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Copper removal by immobilized Microcystis aeruginosa in continuous flow ... Keywords: Adsorption/desorption cycle, bed height, Cu2+ removal, flow rate, ...

World Journal of Microbiology & Biotechnology 17: 829–832, 2001.  2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Copper removal by immobilized Microcystis aeruginosa in continuous flow columns at different bed heights: study of the adsorption/desorption cycle Subhashree Pradhan and L.C. Rai* Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi – 221005, India *Author for correspondence: Tel.: +91-542-367520/367655, Fax: +91-542-368174, E-mail: [email protected] Received 9 April 2001; accepted 6 October 2001

Keywords: Adsorption/desorption cycle, bed height, Cu2þ removal, flow rate, immobilized Microcystis aeruginosa

Summary Microcystis aeruginosa immobilized in a natural polymer was tested for its potential to remove Cu2þ ions from aqueous solution in a continuous, downflow packed columnar reactor. Various parameters like flow rate, bed height and contact time required for maximum removal of test metals by the immobilized Microcystis aeruginosa were optimized. An increase in bed height from 2 to 10 cm resulted in an apparent decrease in biosorption capacity from 8.94 to 5.34 mg g)1, but more Cu2þ solution was purified at the higher bed height. Efficiency of metal recovery from Cu2þ-loaded biomass and its subsequent regeneration was also determined. Immobilized M. aeruginosa was found to be effective in Cu2þ removal from solution for up to 10 cycles of adsorption–desorption and 1 M HCl is very efficient desorbent for regeneration of Microcystis biomass for reuse.

Introduction The toxic metal ions from industrial and mining wastes are a major environmental problem in both developed and developing countries. The natural affinity of biological materials for metallic cations could be instrumental in economically purifying metal-loaded waste water (Chang et al. 1997; Chang & Huang 1998). It is known that for industrial application freely suspended biomass has several disadvantages, like compaction, clogging and washout from the system (Tsezos 1986). In contrast to this, application of immobilized biomass has many advantages including easy separation of cells and effluents, minimal clogging, high biomass loading and repeated use of biomass (Brady et al. 1994). In view of the above characteristics, the potential of immobilized microorganisms to remove metals from the aquatic environment has been widely appreciated (Aloysius et al. 1999; Wilkinson et al. 1990). A preferable process design for biosorption and/or removal with immobilized cells is a fixed-bed reactor that contains the desired type of immobilized biomass (Volesky & Prasetyo 1994; Volesky & Holan 1995). Biosorbents packed in columns appear to be the most appropriate device for effective and continuous removal of heavy metals (Kratochvil & Volesky 1998). Flow rate plays a crucial role for maximum biosorption/removal of different metals. A vast literature has been accumulated on the sequestering and removal of metallic ions by various immobilized microorganisms in packed-bed reactors at fixed bed height. Chang & Huang (1998)

studied the removal of Pb2þ, Cu2þ and Cd2þ from the contaminated water by using alginate-immobilized biomass of Pseudomonas aeruginosa PU 21. Sag et al. (1995) used a biosorption column to remove Cu with an immobilized bacterium Zoogloea ramigera using flow rate and inlet metal ion concentration. Pseudomonas aeruginosa CSU biomass immobilized within polyurethane gel beads was effective for removal of uranium from low concentration acidic waste water (Hu & Reeves 1997). Aksu & Kutsal (1998) made a detailed investigation on Cu removal by the Ca-alginate-immobilized green alga Cladophora sp. in a packed-bed column reactor as a function of flow rate and inlet CuII concentration. In view of the fact that immobilized algal beads show increased mechanical strength, density, resistance to chemical environment and reuse in repeated adsorption and desorption cycles in a packed column reactor, and to ascertain whether observations also apply to our system, unicellular M. aeruginosa was immobilized in a Ca-alginate matrix. Although much work has been conducted on metal removal by various researchers using different microorganisms at fixed bed height, this paper is the first attempt to investigate the metal removal efficiency of M. aeruginosa at different flow rate and bed height. Further, the efficiency of desorption of Cu2þ from the loaded test cyanobacterium and the reusability of the biosorbent in repeated adsorption– desorption operation were also determined to further evaluate the feasibility of using this cyanobacterium in heavy metal removal from metal-contaminated water.

830 Materials and methods The test organism M. aeruginosa collected from Laxmi kund in Varanasi, previously isolated in pure culture was grown in Parker’s medium (Parker 1982) pH 9.2 at 29 ± 2 C under continuous illumination of 72 lmol photon m)1 s)1 light intensity. Preparation of bead Exponentially grown M. aeruginosa was harvested by centrifugation. A thick suspension having a cell density of approximately 3.18 g dry wt. in 100 ml Milli-Q water was subsequently mixed with 3% (w/v) sodium alginate. The sodium alginate–Microcystis mixture was added dropwise into 0.2 M CaCl2 solution with the help of a syringe. The alginate beads (diameter 2 ± 0.2 mm) formed by cross-linking with Ca2þ were harvested after 2 h and evenly packed in a column (19 · 2 cm2) up to a height of 10 cm. Suitable flow rate for maximum removal of Cu at different bed heights In order to determine suitable flow rates for maximum removal of Cu at 2, 4, 8 and 10 cm bed heights, different flow rates (0.25, 0.45, 0.65 and 0.75 ml min)1) were employed. The immobilized M. aeruginosa beads having a dry wt. of 0.161, 0.322, 0.644 and 0.805 g were packed to a height of 2, 4, 8 and 10 cm respectively in order to study the biosorption behaviour at different bed heights. CuCl2 Æ 2H2O solution (50 lg ml)1) was passed downwards through the column with the help of a peristaltic pump and fractions of eluent were collected at regular intervals and the Cu content in aqueous solution was measured in a Perkin-Elmer atomic absorption spectrophotometer model 2380 at 324.8 nm wavelength. Adsorption and desorption cycles For the adsorption/desorption cycle, the column was packed with immobilized biomass to a bed height of 10 cm and 100 ml of 50 lg ml)1 Cu solution was passed through the column in a downward direction. The column was left for the continuous flow of metal ion solution at a flow rate of 0.75 ml min)1. The elution operation was similar to the loading operation. The residual solution was drained from the column and the biomass was rinsed by passing 150 ml of distilled water through the bed. Rinse water was collected for metal analysis. After completion of the adsorption cycle, desorption was carried out by passing 100 ml of 1 M HCl at a flow rate of 5 ml min)1 (in order to avoid shrinkage of the beads due to acid contact) for a period of 20 min. Samples of the eluent leaving the column were collected at intervals of 10 min. After completion of the elution cycle, the column was drained by pumping double distilled water through the column for 30 min to

Subhashree Pradhan and L.C. Rai neutralize and regenerate the immobilized beads for a fresh adsorption experiment. The adsorption–desorption cycle was repeated 10 times to ascertain the reusability of biomass for 10 consecutive cycles.

Results and discussion Figure 1 (A–D) indicates removal of Cu ions at 2, 4, 8 and 10 cm bed height and varying flow rates from 0.25 to 0.75 ml min)1. This figure demonstrated that maximal Cu removal efficiency was largely dependent upon the flow rate at a particular bed height. It was inferred that suitable flow rates for maximum removal of Cu at 2, 4, 8 and 10 cm bed heights were respectively 0.25, 0.45, 0.65 and 0.75 ml min)1. At 2 cm bed height, about 95% Cu2þ was removed at a low flow rate of 0.25 ml min)1 because residence as well as contact time (120 min) between metal ions and biomass was sufficient for maximum removal. However, this removal was decreased at increasing flow rate. This may be attributed to adsorption equilibrium and diffusion of the solute into the biosorbent pores (Sag et al. 1995). With an increase in the bed height from 2 to 10 cm, availability of surface area containing various functional groups for binding of metals was increased because more biomass was loaded in the column, which allowed a progressively higher flow rate for maximum removal of Cu ion. Though percentage removal was almost the same at all the flow rates, metal adsorbed (mg g)1 dry wt.) was decreased from 8.94 to 5.34 mg g)1 dry wt. Various reasons have been suggested to explain the decreased adsorption capacity at increasing biomass including availability of solute, electrostatic interactions, interference between binding sites, and reduced mixing at higher biomass densities (Meikle et al. 1990; Fourest & Roux 1992). Our results are also supported by the findings of Sandau et al. (1996) and Pradhan & Rai (2000). The latter authors observed decreased biosorption of Cu2þ by immobilized Microcystis sp. at increasing biomass. In order to assess the reusability of biomass and efficiency of mineral acid (1 M HCl) to regenerate spent Microcystis, a series of adsorption/desorption experiments were performed in a column. Adsorption of Cu2þ by the test organism in the first adsorption cycle was 5.92 mg g)1 (Table 1). Adsorption efficiency was initially high, but declined slightly over the successive cycles. This may be due to a minimal loss of cells following mechanical shearing (Wilhelmi & Duncan 1996). Chu et al. (1997) and Chang & Huang (1998) tested the suitability of biological materials in continuous column system for successive use by different desorbing agents. In the first adsorption cycle, 95% Cu2þ was adsorbed by the immobilized test organism. Interestingly all the adsorbed Cu (100% desorption) in the first adsorption cycle was desorbed. Further, in all cycles nearly 98– 100% of adsorbed Cu was recovered with 1 M HCl used for desorption (Table 1) and more than 84% of metal was desorbed within 10 min when 50 ml of 1 M HCl

Copper removal by Microcystis aeruginosa

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Figure 1. Effect of different flow rates (0.25, 0.45, 0.65 and 0.75 ml min)1) on the percentage removal of Cu at different bed heights: (A) 2, (B) 4, (C) 8 and (D) 10 cm. Hundred millilitre of 50 lg ml)1 Cu solution was passed downwards through the column.

Table 1. Metal adsorption/desorption from repeated adsorption desorption cycle. Cycle Adsorption of Cu2þ from 50 lg ml)1 Cu solution by 0.805 g of algal bead (mg Cu2þ adsorbed g)1 dry wt.)

1 2 3 4 5 6 7 8 9 10

5.92 5.86 5.78 5.73 5.77 5.82 5.74 5.74 5.72 5.68

Desorption of Cu2þ by 100 ml of 1 M HCl (mg Cu2þdesorbed g)1 dry wt) First 50 ml

Second 50 ml

5.62 5.40 5.20 4.94 4.93 4.85 4.85 4.88 4.77 4.74

0.30 0.51 0.51 0.68 0.83 0.91 0.87 0.86 0.87 0.90

was applied at a flow rate of 5 ml min)1. Complete desorption was obtained after passing 100 ml of 1 M HCl at the same flow rate for 20 min. Our results are also supported by the findings of Huang & Huang (1996) and Wilhelmi & Duncan (1996). According to Huang & Huang (1996), acid treatment did not alter the surface characteristics expect for releasing bound metal from the surface. This study can be of particular interest in continuous removal of Cu from solution through immobilized Microcystis in fixed bed columns. It is also suggested

that before using the biomass for large scale metal removal and recovery, it will be necessary to optimize the flow rate for maximum removal of metal at different bed heights in laboratory conditions. Further, the ability to desorb and recover bound metals and regenerability of the biomass sorbent are the key factors for improving the economy of the process. With the advantages of high metal biosorption capacity, satisfactory recovery efficiencies and persistence on repeated use, biomass of Microcystis appears to hold great potential to be an effective biosorbent for the removal and recovery of heavy metals from polluted waters. Acknowledgements This study was sponsored by grants from the Department of Biotechnology, Ministry of Science & Technology, New Delhi, Pitamber Pant National Environment Fellowship Award from Ministry of Environment and Forest, New Delhi, and Indo-US project from NSF, USA, to L.C. Rai. References Aksu, Z. & Kutsal, T. 1998 Determination of kinetic parameters in the biosorption of Cu(II) on Cladophora sp. in a packed bed column reactor. Process Biochemistry 33, 7–13. Aloysius, R., Karim, M.I.A. & Ariff, A.B. 1999 The mechanism of cadmium removal from aqueous solution by nonmetabolizing free

832 and immobilized live biomass of Rhizopus oligosporus. World Journal of Microbiology and Biotechnology 15, 571–578. Brady, D., Stoll, A. & Duncan, J.R. 1994 Biosorption of heavy metal cations by non-viable yeast biomass. Environmental Technology 15, 429–438. Chang, J.S., Law, R. & Chang, C.C. 1997 Biosorption of lead, copper, and cadmium by biomass of Pseudomonas aeruginosa PU2. Water Research 31, 1651–1658. Chang, J.S. & Huang, J.C. 1998 Selective adsorption/recovery of Pb, Cu and Cd with multiple fixed beds containing immobilized bacterial biomass. Biotechnology Progress 14, 735–741. Chu, A.U., Hashim, K.H., Phang, S.M. & Samuel, V.B. 1997 Biosorption of Cadmium by algal biomass: adsorption and desorption characteristics. Water Science and Technology 35, 115–122. Fourest, E. & Roux, J.C. 1992 Heavy metal biosorption by mycelial by-products: mechanism and influence of pH. Applied Microbiology and Biotechnology 37, 399–403. Kratochvil, D. & Volesky, B. 1998 Advances in the biosorption of heavy metals. Trends in Biotechnology 61, 291–300. Hu, M.Z.C. & Reeves, M. 1997 Biosorption of uranium by Pseudomonas aeruginosa strain CSU immobilized in a novel matrix. Biotechnology Progress 13, 60–70. Huang, C. & Huang, C.P. 1996 Application of Aspergillus oryzae and Rhizopus oryzae for CuII removal. Water Research 30, 1985–1990. Meikle, A.J., Gadd, G.M. & Reed, R.H. 1990 Manipulation of yeast for transport studies: critical assessment of cultural and experimental procedure. Enzyme Microbial technology 12, 865–872.

Subhashree Pradhan and L.C. Rai Parker, D.L. 1982 Improved procedures for the cloning and purification of Microcystis cultures (Cyanophyta). Journal of Phycology 18, 471–477. Pradhan, S. & Rai, L.C. 2000 Optimization of flow rate, initial metal ion concentration and biomass density for maximum removal of Cu2þ by immobilized Microcystis. World Journal of Microbiology and Biotechnology 16, 579–584. Sag, Y., Nourbakhsh, M., Aksu, Z. & Kutsal, T. 1995 Comparison of Ca-alganite and immobilized Zooglea ramigera as sorbents for copper(II) removal. Process Biochemistry 30, 175–181. Sandau, E., Sandau, P., Pulz, O. & Zimmermann, M. 1996 Heavy metal sorption by marine algae and algal by products. Acta Biotechnology 16, 103–119. Tsezos, M. 1986 Adsorption by microbial biomass as process for removal of ions from process or waste solutions. In Immobilization of Ions by Bio-sorption, eds. Eccles, H. & Hunt, S. pp. 201–218, Chichester, UK: Ellis Harwood. ISBN 0-74580003-3. Volesky, B. & Prasetyo, I. 1994 Cadmium removal in a biosorption column. Biotechnology and Bioengineering 43, 1010–1015. Volesky, B. & Holan, Z.R. 1995 Biosorption of heavy metals. Biotechnology Progress 11, 235–250. Wilhelmi, B.S. & Duncan, J.R. 1996 Reusability of immobilized Saccharomyces cerevisiae with successive copper adsorption– desorption cycles. Biotechnology Letters 18, 531–536. Wilkinson, S.C., Goulding, K.H. & Robinson, P.K. 1990 Mercury removal by immobilized algae in batch culture systems. Journal of Applied Phycology 2, 223–230.

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