Toxicity of nickel, zinc, and cadmium to nitrate uptake in free and immobilized cells of Scenedesmus quadricauda

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Abstract

We examined the influences of three trace metals on the accumulation of a major nutrient (NO3) 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 (P<0.001). Free and immobilized states of S. quadricauda responded differently (P<0.05, 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.

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 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 (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 NO3 uptake by imbedded algae more so than that by free cells, thereby creating problems with effluent treatment using imbedded cells.

Section snippets

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 filter-sterilized by passing through Millipore membrane filters (0.45 μm) before addition to the culture medium. Test metals were freshly

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 NO3 uptake by the organism was recorded following the addition of LC50 concentrations of Ni (2.10 μmol L−1), Zn (2.40 μmol L−1), and Cd (2.30 μmol L−1), 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:

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

Acknowledgments

The authors thank the Ministry of Environment & Forest, New Delhi for financial assistance to the project. 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 post-doctoral fellowship.

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