METHOD OF DBTERMIWING THE TOXICITY OF A SAMPLE FLOW, AND APPARATUS THEREFOR

According to a first aspect, the present invention relates to a method for determining the toxicity of a sample flow.

According to a further aspect, the invention relates 5 to an apparatus suitable for performing the method according to the invention. Such an apparatus can for instance be applied as a biosensor.

The use of a microbial fuel cell (MFC) as biosensor for the purpose of determining the biochemical oxygen 0 demand (BOD) is known from the prior art [1,2,3,4,5]. Such BOD biosensors make use of anodophilic microorganisms which can oxidize an organic substrate under anaerobic conditions by making use of an anode as electron acceptor. The electrons are then used at a 5 cathode for the reduction of oxygen. In such biosensors the metabolic activity of the anodophilic micro-organisms is correlated to the BOD. Since the anodophilic microorganisms use the anode as electron acceptor, their metabolic activity can be determined on the basis of the 0 current flowing between the anode and cathode.

In these MFC biosensors described in the prior art the number of Coulomb charges displaced between the anode and cathode is related to the metabolic activity of the micro-organisms in the fuel cell. An integration of the 5 charge displacement through time must hereby take place. This makes it possible to perform real-time measurements. The MFC biosensor known from the prior art is further described only as BOD sensor, and is not particularly suitable for measuring the toxicity of a sample flow. 0 This is because the sensitivity of the centre cannot be readily controlled.

The invention has for its object to provide a solution for at least one of the above stated drawbacks of the prior art. It has been found that the anode in an MFC biosensor can be regulated potentiostatically, and that such a potentiostatic regulation of the anode makes it possible to directly correlate the metabolic activity of the micro-organisms to the amperage of the current flowing in the system between the cathode and the anode. Similarly, potentiostatic regulation of the cathode is also possible. This is particularly useful when microorganisms are applied which are involved in the transfer of electrons from the cathode, as is the case in microbially assisted reduction of oxygen at a cathode. The inventors of the present invention have had the further insight that it is also possible through the potentiostatic regulation of the anode or the cathode to control the sensitivity of the biosensor. It has been found that this is possible by regulating the potential of the anode or the cathode to a predetermined value relative to the equilibrium potential of the oxidation reaction at the anode or the reduction reaction at the cathode. The background hereto will be further elucidated below.

Bond and Lovley [6] describe experiments in which an MFC with anodophilic micro-organisms is operated with a potentiostatic regulation of the anode. The described MFC is not applied as sensor, and the described experiments relate only to an MFC operated at a constant, relatively high regulated potential of the anode (+200 mV relative to an Ag/AgCl electrode at an equilibrium potential of - 0.42 V relative to the Ag/AgCl electrode). This regulated potential is here not related beforehand to the equilibrium potential of the anode system.

Bergel et al . [7] describe experiments in which in a fuel cell microbially assisted reduction of oxygen is applied at the cathode using a biofilm. The described fuel cell is not used as sensor and the experiments are

performed at a regulated potential of the cathode of between -0.1 V and -0.4 V relative to a standard calomel electrode (SCE) . The redox equilibrium potential of the cathode system lies at + 0.20 V relative to the SCE. This regulated potential is here not related beforehand to the equilibrium potential of the cathode system.

According to a first aspect, the present invention provides a method for determining the toxicity of a sample flow, comprising of: - providing a number of microbial fuel cells comprising a number of cathode compartments provided with a cathode, a number of anode compartments provided with an anode, which anode and cathode are electrically connected, and wherein an anode substrate fluid is present in the anode compartment comprising an oxidizable compound which can be oxidized at the anode, a cathode substrate fluid is present in the cathode compartment comprising a reducible compound which can be reduced at the cathode, an electrochemically active microbial population is present on at least one of the anode or cathode which is involved in the transfer of electrons respectively to the anode and from the cathode, and wherein means are further present for supplying a sample flow to at least a part of the microbial population; - supplying the sample flow to at least a part of the microbial population;

- determining the potential toxicity of the sample flow on the basis of a change in the metabolic activity of at least the part of the microbial population relative to a reference measurement on the basis of a change in the electric current flowing between the cathode and the anode .

The method according to the invention is characterized in that at least one of the anode or cathode is connected electrically to a reference electrode, the metabolic activity of at least the part of the microbial population is determined on the basis of

the amperage of the electric current between the cathode and the anode, and that at least one of the anode or cathode connected to a reference electrode is regulated potentiostatically to a regulated potential (V _p ) relative to this reference electrode such that the condition 0 > V _e -V _p > x = -500 mV is satisfied at any given moment for a potentiostatically regulated anode and/or the condition 0 < ^v _e ~ ^v _P ≤ y = +200 mV is satisfied for a potentiostatically regulated cathode, wherein V _e is the value relative to the reference electrode of the equilibrium potential of the oxidation reaction at the potentiostatically regulated anode and respectively the value relative to the reference electrode of the equilibrium potential of the reduction reaction at the potentiostatically regulated cathode. The condition 0 > V _e -V _p > x = -500 mV is satisfied if the regulated potential is a maximum of 500 mV higher than the equilibrium potential. The condition 0 < V _e -V _p < y = +200 mV is satisfied if the regulated potential is a maximum of 200 mV lower than the equilibrium potential.

The method according to the invention relates to determining the (potential) toxicity of a sample flow. It is known that changes in the metabolic activity of organisms and micro-organisms in particular can be used as indication of the presence of substances which have a negative effect on biochemical activity, and are thus possibly toxic. The method according to the invention can for instance be useful in the sample analysis of wastewater or the analysis of samples obtained from drinking water mains systems. Other applications will be apparent to the skilled person.

It should be understood that the method according to the invention cannot provide a definitive answer in respect of the toxicity of the sample flow. Further analysis of the sample flow may be necessary for this purpose. The method according to the invention is however particularly suitable for determining in simple manner

whether a toxic compound is potentially present in the sample flow, and whether further analysis hereof is necessary.

In the method according to the invention the metabolic activity of electrochemically active microorganisms is determined. In the context of the present invention electrochemically active micro-organisms are understood to mean micro-organisms which during the oxidation of a substrate can use an anode, either directly or via a redox mediator, as terminal electron acceptor, or can use a cathode (directly or via a mediator) as electron donor for the reduction of a substrate. Such micro-organisms are known to the skilled person. Anodophilic micro-organisms (which can use an anode as terminal electron acceptor) are for instance selected from one or more of Geobacter sulfurreducens, Geobacter metallireducens, Shewanella putrefaciens, Rhodoferax ferrireducens, including combinations thereof. A cathodic biofilm which can use a cathode as electron donor is described by Bergel et al. [7].

A microbial fuel cell is provided in the method according to the invention. The operation of a microbial fuel cell is known to the skilled person [1,2,3,4,5,6,8]. The skilled person will thus understand that a microbial fuel cell in a suitable form will comprise, among other parts, a number of cathode compartments provided with a cathode, a number of anode compartments provided with an anode, wherein the anode and cathode are electrically connected. Further present in the anode and cathode compartment is a fluid comprising respectively an oxidizable compound and a reducible compound as substrate. Electrochemically active micro-organisms will be further present at the anode and/or the cathode in an MFC. Anodophilic micro-organisms can thus be present at the anode and micro-organisms can be present at the cathode which are involved in the microbially assisted oxygen reduction. The skilled person will further

appreciate that in a working microbial fuel cell an electric current is generated between the cathode and anode as a consequence of the anaerobic oxidation of a number of substrates in the anode compartment making use of the anode as electron acceptor, and the reduction of a reducible compound at the cathode in the cathode department with electrons coming from the anode.

In order to ensure that the anode substrate fluid and/or cathode substrate fluid is refreshed/supplemented in the course of time, they can be fed to respectively the anode compartment and the cathode compartment. If feed of the anode substrate fluid and/or cathode substrate fluid is desirable and/or necessary, means suitable for this purpose are provided. Feed of anode substrate fluid and/or cathode substrate fluid possibly takes place together with the sample flow in respectively a mixed anode mixture or cathode mixture. Feed separately of the sample flow is however also possible, wherein mixing of the anode substrate fluid and the sample flow takes place to form an anode mixture in the anode compartment, or mixing of the cathode substrate fluid and sample flow takes place to form a cathode mixture in the cathode compartment.

The cathode and anode compartments can further be separated by a partition surface which is permeable to protons and which ensures that transport of protons is made possible, while further exchange of reagents is minimized. Although such a partition surface permeable to protons is highly desirable for a microbial fuel cell applied for the purpose of generating electric energy, such a partition surface is only optional in the present invention. This is because exchange of reagents is less critical in the present invention. Examples of proton- conducting materials which can be applied as partition surface are for instance cation-selective membranes such as Nafion® or alternatives thereto.

As known to the skilled person, anodophilic organisms can use a wide variety of organic compounds and mixtures of these compounds as nutrient substrate (oxidizable compound) . Examples of suitable nutrient substrates are lower (C1-C8) alcohols or lower (C1-C8) organic acids such as ethanol, propanol, acetic acid or lactic acid. The nutrient substrates can also be present in complex mixtures of organic' compounds, such as for instance a wastewater flow. Cathodic biofilms and the micro-organisms present therein are thought to be autotrophic, i.e. they can use CO ₂ as carbon source. The energy they need for their anabolic metabolism is obtained from the reduction of a substrate, such as oxygen, with electrons from the cathode.

In the method according to the invention a sample flow to be tested is supplied to at least a part of the microbial population. It must be appreciated that the sample flow can be fed to the microbial population separately of the anode substrate fluid or the cathode substrate fluid, or that it can be mixed here with the anode substrate fluid or the cathode substrate fluid. The (potential) toxicity of the sample flow is determined on the basis of the influence of the sample flow on the metabolic activity of the microbial population to which it is fed. The sample flow will generally be in the form of a fluid, such as a liquid.

The sample flow may or may not comprise an oxidizable and/or reducible substrate for the electrochemically active micro-organisms. In order to prevent fluctuations in the level of oxidizable and/or reducible compounds in the sample flow affecting the metabolic activity of the electrochemically active micro-organisms, the present invention provides a further preferred embodiment. In this embodiment the sample flow is mixed with the anode substrate fluid to form an anode mixture when the microbial population to which it is fed is situated in

the anode compartment (at the anode) , or it is mixed with the cathode substrate fluid to form a cathode mixture when the microbial population to which it is fed is situated in the cathode compartment (at the cathode) . It is ensured here that the oxidizable compound or the reducible compound is present in respectively the anode mixture or the cathode mixture in a quantity such that this oxidizable compound or reducible compound does not limit the metabolic activity of the electrochemically active micro-organisms to which the sample flow is fed.

The contribution of the anode substrate fluid or the cathode substrate fluid is preferably sufficient to ensure substrate-unlimited activity of the microbial population. This has the result that 100% variation in the quantity of any oxidizable or reducible substrate in the sample flow has no effect on the metabolic activity of the electrochemically active micro-organisms to which it is fed. This increases the chance of measured effects being caused by the presence of potentially toxic compounds.

If the sample flow is mixed with the anode substrate fluid, the oxidizable compound in the anode substrate fluid can for instance be acetate. The threshold value for acetate-unlimited activity of anodophilic micro- organisms in a system can for instance be determined by- increasing the acetate concentration in the absence of the sample flow at the highest regulated potential of the anode that is selected, until there is no further increase in the electric current. In the range where there is no further increase in the electric current, acetate as substrate will be non-limiting.

Acetate will generally already be non-limiting as substrate at a concentration of between 2-5 mM, as the skilled person will be aware. Also known to the skilled person will be the concentrations at which other oxidizable substrates, such as glucose, ethanol or lactic acid, are also non-limiting. These concentrations for

reducible compounds such as oxygen will also be known to the skilled person, or can be readily determined analogously to the description in the foregoing.

Compared to the anode substrate fluid or cathode substrate fluid with which it is mixed, the sample flow preferably comprises no biologically oxidizable or reducible compounds, or hardly any. An example of a sample flow with a low content of biologically oxidizable or reducible compounds is for instance drinking water. The volume of the sample flow relative to an anode substrate fluid or cathode substrate fluid with which it is mixed is preferably as great as possible. Suitable ratios for the mixture of sample flow : anode substrate fluid lie within the distribution of 500:1 to 5:1, such as 1000:1, 500:1, 100:1, 10:1. Suitable ratios for the mixture of sample flow with cathode substrate fluid lie within the same distribution.

In the method according to the invention the metabolic activity of the electroactive micro-organisms is determined on the basis of the amperage of the current flowing between the cathode and anode. In contrast to the prior art sensors, in the method according to the invention the metabolic activity of the micro-organisms is not correlated by determining the number of Coulomb charges displaced between the anode and cathode, this requiring that the current be integrated through time. Owing to the potentiostatic regulation of the anode, the amperage between the cathode and anode can instead be used directly to determine the metabolic activity of the anodophilic micro-organisms in the method according to the invention.

Potentiostatic regulation of an electrode is per se known from the field of electrochemistry. Various companies market devices suitable for this purpose, such as for instance Bank Elektronic - Intelligent Controls GmbH. Diverse suitable potentiostats are thus available to the skilled person for application in the present

invention. For the potentiostatic regulation of the anode or cathode, the anode or cathode is coupled by means of a potentiostat to a reference electrode. In the potentiostatic regulation of the anode the cathode will generally be applied as counter electrode. Conversely, in the potentiostatic regulation of the cathode the anode will generally be applied as counter electrode.

As reference electrode can be applied any reference electrode which it is apparent to the skilled person is suitable for application in the present invention, such as an Ag/AgCl reference electrode, a calomel electrode or a standard hydrogen electrode.

Means for determining the current between the cathode and the anode and the method of using these means are known to the skilled person. Use can for instance be made of an ammeter of a suitable type.

Since the metabolic activity of the electroactive micro-organisms can now be directly correlated to the amperage of the current between the anode and cathode, it is possible to perform real-time measurements and thus determine changes in the metabolic activity of the electroactive micro-organisms. These changes can be correlated to changes in the composition of the sample medium fed to the anode compartment or the cathode compartment, for instance as a consequence of the presence of a (potentially) toxic compound.

Similarly to their effect on other (higher) organisms, toxic compounds can inhibit the metabolic activity of electroactive micro-organisms, such as anodophilic micro-organisms. In order to determine the presence of the toxic substances, the metabolic activity of the electroactive micro-organisms can be correlated to a reference measurement. Because it is possible with the method according to the invention to perform real-time measurements, the reference measurement can be a measurement on a sample flow taken at an earlier time.

Real-time determinations can thus be carried out with the method according to the invention. Changes, particularly decreases, in the metabolic activity of the micro-organisms can be an indication of the presence of (potentially) toxic substances in a sample flow. In the case of for instance a fall in the metabolic activity of anodophilic micro-organisms the conversion of the oxidizable compound will decrease, whereby fewer electrons are relinquished to the anode and the amperage of the current between the cathode and anode decreases. Similarly, the amperage of the current between the cathode and anode will decrease if the metabolic activity of a cathodic biofilm decreases.

The metabolic energy available to electroactive micro-organisms is determined by the difference between the equilibrium potential of the oxidation reaction at the anode or of the reduction reaction at the cathode and the potential of the anode or the potential of the cathode. The equilibrium potential of the oxidation reaction at the anode is here equal to that potential of the anode at which current has not yet quite started to flow in the system in a steady state mode. This is because at this equilibrium potential the oxidation of the oxidizable compound at the anode is in equilibrium with reduction of the oxidized products at the anode, whereby no net reaction takes place at the anode.

Analogously herewith, the equilibrium potential of the reduction reaction at the cathode is equal to that potential of the cathode at which current has not yet quite started to flow in the system in a steady state mode. This is because at this equilibrium potential the reduction of the reducible compound at the cathode is in equilibrium with oxidation of the reduced products at the cathode, whereby no net reaction takes place at the cathode.

In order to influence the metabolic energy of the anodophilic micro-organisms, the composition of the anode

fluid or the cathode fluid can thus be changed in respect of the oxidizable compound or the reducible compound. Alternatively, the regulated anode potential or the regulated cathode potential can be changed. Change in the regulated potentials can be easily realized by varying the regulated potential of the anode or that of the cathode. A change in the regulated potential- also has the most direct effect, since when the oxidizable and/or reducible compound is changed other effects, such as transmembrane transport of the compound and induction of appropriate transport systems, can also be a factor here. In the method according to the invention the anode or the cathode is regulated potentiostatically to a regulated potential (V _p ) relative to the reference electrode such that the condition 0 > V _e -V _p > x = -500 mV is satisfied at any given moment for a potentiostatically regulated anode and/or the condition 0 < V _e -V _p < y = +200 mV is satisfied for a potentiostatically regulated cathode, wherein V _e is respectively the value relative to the reference electrode of the equilibrium potential of the oxidation reaction at the potentiostatically regulated anode or the value relative to the reference electrode of the equilibrium potential of the reduction reaction at the potentiostatically regulated cathode. The anode or the cathode are preferably regulated potentiostatically relative to the reference electrode such that in the above relation the limit x has a value of -450 mV, -400 mV, -350 mV, -300 mV, -250 mV, -200 mV, -150 mV, -100 mV, -90 mV, -80 mV, -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, -20 mV, or -10 mV and/or the limit y has a value of +150 mV, +100 mV, +90 mV, +80 mV, +70 mV, +60 mV, +50 mV, +40 mV, +30 mV, +20 mV, or +10 mV. At these values of the potential difference the electrochemically active micro-organisms have relatively little metabolic energy available. This results in a sensor with a high sensitivity.

Without wishing to be bound by this theory, it is thought that micro-organisms which have much metabolic energy available have more possibilities for neutralizing toxic compounds. A sample flow with a relatively low toxicity level can thus already have an effect on microorganisms which have little metabolic energy available, while micro-organisms with much metabolic energy are possibly hardly influenced. A sample flow with a relatively high toxicity can on the other hand affect the metabolic activity of micro-organisms which have little metabolic energy available as well as micro-organisms which have much metabolic energy.

According to a preferred embodiment of the method according to the invention, the potentiostatic regulation of the anode or the cathode relative to the reference electrode, within the given values of 0 > V _e -V _p > x = -500 mV for the anode and/or of 0 < V _e -V _p < y = +200 mV for the cathode, takes a minimum of 15 minutes, such as a minimum of 30 minutes, preferably a minimum of 60 minutes, such as a minimum of 120 minutes, more preferably a minimum of 5 hours, such as a minimum of 10 hours. If the measurement of the sample flow continues for more than a 24-hour period, these times are preferably employed per 24-hour period. It is generally sufficient during these periods of time to perform measurements on the sample flow at a sensitivity related to the given potential differences at the anode and/or cathode. Recommended here however in the case of measurement for a long period is a sensitivity related to the given potential differences. Most preferably therefore, the condition 0 > V _e -V _p > x = -500 mV for the anode and/or 0 < V _e -V _p < y = +200 mV for the cathode is satisfied continuously.

As is apparent from the above, the extent to which the micro-organisms are influenced by a sample flow at different metabolic energy levels can be used as a measure for the toxicity of the sample flow (which is determined for instance by the concentration of a toxic

compound that is present or the extent of toxicity of a compound that is present) . In a preferred embodiment of the method according to the intention the regulated potential of the anode or the cathode is therefore varied in time. The metabolic energy available to the electroactive micro-organisms is hereby varied. By- varying the metabolic energy of the electroactive microorganisms (for instance by varying the potential of the anode if anodophilic organisms are used) , a qualitative and possibly even (semi-) quantitative indication can be obtained of the toxicity of the sample flow that is being measured. It should be noted that, when the regulated potential of the anode is varied in time, the potential difference between the equilibrium potential of the oxidation reaction at the anode and the regulated potential of the anode can at a determined moment exceed the above stated condition of 0 > V _e -V _p > x = -500 mV. The fact that said potential difference amounts at any given moment to between 0 and -500 mV does not of course preclude the possibility of this potential difference being more negative than -500 mV at another moment. With variation of the regulated potential of the cathode it is also the case that the potential difference between the equilibrium potential of the reduction reaction at the cathode and the regulated potential of the cathode can be greater than +200 mV at determined points in time. As the skilled person will appreciate, the potential of the anode and/or cathode can be regulated by changing the current or the resistance in the system. An alternative method of determining the effect of the sample composition on the micro-organisms at different metabolic energy levels forms part of a further preferred embodiment of the invention. In this embodiment there is provided a plurality of microbial fuel cells, of which the regulated potentials of the anodes or the cathodes differ at any point in time. By supplying the anode or the cathode compartments with the same sample

flow the effect of the sample composition on the microorganisms can thus also be determined at different metabolic energy levels. It is possible here for different (separated) anode compartments or cathode compartments to be supplied with the same sample flow, but also for a plurality of anodes or cathodes to be present in a single anode compartment or cathode compartment. It is recommended that measures are taken which ensure that the sample flow 'seen' by the different anodes or cathodes have an identical composition. This is possible for instance by causing the sample flow to flow in parallel flows to the different anodes or cathodes. In this preferred embodiment the regulated potential of the anodes or cathodes can vary in time as well as being constant in time. It should be noted that in this embodiment the potential difference between the regulated potential of a number of anodes and the equilibrium potential of the oxidation reaction taking place thereon may also lie outside the above stated range of 0 to -500 mV. The fact that said potential difference lies at any given moment between 0 and -500 mV in one of the MFCs does not of course preclude this potential difference lying outside this in one or more of the other MFCs. This is also the case for the potential difference between the equilibrium potential of the reduction reaction at the cathode and the regulated potential of a number of cathodes .

It is particularly in this embodiment that it is possible to vary the potential difference between different anodes or cathodes and the respective oxidation reactions or reduction reactions taking place thereon by a variation in the composition of respectively the anode substrate fluid or cathode substrate fluid. This can be done specifically by making a variation in the oxidizable compound or the reducible compound between the individual MFCs.

According to a further preferred embodiment of the method according to the invention, the equilibrium potential of the oxidation reaction at the anode lies between +100 and -600 mV, preferably 0 and -500 mV, more preferably -100 and -500 mV, most preferably -200 and -450 mV relative to an Ag/AgCl electrode.

According to yet another preferred embodiment of the method according to the invention, the equilibrium potential of the reduction reaction at the cathode lies between 0 and +1000 mV, preferably +100 and +800 mV, more preferably +200 and +700 mV relative to an Ag/AgCl electrode.

The redox equilibrium potential of the oxidation reaction at an anode can be determined in simple manner by reducing the potential of the anode in an MFC in a situation in which the oxidation substrate is non- limiting. The potential at which current has just ceased to flow in the system is equal to the equilibrium potential of the oxidation reaction taking place at the anode.

As the skilled person is aware, the equilibrium potential can also be determined theoretically for well- defined media.

The method according to the invention can be applied within a wide temperature range. All that is important is that the applied temperature allows microbial activity and free flow of the sample flow and/or an optionally used anode mixture and/or cathode mixture. The method according to the invention is thus performed at a temperature above the freezing point of the sample flow and/or an optionally applied anode mixture and/or cathode mixture. The upper limit of the applied temperature is determined by the temperature tolerated by the applied micro-organism. Micro-organisms are known which can grow up to a temperature of 120 ^" C (at increased pressure). The method according to the invention can thus be applied at

0-120'C, preferably 5-80'C, more preferably 5-60'C, most preferably 5-45 ^' C.

The invention further relates to an apparatus suitable for performing the method according to the invention. The apparatus can for instance be applied as a biosensor and comprises:

- a number of microbial fuel cells comprising a number of cathode compartments provided with a cathode, a number of anode compartments provided with an anode, wherein at least one of the anode or cathode is suitable for accommodating an electrochemically active microbial population which can be involved in a transfer of electrons respectively to an anode and from a cathode;

- means for supplying a sample flow to at least a part of the microbial population;

- means for determining a change in the electric current flowing between the cathode and the anode relative to a reference measurement.

The apparatus according to the invention is characterized in that at least one of the anode or cathode is connected electrically to a reference electrode, that the apparatus further comprises means for potentiostatic regulation to a regulated potential of at least one of the anode or cathode connected to a reference electrode, and the means for determining a change in the electric current between the cathode and the anode relative to a reference measurement are further adapted to determine a change in the amperage of the electric current between the cathode and the anode. The above technical features of the apparatus according to the invention and of the preferred embodiments thereof will be apparent to the skilled person from the foregoing description of the method according to the invention. The invention is now further elucidated on the basis of the following figures and the accompanying examples,

which illustrate non-limitative embodiments of the invention.

Figure 1 shows schematically how at the anode (A) of a microbial fuel cell organic material (OM) , present as oxidizable compound in the anode fluid, is oxidized anaerobically together with water to form CO ₂ and protons due to anodophilic micro-organisms. The electrons which are here released are relinquished by the anodophilic micro-organisms to anode (A) and flow via a potentiostat (2) to cathode (C) . At cathode (C) the electrons are used for the reduction of oxygen together with protons to form water. The charge balance in the system is maintained in that protons can flow through the proton-conducting material (1) of the anode compartment to the cathode compartment.

Also coupled to potentiostat (2) is an Ag/AgCl reference electrode (3) which is placed in the vicinity of anode (A) in the anode compartment. Potentiostat (2) holds the potential of anode (A) at a set value relative to the potential of the reference electrode. The potential of the anode is regulated to a potential of - 300 mV relative to the Ag/AgCl electrode. The equilibrium potential of the oxidation reaction at the anode is -420 mV relative to the Ag/AgCl electrode. V _e - V _p = -420 - -300 is here therefore -120 mV. It is for instance possible here for the regulated potential of the anode to be varied in time from -415 to -300 mV, but also for instance from -415 to 100 mV relative to the Ag/AgCl electrode. The condition 0 > V _e - V _p ≥ -500 mV will here also be satisfied for a large part of the range (within the range of -415 mV < V _p < +80 mV) .

Owing to the potentiostatic regulation of the anode it is possible to use the momentary amperage in the system as a measure for the metabolic activity of the anodophilic micro-organisms at the anode. The amperage can be measured in simple manner using an ammeter (4) incorporated in the electrical system. The measurement

values from ammeter (4) can be transmitted via a data line (not shown) to a data-processing device (not shown) , such as a programmed microcomputer. This can compare momentary values of the measurement to a value of a reference measurement.

Experiment

- Construction of the reactor

The reactor (figure 2) is made from plexiglass, the electrodes from graphite and the screws from nylon. The reactor has two parts, the anode side and the cathode side. Two graphite plates impregnated with epoxy resin in order to make them airtight act as electrode with a surface area of 22 cm ² . Reference electrode (8) is used to measure or to monitor the anode potential. The anolyte and catholyte are separated from each other with a cation exchange membrane (Fumasep FKS, Fumatech, Germany) . Anolyte: medium with acetate as substrate for the bacteria, catholyte: potassium ferricyanide as electron acceptor. The anolyte and catholyte have a volume of 33 ml.

A potentiostat controls the anode potential or the cell voltage or the current in the reactor. The temperature is controlled with a water jacket (6).

- Medium composition

0 . 816 g/1 NaC ₂ H ₃ 0 ₂ * 3H ₂ O, 0 . 74 g/1 KCl , 0 . 58 g/1 NaCl , 1 . 36 g/1 KH ₂ PO ₄ , 1 . 74 g/1 K ₂ HPO ₄ , 0 . 28 g/1 NH ₄ Cl , 0 . 01 g/1 MgSO ₄ * 7H ₂ O, 0 . 1 g/1 CaCl ₂ *2H ₂ O and 1 ml /1 of trace element mixture [ 9 ] .

- Start-up of the sensor

A mixed culture of micro-organisms is introduced into the reactor, in the anolyte. This inoculum comes from experiments with biofuel cells. Anode (7) and cathode (9) are connected to each other via a fixed resistance (5) of 1000 Ohm. After several hours a biofilm forms on anode

(7). The potentiostat does not yet control anything during start-up. After a time a cell voltage will occur and this increases to a constant value. Once the cell voltage is constant, the electrodes are connected to the potentiostat.

Due to the constant addition of medium with a fixed composition a biofilm will be created after a time which produces a constant electric signal (anode potential or current) . The electric signal will change due to the addition of a toxic substance. This change may for instance give cause to set off an alarm.

The results of adding MTBE are shown in Figure 3. The results show the effect of adding MTBE on the current production by the micro-organisms. The moment of addition of MTBE in the experiment is indicated in the figure by the arrows .

The results of adding potassium ferricyanide are shown in Figure 4. The results show the effect of adding potassium ferricyanide on the current production by microorganisms .

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