Bioelectrochemical detection of cyanobacteria for harmful bloom management

Abstract

High levels of nutrients and increasing temperatures favour the periodic proliferation of harmful cyanobacterial blooms with catastrophic effects for aquatic ecosystems. The production of toxins that occurs naturally as secondary metabolites is a major concern for public health. Effective in situ monitoring of cyanobacterial populations is therefore essential. Various cyanobacteria species have been shown to carry out extracellular electron transfer (EET) to insoluble acceptors – e.g. to manage reductive stress caused by excessive light exposure – and are thus able to produce a measurable electrical current.This PhD project focused on exploring cyanobacterial EET to set foundations for the development of a cyanobacteria-monitoring device for early detection and management of harmful blooms. This technology exploits cyanobacteria’s ability to produce electrical current to monitor these microorganisms in water reservoirs. Yet, occurrence of EET in cyanobacteria is not fully understood. There is little information on the redox potential of the reactions involved and their dependence on i.e. pH and illumination. Furthermore, there is no available method for efficient current collection from a diluted cyanobacteria suspension like those that inhabit water reservoirs.Hence, the following research objectives were pursued: (i) to investigate driving factors for EET occurrence in cyanobacteria, (ii) to elucidate the redox potential of the reactions involved in EET for different cyanobacteria species and their pH dependence, (iii) to develop a cyanobacteria sensor prototype. Three-electrode photoelectrochemical cells were used, with working electrodes poised at +0.6V vs. SHE for chronoamperometric measurements, and potential scans between -0.1 V vs. SHE and 1 V vs. SHE for all voltammetric techniques.The first research objective focused on exploring the effect of increased reductive stress triggered by illumination and absence of electron acceptors such as O2 and CO2, and the effect of pH on current generation. Observations confirmed that EET onto polarised electrodes is triggered by increasing reductive stress, and also by medium pH levels above 7.8 for the cyanobacteria species Microcystis aeruginosa. Current density increased with stepwise pH increases from about 5 mA m-2 at pH 7.8 to 30 mA m-2 at pH 10.5, for dense (0.4 mg mL-1) Microcystis aeruginosa suspensions with dissolved CO2 and O2 approaching equilibrium with atmospheric concentrations. This rise in current density was greater for suspensions subject to higher reductive stress, i.e. with negligible dissolved CO2 and O2. Current density under illumination for these suspensions increased stepwise with pH from 5 mA m-2 at pH 7.8 to 40 mA m-2 at pH 10.2. Electrochemical analysis of EET from Microcystis aeruginosa through cyclic voltammetry reveals a shift of the reaction’s redox potential to lower values with increasing pH, thus supporting a current density rise with pH. A pH-driven current surge was not observed for Anabaena circinalis or Synechocystis sp., suggesting that this phenomenon is not universal for all cyanobacterial species and that different species rely on different methods of extracellular electron transport.Research objective (ii) explored the EET process of different species of cyanobacteria to shed some light onto the redox reactions involved in current generation for each of them. The freshwater species studied, Anabaena circinalis, Microcystis aeruginosa and Synechococcus sp. produce characteristic non-turnover voltammograms with significantly different redox peaks. The midpoint potentials of the redox reactions driving EET at pH 7.8 for Synechococcus sp., Microcystis aeruginosa, and Anabaena circinalis were +0.59 ± 0.01 V vs SHE, +0.61 ± 0.01 V vs SHE for, and +0.75 ± 0.01 V vs SHE, respectively. Furthermore, pH dependency was observed for EET in Microcystis aeruginosa and Synechocuccus sp., both suggesting a 1:1 proton-electron redox reaction.The final stage of this project aimed at developing a sensor prototype that would detect Microcystis aeruginosa at cell densities as low as 2,000 cell mL-1, the recommended WHO alert level cell count for any cyanobacteria in recreational water. In order to overcome the limitations of cell-electrode contact in diluted cell suspensions characteristic of actual cyanobacteria-containing waters, the concept of a filter-electrode was introduced, where a given cell suspension is filtered using a conductive filter (carbon nanotube-coated glass fibre), which acts as a working electrode and is poised at +0.6 V vs. SHE. This method produces current densities that are roughly proportional to the cell load onto the electrode and therefore to the cell density of the initial suspension. The prototype was able to measure a current density of about 0.43 mA m-2 under illumination (200 µmol m-2 s-1 of PAR), from filtering 15L of a diluted sample of 2000 cell mL-1 of Microcystis aeruginosa, which corresponded to a filter-electrode load of 0.36 mg. In summary, the design of an efficient setup for concentrating cells onto an electrode, coupled with a better understanding of the driving factors and characteristics of current generation for different cyanobacteria species drives us closer to developing an in situ sensor, and moreover provide useful information and methodology for understanding current generation in cyanobacteria and other phototrophs.