Screening of natural phenazine producers for electroactivity in bioelectrochemical systems

Abstract

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 μg/ml mediating an anodic current of 15.1 μA/cm² . On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 μA/cm² . Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.

Figure 1

(A) Three‐electrode BES configuration for the exploration of potential EAB through self‐produced phenazines as electron shuttles to mediate electron transfer. Green cells, limited to growing on the anode perform direct electron transfer (green arrow). Planktonic cells (all other colours to show diversity), occupying the whole reactor volume, perform mediated electron transfer to the (distant) anode through successive reduction and oxidation cycles (red and blue arrows) of phenazines. Working, reference and counter electrode (WE, RE and CE respectively). I, V, represent the potentiostat connections. (B) Key steps of the phenazine biosynthesis pathway. 2‐OH‐PHZ, 2‐hydroxy‐phenazine; ADIC, 2‐amino‐2‐deoxyisochorismic acid; AOCHC, (1R,6 S)‐6‐amino‐5‐oxo‐2‐cyclohexene‐1‐carboxylic acid; DHHA, (2 S,3 S)‐2,3‐dihydro‐3‐hydroxy anthranilic acid; DHPCA, dihydro‐phenazine‐1‐carboxylic acid; HHPCD, hexahydrophenazine‐1,6‐dicarboxylic acid; PCA, phenazine‐1‐carboxylic acid; PCN, phenazine‐1‐carboxamide; PYO, pyocyanin.

FIGURE 2

Maximum current density, phenazine concentration, CE and biomass formation in BES. (+) Indicates the addition of 20 μg/ml of PCA to the reactors. Data represent the mean and standard deviation of three independent replicates for experiments with P. chlororaphis subsp. aurantiaca and N. chromatogenes. For P. putida and P. aeruginosa data represent the mean and its deviation. One replicate was done for B. cepacia, S. thioluteus and B. iodinum. Biomass information for P. putida was not calculated.

FIGURE 3

Current generation and phenazine quantification over time by P. chlororaphis subsp. aurantiaca (A) without and (B) with the addition of 20 μg/ml PCA. Inoculation of the reactors and PCA addition occurred after 22 h (dotted line in A and B). Optical density (OD), pH and glucose consumption are also shown. Data represent the mean and standard deviation of three replicates. Representative voltammograms with the peak systems (PS) obtained at the point of the highest electric current generated by P. chlororaphis subsp. aurantiaca (C) without and (D) with the addition of 20 μg/ml PCA.

FIGURE 4

Charge distribution of reducing equivalents derived from glucose oxidation and discharged to the anode or transformed into biomass, metabolites and the unaccounted fraction. The balance was calculated based on the final dried biomass. Data represent the mean and standard deviation of three replicates.

FIGURE 5

Current generation and phenazine quantification over time by N. chromatogenes (A) without and (B) with the addition of 20 μg/ml PCA. Inoculation of the reactors and PCA addition was done after 22 h (dotted line). Optical density (OD), pH and glucose consumption are also shown. Data represent the mean and standard deviation of three replicates. Current drop‐in (B) occurred due to a reactor operation disturbance in one replicate. Representative voltammograms were obtained at the point of the highest electric current generated by N. chromatogenes (C) without and (D) with the addition of 20 μg/ml PCA.

FIGURE 6

Current generation by N. chromatogenes to determine the main mechanism of EET. Current recovery was followed after (A) new electrodes were placed into the original medium and (B) biofilm‐covered electrodes from the original reactors were placed into fresh medium (grey and blue zones respectively). The initial 8 days are identical in both figures since they represent the first days of the current generation in the original reactors. Results show the mean of two replicates and error bars indicate the deviation of the mean.