A Proof-of-Concept Electrochemical Cytosensor Based on Chlamydomonas reinhardtii Functionalized Carbon Black Screen-Printed Electrodes: Detection of Escherichia coli in Wastewater as a Case Study

Herein, we report a proof-of-concept algal cytosensor for the electrochemical quantification of bacteria in wastewater, exploiting the green photosynthetic alga Chlamydomonas reinhardtii immobilized on carbon black (CB) nanomodified screen-printed electrodes. The CB nanoparticles are used as nanomodifiers, as they are able to sense the oxygen produced by the algae and thus the current increases when algae are exposed to increasing concentrations of bacteria. The sensor was tested on both standard solutions and real wastewater samples for the detection Escherichia coli in a linear range of response from 100 to 2000 CFU/100 mL, showing a limit of detection of 92 CFU/100 mL, in agreement with the maximum E. coli concentration established by the Italian law for wastewater (less than 5000 CFU/100 mL). This bacterium was exploited as a case study target of the algal cytosensor to demonstrate its ability as an early warning analytical system to signal heavy loads of pathogens in waters leaving the wastewater treatment plants. Indeed, the cytosensor is not selective towards E. coli but it is capable of sensing all the bacteria that induce the algae oxygen evolution by exploiting the effect of their interaction. Other known toxicants, commonly present in wastewater, were also analyzed to test the cytosensor selectivity, with any significant effect, apart from atrazine, which is a specific target of the D1 protein of the Chlamydomonas photosystem II. However, the latter can also be detected by chlorophyll fluorescence simultaneously to the amperometric measurements. The matrix effect was evaluated, and the recovery values were calculated as 105 ± 8, 83 ± 7, and 88 ± 7% for 1000 CFU/100 mL of E. coli in Lignano, San Giorgio, and Pescara wastewater samples, respectively.


Introduction
Currently, water resources face severe quantitative and qualitative threats due to the industrialization and rapid economic development in many areas worldwide. Issues related to the ecological, health, and hygienic state of the water bodies are well-documented and represent a crucial concern to minimize the human impact on both fresh and saltwater bodies [1]. One crucial point in preserving the quality of bodies of water is wastewater production in the presence of bacteria. Indeed, we demonstrated in our previous article [14] the capability of CB nanoparticles to "sensitively sense changes in algae oxygen evolution during the photosynthetic process". To the best of our knowledge, this is the first cytosensor developed for pathogen detection that uses algae as bioreceptors.

Algae Growth Conditions and Physiological Characterization of the Algal Liquid Cultures
Algae growth and characterization was accomplished according to the protocols described in Ref. [14]. C. reinhardtii F255N was grown under continuous light (50 µL photons m −2 s −1 ), on tris-acetate-phosphate (TAP) culture medium into an orbital shaker at 25 • C, stirring at 150 rpm. At 72 h inoculation, the algae culture was diluted in TAP to an optical density of 0. 15 OD 750 . Then the refreshed culture was grown under the same conditions for all the periods of the physiological characterization, using cell culture in an early mid-exponential growth phase, with Abs 750 0.5 O.D., 10 6 cells/mL, and 5 µg/mL chlorophyll content. Cell number was quantified using a Bio-Rad TC-10 automated counter (Hemel Hempstead, UK), using a 10 µL-volume cell counting slide. Pigment content was spectrophotometrically measured by quantifying the absorbance (O.D.) of the chlorophylls a and b at 652 nm wavelength, once extracted with 80% acetone. The calculation of the total chlorophyll concentration expressed as µg/mL was performed by the equation (O.D. 652 × 1000)/34.5. The photosynthetic profile was assessed by the chlorophyll a fluorescence induction (Kautsky) curves, recorded with a Plant Efficiency Analyzer (PEA) at room temperature after 10 min of dark adaptation and with a 5 s saturating pulse excitation light (3500 µL photons m −2 s −1 ) using an array of six red light-emitting diodes (650 nm peak). Kautsky curves or OJIP curves are defined by a polyphasic fluorescence rise in time, with O as the minimal dark-acclimated fluorescence level (indicating that all QA are oxidised) and P as the maximal level (indicating that all PSII quinone acceptors are fully reduced). The difference in the fluorescence signal of these distinct states helps to evaluate the PSII functionality through the following parameters calculated by the fluorimeter: F 0 or fluorescence in the initial state: minimum fluorescence intensity in the state acclimated to the darkness, recorded when all PSII reaction centres are open (oxidized quinones); F M or maximum fluorescence: maximum fluorescence intensity reached after 10 min of darkness and a subsequent saturating light pulse, recorded when all reaction centres of the PSII are closed (reduced); F V /F M : maximum fluorescence yield of PSII photochemical reaction expressed as a ratio of variable fluorescence (F M − F 0 ) and maximum fluorescence, calculated according to the Equation (1): where F V represents the maximum variable fluorescence calculated as F M -F 0 , F M corresponds to the maximum fluorescence emission and F 0 is the minimum fluorescence emission. It reflects the efficiency of PSII in using light for photochemical conversion and its value is usually at 0.8 in physiological conditions or decreased values under stress. Optical microscopy was conducted on liquid cultures of C. reinhardtii algae cells in TAP medium at different grow stages, using a Leitz Diavert Microscope.

Algae Immobilization Protocol
Algae immobilization was accomplished according to the protocols described in Ref. [14] with a final cell concentration of 0.08 × 10 6 cells/µL for each SPE. C. reinhardtii F255N cell cultures in an early mid-exponential growth phase, with Abs 750 0.7 O.D., 10 7 cells/mL, and 10 µg/mL chlorophyll content, were exploited for the immobilization on carbon black modified screen-printed electrodes (CB-SPEs) purchased from SENS4MED (Rome, Italy). Algae/CB-SPEs were stored in 50 mM Tricine, 20 mM CaCl 2 , 5 mM MgCl 2 , 50 mM NaCl, and 70 mM sucrose pH 7.2 and incubated for 2 h under continuous light (50 µL photons m −2 s −1 ) and 25 • C. SEM analysis was conducted on a ZEISS EVO MA10 scanning electron microscope. Algae were subjected to gold metallization and then dehydration under vacuum before SEM analysis. Microphotographs were provided at a magnification of 2 µm.

Biosensor Prototype
A dual electro-optical transduction prototype was projected and realized to furnish both optical and electrochemical analysis by the company Biosensor Srl ( Figure 1A). The instrument is a portable prototype consisting of 6 module chambers for the insertion of the algal CB-SPEs ( Figure 1B). The chamber is equipped with a LED system (of 350 µL photons m −2 s −1 of red light at a 650 nm wavelength) that provides the algae illumination. The electrochemical set-up is constituted of a DC voltage supply, which provides a bias potential in the range of ±0.800 V between the working and the reference electrodes, and an amperometer to detect the current intensity variation deriving from the algae oxygen evolution process. The biological module, perfectly sealed, hosts the samples under test. Both static and dynamic operations are allowed thanks to an automatically controlled fluidic system equipped with inlet/outlet connections for the electrolytic/washing solution and sample flow. Fifty mM Tricine, 20 mM CaCl 2 , 5 mM MgCl 2 , 50 mM NaCl, 70 mM sucrose, and pH 7.2 was used as the measuring buffer for the electrochemical analysis.

Algae Immobilization Protocol
Algae immobilization was accomplished according to the protocols described in Ref. [14] with a final cell concentration of 0.08 × 10 6 cells/µL for each SPE. C. reinhardtii F255N cell cultures in an early mid-exponential growth phase, with Abs750 0.7 O.D., 10 7 cells/mL, and 10 µg/mL chlorophyll content, were exploited for the immobilization on carbon black modified screen-printed electrodes (CB-SPEs) purchased from SENS4MED (Rome, Italy). Algae/CB-SPEs were stored in 50 mM Tricine, 20 mM CaCl2, 5 mM MgCl2, 50 mM NaCl, and 70 mM sucrose pH 7.2 and incubated for 2 h under continuous light (50 µL photons m −2 s −1 ) and 25 °C. SEM analysis was conducted on a ZEISS EVO MA10 scanning electron microscope. Algae were subjected to gold metallization and then dehydration under vacuum before SEM analysis. Microphotographs were provided at a magnification of 2 µm.

Biosensor Prototype
A dual electro-optical transduction prototype was projected and realized to furnish both optical and electrochemical analysis by the company Biosensor Srl ( Figure 1A). The instrument is a portable prototype consisting of 6 module chambers for the insertion of the algal CB-SPEs ( Figure 1B). The chamber is equipped with a LED system (of 350 µL photons m −2 s −1 of red light at a 650 nm wavelength) that provides the algae illumination. The electrochemical set-up is constituted of a DC voltage supply, which provides a bias potential in the range of ±0.800 V between the working and the reference electrodes, and an amperometer to detect the current intensity variation deriving from the algae oxygen evolution process. The biological module, perfectly sealed, hosts the samples under test. Both static and dynamic operations are allowed thanks to an automatically controlled fluidic system equipped with inlet/outlet connections for the electrolytic/washing solution and sample flow. Fifty mM Tricine, 20 mM CaCl2, 5 mM MgCl2, 50 mM NaCl, 70 mM sucrose, and pH 7.2 was used as the measuring buffer for the electrochemical analysis.

Pathogen Detection
The electrochemical detection of Escherichia coli (E. coli BL21), exploited as a case study pathogen, was provided by following algae oxygen evolution capacity at an applied potential of −0.6 V, using a dual electro-optical transducer prototype (Biosensor Srl, Via degli Olmetti, Rome, Italy). Algae were illuminated by a 350 µL photons m −2 s −1 light with repeated cycles of 30 s light excitation and 10 min dark. An applied potential of −0.6 V was used with an acquirement interval of 0.5 s. Pathogens were added into the electrochemical chamber (200 µL volume containing 50 mM Tricine, 20 mM CaCl2, 5 mM MgCl2, 50 mM NaCl, 70 mM sucrose pH 7.2) in a concentration range from 100 to 2000 CFU/100 mL and the current signals, due to oxygen production on the CB-SPE working electrode, were recorded in dependence to the target analyte concentrations.

Pathogen Detection
The electrochemical detection of Escherichia coli (E. coli BL21), exploited as a case study pathogen, was provided by following algae oxygen evolution capacity at an applied potential of −0.6 V, using a dual electro-optical transducer prototype (Biosensor Srl, Via degli Olmetti, Rome, Italy). Algae were illuminated by a 350 µL photons m −2 s −1 light with repeated cycles of 30 s light excitation and 10 min dark. An applied potential of −0.6 V was used with an acquirement interval of 0.5 s. Pathogens were added into the electrochemical chamber (200 µL volume containing 50 mM Tricine, 20 mM CaCl 2 , 5 mM MgCl 2 , 50 mM NaCl, 70 mM sucrose pH 7.2) in a concentration range from 100 to 2000 CFU/100 mL and the current signals, due to oxygen production on the CB-SPE working electrode, were recorded in dependence to the target analyte concentrations.

Effect of Wastewater Samples on the Alga C. Reinhardtii
With the aim to design an algal cytosensor for pathogen detection, the first step entailed the study of the effect of wastewater samples on the algal physiological parameters including the photosynthetic activity, the growth rate, and the pigment content. The green photosynthetic alga C. reinhardtii was thus grown in different water samples from 3 selected sites in the Adriatic region, i.e., Lignano, San Giorgio, and Pescara depuration plants (DPs).
In detail, optical density, cell number, chlorophyll a fluorescence, and the total chlorophyll content were measured. Results on growth (absorption at 750 nm and cell number/mL, Figure 2A,B) and pigment content ( Figure 2C) evidenced a slight influence of the Lignano water sample on algae cell grow in terms of altered vital processes and variations in the physiological parameters (e.g., cell duplication). On the contrary, a toxic effect from San Giorgio and Pescara water samples was evidenced on both algae growth (Figure 2A,B) and pigment content production (i.e., chlorophylls) ( Figure 2C). The photochemical efficiency of PSII was also evaluated through Kautsky curves as described in Section 2.3 "Algae physiological characterization", following the maximum fluorescence yield of Photosystem II F V /F M during the analyzed period of 9 days ( Figure 2D). In this case, no effect was registered regarding the maximum fluorescence yield, which remains constant during the time.

Effect of Wastewater Samples on the Alga C. Reinhardtii
With the aim to design an algal cytosensor for pathogen detection, the first step entailed the study of the effect of wastewater samples on the algal physiological parameters including the photosynthetic activity, the growth rate, and the pigment content. The green photosynthetic alga C. reinhardtii was thus grown in different water samples from 3 selected sites in the Adriatic region, i.e., Lignano, San Giorgio, and Pescara depuration plants (DPs).
In detail, optical density, cell number, chlorophyll a fluorescence, and the total chlorophyll content were measured. Results on growth (absorption at 750 nm and cell number/mL, Figure 2A,B) and pigment content ( Figure 2C) evidenced a slight influence of the Lignano water sample on algae cell grow in terms of altered vital processes and variations in the physiological parameters (e.g., cell duplication). On the contrary, a toxic effect from San Giorgio and Pescara water samples was evidenced on both algae growth ( Figure  2A,B) and pigment content production (i.e., chlorophylls) ( Figure 2C). The photochemical efficiency of PSII was also evaluated through Kautsky curves as described in Section 2.3 "Algae physiological characterization", following the maximum fluorescence yield of Photosystem II FV/FM during the analyzed period of 9 days ( Figure 2D). In this case, no effect was registered regarding the maximum fluorescence yield, which remains constant during the time.

Set-Up of the Algal Cytosensor and Assessment of the Analytical Parameters
The algal CB-SPEs and CB-SPEs immobilized with algae (using the protocol described in Section 2.3) were observed at Scanning Electron Microscopy (SEM) as reported in Figure 4A, which show microphotographs of CB (left) and algae whole cells entrapped into the calcium/alginate matrix (right) at a magnification of 2 µm. The algal/CB-SPEs were thus inserted into the measurement chamber of the biosensor prototype ( Figure 1) and amperometric measurements were accomplished for the detection of E. coli, a case study pathogen that can be found in wastewater. A scheme of the obtained algal/CB-SPE cytosensor was reported in Figure 4B. Once we have obtained the algal/CB-SPE cytosensor, all the analytical parameters were optimized. The best applied potential was set for monitoring the oxygen reduction signal generated by the algal activity both in the absence and in the presence of the target,

Set-Up of the Algal Cytosensor and Assessment of the Analytical Parameters
The algal CB-SPEs and CB-SPEs immobilized with algae (using the protocol described in Section 2.3) were observed at Scanning Electron Microscopy (SEM) as reported in Figure 4A, which show microphotographs of CB (left) and algae whole cells entrapped into the calcium/alginate matrix (right) at a magnification of 2 µm. The algal/CB-SPEs were thus inserted into the measurement chamber of the biosensor prototype ( Figure 1) and amperometric measurements were accomplished for the detection of E. coli, a case study pathogen that can be found in wastewater. A scheme of the obtained algal/CB-SPE cytosensor was reported in Figure 4B.

Set-Up of the Algal Cytosensor and Assessment of the Analytical Parameters
The algal CB-SPEs and CB-SPEs immobilized with algae (using the protocol described in Section 2.3) were observed at Scanning Electron Microscopy (SEM) as reported in Figure 4A, which show microphotographs of CB (left) and algae whole cells entrapped into the calcium/alginate matrix (right) at a magnification of 2 µm. The algal/CB-SPEs were thus inserted into the measurement chamber of the biosensor prototype ( Figure 1) and amperometric measurements were accomplished for the detection of E. coli, a case study pathogen that can be found in wastewater. A scheme of the obtained algal/CB-SPE cytosensor was reported in Figure 4B. Once we have obtained the algal/CB-SPE cytosensor, all the analytical parameters were optimized. The best applied potential was set for monitoring the oxygen reduction signal generated by the algal activity both in the absence and in the presence of the target, Once we have obtained the algal/CB-SPE cytosensor, all the analytical parameters were optimized. The best applied potential was set for monitoring the oxygen reduction signal generated by the algal activity both in the absence and in the presence of the target, in response to light exposure (λ = 650 nm, 350 µL m −2 s −1 intensity). The algal/CB-SPE was incubated for 10 min in the dark in a reaction volume of 200 mL of measuring buffer, and then stimulated by a light flash of 30 s of red LEDs, optically filtered to a peak wavelength of 650 nm. In this condition, the algal/CB-SPE generated peak current signals from 0.3 to 1.5 µA depending on the potential applied in the range from -0.8 to -0.3 V. As highlighted in Figure 5A, the ad hoc applied potential for the measurements light-induced oxygen evolution was equal to −0.6 V, which resulted in peak currents of 1.5 µA. This value was optimized using the best conditions obtained also for the light intensity and immobilized cell number, which were set to obtain the higher current signals at 350 µL photons m −2 s −1 intensity ( Figure 5B) and 0.8 × 10 6 immobilized cell number ( Figure 5C), respectively. in response to light exposure (λ = 650 nm, 350 µL m −2 s −1 intensity). The algal/CB-SPE was incubated for 10 min in the dark in a reaction volume of 200 mL of measuring buffer, and then stimulated by a light flash of 30 s of red LEDs, optically filtered to a peak wavelength of 650 nm. In this condition, the algal/CB-SPE generated peak current signals from 0.3 to 1.5 µA depending on the potential applied in the range from -0.8 to -0.3 V. As highlighted in Figure 5A, the ad hoc applied potential for the measurements light-induced oxygen evolution was equal to −0.6 V, which resulted in peak currents of 1.5 µA. This value was optimized using the best conditions obtained also for the light intensity and immobilized cell number, which were set to obtain the higher current signals at 350 µL photons m −2 s −1 intensity ( Figure 5B) and 0.8 × 10 6 immobilized cell number ( Figure 5C), respectively.

Algal Cytosensor Analytical Response to Pathogens
Algal/CB-SPEs were incubated in the dark with E. coli at a concentration of 1000 CFU/100 mL from 5 to 60 min, to evaluate the incubation time at which a higher algae oxygen production occurs due to the presence of bacteria, which reduce the photosynthetic oxygen tension within the microenvironment of the algal cells. Indeed, within a short incubation time from 5 and 15 min, an increase of the current signals was observed ( Figure 6A), while at higher incubation time a balance of algal oxygen evolution and oxygen sequestration by bacteria was observed, thus providing current signals comparable to algae oxygen production in the absence of bacteria. Considering the results reported in Figure 6A, an incubation time of 15 min was selected.

Algal Cytosensor Analytical Response to Pathogens
Algal/CB-SPEs were incubated in the dark with E. coli at a concentration of 1000 CFU/100 mL from 5 to 60 min, to evaluate the incubation time at which a higher algae oxygen production occurs due to the presence of bacteria, which reduce the photosynthetic oxygen tension within the microenvironment of the algal cells. Indeed, within a short incubation time from 5 and 15 min, an increase of the current signals was observed ( Figure 6A), while at higher incubation time a balance of algal oxygen evolution and oxygen sequestration by bacteria was observed, thus providing current signals comparable to algae oxygen production in the absence of bacteria. Considering the results reported in Figure 6A, an incubation time of 15 min was selected. in response to light exposure (λ = 650 nm, 350 µL m −2 s −1 intensity). The algal/CB-SPE was incubated for 10 min in the dark in a reaction volume of 200 mL of measuring buffer, and then stimulated by a light flash of 30 s of red LEDs, optically filtered to a peak wavelength of 650 nm. In this condition, the algal/CB-SPE generated peak current signals from 0.3 to 1.5 µA depending on the potential applied in the range from -0.8 to -0.3 V. As highlighted in Figure 5A, the ad hoc applied potential for the measurements light-induced oxygen evolution was equal to −0.6 V, which resulted in peak currents of 1.5 µA. This value was optimized using the best conditions obtained also for the light intensity and immobilized cell number, which were set to obtain the higher current signals at 350 µL photons m −2 s −1 intensity ( Figure 5B) and 0.8 × 10 6 immobilized cell number ( Figure 5C), respectively.

Algal Cytosensor Analytical Response to Pathogens
Algal/CB-SPEs were incubated in the dark with E. coli at a concentration of 1000 CFU/100 mL from 5 to 60 min, to evaluate the incubation time at which a higher algae oxygen production occurs due to the presence of bacteria, which reduce the photosynthetic oxygen tension within the microenvironment of the algal cells. Indeed, within a short incubation time from 5 and 15 min, an increase of the current signals was observed ( Figure 6A), while at higher incubation time a balance of algal oxygen evolution and oxygen sequestration by bacteria was observed, thus providing current signals comparable to algae oxygen production in the absence of bacteria. Considering the results reported in Figure 6A, an incubation time of 15 min was selected. To obtain a calibration curve for the detection of the target bacterium, algal/CB-SPEs were incubated for 15 min in the dark with E. coli in a concentration range from 100 to 2000 CFU/100 mL. Then, a light flash of 30 s of red LEDs (optically filtered to a peak wavelength of 650 nm at an intensity of 350 µL photons m −2 s −1 ) was applied to stimulate the algal photosynthetic light-induced oxygen evolution. An increase of the oxygen evolution and thus of the current signals was registered in the presence of the increasing pathogen concentration ( Figure 6B), obtaining a linear response and allowing for the construction of a calibration curve described by the equation y = 1.530 (±0.059) − 0.00060 (±0.00005) x, with an R 2 = 0.985 ( Figure 6C). A detection limit of 92 CFU/100 mL was achieved (LOD = 3 × sd/slope). The linear range and the LOD found can be considered coherent with the maximum E. coli concentration suggested by Italian law for wastewater (less than 5000 CFU/100 mL) [25].
To test the algal cytosensor in wastewater samples, its selectivity was evaluated analyzing metals (10 ppb arsenic, 1.3 ppb copper, 5 ppb cadmium, 10 ppb lead), pesticides (1 ppb paraoxon), and phenolic compounds (10 ppb bisphenol A) at legal limits established by the European legislations for surface water (where present) as interferents that should be present in wastewater from depuration plants [26]. The results reported in Figure 7A highlighted that the interfering species did not affect the analysis of E. coli at the tested concentrations, unless atrazine, which is, as a photosynthetic herbicide, the specific target of the alga. However, the presence of such a herbicide can also be analyzed by chlorophyll fluorescence simultaneously to the amperometric measurements, exploiting the optical module of the biosensor prototype (Figure 1), thus supporting the amperometric analysis of bacteria. algal photosynthetic light-induced oxygen evolution. An increase of the oxygen evolution and thus of the current signals was registered in the presence of the increasing pathogen concentration ( Figure 6B), obtaining a linear response and allowing for the construction of a calibration curve described by the equation y = 1.530 (±0.059) − 0.00060 (±0.00005) x, with an R 2 = 0.985 ( Figure 6C). A detection limit of 92 CFU/100 mL was achieved (LOD = 3 × sd/slope). The linear range and the LOD found can be considered coherent with the maximum E. coli concentration suggested by Italian law for wastewater (less than 5000 CFU/100 mL) [25].
To test the algal cytosensor in wastewater samples, its selectivity was evaluated analyzing metals (10 ppb arsenic, 1.3 ppb copper, 5 ppb cadmium, 10 ppb lead), pesticides (1 ppb paraoxon), and phenolic compounds (10 ppb bisphenol A) at legal limits established by the European legislations for surface water (where present) as interferents that should be present in wastewater from depuration plants [26]. The results reported in Figure 7A highlighted that the interfering species did not affect the analysis of E. coli at the tested concentrations, unless atrazine, which is, as a photosynthetic herbicide, the specific target of the alga. However, the presence of such a herbicide can also be analyzed by chlorophyll fluorescence simultaneously to the amperometric measurements, exploiting the optical module of the biosensor prototype (Figure 1), thus supporting the amperometric analysis of bacteria.   The calibration curve obtained for the algal cytosensor in real samples was further used to calculate the recovery values of the surface water samples. Recovery values of 105 ± 8, 83 ± 7, and 88 ± 7% were obtained for 1000 CFU/100 mL of E. coli for Lignano, San Giorgio, and Pescara wastewater samples, respectively.
The storage stability of the algal cytosensor was evaluated by storing the algal/CB-SPEs in the measuring buffer at room temperature under continuous light at 50 µL photons m −2 s −1 for 21 days. Amperometric analysis at the defined time intervals of each SPE recorded in a 200 µL volume of measuring buffer showed that detectable loss of the photosynthetic activity occurred after 21 days ( Figure 7C). Working stability was assessed by amperometric measurements run on the algal/CB-SPEs up to 12 h at room temperature, under repeated cycles of 10 min dark and 30 s light of red LEDs, showing 100% intra-electrode repeatability of light-induced oxygen evolution activity. Moreover, the preparation of the algal/CB-SPEs showed a high inter-electrode repeatability with RSD of 1.1% (n = 12) (data not shown).

Discussion and Conclusions
In this work, a cytosensor based on microalgae has been developed for the detection of E. coli presence in water outputs from wastewater treatment plants. The system, constituted of C. reinhardtii whole cells immobilized on the carbon black-modified working electrode of SPEs, detects the oxygen produced by the microalgae during the photosynthetic cycle when a potential of −0.6 V is applied in a chronoamperometry measurement. It has been demonstrated [24] that the presence of bacteria strains in growing microalgae solutions promote oxygen production, with the promotion effect not dependent by the specific bacteria strain. This phenomenon has been used in this case to develop a biosensor capable of quickly determining the presence of bacteria in water downstream from wastewater treatment plants. In particular, the assembled system can detect the presence of E. coli down to a LOD of 92 CFU/100 mL in real wastewater, with a linear response obtained in the range 100 to 2000 CFU/100 mL, after only 15 min of incubation time. The values found are in line with those obtained in similar works in the literature [27][28][29][30][31][32]. It should be underlined that this bacterium was exploited as a case study target of the algal cytosensor to demonstrate its ability as an early warning analytical system to signal heavy loads of pathogens in waters leaving the wastewater treatment plants. Indeed, the cytosensor is not selective towards E. coli but it is capable to sense all the bacteria that induce the algae oxygen evolution by exploiting the effect of their interaction. On the contrary, the cytosensor shows the advantages of not being affected from other pollutants commonly present in wastewaters and, in addition, any pretreatment of the sample is required before the analysis, resulting in an easy integration for on-line microorganism monitoring.