Bacterial biosensors to quantify bioavailable concentration of heavy metals in polluted soils and to predict their risk of transfer to the food chain

P. Corbisier, C. Tibarzawa, D. van der Lelie^* (VITO, Vlaamse Instelling voor Technologisch Onderzoek, Environmental Technology Centre, Boeretang 200, 2400 Mol, Belgium, Tel: +32-14-335166, Fax: +32-14-580523, e-mail: niels.vanderlelie@vito.be);

J. Vangronsveld (Limburgs Universitair Centrum, Environmental Biology, Universitaire Campus, B-3590 Diepenbeek, Belgium);

M. Mensch (INRA, Unité d4Agronomie-BGETA, Bordeaux, France)

Abstract

In this work, naturally heavy metal polluted soils as well as Zn, Cd, Pb, As, Hg, Cr spiked soils of different origins and compositions have been tested using a collection of chemical and biological methods to determine heavy metal bioavailability. Among the biological methods, bacterial biosensors (BIOMET) have been used as fast detection tool for the quantification of bioavailable concentrations. In general, results of bacterial and phytotoxicity tests agree rather well with the data from the physico-chemical analysis. The BIOMET test is a simple and quick test, which can be used for an efficient first screening of the toxicity and bioavailability of a substrate. In case of positive results for this test, organisms of other trophic levels must be tested.

Introduction

It is becoming accepted that the measurement of the total concentration of metals present in a soil, classically measured by chemical methods, is giving an incomplete image of the potential risks for the different biological target present at different trophic levels. The risk of heavy metals in soils is determined by the bioavailabilty of the metals, which depends on a large number of chemical and biological parameters. Since chemical measurements alone do not provide a complete information about the bioavailablity of the metals and since the modelling approach is time-consuming and incomplete, another approach was to directly measure the effect of the heavy metals on different biological target. Therefore a number of biological assays have been developed and standardised. Those assays can be classified on soil microbial tests, soil invertebrate tests and terrestrial plant tests. A panel of specialists has recently evaluated those methods (Fairbrother et al., 1999). One of these test is the BIOMET® assay.

METHODS

The BIOMET® assay (Corbisier et al., 1994) is based on transcriptional fusions between specific heavy metal-induced promoters and the lux operon of Vibrio fischeri. The quantification of the emitted bioluminescence can be easily recorded in a luminometer and compared to an internal calibration realised with known concentration of metal ions (for protocols, see Corbisier et al., 1999). Bacterial sensors specific for Zn, Cd, Cu, Cr, Pb (Corbisier et al., 1999) and Ni (Tibazarwa et al., 2000) have been fully characterised in term of specificity, detection limits and selectivity (Table 1). The BIOMET® assay is available in an easy to use kit format.

Table 1. Properties of the most commonly used BIOMET sensors

Name	Bacterial Host	Metal ions	DL (µM)	DR (µM)	Reference
AE1239	Ralstonia silverii DS185	Cu²⁺	2	2-40	Corbisier et al., 1994
AE1433	Ralstonia metallidurans CH34	Zn²⁺ Cd²⁺	5 5	5-250 5-200	Corbisier et al., 1994
AE2440	Ralstonia metallidurans CH34	Cr⁶⁺	1	1-40	Corbisier et al., 1999
AE2515	Ralstonia metallidurans CH34	Ni²⁺	0.08	0.08-100	Tibazarwa et al., 2000
AE2450	Ralstonia metallidurans CH34	Pb²⁺	0.5	0.5-5	Corbisier et al., 1999

DL : Detection limit giving as signal/noise ratio of 2; DR : Dynamic range of the sensor

RESULTS

An example is given below, where a BIOMET^® sensor reacting specifically to Ni was used to test soils which were amended with dredging sludges polluted by Ni, Zn and Cd. The bioavailable Ni concentration measured with the bacterial sensor was well correlated with the accumulated Ni concentration found in the corn grains as shown on Fig 1.

Figure 1: Correlation between the bioavailable Ni concentration in soils measured with the bacterial sensor and the accumulated Ni found in the corn grains of plants grown on the same soils (Tibazarwa et al., 2000).

Another example is given on Fig 2, where a Cd-sensor was used and Cd bioavailability compared with the accumulation of Cd in potato plants.

Figure 2: Correlation between the bioavailable Cd concentration in soils measured with the bacterial sensor and the accumulated Cd found in the potatoes of plants grown on the same soils (Tibazarwa et al., 2000).

Cu-spiked and aged soils have been tested with a specific Cu-sensor and compared to total Cu concentration present in the soils and Cu accumulation in the 2^nd leaves of beans grown on those soils. The response of Cu-sensor is well proportional to the quantity of copper sulphate that was added to the soil (Fig 3).

Figure 3: Comparison between the bioavailable copper concentration measured with the Cu-BIOMET sensor and the total amount of copper present in the different Cu-spiked soils.

Since the soil present the same soil characteristic, the only factor which varied was the copper concentration. We can also conclude that about 1/6 of the added copper is available for the soil bacteria. The bacterial available copper concentration is also proportional to the quantity of copper which later accumulate in the leaves of beans grown on those soils (Figure 4).

Figure 4: Comparison between the bioavailable copper concentration measured with the Cu-BIOMET sensor in the Cu-spiked soils and accumulation of Cu in leaves of bean plants grown in the same soils.

Conclusions

This work illustrates the potential use of the BIOMET^® biosensors for a quick evaluation of heavy metal bioavailability in polluted soils. A possible use of the BIOMET^® biosensors as a regulatory ecotoxicological tool has been identified with the prospect of providing a fast Indication of bioavailable metal species. The BIOMET test is simple and quick and can be used for an efficient first screening of the toxicity and bioavailability of a substrate. In case of positive results for this test, organisms of other trophic levels must be tested. The system is also a useful tool for the evaluation of heavy metals immobilisation techniques.

Acknowledgements: This work was supported by the European Commission (contract ENV4-CT95-0141), by EFRO (European Fund for Regional Development) and OVAM (Public Authorities of Flanders for Waste).

REFERENCES

Corbisier P. van der Lelie D., Borremans B., Provoost A., de Lorenzo V., Brown N., Lloyd J., Hobman J., Csöregi E., Johansson G, Mattiasson B. (1999) Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Analytica Chimica Acta 387, 235-244.

Corbisier P., Thiry E., Masolijn A., L. Diels, Construction and development of metal ion biosensors in Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects ed A.K. Campbell, LJ Kricka and P.E Stanley. John Wiley & Sons Chichester, New York, Brisbane, Toronto, Singapore (1994) p151-155.

Fairbrother A., Glazebrook P.W., Van Straalen, Tarazona J.V. (1999). Test methods for hazard determination of metals and sparingly soluble metal compounds in soils, SETAC, 1999, ed A. Fairbrother. pp1-24.

Tibazarwa C., Corbisier P., Mench M.., Bossus A., Solda P., Mergeay M., Wyns L., van der Lelie D. (2000) A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants. Environmental Pollution. Submitted.