The microbiological decolorization of an industrial effluent containing a diazo-linked chromophore

War. Res. Vol. 29, No. 7, pp. 1807-1809, 1995

Pergamon

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RAPID C O M M U N I C A T I O N

THE MICROBIOLOGICAL DECOLORIZATION OF AN INDUSTRIAL EFFLUENT CONTAINING A DIAZO-LINKED CHROMOPHORE J. S. K N A P P * a n d P. S. N E W B Y Department of Microbiology, University of Leeds, Leeds LS2 9JT, U.K.

(First received November 1994; accepted December 1994) Abstract--Mixed bacterial cultures from a wide variety of habitats were shown to be able to decolorize the diazo-linked chromophore of a highly coloured industrial effluent from the manufacture of nitrated stilbene sulphonic acid. Decolorization was favoured by strictly anaerobic conditions and highly proteinaeeous media. 10% v/v dilutions of the effluent with initial absorbances of 100 units at ~,~ were rapidly decolorized---ca. 85% in 2 days. When decolorized effluent was exposed to air it slowly developed a different colour which absorbed light more strongly in the visible region. The mechanisms involved in these processes and their implications for treatment of coloured effluents are discussed.

Key words--coloured effluents, chemical industry effluents, azo dyes, decolorization

INTRODUCTION

METHODS AND MATERIALS

Azo dyes are the commonest type of artificial dye and are in regular use ill the textile, paper, food, cosmetics, pharmaceutical and other industries. These dyes are generally resistan~L to attack by aerobic bacteria and are not amenable to aerobic effluent treatment processes. It is however known that the azo bond can be reduced by bacteria, normally under anaerobic conditions. The reduction of azo dyes can be mediated by facultative as well as obligate anaerobes and has been reviewed recently (Chung et al., 1992). The role of microbes in reducing azo dyes to toxic or carcinogenic compounds is of considerable concern (Brown and De Vito, 1993). H a u g et al. (1991) described a process in which a bacterial consortium could reduce mordant yellow 3 under anaerobic conditions and then mineralize the resultant amines under anaerobic conditions. Most of the work reported to date appears to involve use of pure dyes in artificial media. This note reports research on the treatment of a real chemical industry effluent from the production o f nitrated stilbene sulphonic acid (NSSA), an intermediate in the manufacture o f optical brightener,;. This effluent is highly coloured (with an absorbance at 2m,x of up to 1000 units) and the chromophore is an oligomer of diazo linked aminostibene 2,2' disulphonic acid units with terminal nitro groups. We report here the anaerobic decolorization of this effluent.

General chemicals were obtained from Merck Ltd or Aldrich Ltd and were of Analar or the highest available grade. Bacteriological media were obtained from Oxoid Ltd. The decolorization of the NSSA effluent was assessed by monitoring (by scanning spectrophotometer) the reduction in absorbance at 390 nm (2m~x for the chromophore of the effluent). Anaerobic mixed bacterial cultures were obtained from a variety of samples obtained from various sources including human and animal faeces, muds from rivers and household drains, sludge from anaerobic digesters, effluent treatment lagoons and primary clarifiers in sewage works, and laboratory contaminants. Screening for decolorization was done with 1% v/v of effluent in GPP medium (glucose 1% w/v, peptone 0.2 gi - ' and KI-I2PO4 2gl-', pH 7.0). 10 ml of this medium was distributed in loosely capped tubes and incubated in an anaerobic jar (using triple mix gas I-I2-10%/CO2-10%/ Nr--80%) at 30°C. The yeast extract based medium contained the following in g per litre of distilled water: yeast extract 0.5; KH2PO4 0.5; (NH4)2SO4 0.5; KCI 0.5; MgSOc7H20 0.2; CaCI2 0.1, and pH was adjusted to 7.0. Larger scale work was done in an anaerobic bioreactor with a 200 ml working volume which was sparged with oxygen-free nitrogen and incubated at 30°C. Thin layer chromatography was performed using standard methods with micro crystalline cellulose plates (Whatman, Maidstone) and a solvent system containing 63% propan-2-ol in water.

*Author to whom all correspondence should be addressed.

RESULTS AND DISCUSSION In initial screening it was found that with 20 different mixed cultures all were able to cause a high degree of decolorization during 15 days incubation. All cultures reduced A390by at least 77% with the best

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decolorizing by 85%. This compares with a slight decolorization (6%) in uninoculated controls. No significant change (2%) was seen in the colour of inoculated effluent-free media. A further 35 days incubation resulted in only slight improvement, with a maximum of 91% decolorization. A decrease in a second light absorbance peak at 270 nm was also noted but this decreased to a lesser extent--maximally 40-50% reduction. A typical example of light absorption sprectra before and after anaerobic reduction is given in Fig. 1. In a similar experiment with a yeast extract based medium similar results were obtained after 15 days (75-86% decolorization at 390 nm compared to 8% in uninoculated medium) however it was found that on prolonged (51 days) anaerobic incubation, uninoculated medium could itself cause decolorization. Decolorization ofazo dyes has been attributed in some cases to direct (non enzymic) interaction with ravin coenzymes (e.g Dubin and Wright, 1975; Gingell and Walker, 1971) and it may be that this reduction was similarly mediated, with H2 causing reduction of the electron carrier. Tests using two typical cultures (ADM and BW,) with NSSA effluent and the dye chlorazol yellow (an oligomeric diazo dye with aminostilbene sulphonic acid units which is thought to be similar to the NSSA effluent chromophore) showed that although decol-

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Fig. 2. Growth of mixed bacterial culture ADM with decolorization (at 390 and 270 nm) of a 10% dilution of NSSA effluent in Brain Heart Infusion medium.

orization occurred in semi-anaerobic conditions (given by incubation in loosely capped Universal bottles incubated in air), it was much superior in the strictly anaerobic conditions of the anaerobic jar. It was also shown that decolorization was favoured by greater concentrations of peptone (0.4 gl- ') and that in the anaerobic jar higher glucose concentrations (up to 2%) did not improve decolorization and could be inhibitory. This later observation is in agreement with the findings of Chung et al. (1978). Accordingly several bacteriological media with higher content of proteinaceous material were tested. Nutrient Broth and Brain Heart Infusion (BHI) both gave effective decolorization (77 and 80% decolorization respectively) in 3 days incubation. BHI was therefore used in further work. Incubation of NSSA effluent with both media without microbial inoculation resulted in some decolourization but this was much slower than in the presence of microbial cultures. Experiments with a larger scale anaerobic reactor showed that three mixed bacterial cultures were able to decolorize more concentrated NSSA effluent up to 10% v/v, generally giving 70-85°/, decolorization in ca. 3 days. Typical results for mixed culture ADM (originally derived from sludge from an anaerobic digester) are given in Fig. 2. This also shows that the greatest part of the reduction in colour occurs during the period of active bacterial growth. Control cultures in which ADM was incubated in NSSA effluent-free BHI medium showed no comparable changes in light absorbtion spectra and showed very similar growth suggesting that the effluent is not toxic to the culture

Rapid Communication at this concentration, pH was also monitored and it was repeatedly nol:ed that in cultures containing NSSA effluent the pH rose markedly and was generally about 0.8-1 units higher (8.0 cf. 7.0) than in controls. This increase was probably due to the production of aromatic amines which are more basic than azo compounds. The production of 4,4' diaminostilbene 2,2' disulphonic acid (DAS) was not demonstrated unequivocally. However it was noted, using TLC, that spots associated with the chromophore disappeared after anaerobic incubation and that new spots appeared on chromatograms which did not absorb visible light but fluoresced strongly under long wavelength u.v. light. These spots had similar Rf to authentic DAS but gave fluorescence of a slightly different colour. When removed from TLC plates and eluted the material had a similar spectrum and 2m~x(ca. 330 nm) to DAS. It was thought that this material was probably similar if not identical to DAS (which on the basis of studies wil;h pure azo dyes (Chung et al., 1992) would be the predicted product of reduction), it might also be a mixture of similar compounds. The mixed cullure A D M contained at least four distinct microbial strains designated A - D . A, B and C were all facultatively anaerobic spore forming, gram positive rod shaped bacteria and were considered to be members of the genus Bacillus. Strain D was similar to the others except that it was an obligate anaerobe and was considered to be a Clostridium sp. All strains incubated in pure culture could reduce the colour of NSSA effluent or chlorazol yellow but the mixed culture did so more rapidly, suggesting some degree of synergism. Immediately afLer anaerobic decolorization it was noted that NSSA effluent was strongly visually decolorized changing from black to straw yellow. However if samples of decolorized effluent were left in the laboratory over a period of several weeks in loosely capped tubes it was observed that they steadily changed colour--darkening visibly from the top downwards. Tubes showed zones of colour with dark green to black at the top (the air/liquid interface), then a red to brown layer and then the straw yellow colour at the base of the tube. This recolouration clearly occurs in response: to the presence of oxygen and may be due to either a spontaneous or a microbially catalysed oxidation of some of the reduction products of the NSSA effluent. The new chromophore was not simply the result cf reformation of the azo bond as its colour and light absorbtion spectrum was quite different, as shown in Fig. 1. The oxidized products

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have no peak of light absorbance but show much (ca. 2-4 times) higher absorbance across a broad range of the visible region from 350 to 600 nm and were thus much more visible. Aromatic amines (which are thought to be the products of anaerobic decolorization of NSSA effluent) are well known to be unstable and even pure chemicals deteriorate on storage to give coloured products. This recolouration phenomenon is a major draw back probably making an anaerobic decolorization process for this azo dye-containing waste of little value. The production of dark colours during the treatment of effluents containing aromatic amines has been reported by other workers (Kulla et al., 1984). A two stage biotreatment process such as that reported by Haug et al. (199 l) might be applicable to the treatment of NSSA effluent, unfortunately attempts to isolate bacterial cultures capable of mineralising DAS, the putative reduction product, were unsuccessful. The widespread occurrence in the environment of bacteria capable of reducing azo compounds to aromatic amines (which may be considerably more toxic, Brown and De Vito, 1993) coupled with the ready oxidation of the reduction products possibly to coloured compounds heightens the requirement for effective means to degrade these dyestuffs and treat effluents containing them. Acknowledgement--P. S. N. gratefully acknowledges the

provision of a Research Studentship by the Science and Engineering Research Council. REFERENCES

Brown M. A. and De Vito S. C. (1993) Predicting azo dye toxicity. Crit. Rev. Environ. Sci. Tecb. 23, 249-324. Chung K-T., Fulk G. E. and Egan M. (1978) Reduction of azo dyes by intestinal anaerobes Appl. Environ. Microbiol. 35, 558-562. Chung K-T., Stevens S. E. and Cerniglia C. E. (1992) The reduction of azo dyes by the intestinal microflora. Crit. Rev. Microbiol. 18, 175-190. Dubin P. and Wright K. L. (1975) Reduction of azo food dyes in cultures of Proteus vulgaris. Xenobiotica 5, 563-571. Gingell R. and Walker R. (1971) Mechanisms of azo reduction by Streptococcusfaecalis II. The role of soluble flavins. Xenobiotica l, 231-239. Haug W., Schmidt A., N6rtemann B., Hempel D. C., Stolz A. and Knackmuss H-J. (199 l) Mineralization of the sulphonated azo dye mordant yellow 3 by a 6-aminonaphthalene-2-sulphonate-degrading bacterial consortium. Appl. Environ. Microbiol. 57, 3144-3149. Kulla H. G., Krieg R., Zimmermann T. and Leisinger T. (1984) Biodegradation of xenobiotics: experimental evolution of azo dye-degrading bacteria. In Current Perspectives in Microbial Ecology (Edited by Klug M. J. and Reddy C. A.), pp. 663-667. ASM, Washington.