Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 μA cm(-2) with ∼150 μg ml(-1) phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions.

Importance: Microbial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial community to access a solid electrode as an alternative electron acceptor. To better understand the ecological relationships between mediator producers and mediator utilizers, we here present a comparison of the phenazine-dependent electroactivities of three Pseudomonas strains. This work forms the foundation for more complex coculture investigations of mediated electron transfer in microbial fuel cells.

FIG 1

Carbon substrate consumption (a, b, and c) and metabolite formation time profiles (d, e, and f) for three P. aeruginosa strains in oxygen-limited bioelectrochemical experiments. All data are means and standard deviations for three biological replicates, except those for KRP1 with 2,3-BD, which include only duplicates. The lower reported concentrations of ethanol for PAO1 (the initial concentration was also 30 mM) are due to strong evaporation during sampling and HPLC analysis.

FIG 2

Current generation and phenazine production by P. aeruginosa PA14, KRP1, and PAO1 grown with the substrates glucose, 2,3-BD, and ethanol. Data are from three biological replicates, except those for KRP1 with 2,3-BD, which are in duplicates. Note the different current density axis scaling for PA14 with 2,3-BD, KRP1 with glucose, and KRP1 with 2,3-BD.

FIG 3

Carbon substrate uptake by cultures supplied with equimolar glucose and 2,3-BD (a, b, and c) and related electric current generation and phenazine concentrations (d, e, and f) for strains PA14, KRP1, and PAO1. The vertical dashed lines in panels a, b, d, and e represent the time of substrate switch from glucose to 2,3-BD. All data are means and standard deviation for three biological replicates.

FIG 4

Cyclic voltamograms for the three P. aeruginosa strains grown with the three carbon sources. Plots show a representative blank scan (immediately after inoculation), one scan during increasing electrochemical activity, and one scan during maximum electrochemical activity for one representative biological replicate for each condition.

FIG 5

Phenazine utilization assay with defined phenazine concentrations. The bar graphs show the relative increase in current generation (at 0.2 V) with the addition of defined concentrations of phenazines to a resuspension of washed cells of strains PA14 (a), PA14 Δphz (b), KRP1 (c), and PAO1 (d). The dashed horizontal line at 100% highlights the activity of the cells before phenazine addition. Solid bars represent cultures with 30 mM glucose, and crosshatched bars represent cultures without glucose addition. Values at 1 min, 30 min, and 60 min after phenazine addition are reported.

FIG 6

(a) Observed maximum current densities for P. aeruginosa strains PA14, KRP1, and PAO1 grown in the presence of glucose, 2,3-BD, ethanol, or equimolar glucose and 2,3-BD. Values are averages from three independent biological replicates, except those for KRP1 with 2,3-BD, which are in duplicates. (b) Photograph of the bioelectrochemical setup used.