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ScienceDirect Materials Today: Proceedings 3 (2016) 3467–3472
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International Conference on Advances in Bioprocess Engineering and Technology 2016 (ICABET 2016)
Removal of methylene blue dye using immobilized bacillus subtilis in batch & column reactor Ganta Upendara, Susmita Duttaa* , Jitamanyu Chakrabortyb, Pinaki Bhattacharyyac Department of Chemical Engineering, National Institute of Technology Durgapur, Durgapur – 713209, India
a
b c
Department of Chemistry, National Institute of Technology Durgapur, Durgapur – 713209, India
Department of Chemical Engineering, Heritage Institute of Technology Kolkata, Kolkata – 700107, India
Abstract The proper treatment of wastewater to remove dye in an environment benign and economic way is essential for pollution free world. Since Methylene Blue (MB) is used extensively in various industries such as textile, leather, etc., it has been selected as model dye for the present study. Due to several shortcomings associated with conventional techniques, biological removal employing Bacillus subtilis is attempted in the present investigation to remove MB from simulated solution. Bacillus subtilis has been immobilized in calcium alginate bead and used in both batch and continuous reactor for removal of MB © 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016. Keywords: Methylene blue, biosorption, immobilization, microorganism
* Corresponding author. Tel.: +91-343-2754082; fax: +91-343-2547375. E-mail address:
[email protected] 2214-7853© 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016.
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1. Introduction Removal of dye from wastewater in a cost effective and environment friendly manner is the need of the hour. Methylene blue (MB), a common dye, is used extensively for dying silk, cotton and wood. It has adverse effect on human health such as eye burns, breathing problem, gastritis, diarrhoea and vomiting as described by Chatterjee et al. [1] and Bhattacharyya et al [2]. Furthermore, dye contaminated coloured water is detrimental for aquatic plants and animals. Therefore, proper treatment is mandatory to remove dye before being discharged to the environment. Several techniques such as electro chemical techniques as done by Mantzavinos et al [3], oxidation as depicted by Balcioglu et al [4], etc. have been attempted to remove dye from wastewater. However, each technique has its own limitation. In such cases biological method using bacteria may be an alternative approach for dye removal. In the present study Bacillus subtilis has been used for biosorptive removal of MB from simulated wastewater. B. subtilis has been immobilized in calcium alginate bead following standard protocol as described by Kathiravan et al. [5] and used to remove MB from its simulated solution in both batch and continuous contactors. Selection of calcium alginate as carrier matrix is based on the fact that it itself partially adsorbs MB. As a result the overall effect of using this cell matrix conjugate is to get enhanced MB removal efficiency. The cell matrix conjugate has been viewed here as a lumped system. 2. Materials and methods 2.1. Materials All the chemicals unless otherwise mentioned were of AR grade and purchased from MERCK, India. 2.2. Growth of microorganism in laboratory conditions Bacillus subtilis, the bacteria used in the present investigation, was taken from Department National Institute of Technology Durgapur. The bacterial strain was grown in nutrient broth biomass was measured after every two hours interval and growth of the said bacterial strain was of its dry biomass content. Later on bacterial strain was grown in mineral medium containing source.
of Biotechnology, medium Bacterial measured in terms glucose as carbon
2.3. Preparation of calcium alginate immobilized bacillus subtilis B. subtilis was immobilized in calcium alginate bead and utilized for removal of dye. Immobilization was done following standard protocol as described by Kathiravan et al. [5]. To get substantial amount of biomass, B. subtilis was initially grown in mineral media at 30 deg C for 16 hrs. The culture was centrifuged at 360000 rps for 10 min. The biomass was washed thoroughly with sterile distilled water. It was then added to 0.15 L sodium alginate solution having concentration (30 g/L) under aseptic condition. The mixture was then added drop by drop into the sterile and chilled 0.2 (M) CaCl2 solution. The calcium alginate were then added into CaCl2 solution at 4 deg C and left for 24 h for hardening. The beads were stored under water in refrigerator at 4 deg C. 2.4. Utilization of calcium alginate immobilized bacillus subtilis for removal of methylene blue in batch contactor Simulated solution of Methylene Blue having 0.5 g/L was prepared by dissolving 0.25 g MB in 0.5 L distilled water. It was then diluted with requisite amount of distilled water and used in kinetic studies on MB removal using B. subtilis immobilized calcium alginate bead. The flasks were placed in BOD Incubator & Shaker at 30 deg C and shaken at 900 rps for 3 hrs. Three parameters such as initial concentration of dye, weight of beads and temperature were varied during the removal study in a prescribed manner. The samples were collected after a particular time interval and solid was separated from solution by centrifugation. The solution was analysed for MB concentration spectrophotometrically using Spectrophotometer (Genesys 20, Thermo Scientific) at a wavelength of 660 nm.
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2.5. Utilization of calcium alginate immobilized bacillus subtilis for removal of methylene blue in continuous contactor A packed bed glass contactor (internal diameter 23.6 mm and length 100 mm) was used for continuous study of MB removal. Ambient temperature of 35 deg C was maintained during the present study. The alginate immobilized B. subtilis beads were used as packing material in the contactor and the bed volume of 30 cm3 was maintained for different flow rates. MB solution having concentration 0.01 g/L was passed through the fixed bed column at various flow rates (0.06 – 0.09 L/hr.) in up flow mode. The column was operated for 360 min with each flow rate. The effluent samples were collected at regular intervals of time. The samples were then analysed for absorbance of residual MB concentration by Spectrophotometer (Genesys 20, Thermo Scientific) at a wavelength of 660 nm. 3. Results and discussion 3.1 Growth study of bacillus subtilis in nutrient broth The variation of dry biomass content with time is shown in Fig. 1 when B. subtilis has been grown in nutrient broth medium. From the figure it is clear that lag phase exits upto 2 hours and stationary phase starts at 18 hours.
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Fig.1 Growth study of B. subtilis in nutrient broth medium
3.2 Utilization of calcium alginate immobilized bacillus subtilis for removal of methylene blue in batch contactor Removal of MB using immobilized cell is investigated in batch reactor when initial concentration of dye has been varied from 0.02 to 0.06 g/L keeping other parameters constant such as temperature: 30 deg C, adsorbent dose:20 g/L, and shaking speed 900 rps. The percentage removal of MB decreases (96.17 to 90.11%) with increase in initial concentration from 0.02 to 0.06 g/L (Fig. 2). The less removal at higher concentration may be due to the exhaustion of the adsorption capability of beads.
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Fig. 2 Decrease in concentration of dye using immobilized cell with varying initial concentration
Removal of MB using immobilized cell have been studied by varying the adsorbent dose from 20 to 60 g/L, keeping the other parameters constant such as temperature: 30 deg C, rotation speed: 900 rps and initial concentration of MB: 0.02 g/L. The percentage removal of MB is almost same when adsorbent dose increases from 20 to 60 g/L (Fig. 3). As adsorption is surface phenomenon, the increase in amount of adsorbent means availability of more surface area for adsorption.
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3.3 Utilization of calcium alginate immobilized bacillus subtilis for removal of methylene blue in continuous contactor
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The break through time is defined as the time when the effluent concentration reaches 50% of the influent concentration as defined by Tan et al [6]. When effluent concentration reaches 90% of the influent concentration, it is called as exhaustion time. The column is operated for 6 hrs. with each flow rate. The effluent samples have been analysed to determine the residual MB concentration. The breakthrough curves are shown in Fig.4.
From the figure it is seen that for lower flow rate (0.06 L/hr.), breakthrough time is less than that in case of higher flow rate (0.09 L/hr.). This may be due to the more contact time of solution with specific amount of adsorbent at lower flow rate.
4. Conclusion Methylene Blue that is used extensively in dying cotton, silk, etc. has been chosen as model dye in the present study. Biological removal of MB using calcium alginate immobilized Bacillus subtilis has been attempted both in batch and continuous contactor. Selection of calcium alginate as carrier matrix is based on the fact that it itself partially adsorbs MB. As a result the overall effect of using this cell matrix conjugate is to get enhanced MB removal efficiency. The cell matrix conjugate has been viewed here as a lumped system. More than 90% removal has been achieved during kinetic study in batch contactor. MB has successfully been removed in continuous contactor also. Therefore, utilization of calcium alginate immobilized Bacillus subtilis in removal of Methylene Blue may be a feasible option for greener world.
Acknowledgement The authors are grateful to the National Institute of Technology Durgapur, Durgapur, India, for supporting the research work. References 1. 2. 3. 4. 5. 6.
S. Chatterjee, A. Kumar, S. Basu, S. Dutta, Chem. Eng. J. 181-182 (2012) 289-299. A. Bhattacharyya, S. Dutta, A. Ganguly, S. Gupta, S. Basu, Desalination 275 (2011) 26-36. E. Chatzisymeon, N.P. Xekoukoulotakis, A. Coz, N. Kalogerakis, D. Mantzavinos, J. Hazard. Mater. B137 (2006) 998-1007. I.A. Balcioglu, I. Arslan, B.W. Bahnemann, Dyes Pigments 47 (2000) 207-218. M.N. Kathiravan, S.A. Praveen, G.H. Gim, G.H. Han, S.W. Kim, Bioproc. Biosyst. Eng. 37 (2014) 2149-2162. L. Tan, H. Li, S. Ning, B. Xu, Bioresour. Technol. 158 (2014) 321-328.
