Microalgal-luffa sponge immobilized disc - Wiley Online Library

Letters in Applied Microbiology 2003, 37, 149–153

Microalgal-luffa sponge immobilized disc: a new efficient biosorbent for the removal of Ni(II) from aqueous solution N. Akhtar1, J. Iqbal2 and M. Iqbal3 1

Department of Biology, Government Islamia College for Women, 2Department of Botany, University of Punjab, Quaid-e-Azam Campus, and 3Environment Biotechnology Group, Biotechnology and Food Research Center, PCSIR Laboratories Complex, Lahore, Pakistan

2002/384: received 11 December 2002, revised 8 April 2003 and accepted 24 April 2003

ABSTRACT N . A K H T A R , J . I Q B A L A N D M . I Q B A L . 2003.

Aims: The aim was to develop a new, efficient and cost-effective biosorbent for the removal of heavy metals from aqueous solution. Methods and Results: A new biosorbent was developed by immobilizing a unicellular green microalga Chlorella sorokiniana within luffa sponge discs and used for the removal of metal ions from aqueous solution. Microalgal-luffa sponge immobilized discs (MLIDs) removed Ni(II) very rapidly, with 97% of equilibrium loading being reached in 5 min. MLIDs were tested for their potential to remove Ni(II) from aqueous solution in fixed-bed column bioreactor. The regenerated MLIDs retained 92Æ9% of the initial binding capacity for Ni(II) up to five cycles of reuse. Conclusions: In this study for the first time, C. sorokiniana biomass immobilized within luffa sponge disc was successfully used as a metal biosorbent for the removal of Ni(II). It appears that MLIDs can be used as an effective biosorbent for efficient removal of Ni(II) or other metals from aqueous solution. Significance and Impact of the Study: MLIDs biosorption system was shown to have good biosorption properties with respect to Ni(II). Efficient metal removal ability of MLIDs, low cost and simplicity of the technique used for the preparation of MILDs could provide an attractive strategy for developing high-affinity biosorption system for heavy metal removal. Keywords: biosorption, Chlorella sorokiniana, Luffa cylindrica, luffa sponge, microalgae, nickel(II).

INTRODUCTION Industrial effluents, particularly those containing heavy metals, are a cause of serious hazard to human health and other forms of life. Environmental protection laws demand that industrial discharges are duly treated to minimum acceptable limits of various toxic metals. Conventional methods are either cost-prohibitive or not practicable on account of operational shortcomings (Atkinson et al. 1998). Biosorption by algal biomass has been recently projected as an alternative for the remediation of wastewaters containing Correspondence to: Dr Muhammed Iqbal, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK (e-mail: [email protected]).

ª 2003 The Society for Applied Microbiology

toxic metals (Robinson 1998). However, the fragile nature of algal biomass is not suitable for robust wastewater treatment operations (Chu et al. 1997). This has led to the interest in the use of entrapped biomass as immobilized preparations. Several immobilization media, such as alginates, carrageenans and polacrylamide gel have been used for this purpose (Robinson 1998). Immobilization based on these polymeric matrices, however, result in restrictive diffusion because of closed embedding structures with low mechanical strength. The purpose of the present study, therefore, is to provide a new immobilized microalgal biosorption process using a low cost, physically strong, ridged and highly porous immobilization matrix; luffa sponge. The use of luffa sponge for immobilization of algal, fungal and yeast cells has long been reported (Iqbal and Zafar 1993; Ogbonna et al. 1997).

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However, the use of luffa sponge immobilized cells has not been reported for biosorption studies. Thus, the objective of this study was to investigate the use of luffa-immobilized cells as biosorbent for the removal of heavy metals, using Chlorella sorokiniana and Ni(II) as the model organism and metal ion, respectively.

A unicellular green microalga C. sorokiniana, isolated from a wastewater body charged by effluents from electroplating and leather industries was used in this study. Biomass for inoculum was grown to stationary phase in 100 ml Bold’s medium contained in 250 ml Erlenmeyer flasks, shaken in orbital shaker at 100 rev min)1 at 25 ± 2C under continuous illumination with cool white light at an intensity of 50 lE m)2 s)1.

water. The pH of the solution was adjusted to 5Æ0 using 1Æ0 M NaOH. Batch biosorption experiments were carried out by suspending MLIDs containing 100 mg freeze-dried biomass of C. sorokiniana in 100 ml Ni(II) solution of desired concentration (pH 5Æ0) contained in 250 ml Erlenmeyer flasks. The flasks were incubated on orbital shaker at 100 rev min)1 for 30 min at 25 ± 2C. MLIDs were separated from the solution by decantation. Residual concentration of Ni(II) in the metal supernatant solution was determined using atomic absorption spectrophotometer (UNICAM-969, Unicam, Cambridge, UK). For the determination of the rate of metal biosorption by MLIDs, the supernatant was analysed for residual Ni(II) after contact periods of 5, 10, 15, 20, 30, 45 and 60 min. All experiments were performed in triplicate. Metal-free solution and C. sorokiniana-free metal solution containing only luffa discs blanks were used as controls. Statistical analysis of the data was carried out according to the Duncan’s new multiple range test (Steel and Torrie 1996).

Preparation of microalgal-luffa sponge immobilized discs

Continuous removal of Ni(II) by MLIDs packed in fixed-bed column bioreactor

The luffa sponge discs were obtained from matured dried fruit of Luffa cylindrica. The sponge was cut into discs of ca. 2Æ5 cm diameter, 2–3 mm thick, soaked in boiling water for 30 min, thoroughly washed under tap water, and left for 24 h in distilled water, changed three to four times. The luffa discs were oven-dried at 70C, autoclaved for 20 min at 120C at 1Æ06 kg cm)2 pressure, and soaked in Bold’s medium for 5–10 min under aseptic conditions. Four preweighed luffa sponge discs were transferred to 100 ml Bold’s medium contained in 250 ml flasks. Each of these flasks was inoculated with 5 ml, 3–4-week-old stationary phase cultures of C. sorokiniana and incubated for 2 weeks under similar conditions as those for developing inoculum biomass. The luffa discs were removed from the culture flasks, washed thoroughly with fresh culture medium to remove any free algal cells, transferred to 100 ml fresh medium and incubated under the same cultural conditions. The microalgal-luffa sponge immobilized discs (MLIDs) were harvested after 24 days, washed thoroughly with deionized water and freeze-dried for further studies on metal biosorption. Quantity of the immobilized algal biomass was determined as the difference between constant dry weights of the luffa discs, before and after immobilization.

MLIDs were used to investigate the possibility of removal of low levels of Ni(II) in repeated operations. A glass column (2Æ7 cm in diameter and 30 cm in height) was packed with 1Æ52 g C. sorokiniana biomass entrapped within 70 luffa discs, packing height 25 cm. Ni(II) solution (5 mg l)1, pH 5Æ0) was then pumped upwards through the column at a flow rate of 5 ml min)1. Samples were collected at regular intervals from the effluent to measure residual Ni(II) concentrations. As the bed was saturated, the Ni(II) loading was terminated, and the bed was eluted with 0Æ1 M HCl solution to recover the loaded Ni(II) ions. The regenerated bed was washed thoroughly with deionized water before being used for the next adsorption cycle. Three such columns were operated separately, under the same conditions, to check the reproducibility. Separate columns having same specifications were packed with luffa sponge discs, without immobilized algal cells to serve as the control.

M A T E R I A LS A N D M E T H O D S Organism and culture medium

Biosorption studies Desired concentrations of Ni(II) solution were prepared by diluting standard Ni(II) stock solution (Merck Ltd., Poole, UK) of concentration 1000 ± 2 mg l)1 with deionized

RESULTS Microalgal-luffa sponge immobilized discs preparation The fibrous network of the luffa sponge disc was noted to be completely covered by immobilized microalgal cells within 24 days (Fig. 1). The scanning electron microscopy micrograph of C. sorokiniana immobilized on fibres of luffa sponge disc revealed a uniform algal growth along the surface of the fibrous thread indicating that immobilized

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 149–153

MICROALGA-LUFFA IMMOBILIZED DISCS AS A NEW BIOSORBENT

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Ni(II) adsorbed (mg g–1)

60 50 40 30

10 mg I–1

25 mg I–1

50 mg I–1

75 mg I–1

10 mg I–1

150 mg I–1

200 mg I–1

300 mg I–1

20 10 0 0

C. sorokiniana cells are not localized at a single point. This uniform distribution is an important criterion for the proper biosorption of heavy metal ions on the entire surface area of the immobilized algal cells. Thus, immobilization of the cells on the surface of threads of luffa sponge discs could also provide additional advantages over the freely suspended algal cells. In free cultures algal cells form individually distributed spherical clumps. This tight packing of algal cells could also lead to diffusional restriction and less adsorptive sites for heavy metal ions than the luffa sponge-immobilized cells. The amount of immobilized C. sorokiniana in the luffa sponge discs, after 24 days of growth, was 263 ± 13 mg g)1 of dry luffa sponge. Ni(II) binding to microalgal-luffa sponge immobilized discs MLIDs removed the Ni(II) very efficiently from contaminated water containing various concentration of Ni(II). Figure 2 shows the Ni(II) binding capacity of MLIDs as a function of time. The uptake was rapid; MLIDs removed 97% of the metal ion within the first 5 min and reached equilibrium within 15 min of contact of MLIDs with aqueous metal solution. The rate of Ni(II) uptake remained linear only up to 5 min irrespective of the metal concentration used. The magnitude of biosorption of Ni(II) was increased with increase in concentration of Ni(II) at each time interval tested. With the increase in Ni(II) concentration, the uptake of Ni(II) increased rapidly and then slowly up to 150 mg l)1 after which it saturates (Fig. 3). The Ni(II) adsorption capacity of MLIDs was not affected by the presence of luffa sponge used to immobilize the microalgal cells. In addition the Ni(II) sorption capacity of MLIDs was

40

60 80 Time (min)

100

120

Fig. 2 Biosorption of Ni(II) from aqueous solution of different concentrations, pH 5Æ0, by 1 g l)1 microalga-luffa sponge immobilized cells of Chlorellal sorokiniana as related to the time of contact. All values are mean of concurrent triplicate observations

70 Ni(II) absorbed (mg g–1)

Fig. 1 (a) Microalgal-luffa sponge immobilized discs; (b) luffa sponge discs before immobilization of algae showing open structure; (c) luffa sponge disc after immobilization of algae (completely covered with immobilized microalgal cells)

20

60 50 40 30 MLIDs

20

Free microalgal cells

10 0 0

50

100 150 200 250 300 Ni(II) concentration (mg l–1)

350

Fig. 3 Effect of initial concentration of Ni(II) on biosorption by Microalgal-luffa sponge-immobilized discs (MLIDs). The biosorption Ni(II) was calculated on the basis of freeze-dried weight (g) of immobilized biomass of Chlorella sorokiniana and expressed as mg g)1. All values are mean of concurrent triplicate observations

found significantly higher (Duncan’s new multiple range test at P ¼ 0Æ05) than that of free microalgal cells (Fig. 3). Continuous removal of Ni(II) by MLIDs packed in fixed-bed column bioreactor To demonstrate the metal-binding potential of MLIDs in continuous flow system, MLIDs were packed in an up-flow fixed-bed column bioreactor. A Ni(II) solution of 5 mg l)1 adjusted to pH 5Æ0 was pumped through a fixed-bed column at a flow rate of 5 ml min)1. Breakthrough curve obtained exhibited the typical ‘S’ shape curve of a fixed-bed column;


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Ni(II) concentration (mg l–1)

6 5 4 3

MLIDs first experiment MLIDs second experiment MLIDs third experiment LDs

2 1 0 0

5000

10 000 15 000 20 000 25 000 30 000 Ni(II) solution passed (ml)

Fig. 4 Biosorption breakthrough curves for the removal of Ni(II) in fixed column bioreactors packed with luffa sponge discs (LDs) and microalgal-luffa sponge immobilized discs (MLIDs)

an initial zero reading or minimal Ni(II) at column outlet followed by a gradual increase in the effluent Ni(II) concentration that would ultimately reach the initial concentration of feed solution (Fig. 4). At this point, the MLIDs were considered fully saturated with Ni(II). Approximately 100% removal of Ni(II) was obtained in the first 15 l, followed by a gradual increase of the Ni(II) concentration in the effluent. Complete breakthrough was not detected until the 25th litre. The total amount of Ni(II) removal was determined to be 107Æ8 mg by integrated breakthrough curve. In contrast, breakthrough occurred only after 6 l for a similar column with naked luffa sponge discs (Fig. 4). These results clearly indicated the high efficiency of C. sorokiniana for Ni(II) even at ppm levels. To test the reproducibility of the biosorption capacity of MLIDs, the column experiments were repeated three times and results are shown in Fig. 4. Data so obtained from these column experiments were statistically analysed (Duncan’s new multiple range test at P ¼ 0Æ05). No significant difference was observed between the Ni(II) removing capacity of MLIDs used in separate experiments. These observations indicate that the MLIDs, prepared even from different batches of growth, have similar reproducible biosorption capacity. Regeneration of MLIDs In order to access the reusability of the MLIDs, a series of adsorption–desorption experiments were performed in a fixed-bed column bioreactor. The desorption of Ni(II) from MLIDs loaded with Ni(II) was accomplished by passing 500 ml of 0Æ1 M HCl through the column. The regenerated MLIDs were then reused for Ni(II) binding as described above for five adsorption–desorption cycles and results are

Fig. 5 The performance of microalgal-luffa sponge immobilized discs by multiple cycles of regeneration through fixed-bed column bioreactor. All values are mean of concurrent triplicate observations

shown in Fig. 5. It is clear that the adsorption capacity of MLIDs was nearly the same for three cycles and dropped little in the fourth and fifth cycles. The decline in efficiency was not more than 7Æ1%, which shows that the biosorbent has good potential to adsorb metal from aqueous solution. The regeneration and subsequent use of MLIDs may offer an economical method for biosorption of heavy metals from wastewater streams. DISCUSSION An excellent potential for Ni(II) removal from aqueous solution was demonstrated by newly developed MLIDs biosorbent. The maximum adsorption capacity of MLIDs was found to be 58Æ7 ± 1Æ7 and 70Æ9 ± 2Æ6 mg Ni(II) g)1 immobilized biomass in batch and continuous flow biosorption system, respectively. It is higher than those reported for other immobilized systems by Wong and Fung (1997) for Enterobacter sp. (36Æ0 mg g)1), Blanco et al. (1999) for Phorimidium laminosum (16Æ1 mg g)1) and Dias et al. (2002) for Aspergillus terreus (19Æ6 mg g)1). This higher sorption of Ni(II) by MLIDs is mainly because of cell immobilization along the surface of the fibrous threads, no clumping and the reticulated open network of immobilized matrix, together contributing to enhanced surface area and free access of the metal to sorption sites. The higher rate of Ni(II) removal by MLIDs further indicates that no diffusional limitations were presented as noted with other microorganisms immobilized in polymeric matrices where a significant decrease in the rate of metal sorption, in comparison with free cells, occurred (Gourdon et al. 1990; Mahan and Holcombe 1992; Lopez et al. 2002). Surface immobilization of C. sorokiniana on individual threads of luffa sponge providing direct contact of biomass to metal solution is therefore better suited for


MICROALGA-LUFFA IMMOBILIZED DISCS AS A NEW BIOSORBENT

biosorption than enclosed or beaded immobilization in polymeric gel structure. The regeneration of the biosorbent is likely to be a key factor in accessing the potential of the biosorbent for commercial application. The study shows the potential of MLIDs to be used as a biosorption resin (biofilter) for the removal and recovery of metal ions from contaminated waters. Like ion-exchange resins, the MLIDs can be recycled. The Ni ions were sorbed and desorbed over as many as five cycles without any major loss in MLIDs efficiency in a fixed-bed column bioreactor. Furthermore, no leakage of entrapped biomass or physical breakage of MLIDs was observed during this continuous process of adsorption–desorption, as was noted with other polymeric matrices immobilized systems (Hu and Reeves 1997; Robinson 1998) which ultimately resulted in the loss of biosorption capacity of these immobilized systems. Luffa sponge is an inexpensive, easily available material and MLIDs can be easily made using very simple immobilization technique. This innovative technology provides a new reusable excellent Ni(II) biosorbent that is not only biodegradable, but also environmentally friendly for the development of a low-cost biosorption process to be used as a polishing treatment of heavy metal wastes. ACKNOWLEDGEMENT The authors thank the Alexander von Humboldt Foundation, Bonn, Germany for their equipment donations (atomic absorption spectrophotometer and illuminated incubator) during this study. REFERENCES Atkinson, B.W., Bux, F. and Kasan, H.C. (1998) Considerations for application of biosorption technology to remediate metal-contaminated industrial effluents. Water S.A. 24, 129–135.

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