A 96-well high-throughput, rapid-screening platform of extracellular electron transfer in microbial fuel cells

Microbial extracellular electron transfer (EET) stimulates a plethora of intellectual concepts leading to potential applications that offer environmentally sustainable advances in the fields of biofuels, wastewater treatment, bioremediation, desalination, and biosensing. Despite its vast potential and remarkable research efforts to date, bacterial electrogenicity is arguably the most underdeveloped technology used to confront the aforementioned challenges. Severe limitations are placed in the intrinsic energy and electron transfer processes of naturally occurring microorganisms. Significant boosts in this technology can be achieved with the growth of synthetic biology tools that manipulate microbial electron transfer pathways and improve their electrogenic potential. In particular, electrogenic Pseudomonas aeruginosa has been studied with the utility of its complete genome being sequenced coupled with well-established techniques for genetic manipulation. To optimize power density production, a high-throughput, rapid and highly sensitive test array for measuring the electrogenicity of hundreds of genetically engineered P. aeruginosa mutants is needed. This task is not trivial, as the accurate and parallel quantitative measurements of bacterial electrogenicity require long measurement times (~tens of days), continuous introduction of organic fuels (~tends of milliliters), architecturally complex and often inefficient devices, and labor-intensive operation. The overall objective of this work was to enable rapid (<30 min), sensitive (>100-fold improvement), and high-throughput (>96 wells) characterization of bacterial electrogenicity from a single 5 μL culture suspension. This project used paper as a substratum that inherently produces favorable conditions for easy, rapid, and sensitive control of an electrogenic microbial suspension. From 95 isogenic P. aeruginosa mutant, an hmgA mutant generated the highest power density (39 μW/cm²), which is higher than that of wild-type P. aeruginosa and even the strongly electrogenic organism, Shewanella oneidensis (25 μW/cm²). In summary, this work will serve as a springboard for the development of novel paradigms for genetic networks that will help develop mutations or over-expression and synthetic biology constructs to identify genes in P. aeruginosa and other organisms that enhance electrogenic performance in microbial fuel cells (MFCs).