Robust measurement of microbial reduction of graphene oxide nanoparticles using image analysis

<i>Shewanella oneidensis</i> (<i>S. oneidensis</i>) has the capacity to reduce electron acceptors within a medium and is thus used frequently in microbial fuel generation, pollutant breakdown, and nanoparticle fabrication. Microbial fuel setups, however, often require costly or labor-intensive components, thus making optimization of their performance onerous. For rapid optimization of setup conditions, a model reduction assay can be employed to allow simultaneous, large-scale experiments at lower cost and effort. Since <i>S. oneidensis</i> uses different extracellular electron transfer pathways depending on the electron acceptor, it is essential to use a reduction assay that mirrors the pathways employed in the microbial fuel system. For microbial fuel setups that use nanoparticles to stimulate electron transfer, reduction of graphene oxide provides a more accurate model than other commonly used assays as it is a bulk material that forms flocculates in solutions with a large ionic component. However, graphene oxide flocculates can interfere with traditional absorbance-based measurement techniques. This study introduces a novel image analysis method for quantifying graphene oxide reduction, showing improved performance and statistical accuracy over traditional methods. A comparative analysis shows that the image analysis method produces smaller errors between replicates and reveals more statistically significant differences between samples than traditional plate reader measurements under conditions causing graphene oxide flocculation. Image analysis can also detect reduction activity at earlier time points due to its use of larger solution volumes, enhancing color detection. These improvements in accuracy make image analysis a promising method for optimizing microbial fuel cells that use nanoparticles or bulk substrates.IMPORTANCE<i>Shewanella oneidensis</i> (<i>S. oneidensis</i>) is widely used in reduction processes such as microbial fuel generation due to its capacity to reduce electron acceptors. Often, these setups are labor-intensive to operate and require days to produce results, so use of a model assay would reduce the time and expenses needed for optimization. Our research developed a novel digital analysis method for analysis of graphene oxide flocculates that may be utilized as a model assay for reduction platforms featuring nanoparticles. Use of this model reduction assay will enable rapid optimization and drive improvements in the microbial fuel generation sector.