Biocapteurs pour le contrôle de la toxicité des eaux : application des bioélectrodes algales Biosensors for monitoring pollutions of aquatic systems : applications of algal electrodes

Résumé

L'amélioration de la qualité de l'environnement passe par la réalisation de contrôles de toxicité in situ et en continu des sources de pollution ou des milieux contaminés, à l'aide de systèmes automatisés à réponse rapide. Les systèmes donnant une réponse en temps réel permettent d'intervenir immédiatement à la source, d'interrompre le rejet d'un flux toxique et de prévenir ainsi les accidents de pollution. Ce type de stratégie ne peut être développé qu'au moyen de biocapteurs : les méthodes d'essais conventionnelles n'autorisent que des contrôles de toxicité épisodiques, en laboratoire, effectués dans des conditions statiques quelque peu éloignées des conditions dynamiques.

Nous nous sommes intéressés à la mesure de l'activité photosynthétique d'algues unicellulaires immobilisées. La photosynthèse induite par des stimuli lumineux est en effet un processus dont la réponse est immédiate et aisément mesurable à l'aide de transducteurs électrochimiques. Il apparaît donc intéressant d'utiliser ces réactions photosynthétiques pour la détection des polluants.

Deux dispositifs mesurant l'activité photosynthétique d'algues unicellulaires ont été testés. Le premier dispositif mesure le transfert d'électrons le long de la chaîne photosynthétique lors d'une illumination des micro-organismes. Le second système permet de quantifier la production d'oxygène résultant de cette excitation lumineuse.

La mesure du transfert d'électrons photosynthétiques nécessite l'addition d'une substance oxydo-réductible (médiateur) dans le milieu pour capter ces électrons. De la série de médiateurs testés, seuls les dérivés à caractère lipophile (2,6-diméthylbenzoquinone et p-benzoquinone) ont permis de mesurer un transfert d'électrons. Toutefois la durée de vie de ce biocapteur s'est révélée limitée à moins de 24 heures, ce qui exclut toute utilisation en continu.

Le second dispositif développé présente en revanche une longévité d'une semaine, ce qui le rend intéressant en vue d'une utilisation in situ. Les performances de ce capteur à oxygène ont été testées sur des produits de type herbicides, cyanures, métaux et comparées aux valeurs obtenues à l'aide de tests algues classiques ou de méthodes de détection rapide de la toxicité.

Mots-clés:

• Biocapteur,
• pollution,
• chlorophycées,
• photosynthèse,
• électrode à oxygène

Abstract

Environmental monitoring of pollutants with automatic systems, applied on-line and allowing rapid response constitutes one of the most successful ways to improve the quality of the environment. Real time analysis offers the advantage of detecting rapidly sources of pollution and preventing any accidental release of pollutants. Such a strategy is possible only by means of biosensors : current methods, commonly used far toxicity testing are usually carried out in Laboratory in static conditions, making real lime analysis impractical.

Two types of amperometric environmental sensor incorporating eukaryotic algae were investigated for use in monitoring industrial pollution of aquatic systems. Both sensors allowed the monitoring of photosynthetic events.

The first sensor follows photosynthetic electron chain events within the cell resulting in the reduction of mediator acting as terminal electron accepter. Reoxidation of the mediator at the biosensor electrode surface rues in a flow of current, the magnitude of which is proportional to the level of photosynthetic activity of the microalgae.

In the second approach photosynthetic oxygen evolution by the illuminated biocatalyst is measured by reduction at a cathodic electrode. Enzymic systems associated with the water splitting and oxygen evolution are amongst the most fragile components of the photosynthetic apparatus, and the monitoring of algal oxygen production is therefore a useful approach to early detection of toxic environmental pollutants.

Several species of unicellular algae were used for these experiments : Chlorella vulgaris, Scenedesmus subspicatsus and Selenastrum capricomutum. Algal cultures were harvested in the exponential growth phase and diluted to 0.5 O.D. (655 nm); then 1 ml aliquots were centrifuged at 900xg for 3 min. After centrifugation, cells were resuspended in growth medium, LEFEBVRE and CZARDA (LC), and immobilized by aspiration onto a filter disc. This filter disc was placed onto the carbon working electrode surface. Filters were held in place by a fine nylon mesh.

This biosensor is a two electrode system comprising a carbon working electrode and Ag/AgCl reference/counter electrode. Solution was continuously flowed through the electrochemical cell at a flow rate of 2 ml min^-1. Illumination of the algal biocatalyst was supplied by light emitting diodes with a peak wavelength of 635 nm and a light intensity of 125 millicandellas. Periodicity of illumination was chosen in order to obtain a stable photosynthetic response.

Biosensors exploiting direct electron transfer from a biocatalyst to an electrical system are not feasible. Indeed, the tell wall of the biocatalyst act as a barrier to the exchange of electrons between the electrode and the redox intermediates oft the cell. Electroactive compounds (mediators) must be used to shuttle electrons from the photosynthetic electron transfer chain to the electrode. Mediators were added to the flowing solution of LC medium, and a potential of 550 mV applied at the working electrode to reoxidize mediator reduced by the biocatalyst. The mediator must be lipophilic to access the chloroplast electron transport chain of eukaryotic algae. We tested a wide range of mediators but only p-benzoquinone (p-BQ) and 2,6-dimethylbenzoquinone gave measurable responses.

A concentration of 0.2 mM p-BQ (21.5 mg/l) was employed to measure photosynthetic activity. Experiments showed that 15 minutes light period followed by a 15 minutes dark period gave a steady photosynthetic response. However, this high concentration of mediator was toxic for the cells. Static algal tests using Chlorella vulgaris have shown that growth is totally inhibited after 72 hours at a concentration of 5.4 mg/l. The working life of this sensor was therefore very short, less than 24 hours : after 16 hours of continuous monitoring, the recorded photosynthetic current was less than 20 % of initial response. Sensor life was not increased when the probe was used alternately with recovery periods in nutrient medium (4 hours of working period/4 hours of recovery period).

The same apparatus was used for the oxygen electrode based biosensor. The working electrode was coated with a Teflon gas permeable membrane to protect the sensor against poisoning by electrochemically active compounds. Separation of the working and reference/counter electrode requires addition of electrolyte in the flowing solution. With such a semi-protected oxygen electrode, mass transport controlled oxygen reduction currents were obtained when the Teflon covered cathode was poised at -700 mV.

The oxygen biosensor responded more rapidly than the mediated biosensor to changes in the light regime, and alternating light and dark periods of 1 min of light followed by 4 min of dark were used. The sensor also showed good long term stability, with a working life of up to seven days using Chlorella vulgaris or Scenedesmus subspicalus as biocatalysts.

The sensitivity of this oxygen electrode based biosensor was tested on herbicides (isoproturon, propanil and atrazine), cyanide and heavy metals (copper and mercury). Results were compared with chose obtained with three toxicity tests : a standard algal growth inhibition test, the inhibition of photosynthetic activity in spinach leaves and the alga Chlamydomonas reinhardii, and the Microtox test using the luminescent bacterium Photobacterium phosphoreum.

IC₅₀ obtained for isoproturon and atrazine were very similar for the growth inhibition and the oxygen sensor tests. The inhibition of oxygen production by spinach leaves was less sensitive to atrazine; no toxic affect could be detected with the Microtox test. The oxygen sensor was also very sensitive to cyanide but the response of the probe was quite different if Selenastrum capricornutum or Chlorella vulgaris was used.

The sensor allowed metals detection but this detection of toxicity was slow compared to that of herbicides or cyanide. Inhibition growth tests and Microtax test were more sensitive than the algal sensor for copper and mercury.

Keywords:

• Biosensor,
• pollution,
• chlorophycae,
• photosynthesis,
• oxygen electrode