ELECTROCHEMICAL APPROACHES FOR ENERGY HARVESTING FROM WASTE AND FOR POLLUTION MITIGATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1011745
• Project Start Date: Feb 1, 2017
• Project End Date: Jan 31, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Microorganisms can naturally immobilize metals, including toxic metals and radionuclides, via electrical currents. These processes can be harnessed for the development of environmentally-friendly technologies for cleaning up metal contaminants and for generating electricity from wastes. Key to these applications is knowledge of how the microorganisms perform these reactions and how the processes can be scaled up to industrial standards. This is the major objective of this project.

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
403	0210	1103	60%
133	0199	1103	40%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0210 - Water resources; 0199 - Soil and land, general;

Field Of Science
1103 - Other microbiology;

Keywords

bioenergy

bioremediation

biomining

waste reuse

pollution mitigation

Goals / Objectives
Our long-term goal is to understand how Geobacter bacteria transfer electrons to extracellular electron acceptors such as metals and other cells in current-harvesting biofilms. We use this knowledge to harness the natural electrochemical activities of these organisms for applications in bioremediation and bioenergy. The general approach is to identify genetic targets for key electrochemical processes and to genetically engineer strains with improved activities and performance in electrochemical devices. Efforts in the lab have lead to the identification of protein nanowires produced by these bacteria that bind and transfer respiratory electrons to metal and also wire cells in biofilms formed on current-harvesting electrodes. We are also developing genetic engineering tools to needed to construct hybrid devices that integrate proteins nanowires with selectivity to bind various toxic metals and reductively precipitate them to reduce their mobility and potential for spread. More recently, we have begun to explore applications for the electrochemically active biofilms, which we genetically engineer to develop "bioelectrodes" that drive the fermentation of industrial and agricultural wastes into added-value products, a process known as electro-fermentation.Our objectives in this new project are to scale up electrochemical devices and investigate novel applications for the protein nanowires and bioelectrodes in the immobilization of metal and radionuclide contaminants (KA 113) and in waste recycling and reuse (KA 403). To achieve these objectives, we use genetic engineering and adaptive evolution techniques to manipulate cellular components involve in metal binding such as the protein nanowires to generate strains or hybrid devices that are more active and selective at the immobilization of metal contaminants, thus setting the foundation for green technologies for bioremediation and biomining. Similar approaches will be used to develop bioelectrodes of superior performance and to identify microbes that can carry out efficient electro-fermentation with bioelectrodes in scaled-up devices.

Project Methods
The interdisciplinary nature of our work requires the use of a wide range of methods spanning the life and physical sciences and engineering. All projects rely heavily on genetic approaches to manipulate bacteria and their components in order to gain functional insights and identify targets for modification. Because the bacteria we work with perform their functions under anaerobic conditions, microbial physiology methods are routinely use in our lab. The electrical nature of the microorganisms we work with also allows us to apply nanotechnology and engineering techniques to the study of microbial processes. Indeed, we propose to interface protein nanowires and the nanowires subunits with electrodes, an approach that requires the application of recombinant methods for protein mass-production, chemical approaches for protein assembly in vitro and deposition on electrodes, and nanotechnology tools for the probing of conductive nanointerfaces. Similarly, scaling up electro-fermentations involves microbial genetics, methods use in fermentation technology, and application of chemical engineering principles for the development of electrochemical reactors that operate efficiently at industrial scales.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our target audience continues to be basic and applied research scientists (through our peer-reviewed publications and invited talks/poster presentations at scientific meetings). Basic research during the last year focused on harnessing the conductive components of electric microbes, protecting the intellectual property associated with the projects and fostering strong ties with industrial partners and biotech companies to commercialize the technologies. We also initiated projects to expand the environmental roles of electric microbes in the cycling of commercially important metals and in the treatment of organic wastes and fertilizers carried in agricultural water runoffs. We continue to actively disseminate our research to the public. The educational aspects of the work are also reflected in the incorporation of the research in the department's curriculum and the training of students and postdocs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research continued to provide opportunities for training and professional development of: - Two postdocs (a microbiologist and a geomicrobiologist). - Three graduate students: 1) Hunter Dulay (third-year graduate student), 2) Marcela Tabares (second-year graduate student). and 3) Morgen Clark (second-year graduate student). - One undergraduate student, Stavros Hedling, who is a Professorial Assistant in the College of Natural Science (mentored by graduate student Hunter Dulay). How have the results been disseminated to communities of interest?In the form of new peer-reviewed publications, numerous poster presentations and talks, and partnerships with several industry and goverment stakeholders. Examples of poster presentations and talks include an invited talk by student Hunter Dulay (on behalf of the PI) at the annual meeting of the American Geochemical Union (December 2019) and a keynote talk by the PI at the annual meeting of the International Electrochemical Society (September 2020). The PI also served as guest editor of the special issue of the Journal of Industrial Microbiology and Biotechnology entitled "Frontiers of Industrial Microbiology and Biotechnology 2020", which covered select topics and authors who presented at the 2019 Annual Meeting of SIMB in Washington, DC. Our review on the Biology and Biotechnology of Microbial Pilus Nanowires was selected for publication in this special issue. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We are currently focusing on completing the research on cobalt biomineralization by nanowires to submit an article for publication. We also have an invitation from the Office of the Army to submit a white paper to expand the cobalt project to extremophilic environments. Additionally, we are starting a new project to investigate the role of electric microbes in cadmium immobilization and the role that metal detoxification has in the selection of antibiotic resistance in electromicrobiomes. Objective 2: We will focus our efforts on the development of treatment options for wastewater from agricultural runoffs, with a particular emphasis on nitrate and pesticide remediation.

Impacts
What was accomplished under these goals? Objective 1 - "Electrochemical technologies for metal bioremediation and biomining". This project focused on NIFA's Area Priority in Bioenergy, Natural Resources, and Environment. We completed the third and last year of a project funded by NSF-Geobiology to identify the spectrum of nanowire-mediated metal reductive reactions (Subobjective 1.1). We demonstrated high cobalt resistance in the model representative Geobacter sulfurreducens and submitted a paper to Frontiers in Microbiology (under review) describing multiple mechanisms for detoxification, including extracellular mineralization via a respiratory chain of cytochromes. We are now working on a new paper (with graduate students Marcela Tabares and Hunter Dulay) describing the key role that Geobacter pili have in cobalt mineralization and detoxification (Subobjectives 1.2 and 1.3). This result has important environmental implications because, prior to our work, cobalt mineralization was assumed to only involved abiotic reactions. These findings supported a grant proposal to NSF-Geobiology to investigate the respiratory chains responsible for cobalt mineralization in Geobacter sulfurreducens and the environmental role of these bacteria in intertwining biotic and abiotic reactions to cycle cobalt and other metals. As part of this project, we published one paper in Trends in Microbiology that made Geobacter sulfurreducens the "Microbe of the Month" (with graduate students Marcela Tabares and Hunter Dulay). We are also awaiting publication of a review paper currently in press in the Journal of Industrial Microbiology and Biotechnology about the biology and biotechnology of microbial pilus nanowires, which was led by a new graduate student, Morgen Clark. This review paper describes the basic science that inspired nanowire-electrode interfaces developed by my research team (Subobjective 1.4) and near-term applications tfor cobalt bioming and bioreclamation. Morgen is now developing protocols to synthesize protein nanowires for a biotech company interested in incorporating the biomaterials in their proprietary technology. Objective 2 - "Electrochemical technologies for waste recycling and reuse" For the past several years, I received funds from a Talent Grant from the Michigan Economic Corporation and Spartan Innovations to de-risk our electrochemical technologies and bring them closer to the market. Hiring qualified and motivated postdocs for this project became challenging and, after two disappointing hires, I redirected efforts towards joint ventures with companies that could benefit from the electrochemical treatment of wastewater and water reuse. These efforts rely on STTR/SBIR applications and/or cooperative agreements that will fund students and/or technicians in my lab, rather than postdocs. This project was also expanded to the treatment of agricultural runoffs carrying fertilizers and pesticides. These projects capitalize on our research on nitrate ammonification by Geobacter bacteria. A DOE-funded effort with MSU biochemist Eric Hegg to study the nitrate respiratory chain of Geobacter lovleyi led to the discovery of a new, yet widespread subclass of calcium-independent nitrite reductase enzymes that can more efficiently function in metal-rich environments (J Biol Chem. 2020 Aug 14;295(33):11455-11465). Using this bacterium as model representative, my student Marcela Tabares has made seminal discoveries about the environmental signals that allow these microbes to play key roles in nitrate ammonification (paper in preparation). Marcela also discovered that the enrichment of Geobacter species in woodchip bioreactors that effectively remove nitrate and pesticides from agricultural runoffs (manuscript in preparation). This project is part of a collaborative effort with MSU horticulturist Tom Fernandez, a water management expert, to develop cost-effective treatment options for agricultural runoffs. As part of this collaborative effort, we recently published a paper describing a two-stage reactor system for the removal of nutrients and pesticides (Water Res. 2020 Mar 1;170:115311). Together, we will investigate sustainable approaches for nitrate removal from agricultural runoffs with recovery of ammonium and reduction of gas emissions from denitrification. This project aligns with Area Priority A1402 in Plant Systems and Natural Resources.

Publications

• Type: Journal Articles Status: Other Year Published: 2020 Citation: Tabares, D.M. and G. Reguera. Low levels of nitrate rather than high carbon-nitrogen ratio induce dissimilatory nitrate reduction to ammonia in Geobacter lovleyi SZ. (in preparation)
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Clark, M.M. and G. Reguera (2020) Biology and biotechnology of microbial pilus nanowires. J Ind Microbiol Biotechnol (in press)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Voltammetric study of conductive planar assemblies of Geobacter nanowire pilins unmasks their ability to bind and mineralize divalent cobalt. Cosert KM, Reguera G. J Ind Microbiol Biotechnol. 2019 Oct;46(9-10):1239-1249. doi: 10.1007/s10295-019-02167-5. Epub 2019 Apr 5.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: The electrifying physiology of Geobacter bacteria, 30 years on. Reguera G, Kashefi K. Adv Microb Physiol. 2019;74:1-96. doi: 10.1016/bs.ampbs.2019.02.007. Epub 2019 May 15.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Nutrient and pesticide remediation using a two-stage bioreactor-adsorptive system under two hydraulic retention times. Abdi DE, Owen JS Jr, Brindley JC, Birnbaum AC, Wilson PC, Hinz FO, Reguera G, Lee JY, Cregg BM, Kort DR, Fernandez RT. Water Res. 2020 Mar 1;170:115311. doi: 10.1016/j.watres.2019.115311. Epub 2019 Nov 16.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Dulay, H , M. Tabares , K. Kashefi, and G. Reguera. Cobalt resistance via detoxification and mineralization in the iron-reducing bacterium Geobacter sulfurreducens. Front Microbiol ( equal contribution)
• Type: Journal Articles Status: Other Year Published: 2020 Citation: Tabares, D.M., D. Abdi, N. Redekar, J. Parke, R.T. Fernandez, and G. Reguera. Microbial communities controlling the nutrient and pesticide remediation capacity of woodchip bioreactors treating agricultural runoffs. (in preparation)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Bottom-Up Fabrication of Protein Nanowires via Controlled Self-Assembly of Recombinant Geobacter Pilins. Cosert KM, Castro-Forero A, Steidl RJ, Worden RM, Reguera G. mBio. 2019 Dec 10;10(6):e02721-19. doi: 10.1128/mBio.02721-19.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Geobacter sulfurreducens. Tabares DM , Dulay H , Reguera G. Trends Microbiol. 2020 Apr;28(4):327-328. doi: 10.1016/j.tim.2019.11.004. Epub 2019 Dec 18. ( equal contribution)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass. Campeci�o J, Lagishetty S, Wawrzak Z, Sosa Alfaro V, Lehnert N, Reguera G, Hu J, Hegg EL. J Biol Chem. 2020 Aug 14;295(33):11455-11465. doi: 10.1074/jbc.RA120.013981. Epub 2020 Jun 9.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Our target audience continues to be basic and applied research scientists (through our peer-reviewed publicationsand invited talks/poster presentations at scientific meetings). Last year we fostered strong ties with industrial partnersand biotech companies and initiated collaborative efforts to co-apply for STTR grants and/or sign option agreements to some of our patented technologies related to this project.We continue to actively disseminate our research to the public inthe form of podcasts, and radio and media print interviews. The educational aspects of the work are also reflected in theincorporation of the research in the department's curriculum and the training of students and postdocs. Changes/Problems:Objective 1 has been very successful and I was able to recruit very talented students to continue the project. Hiring a postdoc with the interdisciplinaryqualifications required to suceed in objective 2 has been more challenging. The shift towards collaborating with established companies and applying for a STTR will include technicians rather than postdocs hired specifically for the project. What opportunities for training and professional development has the project provided?The research continued to provide opportunities for training and professional development of: - One postdoc(a microbiologist). - Threegraduate student (Krista Cosert, who graduated in 2018 and is now a postdoc at UC Davisl Hunter Dulay, a second-year graduate students; and Marcela Tabares, a first-year graduate students). - Oneundergraduate student who is a Professorial Assistant in the College of Natural Science(mentored by graduate student Hunter Dulay). How have the results been disseminated to communities of interest?In the form of new peer-reviewed publications, numerous poster presentations and talks, and partnerships with several industry and govermentstakeholders. Examples of poster presentations and talks included, for example, those presented by students Marcela Tabares and Hunter Dulay at the General Meeting of the Society for Industrial Microbiology and Biotechnology (SIMB). Hunter's abstract was selected for an oral talk in the Science Slam session at this meeting. Also at this meeting, I served as Chair of the Environmental program committee and organized four sessions (antibiotic in the environment,bioremediation, etc.). I co-convened one of the sessions on Women in Environmental Science, which was supported by a successful conference grant I submitted toDOE-BES,and presented the nanowire work to emphasize the number of female scientists that I have mentored throughout the years and that have made a significant contribution to the advancement of electromicrobiology. What do you plan to do during the next reporting period to accomplish the goals?We are currently focusing on completing the research on cobalt biomineralization to submit an article for publication and a grant proposal renewal to NSF. These efforts will be complemented with thedevelopment of novel nanowire-inspiredbiointerfaces for metal immobilization and reclamation (objective 1). We have shifted our focus for objective 2 to develop electrode pretreatments suitablefor operation with commercially available plug-and-play technologiesfor the treatment of industrial wastewater and promote water recycling and reuse(objective 2).

Impacts
What was accomplished under these goals? Objective 1 - "Electrochemical technologies for metal bioremediation and biomining". We finalized the second year ofa project funded by NSF (Geobiology program) to identify the spectrum of nanowire-mediated metal reductivereactions (Subobjective 1.1), characterize the reduced minerals and pili ligands (Subobjective 1.2), and investigate metalreduction under growth conditions (Subobjective 1.3). As part of this project, we published a paper in the Journal of Industrial Microbiology and Biotechnology led by mygraduate student Krista Cosert, who used bioinspirednanowire-electrode interfaces (Subobjective 1.4) to demonstrate their ability to immobilize and reductively precipitate cobaltfrom solution.A 2018 submission toUSDA Nanotechnology for Agricultural and Food Systems program (area priority A1511) proposed to do thisfor cadmium,was not funded. A resubmission focused on cobalt is planned as a bioremediation strategy to reclaim this metal from contaminated waters andelectronic waste is planned. We also filed a provisional patent andsubmitted a paper for peer reviewed describing the in vitro synthesis of protein nanowires using peptides inspired in the nanowire submit (the pilin) and produced in highly efficient and low cost recombinant platforms. Our goal is to integrate these synthetic nanowires with electrodes to make nanobrushes for environmental sensing, bioremediation and reclamation of cobalt and other toxic metals. In 2018 we initiated in vivo experiments to demonstrate the ability of the cells expressing nanowires toimmobilize and reductively precipitate cadmium to gain energy for growth (Subobjective 1.3), opening opportunities forbioremediation interventions for this toxic and pervasive metal. The discovery with our in vitro nanowire platforms that cobalt can be immobilized as cobalt nanoparticles prompted us to examine the in vivo relevance of this reaction. This is significant because cobalt is considered to be too toxic for its bioremediation via reductive prepitation and abiotic reactions are the only ones believed to contribute to the mineralization of this metal.Two of my graduate students, Marcela Tabares and Hunter Dulay, designed in vivo experiments to investigate if Geobacter sulfurreducens is indeed able to use the nanowires to respire cobalt. They developed a colorimetric assay to measure cobalt removal in cultures, established the cobalt tolerance of the bacterium to be higher than any other bacteria reported in pure culture, and used nanowire mutants to demonstrate the requirement of the cells to produce these electronic conduits to detoxify the cobalt. Transmission electron microscograhs from cobalt culturesconfirmed the production of cobalt nanoparticles along the nanowires, demonstrating for the first time the biological reduction of this metal. We are currently sequencing the transcriptome of cultures under cobalt stress compared to the untreated cultures to gain insights into the complex regulatory networks and mechanisms used by this bacterium to carry out this reaction.The RNAseq data will allow us to build a model for cobalt reduction and detoxification in Geobacter and prepare a proposal to renew our current NSF-Geobiology grant. Our bioremediation research has also led to novel approaches to manipulate the rhizosphere microbiome in order to promotethe reduction of nitrate to ammoniafor assimilation by plant roots. In a grant proposal funded by DOE-BER I collaborated with MSU biochemist Eric Hegg to study the respiratory chain of Geobacter lovleyi that carries out this reaction. We are currently preparing two articles: one describing the unique crystal structure of the nitrite reductase enzyme of these bacteria, which uses a never before described active site to efficiently bind and reduce nitrite to ammonia in metal-rich environmens, and another using culturing approaches and RNAseq profiling of the ntirate transcriptome to reveal the complexity of thenitrate respiratory chain in these organisms and the regulatory and signaling networks that allow it to integrate this reaction with the cycling of other essential elements, including metals. We are planning a grantubmissionto USDAAgricultural Microbiomes in Plant Systems and Natural Resources Program on this topic (Area Priority, A1402). Objective 2 - "Electrochemical technologies for waste recycling and reuse" For the past three years, wereceived funds from a Talent Grant from the Michigan Economic Corporation and Spartan Innovations to de-risk ourelectrochemical technologies and bring them closer to the market. As part of these efforts, we filed a provisional patent to pre-seed electrodes with proprietary methods and bacteria developed in my lab for the electrochemical treatment of industrialwastewater containing of low to high strength (aka, chemical oxygen demand or COD). We started a collaboration with abiotech company in San Diego interested in an option agreement for this technology and together we submitted a successful project pitch (like a preproposal) for an NSF-STTR grant. We are currently preparing the full proposal for submission in December. The joint effort reduces the start-up time needed for commercial bioelectrochemical systems to treate high COD wastewaterfrom the food and beverage (F&B) industryand provides a viable treatment option for the removal of fermentation inhibitors in low COD wastewaters recycled in ethanol plants.This allows for efficient plug-and-play operation of bioelectrochemical units forwater reuse and stimulation of yeast-based ethanologenic fermentations (Subobjective 2.2).

Publications

• Type: Book Chapters Status: Published Year Published: 2019 Citation: Reguera, G. & Kashefi, K. The electrifying physiology of Geobacter bacteria, 30 years on. Adv Microb Physiol 74, 1-96, doi:10.1016/bs.ampbs.2019.02.007 (2019).
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cosert, K. M. & Reguera, G. Voltammetric study of conductive planar assemblies of Geobacter nanowire pilins unmasks their ability to bind and mineralize divalent cobalt. J Ind Microbiol Biotechnol, doi:10.1007/s10295-019-02167-5 (2019).
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Cosert, K. M., A. Castro-Forerob!, R. J. Steidl, R. M. Worden, and G. Reguera. Bottom-up fabrication of protein nanowires via controlled self-assembly of recombinant Geobacter pilins. mBio (in second review for minor revisions)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Abdi DE et al. Enhanced nutrient and pesticide remediation using a two-stage bioreactor-adsorbtive system controlled by hydraulic retention times. Water Research (under review)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Tabares M, H. Dulay & G. Reguera. Geobacter sulfurreducens. Trends in Microbiology (under editorial review)

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our target audience continues to be basic and applied research scientists (through our peer-reviewed publications andinvited talks/poster presentations at scientific meetings). Last year we were particularly active at meeting withindustrial representatives andtech companies, who see value in our technologies to custom-tailor bioprocesses for clean up of theirwaste streams and energy recovery as bioproducts. We continue to actively disseminate our research to the public in the form of podcasts, and radio and media print interviews. The educational aspects of the work are alsoreflected in the incorporation of the research in the department's curriculum andthe training of students and postdocs. Changes/Problems:The development of genetic systems for fermentative bacteria encountered many challenges that caused significant delays. This made us develop alternative approaches to de-risk electro-fermentations for the treatment of and energy harvesting from agricultural and industrial wastes. Our current approach focuses on harnessing the diversity of electric microbes that can be used in bioelectrodes and the genetic tools available for their manipulation. What opportunities for training and professional development has the project provided?The research continued to provide opportunities for training and professional development of: - Two postdocs (a microbiologist who is now a faculty member at Mercy College Health Sciences in Iowa and another currently pursuing a career in industry). - One graduate student (Krista Cosert, who graduated last June and is now a postdoc at Georgia Tech). - Two undergraduate students who graduated last year. How have the results been disseminated to communities of interest?In the form of new peer-reviewed publications, numerous talks, and partnerships with several industry and goverment stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We are currently focusing on developing novel biointerfaces for metal immobilization and reclamation that harness themetal reducing activities of the nanowires (objective 1). We have shifted our focus for objective 2 to develop hybrid bioelectrodes that operate optimally with fermentative bacteria of interest in order to bypass the need todevelopgenetic systems for their manipulation. This will allow us to more rapidly de-risk electro-fermentation technologies toprocess industrial wastes(objective 2).

Impacts
What was accomplished under these goals? Objective 1 - "Electrochemical technologies for metal bioremediation and biomining". We initiated a project funded byNSF (Geobiology program) to identify the spectrum of nanowire-mediatedmetal reductive reactions (Subobjective 1.1), characterize the reduced minerals and pili ligands (Subobjective 1.2), andinvestigate metal reduction under growth conditions (Subobjective 1.3). As part of this project, graduate student Krista Cosert used bioinspired nanowire-electrode interfaces (Subobjective 1.4) to demonstrate their ability to immobilizeand reductively precipitate cobalt from solution. This work is described in a chapter of Krista's dissertation, which she successfully defended on June 22nd. We are reformatting this chapter to submit it for peer review.This platformsets thefoundation of sensors for toxic metals and scaled up systems for metal remediation and reclamation. This work also provided the preliminary results to support an application to USDA Nanotechnology for Agricultural and Food Systems program (area priorityA1511).If funded, this project will exploitthe metal-chelating properties of our biomaterials to develop soil amendments for cadmium immobilization in agricultural lands. We have also initiated in vivo experiments to demonstrate the ability of the cells expressing nanowires to immobilize and reductively precipitate cadmium to gain energy for growth (Subobjective 1.3), opening opportunities for bioremediation interventions for this toxic and pervasive metal. Our bioremediation research has also led to novel approaches to manipulate the rhizosphere microbiome in order to promote the reduction of iron and managanese mineralsand the controlled release of growth-essential nutrients (iron and managanese) for assimilation by plant roots. These studies are described in a grant proposal we submitted to USDA Agricultural Microbiomes in Plant Systems andand Natural Resources Program (Area Priority,A1402) and include a collaboration with Dr. Tom Fernandez, a professor in the department of Horticulture at Michigan State interested in agricultural water management. Objective 2 - "Electrochemical technologies for metal bioremediation and biomining" We have received funds from a Talent Grant from the Michigan Economic Corporation and Spartan Innovations to de-risk our electrochemical technologies and bring them closer to the market. These efforts have also led us to create a universitystartup (BioElectrica Inc.) to commercialize the most promising technologies. As par tof these efforts, we successfully developedhybrid electrodes for the removal of fermentation waste products in water streams generated in bioethanol refineries. This allows for water reuse and stimulation of yeast-based ethanologenic fermentations(Subobjective 2.2). We are also developing hybrid bioelectrodes to operate within fermenters toremove unwanted products, stimulate fermentations of crude industrial waste substrates and generate added value products.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Reguera, G. (2018). Microbial nanowires and electroactive biofilms. FEMS Microbiol. Ecol., 94(7), fiy086-fiy086. doi:10.1093/femsec/fiy086
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Reguera, G. (2018) Harnessing the power of microbial nanowires. Microb. Biotechnol., 0(0), 1-16. doi:10.1111/1751-7915.13280
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Reguera, G. (2018). Biological electron transport goes the extra mile. Proc. Natl. Acad. Sci. USA, 115(22), 5632-5634. doi:10.1073/pnas.1806580115

Progress 02/01/17 to 09/30/17

Outputs
Target Audience:Our target audience continues to be basic and applied research scientists(through our peer-reviewed publications and invited talks and poster presentations at scientific meetings) but has begun to shift more and more towards industrial and tech companies, who seek to address the management of their waste streams and products with our electrochemical technologies. We continue to actively disseminate our researchto the public in the form of educational videos (which we released in YouTube), podcasts, and radio and media print interviews.The educational aspects of the work are also reflected in the incorporation of the research in the department's curriculum and the training of studentsand postdocs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research provided opportunities for training and professional developmentof two postdocs, one graduate student, and two undergraduate students. How have the results been disseminated to communities of interest?In the form of new peer-reviewed publications, numerous talks, and industry and goverment stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We are currently focusing on investigating metal reduction by nanowires (objective 1) anddeveloping genetic systems for fermentative bacteria that can process industrial wastes in electro-fermentations (objective 2).

Impacts
What was accomplished under these goals? Objective 1 - "Electrochemical technologies for metal bioremediation and biomining". We received funding from NSF (Geobiology program) to initiate studies needed to identify the spectrum of nanowire-mediated metal reductive reactions (Subobjective 1.1),characterize the reduced minerals and pili ligands (Subobjective 1.2), and investigate metal reduction under growth conditions (Subobjective 1.3). We published a paper in the journal of Physical Chemistry and Chemical Physics (Cosert et al. 2017) that described the manufacturing and characterization of bioinspired nanowire-electrode interfaces (Subobjective 1.4). This platforms set the foundation of sensors for toxic, precious and rare metals and scaled up systems for metal remediation and reclamation. Objective 2 - "Electrochemical technologies for metal bioremediation and biomining" We published a paper in the Journal of Power Sources (Awate et al. 2017) describing a single-chambered reactor for electro-fermentations (Subobjective 2.1). A paper describing this technology was awarded and published in 2017. We are currently developing genetic systems to metabolically engineer targeted fermentative bacteria that perform well in scaled up electro-fermentations (Subobjective 2.2).

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Awate B, Steidl RJ, Hamlischer T, Reguera G. Stimulation of electro-fermentation in single-chamber microbial electrolysis cells driven by genetically engineered anode biofilms. Journal of Power Sources. 2017;356:510-8.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cosert KM, Steidl RJ, Castro-Forero A, Worden RM, Reguera G. Electronic characterization of Geobacter sulfurreducens pilins in self-assembled monolayers unmasks tunnelling and hopping conduction pathways. Phys Chem Chem Phys. 2017;19(18):11163-72.