Environment International 26 (2000) 7579 www.elsevier.

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Decolorization of Remazol Black-B using a thermotolerant yeast, Kluyveromyces marxianus IMB3

C. Meehan, I.M. Banat*, G. McMullan, P. Nigam, F. Smyth, R. Marchant
Biotechnology Research Group, School of Applied Biological and Chemical Sciences University of Ulster, Coleraine BT52 1SA, Northern Ireland Received 25 January 1999; accepted 6 June 2000

Abstract The ability of Kluyveromyces marxianus IMB3 to decolorize Remazol Black-B dye was investigated. The effect of environmental conditions, such as pH and temperature were examined. No noticeable effects on decolorization were observed when pH varied from 3.05.5. Maximum colour removal, 98%, was achieved at 37 C. Little or no colour removal was detected when K. marxianus IMB3 was incubated under anaerobic conditions. Further investigation, in which decolorization was monitored under extreme temperatures and low pH (to inhibit growth) and using ten fold dense inoculum, revealed that decolorization was due to biosorption to the yeast cells and not due to a metabolic reaction. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Bioremediation; Biosorption; Textile dyes; Decolorization; Pollution removal; Kluyveromyces marxianus; Dye decolorization by yeast

1. Introduction Effluents containing textile dyes are usually discharged in large quantities worldwide into natural water bodies on a daily base. Their pollution potential stems from their possible toxicity and carcinogenicity which is mainly due to components such as benzidine and other aromatic compounds, which might be reformed as a result of microbial metabolism as reported by Clarke and Anliker (1980). Azo- and nitro-compounds, which constitute a main component of many dyes, are reduced in sediments (Weber and Wolfe, 1987) and in the intestinal environment (Chung et al., 1978), resulting in the regeneration of the parent toxic amines. Baughman and Perenich (1988) showed some dyes did bio-accumulate in some algae. Srivastava and Prakash (1991) also detected high concentrations of heavy metal ion components of some dyes in some algae and higher plants exposed to dye containing effluents. Although dyes constitute only a small portion of the total volume of waste discharge from textile processing, typical microbial-based wastewater treatment processes (Brown et al., 1981) do not readily remove them. On the contrary, the dye components in textile wastewater effluent can be detrimental to the microbial population
* Corresponding author. School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine BT52 1SA, UK. Fax: 0044 2870 324906. E-mail address: IM.Banat@ulst.ac.uk (I.M. Banat).

present in such treatment works and may lead to decreased efficiency or treatment failure in such plants (Ogawa et al., 1988). Similar adverse effects have also been detected in aquatic microbial populations (Michaels and Lewis, 1985) and in laboratory cultures exposed to such dyes (Ogawa et al., 1989). In a recent review on decolorization of textile-dye-containing effluent, Banat et al. (1996) reported the lack of and search for economically suitable methods for decolorizing wastewater. Primary treatments, including screening, sedimentation, flotation, and flocculation, were not effective in the removal of dyes without simultaneous chemical treatment. Among the possible physico-chemical techniques are flocculation with Fe(II)/Ca(OH)2, flotation, electrokinetics coagulation, electrochemical destruction, ionexchange, irradiation, precipitation, ozonation, adsorption, and chemical oxidation (Groff, 1993; Mishra and Tripathy, 1993). Activated carbon has also been reported as a suitable dye sorbent; however its manufacturing and regeneration costs are high (Low et al., 1996). Some of these techniques have been shown to be effective, although excess amounts of chemical usage, generation of large volumes of sludge in addition to costly plant requirements and operating expenses aggravate the problem. The economic removal of polluting dyes and other components is gaining great importance as European Community (EC) regulations on industrial effluent dis-

0160-4120/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0160-4120(00)00084-2

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charge are being enforced. Both industries and scientists are actively searching for novel innovative treatments and technologies directed particularly towards the decolorization of dyes in effluents. Dyes usually have a low rate of removal ratio for biochemical oxygen demand (BOD) to chemical oxygen demand (COD) (BOD/ COD less than 0.1) (De Angelis and Rodrigues, 1987). Recent research into textile effluent decolorization and degradation has been focusing on biological techniques due to their general simplicity and inexpensive technologies involved. At the University of Ulster, Nigam and Marchant (1995), Nigam et al. (1995a, 1995b, 1996a, and 1996b) and Banat et al. (1997) investigated the feasibility of such treatments mainly using bacterial and white rot fungi strains and have achieved various degrees of success under laboratory conditions. A potential low cost alternative in colour removal from textile effluent is biosorption, which has been used successfully by Modak et al. (1995) for the removal and recovery of toxic metals from industrial effluent. Strong biosorbent behavior of certain types of microbial biomass toward metallic ions and other pollutants, such as textile dyes, is a function of the chemical makeup of the microbial cells of which the biomass consists. White and Gadd (1986) and White et al. (1995) described several chemical groups in biomass that could attract and sequester charged pollutants such as acetamide groups of chitin and phosphate groups in nucleic acids, amino, amido, sulfhydryl, and carboxyl groups in proteins and hydroxyls in polysaccharides. The use of biomass will lead to sludge generation which may require further treatment such as the use of solid state fermentation (SSF). Many of the fungi capable of carrying out SSF are white rot fungi, which have also been shown to be capable of textile dye decolorization (Kirby et al., 1995); such use however, would require further investigation into the fate of the textile dyes once adsorbed on biomass and the possibility of their break down in SSF prior to recycling as compost or soil conditioner. According to Kwasniewska (1985) and Kakuta et al. (1992 and 1998), some yeast strains are able to degrade synthetic dyes and to act as an effective metal biosorbent. In this study we investigated the ability of K. marxianus IMB3 a thermotolerant yeast to decolorize solutions containing Remazol Black-B dye and whether this is due to metabolic/enzymatic activity or physical adsorptive capabilities and possible future application in dye-containing effluent decolorization. 2. Materials and methods 2.1. Textile dyes tested Remazol Black-B (diazo dye) was selected for testing as it is one of the more common dyes used by local dyeing companies in Northern Ireland and the border-

ing counties of the republic or Ireland. A stock solution of Remazol Black-B dye was prepared at a concentration of 100g/L and diluted as required. 2.2. Organism and Inoculum preparation A thermotolerant yeast strain (Kluyveromyces marxianus IMB3) (Singh et al., 1998), which was isolated at the University of Ulster, was used. Cultures were maintained on malt extract agar (Oxoid, Unipath Ltd., England) at 40 C. An inoculum was prepared in glucose mineral medium as described by Banat et al. (1992) and used either at 50mL/L (considered a light inoculum) or at tenfold concentration (through centrifugation of cell suspension) in some treatments (considered as a heavy inoculum). This was carried out in order to confirm the effective method of colour removal observed using K. marxianus IMB3. Decolorization, therefore, was monitored in dye containing media inoculated with the usual light inoculum (an overnight grown culture) while comparing it to other treatments inoculated with tenfold dense inoculum obtained through centrifugation. The same final volumes of inoculum were used in all cases and some treatments were incubated under conditions that did not allow growth yet had no detrimental effects on viability of the cell biomass. All experiments were carried out in triplicate and results are expressed as the mean values. 2.3. Culture conditions Decolorization studies were carried out in 250mL conical flasks containing 50mL of glucose mineral medium. Cultures were incubated in an orbital shaker (150rpm) at 37 C and pH 5.0 unless otherwise specified. Anaerobic conditions were obtained through gassing suba-sealed flasks with oxygen free nitrogen. Growth and decolorization were monitored at various temperatures and pH values. 2.4. Growth and decolorization measurement Growth was monitored through optical density measurements of culture medium using a Spectronic 20E spectrophotometer at 660nm. Decolorization was determined through measurements of culture supernatant absorption at Remazols max (600nm) using a Shimadzu UV-2101PC spectrophotometer against uninoculated dye-containing medium. Percentage decolorization was calculated from adsorption values obtained against the controls. 3. Results and discussion Both filamentous fungi and yeast have received considerable attention in connection with metal biosorption, particularly because waste fungal biomass arises as a byproduct of several industrial fermentations. Yeast

C. Meehan et al. / Environment International 26 (2000) 7579 Table 1 Decolorization (%) of Remazol Black B by K. marxianus IMB3 after 24h growth under aerobic conditions at different pH values and dye concentrations pH 5.5 5.0 4.5 4.0 3.5 3.0 200mg/L 84 96 96 97 95 78 100mg/L 96 97 94 98 97 97 50mg/L 98 97 97 96 98 96 25mg/L 96 94 96 95 94 97

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has long been known to be capable of rapid uptake of metal from solution, but little work has been carried out investigating the ability of yeast to act as a biosorbent for textile dyes in wastewater effluents. Biosorption of textile dyes on distillery waste containing inactive Saccharomyces cerevisiae biomass and biosorption of uranium ions by K. marxianus biomass have both been recently reported (Bustard et al., 1996, 1997 and 1998). Actively growing K. marxianus yeast cells under aerobic conditions almost completely decolorized Remazol Black-B within 24h at all dye concentrations used (25 200 mg/L) and at various acidic pH values (Table 1). Colour removal varied from 78 to 98% of the original colour present. There were no noticeable effects on percentage decolorization at different initial pH values within the range tested when using 25, 50, and 100 mg/ L dye concentrations. A slight reduction was seen at the lowest (3.0) and highest (5.5) pH values tested when 200 mg/L dye was used. Temperature variation in comparison, however, had a significant effect on the decolorization of Remazol Black-B by K. marxianus IMB3 (Table 2). The highest colour removal was detected at 37 C. This temperature has been reported earlier within the optimum temperature range of growth for this yeast strain. When the yeast cells were incubated under anaerobic conditions, decolorization was dramatically reduced. No significant decolorization occurred at the higher concentration of 200 mg/L Remazol (Table 2). Since K. marxianus IMB3 is capable of growth under aerobic conditions and unTable 2 Decolorization (%) of Remazol Black-B by K. marxianus IMB3 at different temperatures and dye concentrations under aerobic and anaerobic conditions 200mg/L Temp. C 25 30 37 40 45 50 A A 33 35 38 5 60 1 aerobic; An An 0 0 0 8 3 0 100mg/L A 82 44 65 93 93 0 anaerobic. An 12 20 28 25 26 1 50mg/L A 62 70 88 96 97 10 An 21 19 44 50 49 5 25mg/L A 82 81 97 98 98 19 An 29 42 53 59 60 10

Fig. 1. Growth and decolorization of Remazol Black-B by Kluyveromyces marxianus IMB3 at 37 C and pH 5.0; L.I. light inoculum, H.I. heavy inoculum.

able to grow under anaerobic conditions, decolorization seemed to be growth associated. Whether decolorization occurred due to metabolic reactions or due to biosorption to produced biomass under aerobic condition was at this stage uncertain. Under suitable conditions of temperature and pH, K. marxianus IMB3 cells grew after a short lag phase. Decolorization started and was complete within 18h (Fig. 1). When a heavy inoculum was used, immediate decolorization was observed, indicating adsorption to biomass. When growth was inhibited under extreme conditions such as temperatures of 4 C or 60 C (Fig. 2 and 3) or at very low pH of 2.0 (Fig. 4) rapid decolorization was observed only when a heavy inoculum was used and within a time period before any significant growth occurred. This concludes that colour removal by K.

Fig. 2. Growth and decolorization of Remazol Black-B by Kluyveromyces marxianus IMB3 at 37 C and pH 2.0; L.I. light inoculum, H.I. heavy inoculum.

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4. Conclusion To consider the merits of using yeast biomass for the purpose of decolorization of textile dye-containing effluent, several factors have to be taken into consideration. There are limitations on the cost of producing biomass just for the purpose of using in biosorption on an industrial scale as it is economically nonviable. In addition, biosorption based processes have their shortcomings in that they generate large amounts of sludge, which requires safe disposal or further treatment. Therefore, for possible future applications, consideration into linking distilleries yeast biomass waste to textile effluent discharge may be an option.
Fig. 3. Growth and decolorization of Remazol Black-B by Kluyveromyces marxianus IMB3 at 4 C and pH 5.0; L.I. light inoculum, H.I. heavy inoculum.

Acknowledgments We acknowledge financial support from the European Structural Funds INTERREG PROGRAMME administered by the Department of Environment (DOE) Northern Ireland.

marxianus IMB3 was due to physical adsorption of the dye to cellular biomass and not due to any chemical enzymatic activity. The mechanism by which the cellular biomass takes up dye component is unclear, and the main cellular component that may be responsible for such biosorption could be their cell wall. Yeast cell walls are known to be resilient structures and substantial quantities of it are generated as a low-value by-product by many biotechnological processes, which would therefore render it suitable for exploitation by other biosorptive biotechnological processes, particularly those involving toxic effluents. The ability to function under extreme conditions of pH and temperature values and not being growth associated are added advantages.

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