Sorption of mercury, cadmium and lead by microalgae

ScienceAsia 28 (2002) : 253-261 Sorption of mercury, cadmium and lead by microalgae Duangrat Inthorna, *, Nalin Sidtitoona, Suthep Silapanuntakula and Aran Incharoensakdib a Department of Environmental Health Sciences, Faculty of Public Health, Mahidol University. Department of Biochemistry, Faculty of Science, Chulalongkorn University. * Corresponding author, E-mail: [email protected] b Received 15 Mar 2001 Accepted 11 Dec 2001 ABSTRACT Forty-six strains of microalgae from BIOTECH Culture Collection (NSTDA) , Microbiological Resources Center (TISTR) and Institute of Research and Food Development, 3 strains collected from Thai natural and industrial areas and 3 strains from the Gottingen University culture collection were tested for Mercury (Hg), Cadmium (Cd) and Lead (Pb) removal in aqueous solutions. In green algae, the highest Hg removal was by Scenedesmus sp., Chlorococcum sp., Chlorella vulgaris var. vulgaris and Fischerella sp., (97%, 96%, 94% and 92%, respectively). In blue green algae, highest Hg removal was by Lyngbya spiralis, Tolypothirx tenuis, Stigonema sp., Phormidium molle (96%, 94%, 94% and 93%, respectively). For Cd removal in green algae, the highest was by Chlorococcum sp., T5, Fischerella sp., Chlorella vulgaris var. vulgaris and Scenedesmus acutus (94%, 94%, 91%, 89% and 88%, respectively). In blue green algae, highest Cd removal was by Lyngbya heironymusii, Gloeocapsa sp., Phormidium molle, Oscillatoria jasorvensis and Nostoc sp. (97%, 96%, 95%, 94% and 94%, respectively). In green algae, highest Pb removal was by Scenedesmus acutus, Chlorella vulgaris var. vulgaris, Chlorella vulgaris, Scenedesmus vacuolatus and Chlorella vulgaris, (89%, 88%, 85%, 85% and 84%, respectively). In blue green algae, highest Pb removal was by Nostoc punciforme, Oscillatoria agardhii, Gloeocapsa sp., Nostoc piscinale, Nostoc commune and Nostoc paludosum (98%, 96%, 96%, 94%, 94% and 92%, respectively). Scenedesmus acutus had the highest concentration factor (CF) at 3,412, 4,591 and 4,078 for Hg, Cd and Pb, respectively. Tolypothrix tenuis had the highest maximum adsorption capacity of 27 mg Hg/g dry wt. at a minimum concentration of 1.04 mg/l, Scenedesmus acutus had the highest maximum adsorption capacity of 110 mg Cd/g dry wt. at a minimum concentration of 48 mg/l and Chlorella vulgaris had the highest maximum adsorption capacity of 127 mg Pb/g dry wt. at a minimum concentration of 130 mg/l. KEYWORDS: microalgae, concentration factor, heavy metal removal, mercury, cadmium and lead. INTRODUCTION The release of heavy metals from industries into the environment has resulted in many problems for both human health and aquatic ecosystems.1, 2 One strategy to reduce heavy metal solution is to use microorganisms. Microalgae, due to their ubiquitous occurrence in nature, have been studied extensively in this regard. They can sequester heavy metal ions by adsorption and absorption, as do by other microorganisms. This ability may be induced in response to stress by toxic heavy metal exposure.3 The use of microalgae for metal removal has the potential to achieve greater performance at a lower cost than conventional wastewater treatment technologies. This is consistent with the recent trend for growing interest in biosorbent technology for removal of trace amounts of toxic metals from dilute aqueous waste. New biosorbent materials are already being used.4 In this study, a number of microalgal strains potentially suitable for Cd, Pb and Hg removal in aqueous solution were selected and the ranking table as removal index and concentration factor index were also calcu lated. Efficien cy of rem oval, maximum adsorption capacity ( qmax) and binding constants ( Kb) were determined. MATERIALS AND METHODS Microorganisms and culture conditions The strains of algae used and their sources are shown in Table 1. Altogether, 52 strains were tested. Of these, 18 strains were green algae and 34 blue green algae. The experiments were performed as described previously.5 Collection of microalgae Surface water from canals and rivers was sampled in 500 ml sampling bottles. In addition, microalgae were harvested using nylon screens (30 µm) drawn across the surface of the same water body for addition to the same sampling bottle at the same sampling 254 place and time. Some filamentous strains were picked up from rocks or soil at the sampling sites. Temperature and pH were measured at the water surface. Samples were transported to the laboratory and further proceeded within a day and a 2 ml aliquot from each was added to individual test tubes containing 8 ml of modified Medium 18 and incubated at 28oC with illumination (white fluorescent light 4,000 lux) on a 12 hour light and 12 hour dark cycle for 2 weeks. The samples were then streaked on agar modified Medium 18 plates with 1.5 % agar and incubated at 28oC under the same illumination program for 2 weeks or until the appearance of algal colonies. Single algal colonies were selected and re-streaked on agar medium plates several times to obtain unialgal cultures. Single colonies were maintained in 2 ml of modified Medium 18 under the same conditions, and with weekly transfer for stock cultures. Cultivation system Microalgae were in ocu lated in to modified Medium 18 consisting of (per liter) 1.50 g NaNO3, 380 mg MgSO 4.7H2O, 120 mg K2HPO 4, 110 mg CaCl2.2H2O, 70 mg NaCl, 10 mg Fe2(SO4) 3.nH2O, 9 mg Na2MoO4.2H2O, 3 mg H3BO3, 2 mg MnSO4.4H2O, 0.3 mg ZnSO4.7H2O, 0.08 mg CuSO4.5H2O, and 0.04 mg CoCl2.6H2O. The pH was adjusted to 7.5 and the modified Medium 18 was then autoclaved. Microalgae were cultivated in 200 ml test tubes under the conditions previously described (1) for 7- 10 days before use as inoculum for experiments. Chemical reagents Cd(NO 3 ) 2 an d Pb(NO 3 ) 2 were from Un ilab, Australia. HgCl2 , HNO3 90% and NaNO 3 were from Carlo Erba Italy. HNO 3 69%, ZnSO4.7H2O and CoCl2.6H2O were from BDH Laboratory Supplies, England. H 2O 2 , NaOH, MgSO 4 .7H 2 O, K2HPO 4 an d CaCl 2 .2H 2 O were from Merck, German y. Fe2(SO4) 3.nH2O, NaMoO4.2H2O, H3BO3, MnSO4.4H2O and CuSO 4.5H2O were from May and Baker ltd. Dagenham, England. Agar powder (commercial grade) was from Whongtavee Company, Thailand. Methyl alcohol was from the government pharmaceutical organization, Thailand. NaCl was from Riedel-de HaenAG Seeize-Hannover, Germany. Preparation of Hg, Cd and Pb solutions Hg, Cd and Pb were prepared as 100 ml stock solutions of HgCl2, Cd (NO 3) 2 and Pb(NO 3) 2, in water purified by Milli-Q Jr. (Milli-Q UF Plas, ScienceAsia 28 (2002) Millipore, France), sterilized using membrane filter (Millipore comp. 0.22µ) and kept in the refrigerator. Heavy metal concentrations were calculated from the equation; M1V1 = M2V2 where M1 was the stock solution concentration, M2 the required concentration, V1 the volume of the stock solution and V2 the volume of the solution at required concentration. Hg, Cd and Pb removal For Hg, Cd and Pb removal in aqueous solution experiments, a cell concentration of 1 g dry wt./l from inoculum was used. Cells (50 mg dry wt.) were centrifuged and washed twice with deionized water before resuspension in 50 ml of each heavy metal solution at 1 mg/l, pH 7, in 200-ml Erlenmeyer flasks. After mixing at 200 rpm, 28oC, for 30 min, the solution was centrifuged and the supernatant solution sampled for its heavy metal concentration by atomic absorption spectrophotometry. The centrifuged cells were oven dried at 100 oC until constant weight. Heavy metal removal ability was calculated from the equation (Ci-Cf)/ C i x 100 (%), where C i was the initial concentration (mg/l) and Cf the equilibrium (final) heavy metal concentration (mg/l). Selected microalgal strains (50 mg dry wt.) were suspended in 50 ml of various heavy metal solutions. Hg concentrations were 0.5, 1, 2, 5, 10, 20, 40, 80 and 150 mg/l, Cd concentrations were 5, 10, 20, 40, 80, 100, 150 and 200 mg/l and Pb concentrations were 5, 10, 20, 40, 80, 160, 200, 400 and 600 mg/l. Each sample was incubated at 200 rpm, 28oC for 30 min and then sampled for metal concentration using an at om ic absor p t ion sp ect rop h ot om et er. Adsorption of Hg, Cd and Pb, q (mg/g dry wt.), was determined qmax and Kb were calculated by using Langmuir equation as : q= qmax C Kb + C Where q is the heavy metal adsorbed to the solid phase (mg/g dry wt.) qmax is the maximum adsorption capacity (mg/g dry wt.) Kb is the binding constant (mg/l) C is the equilibrium concentration of heavy metal in solution (mg/l) Atomic absorption analysis For Hg analysis, samples were diluted using milliQ water before being measured in the range of 2540 ppb by using the graphite system wavelength at 253.7nm on an atomic absorption spectrophotometer ScienceAsia 28 (2002) (Varian, Spectr AA600, Australia). For Cd and Pb analysis, the sample solution was measured by atomic absorption spectrophotometer (Z-8000 Varience, Hitachi, Japan) using the graphite system wavelength at 228.8 nm. Sample solutions were fixed using 0.05 M HNO3. Dry weight determination To separate cells from aqueous solutions, blue green algae were filtered using a nylon screen size 30 µm while green algae were collected by centrifugation at 4000 rpm for 10 min. Cells were washed 3 times with deionized water and dried in an oven at 100oC for 24 hours or until constant weight. Samples were cooled in a dessicator for 30 min before dry weight was measured. Calculation of concentration factors The concentration factor (CF) was used to compare heavy metal removal ability among different strains of microalgae. It was calculated as follows: CF = µg metal removed/g dry wt. µg metal in solution/ml solution Digestion of microalgal cells Dried algal samples were digested with 1 ml of conc. HNO 3 in a dry thermo bath (Boekel series 02344, USA) until the solution was dry. After cooling, 1 ml of 30% H2O 2 was added. The sample was then further digested for 1 hour or until the solution had evaporated to dryness. This step was repeated 2 times until a white ash was obtained. Five ml of 0.5 N HNO3 was added to the digested sample and heavy metal concentration was measured using an atomic absorption spectrophotometer as described above. All the experiments were performed in three replicates. RESULTS Collection of microalgae Ten strains of microalgae were screened from natural water and industrial areas. Three of them as green algae (T1, T5 and T10) were used in this experiment. Hg, Cd and Pb removal in aqueous solution Hg, Cd and Pb removal capacity by some green algae and blue green algae was high (Table 1). Within the green algae, Hg removal efficiency was highest at 97%, 96%, 94% and 92% for Scenedesmus 255 sp., Chlorococcum sp., Chlorella vulgaris var. vulgaris and Fischerella sp., respectively and for blue green algae, Hg removal efficiency was highest at 96%, 94%, 94%, 93% and 92% for Lyngbya spiralis, Tolypothrix tenuis BBC 100, Stigonema sp. BCC 92, Phormidium molle and Lyngbya heironymusii.Within the green algae, Cd removal efficiency was highest at 94%, 94%, 91%, 89% and 88%, for Chlorococcum sp., T5, Fischerella sp., Chlorella vulgaris var. vulgaris and Scenedesmus acutus and for blue green algae Cd efficiency was highest at 97%, 96%, 95%, 94% and 94% for Lyngbya heironymusii, Gloeocapsa sp., Phormidium molle, Nostoc sp. and Oscillatoria jasorvensis, respectively. Within the green algae, Pb removal efficiency was highest at 89%, 88%, 85% , 85% and 84% for Scenedesmus acutus, Chlorella vulgaris var. vulgaris, Chlorella vulgaris BCC 15, Scenedesmus vacuolatus an d Chlorella vulgaris CCAP211/11B and for blue green algae, Pb removal efficiency was highest at 98%, 96%, 96%, 94%, 94% and 92% for Nostoc punctiforme, Oscillatoria agardhii, Gloeocapsa sp., Nostoc piscinale, Nostoc commune and Nostoc paludosum, respectively. In order to simplify the ability of each microalga to remove each or all th ree h eavy metals, we converted % removal into removal index by dividing % removal by the highest % removal and multiply the value obtained by 10. This would facilitate the comparison of each heavy metal removal ability by various microalgae. At the same time the additive total index in the last column of Table 1 was very helpful as to the effectiveness of each microalga in removing the combined Hg, Cd and Pb in the wastewater. The concentration factor index and the total index given in Table 2 were also obtained in a similar manner to that of Table 1. Concentration factors for Hg, Cd and Pb removal Concentration factors (CF) for heavy metal removal are shown in Table 2. For green algae Chlamydomonas sp., Scenedesmus acutus, Scenedesmus pertoratusand Myxosarcina sp. had the highest CFs for Hg (3,590, 3,412, 2,711 and 2,210, respectively) while Scenedesmus acutus, Scenedesmus pertoratus, Myxosarcina sp. and Chlorella vulgaris BCC 15 had the highest CFs for Cd (4,591, 3,730, 2,742 and 2,601, respectively) and Scenedesmus acutus, Scenedesmus pertoratus, Chlorella vulgaris BCC 15 and T10 had the highest CFs for Pb (4,078, 3,083, 2,293 and 1,442, respectively). For blue green algae, Nostoc punctiforme, Nostoc sp., Tolypothrix tenuis BCC 100 and Phormidium angustissimum had the highest CFs for Hg (4,218, 2,383, 1,976, 1,967, respectively), while Tolypothrix tenuis, 256 ScienceAsia 28 (2002) Table 1. Heavy metals removal in aqueous solution by microalgae. % Removal Green algae Chlorella vulgaris var. vulgaris BCC 15 Scenedesmus acutus TFRPD 1020 Chlorococcum sp. BCC 16 Chlorella vulgaris BCC 15 Chlorella vulgaris CCAP211/11B Scenedesmus pertoratus IFRPD 1010 T5 Scenedesmus vacuolatus CCAP211 Chlorella sp.BCC 13 Kirchnerriella sp. TISTR Chlorella saccharophilla CCAP211/1A T10 T1 Fischerella sp. BCC 22 Scenedesmus sp. BCC 82 Chamydomonas sp. BCC 11 Myxosarcina sp. BCC 59 Chlorella eillipsoidea BCC 12 Blue green algae Phormidium molle BCC 71 Lyngbya heironymusii BCC 41 Oscillatoria jasorvensis BCC 56 Oscillatoria agardhii BCC 52 Tolypothrix tenuis TISTR 8063 Rivularia sp. BCC 80 Lyngbya spiralis BCC 42 Gloeocapsa sp. BCC 25 Nostoc sp. BCC 50 Phormidium angustissimum BCC 68 Tolypothrix tenuis BCC 100 Nostoc punctiforme BCC 48 Stigonema sp. BCC 90 Stigonema sp. BCC 92 Calothrix sp. BCC 8 Calothrix parietina TISTR 8093 Nostoc paludosum BCC 46 Calothrix marchica var. intermedia BCC 6 Calothrix sp. BCC 5 Calothrix sp. TISTR 8130 Hapalosiphon welwitschii BCC 34 Nostoc commune BCC 76 Cylindrospermum sp. BCC 20 Nostoc piscinale BCC 47 Mastogocladus sp. BCC 36 Calothrix sp. BCC 10 Hapalosiphon hibernicus BCC 27 Oscillatoria amoena BCC 53 Anabaena sp. BCC 2 Nostoc punctiforme BCC 49 Stigonema sp. BCC 90 Nostoc micropicum BCC 77 Calothrix marchica BCC 4 Hapalosiphon sp. BCC 30 Removal index* Hg Cd Pb Hg Cd Pb Total index 94 85 96 74 75 79 73 81 71 72 70 70 68 92 97 86 91 81 89 88 94 86 83 83 94 73 88 80 81 81 84 91 58 86 53 84 88 89 71 85 84 80 75 85 77 81 79 74 72 35 58 30 53 23 9.7 8.8 9.9 7.6 7.7 8.1 7.5 8.4 7.3 7.4 7.2 7.2 7.0 9.5 10.0 8.9 9.4 8.4 9.5 9.4 10.0 9.1 8.8 8.8 10.0 7.8 9.4 8.5 8.6 8.6 8.9 9.7 6.2 9.1 5.6 8.9 9.9 10.0 8.0 9.6 9.4 9.0 8.4 9.6 8.7 9.1 8.9 8.3 8.1 3.9 6.5 3.4 6.0 2.6 29.0 28.1 27.9 26.3 26.0 26.0 26.0 25.7 25.3 25.0 24.7 24.1 24.0 23.1 22.7 21.4 21.0 19.9 93 92 89 73 81 86 96 50 86 74 94 66 92 94 86 50 44 92 37 40 85 43 83 22 89 92 84 12 68 46 82 26 84 95 97 94 90 88 88 80 96 94 87 53 73 89 80 88 88 92 87 84 82 75 69 65 82 78 89 90 83 85 84 90 72 57 90 80 85 96 88 76 73 96 58 77 90 98 52 59 59 91 92 43 88 86 47 94 52 94 29 13 13 89 29 51 5 80 20 9.7 9.6 9.3 7.6 8.4 9.0 10.0 5.2 9.0 7.7 9.8 6.9 9.6 9.8 9.0 5.2 4.6 9.6 3.9 4.2 8.9 4.5 8.6 2.3 9.3 9.6 8.8 1.3 7.1 4.8 8.5 2.7 8.8 9.8 10.0 9.7 9.3 9.1 9.1 8.2 9.9 9.7 9.0 5.5 7.5 9.2 8.2 9.1 9.1 9.5 9.0 8.7 8.5 7.7 7.1 6.7 8.5 8.0 9.2 9.3 8.6 8.8 8.7 9.3 7.4 5.9 9.2 8.2 8.7 9.8 9.0 7.8 7.4 9.8 5.9 7.9 9.2 10.0 5.3 6.0 6.0 9.3 9.4 4.4 9.0 8.8 4.8 9.6 5.3 9.6 3.0 1.3 1.3 9.1 3.0 5.2 0.5 8.2 2.0 28.7 27.7 27.6 26.7 26.5 25.8 25.7 24.9 24.6 24.5 24.4 24.4 24.1 24.1 24.1 23.6 23.5 22.9 21.5 21.4 21.4 21.2 20.7 20.3 20.3 20.1 19.4 18.9 18.8 18.7 18.3 18.3 16.7 86 62 11 9.0 6.4 1.1 16.0 Microalgae * Strains of algae and blue green algae arranged in descending order according to additive removal index. The removal index for each metal is the % removal divided by the highest % removal times 10. Total index represents the sum of the removal index of all three metals. 257 ScienceAsia 28 (2002) Hapalosiphon sp., Nostoc micropicum, Anabaena sp. had the highest CFs for Cd (3,028, 2,416, 2,404 and 2,368, respectively) and Gloeocapsa sp., Nostoc commune, Nostoc paludosum and Tolypothrix tenuis had the highest CFs for Pb (4,154, 3,269, 3,022 and 2,963, respectively). Based on resu lts for rem oval ability, h igh concentration factor and high tolerance on agar plate cultures (data not shown), 3 strains of green algae ( Chlorella vulgaris BCC 15, Chlorella vulgaris CCAP 211/11B and Scenedesmus acutus) were selected for the further experiments. In addition, 2 strains of blue green algae ( Tolypothrix tenuis TISTR 8063 and Calothrix parietina) were selected for the further study because they had high removal capacity and were easily separated from water by filtration because of filamentous morphology. This would be amenable for application in simple outdoor conditions. Hg, Cd and Pb removal at various heavy metal concentrations Hg, Cd and Pb removal by the three selected strains of green algae and two selected strains of blue green algae were assessed using the Langmuir equation for maximum adsorption capacities ( qmax) and binding constants ( Kb) as shown in Table 3. This isot h er m can be d escr ibed by t h e Lan gm u ir adsorption isotherm, which is often reported for biological adsorption by other kinds of biomass5. The Langmuir equation is given by q= qmax C Kb + C (1) Where q is the metal adsorption to the solid phase (mg/g dry wt.), qmax is the maximum adsorption capacity (mg/g dry wt.), Kb is the binding constant (mg/l), and C is the equilibrium metal concentration (mg/l). The Langmuir equation 1 can be rearranged as C q = C K + b qmax qmax (2) The adsorption of Hg, Cd and Pb fitted well with the Langmuir equation. From equation 2, the maximum adsorption capacities ( qmax) for Hg by the two blue green algae strains,Tolypothrix tenuis TISTR 8063 and Calothrix parietina were 27 and 19 mg Hg/ g dry wt., respectively. For the green algae, Chlorella vulgaris BCC 15, Chlorella vulgaris CCAP 211/11B and Scenedesmus acutus, they were 18, 16 and 20 mg Hg/g dry wt., respectively. For Cd, the green algae, gave the highest capacity at 110 mg Cd/g dry wt. followed by the two blue green algae, Tolypothrix tenuis TISTR 8063 and Calothrix parietina at 90 and 79 mg Cd/g dry wt. For Pb adsorption the green alga Chlorella vulgaris BCC 15 gave the best capacity at 127 mg Pb/g dry wt. followed by Scenedesmus acutus at 90 mg Pb/g dry wt. With respect to the binding constant value ( Kb), lower values indicated higher affinity for the heavy metal. For Hg, Tolypothrix tenuis TISTR 8063 had the lowest Kb value (0.01) and thus the highest affinity. For Cd, Tolypothrix tenuis and Calothrix parietina had the highest affinity ( Kb = 0.62 and 0.92, respectively) and for Pb, Chlorella vulgaris BCC 15 and Chlorella vulgaris CCAP 211/11B had highest affinity ( Kb = 3.06 and 3.06, respectively) (Table 3). Figure 1 shows the equilibrium isotherm for the adsorption of Hg, Cd and Pb by three microalgae and two blue green algae. Tolypothrix tenuis was the best among other algae, adsorbing Hg at 27 mg Hg/ g dry wt. at a minimum concentration of 1.04 mg/l (Fig. 1 A). For Cd adsorption, Scenedesmus acutus was best at 110 mg Cd/ g dry wt. at a minimum concentration of 48 mg/l (Fig. 1 B). For Pb adsorption, Chlorella vulgaris was best at 127 mg Pb/g dry wt. at a minimum concentration of 130 mg/l (Fig. 1 C). DISCUSSION Hg, Cd and Pb removal in aqueous solutions Our results indicated that both green algae and blue green algae had very high Hg, Cd and Pb removal capacities and that adsorption occurred within the relatively short time of 10-20 min.5 The green alga Chlorella vulgaris is often used to study adsorption of heavy metals6-9 but in this study, the green alga Scenedesmus acutus had a higher removal capacity. In general heavy metals are taken in by blue green algal cells by adsorption followed in sequence by metabolism-dependent intracellular cation intake as applicable to Zn 10, Cu, Cd and Zn 11, Cd 12, Al13, Ni14 and Hg.15 In other studies, blue green algae had lower capacities than those found herein. For example, Kitjaharn 16, 17 showed that the blue green algae Aphanothece halophytica and Spirulina platensis could remove only 22% and 35% Pb, respectively, from battery factory wastewater. In spite of this, we selected blue green algae to test for heavy metal adsorption because they have high growth rates and are easy to separate from solution by simple filtration. 258 ScienceAsia 28 (2002) Table 2. Heavy metals removal in aqueous solution by microalgae calculated as concentration facctor (CF). Microalgae Green algae Scenedesmus acutus IFRPD 1020 Scenedesmus pertoratusIFRPD1010 Chamydomonas sp.BCC 11 Chlorella vulgaris BCC 15 Myxosarcina sp. BCC 59 T10 Scenedesmus sp. BCC 82 Scenedesmus vacuolatus CCAP211 Kirchnerriella sp. TISTR Fischerella sp. BCC 22 T1 Chlorella sp. BCC 13 Chlorella eillipsoidea BCC 12 Chlorella saccharophilla CCAP211/1A Chlorella vulgaris var. vulgarisBCC15 Chlorococcum sp. BCC 16 Chlorella vulgaris CCAP211/11B T5 Blue green algae Nostoc punctiforme BCC 4 Tolypothrix tenuis TISTR 8063 Nostoc paludosum BCC 46 Nostoc sp. BCC 50 Lyngbya spiralis BCC 42 Phormidium angustissimum BCC 68 Anabaena sp. BCC 2 Nostoc commune BCC 76 Gloeocapsa sp. BCC 25 Nostoc micropicum BCC 77 Cylindrospermum sp. BCC 20 Nostoc punctiforme BCC 49 Hapalosiphon sp. BCC 30 Calothrix sp. BCC 8 Tolypothrix tenuis BCC 100 Oscillatoria agardhii BCC 52 Rivularia sp. BCC 80 Lyngbya heironymusii BCC 41 Calothrix sp. TISTR 8130 Calothrix parietina TISTR 8093 Oscillatoria jasorvensis BCC 56 Calothrix sp. BCC 5 Hapalosiphon welwitschii BCC 34 Nostoc piscinale BCC 47 Calothrix marchica var. intermedia BCC 6 Oscillatoria amoena BCC 53 Stigonema sp. BCC 91 Calothrix sp. BCC 10 Stigonema sp. BCC 90 Stigonema sp. BCC 92 Mastogocladus sp. BCC 36 Calothrix marchica BCC 4 Hapalosiphon hibernicus BCC 27 Phormidium molle TISTR 8246 * Concentration Factor (CF) Concentration index* Hg Cd Pb Hg Cd Pb Total index 3412 2711 3590 1785 2210 1460 1262 1274 1066 1656 1060 857 1778 869 1150 1131 488 327 4591 3730 2217 2601 2742 1878 2164 1351 1372 1354 1537 1184 982 1196 805 1086 980 774 4078 3083 796 2293 422 1442 749 1358 1066 405 793 1167 228 895 914 660 863 586 9.5 7.6 10 5.0 6.2 4.1 3.5 3.5 3.0 4.6 3.0 2.4 5.0 2.4 3.2 3.2 1.4 0.9 10 8.1 4.8 5.7 6.0 4.1 4.7 2.9 3.0 2.9 3.3 2.6 2.1 2.6 1.8 2.4 2.1 1.7 10 7.6 2.0 5.6 1.0 3.5 1.8 3.3 2.6 1.0 1.9 2.9 0.6 2.2 2.2 1.6 2.1 1.4 29.5 23.3 16.3 16.3 13.2 11.7 10.0 9.7 8.6 8.5 8.2 7.9 7.7 7.2 7.2 7.2 5.6 4.0 4218 919 1656 2383 1552 1967 1937 819 1424 740 1656 1283 1608 451 1976 1264 1380 1625 825 1096 1923 725 1636 533 1626 269 1155 776 1396 995 1135 1263 718 953 1110 3028 1980 2210 2329 1847 2368 1355 238 2404 1537 2181 2461 1688 1135 1117 1493 1341 1722 1292 1758 1749 1263 1454 1391 1942 15.6 1906 1493 1035 1141 1025 1422 807 2735 2963 3022 1331 1788 1919 780 3269 4154 1767 1866 1301 422 2489 1675 1998 1356 1317 1478 1784 313 1404 954 1764 774 945 489 283 86 1083 427 338 194 799 10.0 2.2 3.9 5.6 3.7 4.7 4.6 1.9 3.4 1.8 3.9 3.0 3.8 1.1 4.7 3.0 3.3 3.9 2.0 2.6 4.6 1.7 3.9 1.3 3.9 0.6 2.7 1.8 3.3 2.4 2.7 3.0 1.7 2.3 3.7 10.0 6.5 7.3 7.7 6.1 7.8 4.5 0.8 7.9 5.1 7.2 8.1 5.6 3.7 3.7 4.9 4.4 5.7 4.3 5.8 5.8 4.2 4.8 4.6 6.4 5.0 6.3 4.9 3.4 3.8 3.4 4.7 2.7 6.6 7.1 7.3 3.2 4.3 4.6 1.9 7.9 10.0 4.3 4.5 3.1 1.0 6.0 4.0 4.8 3.3 3.2 3.6 4.3 0.8 3.4 2.3 4.2 1.9 2.3 1.2 0.7 0.2 2.6 1.0 0.8 0.5 1.9 20.2 19.3 17.7 16.2 15.7 15.4 14.3 14.3 14.2 13.9 13.5 13.4 13.0 12.6 12.5 11.5 11.5 11.5 11.2 11.2 11.1 10.9 10.3 10.3 10.3 9.3 8.9 8.8 8.4 8.4 7.5 7.2 6.9 6.8 Strains of algae and blue green algae arranged in descending order according to additive Concentration Factor index. The CF index for each metal is the CF divided by the highest CF times 10. Total index represents the sum of the CF index of all three metals. 259 ScienceAsia 28 (2002) Table 3. Maximum adsorption capacities ( q max ) and binding constants ( K b ) for various microalgae with Hg, Cd and Pb. Heavy Metal Microalgae Hg Kb Cd q max (mg/g dry wt.) Kb Pb q max (mg/g dry wt.) Kb q max (mg/g dry wt.) Blue green algae Tolypothrix tenuis TISRT 8063 0.01 27 0.62 90 7.83 31 Calothrix parietina TISTR 8093 0.03 19 0.92 79 6.87 45 Green algae Chlorella vulgaris BCC 15 0.15 18 3.83 76 3.06 127 Chlorella vulgaris CCAP211/11B 0.03 16 1.60 62 3.06 39 Scenedesmus acutus IFRPD 1020 0.11 20 1.57 110 9.45 90 q (mg/g dry wt.) 30 A 20 10 0 40 80 120 160 200 C (mg/l) q (mg/g dry wt.) 160 0 B 120 80 40 0 0 40 80 120 160 200 q (mg/g dry wt.) C (mg/l) 160 C 120 80 40 0 0 200 400 C (mg/l) 600 800 Fig 1. Adsorption of Hg (A), Cd (B) and Pb (C) by five microalgae at various heavy metal concentrations. Microalgae cells (0.5 g dry wt.) were suspended in 50 ml solution containing various concentrations of heavy metals. Symbols: ●, Tolypothrix tenuis; , Calothrix parietina; ▲, Chlorella vulgaris: , Chlorella vulgaris (CCAP211/11B); , Scenedesmus acutus. Concentration factors for Hg, Cd and Pb The concentration factor (CF) is the ratio of the metal concentration in dry biomass to that in aqueous solution and it is defined as ( µg metal removed/g dry wt.) /( µ g metal in solu tion /ml solution). It is used to compare heavy metal removal ability among different microbial strains. In our study Chlamydomonas sp. gave a CF of 3,590 for Hg and this was far lower than 9,060 reported for Chlamydomonas sp. by Hassett.18 By contrast, our 260 CF of 4,218 for Hg for the blue green alga Nostoc punctiforme was much higher than 1,950 reported by Hassett for Nostoc sp..18 So also was our CF of 2,404 for Cd for Nostoc micro-picum when compared to reports of 1,140-1,470 µg/g for Nostoc sp..18 Cd removal values reported for other cyanobacteria included 3,250 µg/g for Chro-ococcus paris11 and 1,160-1,310 µg/g for Oscillatoria sp..18 Our value for Oscillatoria sp. was nearly the same at 1,1171,942 µg/g. We found that Nostoc commune gave the highest CF of 3,269 for Pb and this compared to 1,920 in Nostoc sp. by Hassett.18 He used a microplate technique to determine CF for various algal species, under various conditions (eg, metal concentration and type, culture age, pH, etc) and found that green algae and blue green algae accumulated the metals Hg, Cd, Pb and Zn better than green algae, while green algae accumulated Cd better than blue green algae. In other studies, concentration factors for heavy metals in the green alga Scenedesmus obliguus (11 days old, 3 hours, 22 °C in the dark) were 5,040 for Hg and 3,600 for Cd. These differed somewhat from the values reported herein for Scenedesmus acutus at 3,412 for Hg and 4,591 for Cd. For the blue green alga, Nostoc reported concentration factors were 1,950 for Hg and 1,920 for Cd 19 but the CF values herein were only 234 and 343, respectively. Differences probably depended on strain and on experimental conditions such as strain source, culture age, pH and time of exposure. Hg, Cd and Pb removal at various heavy metal concentrations Srikrajib20 reported maximum adsorption capacities ( qmax) of 103 and 95 mg Cd /g dry wt from the isotherm of Cd for Sargassum polycystum dried at 80 °C and 100 °C, respectively. Herein, the highest maximum adsorption capacity ( qmax) for Cd was found with Scenedesmus acutus, at 110 mg Cd/g dry wt. Values for other strains ranged from 62 to 90 mg Cd/g dry wt. Sargassum polycystum and Scenedesmus acutus had similar maximum adsorption capacities ( qmax) although the former comprised dead cells and the latter living cells. Aksu et al21 studied adsorption of lead from waste water using the green alga Chlorella vulgaris in a single stage batch reactor and reported a value for maximum adsorption capacity ( qmax) of 83 mg Pb /g dry wt. They also found that adsorption increased with increasing pH and temperature. Herein, the highest maximum adsorption capacity ( qmax) for lead was found with Chlorella vulgaris, at 127 mg Pb/g dry wt, while values for other strains ranged from 31 to 90 mg Pb/ ScienceAsia 28 (2002) g dry wt. Value differences for Chlorella vulgaris were probably du e to differen ces in experi-men tal conditions. The ranking table in Table 1 and Table 2 were done as % removal index and concentration factor index in order to rank the ability of those microalgae. Some algae had % removal indexes of 9-10 for all three metals but none did so for CF index. Based on these results, we suggested that the mixture of algae should be used to obtain the best result in real wastewater condition which usually contains more than one metal type. For mixed cultures the organisms to be combined should have similar growth requirements and should be physiologically and biochemically compatible under hard-water field conditions or real wastewater system. Strains that will be useful as biosorbents must have the ability to function in hard water. Thus, tests for Cd removal, for example should also be carried out in solutions containing Ca2+ or Mg2+ at concentrations usually present in hard water. Only strains that pass such tests will be suitable for application under outdoor conditions.1 ACKNOWLEDGEMENT This research was supported by the Thailand Research Fund RTA/05/2540 and a grant from Mahidol University. We thank National Science and Tech n o-logy Develop m en t Agen cy, Th ailan d Institute of Scientific and Technological Research, Institute of Research and Food Product Development and Dr Uwe Gert Schlosser from Gottingen University culture collection for providing all microalgae strains in this study. REFERENCES 1. Inthorn D, Nagase H, Isaji Y, Hirata K and Miyamoto K (1996) Rem oval of cad m iu m from aqu eou s solu t ion by t h e filamentous cyanobacterium Tolypothrix tenuis. J Ferment Bioeng 82 , 580-4. 2. Rai LC, Gaur JP, and Kumar HD (1981) Phycology and heavymetal pollution. Biol Rev 56 , 99-103. 3. Gekeler W, Grill E, Winnacker E-L and Zenk MH (1998) Algae sequ ester h eavy metals via syn th esis of ph ytoch elatin complexs. Arch Microbiol 150 , 197-202. 4. Wild EW, Benemann JR (1993) Bioremoval of heavy metals by the use of microalgae. Biotechnol Adv 11 , 781-812. 5. Inthorn D, Incharoensakdi A and Sidtitoon N (2001) Removal of mercury, cadmium and lead in aqueous solution by microalgae. Asian Journal of Microbiology Biotechnology and Environmental Sciences (AJMBES) 3, 109-116. 6. Hans JG and Urbach W(1983) Sorption of cadmium by the green microalgae Chlorella vulgaris, Ankistrodesmus braunii and Eremosphaeraviridis. Z Pflanzenphysiol 109 , 127-41. 7. Ting YP, Lowson F, and Prince IG (1991) Uptake of Cadmium and Zing by the alga Chlorella vulgaris: multi ion situation. J Biotechnol Bioeng 37 , 445-55. ScienceAsia 28 (2002) 8. Cho DY, Lee ST, Park SW and Chung AS (1994) Studies on the bioadsorption of heavy metals onto Chlorella vulgaris. J Environ Sci Health 29 (2),389-409. 9. Wong KH, Chan KY and Ng SL (1979) Cadmium uptake by the unicellular green alga Chlorella salina CU-1 from culture media with high salinity. Chemospheres Nos 11/12 : 887-91. 10.Shehata FHA, and Whitton BA (1982) Zinc tolerance in strains of blue-green alga Anacystis nidulans. Br Phycol J 17 , 5-12. 11.Les A Walker RW (1984) Toxicity and binding of copper, zinc and cadmium by the blue-green alga, Chroococcus paris. Water Air Soil Pollut 23 ,129-39. 12.Singh S P, Yadava V (1985) Cadmium uptake in Anacyatis nidulans: effect of modifying factors. J Gen Appl Microbiol 31 , 39-48. 13.Pettersson A, Hallbom L, Bergman B (1986) Aluminum uptake by Anabaena cylindrica. J Gen Microbiol 132 , 1771-4. 14.Campbell PM, Smith GD (1986) Transport and accumulation of nikel ions in the cyanobacterium Anabaena cylindrica. Arch Biochem Biophys 244 , 470-7. 15.Johnson PE, Shubert LE (1986) Accumulation of mercury and other element by Spirulina (Cyanophyceae). Nutr Rep Int 34 , 1063-70. 16.Kitjaharn P, and Incharoensakdi A (1992) Factors affecting the accumulation of lead by Aphanothece halophytica. J Sci Chula 17 , 141-7. 17.Incharoensakdi A and Kitjaharn P (1998) Removal of lead from aqueous solution by filamentous cyanobacterium, Spirulina platensis. J Sci Res Chula Univ 23 , 38-44. 18.Hassett JM, Jannett JC and Smith JE (1981) Microplate technique for determining accumulation of metals by algae. Appl Environ Microbiol 41 , 1097-106. 19.Sakaguchi T, Tsuji T, Nakajima A and Horikoshi T (1979) Accumulation of cadmium by green microalgae. Eur J Appl Microbiol Biotechnol 8 , 207-15. 20.Srikrajib S (1990)Cadmium removal by the dry biomass of Sargassum polycystum. (M SC thesis). Bangkok: Faculty of Graduted studies, Mahidol University. 21.Aksu Z, Acikel U and Kutsal T (1997) Application of multicomponent adsorption isotherms to simultanneous biosorption of Iron (III) and Chromium (VI) on C.vulgaris. J Chem Technol Biotechnol 70 , 368-78. 261