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ABSTRACT Anaerobic decolorization of two kinds of dye, viz., azo type represented by Methyl Orange (MO) and anthraquinone type represented by Reactive Blue 4 (RB4) was performed using digested sludge under mesophilic (35OC) and thermophilic (55OC) conditions. Decolorization of dye was investigated to compare the efficiency and extent of decolorization, and additionally to evaluate the effect of temperature and dye on decolorizing microorganisms. Glucose was used as an electron donor in terms of co-substrate without the addition of other nutrients. Under thermophilic treatment, high efficiency of decolourisation was shown in both of the dyes at high concentration, 1000 mg·L-1of MO and 600mg·L-1 of RB4 compared with mesophilic treatment. MO, 200mg·L-1, was decolorized 95-98% under both mesophilic and thermophilic conditions. High decolorizing efficiency of RB4, 100mg·L-1, obtained under thermophilic conditions, was 80% when compared with mesophilic conditions (70%). The reduced form of MO showed an auto-oxidizing effect with pink to violet color when exposed to air, while RB4 showed no autooxidizing reaction. The inhibition of MO was effected on sugar conversion to fatty acids, and CH4 productivity resulted in slow reduction of TOC. While RB4 inhibited only on methane productivity, in which TOC reduction was similarly used as control. Due to the increase of temperature, methanogenesis was inhibited and which low CH4 production, whereas an increase of decolorizing efficiency on both dye decolorization and a high rate of TOC reduction was observed.
Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 9 - Comparative decolorizing efficiency of textile dye by mesophilic and thermophilic anaerobic treatments A. Boonyakamol*, T. Imai*, P. Chairattanamanokorn**, R. Watanapokasin***, T. Higuchi* and M. Sekine* * Graduate School of Science and Engineering, Yamaguchi University, Yamaguchi 755-8611 Japan ** Department of Environmental Science, Faculty of Science, Kasetsart University, Bangkok 10900 Thailand *** Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110 Thailand ABSTRACT Anaerobic decolorization of two kinds of dye, viz., azo type represented by Methyl Orange (MO) and anthraquinone type represented by Reactive Blue 4 (RB4) was performed using digested sludge under mesophilic (35 O C) and thermophilic (55 O C) conditions. Decolorization of dye was investigated to compare the efficiency and extent of decolorization, and additionally to evaluate the effect of temperature and dye on decolorizing microorganisms. Glucose was used as an electron donor in terms of co-substrate without the addition of other nutrients. Under thermophilic treatment, high efficiency of decolourisation was shown in both of the dyes at high concentration, 1000 mg·L -1 of MO and 600mg·L -1 of RB4 compared with mesophilic treatment. MO, 200mg·L -1 , was decolorized 95-98% under both mesophilic and thermophilic conditions. High decolorizing efficiency of RB4, 100mg·L -1 , obtained under thermophilic conditions, was 80% when compared with mesophilic conditions (70%). The reduced form of MO showed an auto-oxidizing effect with pink to violet color when exposed to air, while RB4 showed no auto- oxidizing reaction. The inhibition of MO was effected on sugar conversion to fatty acids, and CH 4 productivity resulted in slow reduction of TOC. While RB4 inhibited only on methane productivity, in which TOC reduction was similarly used as control. Due to the increase of temperature, methanogenesis was inhibited and which low CH 4 production, whereas an increase of decolorizing efficiency on both dye decolorization and a high rate of TOC reduction was observed. Keywords: textile dye decolorization; decolorizing efficiency; mesophilic; thermophilic; anaerobic treatment. INTRODUCTION Azo dye, which is widely used in the textile industry, represents the largest and most versatile group of dyes, whose share in industrial application amounts to some 70% of all dyestuff consumed, and anthraquinone dyes are used for coloration of cotton and cellulose fibers as well as of hydrophobic, synthetic materials (McMullan et al., 2001). During dye processing, as much as 2-50% of dyestuffs applied may be lost to waste- water ultimately released into the environment (O’neill et al., 1999). Due to high water solubility of dye and its not being degradable under the typical aerobic conditions found in conventional biological treatment systems, and that it is absorbed very poorly into biological solids, a result is residual color in discharged effluents (Beydilli et al., 2000; Epolito et al., 2005; Pearce et al., 2003). The impact of dyes in waste-water discharged into environment is a concern because; (1) they cause water bodies to become colored, Address correspondence to Tsuyoshi IMAI, Graduate School of Science and Engineering, Yamaguchi University, Email: imai@yamaguchi-u.ac.jp Received January 30, 2008, Accepted February 26, 2008. Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 10 - absorbing and reflecting sunlight, which in turn interferes with photosynthesis and aquatic ecosystem and, (2) a wide range of textile dye toxicity has been reported, which may cause both chronic and acute toxicity (Sloker and LeMarechal, 1998). Dye decolorization can be readily achieved under anaerobic condition, either chemically or by biological treatment. Biological dye decolorization involves unspecific enzymes ubiquitously found in a wide diversity of microorganisms. However, little is known about the microbiological aspects of anaerobic consortia from waste-water treatment plants in the reductive decolorization of azo and anthraquinone dyes. A large proportion of textile waste-waters, mainly from the dyebath and rinsing processes, is discharged at high temperatures (40-70 o C). However, thermophilic anaerobic textile dye decolorization by biological treatment has been only briefly examined (Dos Santos et al., 2004; Willts and Ashbolt, 2000). In this study, two kinds of dye, viz., azo type, represented by Methyl Orange (MO), and anthraquinone type represented by Reactive Blue 4 (RB4) were investigated for comparative decolorizing efficiency by mesophilic (35 o C) and thermophilic (55 o C) anaerobic biological treatments. The contribution of dye reducing microbial consortia was also evaluated in the presence of acidogens and methanogens inhibitors, vancomycin, and 2-bromoethane sulfonic acid, supplemented with glucose and acetate as electron donors. MATERIALS AND METHODS Chemicals Methyl Orange (MO, CI 13025) and Reactive Blue 4 (RB4, IC 61205) represented azo and anthraquinone dye classes, respectively, and used in this study (Figure 1). Vancomycin and 2- bromoethane sulfonic acid (BES) were used as acidogens and methanogens inhibitor, respectively (Dos Santos, 2004). Glucose and sodium acetate were used as co-substrate or electron donor. All chemicals used were purchased from Wako and Sigma-Aldrich, Japan. Decolorizing microorganisms Digested sludge used to provide decolorizing microorganisms was obtained from the anaerobic waste-water treatment plan, Western Purification Center, Ube city, Yamaguchi Prefecture, Japan. The inoculums of 10% (v/v) sludge or approximated 1,300 mgSSL -1 were previously cultivated in serum bottles sealed with rubber and capped with aluminum overnight without nutrient supplement to reduce the nutrients remaining in the sludge solution, and initial pH was adjusted to about 7-8 and purged with Ar gas. The pre-incubate conditions were prepared separately at 35 o C and 55 o C in a temperature controlled incubator without shaking before use in all experiments. Figu re 1 - Chemica l structu res of MO and RB4 Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 11 - Dye decolorization Dye decolorizing efficiency by digested sludge To reveal the decolorizing efficiency of digested sludge on MO and RB4 decolorizations under anaerobic mesophilic and thermophilic conditions, the variation of MO at concentration of 100, 200, 400, 600, 800 and 1,000mg · L -1 and RB4 at concentration of 100, 200, 300, 400, 500 and 600mg · L -1 supplemented with 1g · L -1 glucose as co-substrate without supplementary nutrients was conducted in batch treatment. Initial pH was adjusted to approximate 7.5 without addition of NaHCO 3 as pH buffer before being added to serum bottles that contained pre-incubated digested sludge. Treatment temperatures were at 35 o C and 55 o C. Samples were taken every 6 hours during the treatment period and measured for decrease in color by spectrophotometer at 470 and 598 nm for MO and RB4, respectively. The extent of decolorization was represented as a percentage decolorization. Effect of glucose on dye decolorizing efficiency To understand the effect of glucose on MO and RB4 decolorization, the concentration of glucose was varied at 0, 0.5, 1.0, 2.0 and 3.0g · L -1 in batch treatment without supplementary nutrients. The dye concentration used was 200mg · L -1, and treatment conditions were as described above. Effect of dyes and temperatures on dye decolorization To explain the effect of dyes and temperature, 35 o C and 55 o C, on decolorizing efficiency and decolorizing microorganisms, 200mg · L -1 of MO and 100mg · L -1 of RB4 were used in the batch experiments. Glucose was added at a concentration of 0.1g · L -1 without supplementary nutrients, and pH was in range of 7-8 by addition of 4g · L -1 NaHCO 3 as pH buffer. All mixture solutions were added into serum bottles of pre- incubated sludge. Control consisted of the same treatment without addition of dye in the mixture. Samples were taken to measure the reduction of dye intensity, volatile fatty acids (VFAs), gas production and total organic carbon (TOC). The reduced form of dyes after being treated by digested sludge was studied by monitoring the change of spectrum wavelength between 300-650 nm. The appearance of a new peak would be presented as a reduced form of treated dye compared with the reduction of the original peak for untreated dye. In addition, the contribution of decolorizing microorganisms was monitored in the experiment by the presence of vancomycin (1g · L -1 ) and BES (10.5g · L -1 ). Vancomycin is an inhibitor used to inhibit the activity of acidogenesis microorganism, and BES for methanogenesis microorganism inhibition. MO and RB4 at concentrations of 200 and 100mg · L -1 , respectively, were tested. 0.1g · L -1 of glucose or sodium acetate was added to assess its effect on decolorizing efficiency as a substrate in the organic matter conversion pathway. Analytical methods Dye reduction was determined photometrically by spectrophotometer (Hitachi U-2001). The appropriate dilution was made in distilled water. The absorbance was read at the maximum absorbance wavelength, i.e., MO at 470nm and RB4 at 598nm. The decolorizing efficiency was defined as percentage decolorization. TOC concentration was measured by total organic carbon analyzer (Shimadzu TOC-5000). Gas Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 12 - chromatography, Shimadzu GC-8APT combined with a Shincarbon T 60/80 column, was used for gas composition measurement, and Shimadzu GC-8APF with Packed Column Unisol F-200 30/60 was used for VFAs concentration measurement. RESULTS AND DISCUSSION Dye decolorization Dye decolorizing efficiency by digested sludge The potential of digested sludge on MO and RB4 tested by variation of dye concentration was shown by the different extent of decolorizing efficiency of the tested dyes. MO could be decolorized completely, amounting to greater than 95% decolorization at both of 35 o C and 55 o C (Figure 2). The concentration of dye did not show effect on decolorization which the decolorizing efficiency of maximum concentration, 1,000mgL -1 , also gave over 95% decolorization in both of treatment conditions. It indicated that the decolorization of MO dye could be performed completely under both mesophilic and thermophilic conditions with no difference in rate of decolorization. In addition, treatment at 55 o C showed a faster decolorizing rate when a low concentration of dye was introduced. An increase of RB4 dye concentration at 35 o C caused a decreasing of decolorizing efficiency (Figure 3A), while the extent of decolorization reached 90% when treatment was conducted at 55 o C (Figure3B). The limitation of digested sludge on RB4 decolorization at 35 o C may be because dye component was absorbed into sludge cells, which showed a blue color during the experiment period. The absorbed dye may block the substrate transportation pathway involved in the decolorizing mechanism. However the absorption was not present when decolorizing efficiency reached about 70%. A consequence of the 55 o C treatment may be that the higher temperature caused an increase the size of pores in the cell membrane that supports the substrate transportation and so increased the potential of digested sludge for dye decolorization. These results revealed that biological dye decolorization using anaerobic digested sludge at high temperature could be accelerated and increased. 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour(s) Decolorization ( %) 0 mg/L 0! mg/ L 0 mg/L 0! mg/ L 0 mg/L !!mg/L 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour(s) Decolorization (%) 0! mg/L 0! mg/L 0! mg/L 0! mg/L 0! mg/L !!mg/L A B Figure 2 - Decolorizing efficiencies of MO with variation of dye concentration under mesophilic (A) and thermophilic (B) conditions Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 13 - 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour(s ) Decolorization (% ) mg / L ! m g / L mg / L ! m g / L mg / L ! m g / L 0 10 20 30 40 50 60 70 80 90 10 0 0 6 12 18 24 30 36 42 48 Hour (s) Decolorizat ion ( %) ! m g / L ! m g / L ! m g / L ! m g / L ! m g / L ! m g / L A B Figure 3 - Decolorizing efficiencies of RB4 with variation of dye concentration under mesophilic (A) and thermophilic (B) conditions Effect of glucose on dye decolorizing efficiency Glucose is easily metabolized and taken up into cells, and is known as the best electron donor of dye decolorization. The variation of glucose concentration at 0, 0.05, 0.1, 0.2 and 0.3g · L -1 was tested. The results showed that presence of glucose in an MO decolorizing system did not have much effect on dye decolorization, and control at 0gL - 1 of glucose showed an 80% decolorization result both in treatment at 35 o C and 55 o C (Figure 4). In addition, presence of glucose could accelerate the extent of decolorization and gave higher decolorizing efficiency (above 90%) compared with control; however an increase of glucose concentration did not show a different extent of decolorizing. These results meant that the decolorization of MO did not need much electron donor substrate. By contrast with RB4 decolorization (Figure 5), the presence of glucose in the decolorizing system was needed. In the absence of glucose, only 20% decolorization occurred, which caused cell absorption, while the decolorizing efficiency increased in presence of glucose. An increase of glucose concentration in treatment at 35 o C caused an increase of decolorizing efficiency, while treatment at 55 o C made no further difference. Figure 4 - Decolorizing efficiencies of MO with variation of glucose concentration under mesophilic (A) and thermophilic (B) 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour (s) Decolorization (%) 0 g/L 0.5 g/L 1.0 g/L 2.0 g/L 3.0 g/L 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour (s) Decolorization (%) 0 g/L 0.5 g/L 1.0 g/L 2.0 g/L 3.0 g/L A B Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 14 - Figure 5 - Decolorizing efficiencies of RB4 with variation of glucose concentration under mesophilic (A) and thermophilic (B) Effect of dyes and temperatures on dye decolorization As in the previous described experiments, these results confirmed that decolorization of MO and RB4 could be accelerated by treatment under thermophilic conditions (55 o C) and gave better results for decolorizing potential, especially in respect of RB4 decolorization when compared with mesophilic treatment. However, the effect of dye and temperature on decolorization is still not understood. In this experiment, the decolorizing efficiency was examined, and other factors including concentration of VFAs, gas production, TOC reduction, together with the reduction of dye were investigated. The obtained results of decolorization of 100mgL -1 of RB4 and 200mgL -1 of MO used in the experiment were compared with a control having no dye addition. Results showed that more than 95-98% of MO was decolourised under both mesophilic and thermophilic conditions during 72 hours of incubation. Comparison with the rate of decolorization of MO showed that thermophilic treatment gave a higher rate than under mesophilic conditions (Figure 6). The intermediate or reduced form of MO showed an auto-oxidizing effect when exposed to air in which color was increased in intensity from clear to pink and dark violet at a density of Abs 550 (Figure 7). An increase in color density corresponded to the initial concentration of MO and the time of exposure. However, auto-oxidation could be prevented by an addition of ascorbic acid. The high rate of decolorization that occurred in treatment under thermophilic conditions is an efficient approach that distinctly enhances electron transfer and subsequent dye reduction in anaerobic treatment. For RB4, a high decolorizing efficiency was obtained under thermophilic conditions, i.e., about 80% decolorization compared with mesophilic conditions, and the decolorizing efficiency was 70% when the initial concentration of RB4 was 100 mg · L -1 (Figure 8), which confirmed the acceleration by an increase of treatment temperature. Reducing byproducts of RB4 did not show any auto-oxidizing reaction, and treated waste-water containing RB4 was in light yellow occurred by presenting of unsubstituted antraquinone as its reduced form (Figure 9). The reduction of anthraquinone dye takes place by mechanism of reversible quinone reduction to hydroquinone in a two step, Benzoquinone <=> Semiquinone <=> Hydroquinone form (Zollinger, 1991). In general, under anaerobic conditions a lower rate and extent of decolorization of anthraquinone dyes has been observed as compared to azo dye, which corresponds to other presented results (Delle et al., 1998; Panswad and Luangdilok, 2000). 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour(s) Decolorization (%) 0 g/L 0.5 g/L 1.0 g/L 2.0 g/L 3.0 g/L 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 36 42 48 Hour (s) Decolorization (%) 0 g/L 0.5 g/L 1.0 g/L 2.0 g/L 3.0 g/L A B Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 15 - Figure 6 - Decolorizing efficiencies of MO Figure 7 - Wavelength scanning of under 35 O C ( ■ ) and 55 O C ( □ ) untreated ( ■ ) and treated ( □ ) MO and apparent color Figure 8 - Decolorizing efficiencies of RB4 Figure 9 - Wavelength scanning of under 35 O C ( ■ ) and 55 O C ( □ ) untreated ( ■ ) and treated ( □ ) RB4 and apparent color Inhibition by temperature increase and presence of dye was associated with inhibition of organic matters conversion pathways caused by of the accumulation of volatile fatty acids in the treatment system. In the case of temperature increase, Figure 10 showed the difference of organic matter conversion pathways under mesophlilic and thermophilic conditions. Under mesophilic conditions, the conversion was the degradation of glucose to intermediate products (acetate, butyrate, propionate, etc.) by acedogens and acetogens to be used as substrate for methane production. Whereas under thermophilic conditions, the major pathway was the hydrogen production by hydrogenotrophic microorganism combined with the minor pathway of VFAs production which is indicated by the high volume of H 2 production and low VFAs concentration produced (Table1). For dye itself, sugar conversion to fatty acid and methane (CH 4 ) productivity were inhibited by the presence of MO which resulted in slow reduction of TOC, while the presence of RB4 inhibited methane productivity. TOC reduction of treatment of RB4 was similar to the control (Figure 11). Due to the increase in treatment temperatures (thermophilic conditions), advantages were shown in the increasing of decolorizing efficiency on both dyes. As previously mentioned, the methane producing bacteria were inhibited or inactivated by the presence of dye, but at the same time the intermediate products were utilized by other groups of anaerobic bacteria, such as sulfur reducing -20 -10 0 10 20 30 40 50 60 70 80 90 100 0 122436486072 Hour (s) Decolorization (%) MO(35°C) MO(55°C) 0 10 20 30 40 50 60 70 80 90 100 0 122436486072 Hour (s) Decolorization (%) RB4(35°C) RB4(55°C) 0 0.5 1 1.5 2 2.5 300 350 400 450 500 550 600 650 Wavelength Ab s Va lue Untreated Treated (Auto-ox idant) Untreated MO Treated MO Treated MO (Auto- oxidatio n) 0 0.5 1 1.5 2 2.5 300 350 400 450 500 550 600 650 Wavelength Ab s Va lue Untreated Treated (Auto-ox idant)Untreated Treated (Auto-ox idant) Untreated MO Treated MO Treated MO (Auto- oxidatio n) 0 0.5 1 1.5 2 2.5 300 350 400 450 500 550 600 650 Wavelength Ab s Va lue Untreated Treated Treated RB4 Untreated RB4 0 0.5 1 1.5 2 2.5 300 350 400 450 500 550 600 650 Wavelength Ab s Va lue Untreated TreatedUntreated Treated Treated RB4 Untreated RB4 Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 16 - bacteria. This led to low CH 4 production and a high rate of TOC reduction during the 72 hours of experiment. After dye decolorization occurred, the CH 4 productivity recovered, as shown in Table 1. Inhibition of decolorizing microorganisms by textile dye and its intermediates has been reported. Young (2004) reported that VFAs accumulation was mainly in the form of acetate and propionate with traces of iso-butyric, n-butyric and iso-valeric when RB4 or RB19 was amended in culture (Lee et al., 2004). Melpei et al. (1998) reported that methanogenic culture amended with 250-300mg · L -1 of Brilliant Red Resolin (BLS) showed 78.9% inhibition of specific methane yield and 59.6% production via aceticlastic methanogenesis (Melpai et al., 1998). Table 1 - Results of Batch decolorization of MO and RB4 by anaerobic treatment under mesophilic and thermophilic conditions at the 72 nd and 168 th hour of experimentation 72 nd hour 168 th hour VFAs (mgCODL -1 ) Gas Prod. (mlL -1 ) % Deco. HAc HPr i-HBu n-HBu TOC Removal (%) CH 4 H 2 CH 4 production (mlL -1 )* Mesophilic condition (35 O C) Control - 254.67 220.29 19.35 46.36 72.32 4.8 1.22 13.41 MO 98.73 298.79 289.31 3.71 7.57 52.17 1.6 3.22 14.73 RB4 73.72 336.74 115.70 10.22 151.96 73.15 0.67 2.40 15.43 Thermophilic condition (55 O C) Control - 240.44 17.77 0 7.56 70.56 3.72 12.48 2.64 MO 98.48 109.44 47.76 2.72 3.47 46.25 0.7 8.42 0.18 RB4 83.82 192.28 28.56 9.54 33.14 63.43 0.48 1.31 3.57 * CH 4 production was the total production during the 72 nd to 168 th hour of experiment. Figure 10 - Suggestion of organic matter conversion pathway under 35ºC and 55ºC To reveal the contribution of microorganism group effect on dye decolorization, vancomycin (1g·L -1 ) and 2-bromoethane sulfonic acid (BES, 10.2g·L -1 ) were used as acidogens and methanogens inhibitors, respectively. The results shown in Figure 12 indicate that decolorization of MO was associated with both acidogens and Figure 11 - TOC reductions of controls and treatments under mesophilic and thermophilic condition 0 100 200 300 400 500 600 0 122436486072 Hours TOC (ppm) C35 C55 RB35 RB55 MO35 MO55 Figure 11 - TOC reductions of controls and treatments under mesophilic and thermophilic condition 0 100 200 300 400 500 600 0 122436486072 Hours TOC (ppm) C35 C55 RB35 RB55 MO35 MO55 Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 17 - methanogens. The reduction mechanism of MO not only related to organic matter conversion, but also to enzymatic reduction reported by other researchers. Azoreductase was known as soluble cytoplasmic enzymes catalyse the process of reductive cleavage of the azo linkage. It facilitates the transfer of electrons via soluble flavin to the azodye, which is then reduced (McMullan et al., 2001). However, the main important microorganism in MO decolorization was acidogens due to high decolorizing efficiency compared with methanogens. For RB4 decoloriZation, the results clearly indicated that acidogens were important in decolorizing mechanism in which high decolorizing efficiency occurred when only using glucose as co-substrate. The decolorizing efficiencies of RB4 using acetate as co-substrate for acidogens and in treatments of methanogens (vancomycin addition) occurred by dye absorption or accumulation, as discussed previously. These indicated that the reduction of RB4, anthraqionone form to hydroquinone was related to H + generated from the organic matter conversion process, and reductive transformation of anthraquinone nucleus (Revenga et al., 1994). Figure 12 - Decolorizing efficiencies of MO (A) and RB4 (B) decolorizations under meso- and thermophilic conditions employed with Vancomycin (acidogens inhibitor) and BES (methanogens inhibitor) CONCLUSIONS The efficiency of MO and RB4 decolorization by mesophilic and thermophilic treatment indicated that the increase of treatment temperature could accelerate the decolorizing efficient of both dyes. The presence of high concentrations of dye in batch treatment did not show a limitation in dye decolorization conducted at 55 o C. The presence of glucose or electron donor substrate was needed for decolorizing reaction, especially in the case of RB4. In addition, differentiation aspect of MO and RB4 in need of electron donor substrate indicated that the decolorization of MO may not only associated with electron transfer mechanism of glucose but is also associated with extracellular component excreted from microorganisms. The effect of temperature at 55 o C did not show an inhibition of dye decolorizing efficiency, but the effect on inhibition of methanogens activity was confirmed by the accumulation of VFAs and low CH 4 production with high TOC removal which was utilized by other groups of anaerobic bacteria such as sulfur reducing bacteria. Acidogens were the main microorganism group in the decolorizing process, as confirmed by the employment of acidogens and methanogens inhibitors, vancomycin and BES. The inhibition dyes occurred through the effect on metabolic pathway of organic conversion, however after 0 10 20 30 40 50 60 70 80 90 100 BES Vancomycin Inhibitors % Decolorization Glucose 35C Glucose 55C Acetate 35C Acetate 55C 0 10 20 30 40 50 60 70 80 90 100 BES Vancomycin Inhibitors % Decolorization Glucose 35C Glucose 55C Acetate 35C Acetate 55C A B Decolorization (%) Decolorization (%) Journal of Water and Environment Technology, Vol. 6, No.1, 2008 - 18 - the dye was completely decolorized, microorganism activity could be recovered, as observed from recovery of methane productivity. ACKNOWLEDGEMENT This study was carried out under the JSPS-NRCT Scientific Cooperation Program and supported by a Japanese government scholarship (MOMBUKAGAKUSHO: MEXT). REFERENCES Beydilli I.M., Pavlostathis S.G. and Tincher W.C. (2000) Biological decolorization of the azo dye Reactive Red 2 under various oxidation-reduction conditions. Wa t e r Environ. Res., 72, 692-705. 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Dyes Pigments, 37, 335-356. Willets J.R.M. and Ashbolt N.J. (2000) Understanding anaerobic decolorization of textile dyes wastewater: mechanism and kinetics. Water Sci. Technol., 42, 409-416. Zollinger H. (1991) Color chemistry, 2 nd edn, VHC publishers, Inc.New York, USA. . 254 .67 220. 29 19. 35 46. 36 72.32 4.8 1.22 13.41 MO 98 .73 298 . 79 2 89. 31 3.71 7.57 52.17 1 .6 3.22 14.73 RB4 73.72 3 36. 74 115.70 10.22 151. 96 73.15 0 .67 2.40. - 240.44 17.77 0 7. 56 70. 56 3.72 12.48 2 .64 MO 98 .48 1 09. 44 47. 76 2.72 3.47 46. 25 0.7 8.42 0.18 RB4 83.82 192 .28 28. 56 9. 54 33.14 63 .43 0.48 1.31 3.57