Extracellular electron transfer by Microcystis aeruginosa is solely driven by high pH

Highlights

• Extracellular electron transfer by Microcystis aeruginosa is triggered by high pH.

• Current densities increased from 5 to 40 mA m⁻² as the pH rose from 7 to 10.

• Light intensity does not affect extracellular electron transfer in M. aeruginosa.

Abstract

Extracellular electron transfer (EET) by the cyanobacterium Microcystis aeruginosa was investigated. Observations indicate that EET onto an electrode poised at + 0.6 vs. standard hydrogen electrode (SHE) is triggered by high pH, more evidently at pH levels above 9. Light intensity does not appear to affect electricity generation, indicating that this may not be a “biophotovoltaic” process. The generated current density was amplified with stepwise pH increases from approximately 5 mA m⁻² at pH 7.8 to 30 mA m⁻² at pH 10.5, for dense (0.4 mg mL⁻¹ dry weight) Microcystis aeruginosa suspensions with dissolved CO₂ and O₂ approaching equilibrium with atmospheric concentrations. The upsurge in current density was more pronounced (from 5 mA m⁻² at pH 7.8 to 40 mA m⁻² at pH 10.2) in the absence of the cells’ natural electron acceptors, dissolved CO₂ and O₂. However, the latter effect is more likely due to competition for electrons by oxygen than to reductive stress. EET in this species is therefore a light-independent process that is enhanced by increasing pH, with reasons that are still unknown, but either related to the involvement of protons in the last step of electron transfer, or to intracellular pH control.

Introduction

Cyanobacteria were the first microorganisms on Earth to perform photosynthesis and continue to thrive in ever-changing environmental conditions. In present times, they account for 20–30% of global photosynthetic productivity converting solar energy into chemical energy [1].

Like many other microorganisms, they were found to be able to transfer electrons onto insoluble electron acceptors, a phenomenon called extracellular electron transfer (EET) [2]. EET can be quantified in the form of anodic electrical current when a conductive solid material, or electrode, is used as the terminal electron acceptor[3]. This capacity has allowed for their use in biophotovoltaics, emerging technologies for sustainable energy generation, where photosynthetic biomaterials are used to convert solar energy into electrical energy [4].

Although electricity generation from cyanobacteria had been reported since 1980s, there is a significant gap in the mechanistic understanding of the underlying electron transfer pathways that result in electricity generation [5]. Several attempts have been made to elucidate the EET pathways in cyanobacteria by means of site-specific photosynthesis inhibitors [6], [7], [8]. Cyanobacteria can generate electric current from both photosynthesis and respiration processes and can therefore produce current both under illumination and in the dark (see Schematic 1) [1]. They possess a diverse range of electron transport pathways which are extended by the requirement to dissipate overflows of electrons caused by excessive illumination and a variety of other environmental factors [9]. Light-dependent reactions occur in the photosynthetic electron transfer chain (PETC) located in thylakoid membranes, and use inorganic carbon in the form of CO₂ as the main electron acceptor for the electrons generated in the photolysis of water [10]. On the other hand, the respiratory electron transport chain (RETC) occurs both in the thylakoid and cytoplasmic membranes with O₂ as the main electron acceptor. PETC and the RETC occur beside each other in the thylakoid membranes and share redox components, such as plastoquinone, cytochrome b6f and plastocyanin, and are further connected through soluble charge carriers that diffuse and can coexist in the thylakoid membranes [4], [5]. This is of great value to cyanobacteria’s adaptability since at high light intensities or low CO₂, the over-reduced PETC can be re-oxidized by RETC, which assists in preventing photo-induced damage to the photosynthetic machinery [5].

There is to date no agreement on the reasons behind the occurrence of EET. One hypothesis correlates EET with reductive stress caused by excessive illumination and the lack of electron acceptors for PETC and RETC [11]. In fact, marine phototrophic consortia were shown to be able to use an external electron acceptor as an electron sink, as a response to the absence of inorganic carbon [10], suggesting that EET could be related to an increase in reductive stress caused by the absence of CO₂. Furthermore, photosynthetically evolved O₂ was shown to help alleviate reductive stress by re-oxidising electron carriers, and its removal shown to enhance anodic current generation [3].

Another hypothesis links EET to the need for cyanobacteria to reduce extracellular ferric minerals to more soluble ferrous iron for assimilatory uptake. EET has been correlated with the production of organic exudates that contain reducing moieties for Fe(III) by Microcystis aeruginosa, possibly to facilitate iron uptake [12], [13]. In fact, cyanobacteria Synechococcus elongatus PCC942 shows enhanced capability to reduce ferricyanide, a widely-used soluble redox mediator, when grown under iron-limiting conditions in comparison to iron sufficient conditions [14]. Ferricyanide is reduced by ferric reductases in the cytoplasmic membrane which are overexpressed in iron-limited growth and operate optimally at an acidic pH [15]. Therefore, ferricyanide reduction and consequent current generation decrease at higher pH [14].

The wide range of external factors correlated with the occurrence of EET in cyanobacteria is a consequence of the complexity if this process. This work further elucidates the effects of environmental factors on EET occurrence. Microcystis aeruginosa is one of the most common harmful bloom-forming cyanobacteria, and like most bloom-forming cyanobacteria, it is resilient and dominate in blooming events where photosynthetic activity depletes CO₂, causing a significant rise in pH and making bicarbonate the most abundant inorganic carbon species up to pH 10 [16]. This study aimed to elucidate the effect of alkaline pH conditions and reductive stress induced by CO₂ limitation on anodic current production by Microcystis aeruginosa, using a graphite rod as electrode and an osmium polymer immobilized onto it as a redox mediator.

Osmium redox polymers have been attracting attention due to their synthetic flexibility and thus possibility to manipulate their formal potential and hydrophilicity/ hydrophobicity and electron shuttling properties. Furthermore, they overcome problems associated with the use of soluble mediators [17], [18] such as the need for continuous replacement and in some cases even potential harm to the environment [19], and are therefore better suited for an in situ sensing device.