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The potential of a sequential reactor system was evaluated using an Up-flow Column reactor (UFCR) containing the fungal strain, Aspergillus niger SA1 (immobilized on support material Scotch-BriteTM) and Aerobic Stirred Tank Reactor (STR) containing activated sludge for the bioremediation of a dye, Drimarene blue (Db) K2RL, in simulated textile effluent.The UFCR was operated at flow rate of 10 mL -1 with hydraulic retention (HRT) time of 10 h. The effluent of UFCR was fed continuously to STR (HRT: 144 h). Using UFCR (Run time: 240 h), decolorization and COD reduction of the effluent was observed maximum i.e., 100% decolorization and 63% COD reduction at 100 ppm of dye; however, further 13% COD reduction was observed in STR (Run time: 168 h). The UFCR and STR treatment leads to COD removal of 76%. While at 500 ppm of dye, UFCR resulted in 80% decolorization and 50% COD removal; however, further treatment in STR resulted in 1% color removal and 27% COD reduction. The combination of UFCR and STR treatment resulted in 81% decolorization and 77% COD removal. The results elucidated that majority of decolorization and COD reductions were achieved in UFCR, while the sludge in STR has limited capability of further dye treatment under aerobic conditions.
Keywords: Sequential reactor; Upflow column reactor; Stirred tank reactor; Aspergillus niger; Activated Sludge; Anthraquinone dye; decolorization; COD
Uncontrolled discharge of dye wastewaters into the environment can be harmful to humans, animals, plants and to urban ecosystems. Various types of pigments and synthetic dyes are widely used (Approximately 100,000 tons/year) worldwide in textile industries (Park et al., 2006). The effluents of these industries are extremely colored and the release of these wastes into receiving waters cause damages to the environment (Dogan et al., 2009). Color is normally noticeable at a dye concentration higher than 1 mg/l (Couto, 2009). Batik textile dye homemade industry is very well famous in Malaysia in the East Cost of Peninsular Malaysia and Sarawak. This industry has become very commercialized and contributed positively to the economic growth of Malaysia (Ali and suhaimi, 2009; Ahmad et al 2006). However color of batik textile dye effluent is unacceptable under Malaysian environmental regulation (Ali and suhaimi, 2009).
Reactive dyes are greatly water soluble, are nondegradable under the aerobic conditions found in conventional, biological treatment, and adsorb very poorly, resulting in remaining color in discharged effluents [Aspland, 2009]. Reactive dyes discharged into the environment are a source of concern because they: (1) tend water bodies to become colored, absorbing and reflecting sunlight, which in turn interferes with the aquatic life; and (2) may cause chronic and acute toxicity [Verma, 2008].
A variety of treatment methods have been developed for the elimination of dye from water, including coagulation (Salari et al. 2009), adsorption (Iqbal and Saeed, 2009), Ozonation (Khadhraoui et al, 2009), Flotation, reverse osmosis, ion exchange, membrane filteration and flocculation (Kim et al. 2004). Although physicochemical treatment methods are useful in dye removal, but the overall cost, regeneration problem, secondary pollutants, limit of versatility, interference with wastewater constituents and residual sludge limit their usage (Ertugrul et al., 2009; Kaushik and Malik, 2009).
In recent years a number of studies have been focused on some micro and macro-organisms that are capable to biosorb and biodegrade dyes in wastewaters. A sufficient variety of organisms are capable to decolorize a wide range of dyes include bacteria (Wu et al. 2009); fungi (Kaushik and Malik, 2009); yeasts (Ertugrul et al., 2009); and algae (Daneshvar et al., 2007; Khataee et al., 2009). Dyes are removed by fungi by biosorption [Ali et al. 2008], biodegradation [You et al. 2010] and enzymatic mineralization [Franciscon et al 2010].
Among the fungi, Aspergillus niger exhibit excellent adsorption capacities in removing dyes such as reactive dye C.I. Direct blue 199 ( Xiong et al. 2010) Drimarene blue K2RL (Siddiqui et al., 2009), azo dyes; acid red (AR) 151 and orange (Or) II ( Ali et al 2009) , and reactive black 8 [Kumari and Ibraham, 2007].
Activated sludge treatment of wastes is often an effective and economic system for reducing organic pollutants in wastewater (Zhang et al. 2007; Worch et al. 2002). A fair amount of research has been conducted assessing the viability of using activated sludge to treat textile effluents [Mohanty et al. 2006; Pagga and Brown, 1986].
Microbial cells immobilization has received increasing interest in the field of waste treatment. It offers a tremendous potential for the improvement of the efficiency of biological treatment process. Immobilized cells compared with free cells have various advantages (Wang, 2002): (1) it can increase the treatment rate through a higher cell loading; (2) the process can be controlled more easily; (3) the continuous process can take place at a high dilution rate without washout; (4) the stability of biocatalysts as well as the tolerance against toxic compounds can be improved. [Siddiqui et al. 2009]. Immobilized cell systems can degrade toxic compounds faster than conventional wastewater treatment systems, because high densities of microorganisms are used in immobilized cell systems. Application of immobilized living fungal strains have been proved more practical than the cell-free system, specifically when they expected to show adsorption as well as enzymatic capabilities of dyes degradation [Gao et al. 2008; Roject et al. 2004].
Several types of bioreactors have been used for treatment of dyes, however, most efficient decolorization achieved when fungal mycelium was immobilized in the reactor [Hao et al 2000; Melo and Oliveira, 2001]. Selecting a suitable reactor is vital in improving the economy and efficiency of immobilized cell process [Chen et al. 2003]. There is need of bioreactor system that can maintain production of high level of enzymes for long period together with controlled growth of fungi [Chang et al 2001; Zheng et al. 1999].
A considerable amount of research has already been done on the decolorization/degradation of azo dyes and their related products [Perey et al 2002], however, limited information exists in case of reactive anthraquinone dyes [Kaushik and Malik, 2009, Epolito et al 2005]. Therefore, there is need to explore effective and efficient treatment systems of microbial stains for the decolorization of anthraquinone dyes.
In present study a reactive Anthraquinone dye C.I. Reactive Blue 4 was used, which is known for it's markedly usage in textile industry. Due to their poor adsorbability to textile fiber has a higher exhaustion rate in wastewater. The main objective of our research work was to evaluate the potential of an Up-flow Column reactor (UFCR) containing the fungal strain, Aspergillus niger immobilized on support material Scotch-BriteTM (80% polyester and 20% nylon) and Activated sludge entraped in polyvinyl Alcohol gel ( PVA) for the treatment of a dye, C.I. Reactive blue 4, in simulated textile effluent.
2. Material and Methods
The majority of chemical compounds and investigated commercial dye C.I. Reactive Blue 4 (Fig. 1) were procured from Buch Sigma chemicals Co; St, Lois, E-Merck (Darmstadt, Germany).
Figure 1. Molecular structure of dye C.I. Reactive blue 4
2.2. Saboraud dextrose broth
Saboraud dextrose broth (Merck) was used for immobilization of fungal strains. It was made by adding per litre of distilled water; dextrose 40 g, and peptone 10 g. pH (5) of broth medium was adjusted by using 0.1 M HCl and NaOH. Agar (15 g l-1) was used as solidifying agent in the media when required in the experiments.
2.3. Composition of Simulated Textile Effluent
Simulated Textile Effluent (STE) was made by adding per liter of distilled water; Acetic Acid (99.9%) 0.15 ml, (NH2)2CO 108.0 mg, KH2PO4 67.0 mg, NaHCO3 840.0 mg, MgSO4. 7H2O 38.0 mg, CaCl2 21.0 mg, FeCl3 .6H2O 7.0 mg, glucose 860 mg [Luangdilok and Panswad, 2000], and C.I. Reactive blue 4 10 mg . pH (5) of effluent was adjusted by using 0.1 M HCl and NaOH.
2.4. Fungal and Immobilization support material
Fungal strain, Aspergillus niger local isolate was refreshed on Sabouraud dextrose agar medium at pH 5. Scotch-BriteTM (Spain) was used as immobilization support material (80% polyester and 20% nylon, color green).
2.6. Fungal Inoculum Preparation for Bioreactor
Fungal strain was grown on sabouraud dextrose agar (pH 5) plate for a week at 28 °C. The fungal spores were scratched and picked up with a loop from mature colony of fungal strain. The loop containing fungal spores was then dipped and mixed 15 times in 100 ml autoclaved distilled water containing 0.05 % Tween 80 soultion. After vigorous shaking, 1ml of the inoculum was poured on the hemocytometer and was observed under microscope. The observation showed average 7.35 x 103 spores per ml of inoculum of the strain. This inoculum was used for further experimentation. The whole process was carried out in aseptic conditions in laminar flow hood. The inoculum was stored in refrigerator at 4 °C.
2.7. Immobilization of fungal strain on support material
Scotch-BriteTM (Spain) was used as immobilization support material. Small pieces (size: 3x3 cm, thickness: 0.8 mm) of Scotch-BriteTM (Spain) were used as immobilization support material [Rodriguez, 2004]. These pieces were thoroughly washed with distilled water and sterilized in autoclave prior to use. Flask having 150 ml Sabouraud Dextrose broth (pH 5) and 15 pieces of Scotch-BriteTM (Spain) were added to it. It was inoculated with 10 ml spores suspension of Aspergillus niger and was placed in rotary shaker incubator (INNOVA TM 4330, New Brunswick Scientific) at 30 °C, 120 rpm for 1 week. The immobilized Scotch brite and without immobilization are shown in fig. 2.
Figure. 2. (A) Immobilized Aspergillus niger on Scotch brite (B) Scotch brite without
2.8. Sludge Sample collection, activation and immobilization
The activated sludge used this study was collected from aeration tank, in sterilized reagent bottle (Pyrex) having 1 L capacity from Kuala municipal sewage treatment plant in Kuala Perlis (Malaysia).
Ten gram of polyvinyl alcohol (PVA, nominal approx. molecular weight 75000- 80000) and 1 g of sodium alginate were dissolved in 50 ml of distilled water, the solution was cooled down to 40°C and then mixed thoroughly with 50 ml of concentrated activated sludge. The resulting mixture contained 10% (w/v) PVA, 1.0% (w/v) sodium alginate, and about 20 g/L of microorganisms. The following gelating solution was used to form gel beads (about 2 mm in diameter): the mixture was dropped into saturated boric acid and CaCl2 (1% w/v) solution and kept for 1 h to form gel beads, then transferring to 0.5 mol/L sodium orthophosphate solution and immersing for 1 h (Zhang et al. 2007) . The formed particles were washed with physiological saline solution for 1 h and then stored in distilled water at 4°C until further use.
2.9. Reactor configuration and Decolorization assays
A column reactor system was made, using the glass column. It was filled with immobilized pieces (Scotch Brite) of Aspergillus niger (45 Scotch Brite pieces) to a bed height of 7 inches. Column was connected at lower side inlet to tubing (Silicon, Sigma Aldrich) attached to feed tank and outlet was connected to tubing that was attached to a sample collection tank (sedimentation tank). The UFCR was fed in from feed tank containing STE in Up-flow mode by a peristaltic pump (EYELA-microtube pump MP3, Tokyo) at an average flow rate of 10 mlh-1 with an average hydraulic retention time (HRT) of 10h. The UFCR was operated continuously for 240 hours.
Column reactor experiments were carried out for the decolorization of C.I. Reactive blue 4 under different physicochemical conditions for 240h. To evaluate the effects of operation and environmental factors on the efficiency of dye removal, the experiments were carried out at different initial pH values (5-9), temperature (25,30,35,40, 450C). Further, different concentrations of dye (10, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400 and 500 mg l-1) were used to check the maximum decolorization abilities. After that the concentration of glucose (1-10 g l-1). Repeated Batch operations on textile dye wastewater were performed by replacing with fresh dye wastewater after regeneration of the biomass used with 0.1 M NaOH and then washing three times with deionized water.
A control column reactor was also run, in which pieces of Scotch-BriteTM (Spain) were checked for their ability to adsorb the dye by overnight incubation in Column reactor having STE containing dye Drimarene blue K2RL to check the abiotic loss of dye . The apparent dye removal by the fungal strain was critically examined into/onto the hyphae by microscope.
Figure 3. Decolorization (reduction) of C.I Reactive blue 4 after biosorption/bioadsorption by A. niger
2.10.1. Spectrophotometric analysis
Sampling during experiments was carried out after every 24 h interval in epindorff tubes (size 2.5 ml). Samples were initially filtered through Whattman filter paper No 1. Samples collected (2ml) from different experiments were centrifuged (Beckman Coulter TM, Germany) at 12000 rpm for 10 minutes. The supernatants collected from centrifuged samples was read at 598nm (λ max of C.I. Reactive blue 4) using spectrophotometer (Agilent spectrophotometer).The dye free Simulated Textile Effluent was used as a blank. Standard curves of known concentrations of dye were made for measuring its concentration in the samples. Percent removal of dye in Simulated Textile Effluent was determined as the percentage ratio of decolorized dye concentration to that of initial one.
2.10.2. Chemical Oxygen Demand (COD)
Chemical Oxygen Demand (COD) of treated Samples were analyzed by Closed Reflux Colorimetric method (APHA, 5220 D). COD was estimated taking absorbance at 600 nm. COD was measured as COD mg O2 /l.
3. RESULTS AND DISCUSSION
Present researchwork has appreciably validated the role of a fungus Aspergillus niger and activated sludge in the Decolorization of an important reactive dye C.I. Reactive blue 4. For this purpose immobilized fungus Aspergillus niger and Activated sludge were tested in column reactor system in decolorization/degradation experiments.
Application of Immobilized column reactor revealed biosorption/bioadsorption to be the one of the dye removal phenomenon. Apparently, dye removal in the present study was merely seen due to biosorption/bioadsorption of fungal hyphae. Likewise, few other studies have also clearly mentioned biosorption/bioadsorption of certain brown rot fungi (A. niger and A. foetidus) [26, 9, 32, 33] as the primary dyes removal phenomenon coupled with electrostatic pull between the positively charged cell wall and negatively charge dyes [34, 35]. Dyes removal by A. niger SA1 was microscopically found more due to biosorption/bioadsorption into/onto fungal hyphae as was reported by Fu and Viraraghavan .
Concentration of Dye
Different concentrations of dye in the effluent were treated through the sequential reactor system. Using UFCR, decolorization and Chemical Oxygen Demand (COD) reduction of the effluent was observed maximum i.e., 100% decolorization and 63% COD reduction at 100 ppm of dye; however, further 13% COD reduction was observed in STR (Fig. 4). The combination of UFCR and STR treatment leads to COD removal of 76%. While at 500 ppm of dye UFCR resulted in 80% decolorization and 50% COD removal; however, further treatment in STR resulted in 1% color removal and 27% COD reduction. The combination of UFCR and STR treatment resulted in 81% decolorization and 77% COD removal (Fig. 5). In several cases, the applied dye concentrations largely exceed the 10-25 mg/l range of normal concentrations in dyehouse effluents . This happens due to the high dye concentration, which may negatively affect the color removal efficiently, either by exceeding the reactors biological dye capacity or by causing toxicity to the biomass . Sequential reactor system based on mix culture of bacteria, for the treatment of azo dyes with the combination anaerobic and aerobic process showed that majority of color was removed in anaerobic process and majority of COD was removed in aerobic process . In our study the Immobilized Aspergillus niger SA1 in column reactor revealed that it has higher capacity for the treatment of dye in context of decolorization and Chemical Oxygen Demand Reduction (COD), while further treatment in stirred tank reactor using sludge have limited potential. The major COD in synthetic wastewater are due to soluble starch, acetic acid and yeast extract, all of which are known as anaerobically and aerobically biodegradable . With different types of activated sludge treatment methods, the following removal is normally achieved: about 90 % of BOD, 40 -50 % of COD and 10-30 % of color .
The color removal efficiency is poor in stirred tank reactor and it could be due to biosorption alone. A fair amount of research has been conducted assessing the viability of using sludge to treat textile effluents . Only few studies have described the successful usage of aerobic sludge for color removal. The successful removal of color was reported in a study by aerobic sludge system with color reduction of 75 and 85 percent respectively . Primary mechanism for removal of dyes on sludge systems may occur by adsorption onto the cell wall of microbes .
The sequential reactor system using anoxic UFCR and aerobic STR achieved 100% decolorization and up to 76% COD reduction at 100 ppm of dye, while at 500 ppm of dye, 81% decolorization and 77% COD reduction were achieved.
The study revealed that majority of decolorization and COD reductions were achieved in UFCR, while the sludge in STR has the capability of further treatment of the dye. No significant color removal was observed in STR under aerobic conditions.
Further studies will be performed to validate on whether the removal is due to biotransformation or biosorption and additional information regarding the possibility of microbial contamination might be needed.