UC Berkeley

Certain microbes have the remarkable ability to form direct electronic connections with their extracellular environment, harnessing existing redox gradients to drive their own life processes. The recognition of extracellular electron transfer (EET) as a widespread microbial phenomenon that plays an important role in biogeochemistry is recent, and is accompanied by new understanding of how this process impacts physical and chemical characteristics of the environment. Our current knowledge of the mechanisms and the microbial physiology behind EET is based largely on studies of two well studied isolates, Geobacter sulfurreducens and Shewanella oneidensis. Nevertheless, nature is not well represented by isolated organisms. Identification of the larger set of “electroactive” microbes from across much of the phylogenetic spectrum is critical for complete understanding of microbial controls on redox chemistry in natural environments. Genomic information for these organisms has the potential to expand our understanding of the mechanisms of growth that rely upon EET. This information should be particularly useful if it is obtained in an experimental setting that enables manipulation of redox potential.

In this doctoral dissertation, microbial electrochemical cells (MXCs) are employed as a tool to select for and characterize the subset of electroactive microbial community members. The main advantage of MXCs is that they use a solid, inert electrode in place of natural minerals as the terminal electron acceptor (TEA) for microbial metabolism. This enables direct control over the redox potential and overcomes the chemical heterogeneity and variation in surface reactivity of natural minerals. Experiments are inoculated using microbial communities from subsurface sediments sampled from a major Department of Energy research site near the town of Rifle, Colorado, USA. This site has general relevance as a riparian zone aquifer; prior research has generated thousands of microbial genomes from samples collected across multiple subsurface environment types and impacted by a variety of experimental manipulations (including acetate amendment). Complex electroactive microbial communities, including anode-associated biofilms, are cultivated in the MXC experiments using the sediment inoculum and acetate as the electron donor. Experiments include two independent MXCs with duplicate compartments.

In Chapter 1, we subjected the biofilms and associated planktonic cells to large changes in anode redox potentials and characterized the responses of these organisms using electrochemical methods. Community composition was documented using 16S rRNA gene-based surveys. Our results strongly suggested that TEA redox potential is an important determinant of the makeup of mineral-respiring communities.

Chapter 2 tested the hypothesis that microbes populating anode-associated biofilms electrochemically adapt to imposed redox conditions on a short timescale (minutes). Through a series of cyclic voltammetry experiments, the overall biofilm responses to changing redox potential were observed. The voltammetry data suggested inherent redox flexibility in the microbial EET network, very likely conferred by multiheme c-type cytochromes known to be critical for extracellular respiration in most known electroactive organisms. We performed a series of experiments that allowed us to rule out within-biofilm pH changes as the cause for the observed shifts.

In Chapter 3, metagenomic methods were employed to genomically characterize the MXC microbial communities. Genome-resolved metagenomics enabled the recovery of comprehensive genetic information for the majority of community members without the requirement for isolation of individual organisms. Hundreds of near-complete genomes were reconstructed and used to compare biofilm and planktonic organisms across experimental systems. Specifically, biofilm vs. planktonic growth preferences for most organisms were defined. Importantly, genomes were used to predict genes that could enable growth on electrodes. Biofilm-associated bacteria are known to have genomes that are strongly enriched in multiheme cytochromes and to have associated outer-membrane porins implicated in electron transfer complexes. In addition to organisms related to known electroactive species, several novel organisms (from an expected undiversity of phyla) were implicated in electrode growth. The extensive metagenomic data assembled previously from the Rifle site was used to link specific organisms enriched in the MXCs to those from Rifle, and thereby yielding insights into their roles in the natural environment.

Coupling electrochemical methods with genomic and metagenomic DNA sequencing provided new information about microbial growth in MXCs and the response of the overall microbial community to imposed changes in redox potential. The hypothesis that TEA (and by proxy, mineral) redox potential shapes community composition was supported. The question of adaptation of microbes to changing environmental redox conditions was investigated in community context, without reliance on isolation methods. The findings suggested that large multiheme cytochromes evolved to cope with intrinsic mineral redox heterogeneity and fluctuating redox conditions. Finally, genome-resolved metagenomics enabled the identification of novel, putatively electroactive organisms functioning as part of complex microbial consortia, with widely different metabolisms from those of the well-studied type strains. This contributes to basic knowledge of EET in natural systems, and opens new avenues for the application of electroactive organisms in biologically-based technology, engineering and remediation.