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Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery
Bioresource Technology 102 (2011) 4191–4195
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery Nabanita Chakraborty, Amita Banerjee, Ruma Pal ⇑ Phycology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India
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Article history: Received 6 April 2010 Received in revised form 5 December 2010 Accepted 6 December 2010 Available online 13 December 2010 Keywords: Accumulation Bio-filter Lead Lyngbya majuscula Spirulina subsalsa
a b s t r a c t Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1–20 mg L 1 of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g 1) than S. subsalsa (1.32 mg g 1) at pH 6 within 3 h of exposure to 20 mg L 1 Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 lM) did not influence Pb accumulation whereas 100 lM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L 1 Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption–desorption is possible. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Lead occurs naturally in the earth’s crust at average concentrations of 15 mg kg 1, in soils from 5 to 25 mg kg 1 and in groundwater <0.45–14 lg L 1 (Smedley et al., 2002). Elevated Pb levels in water are generally caused by human activities such as battery manufacturing, printing, painting, dying and other industries (Eick et al., 1999). Since Pb can cause damage to organisms (Srivastava et al., 2004) and is a potential carcinogen (Silbergeld et al., 2000), EPA and WHO drinking water standard for lead have been set at 0.05 mg L 1 and 10 lg L 1, respectively. It is therefore, essential to remove Pb(II) from wastewater before disposal. A number of reports are available regarding Pb uptake by higher plants (Huang and Cunningham, 1996), and a few studies on Pb uptake, transport and detoxification have also been conducted with cyanobacteria such as Lyngbya, Anabaena, Nostoc, Spirulina, Gloeocapsa and Synechococcus (Klimmek et al., 2001; Heng et al., 2004; El-Sheekh et al., 2005; Gong et al., 2005; Raungsomboon et al., 2008; Shen et al., 2008) and eukaryotic microalgae belonging to the genera Scenedesmus, Chlorella, Cladophora (Fayed et al., 1983; Golab and Smith, 1992; Lamaia et al., 2005). Some of these biological systems have been reported to accumulate 15–75 mg Pb per gram biomass and are therefore as efficient as commonly used chemical adsorbents such as activated charcoal (Gupta and Rastogi, 2008), carbon aerogel (Goel et al., 2005) and carbon nanotubes (Li et al., 2002). ⇑ Corresponding author. Address: Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, West Bengal, India. Tel.: +91 033 24615445x312; fax: +91 033 2476 4419. E-mail address: [email protected] (R. Pal). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.028
Recovery of metal from exposed algal biomass can be achieved by washing with solutions containing chelators, organic and inorganic acids and other salts (Mehta and Gaur, 2005). The accumulation of heavy metals in algae involves metabolism independent passive adsorption and metabolism dependent active accumulation. In the first process, metal ions are rapidly adsorbed onto the cell surface and can easily be recovered by washing, where as in the second process, metal ions are absorbed across the cell membrane and sequestered within the cytoplasm at much slower rate (Swift and Forciniti, 1997). Moreover, metal accumulation depends upon different factors like, concentration of metals, pH of the metal solution, exposure time, presence or absence of chelators etc. Several ions of metal ligands on the cell surface are also responsible for controlling the metal uptake process (Mehta and Gaur, 2005). In this study, influence of metal concentration, pH, exposure time and chelators on Pb(II) accumulation by two cyanobacteria, Spirulina subsalsa and Lyngbya majuscula, was determined. These two cyanobacteria were chosen because they are easily cultured and were known as efficient Pb accumulators (Mehta and Gaur, 2005). The cyanobacteria were studied in free-living state and immobilized on glass wool. Recovery of bound Pb by EDTA was also measured.
2. Methods 2.1. Characterisation of cyanobacteria The marine cyanobacteria, Spirulina subsalsa and Lyngbya majuscula were collected from National Facility for Marine
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Cyanobacteria (NFMC), Trichy, Tamilnadu, India. The cyanobacteria are filamentous, multicellular organisms showing stacks of coin like cells tightly coiled into a more or less regular spiral, broader than 3 l in S. subsalsa and 16–60 l broad mature filaments with thick, hyaline, firm, mucilaginous sheath in L. majuscula. 2.2. Culturing of cyanobacteria Cyanobacterial cultures were grown and maintained at 20 °C in a 16:8 h light/dark cycle under cool fluorescent light with an intensity of 20–30 l Em 2 s 1 in Artificial Sea Nutrient III medium (Ott, 1965) containing (g L 1) NaCl, 25; MgCl26H2O, 2; KCl, 0.5; NaNO3, 0.75; K2HPO43H2O, 0.02; MgSO4.7H2O, 3.5; CaCl2, 0.5; citric acid, 0.003; ferric ammonium citrate, 0.003; EDTA, 0.0005 and Na2CO3, 0.02. The medium was supplemented with 1 mL L 1 trace metal mix (A5) containing (mg mL 1) H3BO3, 2.86; MnCl24H20, 1.81; Na2MoO42H2O, 0.390; ZnSO47H2O, 0.222; CuSO45H2O, 0.079 and Co(NO3)26H2O, 0.0494 pH of the medium was 7.5. 2.3. Chemicals All chemicals used in this study were of analytical grade obtained either from Merck, Germany or Sisco Research Laboratory, India. Purified water was prepared using a Millipore Milli-Q water purification system. Standard solution of Pb(II) (1000 mg L 1) for atomic adsorption spectrophotometer was obtained from Merck, Germany. Standard acid and base solutions (0.1 N HCl and 0.1 N NaOH) were used for pH adjustments. 2.4. Experimental 2.4.1. Metal accumulation in varying factors Lead nitrate salt (0.160 g) was dissolved in 1 L doubled distilled water to prepare 100 mg L 1 stock solution of Pb(II) and dilutions were prepared in modified ASN III medium containing only NaCl and NaNO3. Biomass was obtained by centrifugation of exponentially growing cultures at 1500 rpm (800 g) for 10 min and washing in double distilled water. Excess water was removed by placement of filter paper. Approximately 1 g of fresh biomass was used for each set of experiment. To measure the effect of metal concentration on Pb uptake, the biomasses were exposed to 50 mL of 1, 2.5, 5, 10 and 20 mg L 1 of Pb(II) solution, kept in 100 mL Erlenmeyer flasks without pH adjustment. The suspensions were agitated on a rotary shaker at 150 rpm for 3 h at room temperature. The effect of pH on Pb accumulation was studied by exposing cells for 3 h to the modified ASN III medium containing 5 mg L 1 of Pb(II) at room temperature. The pH was adjusted to pH 6, 7 or 8 with 0.1 M HCl or 0.1 M NaOH using a pH meter (Global model DPH 500, India). A magnetic stirrer was used to agitate the solution continuously. The time dependence of Pb accumulation was determined by exposing the algal biomass in 20 mg L 1 Pb solution at pH 6 for 10, 20, 30, 40, 50 min and 1, 3, 24 and 48 h. For the studies on effect of chelators in metal accumulation process 2 lM EDTA and 100 lM citrate solutions were added separately to medium containing 20 mg L 1 Pb and cyanobacteria were exposed for 3 h. After each experiment the cell suspensions were subjected to centrifugation at 1500 rpm for 10 min. Blank sets were prepared for each case without biomass. Each experiment was run in triplicate and mean values are reported. Standard deviations calculated were within ±1.3%. 2.4.2. Metal uptake modeling Biomass was exposed to 25, 50, 100, 150, 200 and 250 mg L 1 Pb(II) solution (initial concentration, Ci) at 25 °C, equilibrium metal ion concentration in solution (mg L 1) was measured and the
values were used to calculate equilibrium metal ion concentration in adsorbent (mg g 1). Values for these parameters calculated from experimental results were compared with the Langmuir equation qe = qmax b Ce/1 + b Ce (Langmuir, 1918), where, qmax represents the maximum adsorption capacity of the adsorbent (mg g 1), Ce is equilibrium metal ion concentration in solution, qe is equilibrium metal ion concentration in adsorbent and b, the Langmuir constant is an affinity parameter, related to the energy of biosorption. Constant b was calculated from the y intercept of a 1/Ce vs. 1/qe plot.
2.4.3. Recovery of the metals from exposed biomass Biomass showing maximum accumulation after 3 h exposure to 20 mg L 1 Pb(II) in pH 6 was separated from the liquid phase by centrifugation and divided equally. One part was washed with double distilled water and the other with 0.1 M EDTA solution for 30 min. The washed biomass was centrifuged and Pb content was determined to assess the recovery of Pb with respect to the total accumulation in the biomass.
2.4.4. Quantification of accumulated Pb by AAS Pre-weighed cyanobacterial samples (0.5 g) were collected, thoroughly washed with distilled water, oven dried, ground with a mortar and pestle and sieved to pass a 100 mesh screen. Digestion was carried out with concentrated HNO3 followed by concentrated HCl in a digestion chamber until the contents became clear. Digested materials were filtered through Whatman-41 filter paper and the volume of the filtrate was adjusted to 20 mL with double glass distilled water. Pb analysis was done at 217 nm with an flame atomic absorption spectrophotometer (Varian Techtron model AA-575 ABQ). All measurements were carried out in an air/acetylene flame. The concentration of Pb(II) remaining in the exposure solution, after removal of the biomass, was also determined by the same method mentioned above. Mean values were calculated from three replicates. The calibration was performed within the calibration range of Pb and the correlation coefficients for the calibration curves were 0.98 or better. Control Pb solutions were run to detect any possible metal precipitation or contamination and all the experimental samples were compared to the respective standards.
2.4.5. Accumulation and recovery of Pb using immobilized biomass Immobilized live L. majuscula biomass was used in vertical column to design a bio-filter. A glass column of 45.72 cm length and 6.35 cm diameter was packed with commercially available glass wool and ASNIII medium along with live L. majuscula thallus as inoculum and maintained at 20 °C in a 16:8 h light/dark cycle under cool fluorescent light having light intensity of 20–30 l Einstein m 2 s 1. After 30 days actively growing cyanobacterial filaments enmeshed in glass wool and turned into deep blue green algal mat. The medium was removed and the column was washed with double distilled water. Five hundred mL aqueous Pb solution (5 mg L 1) was added, illumination was continued and after 30 min the column was drained (elute I). The column was washed with 0.1 M EDTA (elute II) and successive repetition of the process for a second time generated elute III and IV, respectively. Lead accumulation by dead cyanobacteria was measured with immobilized cells killed at 105 °C for 24 h and exposed to 5 mg L 1 Pb solution. The column was drained to obtain elute V. Glass wool without biomass was exposed to the Pb solution to determine Pb(II) binding to the support. Lead contents of the elutes were measured separately by AAS.
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3. Results and discussion 3.1. Metal accumulation in varying factors 3.1.1. Effect of metal concentration L. majuscula showed higher Pb accumulation than S. subsalsa (Table 1). The absorption was concentration dependant. L. majuscula accumulated 15 times more Pb when exposed to 20 mg L 1 Pb solution for 3 h than that to 1 mg L 1 while a 2.5 to 3-fold increase in accumulation of Pb was observed in S. subsalsa under the same experimental conditions. It is known that metal accumulation (absorption and adsorption) is directly proportional to the metal concentration up to a point when equilibrium is reached (Da Costa and Leite, 1991) as algal cell surface has several functional groups with varying affinity for metal ions. In the present study also a gradual increase in Pb accumulation with gradual increase in Pb(II) ions in the solution up to 20 mg L 1 indicates the presence of both low and high affinity functional groups in both the cyanobacteria. 3.1.2. Effect of pH In L. majuscula accumulation of Pb was 3.5, 3.0 and 2.89 mg g 1 at pH 6, 7 and 8 respectively. The corresponding values were 1.07, 1.05 and 0.9 mg g 1 for S. subsalsa. Although cation binding is expected to increase with increasing pH as deprotonation makes sites available for metal binding, Pb(II) accumulation by the cyanobacteria decreased with increasing pH. This phenomenon could have been due to binding or bonding of Pb to anions or neutral molecules thereby decreasing its bioavailability in higher pH (Brown and Lemay, 1981) and/or the formation of hydroxyl compounds precipitating in the solution. Optimum pH for maximum Pb accumulation in Lyngbya taylorii and Spirulina maxima was reported to be pH 3–7 and pH 5.5, respectively (Klimmek et al., 2001; Gong et al., 2005). Chojnacka et al. (2005) reported the main involvement of carboxyl group in binding of Pb onto cyanobacterial cell surface for pH ranges 3–6. In the present study also predominance of carboxyl groups in biomass exposed to Pb in pH 6 was found by FTIR analysis (result not shown).
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indicated a major metabolism independent passive adsorption onto the cell surface within a short span of time. This was further supported by the result of washing the biomass with 0.1 M EDTA solution which removed surface bound metal, leaving behind the intracellular fraction. L. majuscula and S. subsalsa showed nearly 3.5 and 1.5-fold increase in Pb sorption within 3 h compared to those of in the very first hour (Table 1). Similar results were also found by Hussain et al. (2009) in Spirogyra neglecta. It is known that vacant cell surface sites are gradually occupied by metal ions till the equilibrium is reached and the variation in metal accumulation by different algae depends on the compositional differences of their cell walls. De Philippis et al. (2003) demonstrated higher metal binding capacity of a cyanobacterial strain with thick capsule than strains devoid of or with only a thin layer of capsule. Similarly in the present study thick structural polysaccharide sheath surrounding L. majuscula may have allowed for the higher Pb adsorption than S. subsalsa. The importance of extracellular polysaccharide in metal removal process was also demonstrated by Freire-Nordi et al. (2005) for Anabaena spiroides.
3.1.4. Effect of chelators EDTA decreased accumulation in both species (Fig. 1). EDTA acts as solubiliser of Pb and there are reports on significant increase in Pb uptake in plants after EDTA application (Wu et al., 1999). In the present study less uptake of Pb from solution in presence of EDTA suggests the chelation of Pb by EDTA preventing its binding to cell surface. Decreased Pb uptake after EDTA application was earlier reported by Athalye et al. (1995). S. subsalsa exhibited nearly a 6 to 8-fold increase in Pb accumulation during the first 3 h in presence of 100 lM citrate where as
3.1.3. Effects of contact time The biosorption of Pb by the cyanobacteria increased with exposure time. A quick accumulation within 10 min, accumulation maxima at 3 h of exposure followed by equilibrium within 48 h
Table 1 Accumulation of lead by two cyanobacteria in different experimental conditions. Experimental conditions
Concentration of Pb(II) (mg L 1)
pH of media
Exposure time (h)
Pb(II) Accumulation (mg g
1
)
1 2.5 5 10 20
L majuscula 0.88 ± 0.32 2.0 ± 0.4 3.2 ± 0.38 6.6 ± 0.5 13.5 ± 0.8
S. subsalsa 0.49 ± 0.2 0.83 ± 0.08 1.10 ± 0.14 1.20 ± 0.06 1.32 ± 0.04
6 7 8
3.4 ± 0.2 3.0 ± 0.09 2.89 ± 0.135
1.07 ± 0.07 1.05 ± 0.03 0.90 ± 0.05
4.4 6.25 9 10.5 11.5 12.6 13.5 13.06 13.2
0 0.29 0.458 0.65 0.8 0.9 1.03 1.02 1.03
0.166 0.33 0.5 0.66 0.83 1 3 24 48
Fig. 1. Effect of EDTA and citrate as chelators on lead accumulation in L. majuscula (a) and S. subsalsa (b) after 1–3 h exposure in 20 mg L 1 solution at pH 6.
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accumulation was lower for L. majuscula than in the absence of citrate. Complexation of Pb(II) ion by a citrate ligand would be expected to decrease its bioavailability, and such a decrease was noted for L. majuscula. The increase in Pb accumulation by S. subsalsa in presence of citrate could have possibly been due to transport of metal–ligand complex across cell membrane as suggested by Errecalde et al. (1998). 3.2. Metal uptake modeling The accumulation pattern of Pb fitted well with Langmuir Isotherm model since the test samples showed a gradual increase in metal accumulation with increasing metal concentrations. The equilibrium relationship between metal adsorption (qe) and residual metal (Ce) is typically hyperbolic. Ce plotted against Ce/qe gave a straight line that was fitted by regression and values of b were calculated (data not shown). 1/Ceq vs. 1/qeq plots gave straight-line curves with more than 97–99% significance in both the species (Fig. 2). High R2 values (0.9896-L. majuscula, 0.9809-S. subsalsa) signified greater affinity between metal ion concentration in media and metal accumulated by cyanobacteria. The qmax values for fresh L. majuscula and S. subsalsa were found to be 20.96 mg g 1 and 16.72 mg g 1, respectively in 250 mg L 1 Pb solution. The fit with the Langmuir isotherm model indicates monolayer sorption of Pb to the binding sites. This finding parallels those made for Pb adsorption by cyanobacteria Spirulina sp. (Arthrospira) and Gloeocapsa sp. (Hernandez and Olguin, 2002; Raungsomboon et al., 2008) and other fresh water eukaryotic micro algae Cladophora crispata and Spirogyra neglecta (Ozer et al. 1994; Singh et al., 2007).
Fig. 3. Recovery of lead by 0.1 M EDTA from L. majuscula and S. subsalsa biomass exposed to 20 mg L 1 lead solution for 1–3 h at pH 6.
3.3. Recovery of metals Washing of Pb exposed biomass with 0.1 M EDTA solution recovered 74% and 51.2% of accumulated Pb from L. majuscula and S. subsalsa respectively (Fig. 3). Previously Gong et al. (2005) reported 91% recovery of adsorbed Pb from S. maxima using 0.1 M EDTA. Efficacy of L. majuscula in Pb removal and recovery process leads us to use it as a suitable biosorbent in a proposed columnar bio-filter for metal pollutants. 3.4. Model for bioremoval of metals The column packed with glass wool only retained 18–20% Pb from 5 mg L 1 solution, but 95.8% Pb was adsorbed by the column packed with growing cyanobacteria filaments and only 4.2% Pb was found in Elute I. After the first wash with 0.1 M EDTA, 92.5% of accumulated Pb was recovered in Elute II. A second exposure to 5 mg L 1 Pb solution showed 59–60% removal of Pb (40% remaining in Elute III) indicating its ability to be used in repeated Pb removal process. When heat killed biomass was used as biosorbent, almost 34% Pb was adsorbed by the column, rest 67% recovered in the elute (Elute IV). Therefore, immobilised live L. majuscula biomass is superior to dead biomass and to non-immobilised biomass which removed 64% of the Pb from a 5 mg L 1 Pb solution. The results obtained with immobilized cells parallel those obtained by Bender et al. (1994) who used cyanobacterial mats packed in a glass column to remove Zinc and Manganese from contaminated water with a removal of 96% Zn and 86% Mn within 3 h could be removed by the column. 4. Conclusions
Fig. 2. Linearised Langmuir Isotherm model of lead accumulation (at 25 °C) by two cyanobacteria (a) L. majuscula, (b) S. subsalsa.
Of the two cyanobacteria examined, L. majuscula was the superior Pb(II) accumulator, presumably because of its polysaccharide sheath structure surrounding the filaments. A packed bed column with immobilized L. majuscula filaments would be highly effective in Pb removal from contaminated effluent with almost 96% removal capacity. Although the use of cyanobacteria for heavy metal removal would be ecofriendly and cost effective in comparison to traditional methods, the feasibility of this process on a large scale remains to be demonstrated.
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Acknowledgements The authors would like to thank DAE-BRNS, Government of India, for funding.
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Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery Nabanita Chakraborty, Amita Banerjee, Ruma Pal ⇑ Phycology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India
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Article history: Received 6 April 2010 Received in revised form 5 December 2010 Accepted 6 December 2010 Available online 13 December 2010 Keywords: Accumulation Bio-filter Lead Lyngbya majuscula Spirulina subsalsa
a b s t r a c t Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1–20 mg L 1 of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g 1) than S. subsalsa (1.32 mg g 1) at pH 6 within 3 h of exposure to 20 mg L 1 Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 lM) did not influence Pb accumulation whereas 100 lM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L 1 Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption–desorption is possible. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Lead occurs naturally in the earth’s crust at average concentrations of 15 mg kg 1, in soils from 5 to 25 mg kg 1 and in groundwater <0.45–14 lg L 1 (Smedley et al., 2002). Elevated Pb levels in water are generally caused by human activities such as battery manufacturing, printing, painting, dying and other industries (Eick et al., 1999). Since Pb can cause damage to organisms (Srivastava et al., 2004) and is a potential carcinogen (Silbergeld et al., 2000), EPA and WHO drinking water standard for lead have been set at 0.05 mg L 1 and 10 lg L 1, respectively. It is therefore, essential to remove Pb(II) from wastewater before disposal. A number of reports are available regarding Pb uptake by higher plants (Huang and Cunningham, 1996), and a few studies on Pb uptake, transport and detoxification have also been conducted with cyanobacteria such as Lyngbya, Anabaena, Nostoc, Spirulina, Gloeocapsa and Synechococcus (Klimmek et al., 2001; Heng et al., 2004; El-Sheekh et al., 2005; Gong et al., 2005; Raungsomboon et al., 2008; Shen et al., 2008) and eukaryotic microalgae belonging to the genera Scenedesmus, Chlorella, Cladophora (Fayed et al., 1983; Golab and Smith, 1992; Lamaia et al., 2005). Some of these biological systems have been reported to accumulate 15–75 mg Pb per gram biomass and are therefore as efficient as commonly used chemical adsorbents such as activated charcoal (Gupta and Rastogi, 2008), carbon aerogel (Goel et al., 2005) and carbon nanotubes (Li et al., 2002). ⇑ Corresponding author. Address: Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, West Bengal, India. Tel.: +91 033 24615445x312; fax: +91 033 2476 4419. E-mail address: [email protected] (R. Pal). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.028
Recovery of metal from exposed algal biomass can be achieved by washing with solutions containing chelators, organic and inorganic acids and other salts (Mehta and Gaur, 2005). The accumulation of heavy metals in algae involves metabolism independent passive adsorption and metabolism dependent active accumulation. In the first process, metal ions are rapidly adsorbed onto the cell surface and can easily be recovered by washing, where as in the second process, metal ions are absorbed across the cell membrane and sequestered within the cytoplasm at much slower rate (Swift and Forciniti, 1997). Moreover, metal accumulation depends upon different factors like, concentration of metals, pH of the metal solution, exposure time, presence or absence of chelators etc. Several ions of metal ligands on the cell surface are also responsible for controlling the metal uptake process (Mehta and Gaur, 2005). In this study, influence of metal concentration, pH, exposure time and chelators on Pb(II) accumulation by two cyanobacteria, Spirulina subsalsa and Lyngbya majuscula, was determined. These two cyanobacteria were chosen because they are easily cultured and were known as efficient Pb accumulators (Mehta and Gaur, 2005). The cyanobacteria were studied in free-living state and immobilized on glass wool. Recovery of bound Pb by EDTA was also measured.
2. Methods 2.1. Characterisation of cyanobacteria The marine cyanobacteria, Spirulina subsalsa and Lyngbya majuscula were collected from National Facility for Marine
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Cyanobacteria (NFMC), Trichy, Tamilnadu, India. The cyanobacteria are filamentous, multicellular organisms showing stacks of coin like cells tightly coiled into a more or less regular spiral, broader than 3 l in S. subsalsa and 16–60 l broad mature filaments with thick, hyaline, firm, mucilaginous sheath in L. majuscula. 2.2. Culturing of cyanobacteria Cyanobacterial cultures were grown and maintained at 20 °C in a 16:8 h light/dark cycle under cool fluorescent light with an intensity of 20–30 l Em 2 s 1 in Artificial Sea Nutrient III medium (Ott, 1965) containing (g L 1) NaCl, 25; MgCl26H2O, 2; KCl, 0.5; NaNO3, 0.75; K2HPO43H2O, 0.02; MgSO4.7H2O, 3.5; CaCl2, 0.5; citric acid, 0.003; ferric ammonium citrate, 0.003; EDTA, 0.0005 and Na2CO3, 0.02. The medium was supplemented with 1 mL L 1 trace metal mix (A5) containing (mg mL 1) H3BO3, 2.86; MnCl24H20, 1.81; Na2MoO42H2O, 0.390; ZnSO47H2O, 0.222; CuSO45H2O, 0.079 and Co(NO3)26H2O, 0.0494 pH of the medium was 7.5. 2.3. Chemicals All chemicals used in this study were of analytical grade obtained either from Merck, Germany or Sisco Research Laboratory, India. Purified water was prepared using a Millipore Milli-Q water purification system. Standard solution of Pb(II) (1000 mg L 1) for atomic adsorption spectrophotometer was obtained from Merck, Germany. Standard acid and base solutions (0.1 N HCl and 0.1 N NaOH) were used for pH adjustments. 2.4. Experimental 2.4.1. Metal accumulation in varying factors Lead nitrate salt (0.160 g) was dissolved in 1 L doubled distilled water to prepare 100 mg L 1 stock solution of Pb(II) and dilutions were prepared in modified ASN III medium containing only NaCl and NaNO3. Biomass was obtained by centrifugation of exponentially growing cultures at 1500 rpm (800 g) for 10 min and washing in double distilled water. Excess water was removed by placement of filter paper. Approximately 1 g of fresh biomass was used for each set of experiment. To measure the effect of metal concentration on Pb uptake, the biomasses were exposed to 50 mL of 1, 2.5, 5, 10 and 20 mg L 1 of Pb(II) solution, kept in 100 mL Erlenmeyer flasks without pH adjustment. The suspensions were agitated on a rotary shaker at 150 rpm for 3 h at room temperature. The effect of pH on Pb accumulation was studied by exposing cells for 3 h to the modified ASN III medium containing 5 mg L 1 of Pb(II) at room temperature. The pH was adjusted to pH 6, 7 or 8 with 0.1 M HCl or 0.1 M NaOH using a pH meter (Global model DPH 500, India). A magnetic stirrer was used to agitate the solution continuously. The time dependence of Pb accumulation was determined by exposing the algal biomass in 20 mg L 1 Pb solution at pH 6 for 10, 20, 30, 40, 50 min and 1, 3, 24 and 48 h. For the studies on effect of chelators in metal accumulation process 2 lM EDTA and 100 lM citrate solutions were added separately to medium containing 20 mg L 1 Pb and cyanobacteria were exposed for 3 h. After each experiment the cell suspensions were subjected to centrifugation at 1500 rpm for 10 min. Blank sets were prepared for each case without biomass. Each experiment was run in triplicate and mean values are reported. Standard deviations calculated were within ±1.3%. 2.4.2. Metal uptake modeling Biomass was exposed to 25, 50, 100, 150, 200 and 250 mg L 1 Pb(II) solution (initial concentration, Ci) at 25 °C, equilibrium metal ion concentration in solution (mg L 1) was measured and the
values were used to calculate equilibrium metal ion concentration in adsorbent (mg g 1). Values for these parameters calculated from experimental results were compared with the Langmuir equation qe = qmax b Ce/1 + b Ce (Langmuir, 1918), where, qmax represents the maximum adsorption capacity of the adsorbent (mg g 1), Ce is equilibrium metal ion concentration in solution, qe is equilibrium metal ion concentration in adsorbent and b, the Langmuir constant is an affinity parameter, related to the energy of biosorption. Constant b was calculated from the y intercept of a 1/Ce vs. 1/qe plot.
2.4.3. Recovery of the metals from exposed biomass Biomass showing maximum accumulation after 3 h exposure to 20 mg L 1 Pb(II) in pH 6 was separated from the liquid phase by centrifugation and divided equally. One part was washed with double distilled water and the other with 0.1 M EDTA solution for 30 min. The washed biomass was centrifuged and Pb content was determined to assess the recovery of Pb with respect to the total accumulation in the biomass.
2.4.4. Quantification of accumulated Pb by AAS Pre-weighed cyanobacterial samples (0.5 g) were collected, thoroughly washed with distilled water, oven dried, ground with a mortar and pestle and sieved to pass a 100 mesh screen. Digestion was carried out with concentrated HNO3 followed by concentrated HCl in a digestion chamber until the contents became clear. Digested materials were filtered through Whatman-41 filter paper and the volume of the filtrate was adjusted to 20 mL with double glass distilled water. Pb analysis was done at 217 nm with an flame atomic absorption spectrophotometer (Varian Techtron model AA-575 ABQ). All measurements were carried out in an air/acetylene flame. The concentration of Pb(II) remaining in the exposure solution, after removal of the biomass, was also determined by the same method mentioned above. Mean values were calculated from three replicates. The calibration was performed within the calibration range of Pb and the correlation coefficients for the calibration curves were 0.98 or better. Control Pb solutions were run to detect any possible metal precipitation or contamination and all the experimental samples were compared to the respective standards.
2.4.5. Accumulation and recovery of Pb using immobilized biomass Immobilized live L. majuscula biomass was used in vertical column to design a bio-filter. A glass column of 45.72 cm length and 6.35 cm diameter was packed with commercially available glass wool and ASNIII medium along with live L. majuscula thallus as inoculum and maintained at 20 °C in a 16:8 h light/dark cycle under cool fluorescent light having light intensity of 20–30 l Einstein m 2 s 1. After 30 days actively growing cyanobacterial filaments enmeshed in glass wool and turned into deep blue green algal mat. The medium was removed and the column was washed with double distilled water. Five hundred mL aqueous Pb solution (5 mg L 1) was added, illumination was continued and after 30 min the column was drained (elute I). The column was washed with 0.1 M EDTA (elute II) and successive repetition of the process for a second time generated elute III and IV, respectively. Lead accumulation by dead cyanobacteria was measured with immobilized cells killed at 105 °C for 24 h and exposed to 5 mg L 1 Pb solution. The column was drained to obtain elute V. Glass wool without biomass was exposed to the Pb solution to determine Pb(II) binding to the support. Lead contents of the elutes were measured separately by AAS.
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3. Results and discussion 3.1. Metal accumulation in varying factors 3.1.1. Effect of metal concentration L. majuscula showed higher Pb accumulation than S. subsalsa (Table 1). The absorption was concentration dependant. L. majuscula accumulated 15 times more Pb when exposed to 20 mg L 1 Pb solution for 3 h than that to 1 mg L 1 while a 2.5 to 3-fold increase in accumulation of Pb was observed in S. subsalsa under the same experimental conditions. It is known that metal accumulation (absorption and adsorption) is directly proportional to the metal concentration up to a point when equilibrium is reached (Da Costa and Leite, 1991) as algal cell surface has several functional groups with varying affinity for metal ions. In the present study also a gradual increase in Pb accumulation with gradual increase in Pb(II) ions in the solution up to 20 mg L 1 indicates the presence of both low and high affinity functional groups in both the cyanobacteria. 3.1.2. Effect of pH In L. majuscula accumulation of Pb was 3.5, 3.0 and 2.89 mg g 1 at pH 6, 7 and 8 respectively. The corresponding values were 1.07, 1.05 and 0.9 mg g 1 for S. subsalsa. Although cation binding is expected to increase with increasing pH as deprotonation makes sites available for metal binding, Pb(II) accumulation by the cyanobacteria decreased with increasing pH. This phenomenon could have been due to binding or bonding of Pb to anions or neutral molecules thereby decreasing its bioavailability in higher pH (Brown and Lemay, 1981) and/or the formation of hydroxyl compounds precipitating in the solution. Optimum pH for maximum Pb accumulation in Lyngbya taylorii and Spirulina maxima was reported to be pH 3–7 and pH 5.5, respectively (Klimmek et al., 2001; Gong et al., 2005). Chojnacka et al. (2005) reported the main involvement of carboxyl group in binding of Pb onto cyanobacterial cell surface for pH ranges 3–6. In the present study also predominance of carboxyl groups in biomass exposed to Pb in pH 6 was found by FTIR analysis (result not shown).
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indicated a major metabolism independent passive adsorption onto the cell surface within a short span of time. This was further supported by the result of washing the biomass with 0.1 M EDTA solution which removed surface bound metal, leaving behind the intracellular fraction. L. majuscula and S. subsalsa showed nearly 3.5 and 1.5-fold increase in Pb sorption within 3 h compared to those of in the very first hour (Table 1). Similar results were also found by Hussain et al. (2009) in Spirogyra neglecta. It is known that vacant cell surface sites are gradually occupied by metal ions till the equilibrium is reached and the variation in metal accumulation by different algae depends on the compositional differences of their cell walls. De Philippis et al. (2003) demonstrated higher metal binding capacity of a cyanobacterial strain with thick capsule than strains devoid of or with only a thin layer of capsule. Similarly in the present study thick structural polysaccharide sheath surrounding L. majuscula may have allowed for the higher Pb adsorption than S. subsalsa. The importance of extracellular polysaccharide in metal removal process was also demonstrated by Freire-Nordi et al. (2005) for Anabaena spiroides.
3.1.4. Effect of chelators EDTA decreased accumulation in both species (Fig. 1). EDTA acts as solubiliser of Pb and there are reports on significant increase in Pb uptake in plants after EDTA application (Wu et al., 1999). In the present study less uptake of Pb from solution in presence of EDTA suggests the chelation of Pb by EDTA preventing its binding to cell surface. Decreased Pb uptake after EDTA application was earlier reported by Athalye et al. (1995). S. subsalsa exhibited nearly a 6 to 8-fold increase in Pb accumulation during the first 3 h in presence of 100 lM citrate where as
3.1.3. Effects of contact time The biosorption of Pb by the cyanobacteria increased with exposure time. A quick accumulation within 10 min, accumulation maxima at 3 h of exposure followed by equilibrium within 48 h
Table 1 Accumulation of lead by two cyanobacteria in different experimental conditions. Experimental conditions
Concentration of Pb(II) (mg L 1)
pH of media
Exposure time (h)
Pb(II) Accumulation (mg g
1
)
1 2.5 5 10 20
L majuscula 0.88 ± 0.32 2.0 ± 0.4 3.2 ± 0.38 6.6 ± 0.5 13.5 ± 0.8
S. subsalsa 0.49 ± 0.2 0.83 ± 0.08 1.10 ± 0.14 1.20 ± 0.06 1.32 ± 0.04
6 7 8
3.4 ± 0.2 3.0 ± 0.09 2.89 ± 0.135
1.07 ± 0.07 1.05 ± 0.03 0.90 ± 0.05
4.4 6.25 9 10.5 11.5 12.6 13.5 13.06 13.2
0 0.29 0.458 0.65 0.8 0.9 1.03 1.02 1.03
0.166 0.33 0.5 0.66 0.83 1 3 24 48
Fig. 1. Effect of EDTA and citrate as chelators on lead accumulation in L. majuscula (a) and S. subsalsa (b) after 1–3 h exposure in 20 mg L 1 solution at pH 6.
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accumulation was lower for L. majuscula than in the absence of citrate. Complexation of Pb(II) ion by a citrate ligand would be expected to decrease its bioavailability, and such a decrease was noted for L. majuscula. The increase in Pb accumulation by S. subsalsa in presence of citrate could have possibly been due to transport of metal–ligand complex across cell membrane as suggested by Errecalde et al. (1998). 3.2. Metal uptake modeling The accumulation pattern of Pb fitted well with Langmuir Isotherm model since the test samples showed a gradual increase in metal accumulation with increasing metal concentrations. The equilibrium relationship between metal adsorption (qe) and residual metal (Ce) is typically hyperbolic. Ce plotted against Ce/qe gave a straight line that was fitted by regression and values of b were calculated (data not shown). 1/Ceq vs. 1/qeq plots gave straight-line curves with more than 97–99% significance in both the species (Fig. 2). High R2 values (0.9896-L. majuscula, 0.9809-S. subsalsa) signified greater affinity between metal ion concentration in media and metal accumulated by cyanobacteria. The qmax values for fresh L. majuscula and S. subsalsa were found to be 20.96 mg g 1 and 16.72 mg g 1, respectively in 250 mg L 1 Pb solution. The fit with the Langmuir isotherm model indicates monolayer sorption of Pb to the binding sites. This finding parallels those made for Pb adsorption by cyanobacteria Spirulina sp. (Arthrospira) and Gloeocapsa sp. (Hernandez and Olguin, 2002; Raungsomboon et al., 2008) and other fresh water eukaryotic micro algae Cladophora crispata and Spirogyra neglecta (Ozer et al. 1994; Singh et al., 2007).
Fig. 3. Recovery of lead by 0.1 M EDTA from L. majuscula and S. subsalsa biomass exposed to 20 mg L 1 lead solution for 1–3 h at pH 6.
3.3. Recovery of metals Washing of Pb exposed biomass with 0.1 M EDTA solution recovered 74% and 51.2% of accumulated Pb from L. majuscula and S. subsalsa respectively (Fig. 3). Previously Gong et al. (2005) reported 91% recovery of adsorbed Pb from S. maxima using 0.1 M EDTA. Efficacy of L. majuscula in Pb removal and recovery process leads us to use it as a suitable biosorbent in a proposed columnar bio-filter for metal pollutants. 3.4. Model for bioremoval of metals The column packed with glass wool only retained 18–20% Pb from 5 mg L 1 solution, but 95.8% Pb was adsorbed by the column packed with growing cyanobacteria filaments and only 4.2% Pb was found in Elute I. After the first wash with 0.1 M EDTA, 92.5% of accumulated Pb was recovered in Elute II. A second exposure to 5 mg L 1 Pb solution showed 59–60% removal of Pb (40% remaining in Elute III) indicating its ability to be used in repeated Pb removal process. When heat killed biomass was used as biosorbent, almost 34% Pb was adsorbed by the column, rest 67% recovered in the elute (Elute IV). Therefore, immobilised live L. majuscula biomass is superior to dead biomass and to non-immobilised biomass which removed 64% of the Pb from a 5 mg L 1 Pb solution. The results obtained with immobilized cells parallel those obtained by Bender et al. (1994) who used cyanobacterial mats packed in a glass column to remove Zinc and Manganese from contaminated water with a removal of 96% Zn and 86% Mn within 3 h could be removed by the column. 4. Conclusions
Fig. 2. Linearised Langmuir Isotherm model of lead accumulation (at 25 °C) by two cyanobacteria (a) L. majuscula, (b) S. subsalsa.
Of the two cyanobacteria examined, L. majuscula was the superior Pb(II) accumulator, presumably because of its polysaccharide sheath structure surrounding the filaments. A packed bed column with immobilized L. majuscula filaments would be highly effective in Pb removal from contaminated effluent with almost 96% removal capacity. Although the use of cyanobacteria for heavy metal removal would be ecofriendly and cost effective in comparison to traditional methods, the feasibility of this process on a large scale remains to be demonstrated.
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Acknowledgements The authors would like to thank DAE-BRNS, Government of India, for funding.
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