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