Biofilm-based electrochemical sensor device for environmental conditions and method for the detection of environmental conditions using said device

The present invention relates to the detection of environmental conditions, particularly the detection of the growth of associations of micro-organisms adhered to surfaces (biofllm), as well as the use of the thus-detected information for eco-toxicological and technological purposes.

More specifically, the invention relates to a sensor device for environmental conditions in an aqueous environment, and a method for the detection of environmental conditions in an aqueous environment making use of said device.

In the last decades, environmental monitoring gained an increasing strategic importance for the implementation of environmental and territory management systems.

Environmental management requires, besides the specific fundamental scientific knowledge, operative tools that can be easily used by the operators in the field, which are able to provide, in a relatively short period of time, integrated responses about the contamination and pollution levels and the ecological consequences on the different compartments of the ecosystems involved.

Given the high number of the potentially toxic substances known (over 10,000), the continuous chemical analysis of the individual substances results to be impracticable.

Therefore, a series of toxicity tests based on the observation of the effects on organisms exposed to a toxic environment (bioassays) has been standardized. Such bioassays provide an integrated datum on the potential toxicity of the substances and/or environmental samples on the aquatic compartment.

However, the toxicological tests on the organisms, particularly the aquatic ones, require very long observation times. Several months are often needed, and also years, in the case of

chronic toxicity, to get meaningful responses.

In this context, in the field of the water resources, many efforts have been made by the scientific community to develop innovative investigation tools capable of meeting the operative needs set forth above. Lately, investigation tools capable of combining biological events of high ecological relevance with biosensor devices with a high technological content, capable of making the application thereof easier, have raised high interest among the environmental operators.

Particularly, hybrid biosensors are known, which use both non-cell (macromolecular) and cell constructs, integrated with a wide range of transducer devices.

The highly specific biological reaction occurs, for example, between an enzyme and the device substrate, between an antibody and the corresponding antigen, between a receptor and the corresponding hormone.

Such devices carry out easily interpretable diagnoses and real-time measurements, with minimum dimensions.

The broad spectrum of reactions employed, and the high sensibility and selectivity, make such biosensors potentially suitable for multiple application fields. However, the high specificity thereof results to be a drawback in systems the object of which is to provide the fastest possible signalling of non-specific environmental contamination events (BEWS, Biological Early Warning Systems), hi fact, due to the non-specificity of the contamination searched for, the simultaneous use of a high number of different specific sensors of the above-mentioned type would be required, which, thus far, make these technologies unfeasible in the sector of the in-field environmental monitoring.

In the environmental field, eco-toxicology kits are known and used thus far, based on su- percellular level (organisms) transducers adapted to provide a more integrated response, since it comes from an alteration at the level of the whole organism and/or population.

Such kits base their functioning on the measurement of physiological parameters of an individual species (e.g., crustaceans, bio-luminescent bacteria, bivalves, etc.), making the responses not necessarily representative of complex ecosystems. Furthermore, while ensuring the carrying out of rapid and easily interpretable toxicological tests, they generally do not allow an on-line acquisition of the responses.

The present invention has as its object the provision of a biosensor capable of operating in any aquatic environment, and adapted to provide information in a continuous manner about the potential environmental toxicity, avoiding the drawbacks of the prior art.

According to the present invention, that object is achieved thanks to an electrochemical sensor device having the characteristics reported in claim 1.

It is a further subject of the invention a detection method having the characteristics reported in claim 12.

Particular embodiments are the subject of the dependant claims, the content of which is to be meant as an integral and integrating part of the present description.

In brief, the present invention is based on the principle of employing an electrochemical sensor capable of monitoring the development of a biofilm for the detection of environmental conditions that are favourable or unfavourable to the development thereof, deriving from this measurement information that are useful, for example, for the detection of eco- toxic events.

It is known that, in a natural environment, the micro-organisms tend not to remain as isolated, individual cells, but to organize into true communities (films, flocculates, and biological wastewater), distributing themselves ubiquitously, both in a terrestrial and an aquatic environment. These aggregates grow and develop, colonizing any available surfaces, both natural and artificial, adapting to even extreme environmental conditions.

The term biofilm is commonly used to indicate all these types of microbial association, independently from the biological structuring and the environment in which they form.

Therefore, the biofilm can be defined as an organic matrix formed by not very water- soluble extra-cellular bio-polymers (EPS, extracellular polymeric substances), in which micro-organisms live and reproduce, which follow one another over time, and compete until establishing,' in a dynamic balance, a biological layer which, over time, is intended to cover any non-toxic surface exposed to the environment. Therefore, the biofilm formation is a spontaneous phenomenon in any natural environment.

The biofilm growth and build-up at the solid-liquid interface is an event that, following an initial, relatively slow, triggering phase, then proceeds quickly, in an almost exponential way, until reaching a pseudostationary final level. In seawater, for example, the biofilm- colonizing pioneer micro-organisms (mainly bacteria) start the surface colonization already after a short immersion period (minutes-hours), but periods of time of the order of some days or weeks are needed (in accordance to the temperature and the immersion environment) for the specific biodiversity to increase and the biofilm layer to become already visually observed.

Studies initially performed in the field of the microbially influenced corrosion (MIC) showed that the biofilm formation on determined metal surfaces exposed to natural waters causes an alteration of the kinetics of some reactions evolving on the metal surface, which variations can be detected by electro-chemical techniques.

Biofilm sensors based on this effect are marketed (Bio-George, Biox, Biogard, ...), each of which utilizes a specific electro-chemical technique, always applied in the same mode during the operation. Such sensors provide information about the biofilm growth, in real time, on-line, with high sensibility and, moreover, they do not need frequent maintenance operations.

By observing the biofilm development (growth and/or destruction) in real time, in situ, and

with high sensibility, it is possible to point out a possible alteration or abnormality of the development thereof, - also known or, anyhow, predictable under standard conditions - which is an indication of phenomena that are adverse to the normal development cycle (toxicological end-point), for example, because it is induced by the presence of toxic substances in the aquatic environment.

Specifically, the sensible active element is represented by the association of a metallic substrate and a biological micro-ecosystem (biofilm) comprising a community of microorganisms that spontaneously forms in any aquatic environment, and the growth of which can be spontaneous or conditioned.

The transduction is based on the measurement of the electrochemical activity of the biofilm present on a metal working electrode of the sensor.

Analysis methods based on intensiostatic or potentiostatic techniques are capable of providing real-time information about the development and growth degree of the microorganisms forming the biofilm relative to the total extent of the sensible surface. This datum is directly related to the biological activity of the same biofilm. Such activity is, in turn, strictly related to and affected by the possible presence of toxic substances in the environment and by the concentration thereof.

The sensor device can be used, according to an intensiostatic technique, through the application of a predetermined current and the reading of a voltage electric (more precisely, bio- electro-chemical) signal, i.e. a potential difference between the same electrode and a reference electrode, variable as a function of the development conditions of the biofilm belonging to the sensor and grown on the relative metallic substrate, preferably for biofouling control applications.

The sensor device can be used, according to a potentiostatic technique, through the application of a predetermined potential difference between the working electrode and a reference electrode and the reading of a current electric (more precisely, bio-electro-chemical) signal,

variable as a function of the development conditions of the biofilm belonging to the sensor and grown on the relative metallic substrate, preferably for applications of ecotoxicity conditions assessment.

Advantageously, by using a relatively short life cycle, such as that of the micro-organisms, it is possible to considerably reduce the analysis times, so as to be able to know in real time the toxicity level of an unknown sample, or to detect variations in the toxicity level of a given environment, also a natural one, so as to be able to promptly intervene, above all in the case of environmental alert problems.

Advantageously, the invention implements a new electrochemical sensor which combines the advantages of the already known electro-chemical sensors and the further advantages to be able to vary, also during the operations, the level of sensibility to the biofilm, to change the electro-chemical measurement technique according to the advantage suggested by a specific application, to give the sensible element the possibility to self-clean from the biofilm, thus restoring the sensor initial status, providing detailed information about the biofilm growth rate and the instantaneous development degree.

Suitably, the sensible element does not need specific preparations, and it produces itself directly in the investigation environment, with a high ecological relevance.

Since the sensible element (the biofilm) is formed by an heterogeneous assembly of microorganisms (bacteria, fungi, algae, and protozoa), it provides an integrated datum on the environmental toxicity toward a complex, autochthonous system (micro-ecosystem), which is an extremely important condition for a biological early warning system, unlike what occurs in other bio-sensors, in which there are responses relative to the individual species or an individual parameter.

The same nature of the measurement system of the bio-electro-chemical signal has characteristics of extremely reduced electric consumption, such as to allow the integration thereof into devices supplied by battery or via solar panels.

Finally, the reduced maintenance need due to the auto-maintenance characteristics of the biologically active layer (biofilm) allows to use such technology directly in-field, thereby obtaining a system that can be applied on a large geographical scale.

Therefore, the device according to the invention lends itself to the development of online monitoring and remote acquisition networks of the data indicative of the local environmental conditions monitored, by means of a plurality of biosensors suitably located in a given area to be monitored.

An early warning system based on the device according to the invention may be developed both for fresh and sea waters, exploiting the growth of the biofilm, which develops in both environments.

Compared to the prior arts, which are based on the use of individual species, the sensor of the present invention utilizes an heterogeneous community of spontaneously and ubiquitously present micro-organisms, the biofilm, in perfect balance with the local environment (ecosystem), therefore being representative thereof.

Further characteristics and advantages of the invention will be set forth in more detail in the following detailed description of an embodiment thereof, given by way of non-limiting example, with reference to the annexed drawings, in which:

Fig. 1 schematically shows a sensor device according to the invention;

Fig. 2 is a circuit block diagram of the device of Fig. 1;

Fig. 3 shows the trend over time of the oxygen reduction curve as a function of the surface covering factor of the working electrode by the biofilm; and

Figs. 4a and 4b show the trend of the potential at the working electrode as a function of the current and time, respectively.

hi Fig. 1, a sensor device according to the invention is shown, generally indicated with 10, which can be part of a sensor array in the implementation of an environmental monitoring system. The device 10 comprises a sensor element 12 formed by a first metal working elec-

trode 14, for example composed of a "passive" alloy, such as stainless steel, titanium, etc., adapted to be arranged in an aqueous solution A, and a biofllm layer 16 spontaneously grown on such electrode when it is immersed in the solution constituting the environment to be monitored. Along with the first electrode, a second electrode 18 is put in solution as a "counter electrode" and adapted to act as a "drain" or "source" of electrons, to impose a working current on the electrode 14.

A reference electrode 19, relative to which the measurement of the working electrode potential is defined, is also put in the aqueous solution.

In the case where the counter electrode 18 is made of a material of the "sacrificial anode" type (for example, zinc), the same counter electrode can be, in practice, concurrently used as a pseudo-"reference" electrode instead of the distinct reference electrode 19.

Generally, there are no particular constraints on the working electrode and the auxiliary electrodes design; therefore, the sensor shape and dimensions can be tailored to the particular environment in which it has to be used.

A programmable electronic control unit 20 (Fig. 2) is coupled to the electrodes of the sensor device, and it is arranged to control the operation of the sensor 10 in a potentiostatic or intensiostatic mode.

In the first case, the unit 20 is arranged to impose a preset constant potential to the working electrode, providing to this aim the required current, even if, due to the effect of the gradual biofllm formation, the required current varies over time.

In the second case, the unit 20 is arranged to impose a preset constant current on the working electrode by applying to this aim the required voltage, even if it is variable over time.

Furthermore, the unit 20 is adapted to perform the following functionalities:

- programming the current/voltage profile over time so as to modify the operative

modes of the device and the working conditions thereof, so as to autonomously perform and automatically carry out sequences of measurements at a fixed or variable current/voltage according to user-programmed or default protocols, for example, in order to locally interpret in real time the development of the biofilm activity and to suitably react on the basis of the set modes, alternatively:

- by sequentially operating at discrete current/voltage values;

- by operating at values of current/voltage which is variable over time with continuity;

- by supplying predetermined current profiles which are variable over time between the electrodes, in order to generate an environment favourable to the biofilm formation, for example, an alkaline reducing environment favourable to the formation of the extracellular exopolymers, and consequently favourable to the proliferation of the bacterial colonies forming the biofilm;

- by supplying predetermined current profiles which are variable over time between the electrodes, both in the anodic and cathodic form, in order to hinder or inhibit the biofilm formation or eliminate it, if it is already present, for example, through the creation of an acid oxidizing environment adverse to the biofilm survival.

The latter characteristic allows restoring the sensor status to an initial condition (reset function) in a very short period of time. Upon restoring the operative original mode, also the optimal conditions for the new settlement of the biofilm are restored.

Furthermore, the unit 20 is arranged to:

- modify, when desired or as programmed by the user, the value of the parameter imposed (potential for potentiostatic operation, current for intensiostatic operation);

- measure, with a predetermined or user-programmable sampling frequency, the dependant variable (current for potentiostatic operation, potential for intensiostatic operation);

- activate a self-cleaning function of the working electrode, consisting in supplying for a short period of time a strong anodic or cathodic current on the working electrode in order to hinder or inhibit the biofilm formation, or eliminate it, if it is already present. The

latter characteristic allows restoring the sensor status to an initial condition (reset function) in a very short period of time.

Furthermore, the unit 20 is arranged to convert the obtained measurements into digital format data, adapted to be transmitted to a remote monitoring station. For example, it is adapted to communicate the acquired and/or processed data, and each functional parameter, through heterogeneous field buses (RS485, canbus, proflbus, ethernet, GPRS, GSM, ISDN, etc.) through suitable protocols both on external event (query), and based on events generated by the detection of toxic substances, and on a regular and programmed basis.

In detail, with reference to Fig. 2, an architecture example of the control unit 20 according to the invention is shown. An analog front-end stage 30 includes, in the exemplary case of operation of the sensor in intensiostatic mode, a programmable current generator 32, adapted to produce a controlled flow of electric current to the counter electrode 18, and a sense amplifier 34, coupled to the working electrode 14.

A data acquisition stage 40 downstream the front-end stage 30 comprises a control device for the current flow 42 direction, and a digital/analog converter 44 adapted to convert into the analog format the information of current intensity determined at a microcontroller 50 of a data control and analysis logic unit 52.

The microcontroller 50 is adapted to receive the measurement data detected by the sense amplifier 34 through an analog/digital converter 46.

In the data processing chain, a communication stage 60 is arranged downstream the control and analysis unit 52 for the transmission of the measurement data or the control commands to/from a remote station (not shown), for example, through a GSM/GPRS modem, or the like 62, or on a local network 64. The communication stage 60 is interfaced with the data control and analysis unit 52 through a serial bus driver 70 (for the transmission via GSM/GPRS) or an Ethernet controller 72 of an interface stage 74.

In a preferred embodiment, in which a detection system is adopted, which comprises a plurality of sensor devices 10, the electronic control unit 20 can advantageously be a single one, and common for all the devices, and arranged for the independent control thereof, for example, according to predefined operative schemes. Suitably, in this case, the system comprises a plurality of sensor devices, each of which has a local biofilm development stage which is different from the biofilm development stage of the other devices.

The data processing criterion to obtain information about the biofilm development will be now illustrated with an example relative to the detection of the first development stages of a biofilm formed on surfaces exposed to aerated natural waters.

Studies carried out in the corrosion field have shown that the biofilm formation on active- passive alloys (such as, for example, inox steels and titanium alloys) exposed to aerated natural waters causes an alteration of the oxygen reduction kinetics on the above- mentioned alloys, therefore a variation in the shape of the curve which, in a potential- current diagram, describes the oxygen reduction kinetics (cathodic curve).

As schematically reported in Fig. 3, as the surface covering factor θ of the working electrode by the biofilm increases, there is therefore a gradual alteration of the oxygen reduction curve.

For the assessment of the electrochemical activity of the settled micro-organisms, the sensor device according to the invention is able to measure the shift over time of specific points of the cathodic curve selected as meaningful, and to correlate such shift to the biofilm development and growth on the working electrode.

If, for example, it has been chosen to operate the sensor in a potentiostatic mode (working electrode kept to a suitable constant potential), the current required to keep such potential fixed will increase over time strictly according to the biofilm growth (Fig. 3). Therefore, suitable algorithms can convert the current datum into a datum indicative of the biofilm covering, θ, of the working electrode.

Finally, Figs. 4a and 4b show the data processing criterion in the case where the sensor is set to operate under intensiostatic conditions (fixed cathodic current imposed on the working electrode).

As it shall be appreciated from the scheme of Fig. 4a, when a predetermined current value U is imposed, the working electrode potential remains constant until when the biofilm covering reaches, over time, a value θl such that the current ii is supported by a higher potential.

In Fig. 4b, the trend of the potential difference between the working electrode and the reference electrode is reported, by way of example, as a function of the time: the rapid rise of the potential above a base value indicates that a biofilm covering condition has been reached, corresponding to what has been shown in Fig. 4a.

It is possible to vary the imposed current value in order to modify the sensibility of the sensor to the biofilm. Biofilms covering surface percentages of the order of 1% are clearly detectable.

hi the case where the biofilm present on the working electrode contacts a toxic substance, there will be a progressive macroscopic alteration of the electrochemical activity thereof, and a degeneration of the bacterial colonies, as a function of the toxic substance concentration. The decay of the electro-chemical signal, following a classic dose/effect relationship, allows an easy indicization of the response through the classical methods used in ecotoxi- cology (e.g., median effective concentration, EC50).

Therefore, the operative simplicity of the biosensor according to the invention allows the development of an easily applicable tool capable of pointing out possible non-specific toxicity effects even occasionally present in the aquatic compartment by exploiting the growth inhibition of a natural community of micro-organisms.

Advantageously, the monitoring electro-chemical system according to the invention can be

also used for a number of other applications.

It can be used, for example, to:

- monitor the sterility degree of particular environments (for example, potable waters, conditioning plants, etc.),

- measure, also at a level of laboratory studies, the efficacy of xenobiotic substances, natural toxins, antibiotics, etc.

- assess the effects induced on the biofϊlm by biocide treatments intentionally performed in plants.

As regards the latter possible application of the sensor according to the invention, we observe what follows.

When the biofilm development affects materials used in any man-made artificial apparatus, as it is known, serious technological problems may arise, often with high economic impacts. The terms biofouling or microfouling do represent, in fact, a highlighting of a particular interference of the biofilm on human technological activities; the term biofouling comes from the terminology used in the field of the technologies dedicated to the thermal exchangers, where the term fouling generally indicates an undesired deposit of material on the surfaces, which can be of mineral, organic, or, as in our case, biological origin, and which can strongly reduce the plant yield.

hi fact, it is known that the formation of biofϊlms, for example, on the walls of heat exchangers, beside promoting the triggering and propagation of corrosive etches (Microbially Induced Corrosion, MIC), causes a lower thermal exchange efficiency and a higher expense in terms of power used to pump the cooling water; consequently, the high cost of the damage amenable to the biofilm makes the use of antifouling systems necessary, which are often based on the addition of biocides in the processing waters.

In order to optimize the treatments, and therefore to keep a high efficiency of the plant while minimizing the use of biocides, it would be useful to know in real time the effect on the biofilm

of the applied treatments: the electro-chemical sensors, to the family of which the sensor according to the invention belongs, well lend themselves to such use, exactly because they provide information on-line, in real time, with high sensibility to the biofilm and, moreover, they do not require particular maintenance operations.

It shall be apparent, the principle of the invention being the same, that the embodiments and the implementation details will be able to be widely varied relative to what has been described and illustrated by way of non-limiting example only, without for this departing from the scope of the present invention, defined by the annexed claims.