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Continuous measurement of acute toxicity in water using a solid state microrespirometer
Sensors and Actuators B 126 (2007) 515–521
Continuous measurement of acute toxicity in water using a solid state microrespirometer Fco. Javier Del Campo a,∗ , Olga Ordeig b , Nuria Vigu´es c , Neus Godino b , Jordi Mas c , Francesc Xavier Mu˜noz b a
Institut de Biotecnologia i Biomedicina (IBB-UAB), Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain b Instituto de Microelectr´ onica de Barcelona (IMB-CNM), CSIC (Esfera UAB), Campus Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain c Departamento de Gen´ etica y Microbiolog´ıa, Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain Received 19 January 2007; accepted 29 March 2007 Available online 4 April 2007
Abstract This work presents the first true solid-state microrespirometer. It consists on a naturally developed biofilm of Pseudomonas aeruginosa over a Nafion modified array of gold microdisc electrodes. Such a device can be used to monitor acute toxicity in water streams based on the oxygen reduction current recorded at the microelectrode array. When a healthy biofilm is exposed to a toxic stream the bacteria conforming stop breathing which results in an immediate increase of the oxygen supply reaching the microelectrode array. The fabrication and use of such a microrespirometer is described, and a possible operation mode for a real application is presented. Given that different waters may contain toxic compounds of diverse nature, the respirometer presented here provides qualitative information only. © 2007 Elsevier B.V. All rights reserved. Keywords: Microrespirometry; Ultramicroelectrode arrays; Oxygen reduction; Linear sweep voltammetry; Biofilm
1. Introduction This work presents the development of a new solid-state microrespirometer for the continuous monitoring of acute toxicity in drinking water supply lines. Toxicity of drinking water may be determined by different means. Amongst these, the use of assays based on the response of fish [1], algae [2], or even bacteria is commonplace. Such assays diagnose toxicity through the observation of alterations in the physiology of the subject organisms [3]. Biofilms are aggregates of bacteria, which spontaneously associate on top of surfaces in order to improve their survival rate in certain environments [4,5]. In this work, stable biofilms were grown from a culture broth on top of the electrode system. The growth process takes between 10 and 15 days, after which biofilms remain stable. Because the oxygen consumption of biofilms is heavily influenced by environmental parameters, par-
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Corresponding author. Tel.: +34 93 594 77 00x1313; fax: +34 93 580 14 96. E-mail address: [email protected] (Fco.J. Del Campo).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.03.038
ticularly by the presence of nutrients and toxics, their respiration rate may be used as an indicator of medium toxicity. The method described here has its foundations in the amperometric monitoring of oxygen [6]. The idea of monitoring biofilm respiration using oxygen electrodes is not new [2]. Individual microelectrodes have been used in the past to study oxygen concentration profiles within a biofilm [7]. The novel of our approach is that a biofilm is allowed to grow spontaneously on top of an array of ultramicroelectrodes and measuring the oxygen flux through it, thus achieving a true solid-state microrespirometer. This is in contrast to past micro-respirometry work where cells have been trapped inside a filter membrane which is later placed in the vicinity or in close contact with a Clark oxygen electrode [7,8]. This work shows how a biofilm based micro-respirometer device enables the semi-continuous monitoring of acute water toxicity. All measurements involving biofilms were performed in a carbon source free environment. Thus, in the absence of nutrients, a biofilm behaves purely as a diffusion barrier because the biofilm does not require a significant amount of oxygen for its survival. However, as soon as nutrients are available, oxygen
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consumption shoots up as a result of bacterial metabolic activity. The rate of oxygen consumption is affected by the presence of toxic compounds which, if in sufficiently high concentrations, can kill a very large fraction of the bacteria forming the biofilm [4]. In spite of this, it may take several hours to kill all bacteria within a mature biofilm because the biocide is neutralized by reaction with biofilm constituents faster than it diffuses into the biofilm [9] (sic.). Thus, when bacteria are forming a biofilm they become much more resistant to antibiotics and environmental changes, but they can nevertheless be killed by toxics such as bleach or formaldehyde. This can actually be turned to our advantage. The fraction of cells poisoned, which are expected to be the most superficial ones, eventually dislodge from the biofilm as the inner cells multiply and promote the spontaneous regeneration of the biofilm. The implication is that the same biofilm modified electrode may be employed for very long time in spite of its being placed in contact with a disinfecting agent. All in all, the system is able to provide BOD information, or to serve as a toxicity indicator, the results being expressed as percent inhibition of respiratory activity. The device presented here can be used for toxicity control in raw water sources for human use, and also in the management of wastewater treatment plants. 2. Experimental
pared using high purity deionised water of resistivity not less than 18 M cm. Solutions containing dissolved oxygen were achieved by means of an in-house-built system consisting of air and nitrogen lines which flow could be controlled by means of individual gas rotameters. Backflow stopper valves (Swagelock) were used to avoid pressure unbalances, which could lead to contamination and erroneous gas flow readings. The experimental set-up is schematised in Fig. 1. A bioreactor for biofilm formation has been designed. A pure culture of Pseudomonas putida was grown in a continuous culture with controlled oxygen supply at 37 ◦ C. The bacteria grew in a minimum medium—a medium only composed by salts and glucose as the nutrient, to control their growth [10]. In this case, Minimum Medium AB (MMAB) was prepared as indicated in the literature [11]. Biofilms developed vigorously on the solid-state microrespirometer surface. All electrochemical measurements were carried out using a -Autolab III (Eco-Chemie, The Netherlands) connected to a PC computer using version 4 of the GPES software for Windows® . All measurements were performed using a three-electrode cell configuration. The reference electrode consisted of an Ag/AgCl (3 M KCl) from Crison. A Pt ring electrode, also from Crison, was used as auxiliary electrode. The pH of the various solutions was monitored using a Hanna Instruments HI-300 pH meter. Oxygen concentrations were measured using an optical HACH HQ10 oxygen handheld probe with temperature control.
2.1. Reagents and instrumentation 2.2. Electrode preparation The following reagents were ACS analytical grade and were used as received from Sigma–Aldrich without any further purification: KCl (99%), K4 Fe(CN)6 ·3H2 O (99%), Nafion 117 solution (5% in lower aliphatic alcohols), formaldehyde (36.5–38% in H2 O) and d-glucose (>98%, Fluka). Sterile saline solutions were purchased from Sigma–Aldrich and used as received without further purification. All solutions were pre-
The Au micro disc arrays used in this study have been described previously [12]. They were made up by 256 discs, 10 m in diameter, and arranged under a cubic distribution with an inter-centre distance of 100 m. Because not all the electrodes of an array are active, the actual number of discs for each individual array used in the study has been determined following
Fig. 1. Diagrammatic representation (not to scale) of the experimental set-up. Two back-flow stopper valves after the rotameters allow controlled mixing while avoiding contamination of the gases. WE represents the biofilm oxygen sensor, the reference (RE) and counter (CE) electrodes are also depicted.
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the approach described in [13]. Oxygen free, 1 mM ferrocyanide in 0.1 M KCl solutions were used for the voltammetric characterisation of each of the arrays used in this study.
neutral/basic pH):
2.2.1. Nafion modified arrays In order to avoid the fouling of the electrode as a result of direct contact with the bacteria, the microelectrode array was protected by a spin coated thin layer of Nafion. The coating was carried out in a POLOS MCM-TFM spinner in a class 100000 clean room. Individually mounted chips were placed in the spinner and 10 L of the Nafion solution was pipetted on top of the chip. Next, the array was spinned for 1 min at 800 rpm and with an initial acceleration of 1000 rpm2 . This operation was repeated three times with waiting intervals of 3 min to allow evaporation of the ethanol. Surface topology of the Nafion layer was characterised in three stages by perfilometry using a KLA-Tencor P15 perfilometer controlled by Profiler v7 software run on Windows XP. First, a profile of the clean and bare microelectrode array was obtained and used as the baseline. Next, the Nafion was deposited as describe above and left to cure for not less than 60 min. The deposited layer is so thin that 60 min is time enough for the solvent to evaporate. Finally, the Nafion membranes were hydrated by immersion in water for another 60 min, after which a new profile measurement was performed. The Nafion layer (unhydrated) was ca. 300 nm thick and it homogeneously followed the ultramicroelectrode array profile. Once hydrated, the Nafion layer reached a thickness of ca. 450 nm, which represents a 50% volume increase.
Although we did observe two waves, all the currents used in the construction of calibration plots or the monitoring of oxygen corresponded to the complete process; the four-electron reduction. Unless otherwise stated, all the currents quoted in this work were measured at −1 V versus Ag/AgCl (3 M KCl). First, the response of the gold microelectrode arrays to oxygen was evaluated. It is worth noting that the oxygen signal measured at −1 V versus Ag/AgCl is not affected by pH changes in the range 6 < pH < 8.5, as reported elsewhere [15]. The procedure is described next. Following the electrochemical activation and characterisation of the arrays, the voltammetry of dissolved oxygen was studied. A stream of air was bubbled through 25 mL of 0.1 M Na2 SO4 at pH 5.5 to ensure a constant and controlled level of dissolved oxygen. Moreover, the concentration of oxygen was measured before and after the electrochemical measurements using a HACH optical oxygen probe. Fig. 2a shows a typical voltammogram of oxygen at a 256 ultramicroelectrode array. The linearity of the electrochemical response was checked by bubbling a series of nitrogen/air gas mixtures through the electrolyte solution. Fig. 2b demonstrates that the sensor response is highly linear (unmodified electrode: 0.095 ± 0.013 A ppm−1 , 0.011 ± 0.007 A; LOD3 = 0.29 ppm, R = 0.999, n = 8; modified electrode: 0.070 ± 0.018 A ppm−1 , 0.035 ± 0.01 A; LOD3 = 0.39 ppm, R = 0.998, n = 7) over the range of 2– 8.5 mg L−1 . In general, these curves were recorded starting from the highest concentration and proceeding towards lower concentrations of oxygen. For each point, the gas mixture was allowed to bubble through the solution for no less than 15 min in order to achieve equilibrium. This gas system was used in combination with the HACH optical oxygen probe to verify the
2.2.2. Biofilm modified electrodes Bare and Nafion modified arrays were immersed in a culture as described above for several days (over a month in some instances). Every morning the chips were extracted from the broth in order to measure the oxygen reduction current from an oxygen saturated saline solution. Although it was possible to observe the formation of biofilm on top of the electrodes by sight after the second day, the electrochemical measurements did not show a significant current change on addition of glucose until after the 5th day. By the end of the study, some 30 days later, the chip and its PCB substrate were completely covered in thick biofilm presenting a bone-yellow colour. Typical biofilm thicknesses ranged from 15 up to 60 m, depending on ageing, as determined by confocal microscopy.
O2 + 2e− + H2 O → HO2 − + OH− HO2 − + 2e− + H2 O → 3OH−
(2)
3. Results and discussion 3.1. Amperometric oxygen determination The overall reaction for the reduction of oxygen, in a neutral to basic environment is: O2 + 4e− + 2H2 O → 4OH−
(1)
In reality, the actual mechanism is rather complex [14] and a detailed discussion of it is out of the scope of the present work. However, it is often possible to discern not a single fourelectron wave, but two related to the following sequence (under
Fig. 2. (a) Linear sweep voltammograms of an oxygen saturated saline solution (8.1 ppm O2 , as measured by the HACH optical probe) at a bare gold microelectrode array, composed of 256 10 m diameter discs, before (dashed line) and after (solid line) being covered by a thin Nafion membrane. (b) Calibration plots obtained for the reduction of oxygen at the same microelectrode array, before and after being protected with a Nafion coating. To construct the plot, currents measured at −1 V vs. Ag/AgCl (3 M KCl) were used.
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Fig. 3. Diagram representing (not to scale) the interfacial structure of the microrespirometer in solution. From the point of view of oxygen, the Nafion coating behaves as a diffusion barrier of thickness d1 . The biofilm, of d2 thickness, on the other hand, may consume oxygen in addition to providing a diffusional barrier to its transport. Outside the biofilm a normal diffusion layer, δ, develops. Due to the different solubility of oxygen in water and in Nafion, there is bound to be a discontinuity in its concentration profile across the Nafion–biofilm interface.
working condition of both bare and Nafion modified electrodes, as shown in Fig. 2. The diffusion coefficient of oxygen in the saline solution at 273 K was calculated from our experimental voltammograms using the same ultramicroelectrode arrays and the concentration data given by the HACH photometer. The value of D = (2.2 ± 0.3) × 10−5 cm2 s−1 , obtained in a 0.154 M NaCl saline solution by chronoamperometry, is in good agreement with other values provided in the literature [16,17]. Due to the nature of our system, schematised in Fig. 3, it would be advisable to know the solubility and the diffusion coefficient values of oxygen in Nafion. That would later enable a better quantification of the biofilm activity. These properties have been extensively documented in the literature [17,18] (and references therein). The difficulty in the estimation of both the solubility and diffusion coefficient of oxygen in Nafion is reflected by the wide value ranges found. Thus, the diffusion coefficient is reported to range between 0.2 and 1.9 × 10−6 cm2 s−1 whilst solubility values between 7.2 and 26 mol m−3 can be found [17]. These differences are commonly attributed to the level of hydration of the Nafion membrane used in the measurements, despite the fact that most of them refer to measurements involving Nafion immersed in water or an electrolyte solution. Work is currently in progress in our laboratory to determine both the diffusion coefficient and the solubility of oxygen in Nafion using microelectrodes covered by a thick layer of Nafion. Our aim is to present this work in a separate article where the entire microrespirometer system and the biofilm behaviour are modelled using a finite element method approach. Chronoamperometric measurements will be compared to the model, which should enable us to gain valuable insights to the various transport phenomena occurring within our system before growing a biofilm on top of it. The following sections describe the use and behaviour of such an oxygen sensor when coupled to a biofilm. 3.2. Biofilm respiration monitoring Biofilm growth was followed electrochemically on a daily basis. The chips were extracted from the culture broth and immersed in a saline solution through which oxygen was being bubbled to ensure a sufficient oxygen supply from the solution. Under these conditions, the dissolved oxygen concentration was always 8.2 ± 0.2 ppm (ca. 0.25 mM). Biofilms typically took 5
days to grow to a sufficient thickness and population as to make the respiration measurements significant. Due to the extended duration of the tests it was necessary to verify that the observed current changes were due to respiration of the biofilm and not to fouling of the electrode. Therefore a series of controls were also performed. These control tests included the monitoring the response to oxygen of a series of biofilm-free sensors on a daily basis and over a period of 2 weeks. As expected, the signal kept its original level with random fluctuations below 3%. These fluctuations are accepted as experimental error. Additional controls were also performed to check that the addition of glucose and formaldehyde had no net effects on the current recorded at −1 V versus Ag/AgCl (3 M KCl). These tests were as follows: a gold microelectrode array was characterised as described above and inserted in 25 mL of oxygen saturated saline solution. Linear sweep voltammograms were carried out to and the oxygen reduction currents were recorded. Next, 250 L of either glucose (4%) or pure formaldehyde was added to the solution and the linear sweep voltammetry was repeated. The oxygen reduction current remained unchanged both at bare and Nafion modified electrodes after addition of these two substances. It was thus demonstrated that glucose and formaldehyde do not interfere in the measurement at the working potential range of our application. This implies that any changes recorded at a biofilm modified electrode on addition of these substances would be due to changes in metabolic activity. The functioning of the microrespirometer was monitored over a period of 30 consecutive days. The objective was to determine the reproducibility and stability of the system. The procedure employed to monitor biofilm respiration was as follows. The dissolved oxygen concentration was followed from linear sweep voltammograms recorded at 100 mV s−1 and performed at ca. 1 min intervals. This sweep rate is chosen because it ensures the diffusional independence of each microelectrode in the arrays employed [12]. In general the optimum sweep rate depends on electrode size and on the inter-electrode distance of each array as well as on the analyte diffusion coefficient. Because oxygen is depleted during the electrochemical measurement, we simulated the biofilm behaviour using parameters extracted from the literature [9,19]. Oxygen depletion and reequilibration were studied by numerical modelling. This kind of analysis provided only a qualitative picture, but it gave us a better understanding of the system behaviour. A more in-depth
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description of our modelling will be presented in a separate paper. Thus, assuming a large permeability to oxygen on the part of both the biofilm and the Nafion membrane, leaving a rest period of ca. 1 min between sweeps should enable the re-equilibration of the oxygen concentration near the electrode, which helps maintain the reproducibility of the measurements. The first voltammogram recorded immediately after extraction of the chip from the culture broth systematically yields low currents and an electrochemical activation step is needed to recover adequate sensor performance. This activation step consists in setting the electrode potential down to −1.4 V versus Ag/AgCl (3 M KCl) for 5 min in a NaCl solution. This potential is enough to activate the electrode without bringing about hydrogen evolution at the electrode, which would otherwise result in irreversible damage to the Nafion layer and the biofilm. Following this step, the recorded oxygen currents increase and become reproducible. We believe that regular electrode activation is needed because the biofilm (the viable bacteria constituting it) segregate products, which are able to cross the Nafion membrane and adsorb on the electrode surface, passivating it. A few minutes after the oxygen reduction current has stabilised, an excess of glucose is pipetted into the electrolyte solution while the voltammograms continue to be recorded. A sharp drop in the oxygen current is observed. The current reaches a new constant value after 2 or 3 min. If the nutrients are removed from the solution, the oxygen reduction current recovers its initial levels. This is shown in Fig. 4, where a series of such changes is presented. The magnitude of the current change depends mainly on the number of viable cells present in the biofilm. This has enabled us to follow the development and growth of biofilms and realise that the process seems to undergo three distinct stages. Fig. 5 shows that during the first couple of days there are no significant changes in the oxygen reduction current on addition of glucose.
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Fig. 5. The three stages of biofilm development as observed by respirometry. The y-axis represents the variation observed in the current, relative to the baseline oxygen reading in the absence of nutrients, after the addition of glucose to the system. Once the biofilm reaches its maturity, the measured signal represents ca. 20% of the baseline current.
This period probably corresponds to a nucleation stage during which a few bacteria adhere on the Nafion. Next, there is a short period of fast growth when a maximum current change can be seen on addition of nutrients. This is the growth period, which typically lasts for about 4–5 days. Last, the biofilm reaches its maturity and the current change on addition of glucose remains close to around 15–20% until the biofilm is killed. It is important to point out that biofilms do not grow indefinitely, but that they reach a maximum thickness and then a dynamic equilibrium is established. There are bacteria leaving the biofilm to go into solution and suspended bacteria that join the biofilm. This equilibrium situation explains the constant currents recorded over such long periods of time. This is addressed in the following section. 3.3. Toxicity determination: poisoning of the biofilm
Fig. 4. Oxygen currents recorded at the microrespirometer. The dashed line outlines the current level in the absence of nutrients. As glucose is added to the system, the biofilm starts to consume oxygen, which causes the current to drop. The system behaviour is reversible. As soon as glucose is removed from the system the current returns to its initial levels.
The ultimate purpose of our system is to warn against the presence of toxic substances in the environment where it is placed. Once we were satisfied that the microrespirometer performance was adequate, a further experiment was carried out to check that the oxygen reduction current increased on addition of a toxic substance. The procedures consisted in adding known toxics or disinfectants to the electrolyte solution in the presence of glucose, and then monitor any changes produced in the oxygen reduction current as a result. A gradual increase in the current was expected as a direct consequence of the death of bacteria in the biofilm. This was indeed the case for formaldehyde and hypochlorite, although the current responses were very different. Fig. 6 shows the transient before the addition of formaldehyde and how the current gradually drops down to its basal level before the addition of glucose, when the biofilm only opposes a diffusional barrier to oxygen. Hypochlorite can also be used to kill a biofilm, but its working mechanism is different from that of formaldehyde. Formaldehyde kills a biofilm by poisoning its bacteria but it leaves the
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to determine the respirator activity and avoid erroneous interpretations of the data. Possibly the easiest and most reliable configuration, which involves working offline is as described next. This means using a by-pass line or draining samples from the main stream and then perform a differential measurement similar to what has been described above. The solution may be saturated with oxygen if needed, or at least the oxygen level must be measured either optically or electrochemically. This provides the analyst with an indication of the sort of currents that are to be expected from the microrespirometer in the absence of nutrients. Then glucose may be added and, while the signal of the oxygen sensor should remain unaffected, the microrespirometer then provides the sought toxicity information. 4. Conclusions and outlook Fig. 6. Oxygen currents recorded at −1 V vs. Ag/AgCl (3 M KCl) in an oxygen saturated (ca. 8 ppm) saline solution. As glucose is added to the solution, the oxygen reduction current drops indicating that the biofilm is active. On addition of formaldehyde, the oxygen signal gradually increases until it reaches its initial level once the bacteria are dead.
biofilm structure intact. In contrast, hypochlorite is a strong oxidising agent, which works by attacking the bacterial outer membrane and the proteic structure of the biofilm. This causes a great deal of structural damage on the biofilm and perhaps even on the Nafion coating underneath it too. In principle, hypochlorite should not be able to reach the electrode because sulphonate groups attached to the Nafion polymer backbone provide it with an overall internal negative charge and coulombic forces prevent the diffusion of negatively charged ions through it. However, if the concentration of hypochlorite is too high or the exposure time too long, the electrode protecting Nafion layer may be also removed by the oxidative action of hypochlorite. This extreme case is easy enough to spot (the reduction current boosts due to the combined reduction of hypochlorite and oxygen, which now has no diffusional barriers on its way from the bulk of the solution to the electrode surface). On the other hand, such damaged electrodes need to be completely reconstructed, e.g.: in addition to having a new biofilm grown, these electrodes also need that a new Nafion layer be deposited on them first. Work is currently in progress to check whether the biofilm in a respirometer may be regenerated after having been exposed to a toxic substance, but preliminary data suggest that such is the case. From our experience, it is possible to use the same chip several times as long as the Nafion membrane protecting the electrodes is not damaged. This would be a considerable advantage since it means that the lifetime of the microrespirometer could be extended almost indefinitely. Although our system has been shown to work under conditions of controlled oxygen concentration and nutrients, there are no reasons to believe that it should not work in a real system. For example, two different electrodes could be used to monitor the level of dissolved oxygen. In addition to the microrespirometer, whose basal response must be known, a bare gold electrode would supply information regarding the actual level of dissolved oxygen. This information may be used in combination to that provided by the biofilm modified electrode
This work has shown proof of concept for a microelectrode array based microrespirometer as a potentially useful means to monitor acute water toxicity. The use of a Nafion membrane directly on top of the electrode meets a double purpose. First, it prevents passivation due to the adsorption of proteins or bacterial waste products. Second, it difficults or prevents the access of negatively charged species which, in the case of being electroactive, may affect the readings. Although the system is not yet ready for continuous measurements it has been shown to perform well in semi continuous mode with a time resolution of 1 min. The response time in the presence of toxics is very rapid, and although the biofilm may take several minutes to be completely killed, the toxicity of a sample can be established within the first 5 min. The microelectrode array based respirometer is also very robust; biofilms may be regenerated and the same chip used for as long as the Nafion membrane remains in place. Under normal circumstances this can be a very long period, especially if the source of toxicity is neither a strong oxidising nor reducing agent. Future work will aim to determine the sensitivity of the microrespirometer towards a series of known and common toxics. This will help to establish the adequacy of the method in real applications. In case the system responds within the levels formally established by law, then studies will be performed in real samples. Acknowledgements The authors would like to acknowledge funding from the Spanish Ministry of Education via the MICROBIOTOX project. Olga Ordeig and Neus Godino are funded by I3P grants and F. Javier del Campo by the Ram´on y Cajal Program. References [1] J.B. Sprague, Measurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity, Water Res. 3 (1969) 793. [2] P. Pandard, P. Vasseur, D.M. Rawson, Comparison of 2 types of sensors using eukaryotic algae to monitor pollution of aquatic systems, Water Res. 27 (1993) 427.
Fco.J. Del Campo et al. / Sensors and Actuators B 126 (2007) 515–521 [3] R.D. Handy, M.H. Depledge, Physiological responses: their measurement and use as environmental biomarkers in ecotoxicology, Ecotoxicology 8 (1999) 329. [4] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science 284 (1999) 1318. [5] K.K. Jefferson, What drives bacteria to produce a biofilm? FEMS Microbiol. Lett. 236 (2004) 163. [6] R.W. Zurilla, R.K. Sen, E. Yeager, Kinetics of oxygen reduction reaction on gold in alkaline-solution, J. Electrochem. Soc. 125 (1978) 1103. [7] N.P. Revsbech, An oxygen microsensor with a guard cathode, Limnol. Oceanogr. 34 (1989) 474. [8] S. Haraguchi, M. Yoshino, K. Iyasu, M. Kaneko, Y. Ishimori, M. Fujisawa, A. Shirota, Harmful substance detecting method and device, JP20040208234 20040715 (2006) 12. [9] P.S. Stewart, L. Grab, J.A. Diemer, Analysis of biocide transport limitation in an artificial biofilm system, J. Appl. Microbiol. 85 (1998) 495. [10] M.T. Madigan, J. Martinko, J. Parker, Brock Biology of Microorganisms, Prentice Hall, 2002, p. 1104. [11] D. Balestrino, J.A.J. Haagensen, C. Rich, C. Forestier, Characterization of type 2 quorum sensing in Klebsiella pneumoniae and relationship with biofilm formation, J. Bacteriol. 187 (2005) 2870. [12] T.J. Davies, S. Ward-Jones, C.E. Banks, J. del Campo, R. Mas, F.X. Munoz, R.G. Compton, The cyclic and linear sweep voltammetry of regular arrays of microdisc electrodes: fitting of experimental data, J. Electroanal. Chem. 585 (2005) 51. [13] O. Ordeig, C.E. Banks, T.J. Davies, J. del Campo, R. Mas, F.X. Munoz, R.G. Compton, Regular arrays of microdisc electrodes: simulation quantifies the fraction of ‘dead’ electrodes, Analyst 131 (2006) 440. [14] C. Paliteiro, (100)-Type behavior of polycrystalline gold towards O-2 reduction, Electrochim. Acta 39 (1994) 1633. [15] F.J. Del Campo, O. Ordeig, F.J. Munoz, Improved free chlorine amperometric sensor chip for drinking water applications, Anal. Chim. Acta 554 (2005) 98. [16] G.W. Hung, R.H. Dinius, Diffusivity of oxygen in electrolyte solutions, J. Chem. Eng. Data 17 (1972) 449. [17] A.T. Haug, R.E. White, Oxygen diffusion coefficient and solubility in a new proton exchange membrane, J. Electrochem. Soc. 147 (2000) 980. [18] A. Parthasarathy, C.R. Martin, S. Srinivasan, Investigations of the O-2 reduction reaction at the platinum Nafion interface using a solid-state electrochemical-cell, J. Electrochem. Soc. 138 (1991) 916. [19] P.S. Stewart, A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms, Biotechnol. Bioeng. 59 (1998) 261.
Biographies Fco. Javier Del Campo received a DPhil in electrochemistry from Oxford University in 2001. He joined Centro Nacional de Microelectr´onica as post-doctoral
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researcher in early 2004. In 2006 he won a Ram´on y Cajal fellowship at the Biotechnology and Biomedicine Institute of Barcelona (University Autonomous of Barcelona) to develop novel biosensing systems based on state-of-the-art micro and nano-fabrication technologies. His main research interests involve the development of biosensors for pathogen detection and the design and fabrication of ultramicroelectrodes. Olga Ordeig received her BSc in chemical engineering from Universitat of Barcelona in 2003. She is currently pursuing her PhD at Centro Nacional de Microelectr´onica under the supervision of Dr. F.X. Mu˜noz and Dr. F.J. del Campo. Her research interests lie in the field of ultramicroelectrode arrays and the application of such systems to environmental diagnostics. Nuria Vigu´es received the MSc degree in microbiology in 2005 from the Autonomous University of Barcelona, where she is at the moment pursuing her doctorate in microbiology. Her main research topics are biofilm formation in different species and materials, transport and survival of microorganisms in soil. Neus Godino received her MSc degree in physics in 2001 and in electronic engineer in 2004 from the University of Barcelona. She is currently pursuing her PhD at Centro Nacional de Microelectr´onica of Barcelona (IMB-CNM). Her main research interests are modelation and fabrication of microelectronic devices. Jordi Mas received the PhD in biology in 1985 from the Autonomous University of Barcelona, and got his position as a professor in microbiology at the Autonomous University of Barcelona in 1989. He leads the Environmental Microbiology group in the Department of Genetics and Microbiology and he is actively involved in the development of microbial biofilms for applied purposes as well as in the development and characterisation of microchip-based microbial sensors. ˜ received the PhD degree in physical chemistry from the Francesc Xavier Munoz University Autonomous of Barcelona in 1990. During 1990–1992 he performed postdoctoral research work in the Biosensors Group of the MESA Institute at the University of Twente. In 1992 he joined the Centro Nacional de Microelectronica of Barcelona (CNM-CSIC), where he has been working in the development of new chemical and biochemical microsensors. In 1997 he joined the CNM’s Department of Microsystems as an associated researcher scientist. His areas of interest are silicon micromachining technologies, polymer micro-fabrication and their application to integrated chemical microsensors and biosensors. He has participated and has been project leader of different national and European projects in this field. Currently he is responsible at CNM for two FP6 projects (Bugcheck & Immunolegio) based on the application of immunosensors and BioMEMs to the detection of pathogen bacteria, as well as national projects and contracts focused on the integration of microfluidics and bio-micro/ nanotechnologies.
Continuous measurement of acute toxicity in water using a solid state microrespirometer Fco. Javier Del Campo a,∗ , Olga Ordeig b , Nuria Vigu´es c , Neus Godino b , Jordi Mas c , Francesc Xavier Mu˜noz b a
Institut de Biotecnologia i Biomedicina (IBB-UAB), Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain b Instituto de Microelectr´ onica de Barcelona (IMB-CNM), CSIC (Esfera UAB), Campus Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain c Departamento de Gen´ etica y Microbiolog´ıa, Universitat Aut`onoma de Barcelona, Barcelona 08193, Spain Received 19 January 2007; accepted 29 March 2007 Available online 4 April 2007
Abstract This work presents the first true solid-state microrespirometer. It consists on a naturally developed biofilm of Pseudomonas aeruginosa over a Nafion modified array of gold microdisc electrodes. Such a device can be used to monitor acute toxicity in water streams based on the oxygen reduction current recorded at the microelectrode array. When a healthy biofilm is exposed to a toxic stream the bacteria conforming stop breathing which results in an immediate increase of the oxygen supply reaching the microelectrode array. The fabrication and use of such a microrespirometer is described, and a possible operation mode for a real application is presented. Given that different waters may contain toxic compounds of diverse nature, the respirometer presented here provides qualitative information only. © 2007 Elsevier B.V. All rights reserved. Keywords: Microrespirometry; Ultramicroelectrode arrays; Oxygen reduction; Linear sweep voltammetry; Biofilm
1. Introduction This work presents the development of a new solid-state microrespirometer for the continuous monitoring of acute toxicity in drinking water supply lines. Toxicity of drinking water may be determined by different means. Amongst these, the use of assays based on the response of fish [1], algae [2], or even bacteria is commonplace. Such assays diagnose toxicity through the observation of alterations in the physiology of the subject organisms [3]. Biofilms are aggregates of bacteria, which spontaneously associate on top of surfaces in order to improve their survival rate in certain environments [4,5]. In this work, stable biofilms were grown from a culture broth on top of the electrode system. The growth process takes between 10 and 15 days, after which biofilms remain stable. Because the oxygen consumption of biofilms is heavily influenced by environmental parameters, par-
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Corresponding author. Tel.: +34 93 594 77 00x1313; fax: +34 93 580 14 96. E-mail address: [email protected] (Fco.J. Del Campo).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.03.038
ticularly by the presence of nutrients and toxics, their respiration rate may be used as an indicator of medium toxicity. The method described here has its foundations in the amperometric monitoring of oxygen [6]. The idea of monitoring biofilm respiration using oxygen electrodes is not new [2]. Individual microelectrodes have been used in the past to study oxygen concentration profiles within a biofilm [7]. The novel of our approach is that a biofilm is allowed to grow spontaneously on top of an array of ultramicroelectrodes and measuring the oxygen flux through it, thus achieving a true solid-state microrespirometer. This is in contrast to past micro-respirometry work where cells have been trapped inside a filter membrane which is later placed in the vicinity or in close contact with a Clark oxygen electrode [7,8]. This work shows how a biofilm based micro-respirometer device enables the semi-continuous monitoring of acute water toxicity. All measurements involving biofilms were performed in a carbon source free environment. Thus, in the absence of nutrients, a biofilm behaves purely as a diffusion barrier because the biofilm does not require a significant amount of oxygen for its survival. However, as soon as nutrients are available, oxygen
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consumption shoots up as a result of bacterial metabolic activity. The rate of oxygen consumption is affected by the presence of toxic compounds which, if in sufficiently high concentrations, can kill a very large fraction of the bacteria forming the biofilm [4]. In spite of this, it may take several hours to kill all bacteria within a mature biofilm because the biocide is neutralized by reaction with biofilm constituents faster than it diffuses into the biofilm [9] (sic.). Thus, when bacteria are forming a biofilm they become much more resistant to antibiotics and environmental changes, but they can nevertheless be killed by toxics such as bleach or formaldehyde. This can actually be turned to our advantage. The fraction of cells poisoned, which are expected to be the most superficial ones, eventually dislodge from the biofilm as the inner cells multiply and promote the spontaneous regeneration of the biofilm. The implication is that the same biofilm modified electrode may be employed for very long time in spite of its being placed in contact with a disinfecting agent. All in all, the system is able to provide BOD information, or to serve as a toxicity indicator, the results being expressed as percent inhibition of respiratory activity. The device presented here can be used for toxicity control in raw water sources for human use, and also in the management of wastewater treatment plants. 2. Experimental
pared using high purity deionised water of resistivity not less than 18 M cm. Solutions containing dissolved oxygen were achieved by means of an in-house-built system consisting of air and nitrogen lines which flow could be controlled by means of individual gas rotameters. Backflow stopper valves (Swagelock) were used to avoid pressure unbalances, which could lead to contamination and erroneous gas flow readings. The experimental set-up is schematised in Fig. 1. A bioreactor for biofilm formation has been designed. A pure culture of Pseudomonas putida was grown in a continuous culture with controlled oxygen supply at 37 ◦ C. The bacteria grew in a minimum medium—a medium only composed by salts and glucose as the nutrient, to control their growth [10]. In this case, Minimum Medium AB (MMAB) was prepared as indicated in the literature [11]. Biofilms developed vigorously on the solid-state microrespirometer surface. All electrochemical measurements were carried out using a -Autolab III (Eco-Chemie, The Netherlands) connected to a PC computer using version 4 of the GPES software for Windows® . All measurements were performed using a three-electrode cell configuration. The reference electrode consisted of an Ag/AgCl (3 M KCl) from Crison. A Pt ring electrode, also from Crison, was used as auxiliary electrode. The pH of the various solutions was monitored using a Hanna Instruments HI-300 pH meter. Oxygen concentrations were measured using an optical HACH HQ10 oxygen handheld probe with temperature control.
2.1. Reagents and instrumentation 2.2. Electrode preparation The following reagents were ACS analytical grade and were used as received from Sigma–Aldrich without any further purification: KCl (99%), K4 Fe(CN)6 ·3H2 O (99%), Nafion 117 solution (5% in lower aliphatic alcohols), formaldehyde (36.5–38% in H2 O) and d-glucose (>98%, Fluka). Sterile saline solutions were purchased from Sigma–Aldrich and used as received without further purification. All solutions were pre-
The Au micro disc arrays used in this study have been described previously [12]. They were made up by 256 discs, 10 m in diameter, and arranged under a cubic distribution with an inter-centre distance of 100 m. Because not all the electrodes of an array are active, the actual number of discs for each individual array used in the study has been determined following
Fig. 1. Diagrammatic representation (not to scale) of the experimental set-up. Two back-flow stopper valves after the rotameters allow controlled mixing while avoiding contamination of the gases. WE represents the biofilm oxygen sensor, the reference (RE) and counter (CE) electrodes are also depicted.
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the approach described in [13]. Oxygen free, 1 mM ferrocyanide in 0.1 M KCl solutions were used for the voltammetric characterisation of each of the arrays used in this study.
neutral/basic pH):
2.2.1. Nafion modified arrays In order to avoid the fouling of the electrode as a result of direct contact with the bacteria, the microelectrode array was protected by a spin coated thin layer of Nafion. The coating was carried out in a POLOS MCM-TFM spinner in a class 100000 clean room. Individually mounted chips were placed in the spinner and 10 L of the Nafion solution was pipetted on top of the chip. Next, the array was spinned for 1 min at 800 rpm and with an initial acceleration of 1000 rpm2 . This operation was repeated three times with waiting intervals of 3 min to allow evaporation of the ethanol. Surface topology of the Nafion layer was characterised in three stages by perfilometry using a KLA-Tencor P15 perfilometer controlled by Profiler v7 software run on Windows XP. First, a profile of the clean and bare microelectrode array was obtained and used as the baseline. Next, the Nafion was deposited as describe above and left to cure for not less than 60 min. The deposited layer is so thin that 60 min is time enough for the solvent to evaporate. Finally, the Nafion membranes were hydrated by immersion in water for another 60 min, after which a new profile measurement was performed. The Nafion layer (unhydrated) was ca. 300 nm thick and it homogeneously followed the ultramicroelectrode array profile. Once hydrated, the Nafion layer reached a thickness of ca. 450 nm, which represents a 50% volume increase.
Although we did observe two waves, all the currents used in the construction of calibration plots or the monitoring of oxygen corresponded to the complete process; the four-electron reduction. Unless otherwise stated, all the currents quoted in this work were measured at −1 V versus Ag/AgCl (3 M KCl). First, the response of the gold microelectrode arrays to oxygen was evaluated. It is worth noting that the oxygen signal measured at −1 V versus Ag/AgCl is not affected by pH changes in the range 6 < pH < 8.5, as reported elsewhere [15]. The procedure is described next. Following the electrochemical activation and characterisation of the arrays, the voltammetry of dissolved oxygen was studied. A stream of air was bubbled through 25 mL of 0.1 M Na2 SO4 at pH 5.5 to ensure a constant and controlled level of dissolved oxygen. Moreover, the concentration of oxygen was measured before and after the electrochemical measurements using a HACH optical oxygen probe. Fig. 2a shows a typical voltammogram of oxygen at a 256 ultramicroelectrode array. The linearity of the electrochemical response was checked by bubbling a series of nitrogen/air gas mixtures through the electrolyte solution. Fig. 2b demonstrates that the sensor response is highly linear (unmodified electrode: 0.095 ± 0.013 A ppm−1 , 0.011 ± 0.007 A; LOD3 = 0.29 ppm, R = 0.999, n = 8; modified electrode: 0.070 ± 0.018 A ppm−1 , 0.035 ± 0.01 A; LOD3 = 0.39 ppm, R = 0.998, n = 7) over the range of 2– 8.5 mg L−1 . In general, these curves were recorded starting from the highest concentration and proceeding towards lower concentrations of oxygen. For each point, the gas mixture was allowed to bubble through the solution for no less than 15 min in order to achieve equilibrium. This gas system was used in combination with the HACH optical oxygen probe to verify the
2.2.2. Biofilm modified electrodes Bare and Nafion modified arrays were immersed in a culture as described above for several days (over a month in some instances). Every morning the chips were extracted from the broth in order to measure the oxygen reduction current from an oxygen saturated saline solution. Although it was possible to observe the formation of biofilm on top of the electrodes by sight after the second day, the electrochemical measurements did not show a significant current change on addition of glucose until after the 5th day. By the end of the study, some 30 days later, the chip and its PCB substrate were completely covered in thick biofilm presenting a bone-yellow colour. Typical biofilm thicknesses ranged from 15 up to 60 m, depending on ageing, as determined by confocal microscopy.
O2 + 2e− + H2 O → HO2 − + OH− HO2 − + 2e− + H2 O → 3OH−
(2)
3. Results and discussion 3.1. Amperometric oxygen determination The overall reaction for the reduction of oxygen, in a neutral to basic environment is: O2 + 4e− + 2H2 O → 4OH−
(1)
In reality, the actual mechanism is rather complex [14] and a detailed discussion of it is out of the scope of the present work. However, it is often possible to discern not a single fourelectron wave, but two related to the following sequence (under
Fig. 2. (a) Linear sweep voltammograms of an oxygen saturated saline solution (8.1 ppm O2 , as measured by the HACH optical probe) at a bare gold microelectrode array, composed of 256 10 m diameter discs, before (dashed line) and after (solid line) being covered by a thin Nafion membrane. (b) Calibration plots obtained for the reduction of oxygen at the same microelectrode array, before and after being protected with a Nafion coating. To construct the plot, currents measured at −1 V vs. Ag/AgCl (3 M KCl) were used.
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Fig. 3. Diagram representing (not to scale) the interfacial structure of the microrespirometer in solution. From the point of view of oxygen, the Nafion coating behaves as a diffusion barrier of thickness d1 . The biofilm, of d2 thickness, on the other hand, may consume oxygen in addition to providing a diffusional barrier to its transport. Outside the biofilm a normal diffusion layer, δ, develops. Due to the different solubility of oxygen in water and in Nafion, there is bound to be a discontinuity in its concentration profile across the Nafion–biofilm interface.
working condition of both bare and Nafion modified electrodes, as shown in Fig. 2. The diffusion coefficient of oxygen in the saline solution at 273 K was calculated from our experimental voltammograms using the same ultramicroelectrode arrays and the concentration data given by the HACH photometer. The value of D = (2.2 ± 0.3) × 10−5 cm2 s−1 , obtained in a 0.154 M NaCl saline solution by chronoamperometry, is in good agreement with other values provided in the literature [16,17]. Due to the nature of our system, schematised in Fig. 3, it would be advisable to know the solubility and the diffusion coefficient values of oxygen in Nafion. That would later enable a better quantification of the biofilm activity. These properties have been extensively documented in the literature [17,18] (and references therein). The difficulty in the estimation of both the solubility and diffusion coefficient of oxygen in Nafion is reflected by the wide value ranges found. Thus, the diffusion coefficient is reported to range between 0.2 and 1.9 × 10−6 cm2 s−1 whilst solubility values between 7.2 and 26 mol m−3 can be found [17]. These differences are commonly attributed to the level of hydration of the Nafion membrane used in the measurements, despite the fact that most of them refer to measurements involving Nafion immersed in water or an electrolyte solution. Work is currently in progress in our laboratory to determine both the diffusion coefficient and the solubility of oxygen in Nafion using microelectrodes covered by a thick layer of Nafion. Our aim is to present this work in a separate article where the entire microrespirometer system and the biofilm behaviour are modelled using a finite element method approach. Chronoamperometric measurements will be compared to the model, which should enable us to gain valuable insights to the various transport phenomena occurring within our system before growing a biofilm on top of it. The following sections describe the use and behaviour of such an oxygen sensor when coupled to a biofilm. 3.2. Biofilm respiration monitoring Biofilm growth was followed electrochemically on a daily basis. The chips were extracted from the culture broth and immersed in a saline solution through which oxygen was being bubbled to ensure a sufficient oxygen supply from the solution. Under these conditions, the dissolved oxygen concentration was always 8.2 ± 0.2 ppm (ca. 0.25 mM). Biofilms typically took 5
days to grow to a sufficient thickness and population as to make the respiration measurements significant. Due to the extended duration of the tests it was necessary to verify that the observed current changes were due to respiration of the biofilm and not to fouling of the electrode. Therefore a series of controls were also performed. These control tests included the monitoring the response to oxygen of a series of biofilm-free sensors on a daily basis and over a period of 2 weeks. As expected, the signal kept its original level with random fluctuations below 3%. These fluctuations are accepted as experimental error. Additional controls were also performed to check that the addition of glucose and formaldehyde had no net effects on the current recorded at −1 V versus Ag/AgCl (3 M KCl). These tests were as follows: a gold microelectrode array was characterised as described above and inserted in 25 mL of oxygen saturated saline solution. Linear sweep voltammograms were carried out to and the oxygen reduction currents were recorded. Next, 250 L of either glucose (4%) or pure formaldehyde was added to the solution and the linear sweep voltammetry was repeated. The oxygen reduction current remained unchanged both at bare and Nafion modified electrodes after addition of these two substances. It was thus demonstrated that glucose and formaldehyde do not interfere in the measurement at the working potential range of our application. This implies that any changes recorded at a biofilm modified electrode on addition of these substances would be due to changes in metabolic activity. The functioning of the microrespirometer was monitored over a period of 30 consecutive days. The objective was to determine the reproducibility and stability of the system. The procedure employed to monitor biofilm respiration was as follows. The dissolved oxygen concentration was followed from linear sweep voltammograms recorded at 100 mV s−1 and performed at ca. 1 min intervals. This sweep rate is chosen because it ensures the diffusional independence of each microelectrode in the arrays employed [12]. In general the optimum sweep rate depends on electrode size and on the inter-electrode distance of each array as well as on the analyte diffusion coefficient. Because oxygen is depleted during the electrochemical measurement, we simulated the biofilm behaviour using parameters extracted from the literature [9,19]. Oxygen depletion and reequilibration were studied by numerical modelling. This kind of analysis provided only a qualitative picture, but it gave us a better understanding of the system behaviour. A more in-depth
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description of our modelling will be presented in a separate paper. Thus, assuming a large permeability to oxygen on the part of both the biofilm and the Nafion membrane, leaving a rest period of ca. 1 min between sweeps should enable the re-equilibration of the oxygen concentration near the electrode, which helps maintain the reproducibility of the measurements. The first voltammogram recorded immediately after extraction of the chip from the culture broth systematically yields low currents and an electrochemical activation step is needed to recover adequate sensor performance. This activation step consists in setting the electrode potential down to −1.4 V versus Ag/AgCl (3 M KCl) for 5 min in a NaCl solution. This potential is enough to activate the electrode without bringing about hydrogen evolution at the electrode, which would otherwise result in irreversible damage to the Nafion layer and the biofilm. Following this step, the recorded oxygen currents increase and become reproducible. We believe that regular electrode activation is needed because the biofilm (the viable bacteria constituting it) segregate products, which are able to cross the Nafion membrane and adsorb on the electrode surface, passivating it. A few minutes after the oxygen reduction current has stabilised, an excess of glucose is pipetted into the electrolyte solution while the voltammograms continue to be recorded. A sharp drop in the oxygen current is observed. The current reaches a new constant value after 2 or 3 min. If the nutrients are removed from the solution, the oxygen reduction current recovers its initial levels. This is shown in Fig. 4, where a series of such changes is presented. The magnitude of the current change depends mainly on the number of viable cells present in the biofilm. This has enabled us to follow the development and growth of biofilms and realise that the process seems to undergo three distinct stages. Fig. 5 shows that during the first couple of days there are no significant changes in the oxygen reduction current on addition of glucose.
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Fig. 5. The three stages of biofilm development as observed by respirometry. The y-axis represents the variation observed in the current, relative to the baseline oxygen reading in the absence of nutrients, after the addition of glucose to the system. Once the biofilm reaches its maturity, the measured signal represents ca. 20% of the baseline current.
This period probably corresponds to a nucleation stage during which a few bacteria adhere on the Nafion. Next, there is a short period of fast growth when a maximum current change can be seen on addition of nutrients. This is the growth period, which typically lasts for about 4–5 days. Last, the biofilm reaches its maturity and the current change on addition of glucose remains close to around 15–20% until the biofilm is killed. It is important to point out that biofilms do not grow indefinitely, but that they reach a maximum thickness and then a dynamic equilibrium is established. There are bacteria leaving the biofilm to go into solution and suspended bacteria that join the biofilm. This equilibrium situation explains the constant currents recorded over such long periods of time. This is addressed in the following section. 3.3. Toxicity determination: poisoning of the biofilm
Fig. 4. Oxygen currents recorded at the microrespirometer. The dashed line outlines the current level in the absence of nutrients. As glucose is added to the system, the biofilm starts to consume oxygen, which causes the current to drop. The system behaviour is reversible. As soon as glucose is removed from the system the current returns to its initial levels.
The ultimate purpose of our system is to warn against the presence of toxic substances in the environment where it is placed. Once we were satisfied that the microrespirometer performance was adequate, a further experiment was carried out to check that the oxygen reduction current increased on addition of a toxic substance. The procedures consisted in adding known toxics or disinfectants to the electrolyte solution in the presence of glucose, and then monitor any changes produced in the oxygen reduction current as a result. A gradual increase in the current was expected as a direct consequence of the death of bacteria in the biofilm. This was indeed the case for formaldehyde and hypochlorite, although the current responses were very different. Fig. 6 shows the transient before the addition of formaldehyde and how the current gradually drops down to its basal level before the addition of glucose, when the biofilm only opposes a diffusional barrier to oxygen. Hypochlorite can also be used to kill a biofilm, but its working mechanism is different from that of formaldehyde. Formaldehyde kills a biofilm by poisoning its bacteria but it leaves the
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to determine the respirator activity and avoid erroneous interpretations of the data. Possibly the easiest and most reliable configuration, which involves working offline is as described next. This means using a by-pass line or draining samples from the main stream and then perform a differential measurement similar to what has been described above. The solution may be saturated with oxygen if needed, or at least the oxygen level must be measured either optically or electrochemically. This provides the analyst with an indication of the sort of currents that are to be expected from the microrespirometer in the absence of nutrients. Then glucose may be added and, while the signal of the oxygen sensor should remain unaffected, the microrespirometer then provides the sought toxicity information. 4. Conclusions and outlook Fig. 6. Oxygen currents recorded at −1 V vs. Ag/AgCl (3 M KCl) in an oxygen saturated (ca. 8 ppm) saline solution. As glucose is added to the solution, the oxygen reduction current drops indicating that the biofilm is active. On addition of formaldehyde, the oxygen signal gradually increases until it reaches its initial level once the bacteria are dead.
biofilm structure intact. In contrast, hypochlorite is a strong oxidising agent, which works by attacking the bacterial outer membrane and the proteic structure of the biofilm. This causes a great deal of structural damage on the biofilm and perhaps even on the Nafion coating underneath it too. In principle, hypochlorite should not be able to reach the electrode because sulphonate groups attached to the Nafion polymer backbone provide it with an overall internal negative charge and coulombic forces prevent the diffusion of negatively charged ions through it. However, if the concentration of hypochlorite is too high or the exposure time too long, the electrode protecting Nafion layer may be also removed by the oxidative action of hypochlorite. This extreme case is easy enough to spot (the reduction current boosts due to the combined reduction of hypochlorite and oxygen, which now has no diffusional barriers on its way from the bulk of the solution to the electrode surface). On the other hand, such damaged electrodes need to be completely reconstructed, e.g.: in addition to having a new biofilm grown, these electrodes also need that a new Nafion layer be deposited on them first. Work is currently in progress to check whether the biofilm in a respirometer may be regenerated after having been exposed to a toxic substance, but preliminary data suggest that such is the case. From our experience, it is possible to use the same chip several times as long as the Nafion membrane protecting the electrodes is not damaged. This would be a considerable advantage since it means that the lifetime of the microrespirometer could be extended almost indefinitely. Although our system has been shown to work under conditions of controlled oxygen concentration and nutrients, there are no reasons to believe that it should not work in a real system. For example, two different electrodes could be used to monitor the level of dissolved oxygen. In addition to the microrespirometer, whose basal response must be known, a bare gold electrode would supply information regarding the actual level of dissolved oxygen. This information may be used in combination to that provided by the biofilm modified electrode
This work has shown proof of concept for a microelectrode array based microrespirometer as a potentially useful means to monitor acute water toxicity. The use of a Nafion membrane directly on top of the electrode meets a double purpose. First, it prevents passivation due to the adsorption of proteins or bacterial waste products. Second, it difficults or prevents the access of negatively charged species which, in the case of being electroactive, may affect the readings. Although the system is not yet ready for continuous measurements it has been shown to perform well in semi continuous mode with a time resolution of 1 min. The response time in the presence of toxics is very rapid, and although the biofilm may take several minutes to be completely killed, the toxicity of a sample can be established within the first 5 min. The microelectrode array based respirometer is also very robust; biofilms may be regenerated and the same chip used for as long as the Nafion membrane remains in place. Under normal circumstances this can be a very long period, especially if the source of toxicity is neither a strong oxidising nor reducing agent. Future work will aim to determine the sensitivity of the microrespirometer towards a series of known and common toxics. This will help to establish the adequacy of the method in real applications. In case the system responds within the levels formally established by law, then studies will be performed in real samples. Acknowledgements The authors would like to acknowledge funding from the Spanish Ministry of Education via the MICROBIOTOX project. Olga Ordeig and Neus Godino are funded by I3P grants and F. Javier del Campo by the Ram´on y Cajal Program. References [1] J.B. Sprague, Measurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity, Water Res. 3 (1969) 793. [2] P. Pandard, P. Vasseur, D.M. Rawson, Comparison of 2 types of sensors using eukaryotic algae to monitor pollution of aquatic systems, Water Res. 27 (1993) 427.
Fco.J. Del Campo et al. / Sensors and Actuators B 126 (2007) 515–521 [3] R.D. Handy, M.H. Depledge, Physiological responses: their measurement and use as environmental biomarkers in ecotoxicology, Ecotoxicology 8 (1999) 329. [4] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science 284 (1999) 1318. [5] K.K. Jefferson, What drives bacteria to produce a biofilm? FEMS Microbiol. Lett. 236 (2004) 163. [6] R.W. Zurilla, R.K. Sen, E. Yeager, Kinetics of oxygen reduction reaction on gold in alkaline-solution, J. Electrochem. Soc. 125 (1978) 1103. [7] N.P. Revsbech, An oxygen microsensor with a guard cathode, Limnol. Oceanogr. 34 (1989) 474. [8] S. Haraguchi, M. Yoshino, K. Iyasu, M. Kaneko, Y. Ishimori, M. Fujisawa, A. Shirota, Harmful substance detecting method and device, JP20040208234 20040715 (2006) 12. [9] P.S. Stewart, L. Grab, J.A. Diemer, Analysis of biocide transport limitation in an artificial biofilm system, J. Appl. Microbiol. 85 (1998) 495. [10] M.T. Madigan, J. Martinko, J. Parker, Brock Biology of Microorganisms, Prentice Hall, 2002, p. 1104. [11] D. Balestrino, J.A.J. Haagensen, C. Rich, C. Forestier, Characterization of type 2 quorum sensing in Klebsiella pneumoniae and relationship with biofilm formation, J. Bacteriol. 187 (2005) 2870. [12] T.J. Davies, S. Ward-Jones, C.E. Banks, J. del Campo, R. Mas, F.X. Munoz, R.G. Compton, The cyclic and linear sweep voltammetry of regular arrays of microdisc electrodes: fitting of experimental data, J. Electroanal. Chem. 585 (2005) 51. [13] O. Ordeig, C.E. Banks, T.J. Davies, J. del Campo, R. Mas, F.X. Munoz, R.G. Compton, Regular arrays of microdisc electrodes: simulation quantifies the fraction of ‘dead’ electrodes, Analyst 131 (2006) 440. [14] C. Paliteiro, (100)-Type behavior of polycrystalline gold towards O-2 reduction, Electrochim. Acta 39 (1994) 1633. [15] F.J. Del Campo, O. Ordeig, F.J. Munoz, Improved free chlorine amperometric sensor chip for drinking water applications, Anal. Chim. Acta 554 (2005) 98. [16] G.W. Hung, R.H. Dinius, Diffusivity of oxygen in electrolyte solutions, J. Chem. Eng. Data 17 (1972) 449. [17] A.T. Haug, R.E. White, Oxygen diffusion coefficient and solubility in a new proton exchange membrane, J. Electrochem. Soc. 147 (2000) 980. [18] A. Parthasarathy, C.R. Martin, S. Srinivasan, Investigations of the O-2 reduction reaction at the platinum Nafion interface using a solid-state electrochemical-cell, J. Electrochem. Soc. 138 (1991) 916. [19] P.S. Stewart, A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms, Biotechnol. Bioeng. 59 (1998) 261.
Biographies Fco. Javier Del Campo received a DPhil in electrochemistry from Oxford University in 2001. He joined Centro Nacional de Microelectr´onica as post-doctoral
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researcher in early 2004. In 2006 he won a Ram´on y Cajal fellowship at the Biotechnology and Biomedicine Institute of Barcelona (University Autonomous of Barcelona) to develop novel biosensing systems based on state-of-the-art micro and nano-fabrication technologies. His main research interests involve the development of biosensors for pathogen detection and the design and fabrication of ultramicroelectrodes. Olga Ordeig received her BSc in chemical engineering from Universitat of Barcelona in 2003. She is currently pursuing her PhD at Centro Nacional de Microelectr´onica under the supervision of Dr. F.X. Mu˜noz and Dr. F.J. del Campo. Her research interests lie in the field of ultramicroelectrode arrays and the application of such systems to environmental diagnostics. Nuria Vigu´es received the MSc degree in microbiology in 2005 from the Autonomous University of Barcelona, where she is at the moment pursuing her doctorate in microbiology. Her main research topics are biofilm formation in different species and materials, transport and survival of microorganisms in soil. Neus Godino received her MSc degree in physics in 2001 and in electronic engineer in 2004 from the University of Barcelona. She is currently pursuing her PhD at Centro Nacional de Microelectr´onica of Barcelona (IMB-CNM). Her main research interests are modelation and fabrication of microelectronic devices. Jordi Mas received the PhD in biology in 1985 from the Autonomous University of Barcelona, and got his position as a professor in microbiology at the Autonomous University of Barcelona in 1989. He leads the Environmental Microbiology group in the Department of Genetics and Microbiology and he is actively involved in the development of microbial biofilms for applied purposes as well as in the development and characterisation of microchip-based microbial sensors. ˜ received the PhD degree in physical chemistry from the Francesc Xavier Munoz University Autonomous of Barcelona in 1990. During 1990–1992 he performed postdoctoral research work in the Biosensors Group of the MESA Institute at the University of Twente. In 1992 he joined the Centro Nacional de Microelectronica of Barcelona (CNM-CSIC), where he has been working in the development of new chemical and biochemical microsensors. In 1997 he joined the CNM’s Department of Microsystems as an associated researcher scientist. His areas of interest are silicon micromachining technologies, polymer micro-fabrication and their application to integrated chemical microsensors and biosensors. He has participated and has been project leader of different national and European projects in this field. Currently he is responsible at CNM for two FP6 projects (Bugcheck & Immunolegio) based on the application of immunosensors and BioMEMs to the detection of pathogen bacteria, as well as national projects and contracts focused on the integration of microfluidics and bio-micro/ nanotechnologies.