Bioelectronic Sensor using Synthetically Engineered and Electroactive Bacteria for Detection of Aquatic Nutrients

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Across the globe, freshwater and marine ecosystems are threatened by the effects of multiple, co-occurring environmental pressures including pollutants, invasive species, climate change, acidification, and excess nutrients. Ecologists strive to monitor, understand, and model the effects of excess nutrients, including phosphates and nitrates, in combination with other human threats. Understanding dynamic spaces with complex and interdependent factors will require a new generation of sensors. Biology-enabled sensors have significant advantages with respect to sensitivity, error-tolerance, scalability, selectivity, and versatility. However, readout and long-range interconnectivity are currently problematic, if not impossible, with biology alone. Biohybrid devices can exploit and tune the strengths of both electronic and biological sensing through a dynamic bioelectronic interface. To achieve it, electroactive bacteria (dissimilatory metal/sulfate reducing bacteria) and their extracellular electron transport mechanisms are employed to transduce their environmental response to measurable biocurrent. A three- dimensional nanofabricated electrode can collect a bacterially derived current for signal processing. The response to a target in the environment is more precisely selected and intensified by the collective response of engineered and highly versatile Escherichia coli. By distributing the sensing and actuation roles between synthetic E. coli and the dissimilatory reducing powerhouse Shewanella oneidensis MR-1, confidence in the presence of a select target is enhanced with the aggregation of individual responses from large number of E. coli bacteria to the community of electroactive bacteria. The bioelectronic interface design is a foundational step toward a new generation of sensing hardware that can meet the vast and expanding promise of machine learning and artificial intelligence. Cross-disciplinary training modules will be developed to designed by the on-campus community of researchers including graduate and undergraduate students. By including new researchers, accessible content is created for the local and international GK-12 community. Local students from the Independent Sanctuary for Independent Media Nature Lab will create informative and creative content for the international cohort starting with the H20 Virtual Academy at the Karada Mixed Secondary School in Kisumu, Kenya. The international connection of gifted local and international students, a flow of stimuli, to convey state-of- the-art knowledge about the environment around them reflect the goals of research.In the proposed work, a mechanism for the detection of phosphate will be investigated using an interconnected network of E. coli and S. oneidensis MR-1. Phosphate detection is essential to understanding ecological dynamics in aquatic ecosystems. A dynamic bioelectronic interface will be created for its potential use in the detection of multiple small molecule targets by engineered bacteria. The system design is inherently modular where electroactive S. oneidensis serves as the bioelectronic interface while E.coli is the easily engineered front end. Electroactive bacteria (dissimilatory metal/sulfate reducing bacteria) and their extracellular electron transport mechanisms will be employed to transduce their environmental response to achieve a measurable biocurrent. A three-dimensional nanofabricated electrode, consisting of a nanomaterial-decorated graphene foam and the two bacteria will generate and transduce the biocurrent for signal processing. The response to a target in the environment is more precisely selected and intensified by the collective response of engineered E. coli. Modules will be linked via chemical quorum sensing and tuned with respect to transfer function and signal amplification using synthetic biology approaches. The co-cultured bacteria configuration will be optimized for cell-viability and signal transport. The research is accomplished through the following objectives: Objective 1: Design and fabricate a bioelectronic backend interface from quorum sensing signal to S. oneidensis biocurrent for signal transduction; Objective 2: Design and fabricate a bioelectronic frontend interface from target (phosphate) to E. coli quorum sensing signal; Objective 3: Integrate objectives 1 and 2, and fabricate, test, and validate bioelectronic sensor from target (phosphate) to biocurrent signal.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.