We integrate microbial physiology, molecular biology, genetics, electrochemistry, genomics, and bioinformatics to unravel the complexities of electron transfer at the core of applications such as water treatment, desalination, and sensing.
Specialized microbes such as Geobacter sulfurreducens generate energy by transferring electrons to acceptors outside their cell membranes. Reduction of minerals can cause mobilization of toxic heavy metals such as arsenic, or precipitation of uranium. In microbial communities, Geobacter species can drive the reduction of CO2 to methane by providing electrons to methanogenic archaea. In engineered bioelectrochemical systems such as microbial fuel cells, Geobacter species form as the interface between biology and electrochemistry, converting waste into electricity and other valuable chemical compounds.
Our work has shown that at almost every level, Geobacter uses different cytochromes and molecular systems to tune its electron transfer machinery to meet the challenges posed by these different environments and electron acceptors.
Genetics of extracellular respiration
Recently, we described new genetic tools that allow removal and control of Geobacter respiratory processes at the molecular level. For example, by controlling production of either the inner membrane cytochrome required for growth with high-potential acceptors (Levar et al, ImcH), or the inner membrane cytochrome required for growth with low-potential acceptors (Zacharoff et al, CbcL) electron transfer to electrodes can be easily triggered (Chan et al, 2015). By deep sequencing of random mutant libraries using Tn-Seq, we can identify new loci in the G. sulfurreducens genome essential for reduction of electrodes, metals, and other extracellular terminal acceptors (read the Tn-Seq paper or download the raw data here).
Tn-Seq identified a chemosensory-like gene cluster and two-complement system we termed the ‘electrode sensing network’ (esn) that appears essential for growth using an electrode, but is dispensable for growth using metals. Out of over 70 MCP and Che homologs encoded in the genome, only these esn family proteins were essential in electrode experiments. Deletions of key esn components—a methyl-accepting chemotaxis protein (MCP) (EsnA), a CheW-like scaffolding protein (EsnB), and a CheA-like histidine kinase (EsnC)—severely diminish electrode growth.
Our working hypothesis is that the Esn network is central to whether cells will reduce insoluble particles such as Fe(III) oxides, or form conductive biofilms on surfaces. This, combined with data showing different conduits, inner membrane cytochromes, and extracellular proteins are involved in electron transfer to metals vs. electrodes, shows how the pathways of electron transfer differ depending on the extracellular condition.
New cytochrome conduits that move electrons across membranes
Protein complexes known as ‘porin-cytochrome’ electron conduits allow for electrons to be transported across outer membranes. These complexes consist of an integral β-barrel through which at least two multiheme c-type cytochromes, one from the periplasm and one lipoprotein on the extracellular leaflet of the outer membrane, can interact. The combination of high heme density throughout the complex and the arrangement of these cofactors results in electron flow through the complex.
We identified several new putative conduit complexes in the genome of G. sulfurreducens, via Tn-Seq and genetic screens. As described by Jiménez-Otero et al, By deleting all possible combinations of these loci, each of these electron conduit clusters was found to represent a substrate-dependent pathway specific to respiration of different electron acceptors. For example, the commonly studied omcB-based conduit is only utilized in electron transfer to metals, while a new cluster we have named extABCD is the primary conduit for electron transfer to electrodes. Strains expressing only the extABCD conduit cluster grow faster and to higher current densities on electrodes.
This advancement shows the specific nature of Geobacter’s metal reduction pathway, and could enhance our ability to design biological systems focused on specific metal or electrode reduction applications.
Sequencing microbial communities to understand extracellular electron transfer in diverse environments
Genomic sequencing of enrichments from unique environments can bypass the requirement for pure culture isolation, as we seek to understand the evolutionary diversity of extracellular electron transfer. For example, the Soudan Iron Mine (located near Ely, MN) hosts microbial communities a half mile underground that thrive in fractured rock brines up to three times saltier than seawater. We are defining these communities via shotgun metagenomic sequencing supported by the Deep Carbon Observatory‘s Census of Deep Life. In parallel, we enrich microbial communities that perform extracellular electron transfer under elevated salinity and/or temperature. Through long read/single molecule sequencing we can identify otherwise elusive signatures of horizontal gene transfer, gene duplication, and phage infection, all of which appear to play key roles in shuffling the deck of respiratory flexibility in metal-reducing microbes.
Custom bioreactor designs for reliable microbial electrochemistry
Over the last decade, we have invented custom electrochemical reactors to study extracellular electron transfer. The design of these reactors evolved from large dual-chambered reactors to small conical reactors, in order to minimize diffusion and charge transfer issues, while allowing easy operation of multiple replicates. Our current design uses either a 15 or 100 ml reactor fitted with custom-machined tops.
The latest design consists of PEEK top, sealed using gasketed compression fittings and threaded steel screws. PEEK is oxygen impermeable and temperature resistant, allowing use of threaded stainless fittings for all ports. Reactors built this way can accommodate long-term enrichments, growth of anodic or cathodic strains, can be modified to be used as a chemostat, modified to vent gases produced at the counter electrode to the outside, and be be scaled to house multiple electrodes. CAD drawings and instructions for constructing your own are available here.