The chemical compounds and dye Drimarene blue K2RL (Fig. 1) were procured from Buch Sigma chemicals Co; St, Lois, E-Merck (Darmstadt, Germany). Saboraud Dextrose Broth was used for immobilization of A. niger, broth was prepared by adding per litre of distilled water; dextrose 40 g, and peptone 10 g. A pH value of 5 of medium was adjusted 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. 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 Drimarene blue K2RL 10 mg .
2.2. A. niger Immobilization
A local isolate A. niger was refreshed on sabouraud dextrose agar medium at pH value of 5. Scotch-BriteTM (Spain) was used as immobilization support material (80% polyester and 20% nylon, color green). A. niger was grown on sabouraud dextrose agar at pH value of 5 for a week at 28 °C. The spores were scratched and picked up with a loop from mature colony, then loop containing fungal spores were mixed 100 ml autoclaved distilled water containing 0.05 % Tween 80 soultion. After vigorous shaking, 1ml of the inoculum was poured on the hemocytometer and spore were observed under microscope. The observation showed average spores approximately 7.35 x 103 per ml. The whole process was carried out in aseptic conditions in laminar flow hood. The inoculum was stored in refrigerator at 4 °C. Small pieces of Scotch-BriteTM size: 3x3 cm, thickness: 0.8 mm were used as immobilization support material [Rodriguez, 2004]. These pieces were thoroughly washed with distilled water and sterilized in autoclave prior to further use. Fifteen pieces of Scotch-BriteTM were added to the flask having 150 ml sabouraud dextrose broth. It was inoculated with 10 ml spores suspension of A. niger and was placed in rotary shaker incubator (INNOVA TM 4330, New Brunswick Scientific) at 30 °C, 120 rpm for 1 week. Immobilized and non- immobilized Scotch brite are shown in figure 2.
2.3. Activated Sludge immobilization
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The activated sludge used in 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.
2.4. Experimental design
The Up-flow Column reactor was built from borosilicate glass column (14 x 1.6//) . One of the glass column reactors was filled with immobilized pieces (Scotch Brite) of Aspergillus niger SA1 (45 Scotch Brite pieces) and another Column was filled with immobilized beads of Activated sludge to a bed height of 8 inch. Column reactors were connected at lower side inlet to a tubing (Silicon, Sigma Aldrich) attached to feed tank and outlet was connected to a tubing that was attached to a sample collection tank (sedimentation tank). The UFCR was fed in from feed tank containing STE in Upflow mode by a peristaltic pump (EYELA-microtube pump MP3, Tokyo) (Figure. 3) at an average flow rate of 10 mlh-1 with an average hydraulic retention time (HRT) of 10 h. Column reactor experiments were carried out for the decolorization of Drimarene blue K2RL under different physicochemical conditions for 6 days. 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 (3,4, 5, 6, 7, 8, 9). Further, different concentrations of dye (10, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500 mg l-1) were used to check the maximum decolorization abilities. After that, the concentration of glucose (1, 2, 4, 6, 8, 10 g l-1) and repeated Batch operations on textile dye wastewater were performed by replacing with fresh dye wastewater after regeneration of the biomass with 0.1 M NaOH and then washing three times with deionized water. An experiment was also performed using optimized conditions, to test the continuous run of both immobilized reactors for 30 days. After the dye treatment experiments using immobilized fungus and immobilized activated sludge, two control column reactors were also run, to check the abiotic loss of dye [Rodriguez et al 2004]. The apparent dye removal by the A. niger and A. sludge was critically examined into/onto the cells by microscope.
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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. 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.
Data obtained during experiments was statistically analyzed by ANOVA (Single factor) and LSD test using Microsoft excel and MSTAT, while correlation r value between different parameters was calculated by SPSS software. Probability (p-value 0.05 consider as strong significant and 0.01 was considered less significan respectively. The results were expressed in terms of Means and Standard error (SE±).
3. RESULTS AND DISCUSSION
This research validated the application of immobilized A. niger and Immobilized A.Sludge in column reactors for the treatment of STE containing dye Drimarene blue K2RL.
Effect of pH
The decolorization and COD reduction of STE containing Drimarene blue K2RL was carried out at a pH range of 3 to 9 by immobilized A. niger and immobilized activated sludge. Using A.niger and A.Sludge the decolorizaton was maximum i.e., 95.45% at pH value of 5 and 59.65% at pH value of 4, respectively. However, it reduced to 55.43% and 43.09% at pH value of 9. It can be seen, that when pH was in the range of 3-5, the decolorization ability was increased, but increase in pH from 5-9, resulted a decline in decolorization by A.niger. Decolorization was apparently seemed to be associated with hyphal uptake phenomenon of fungus as biosorption/bioadsorption and it was confirmed through microscopic examination. While using Immobilized A. niger and A.Sludge the COD reduction was maximum ie.. 59.65% at pH 5 and 83.23% at pH 5. Increase in pH from 5-9 resulted a decline in decolorization and COD reduction. (Figure). These results provide informations that acidic pH is required for decolorization and COD reduction by A. niger and A. Sludge.
Effect of dye concentration
Experiments were sequentially conducted from lower to higher (10-500 mg l-1) concentrations of dye Drimarene blue K2RL. Generally, increase in concentration of dye in different experiments proved to have an inverse effect on decolorization and COD reduction by immobilized A.niger and A. Sludge. However, a direct (positive) correlation was observed between decolorization of dye at different concentrations. In case of Drimarene blue K2RL by A. niger, maximum decolorization (98.10%) was observed at a concentration of 100 mg l-1 , though it drastically reduced to 79.31% at 300 mg l-1 and almost 50.65% at 500 mg l-1. On the other hand, decolorization by immobilized A. Sludge, maximum decolorization (30.50%) was observed at a concentration of 10 mgl-1 and it reduced to 3.65% at 500 mg l-1 (Figure). While using A.niger maximum COD reduction of 85.54% at 10 mgl-1 of dye and it was reduced to 37.33% at dye concentration of 500 mgl-1. The COD reduction using A. Sludge was maximum 81.09% at 10 mgl-1 and it was declined to 34.53% at dye concentration of 500 mgl-1.
Effect of Glucose
Different concentrations of glucose (1-10 mgl-1) as additional carbon source were used for decolorization and COD reduction. The optimum concentration of glucose was found to be 6 gm l-1 where maximum decolorization of Drimarene blue K2RL (98.12%) and COD reduction (73.06%) were observed by A. niger. Decolorization was ≥90% from 6-10 gm l-1 glucose concentration. At 4 gm l-1 of glucose, there was almost 70% decolorization and below this level of glucose (gm l-1), there occurred a significant decline in decolorization (Figure). General increase in glucose concentration from 1-10 gm l-1, proved to have a strong positive effect on decolorization of Drimarene blue K2RL. While COD reduction was ≥50% from 6-10 gm l-1 of glucose concentration and increase or decrease concentration from this level declined the COD reduction. Moreover, decolorization and COD reduction were positively correlated (r=) at varying concentrations of glucose in different experiments.
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While on other hand, using A. sludge maximum decolorization (30.23%) of dye and COD reduction (83.33%) was achieved using 2 gml-1. Further increase in glucose concentration (4-10 gml-1) resulted in decreased removal of decolorization (23.56-4.21%) and COD reduction (55.76-18.39%). However, the lower concentration of glucose (1 gml-1 ) also declined the rate of decolorization (26.23%) and COD reduction (18.39%), respectively.
Effect of Repeated Uses
In this research work, repeated batch experiments were performed to study the reusability of immobilized Aspergillus niger and immobilized activated sludge for the treatment of dye wastewater containing dye Drimarene blue K2RL. The data in table shows, that during 4 repeated runs, immobilized fungus and immobilized activated sludge approximately resulted in same biosorption rate that was obtained from the first run. This might be attributed to an adaptation effect, since the fungal and activated sludge biomasss were repeatedly exposed to the dye. Brahimi-Horn et al. (1992) observed that methanol-treated cells of Microthecium verrucaria still effluent possessed to a large extent the capacity to absorb dyes. They suggested that methanol might influence the hydrophobic/ hydrophilic interaction between the dyes and the biomass. Also, Zhous and Banks (1993) showed that the R. arrihizus biomass, which regenerated by 0.1 M NaOH, could be used for several sorption/desorption cycles with similar efficiency. The Present results indicate that immobilized fungus Aspergillus niger and immobilized activated sludge hold great reusability in repetitive biological treatment operation.
Effect of Continuous Run
Both reactors were run continuously to check the performance for about 30 days.