- Email: [email protected]
Toxicity of nickel, zinc, and cadmium to nitrate uptake in free and immobilized cells of Scenedesmus quadricauda
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 61 (2005) 268–272 www.elsevier.com/locate/ecoenv
Toxicity of nickel, zinc, and cadmium to nitrate uptake in free and immobilized cells of Scenedesmus quadricauda Mamta Awasthia,, Lal Chand Raib a
Department of Zoology, Arunachal University, Itanagar 791 112, India Department of Botany, Banaras Hindu University, Varanasi 221005, India
b
Received 26 May 2004; received in revised form 17 December 2004; accepted 20 December 2004 Available online 3 February 2005
Abstract We examined the influences of three trace metals on the accumulation of a major nutrient (NO 3 ) in Scenedesmus quadricauda. A comparative study on metal–nutrient interaction in free and immobilized states of algal cells was conducted. The effect due to interaction between different variables (cell state type, metal type, and metal dose) was studied to assess the variation in the nitrate uptake by free and immobilized cells. The results analyzed by ANOVA (three-way) (components: cell state type, metal type, and metal dose) confirmed that the inhibition of nitrate uptake by test metals was highly significant (Po0.001). Free and immobilized states of S. quadricauda responded differently (Po0.05, ANOVA) to the types of metal added. Uptake kinetics was studied by monitoring short-term uptake rates at different nutrient levels. Free and immobilized cells of the organism displayed noncompetitive modes of inhibition for Ni and Zn while a competitive mode of inhibition by Cd was observed in both free and immobilized states of the organism. r 2005 Elsevier Inc. All rights reserved. Keywords: Heavy metal; Immobilized; Scenedesmus quadricauda; Nitrate uptake
1. Introduction Freshwater ecosystems are influenced by heavy metal pollution (Baun et al., 1998). The chemical forms of these metals in water are accessible to the biota through significant accumulation in the food chain. Toxicity of heavy metals to algae has been reviewed earlier (Gaur and Rai, 1994). In addition to heavy metal pollution, excess nitrate discharge is also attracting attention. The nitrate threat to ground water comes from various sources including nitrogen-based fertilizers, waste from dairy and other livestock operations, and septic tank systems, both residential and industrial (Bier, 2002). Many organisms including algae possess the ability to incorporate nutrients very rapidly from the external medium (Forni et al., 2001). For total removal of Corresponding author.
E-mail address: [email protected] (M. Awasthi). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.12.018
nitrogen, a biological process is more suitable than any conventional technique. However, the use of algae as a biological material is restricted since it is difficult to harvest algae for its use in sewage treatment. Recently, this problem was overcome by the use of immobilized algae (Lau et al., 1998). Immobilized biomass offers many advantages including better reusability, high biomass loading, and minimal clogging in continuous flow systems (Zhang et al., 1998; Tam et al., 1998). Nitrate uptake is an interaction between cells and substrate similar to the interaction between enzymes and substrate in any biochemical process. In the present study, nitrate serves as a substrate and is converted into amine form (–NH2). During this process, many substances (including heavy metals) may alter the uptake of nutrients by combining with them in a way that influences the binding of substrate by algal cells thus they act as inhibitors. An inhibitor can compete directly with the substrate for an enzymatic-binding site
ARTICLE IN PRESS M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
(competitive inhibition) or can bind to either the free enzyme or the enzyme–substrate complex (noncompetitive inhibition). The study of nitrate uptake by algae becomes important since algae can be used as an excellent nutrient stripper, if properly managed. The present analysis will enable assessment of the extent and mode of inhibition of nitrate uptake in the immobilized state of algal cells by heavy metals. A comparative study of nutrient uptake kinetics in free and immobilized cells is also significant for assessing the superiority of immobilized cells, if any, over free cells. The presence of heavy metals together with excess nutrients can cause interference in the nutrient removal process by algae. The study was designed to evaluate whether metals present in effluents inhibit NO 3 uptake by imbedded algae more so than that by free cells, thereby creating problems with effluent treatment using imbedded cells.
2. Material and methods 2.1. Isolation, purification, and culture Eukaryotic green algae Scenedesmus quadricauda (local isolate, Banaras Hindu University) was grown in modified CHU-10 medium (Gerloff et al., 1950). The pH was maintained at 7.0 by using 2.0 mM Tris (hydroxymethyl) methylene/HCl. Cultures from the logarithmic phase were used for toxicity tests. Stock solutions of NiCl2 6H2O, ZnSO4 H2O and CdSO4 H2O were filtersterilized by passing through Millipore membrane filters (0.45 mm) before addition to the culture medium. Test metals were freshly prepared and the actual metal concentrations used were the LC50 values determined previously by the plate/colony count method based on the survival of the test alga to 50%. The concentrations were as follows: Ni, 1.10, 2.10 (LC50), and 3.10 mmol L1; Zn, 1.40, 2.40 (LC50), and 3.40 mmol L1; Cd, 1.30, 2.30 (LC50), and 3.30 mmol L1. The culture received 72 mmol photons m2 s1 PAR light intensity at 2472 1C. Standard microbial techniques were employed for selection, isolation, and cloning of algae in pure culture. Protein value was estimated following the method of Herbert et al. (1971). 2.2. Bubble column reactor A glass tube containing a ground-glass filter at the bottom and sealed with a rubber stopper at the top was used. A hollow rod was inserted through the stopper to reach the bottom of the column. The tube was used to pump air in the reactor. The column was aerated at 250–300 cc/min with an air bubbler. A hollow tube was
269
also inserted sideways at the bottom to remove the solution from the reactor at different time intervals. 2.3. Cell immobilization Most methods for immobilization of biomaterials in alginate beads basically involve two main steps. First, there is the internal phase where the alginate solution containing biological materials is dispersed into small droplets. Secondly, the droplets are solidified by gelling or membrane formation at the droplet surface. This is termed the dialysis/diffusion method (diffusion setting) as the alginate solution is gelled by diffusion of gelling ions from an outer reservoir. The procedure is as follows: Dissolve 30 g of sodium alginate in 1 L to make a 3% solution. Mix approximately 10 mL of desired protein value algal culture with 10 mL of 3% (wt.) sodium alginate solution. The concentration of sodium alginate can be varied between 6% and 12% depending on the desired hardness. The beads are formed by dripping the polymer solution from a height of approximately 20 cm into an excess (100 mL) of stirred 0.2 M CaCl2 solutions with a syringe and a needle at room temperature. Pump pressure and the needle gauge can control the bead size. A typical hypodermic needle produces beads of 0.5–2 mm in diameter. Other shapes can be obtained by using a mold whose wall is permeable to calcium ions. Leave the beads in the calcium solution to cure for 0.5–3 h. In the present study, exponentially grown algal cells (500 mg protein/mL) obtained by centrifugation and repeated washings were suspended in 3% (w/v) solution of sodium alginate (Sigma). The mixture was pumped dropwise into CaCl2 (0.2 M) solution. The beads thus formed contained algal cells in an amount of 60 mg protein per bead. The beads were washed several times with sterile deionized double-distilled water and resuspended in a 200-mL growth medium for autotrophic growth under culture room conditions. Free cells were also cultured under similar conditions at the same time. 2.4. Estimation of NO3 For studying the effect of metals on NO 3 uptake, cultures were incubated in KNO3 (1.5–20.0 mM). A bubble column reactor was used for NO 3 uptake. The column was aerated at 250–300 cc/min with an air bubbler. Beads were placed into a bubble column reactor in 500 mL growth medium for 24 h. At that time, the medium was pumped out and the beads were washed with 500 mL of double-distilled water. The column reactor was exposed to a NO 3 uptake medium for 30 min; 3-mL samples were taken every 10 min and tested for NO 3 depletion. NO3 in the medium was estimated spectrophotometrically by
ARTICLE IN PRESS M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
Immobilized
0
2.0070.01
2.4070.15 (20.0)a
Nickel
3.1 2.1 1.1
0.9070.02 (55.0) 1.1070.01 (45.0) 1.5070.02 (25.0)
1.2070.06 (40.0) 1.3070.02 (35.0) 1.6070.03 (20.0)
Zinc
3.4 2.4 1.4
0.6070.01 (70.0) 1.0070.06 (50.0) 1.2070.05 (40.0)
1.3070.04 (35.0) 1.4070.01 (30.0) 1.6070.02 (20.0)
Cadmium
3.3 2.3 1.3
0.7070.04 (65.0) 1.1070.01 (45.0) 1.2070.02 (40.0)
1.1070.02 (45.0) 1.4070.01 (30.0) 1.5070.05 (25.0)
All the values are mean7SE. Data in parentheses denote % inhibition. All the treatments are significantly different (Po0.001) from their respective control according to Student t test. a Shows stimulation over control (free cells).
3. Results A concentration-dependent inhibition of nitrate uptake in both free and immobilized cells (Table 1) was observed. Approximately 45%, 50%, and 45% inhibition of NO 3 uptake by the organism was recorded following the addition of LC50 concentrations of Ni (2.10 mmol L1), Zn (2.40 mmol L1), and Cd 1 (2.30 mmol L ), respectively; 35%, 30%, and 30% inhibition was recorded in the presence of Ni, Zn, and Cd, respectively, in the immobilized organism. The results analyzed by ANOVA (three-way) (components: cell state type, metal type, and metal dose) confirmed that the inhibition of nitrate uptake by test metals was highly significant (Po0.001). The two states of cells (free/immobilized) responded differently (Po0.05, ANOVA) with different test metals. The nutrient uptake kinetics was studied and the values for V max and half-saturation constant (K m ) for uptake were obtained employing the Lineweaver–Burk double-reciprocal plot. Free (Fig. 1) and immobilized (Fig. 2) cells of the organism displayed noncompetitive modes of inhibition for Ni and Zn. In the noncompetitive mode of inhibition, V max 1 decreased from 1.33 to 1.11 and to 1.0 mmol NO1 3 mg 1 protein h , respectively, in the medium supplemented
Free living Control
-1
Uptake kinetics are typically described using the analogous enzyme kinetic parameters; V max ; which is determined as the mean of uptake rates obtained at saturating substrate concentrations, and K m ; which is the nutrient concentration at which the uptake rate is half maximum. The rate equation is given by the Michaelis–Menten equation, V ¼ V max S=ðK m þ SÞ; where V is the initial velocity when no product is formed, V max is the maximum reaction rate, K m is the Michaelis–Menten constant, and S is the substrate concentration. To obtain a linear plot, the above formula is inverted and a modified equation (Lineweaver–Burk plot) for experimental determination of V max and K m is obtained. The Lineweaver–Burke plot is a plot of 1=V max (the reciprocal of the reaction rate) against 1/S (the reciprocal of the substrate concentration). Noncompetitive inhibition results in intersection at the x-axis (1=K m ¼ constant) and competitive inhibition results in intersection at the y-axis (1=V max ¼ constant) in the presence of the inhibitor. Here nitrate uptake rate is plotted against the substrate concentration (nitrate) as a Lineweaver–Burke plot.
1 –1 protein) NO 3 uptake (mmol NO3 mg
Metal concentration (mmol L1)
3.5
-0.3
-0.2
Control -1
Nickel (2.1 µmol L ) -1
3.0
Zinc (2.4 µmol L ) Linear regression
2.5
-1
2.5. Uptake kinetics
Table 1 Differential toxicity of Ni, Zn and Cd to nitrate uptake (supplemented with 5 mM KNO3) after 4 h of metal treatment
2.0
-1
the brucine–sulfuric acid method (Nicholas and Nason, 1957). The color so developed was measured at 410 nm.
1/V (µmol NO3 µg protein h-1)
270
1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
Fig. 1. Nitrate uptake and inhibition by Ni and Zn in free state of S. quadricauda.
with LC50 concentration of Ni and Zn in the free state of cells. In the immobilized organism, V max declined from 1 1.81 to 1.25 and to 1.43 mmol NO1 protein h1, 3 mg respectively, in the presence of Ni and Zn. Conversely, the K m remained unchanged at 4.34 mM in the immobilized cells (Fig. 2), whereas it remained at 5.55 mM in the free cells (Fig. 1). A competitive mode of inhibition by Cd was observed in both the free (Fig. 3) and the immobilized (Fig. 4) states of the organism. Here, K m values increased from 5.71 to 8.33 mM in the free cells and from 5.0 to 6.66 mM in the immobilized cells of the test organism. However, the value for V max remained unchanged, i.e., 1 1.33 and 1.53 mmol NO1 protein h1 in the cases 3 mg of free and immobilized cells, respectively.
ARTICLE IN PRESS 1/V (µmol NO3-1 µg-1 protein h-1)-1
2.5
Control -1
Zinc (2.4 µmol L ) -1 Nickel (2.1 µmol L )
2.0
Linear regression
-1
-1
1/V (µmol NO3 µg protein h )
-1 -1
M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
-0.3
-0.2
1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-0.2
3.0 2.5
271
Control -1 Cadmium (2.3 µmol L ) Linear regression
2.0 1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
1/(S) mM
-1
Fig. 2. Nitrate uptake and inhibition by Ni and Zn in the immobilized state of S. quadricauda.
1/V (µmol NO3-1 µg-1 protein h-1)-1
3.5
-0.3
-0.2
3.0
Control -1 Cadmium (2.3 µmol L ) Linear regression
2.5 2.0 1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
Fig. 3. Nitrate uptake as inhibited by Cd in free state of S. quadricauda.
4. Discussion This investigation indicates the potential for the use of immobilized cells in the effluent treatment when the target pollutants are both heavy metals and nitrate. The heavy metal component seems to compete with the nitrate for the selective binding sites present on the extracellular surface (Zhang and Majidi, 1994; Gardea-Torresdey et al., 1990). In addition to passive uptake, there is another mechanism for metal uptake which requires energy (Silver, 1991), and since nutrient uptake is also an energy-dependent process (Peckol et al., 1994), it may compete with metal for energy. The algae also exhibited a varied response to different metals. This may be due to different chemical properties, speciation, and affinity of metals for the algae when present in liquid medium (Borgmann, 2000).
Fig. 4. Nitrate uptake and inhibition by Cd in the immobilized state of S. quadricauda.
The noncompetitive mode of inhibition of NO 3 suggests a direct involvement of energy. In this kind of inhibition, the substrate and the inhibitor bind to different sites on the carrier (Hofer and Hoggett, 1981). The heavy metals cause reduction of photosynthesis (Reiriz et al., 1994) which points toward an alternation in the structure of the enzyme by binding of the metal with the sulfydryl group (De Filippis and Pallaghy, 1994). Apart from the metal-induced depletion of ATP and reductant pools, exhaustion of energy-yielding substrates and competition of metals with essential micronutrients may result in the noncompetitive inhibition of nutrient uptake by phototrophic organisms. The competitive mode of inhibition is indicative of the common entry site for metals and nutrients. It rules out the direct involvement of energy. This was further confirmed when the toxic effects were counteracted by increasing substrate concentration. The higher uptake of nitrate by immobilized cells than by free cells could be due to increased cell wall permeability as observed by Brouers et al. (1989). Nevertheless, the higher energy state of the immobilized system could be another factor for the facilitated nutrient uptake (Awasthi and Das, 2005). A lower inhibition of nitrate uptake in the immobilized cells in the presence of metals shows their superiority over free cells. Immobilized S. quadricauda exhibit the lowest inhibition for nitrate uptake in the presence of Zn and Cd. The capacity of immobilized cells to bind nutrients even in the presence of metals shows the potency to do further research in this area.
Acknowledgments The authors thank the Ministry of Environment & Forest, New Delhi for financial assistance to the project.
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Thanks are also due to The Head, Department of Botany, Banaras Hindu University for providing laboratory and other infrastructure facilities during the experiment. The first author also thanks the Council of Scientific & Industrial Research, New Delhi for a postdoctoral fellowship. References Awasthi, M., Das, D.N., 2005. Impact of Ni, Zn and Cd on growth rate, photosynthetic activity, nitrate reductase and alkaline phosphatase activity of free and immobilized Scenedesmus quadricauda. Algol. Stud. 115 (in press). Baun, A., Bussarawit, N., Nyholm, N., 1998. Screening of pesticide toxicity in surface water from an agricultural area at Phuket Island (Thailand). Environ. Pollut. 102, 185–190. Bier, J., 2002. Nitrate in groundwater: sources, impacts and solutions. Sixth Symposium on Groundwater contaminants, November 12 and 13, Fresno, CA. Borgmann, U., 2000. Methods for assessing the toxicological significance of metals in aquatic ecosystems: bio-accumulation– toxicity relationships, water concentrations and sediment spiking approaches. Aquat. Ecosyst. Health Manage. 3, 277–289. Brouers, M., De Jong, H., Shi, D.J., Hall, D.O., 1989. Immobilized cells: an appraisal of the methods and applications of cell immobilization techniques. In: Creswell, R.C., Rees, T.A.V., Shah, N. (Eds.), Algae and Cyanobacterial Biotechnology. Longman Scientific and Technical Publishers, USA, pp. 272–290. De Filippis, L.F., Pallaghy, C.K., 1994. Heavy metals: sources and biological effects. In: Rai, L.C., Gaur, J.P., Soeder, C.J. (Eds.), Algae and Water Pollution. E. Schweitzerbart’sche, Verlagsbuchhandlung, Stuttgart, Germany, pp. 31–77. Forni, C., Chen, J., Tancioni, L., Caiola, M.G., 2001. Evaluation of the fern Azolla for growth, nitrogen and phosphorus removal from wastewater. Water Res. 35, 1592–1598. Gardea-Torresdey, J.L., Becker-Hapak, M.K., Hosea, J.M., Darnell, D.W., 1990. Effect of chemical modification of algal carboxyl groups on metal ion binding. Environ. Sci. Technol. 19, 1372–1379.
Gaur, J.P., Rai, L.C., 1994. Introduction. In: Rai, L.C., Gaur, J.P., Soeder, C.J. (Eds.), Algae and Water Pollution. E. Schweitzer Bart’ sche, Verlagsbuchhandlung, Stuttgart, Germany, p. 304. Gerloff, G.C., Fitzerald, G.P., Skoog, F., 1950. The isolation, purification, and culture of blue-green algae. Am. J. Bot. 27, 216–218. Herbert, D., Phipps, P.J., Strange, R.E., 1971. Chemical analysis of microbial Cells. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. Academic Press, London, pp. 209–334. Hofer, M., Hoggett, J.G. (Eds.), 1981, Transport Across Biological Membranes. Pitman Publishing, Ltd., GB, p. 183. Lau, P.S., Tam, N.F.Y., Wong, Y.S., 1998. Operational optimization of batchwise nutrient removal from wastewater by carrageenan immobilized Chlorella vulgaris. 19th Biennial Conference of the International Association on Water Quality, Vancouver, Canada. Nicholas, D.J., Nason, A., 1957. Determination of nitrate and nitrite. Methods Enzymol. 111, 320–343. Peckol, P., DeMeo-Anderson, B., Rivers, J., Valiela, I., Maldonado, M., Yates, J., 1994. Growth, nutrient uptake capacities and tissue constituents of the macroalgae Cladophora vagabunda and Gracilaria tikvahiae related to site-specific nitrogen loading rates. Mar. Biol. 121, 175–185. Reiriz, S., Cid, A., Torres, E., Abalde, J., Herrero, C., 1994. Different responses of the marine diatom Phaeodactylum tricornutum to copper toxicity. Microbiology 10 (3), 263–272. Silver, S., 1991. Bacterial heavy metal resistance systems and possibility of bioremediation. In: Biotechnology, Bridging Research and Applications. Kluwer Academic Publishers, London, pp. 265–287. Tam, N.F.Y., Wong, Y.S., Simpson, C.G., 1998. Repeated removal of copper by alginate beads and the enhancement by microalgae. Biotech. Technol. 12, 187–190. Zhang, L., Huang, G., Yu, Y., 1998. Immobilization of microalgae for biosorption and degradation of butyltin chlorides. Artif. Cells Blood Substit. Immobil. Biotechnol. 26, 399–410. Zhang, W., Majidi, V., 1994. Monitoring the cellular response of Stichococcus bacillaris to exposure of several different metals using in vivo 31P NMR and other spectroscopic techniques. Environ. Sci. Technol. 28, 1577–1581.
Ecotoxicology and Environmental Safety 61 (2005) 268–272 www.elsevier.com/locate/ecoenv
Toxicity of nickel, zinc, and cadmium to nitrate uptake in free and immobilized cells of Scenedesmus quadricauda Mamta Awasthia,, Lal Chand Raib a
Department of Zoology, Arunachal University, Itanagar 791 112, India Department of Botany, Banaras Hindu University, Varanasi 221005, India
b
Received 26 May 2004; received in revised form 17 December 2004; accepted 20 December 2004 Available online 3 February 2005
Abstract We examined the influences of three trace metals on the accumulation of a major nutrient (NO 3 ) in Scenedesmus quadricauda. A comparative study on metal–nutrient interaction in free and immobilized states of algal cells was conducted. The effect due to interaction between different variables (cell state type, metal type, and metal dose) was studied to assess the variation in the nitrate uptake by free and immobilized cells. The results analyzed by ANOVA (three-way) (components: cell state type, metal type, and metal dose) confirmed that the inhibition of nitrate uptake by test metals was highly significant (Po0.001). Free and immobilized states of S. quadricauda responded differently (Po0.05, ANOVA) to the types of metal added. Uptake kinetics was studied by monitoring short-term uptake rates at different nutrient levels. Free and immobilized cells of the organism displayed noncompetitive modes of inhibition for Ni and Zn while a competitive mode of inhibition by Cd was observed in both free and immobilized states of the organism. r 2005 Elsevier Inc. All rights reserved. Keywords: Heavy metal; Immobilized; Scenedesmus quadricauda; Nitrate uptake
1. Introduction Freshwater ecosystems are influenced by heavy metal pollution (Baun et al., 1998). The chemical forms of these metals in water are accessible to the biota through significant accumulation in the food chain. Toxicity of heavy metals to algae has been reviewed earlier (Gaur and Rai, 1994). In addition to heavy metal pollution, excess nitrate discharge is also attracting attention. The nitrate threat to ground water comes from various sources including nitrogen-based fertilizers, waste from dairy and other livestock operations, and septic tank systems, both residential and industrial (Bier, 2002). Many organisms including algae possess the ability to incorporate nutrients very rapidly from the external medium (Forni et al., 2001). For total removal of Corresponding author.
E-mail address: [email protected] (M. Awasthi). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.12.018
nitrogen, a biological process is more suitable than any conventional technique. However, the use of algae as a biological material is restricted since it is difficult to harvest algae for its use in sewage treatment. Recently, this problem was overcome by the use of immobilized algae (Lau et al., 1998). Immobilized biomass offers many advantages including better reusability, high biomass loading, and minimal clogging in continuous flow systems (Zhang et al., 1998; Tam et al., 1998). Nitrate uptake is an interaction between cells and substrate similar to the interaction between enzymes and substrate in any biochemical process. In the present study, nitrate serves as a substrate and is converted into amine form (–NH2). During this process, many substances (including heavy metals) may alter the uptake of nutrients by combining with them in a way that influences the binding of substrate by algal cells thus they act as inhibitors. An inhibitor can compete directly with the substrate for an enzymatic-binding site
ARTICLE IN PRESS M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
(competitive inhibition) or can bind to either the free enzyme or the enzyme–substrate complex (noncompetitive inhibition). The study of nitrate uptake by algae becomes important since algae can be used as an excellent nutrient stripper, if properly managed. The present analysis will enable assessment of the extent and mode of inhibition of nitrate uptake in the immobilized state of algal cells by heavy metals. A comparative study of nutrient uptake kinetics in free and immobilized cells is also significant for assessing the superiority of immobilized cells, if any, over free cells. The presence of heavy metals together with excess nutrients can cause interference in the nutrient removal process by algae. The study was designed to evaluate whether metals present in effluents inhibit NO 3 uptake by imbedded algae more so than that by free cells, thereby creating problems with effluent treatment using imbedded cells.
2. Material and methods 2.1. Isolation, purification, and culture Eukaryotic green algae Scenedesmus quadricauda (local isolate, Banaras Hindu University) was grown in modified CHU-10 medium (Gerloff et al., 1950). The pH was maintained at 7.0 by using 2.0 mM Tris (hydroxymethyl) methylene/HCl. Cultures from the logarithmic phase were used for toxicity tests. Stock solutions of NiCl2 6H2O, ZnSO4 H2O and CdSO4 H2O were filtersterilized by passing through Millipore membrane filters (0.45 mm) before addition to the culture medium. Test metals were freshly prepared and the actual metal concentrations used were the LC50 values determined previously by the plate/colony count method based on the survival of the test alga to 50%. The concentrations were as follows: Ni, 1.10, 2.10 (LC50), and 3.10 mmol L1; Zn, 1.40, 2.40 (LC50), and 3.40 mmol L1; Cd, 1.30, 2.30 (LC50), and 3.30 mmol L1. The culture received 72 mmol photons m2 s1 PAR light intensity at 2472 1C. Standard microbial techniques were employed for selection, isolation, and cloning of algae in pure culture. Protein value was estimated following the method of Herbert et al. (1971). 2.2. Bubble column reactor A glass tube containing a ground-glass filter at the bottom and sealed with a rubber stopper at the top was used. A hollow rod was inserted through the stopper to reach the bottom of the column. The tube was used to pump air in the reactor. The column was aerated at 250–300 cc/min with an air bubbler. A hollow tube was
269
also inserted sideways at the bottom to remove the solution from the reactor at different time intervals. 2.3. Cell immobilization Most methods for immobilization of biomaterials in alginate beads basically involve two main steps. First, there is the internal phase where the alginate solution containing biological materials is dispersed into small droplets. Secondly, the droplets are solidified by gelling or membrane formation at the droplet surface. This is termed the dialysis/diffusion method (diffusion setting) as the alginate solution is gelled by diffusion of gelling ions from an outer reservoir. The procedure is as follows: Dissolve 30 g of sodium alginate in 1 L to make a 3% solution. Mix approximately 10 mL of desired protein value algal culture with 10 mL of 3% (wt.) sodium alginate solution. The concentration of sodium alginate can be varied between 6% and 12% depending on the desired hardness. The beads are formed by dripping the polymer solution from a height of approximately 20 cm into an excess (100 mL) of stirred 0.2 M CaCl2 solutions with a syringe and a needle at room temperature. Pump pressure and the needle gauge can control the bead size. A typical hypodermic needle produces beads of 0.5–2 mm in diameter. Other shapes can be obtained by using a mold whose wall is permeable to calcium ions. Leave the beads in the calcium solution to cure for 0.5–3 h. In the present study, exponentially grown algal cells (500 mg protein/mL) obtained by centrifugation and repeated washings were suspended in 3% (w/v) solution of sodium alginate (Sigma). The mixture was pumped dropwise into CaCl2 (0.2 M) solution. The beads thus formed contained algal cells in an amount of 60 mg protein per bead. The beads were washed several times with sterile deionized double-distilled water and resuspended in a 200-mL growth medium for autotrophic growth under culture room conditions. Free cells were also cultured under similar conditions at the same time. 2.4. Estimation of NO3 For studying the effect of metals on NO 3 uptake, cultures were incubated in KNO3 (1.5–20.0 mM). A bubble column reactor was used for NO 3 uptake. The column was aerated at 250–300 cc/min with an air bubbler. Beads were placed into a bubble column reactor in 500 mL growth medium for 24 h. At that time, the medium was pumped out and the beads were washed with 500 mL of double-distilled water. The column reactor was exposed to a NO 3 uptake medium for 30 min; 3-mL samples were taken every 10 min and tested for NO 3 depletion. NO3 in the medium was estimated spectrophotometrically by
ARTICLE IN PRESS M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
Immobilized
0
2.0070.01
2.4070.15 (20.0)a
Nickel
3.1 2.1 1.1
0.9070.02 (55.0) 1.1070.01 (45.0) 1.5070.02 (25.0)
1.2070.06 (40.0) 1.3070.02 (35.0) 1.6070.03 (20.0)
Zinc
3.4 2.4 1.4
0.6070.01 (70.0) 1.0070.06 (50.0) 1.2070.05 (40.0)
1.3070.04 (35.0) 1.4070.01 (30.0) 1.6070.02 (20.0)
Cadmium
3.3 2.3 1.3
0.7070.04 (65.0) 1.1070.01 (45.0) 1.2070.02 (40.0)
1.1070.02 (45.0) 1.4070.01 (30.0) 1.5070.05 (25.0)
All the values are mean7SE. Data in parentheses denote % inhibition. All the treatments are significantly different (Po0.001) from their respective control according to Student t test. a Shows stimulation over control (free cells).
3. Results A concentration-dependent inhibition of nitrate uptake in both free and immobilized cells (Table 1) was observed. Approximately 45%, 50%, and 45% inhibition of NO 3 uptake by the organism was recorded following the addition of LC50 concentrations of Ni (2.10 mmol L1), Zn (2.40 mmol L1), and Cd 1 (2.30 mmol L ), respectively; 35%, 30%, and 30% inhibition was recorded in the presence of Ni, Zn, and Cd, respectively, in the immobilized organism. The results analyzed by ANOVA (three-way) (components: cell state type, metal type, and metal dose) confirmed that the inhibition of nitrate uptake by test metals was highly significant (Po0.001). The two states of cells (free/immobilized) responded differently (Po0.05, ANOVA) with different test metals. The nutrient uptake kinetics was studied and the values for V max and half-saturation constant (K m ) for uptake were obtained employing the Lineweaver–Burk double-reciprocal plot. Free (Fig. 1) and immobilized (Fig. 2) cells of the organism displayed noncompetitive modes of inhibition for Ni and Zn. In the noncompetitive mode of inhibition, V max 1 decreased from 1.33 to 1.11 and to 1.0 mmol NO1 3 mg 1 protein h , respectively, in the medium supplemented
Free living Control
-1
Uptake kinetics are typically described using the analogous enzyme kinetic parameters; V max ; which is determined as the mean of uptake rates obtained at saturating substrate concentrations, and K m ; which is the nutrient concentration at which the uptake rate is half maximum. The rate equation is given by the Michaelis–Menten equation, V ¼ V max S=ðK m þ SÞ; where V is the initial velocity when no product is formed, V max is the maximum reaction rate, K m is the Michaelis–Menten constant, and S is the substrate concentration. To obtain a linear plot, the above formula is inverted and a modified equation (Lineweaver–Burk plot) for experimental determination of V max and K m is obtained. The Lineweaver–Burke plot is a plot of 1=V max (the reciprocal of the reaction rate) against 1/S (the reciprocal of the substrate concentration). Noncompetitive inhibition results in intersection at the x-axis (1=K m ¼ constant) and competitive inhibition results in intersection at the y-axis (1=V max ¼ constant) in the presence of the inhibitor. Here nitrate uptake rate is plotted against the substrate concentration (nitrate) as a Lineweaver–Burke plot.
1 –1 protein) NO 3 uptake (mmol NO3 mg
Metal concentration (mmol L1)
3.5
-0.3
-0.2
Control -1
Nickel (2.1 µmol L ) -1
3.0
Zinc (2.4 µmol L ) Linear regression
2.5
-1
2.5. Uptake kinetics
Table 1 Differential toxicity of Ni, Zn and Cd to nitrate uptake (supplemented with 5 mM KNO3) after 4 h of metal treatment
2.0
-1
the brucine–sulfuric acid method (Nicholas and Nason, 1957). The color so developed was measured at 410 nm.
1/V (µmol NO3 µg protein h-1)
270
1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
Fig. 1. Nitrate uptake and inhibition by Ni and Zn in free state of S. quadricauda.
with LC50 concentration of Ni and Zn in the free state of cells. In the immobilized organism, V max declined from 1 1.81 to 1.25 and to 1.43 mmol NO1 protein h1, 3 mg respectively, in the presence of Ni and Zn. Conversely, the K m remained unchanged at 4.34 mM in the immobilized cells (Fig. 2), whereas it remained at 5.55 mM in the free cells (Fig. 1). A competitive mode of inhibition by Cd was observed in both the free (Fig. 3) and the immobilized (Fig. 4) states of the organism. Here, K m values increased from 5.71 to 8.33 mM in the free cells and from 5.0 to 6.66 mM in the immobilized cells of the test organism. However, the value for V max remained unchanged, i.e., 1 1.33 and 1.53 mmol NO1 protein h1 in the cases 3 mg of free and immobilized cells, respectively.
ARTICLE IN PRESS 1/V (µmol NO3-1 µg-1 protein h-1)-1
2.5
Control -1
Zinc (2.4 µmol L ) -1 Nickel (2.1 µmol L )
2.0
Linear regression
-1
-1
1/V (µmol NO3 µg protein h )
-1 -1
M. Awasthi, L.C. Rai / Ecotoxicology and Environmental Safety 61 (2005) 268–272
-0.3
-0.2
1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-0.2
3.0 2.5
271
Control -1 Cadmium (2.3 µmol L ) Linear regression
2.0 1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
1/(S) mM
-1
Fig. 2. Nitrate uptake and inhibition by Ni and Zn in the immobilized state of S. quadricauda.
1/V (µmol NO3-1 µg-1 protein h-1)-1
3.5
-0.3
-0.2
3.0
Control -1 Cadmium (2.3 µmol L ) Linear regression
2.5 2.0 1.5 1.0 0.5
-0.1
0.1
0.2
0.3
0.4
0.5
-1
1/(S) mM
Fig. 3. Nitrate uptake as inhibited by Cd in free state of S. quadricauda.
4. Discussion This investigation indicates the potential for the use of immobilized cells in the effluent treatment when the target pollutants are both heavy metals and nitrate. The heavy metal component seems to compete with the nitrate for the selective binding sites present on the extracellular surface (Zhang and Majidi, 1994; Gardea-Torresdey et al., 1990). In addition to passive uptake, there is another mechanism for metal uptake which requires energy (Silver, 1991), and since nutrient uptake is also an energy-dependent process (Peckol et al., 1994), it may compete with metal for energy. The algae also exhibited a varied response to different metals. This may be due to different chemical properties, speciation, and affinity of metals for the algae when present in liquid medium (Borgmann, 2000).
Fig. 4. Nitrate uptake and inhibition by Cd in the immobilized state of S. quadricauda.
The noncompetitive mode of inhibition of NO 3 suggests a direct involvement of energy. In this kind of inhibition, the substrate and the inhibitor bind to different sites on the carrier (Hofer and Hoggett, 1981). The heavy metals cause reduction of photosynthesis (Reiriz et al., 1994) which points toward an alternation in the structure of the enzyme by binding of the metal with the sulfydryl group (De Filippis and Pallaghy, 1994). Apart from the metal-induced depletion of ATP and reductant pools, exhaustion of energy-yielding substrates and competition of metals with essential micronutrients may result in the noncompetitive inhibition of nutrient uptake by phototrophic organisms. The competitive mode of inhibition is indicative of the common entry site for metals and nutrients. It rules out the direct involvement of energy. This was further confirmed when the toxic effects were counteracted by increasing substrate concentration. The higher uptake of nitrate by immobilized cells than by free cells could be due to increased cell wall permeability as observed by Brouers et al. (1989). Nevertheless, the higher energy state of the immobilized system could be another factor for the facilitated nutrient uptake (Awasthi and Das, 2005). A lower inhibition of nitrate uptake in the immobilized cells in the presence of metals shows their superiority over free cells. Immobilized S. quadricauda exhibit the lowest inhibition for nitrate uptake in the presence of Zn and Cd. The capacity of immobilized cells to bind nutrients even in the presence of metals shows the potency to do further research in this area.
Acknowledgments The authors thank the Ministry of Environment & Forest, New Delhi for financial assistance to the project.
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