerobic digestion ofthe textile wastewater (Table 2) was repeated using this timeacetic acid solution and the acclimatized acetate–consumingbacteria as with the previously described dye-solutions ex-periments. Anaerobic (reductive) conditions were developedvery fast (redox = −350 mV) leading to almost completewastewater decolorization in 4 days of experiment (Fig. 5).Due to the regular supply of acetic acid (as a substrate), acontinuously active anaerobic biomass was maintained at alltimes (Fig. 5).

This work comprised a preliminary study for the investi-gation of cotton-textile wastewater decolorization using ananaerobic digestion process at a pilot plant-scale.

4. Conclusions

Anaerobic digestion of azo-reactive-dye aqueous so-lutions in a batch-mode reactor utilizing acetic acidas an external supply of substrate (electron-donor) andacetate-consuming bacteria lead to the complete decol-orization of the dye solutions in 4–5 days of experiment.Moreover, anaerobic digestion of cotton-textile wastew-ater without the addition of external substrate lead topoor decolorization results. However, the anaerobic di-gestion of the same wastewater utilizing acetic acid as anexternal supply of substrate and acetate-consuming bac-teria lead to the complete wastewater decolorization in 4days of experiment. As a result, anaerobic digestion usingacetic acid and acetate-consuming bacteria seems to be avery promising technique for the decolorization of textilewastewater.

References

[1] R. Junkins, Textile wastes, case history: pretreatment of textilewastewater, in: Proceedings of the International Conference onIndustrial Waste 37, Purdue University, Ann Arbor Science, 1982,pp. 139–146.

[2] R.C. Kertell, F.G. Hill, Textile dyehouse wastewater treatment: a casehistory, in: Proceedings of the International Conference on IndustrialWaste 37, Purdue University, Ann Arbor Science, 1982, pp. 147–156.

[3] P. Grau, Textile industry wastewater treatment, Water Sci. Technol.24 (1) (1991) 97–103.

[4] U. Meyer, Biodegradation of synthetic organic colorants, in:Microbial Degradation of Xenobiotics and Recalcitrant Compounds,FEMS Symposium, No. 12, 1981, pp. 371–378.

[5] A.R. Larson, E.J. Weber, Reaction Mechanisms in EnvironmentalOrganic Chemistry, Lewis Publishers, CRS Press Inc., USA, 1994.

[6] H.L. Sheng, L.C. Ming, Treatment of textile wastewater by chemicalmethods for reuse, Water Res. 31 (4) (1997) 868–876.

[7] Y.M. Slokar, A. Le Marechal, Methods of decoloration of textilewastewaters, Dyes Pigments 37 (4) (1998) 335–356.

[8] W.J. Eilbeck, G. Mattock, Chemical Processes in WastewaterTreatment, Ellis Horwood Ltd., 1987.

[9] W. Stumm, J.J. Morgan, Chemical Aspects of Coagulation, AmericanWater Works Association, 1962, pp. 971–992.

[10] G. Tchobanoglous, F.L. Burton, Wastewater Engineering, McGraw-Hill Inc., 1991.

[11] R. Venkatandri, W.R. Peters, Chemical oxidation technologies:ultraviolet light/hydrogen peroxide, Fenton’s reagent, and titaniumdioxide-assisted photocatalysis, Hazard. Waste Hazard. Mater. 10 (2)(1993) 107–149.

[12] G.M. Colonna, T. Caronna, B. Marcandalli, Oxidative degradation ofdyes by ultraviolet radiation in the presence of hydrogen peroxide,Dyes Pigments 41 (1999) 211–220.

[13] C. Galindo, A. Kalt, UV/H2O2 oxidation of azo dyes in aqueousmedia: evidence of a structure-degradability relationship, DyesPigments 42 (1999) 199–207.

[14] D. Georgiou, P. Melidis, A. Aivasidis, K. Gimouhopoulos,Degradation of azo-reactive dyes by ultraviolet radiation in thepresence of hydrogen peroxide, Dyes Pigments 52 (2002) 69–78.

D. Georgiou et al. / Biochemical Engineering Journal 19 (2004) 75–79 79

[15] K. Chung, B.E. Fulk, M. Egan, Reduction of azo dyes by intestinalanaerobes, Appl. Environ. Microbiol. 35 (1978) 558–562.

[16] P. Dubin, K.L. Wright, Reduction of azo food dyes in cultures ofproteus vulgaris, Xenobiotica 5 (1975) 563–571.

[17] W. Luangdilok, T. Panswad, Effect of chemical structures of reactivedyes on color removal by an anaerobic–aerobic process, Water Sci.Technol. 42 (3/4) (2000) 377–382.

[18] K.C.A. Bromley-Challenor, J.S. Knapp, Z. Zhang, N.C.C. Gray,M.J. Hetheridge, M.R. Evans, Decolorization of an azo dye byunacclimated activated sludge under anaerobic conditions, Water Res.34 (18) (2000) 4410–4418.

[19] A. Glasser, U. Liebelt, D.C. Hempel, Design of a two-stage processfor total degradation of azo dyes, DECHEMA Biotechnol. Conf. 5(1992) 1085–1088.

[20] M. Kudlich, P.L. Bishop, H.-J. Knackmuss, A. Stolz, Simultaneousanaerobic and aerobic degradation of the sulfonated azo dye mordantyellow 3 by immobilized cells from a naphatlenesulfonate-degradingmixed culture, Appl. Microbiol. Biotechnol. 46 (1996) 597–603.

[21] C. O’Neill, F.R. Hawkes, D.L. Hawkes, S. Esteves, S.J.Wilcox, Anaerobic–aerobic biotreatment of simulated textile effluentcontaining varied ratios of starch and azo dye, Water Res. 34 (8)(2000) 2355–2361.

[22] S. Chinwetkitvanich, M. Tuntoolvest, T. Panswad, Anaerobicdecolorization of reactive dyebath effluents by a two-stage UASBsystem with tapioca as a co-substrate, Water Res. 34 (8) (2000)2223–2232.