C Durrieu

C Tran-Minh

J M Chovelon

L Barthet

C Chouteau

C Védrine

Algal biosensors for aquatic ecosystems monitoring

Keywords: 87, 80, Tq Biological signal processing and instrumentation

The harmful effect of toxic chemicals on natural ecosystems has led to an increasing demand for early-warning systems to detect those toxicants at very low concentrations levels. Whole-cell biosensors based either on chlorophyll fluorescence or enzyme (phosphatase and esterase) inhibition are constructed for real-time detection and on-line monitoring. Results show that these devices are sensitive to heavy metals and pesticides. The system allows the cells to operate in their natural environment which favours long term stability and reflects the toxic action mechanism providing therefore an ecological interest.

Introduction

Aquatic ecosystems monitoring has currently referred to sensitive and reliable methods based on spectrometric and electrochemical techniques (ICP-MS, GC-MS, SAA…) that ensures detection of specific chemicals at low concentration levels [START_REF] Barcelo | Environmental Analysis, Techniques, Applications and Quality assurance[END_REF][START_REF] Capiello | [END_REF]. However, those techniques are costly, time-consuming and limited to a restricted number of species. Additionally, they have to face long delays after sampling to produce expected results. Therefore, continuous detection and on-site monitoring are in great demand in aquatic ecosystems management. This could be achieved with biological sensors permanently settled in the areas under control. Whole-cell biosensors presented hereafter are based on metabolic perturbation of immobilized algal cells in the presence of toxicants. Algal cells are chosen for their high sensitivity and their place in the ecosystem: being at the very beginning of the trophic chain, they represent a good biological marker of ecosystem pollution and an early-warning system to prevent irreversible effects [3,4,5,6]. The work presented here concerns the construction of a biosensor from unicellular green algae, not having undergone any genetic modification. This tool can thus be placed in situ and makes it possible to evaluate the response of the algae in their natural environment. In this paper, the objective is to use two different enzymes to screen two different families of toxicants and mainly to detect pesticides and heavy metals at the same time.

General principle of biosensors

A biosensor can be considered as a combination of a bioreceptor, the biological component, and a transducer, the detection method. The total effect of a biosensor is to transform a biological event into an electrical signal. The first link of a biosensor is the bioreceptor, which has a particularly selective site that identifies the analyte. The bioreceptor ensures molecular a e-mail: Claude.durrieu@entpe.fr recognition, and may transform the analyte in some way [7]. This localized modification is generally made via an immobilized enzyme, which transforms the analyte into a product that is detectable by the transducer [8,9]. This is the case for enzyme sensors. Sometimes, however, the enzyme is only stable in its natural environment, which cannot be modified, and the whole cell or microorganism is immobilized on the biosensor [10].

The other component of a biosensor is the transducer, which exploits the biochemical modification of the substrate by the bioreceptor by transforming it into an electrical signal (Figure 1). We could say that the transducer converts one type of energy into another. The choice of transducer depends on the type of biochemical modification, it should make optimal use of the product of the bioreceptor and give a signal that is sensitive, easily monitored, and has minimal background noise. Low background noise reduces the detection limit and improves the biosensor performance.

The combination of any bioreceptor such as enzymes [11,12,13], immuno-agents [14], tissues [START_REF] Arnold | Biosensors Fondamentals and Applications[END_REF] or cells [START_REF] Pandard | [END_REF]17,18] with any transducer leads to a large number of biosensors (Table 1). Electrochemical transducers couple relatively easily with enzymes, and so such biosensors were first reported [19].

3-Biosensors applications

Biosensors have many commercial applications in a large range of activities. The most important applications are in medicine (in hospitals or in the home) and in the food produce industry for the control of manufacturing processes. More recently, many biosensors have been applied to environmental control: the bioreceptor being purified enzymes or whole cells directly immobilized on a transducer.

Biosensors based on enzyme inhibition

The biosensor makes use of an enzyme layer and a transducer, in close contact with each other, to form a single unit for detection of heavy metals, organophosphorous or organochlorinated pesticides [12,20,21]. Enzymes are commonly used in their purified form to achieve specific detection of a toxic analyte by enzyme inhibition. The percentage of inhibition is directly correlated to analyte concentration [22,23]. For pesticides determination, cholinesterase and urease have been used in conjunction with electrochemical transducers. Recently, a conductometric acetylcholinesterase biosensor was constructed for assessment of toxicity of methyl parathion and its photodegradation products in water [24]. Since a wide range of enzymes (urease, glucose oxydase, invertase) are inhibited by heavy metal ions at low concentrations, they were immobilized onto different transducers for metal ions determination [25,26]. However, those enzyme sensors have the inherent drawbacks of purified enzymes whose easy denaturation hinders their use in pollution control.

Biosensors based on whole cells activity

Microbial species can provide a valuable source of biocatalyst material for whole cell biosensor design and have proven to be amenable to immobilization in biosensor configuration. Microbes have a number of advantages as biological sensing materials in the biosensor design : they are present ubiquitously and are able to metabolise a wide range of chemical compounds. Microorganims have a great capacity to adapt to adverse conditions and to develop the ability to degrade new molecules with time. Biosensor electrodes have been developed that incorporate a range of bacterial, yeast and algal cell types [START_REF] Pandard | [END_REF]27]. Compared to biosensors using purified enzyme , whole-cell biosensors are more resistant to the activity loss because their enzymes and cofactors are hosted in an environment optimized by nature. Therefore, these biosensors are more suitable to meet all the requirements for environmental surveillance [18,28]: they can identify in situ the presence of a toxic compound as soon as it is released in waste water or aquatic environment. Other whole cell biosensors were constructed from genetically modified cells [21]. Those techniques may improve the biosensor sensitivity and selectivity but are no longer able to reflect the ecosystem operating conditions. In the present work, only native cells have been used to preserve the ecological aspect of the media under study.

Algal biosensors

First algal biosensors made use of an oxygen electrode to detect the oxygen production [START_REF] Pandard | [END_REF] that results from the photosynthetic activity commonly observed in all plants. However, this corresponds to an unspecific sensing since many pollutants are more or less inhibitors of the oxygen emission. Algal biosensors developed in this study have the ability to detect a group of pollutants provided they affect a particular alga metabolic pathway. This is the case of pesticides and heavy metals which are strong inhibitors of acetylcholinesterase and alkaline phosphatase, both are located in Chlorella vulgaris. This green alga belongs to the Chlorophycea group and it was selected for its greater stability in producing biological signals. The chlorophyll fluorescence emitted from its photosynthetic activity enables pesticides detection [18] while inhibition of its alkaline phosphatase and esterase allows determination of heavy metals [29] and organophosphorous insecticides [30] respectively. The corresponding signals are obtained with optical and conductometric transducers. This tool has been designed to monitor simultaneously several metabolic activities of immobilized algal cells (chlorophyll fluorescence and alkaline phosphatase / esterase activities). Since they are not genetically modified, they can be used for real-time screening of the various families of pollutants coexisting on the same site. The response of algae under chemical stress can then be analyzed directly from their original medium.

Construction of an optical algal biosensor

The algal strain used in this study was Chlorella vulgaris (CCAP 211/12) purchased from The Culture Collection of Algae and Protozoa at Cumbria, United Kindom. The axenic algal strain was grown in the culture medium and under conditions described by the International Organization for Standardization (ISO,1989). The active membrane was obtained by physical entrapment of the algal cells onto a porous matrix. Immobilization was achieved by simple filtration of an algal suspension on a glass fiber or quartz membrane. The optical biosensor was constructed with the tip of an optical fiber bundle placed in front of the algal membrane at a few millimeters distance in order to allow the sample solution to circulate between them (Figure 2). The various biological signal results from: -chlorophyll fluorescence produced by the photosynthetic activity -esterase activity involving in photosynthesis -phosphatase activity essential to phosphorous metabolism in algal growth Optical signals are obtained directly with chlorophyll fluorescence measurement [18] or after injection of fluorescent substrates with enzyme activity measurement [28]. Toxicant concentrations are determined from the variation of fluorescence amplitudes. Alkaline phosphatase activity in Chlorella vulgaris exhibits a good stability during 30 days, whereas a drift of esterase activity and chlorophyll fluorescence was observed after 4 to 5 days (unpublished results). In this case, the membrane needs to be changed after 5 days for the biosensor to keep its optimal response. An optical biosensor associated with a fluorimeter may result in a rather cumbersome equipment. Conversely, a conductometric transducer connected to its measuring system can easily be miniaturized and the reduced cost will allow this biosensor to be placed on several sites under monitoring.

Construction of a micro-conductometric algal biosensor

This biosensor is based on the local changes in conductivity of the "bio-membrane" resulting from alkaline phosphatase and esterase activities which produce ionic species. Algal cells are immobilized on a pair of interdigitated platinum electrodes printed on a Si/SiO 2 substrate of 1 mm thickness (dimensions 5 mm x 30 mm) which were fabricated at the Institute of Chemoand Biosensorics (Muenster, Germany) [31]. Each finger of the electrode was 10 µm wide and 1 mm long, with 10 µm spacing between fingers (Fig. 3). Another similar pair of electrodes is used as a reference. The sensitive area of each electrode pair was about 1 mm x 1.5 mm. The in-phase differential signal between the pair of electrodes was registered by a "home made" conductometric laboratory amplifier in which a small-amplitude alternative voltage of 10 mV with a high frequency of 100 kHz were applied. Under these conditions, it has been demonstrated [32] that the transfer resistance as well as the Warburg impedance can be neglected so that the output signal is directly proportional to the resistance (or conductance) within the "bio-membrane". In this paper, the results were obtained under the same conditions. The algal cells are entrapped in bovine serum albumin (BSA) crosslinked with glutaraldehyde [7,17]. This micro-biosensor is so small and inexpensive (the miniaturization allows a mass production at low cost) that it can easily work on the field with a portable conductimeter.

Results

Figure 4 shows various responses of a conductometric alkaline phosphatase biosensor to pnitrophenyl phosphate (pNPP) used as a substrate. Similar curves are obtained with an optical algal biosensor. The results agree with those carried out in test tubes with non-immobilized algae, which confirms the possibility to use algal biosensors to monitor their metabolic activities. Determination of alkaline phosphatase activity (APA) can also be carried out with methylumbelliferyl phosphate (MUP) as a substrate. The reaction product methylumbelliferone (MUF) is fluorescent. APA can easily be measured from the MUF fluorescence emission (460 nm) under excitation light (350 nm) when the MUP solution is brought into contact with the enzyme. Figure 5 gives an example of the algal APA inhibition measured with an optical algal biosensor before (A) and after (B) injection of a municipal solid waste effluent in a flow system. MUP is injected in the flow as a substrate to produce the optical response. Chemical analysis of these effluents showed high concentrations of heavy metals (more than 1mg/L), which can explain the inhibition effect observed on algae phosphatase activity Tableau 2 shows results obtained with the optical biosensor for herbicides detection. The biosensor was tested in the absence of toxic compounds and in the presence of herbicides (atrazine, simazine, isoproturon and diuron) to compare their effects on fluorescence induction. The presence of these herbicides increases the fluorescence emission. The detection limit, the toxic concentration EC 50 that affects 50% of the response, and the reversibility of action of these pollutants are listed in Table 2. Detection of herbicides was always achieved with concentration level down to 1µg/L The fluorescence based-biosensor using algae cells seems to be particularly suitable for detection of herbicides. Detection limits of this biosensor are compatible for aquatic environment quality monitoring. Compared to other previous paper [17,28,30], the results obtained here show that a single system can be used to assess the level of pollution with the possibility to identify the corresponding family of toxicants since the optical or conductometric specific responses of these biosensors enables to target the corresponding pesticides or heavy metals present in aquatic environment.

Conclusion

Whole-cell algal biosensors have proven to be successful as early warning systems to identify the presence of a pollutant before it causes damage to the ecosystem. These devices can be adopted by industry to monitor effluents or sewage treatment plants and by decision-makers in charge of aquatic ecosystems surveillance. The two types of biosensors presented in this study exhibit similar detection limits. An advantage of miniaturization of conductometric biosensors is to produce a large number of low-cost field devices to be placed in the areas under monitoring.

Figures and table captions

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 12345 Fig.1. The various stages of determination with a biosensor[7]

Fig. 1 .

 1 Fig.1. The various stages of determination with a biosensor[7]

Fig. 2 .Fig. 3 .Fig. 4 .

 234 Fig. 2. Optical algal biosensor

Fig. 5 .

 5 Fig. 5. Alkaline Phosphatase detection with algal optical biosensor in the absence (A) and in the presence (B) of a municipal solid waste effluent in the carrier flow. Substrate is injected in the flow to produce optical response.

Table 1 .

 1 Typical combinations of bioreceptors and transducers in environmental monitoring

Table 2 .

 2 Characteristics of a biosensor response to herbicides based on chlorophyll fluorescence

Table 2 .

 2 Characteristics of a biosensor response to herbicides based on chlorophyll fluorescence

	Herbicides	detection limits	EC 50	Reversibility
		(µg/l)	(µg/l)
	Atrazine	0,25	96 ± 4	Yes
	Simazine	0,5	127 ± 4	Yes
	Isoproturon	0,025	35 ± 2	Yes
	Diuron	0,025	39 ± 3	Yes


Claude Durrieu

Ce ´line Chouteau

Lucile Barthet

Jean-Marc Chovelon

Canh Tran-Minh

A Bi-Enzymatic Whole-Cell Algal Biosensor for Monitoring Waste Water Pollutants

Keywords: Biosensors, Algae, Alkaline phosphatase, Esterase, Heavy metals, Pesticides, Toxicity, Early warning systems

Two algal whole cells biosensors are developed to measure specific toxicity of freshwater pollutants. Both optical and conductometric biosensors are based on inhibition of algal alkaline phosphatase (AP) and esterase activities. Chlorella vulgaris cells are immobilised on a membrane placed in front of an optical fiber bundle for optical sensing or deposited on the surface of an electrode for conductometric sensing. Phosphatase activity of the biosensor is strongly inhibited by heavy metal ions (60% loss of

INTRODUCTION

The ever-growing discharge of chemicals in the environment leads to the requirement of early warning systems (EWS) to detect toxic compounds in order to react quickly in case of accidental pollution. Biosensors for pollutants determination can act as EWS thanks to their unique characteristics which include their sensitivity, low cost, and easy adaptation for on-line monitoring. [START_REF] Rogers | Biosensors for environmental application[END_REF] The aim of this study is to design two types of biosensors, optical and conductometric, based on enzyme activity inhibition directly on algal whole cells. The principle of algal biosensor [START_REF] Pandard | Comparison of two types of sensors using eukariotic algae to monitor polltion of aquatic systems[END_REF] was adopted here to provide inexpensive biosensors for environmental control. Enzyme purification step is avoided and longer lifetime obtained with enzymes kept in their natural environment. [START_REF] Samson | Use of algal fluorescence for determination of phytotoxicity of heavy metals and pesticides as environmental pollutants[END_REF][START_REF] Rechnitz | Biosensors based on cell and tissue material[END_REF] This technique makes use of alkaline phosphatase (AP) and esterase enzymes located on the external membrane of the whole cell Chlorella vulgaris for direct determination of heavy metals and pesticides. In vivo inhibition of these enzymes reflects the natural deleterious effects of toxicants on microalgae and represents a real ecological interest since algae are involved in the primary step of the food chain.

EXPERIMENTAL Algal Cultivation

The C. vulgaris strain (CCAP 211/12) was purchased from The Culture Collection of Algae and Protozoa at Cumbria, United Kingdom. The axenic algal strain was grown in the culture medium and under conditions described by the International Organization for Standardization. [START_REF]ISO 8692 Water Quality: Fresh Water Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum[END_REF] In order to produce high AP concentration in the algal cell, the algal solution was centrifuged and the pellets were resuspended in phosphate-free medium to starve the algal cells for 25 days which gives a maximal enzyme activity. This resuspension was carried out under the same conditions and with the same culture medium as that used previously for the growth of this strain except there were no phosphate ions. Although the medium did not contain any phosphate, an increase in cells density was observed, particularly at the beginning of the subculture period, due to the consumption of the phosphate remaining in the cell. This phosphate consumption induced an increase in AP specific activity. [START_REF] Durrieu | Optical algal biosensor using alkaline phosphatase for determination of heavy metals[END_REF] Esterase activity (EA) measurements were made directly on algal cells in exponential growth according to a previously published method. [START_REF] Gilbert | Rapid assessment of metabolic activity in marine algae. Application in ecotoxicological test and evaluation of water quality[END_REF] Construction of an Optical Biosensor

The biosensor was constructed with a removable algal membrane placed in a 1-mL home-made flow cell. [START_REF] Naessens | Fiber optic biosensor using Chlorella vulgaris for determination of toxic compounds[END_REF] The active membrane was constructed by physical entrapment of the algal cells onto a porous matrix. Immobilisation was achieved by simple filtration of an algal suspension on a glass microfiber filter (GF/C Whatman, 45.7-mm filter diameter, 1.2 mm pore diameter). Punching this filter has provided small disks (8 mm in diameter) which can be fitted in front of the tip of an optical fibre bundle. The incident light hit the upper part of the membrane and the resulting fluorescence radiation was collected through an optical fiber up to the Varian fluorometer equipped with a microcomputer for data recording. This device was quite suitable for the assay under flow injection analysis (FIA) conditions which required a very small amount of substrate for measurement of enzyme activity.

Determination of AP activity (APA) was carried out with methylumbelliferyl phosphate (MUP) as substrate dissolved at various concentrations in a Tris -HCl (0.1 M, pH 8.5) buffer solution containing 1 mM MgCl 2 . The reaction product methylumbelliferone (MUF) is fluorescent. APA can easily be measured from the MUF fluorescence emission (460 nm) under excitation light (350 nm) when the MUP solution is brought into contact with the enzyme.

Determination of EA was carried out with fluoresceine diacetate (FDA) as substrate dissolved at various concentrations in a citrate (0.1 M, pH 5.4) buffer solution. The reaction product fluoresceine emits fluorescence at 538 nm under 480 nm excitation light. Both MUP and FDA were purchased from Sigma.

Construction of a Conductometric Biosensor

The conductometric transducers were fabricated at the Institute of Chemo-and Biosensorics (Munster, Germany). Two pairs of Pt (150 nm thick) interdigitated electrodes were made by the lift-off process on Pyrex glass substrate. The central part of the sensor chip was passivated by Si 3 N 4 layer to define the electrodes working area. The sensitive part of each electrode was about 1 mm 2 . Measurements are based on the detection of conductance variations inside active membranes. Many enzymes can effectively catalyse consumption and production of ionic species resulting in conductivity changes.

The active membrane on the sensitive area of the first electrode was obtained by cross-linking algal cells with bovine serum albumin (BSA) in saturated glutaraldehyde (GA) vapours. [START_REF] Dzyadevych | Thin-film conductometric sensors for enzyme biotransducers[END_REF] A mixture containing 100 mL algae solution and 10% (w/v) BSA was deposited on the sensing electrode surface using a drop method. Another mixture of 100 mL phosphate-free medium and 10% (w/v) BSA was deposited on a second electrode used as a reference. Both sensors were placed in saturated GA vapours for 30 min and then dried in air for 15-30 min.

All measurements with conductometric biosensors were carried out in daylight at room temperature with a 5 mL glass cell. Enzyme activities were measured in a stirred medium with Tris-HCl buffer (10 mM, pH 8.5) and MgCl 2 (1 mM) for APA and KH 2 PO 4 buffer (2.5 mM, pH 8) for EA. After stabilisation of the output signal, different aliquots of the stock solution of substrate were added into the vessel. MUP (0-20mM) and paranitrophenyl-phosphate (pNPP, 0-0.86 mM) were used as substrates for APA, while acetylcholine chloride (AChCl, 0-10 mM) was used as a substrate for EA. The differential output signal was recorded with a "home made" conductometric equipment [START_REF] Dzyadevych | Assessment of the toxicity of methyl parathion and its photodegradation products in water samples using conductometric enzyme biosensors[END_REF] and the biosensor responses were plotted as a function of substrate concentration.

For toxicity assays with heavy metals, the decrease in the response to pNPP following 30 or 120 min exposures to Cd 2þ or Zn 2þ solutions (from 10 ppb to 1 ppm) was used to assess the enzyme inhibition. Measurements were carried out with 0.86 mM pNPP corresponding to a substrate saturation concentration. The same protocol was adopted for pesticides determination with 10 mM AChCl as substrate. The percentage of inhibition was obtained by comparing the biosensor responses dS before and after exposure to toxicants: 100 1 À dS after exposure dS before exposure %:

RESULTS AND DISCUSSION

Responses to Substrates

APA and EA activities were determined with algae immobilized on the transducers. For APA measurements, two substrates MUP and pNPP were tested to get optimal signals corresponding to the detection method. MUP was chosen for its higher sensitivity and its simplicity in optical biosensor design. This substrate was placed in a Tris buffer solution (0.1 M, pH 8.5) containing 1 mM MgCl 2 as an enzyme activator, and AP activity was measured in terms of fluorescence intensity from the biosensor response. Various MUP solutions were injected in the FIA system (Fig. 1): a good compromise for an acceptable response time and good repeatability was obtained with 0.5 mL injected volume and 8.4 mL min [START_REF] Naessens | Fiber optic biosensor using Chlorella vulgaris for determination of toxic compounds[END_REF] flow rate. pNPP is a classical substrate for electrochemical measurements. [START_REF] Pariente | Amperometric sensor based on the alkaline phosphatase activity[END_REF] In a recent study in which a conductometric biosensor was used, [12] it has been shown that pNPP gave much higher conductivity variations than MUP. Therefore, pNPP was preferred for conductometric experiments. Fluorescence evolution (for APA) or local conductivity variations (for EA) are given in Figs. 2 and3. Both calibration curves exhibit typical Michaelis -Menten kinetics. The relatively low response time of about 5 min observed for those substrates could be attributed to the corresponding enzymes located on the algal cell surface, so that their activity is readily available without requiring the substrates to diffuse across the cell membrane.

Determination of Heavy Metal Ions

The effects of toxic chemicals on APA and EA were tested with cadmium and lead for the optical biosensor and with cadmium and zinc for the conductometric biosensor. Enzymatic activities were determined after a definite exposure time to metal ion solutions. Fluorescence measurements with the optical biosensor was conducted with 0.1 mM MUP and 3 mM of FDA solutions which corresponds to the maximal velocity of APA and EA, respectively. When a metal ion solution was injected, APA was inhibited as indicated by the fluorescence reduction but no significant inhibition of EA was observed. Inhibition is considered as significant when the percentage of inhibition is above 15%. A comparison between the APA optical biosensor and a microplate reader based on APA inhibition showed similar results in Cd 2þ and Pb 2þ detection (Fig. 4). Conductometric biosensors based on APA inhibition were used for the determination of Cd 2þ and Zn 2þ (Table 1). It appears that Cd 2þ gives higher inhibition rates than Zn 2þ in agreement with the results obtained with both optical biosensor and microplate reader.

Since assays with a microplate reader showed that nickel, mercury, copper, and chromium are also inhibitors of C. vulgaris APA, [START_REF] Durrieu | A dialysis system with phytoplankton for monitoring chemical pollution in freshwater ecosystems by alkaline phosphatase assay[END_REF] these compounds are now tested with our optical and conductometric biosensors. The biosensor response depends on substrate concentration for a given concentration of heavy metal ions used as inhibitors. High substrate concentrations often reduce enzyme dependence upon substrate concentration. However, such concentrations cannot be used with enzymes that are inhibited by an excess of substrate, which interferes with the metal detection also based on enzyme inhibition. An optimal substrate concentration should then be determined from the activity -substrate concentration curve.

Although heavy metal ions also inhibit algal growth, [START_REF] Vasseur | Influence of some experimental factors on metal toxicity to Selenastrum capricornutum Toxic[END_REF] this inhibition does not interfere in this enzyme inhibition-based method because only the ratio of the peak areas in the presence and absence of heavy metals is concerned in the use of a single algal membrane.

Determination of Pesticides

Experiments were carried out with organophosphorous compounds to determine EA inhibition using conductometric biosensors. An EA inhibition of 30 -40% was observed with 100 ppb of methyl paraoxon with a detection limit of 10 ppb after a 30 min contact with the enzyme. These results have to be extended to other concentrations. Since a same algal biosensor can measure both APA and EA, a multi-detection system is possible.

Toxicity Assessment of a Contaminated Effluent

In most cases, inhibition of enzymes is not really specific. The activity of a same enzyme may be affected by various types of inhibitors which could be competitive or not depending on their chemical structures. [START_REF] Tran-Minh | Immobilized enzyme probes for determining inhibitors[END_REF] This is the case for APA and EA which are also inhibited by other contaminants [15 -17] and already been used to produce biosensors which could be potentiometric, [START_REF] Tran-Minh | Immobilized enzyme probes for determining inhibitors[END_REF][START_REF] Tran-Minh | Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase[END_REF] amperometric, [START_REF] Cremisini | Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor[END_REF] or optical. [START_REF] Ayyagari | Biosensors for pesticide detection based on alkaline phosphatase-catalyzed chemiluminescence[END_REF][START_REF] Garcı ´a Sa ´nchez | Free and sol-gel immobilized alkaline phosphatase-based biosensor for the determination of pesticides and inorganic compounds[END_REF] Therefore, biosensors using those enzymes are quite adapted for assessment of overall toxicity of waste water.

The detection limits of these optical and conductometric biosensors for heavy metals indicate that they are more suitable when those compounds are present in wastewater rather than surface water. Figure 5 gives an example of APA inhibition measured with an optical biosensor after injection of an effluent containing Cd 2þ or Zn 2þ in the FIA carrier flow. The presence of other toxicants in effluents could interfere. In this case, the biosensor reflects a disturbance of the algal metabolism and this could be used to monitor deleterious effects of pollutants on algal cells.

Experiments using conductometric biosensors were carried out with natural effluents from French or Vietnamese rivers to determine the percentage of APA inhibition. In both cases, APA was inhibited to at least 50%, which suggests that these effluents contained APA inhibitors. The presence of heavy metal ions has been confirmed with atomic absorption spectrometry.

As for EA, APA inhibition on algal cells has been found irreversible even after several washing of the algal membrane. However, it has been reported that purified APA could be reactivated with 10 mM EDTA. [START_REF] Conyers | The action of EDTA on human alkaline phosphatases[END_REF] When immobilized in the whole cells, this cannot be achieved and the membrane has to be changed after use. The biosensor can be used up to 20 days with 90% remaining activity.

CONCLUSION

Biosensors using enzymes present on the external membrane of the whole cells have potential advantages for determination of toxic compounds acting as APA or EA inhibitors. The enzymes remain in their natural environment which favours long-term stability and reflects the mechanism of toxic inhibition, being therefore of ecological interest. Algal AP is mainly sensitive to heavy metal ions while algal esterase is mainly sensitive to organophosphorus pesticides. If their activities are measured simultaneously, a multidetection of these compounds is possible. Such biosensors complement those based on photosynthesis inhibition designed for herbicide determination [START_REF] Naessens | Fiber optic biosensor using Chlorella vulgaris for determination of toxic compounds[END_REF][START_REF] Ve ´drine | Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides[END_REF] and those based on other transducers. Optical detection has the advantage of being insensitive to electrical disturbances while conductometric detection is quite suitable for a turbid medium.

Optical and conductometric algal biosensors appear to be alternative tools to monitor pollutants in aqueous media and thus be used as EWSs. Further EQ1: Please supply new art as this is incorrect (arrows in wrong position) investigations with different immobilised unicellular micro-organisms, such as bacteria or yeast, would also provide insights into the effect of pollutants on aquatic ecosystems.

Figure 1 .

 1 Figure1. FIA manifold using an algal biosensor for determination of algal APA.

Figure 2 .

 2 Figure 2. Response of an algal AP optical biosensor to MUP as substrate.

Figure 3 .

 3 Figure 3. Response of an algal esterase conductometric biosensor to AChCl as substrate.

Figure 4 .

 4 Figure 4. Comparison between micro-plate reader and optical biosensor responses to cadmium and lead concentrations in terms of percentage of inhibition (I%) of AP.

Figure 5 .

 5 Figure 5. FIA of algal APA inhibition with optical biosensor in the abscence (A) EQ and in the presence (B) of a toxic effluent in the carrier flow. MUP is injected in the flow as a substrate to produce the optical response.

Table 1 .

 1 APA inhibition rates with conductometric biosensor after exposure to Cd 2þ or Zn 2þ .

		10 ppb		100 ppb		1 ppm
		Cd 2þ	Zn 2þ	Cd 2þ	Zn 2þ	Cd 2þ	Zn 2þ
	30 min	15	,10	40	25	50	30
	120 min	60	60	100	90	nd	nd

ACKNOWLEDGMENTS

We greatly acknowledge CNRS (ACI) and Region Rhone-Alpes (Thermatiques prioritaires) for their financial support.


Ioannis Ignatiadis

Caroline Michel

Fabienne Battaglia-Brunet

Mireille Bruschi

Pierre Bianco

Elizabeth Lojou

Canh Tran-Minh

Development of an enzymatic amperometric biosensor using cytochromes C3 for the fast quantification of chromate bio-availability