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Removal of a copper-phthalocyanine dye from wastewater by acclimated sludge under anaerobic or aerobic conditions
Process Biochemistry 37 (2002) 1151– 1156 www.elsevier.com/locate/procbio
Removal of a copper-phthalocyanine dye from wastewater by acclimated sludge under anaerobic or aerobic conditions Li-yan Fu, Xiang-hua Wen *, Li-jie Xu, Yi Qian En6ironmental Simulation and Pollution Control, Department of En6ironmental Science and Engineering, Tsinghua Uni6ersity, Beijing 100084, People’s Republic of China Received 21 July 2001; received in revised form 29 October 2001; accepted 8 November 2001
Abstract Using acclimated sludge, the removal of a copper-phthalocyanine dye (Reactive Turquoise Blue KN-G, RTB) from wastewater was studied under anaerobic and aerobic conditions to assess the fate of the RTB in wastewater treatment facilities. An immediately sharp reduction of the RTB concentration in solution was observed after the influent was fed into the reactor, which reflects rapid biosorption. Aerobic microorganisms were capable of utilizing RTB as the sole carbon and energy source for proliferation while anaerobic microorganisms were not, however, sufficient glucose as ancillary carbon source was essential for both systems to maintain long-term, stable and efficient performance. Glucose promoted the growth of biomass and microbial activity. Sufficient glucose prevented aerobic microorganisms decreasing activity due to high-concentration RTB. The cooperation of biosorption and biodegradation were considered to be the mechanisms for RTB removal from wastewater. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phthalocyanine dye; Reactive Turquoise Blue; Anaerobic biological treatment; Aerobic biological treatment; Biosorption; Biodegradation
1. Introduction Dyestuffs are generally synthetic organic compounds with complex molecular structures and large molecular weights. These properties augment treatment difficulties of dye wastewater [1]. Furthermore, it has been reported that dye wastewater is poisonous, carcinogenic and teratogenic to human beings [2]. Therefore, the efficient disposal of dyestuff in wastewater has attracted wide attention [3]. Both anaerobic biological treatment and aerobic biological treatment have been applied in treating dye wastewater [4 – 7]. In recent years, water-soluble reactive dyes have become one of the most important dyes applied to cotton fabric. Its production has shown a continuous increase abroad and accounts for 15 to 25 percent of the total output in some countries. In China, domestic reactive dyes are used in the textile industry and dye printing; * Corresponding author. Tel.: + 86-10-627-85684; fax: + 86-10627-71472. E-mail address: [email protected] (X.-h. Wen).
moreover, the amount of imported reactive dyes is increasing rapidly and heads the list of all imported dyes. The increased application of reactive dyes has propelled the study on the treatment of reactive dye wastewater and a series of work has been performed on the effects of biological treatment [8,9]. Up to date, biological decolorization of azo-type and anthraquinone-type reactive dyes have been extensively documented [6,10], while studies on another important type of reactive dyes, phthalocyanine-type reactive dyes, have seldom been reported. Therefore, the fate of a phthalocyanine dye in a wastewater treatment facility remained unclear. Reactive Turquoise Blue KN-G (C. I. Reactive Blue 21, RTB), which is utilized as a major dyestuff at a textile mill in Beijing, was employed in this study to investigate the biological decolorization of a phthalocyanine dye. The decolorization of RTB by biodegradation had been reported by Conneely et al. [11]. A strain of fungus was used in his study. Our attention was focused on the decolorization of RTB by the microorganisms in the sludge collected from the
0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 3 3 4 - X
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
1152
existing wastewater treatment system. In this study, batch tests simulating an actual biological treatment process were carried out under anaerobic and aerobic conditions to observe if RTB can be removed from wastewater and to determine the appropriate conditions for RTB removal. The mechanism for RTB removal was also a concern of this paper. 2. Methods and materials
2.1. Dyestuff property Reactive Turquoise Blue KN-G has a structure similar to that shown in Fig. 1. Its parent moiety is copperphthalocyanine (denoted by CuPc in the structural formula). The exact structure of x and y remains unknown. It is miscible in water and the solubility is above 50 g/l at 50 °C.
2.2. Sludge Aerobic sludge was obtained from the final sedimentation tank of the wastewater treatment facilities in the above-mentioned textile mill in Beijing and anaerobic sludge was collected from the mesophilic anaerobic digester at the Gao Bei Dian municipal sewage treatment plant. Both sludges had been acclimated for 90 days with RTB containing wastewater before they were seeded to each reaction flask to make 4 g/l biomass concentration.
2.3. Influent composition The synthetic wastewater contained RTB, glucose as supplemental carbon sources, KH2PO4 (47.5 mg/l) as phosphorus source, urea (40 mg/l) as nitrogen source and some mineral salts including 59.3 mg/l MgSO4, 5.7 mg/l MnSO4, 0.3 mg/l FeSO4, 5.7 mg/l CaCl2 and 100 mg/l NaHCO3.
2.4. Experimental system Tests under anaerobic conditions were performed in static conical flasks. Aerobic shake flask tests took place in an incubator shaker set at 160 rpm, and the dissolved oxygen concentration in the shallow reaction mixture was maintained at 6.6– 6.8 mg/l with the help of the air exchange through gauze flask covers during shaking. The temperature of both anaerobic and aerobic tests was held at 30 °C.
Fig. 1. The structure of RTB.
2.5. Analysis Daily analyses included the RTB concentration in the samples of raw and treated wastewater. RTB concentration was determined by measuring the absorbance at 623 nm using a Shimadzu UV-1200V Spectrophotometer. A calibration curve relating RTB concentration and absorbance was constructed. All samples were measured after filtration through a 0.45 mm filter, which would separate any suspended solid from the supernatant. An ultraviolet–visible scan indicated that no other dissolved component in the influent interfered with RTB measurements. Volatile suspended solid (VSS) measured by calculating the loss of sludge before and after heating at 550 °C was assumed to be biomass.
2.6. Time-dependent decolorization of RTB In this test, the decolorization process of RTB was observed under anaerobic and aerobic conditions by measuring the time-varying RTB concentration in the supernatant. Each reaction flask contained 200 ml mixed liquor consisting of the influent and the sludge. RTB (20 mg/l) was the sole carbon source in the influent, and it was added only at the start-up of the test. Other nutrients were supplied to microorganisms each day. The first sample was taken 1 min after the start-up. Other samples were taken at intervals of 0.5 days within the first 2.5 days, and then at intervals of 1 day over the subsequent 7 days.
2.7. Influence of carbon source on the growth rate of biomass A batch test was conducted to observe the relationship between the growth rate of biomass and carbon source. Each reaction flask, which contained 120 ml of the mixed liquor, was operated in a 24-h cycle consisting of instantaneous filling, 20 h reaction, 4 h settling and instantaneous drainage. Forty milliliters of supernatant was changed with fresh wastewater at the start of each cycle. Three test groups separately treated the influent containing 0, 0.2 or 0.5 g/l glucose. RTB concentration in the influent was strictly controlled to guarantee an equal effect on the microorganisms of each test group, so that any difference in the biomass growth rate resulted from the different glucose concentration. The test lasted for 21 days. VSS was measured before and after the test to calculate the growth rate of biomass. The gross amount of RTB added to the reactor was less than 2% of the seed biomass, therefore, the possible interference on VSS measurement that resulted from the adsorption of RTB on the sludge was omitted.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156 Table 1 Test conditions for determining the influence of glucose and RTB concentrations on RTB removal No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Anaerobic conditions
Aerobic conditions
RTB (mg/l)
Glucose (mg/l)
RTB (mg/l)
Glucose (mg/l)
20 20 20 20 50 50 50 50 75 75 75 75 100 100 100 100
50 400 800 1200 50 400 800 1200 50 400 800 1200 50 400 800 1200
20 20 20 20 50 50 50 50 75 75 75 75 100 100 100 100
500 800 1200 1500 500 800 1200 1500 500 800 1200 1500 500 800 1200 1500
1153
test. The observation shown in Fig. 2 was in fact a process from adsorption to adsorption equilibrium, and then to desorption. Desorption took place probably because insufficient carbon source caused an impairment of the activity of the microorganisms including adsorbability. The RTB adsorbed on the anaerobic sludge came not only from this test, but also from the process of sludge acclimation, since there was some RTB present on the seed sludge after the 90-day acclimation and before it was added to the reactor. As a result, the actual amount of RTB released due to desorption by the inactivated microorganisms exceeded the adsorbance from the influent of this test, which eventually led to the RTB concentration in the liquid phase increasing above the initial value. In contrast, the RTB concentration in the liquid phase decreased significantly in the aerobic test. It fell by 37.5% within 24 h and by 54.8% at the end of the 48th hour, which was followed by a temporary equilibrium from the 48th to the 60th hour. Then, RTB concentration continued to decrease until in excess of 80% of RTB was removed from the liquid phase. This
2.8. Influence of glucose and RTB concentrations on RTB remo6al Two-factor and four-level comprehensive tests (16 groups) were separately carried out under anaerobic and aerobic conditions. The operating cycle was the same as that described in Section 2.7. The test conditions are listed in Table 1. Each test condition was repeatedly conducted until the experimental system reached the steady state and produced almost consistent effluent, which in general needed 8–9 cycles. The mean of the RTB removal rates during steady-state performance was employed to analyze the influences of glucose and RTB concentrations on RTB removal.
3. Results and discussion
3.1. RTB decolorization under anaerobic and aerobic conditions Fig. 2 shows the concentration profiles of RTB in the liquid phase with time during a 10-day test. Under anaerobic conditions, the RTB concentration was lowered by 29.5%, 12 h after start-up and remained stable for the following 48 h. However, it increased gradually from the 84th hour to greater than the initial value. Due to the complex molecular structure of RTB, it is difficult for the microorganisms to utilize RTB as carbon and energy sources in a short time. Therefore, biosorption is the main reason for the reduction of RTB concentration in the early stages of the anaerobic
Fig. 2. The time-dependent removal of RTB under anaerobic and aerobic conditions.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
1154
Fig. 3. The growth rate of biomass at different glucose concentrations.
phenomenon illustrated that aerobic sludge was effective with RTB as the sole carbon source. The early decrease of RTB concentration was due to biosorption, while its further reduction after adsorption equilibrium probably resulted from the utilization by aerobic microorganisms. This indicated that RTB was biodegradable under aerobic conditions as opposed to anaerobic conditions.
3.2. Influence of carbon source on the growth rate of biomass The effect of carbon source on microbial growth was studied in a 21-cycle batch test. The growth rate of biomass was calculated according to the following equation. R=
Xi − X0 X0
In the equation, R is the growth rate of biomass; X0 and Xi, respectively, denote the biomass before and after the batch test of 21 cycles. The biomass in the aerobic sludge increased when there was only RTB in the influent, which proved that aerobic microorganisms could utilize RTB as the sole carbon and energy source for proliferation (Fig. 3). In other words, RTB could be degraded under aerobic conditions and this agrees with the results reported in Section 3.1. In addition, glucose as additional carbon source could increase the growth of aerobic microorganisms. The growth of biomass in anaerobic sludge, however, was strongly dependent on glucose. No glucose and even low-concentrations of glucose led to lower biomass, from which it was concluded that the anaerobic microorganisms could not maintain good activity without sufficient glucose in the RTB containing wastewater.
Fig. 4. The influence of glucose concentration on RTB removal rate under anaerobic conditions.
3.3. Influence of glucose and RTB concentrations on RTB remo6al 3.3.1. Influence of glucose concentration on RTB remo6al efficiency From the analyses in Sections 3.1 and 3.2, when RTB was the sole carbon source in the wastewater, it is clear that a long-term run was not possible in an anaerobic treatment facility. Although aerobic sludge could decolorize and even utilize RTB, it would take too much time to achieve a high removal rate. Addition of an extra carbon source might help to obtain an increased RTB removal rate and maintain a continuous and steady run. Glucose was used in this test to observe the effect of an extra carbon source on RTB removal. Figs. 4 and 5, respectively, show the relationship between glucose concentration and RTB removal rate for anaerobic and aerobic sludge. The RTB removal rate was directly proportional to glucose concentration
Fig. 5. The influence of glucose concentration on RTB removal rate under aerobic conditions.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
Fig. 6. The biological activity of anaerobic sludge on RTB removal.
at each RTB concentration, that is, the enhancement of glucose concentration led to an increased RTB removal rate under both anaerobic and aerobic conditions. Glucose is a readily biodegradable carbon and energy source and sufficient glucose promoted microbial proliferation, thus producing more bacterial cells capable of removing RTB. Another possible effect of glucose was that it could enhance microbial activity including biosorption and even utilization of RTB. Both of the above causes resulted in increased RTB removal rate.
3.3.2. Influence of RTB concentration on microbial acti6ity The system treating RTB containing wastewater will fail in long-term stability if RTB has a negative effect on microbial activity. The specific removal of RTB (RTB removed per unit sludge) was taken as the indicator for microbial activity at different glucose and RTB concentrations.
Fig. 7. The biological activity of aerobic sludge on RTB removal.
1155
Fig. 6 shows a step-up curve for specific RTB removal versus RTB concentration. At glucose concentrations of higher than and equal to 0.5 g/l, no inhibition of microbial activity was discovered in the anaerobic process treating the influent containing 20– 100 mg/l RTB. With aerobic sludge (see Fig. 7), 100 mg/l RTB was observed to inhibit biological activity at lower glucose concentrations such as 0.05 and 0.4 g/l, while such inhibition disappeared at higher glucose concentration. From this phenomenon, it was concluded that sufficient carbon source was necessary for the maintenance of the aerobic system treating a highconcentration RTB wastewater.
3.3.3. Comparison of RTB remo6al capability between anaerobic and aerobic sludge Specific RTB removal of anaerobic and aerobic sludge at the same RTB and glucose concentration is shown in Fig. 8. Anaerobic sludge was of higher capability on RTB removal than aerobic sludge. Only aerobic sludge had been shown to be able to utilize RTB as carbon source, and there was still no actual evidence indicating that RTB was biodegradable under anaerobic conditions. However, it was possible that anaerobic microorganisms were capable of decomposing RTB with the help of an additional carbon and energy source, since anaerobic sludge maintained stable and high RTB removal rates in the multicyclic test. Further study was necessary to test this assumption.
4. Conclusions Laboratory tests were carried out to simulate the actual process after RTB was fed into an existing wastewater treatment system. It was feasible for both anaerobic and aerobic treatment to achieve stable and efficient removal of RTB in long-term operation, but there must be sufficient ancillary carbon and energy source in the wastewater. The amount of ancillary carbon source depended on the RTB concentration in the influent and the requirement for effluent quality. Biosorption is the first stage of the process of removing RTB, since a relatively long time was needed in the event of possible degradation due to the complex structure of RTB. However, the time-consuming biodegradation on RTB was not negligible for maintaining stable and efficient treatment. The cooperation of biosorption and utilization in RTB degradation occurred in a long-term-run system.
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Fig. 8. The biological activity of RTB removal in anaerobic and aerobic sludges.
Acknowledgements This work is supported by the Natural Science Foundation of China (59878025).
References [1] Banat IM, Nigam P, Singh D, Marchant R. Microbial decolorization of textile-dye-containing effluents: a review. Bioresour Technol 1996;58:217 –27. [2] Azmi Wamik, Sani Rajesh Kumar, Banerjee Uttam Chand. Biodegradation of triphenylmethane dyes. Enzyme Microb Technol 1998;22:185 – 91. [3] Walker GM, Weatherley LR. Biodegradation and biosorption of acid anthraquinone dye. Environ Pollut 2000;108(2):219 – 23. [4] Brown D. The degradation of dyestuffs: Part I Primary biodegradation under anaerobic condition. Chemosphere 1983;12(3):397 – 404. [5] Zissi U, Lyberatos G. Azo-dye biodegradation under anoxic
conditions. Water Sci Technol 1996;34(5-6):495 – 500. [6] Beydilli MI, Pavlostathis SG, Tincher WC. Decolorization and toxicity screening of selected reactive azo dyes under methanoaenic conditions. Water Sci Technol 1998;38(4-5):225 – 32. [7] Wei Chao-hai, Xie Bo, Wu Chao-fei. High-concentration dyeing wastewater treatment engineering: process condition and case analysis. Chongqing Environ Sci 1998;20(5):14 – 7 (in Chinese). [8] Gu Ping, Yu Zhensheng, Yang Zaoyan. Biodegradation of reactive dye KBR using batch reactor under anaerobic condition. Acta Sci Circumstanttiae 1999;19(1):105 – 9 (in Chinese). [9] Zhang Bo, Zhang Su-rong. Decoloration of photosynthetic bacteria on reactive red X-3B and its relation with plasmid. Urban Environ Urban Ecol 1999;12(1):13 – 5 (in Chinese). [10] Peralta-Zamora Patricio, Kunz Airton, Gomes de Moraes Sandra, Ronaldo, Pelegrini, et al. Degradation of reactive dyes I. A comparative study of ozonation, enzymatic and photochemical processes. Chemosphere 1999;38(4):835 – 52. [11] Conneely A, Smyth WF, McMullan G. Metabolism of the phthalocyanine textile dye remazol turquoise blue by Phanerochaete chrysosporium. FEMS Microbiol Lett 1999;179(2):333 – 7.
Removal of a copper-phthalocyanine dye from wastewater by acclimated sludge under anaerobic or aerobic conditions Li-yan Fu, Xiang-hua Wen *, Li-jie Xu, Yi Qian En6ironmental Simulation and Pollution Control, Department of En6ironmental Science and Engineering, Tsinghua Uni6ersity, Beijing 100084, People’s Republic of China Received 21 July 2001; received in revised form 29 October 2001; accepted 8 November 2001
Abstract Using acclimated sludge, the removal of a copper-phthalocyanine dye (Reactive Turquoise Blue KN-G, RTB) from wastewater was studied under anaerobic and aerobic conditions to assess the fate of the RTB in wastewater treatment facilities. An immediately sharp reduction of the RTB concentration in solution was observed after the influent was fed into the reactor, which reflects rapid biosorption. Aerobic microorganisms were capable of utilizing RTB as the sole carbon and energy source for proliferation while anaerobic microorganisms were not, however, sufficient glucose as ancillary carbon source was essential for both systems to maintain long-term, stable and efficient performance. Glucose promoted the growth of biomass and microbial activity. Sufficient glucose prevented aerobic microorganisms decreasing activity due to high-concentration RTB. The cooperation of biosorption and biodegradation were considered to be the mechanisms for RTB removal from wastewater. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phthalocyanine dye; Reactive Turquoise Blue; Anaerobic biological treatment; Aerobic biological treatment; Biosorption; Biodegradation
1. Introduction Dyestuffs are generally synthetic organic compounds with complex molecular structures and large molecular weights. These properties augment treatment difficulties of dye wastewater [1]. Furthermore, it has been reported that dye wastewater is poisonous, carcinogenic and teratogenic to human beings [2]. Therefore, the efficient disposal of dyestuff in wastewater has attracted wide attention [3]. Both anaerobic biological treatment and aerobic biological treatment have been applied in treating dye wastewater [4 – 7]. In recent years, water-soluble reactive dyes have become one of the most important dyes applied to cotton fabric. Its production has shown a continuous increase abroad and accounts for 15 to 25 percent of the total output in some countries. In China, domestic reactive dyes are used in the textile industry and dye printing; * Corresponding author. Tel.: + 86-10-627-85684; fax: + 86-10627-71472. E-mail address: [email protected] (X.-h. Wen).
moreover, the amount of imported reactive dyes is increasing rapidly and heads the list of all imported dyes. The increased application of reactive dyes has propelled the study on the treatment of reactive dye wastewater and a series of work has been performed on the effects of biological treatment [8,9]. Up to date, biological decolorization of azo-type and anthraquinone-type reactive dyes have been extensively documented [6,10], while studies on another important type of reactive dyes, phthalocyanine-type reactive dyes, have seldom been reported. Therefore, the fate of a phthalocyanine dye in a wastewater treatment facility remained unclear. Reactive Turquoise Blue KN-G (C. I. Reactive Blue 21, RTB), which is utilized as a major dyestuff at a textile mill in Beijing, was employed in this study to investigate the biological decolorization of a phthalocyanine dye. The decolorization of RTB by biodegradation had been reported by Conneely et al. [11]. A strain of fungus was used in his study. Our attention was focused on the decolorization of RTB by the microorganisms in the sludge collected from the
0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 3 3 4 - X
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
1152
existing wastewater treatment system. In this study, batch tests simulating an actual biological treatment process were carried out under anaerobic and aerobic conditions to observe if RTB can be removed from wastewater and to determine the appropriate conditions for RTB removal. The mechanism for RTB removal was also a concern of this paper. 2. Methods and materials
2.1. Dyestuff property Reactive Turquoise Blue KN-G has a structure similar to that shown in Fig. 1. Its parent moiety is copperphthalocyanine (denoted by CuPc in the structural formula). The exact structure of x and y remains unknown. It is miscible in water and the solubility is above 50 g/l at 50 °C.
2.2. Sludge Aerobic sludge was obtained from the final sedimentation tank of the wastewater treatment facilities in the above-mentioned textile mill in Beijing and anaerobic sludge was collected from the mesophilic anaerobic digester at the Gao Bei Dian municipal sewage treatment plant. Both sludges had been acclimated for 90 days with RTB containing wastewater before they were seeded to each reaction flask to make 4 g/l biomass concentration.
2.3. Influent composition The synthetic wastewater contained RTB, glucose as supplemental carbon sources, KH2PO4 (47.5 mg/l) as phosphorus source, urea (40 mg/l) as nitrogen source and some mineral salts including 59.3 mg/l MgSO4, 5.7 mg/l MnSO4, 0.3 mg/l FeSO4, 5.7 mg/l CaCl2 and 100 mg/l NaHCO3.
2.4. Experimental system Tests under anaerobic conditions were performed in static conical flasks. Aerobic shake flask tests took place in an incubator shaker set at 160 rpm, and the dissolved oxygen concentration in the shallow reaction mixture was maintained at 6.6– 6.8 mg/l with the help of the air exchange through gauze flask covers during shaking. The temperature of both anaerobic and aerobic tests was held at 30 °C.
Fig. 1. The structure of RTB.
2.5. Analysis Daily analyses included the RTB concentration in the samples of raw and treated wastewater. RTB concentration was determined by measuring the absorbance at 623 nm using a Shimadzu UV-1200V Spectrophotometer. A calibration curve relating RTB concentration and absorbance was constructed. All samples were measured after filtration through a 0.45 mm filter, which would separate any suspended solid from the supernatant. An ultraviolet–visible scan indicated that no other dissolved component in the influent interfered with RTB measurements. Volatile suspended solid (VSS) measured by calculating the loss of sludge before and after heating at 550 °C was assumed to be biomass.
2.6. Time-dependent decolorization of RTB In this test, the decolorization process of RTB was observed under anaerobic and aerobic conditions by measuring the time-varying RTB concentration in the supernatant. Each reaction flask contained 200 ml mixed liquor consisting of the influent and the sludge. RTB (20 mg/l) was the sole carbon source in the influent, and it was added only at the start-up of the test. Other nutrients were supplied to microorganisms each day. The first sample was taken 1 min after the start-up. Other samples were taken at intervals of 0.5 days within the first 2.5 days, and then at intervals of 1 day over the subsequent 7 days.
2.7. Influence of carbon source on the growth rate of biomass A batch test was conducted to observe the relationship between the growth rate of biomass and carbon source. Each reaction flask, which contained 120 ml of the mixed liquor, was operated in a 24-h cycle consisting of instantaneous filling, 20 h reaction, 4 h settling and instantaneous drainage. Forty milliliters of supernatant was changed with fresh wastewater at the start of each cycle. Three test groups separately treated the influent containing 0, 0.2 or 0.5 g/l glucose. RTB concentration in the influent was strictly controlled to guarantee an equal effect on the microorganisms of each test group, so that any difference in the biomass growth rate resulted from the different glucose concentration. The test lasted for 21 days. VSS was measured before and after the test to calculate the growth rate of biomass. The gross amount of RTB added to the reactor was less than 2% of the seed biomass, therefore, the possible interference on VSS measurement that resulted from the adsorption of RTB on the sludge was omitted.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156 Table 1 Test conditions for determining the influence of glucose and RTB concentrations on RTB removal No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Anaerobic conditions
Aerobic conditions
RTB (mg/l)
Glucose (mg/l)
RTB (mg/l)
Glucose (mg/l)
20 20 20 20 50 50 50 50 75 75 75 75 100 100 100 100
50 400 800 1200 50 400 800 1200 50 400 800 1200 50 400 800 1200
20 20 20 20 50 50 50 50 75 75 75 75 100 100 100 100
500 800 1200 1500 500 800 1200 1500 500 800 1200 1500 500 800 1200 1500
1153
test. The observation shown in Fig. 2 was in fact a process from adsorption to adsorption equilibrium, and then to desorption. Desorption took place probably because insufficient carbon source caused an impairment of the activity of the microorganisms including adsorbability. The RTB adsorbed on the anaerobic sludge came not only from this test, but also from the process of sludge acclimation, since there was some RTB present on the seed sludge after the 90-day acclimation and before it was added to the reactor. As a result, the actual amount of RTB released due to desorption by the inactivated microorganisms exceeded the adsorbance from the influent of this test, which eventually led to the RTB concentration in the liquid phase increasing above the initial value. In contrast, the RTB concentration in the liquid phase decreased significantly in the aerobic test. It fell by 37.5% within 24 h and by 54.8% at the end of the 48th hour, which was followed by a temporary equilibrium from the 48th to the 60th hour. Then, RTB concentration continued to decrease until in excess of 80% of RTB was removed from the liquid phase. This
2.8. Influence of glucose and RTB concentrations on RTB remo6al Two-factor and four-level comprehensive tests (16 groups) were separately carried out under anaerobic and aerobic conditions. The operating cycle was the same as that described in Section 2.7. The test conditions are listed in Table 1. Each test condition was repeatedly conducted until the experimental system reached the steady state and produced almost consistent effluent, which in general needed 8–9 cycles. The mean of the RTB removal rates during steady-state performance was employed to analyze the influences of glucose and RTB concentrations on RTB removal.
3. Results and discussion
3.1. RTB decolorization under anaerobic and aerobic conditions Fig. 2 shows the concentration profiles of RTB in the liquid phase with time during a 10-day test. Under anaerobic conditions, the RTB concentration was lowered by 29.5%, 12 h after start-up and remained stable for the following 48 h. However, it increased gradually from the 84th hour to greater than the initial value. Due to the complex molecular structure of RTB, it is difficult for the microorganisms to utilize RTB as carbon and energy sources in a short time. Therefore, biosorption is the main reason for the reduction of RTB concentration in the early stages of the anaerobic
Fig. 2. The time-dependent removal of RTB under anaerobic and aerobic conditions.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
1154
Fig. 3. The growth rate of biomass at different glucose concentrations.
phenomenon illustrated that aerobic sludge was effective with RTB as the sole carbon source. The early decrease of RTB concentration was due to biosorption, while its further reduction after adsorption equilibrium probably resulted from the utilization by aerobic microorganisms. This indicated that RTB was biodegradable under aerobic conditions as opposed to anaerobic conditions.
3.2. Influence of carbon source on the growth rate of biomass The effect of carbon source on microbial growth was studied in a 21-cycle batch test. The growth rate of biomass was calculated according to the following equation. R=
Xi − X0 X0
In the equation, R is the growth rate of biomass; X0 and Xi, respectively, denote the biomass before and after the batch test of 21 cycles. The biomass in the aerobic sludge increased when there was only RTB in the influent, which proved that aerobic microorganisms could utilize RTB as the sole carbon and energy source for proliferation (Fig. 3). In other words, RTB could be degraded under aerobic conditions and this agrees with the results reported in Section 3.1. In addition, glucose as additional carbon source could increase the growth of aerobic microorganisms. The growth of biomass in anaerobic sludge, however, was strongly dependent on glucose. No glucose and even low-concentrations of glucose led to lower biomass, from which it was concluded that the anaerobic microorganisms could not maintain good activity without sufficient glucose in the RTB containing wastewater.
Fig. 4. The influence of glucose concentration on RTB removal rate under anaerobic conditions.
3.3. Influence of glucose and RTB concentrations on RTB remo6al 3.3.1. Influence of glucose concentration on RTB remo6al efficiency From the analyses in Sections 3.1 and 3.2, when RTB was the sole carbon source in the wastewater, it is clear that a long-term run was not possible in an anaerobic treatment facility. Although aerobic sludge could decolorize and even utilize RTB, it would take too much time to achieve a high removal rate. Addition of an extra carbon source might help to obtain an increased RTB removal rate and maintain a continuous and steady run. Glucose was used in this test to observe the effect of an extra carbon source on RTB removal. Figs. 4 and 5, respectively, show the relationship between glucose concentration and RTB removal rate for anaerobic and aerobic sludge. The RTB removal rate was directly proportional to glucose concentration
Fig. 5. The influence of glucose concentration on RTB removal rate under aerobic conditions.
L.-y. Fu et al. / Process Biochemistry 37 (2002) 1151–1156
Fig. 6. The biological activity of anaerobic sludge on RTB removal.
at each RTB concentration, that is, the enhancement of glucose concentration led to an increased RTB removal rate under both anaerobic and aerobic conditions. Glucose is a readily biodegradable carbon and energy source and sufficient glucose promoted microbial proliferation, thus producing more bacterial cells capable of removing RTB. Another possible effect of glucose was that it could enhance microbial activity including biosorption and even utilization of RTB. Both of the above causes resulted in increased RTB removal rate.
3.3.2. Influence of RTB concentration on microbial acti6ity The system treating RTB containing wastewater will fail in long-term stability if RTB has a negative effect on microbial activity. The specific removal of RTB (RTB removed per unit sludge) was taken as the indicator for microbial activity at different glucose and RTB concentrations.
Fig. 7. The biological activity of aerobic sludge on RTB removal.
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Fig. 6 shows a step-up curve for specific RTB removal versus RTB concentration. At glucose concentrations of higher than and equal to 0.5 g/l, no inhibition of microbial activity was discovered in the anaerobic process treating the influent containing 20– 100 mg/l RTB. With aerobic sludge (see Fig. 7), 100 mg/l RTB was observed to inhibit biological activity at lower glucose concentrations such as 0.05 and 0.4 g/l, while such inhibition disappeared at higher glucose concentration. From this phenomenon, it was concluded that sufficient carbon source was necessary for the maintenance of the aerobic system treating a highconcentration RTB wastewater.
3.3.3. Comparison of RTB remo6al capability between anaerobic and aerobic sludge Specific RTB removal of anaerobic and aerobic sludge at the same RTB and glucose concentration is shown in Fig. 8. Anaerobic sludge was of higher capability on RTB removal than aerobic sludge. Only aerobic sludge had been shown to be able to utilize RTB as carbon source, and there was still no actual evidence indicating that RTB was biodegradable under anaerobic conditions. However, it was possible that anaerobic microorganisms were capable of decomposing RTB with the help of an additional carbon and energy source, since anaerobic sludge maintained stable and high RTB removal rates in the multicyclic test. Further study was necessary to test this assumption.
4. Conclusions Laboratory tests were carried out to simulate the actual process after RTB was fed into an existing wastewater treatment system. It was feasible for both anaerobic and aerobic treatment to achieve stable and efficient removal of RTB in long-term operation, but there must be sufficient ancillary carbon and energy source in the wastewater. The amount of ancillary carbon source depended on the RTB concentration in the influent and the requirement for effluent quality. Biosorption is the first stage of the process of removing RTB, since a relatively long time was needed in the event of possible degradation due to the complex structure of RTB. However, the time-consuming biodegradation on RTB was not negligible for maintaining stable and efficient treatment. The cooperation of biosorption and utilization in RTB degradation occurred in a long-term-run system.
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Fig. 8. The biological activity of RTB removal in anaerobic and aerobic sludges.
Acknowledgements This work is supported by the Natural Science Foundation of China (59878025).
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