Exploiting algal NADPH oxidase for biophotovoltaic energy

Abstract

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

Keywords: Chlamydomonas; NADPH oxidase; alga; biophotovoltaic; energy.

Figure 1

BPV design and function. (a) Image of assembled BPV device containing Chlamydomonas reinhardtii cells in the anodic chamber. Scale bar, 1 cm. (b) Exploded schematic of the anodic chamber. The front clamp (i) permits light to enter via the anode (ii) into the algal chamber (iii) and the back clamp (v) has a cut‐out to allow oxygen contact with the cathode (iv). (c) Principles of operation of a BPV device. Upon illumination, cells (green ovoid) within the algal chamber release electrons (e⁻) which reduce extracellular ferricyanide ([Fe(CN)₆]⁴⁻) to ferrocyanide ([Fe(CN)₆]³⁻). Ferrocyanide shuttles electrons to the anode (red rectangle, ii) into the external circuit via an external resister and multimeter (V) through to the cathode (blue rectangle, iv). Simultaneously, protons (H⁺) diffuse from the chamber via a dialysis and Nafion membrane (dotted line) to the cathode combining with electrons and oxygen to form water. Cells are kept in suspension by a magnetic stirrer (S) and evaporation limited by sealing chamber (B). Wires are connected via crocodile clips to two stainless steel strips. Numbers above selected components in (b) correspond to those shown in (c).

Figure 2

Comparison of plant and algal NADPH oxidase structures. (a) Plant NADPH oxidase (RBOH) structure based on sequence data from Arabidopsis thaliana and other plant NOX described in Torres et al. (1998) and Kawahara et al. (2007). (b) Algal RBO structure based on sequence data from Chondrus crispus, Cyanidioschyzon merolae, Porphyra Yezoensis and Phaeodactylum tricornutum described in Hervé et al. (2005). NOX in these algae have an additional four transmembrane domains in between the NADPH‐binding domain. These are absent from the Chlamydomonas RBO. (c) Schematic of RBO proteins CrRBO1 and CrRBO2 compared to Arabidopsis thaliana AtRBOHC. Abbreviations: EF, EF hand; F, FAD‐binding domain; N, NADPH‐binding domains; TM, transmembrane domain. Dashed TM domains indicate the presence of conserved haem‐binding histidine residues. Proteins are orientated with the cytosolic side at bottom of image. Orange cylinders represent transmembrane domains, green circles represent EF hands, and yellow diamonds represent haem molecules. Binding regions for FAD and NADPH are highlighted.

Figure 3

Chlamydomonas reinhardtii extracellular superoxide anion production is light‐dependent and DPI sensitive. Time course of ${\mathrm{O}}_2^{-}$ production by mid‐logarithmic cells of the Chlamydomonas reinhardtii strain cw92, determined using XTT reduction. Mean ± SEM ${\mathrm{O}}_2^{-}$ production was prevented by dark incubation and inhibited in the light by DPI (20 μm) as a NOX inhibitor. The equivalent dimethylsulphoxide (DMSO) concentration was used as the DPI solvent control (0.0075% v/v). Asterisks denote significant difference (P < 0.05) from control (Student's t‐test; n/s, no significance; n = 3).

Figure 4

Chlamydomonas reinhardtii extracellular superoxide anion production requires RBO1. (a) Time course of ${\mathrm{O}}_2^{-}$ production by mid‐logarithmic cells of the Chlamydomonas reinhardtii cell wall‐deficient strains cw15, cw92 (both STA6RBO1) and the RBO1‐deficient sta6rbo1 mutant, determined using XTT reduction. Levels of ${\mathrm{O}}_2^{-}$ production were indistinguishable between both cell wall‐less strains cw15 and cw92. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3). (b) DPI (20 μm) inhibited light‐dependent ${\mathrm{O}}_2^{-}$ production by all strains. Values are for 120 min. (one‐way ANOVA, P < 0.05; mean ± SEM, n = 3). There was no effect of equivalent concentration (v/v) of DMSO as the solvent for DPI. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3).

Figure 5

Superoxide anion production is restored by RBO1. (a) Validation of pSL18_RBO1 insertion into sta6rbo1 (CC‐4348) genome. To verify integration of the RBO1‐containing construct, PCRs were performed using primers specific to the plasmid. The predicted amplicon size is 1979 bp. (b) Mean ± SEM ${\mathrm{O}}_2^{-}$ production in light (120 min) from cw15, RBO1‐deficient sta6, two lines of sta6rbo1(STA6) (Blaby et al., 2013) and the three RBO1‐complemented lines shown in (a), determined using XTT (n ≤ 5). Only the presence of RBO1 was effective in restoring ${\mathrm{O}}_2^{-}$ production. Asterisks denote significant difference (P < 0.05) from indicated control (Student's t‐test; n/s, no significance).

Figure 6

RBO1‐dependent current from Chlamydomonas reinhardtii. (a) Example current from the cw15 parental strain in the BPV device. Yellow indicates light application; LE1 and LE2 denote the first and second light effect (LE) current. (b) Mean ± SEM of LE1 and LE2 currents from Chlamydomonas reinhardtii cultures (n = 3–4), standardized to chlorophyll (Chl) content. sta6rbo1(RBO1)‐c5 is one of the three strains complemented with RBO1 and exhibiting normal ${\mathrm{O}}_2^{-}$ production (Figure 5b). Letters denote significant differences as determined by one‐way ANOVA (P < 0.05).