High Throughput Screening of Electroactive Bacteria Using a Cross-Bar Architecture in a 3D Design Array

Abstract

Promoting extracellular electron transfer (EET) in bacteria has many widespread applications including wastewater treatment and environmental remediation. With the development of synthetic biology technologies that can alter microbial electron transfer routes and enhance their electrogenic capacity, there is a need for high throughput systems to identify the responsible genes and the consequent metabolic pathways in an expeditious way. Conventional platforms utilizing single electrodes, or electrochemical cells, and colorimetric detection suffer from low sensitivity and low throughput. Moreover, they require bulky equipment for readout and fluid handling. Moving towards a nuanced miniaturized electrochemical detection, we propose individually addressable microwells used to monitor electron flux from EET-capable bacteria with the ability to screen a large number of electroactive bacteria for various applications. Materials and method : The device is simple and fabricated with cost-effective electrodes. Carbon felt is used as the working electrode to improve bacterial entrapment and assist in capturing maximum electrons donated by the electroactive bacteria. Silver-silver chloride ink (Ag/AgCl) as the reference electrode will help maintain the potential of with respect to the working electrode. Conductive carbon ink as the counter electrode will promote current collection. The proposed device has the elements of a traditional three electrode system arranged in two different planes resulting in a 3D configuration of electrodes. The reference and counter electrode are on the bottom plane with acrylic wells housed right above each electrode pair, while the working electrode is used in the cross-bar architecture from the top plane as shown in Figure 1(a). The fabrication steps are illustrated in Figure 1(b). This system can be scaled to have 10000 microwells on a single platform with individual addressability of each micro-well array. With this layout, connections from the innermost wells can be drawn effortlessly without any signal damping from the surrounding wells. As a proof of concept, we have fabricated a 9 by 9 array (20cm by 20 cm) as shown in Figure 1(a). Five strains of Shewanella oneidensis MR-1, the wild type organism and four modified strains overexpressing variants of MtrA (unmodified MtrA, IV-205, IV-261 and an empty vector), were screened for their current producing capacity [1]. Results:A bacterial load of OD600= 0.065 was sufficient to give a reliable current signal. For each strain, the working electrode was biased at 0.205 V with respect to the reference electrode (Ag/AgCl), and the resulting chronoamperometry signal was recorded as shown in Figure 1(c). The values obtained helped identify the current-producing capacity of each mutant. Among them, the highest peak current was given by mtr A+ (20 μA), and IV – 261 gave a low peak current (2 μA) within 3 minutes, validating the difference in the genetic make-up and EET capabilities. Technical and biological replicates were also conducted for all the 5 strains. The average standard deviation for the technical and biological replicates (n=3, OD600=0.065) was 0.09 μA and 0.56 μA respectively. A similar current trend for the same set of strains was obtained by Ian et al., [1] using a traditional bioelectrochemical system, thus validating our platform’s functionality and integrity. This system allows for parallel screening of wild type and mutant variations of multiple electroactive bacteria as demonstrated. The high throughput feature enabled by this device can also find application to characterize mutants generated by directed evolution workflows. Additionally, mechanical and electrical multiplexing can further improve the electronic instrumentation and connections between the wells for parallel readout and will also minimize human intervention during the current measurements. Chronoamperometry measurements is an important technique to characterize EET. Thus, a low-cost system like ours will help do that in a shorter time frame with minimal bacterial inoculum and for a larger number of electroactive bacterial species. In conclusion, we aim to show that a 3D carbon felt platform is miniaturized, and can be scaled to understand and characterize EET from electroactive bacteria in a high throughput manner. References Ian J. Campbell, Joshua T. Atkinson, Matthew D. Carpenter, Dru Myerscough, Lin Su, Caroline M. Ajo-Franklin, and Jonathan J. Silberg Biochemistry 2022 61 (13), 1337-1350 Figure 1