Harold D. May

Polychlorinated biphenyls (PCBs) and chlorinated ethenes, such as perchloroethene (PCE), are persistent environmental contaminants that pose significant ecological and health risks. Dehalobium chlorocoercia strain DF-1 can reductively dechlorinate both PCEs and PCBs, offering a sustainable remediation strategy. However, its large-scale cultivation has been limited by dependence on an undefined growth-stimulatory factor produced by Pseudodesulfovibrio sp. (DSV). Filtrates of DSV-autoclaved cultures passed through a 10-kDa cutoff filter enhanced growth-linked dechlorination of DF-1, indicating that the active component is a small-molecular-weight, nonsecretory, thermostable compound. Acid precipitation of the autoclaved DSV extract yielded a glycolipid-enriched fraction that also supported DF-1 growth; in parallel, the model biosurfactant surfactin stimulated DF-1 activity, consistent with a biosurfactant-mediated effect. Liquid chromatography coupled to high-resolution tandem mass spectrometry analysis of the acid-precipitated fraction detected ions and fragmentation patterns consistent with multiple rhamnolipid congeners, including mono- and di-rhamnolipids with C8–C12 acyl chains. Both glycolipid-enriched extract and commercial rhamnolipids substituted for DSV extracts, stimulating DF-1 growth-linked reductive dechlorination of PCE, 2,3,4,5-tetrachlorobiphenyl (PCB 61), and 2,2′,3,3′,4,5,5′,6-Octachlorobiphenyl (PCB 199) in a concentration-dependent manner. Maximal DF-1 growth-linked dechlorination rates were observed at submicellar rhamnolipid concentrations, whereas higher concentrations were inhibitory. Rhamnolipid supplementation reduced growth lag and increased DF-1 biomass and dechlorination rates by solubilizing hydrophobic electron acceptors, thereby increasing their bioavailability in the medium. These findings suggest that rhamnolipid-type biosurfactants in Pseudodesulfovibrio sp. extracts have a role in enhancing the growth of DF-1 and demonstrate that commercial rhamnolipid supplementation can replace undefined coculture extracts, enabling reproducible large-scale cultivation of DF-1 for bioaugmentation of PCB- and PCE-contaminated environments. IMPORTANCE: Polychlorinated biphenyls (PCBs) and chlorinated ethenes remain widespread contaminants that are difficult to remove with conventional technologies, creating a need for scalable bioremediation strategies. Dehalobium chlorocoercia DF-1 is one of the few isolates available that can reductively dechlorinate both PCBs and PCE, but its application has been hindered by reliance on undefined extracts from a Pseudodesulfovibrio sp. This study shows that the key stimulatory activity is associated with rhamnolipid-type glycolipid biosurfactants released from Pseudodesulfovibrio sp. cells upon lysis. Glycolipid-enriched extracts and defined commercial rhamnolipids enhance DF-1 growth-linked dechlorination of PCE and PCB congeners in a dose-dependent manner, primarily by increasing the bioavailability of hydrophobic electron acceptors. By replacing complex coculture extracts with a defined biosurfactant supplement, these findings remove a major barrier to reproducible, large-scale cultivation of DF-1 and advance the feasibility of bioaugmentation for PCB- and PCE-contaminated environments.

Lactic acid bacteria can use an electron transport system to pass reducing equivalents to extracellular mediators. This redox flexibility decouples carbon and electron balances and presents the opportunity to use electrodes to engineer fermentations. We show that growth with an anode may be a two-edged sword for the fitness of the model lactic acid bacterium Lactiplantibacillus plantarum grown in a chemically defined medium supplemented with quinones, depending on substrate and cultivation regime. In batch culture on glucose, the anode slows growth despite yielding an over four-fold higher ratio of acetate to lactate than the open-circuit control, a fermentation profile associated with higher ATP yield. In batch culture on the more reduced substrate mannitol, the anode facilitates growth by enabling the dissipation of reducing equivalents. However, when acetate is also present as an alternate electron sink, the anode prolongs the lag phase while maintaining a growth rate similar to the open-circuit control, despite the expected ATP cost of acetate-to-ethanol reduction. Under semi-continuous cultivation on mannitol in the absence of acetate, anode polarization reduces growth yield relative to open-circuit conditions. Although the anode promotes more energetically favorable fermentation patterns during batch growth on both glucose and mannitol, these are likely counterbalanced by prophage induction to affect population growth, as confirmed by transcriptomic analysis, extracellular DNA measurement, and transmission electron microscopy. Together, these results indicate that extracellular electron transfer associated with an anode is not necessarily beneficial for fermenters and prompt further inquiry into the context-dependent nature of this style of metabolism. IMPORTANCE: A hybrid metabolism has been described in widespread fermenters where energy conservation through substrate level phosphorylation is coupled with electron transfer to external electron acceptors via extracellular redox mediators. This mechanism shapes interactions in microbial systems and presents opportunities in biotechnology. The variable physiological effects observed here of using an electrode as an electron sink highlight both the potential and challenges of applying electrodes to regulate fermentations. We show that polarization of an anode can hinder Lactiplantibacillus plantarum growth and is associated with induction of prophages, increasing the rate of lysis and thereby possibly counteracting the benefits of metabolic flexibility and more energetic fermentation patterns. These results make a new connection between electron balancing and a mobile genetic element and suggest a dynamic, context-dependent role of these mediators as public goods.

Acetogens are anaerobic bacteria capable of fixing CO₂or CO to produce acetyl-CoA and ultimately acetate using the Wood-Ljungdahl pathway (WLP). This autotrophic metabolism plays a major role in the global carbon cycle.Acetobacterium woodii, which is a member of theEubacteriaceaefamily and type strain of theAcetobacteriumgenus, has been critical for understanding the biochemistry and energy conservation in acetogens. Other members of theAcetobacteriumgenus have been isolated from a variety of environments or have had genomes recovered from metagenome data, but no systematic investigation has been done into the unique and varying metabolisms of the genus. Using the 4 sequenced isolates and 5 metagenome-assembled genomes available, we sequenced the genomes of an additional 4 isolates (A. fimetarium, A. malicum, A. paludosum,andA. tundrae) and conducted a comparative genome analysis of 13 differentAcetobacteriumgenomes to obtain better phylogenomic resolution and understand the metabolic diversity of theAcetobacteriumgenus. Our findings suggest that outside of the reductive acetyl-CoA (Wood-Ljungdahl) pathway, theAcetobacteriumgenus is more phylogenetically and metabolically diverse than expected, with metabolism of fructose, lactate, and H₂:CO₂constant across the genus, and ethanol, methanol, caffeate, and 2,3-butanediol varying across the genus. While the gene arrangement and predicted proteins of the methyl (Cluster II) and carbonyl (Cluster III) branches of the Wood Ljungdahl pathway are highly conserved across all sequencedAcetobacteriumgenomes, Cluster 1, encoding the formate dehydrogenase, is not. Furthermore, the accessory WLP components, including the Rnf cluster and electron bifurcating hydrogenase, were also well conserved, though all but four strains encode for two Rnf clusters. Additionally, comparative genomics revealed clade-specific potential functional capabilities, such as amino acid transport and metabolism in the psychrophilic group, and biofilm formation in theA. wieringaeclade, which may afford these groups an advantage in low-temperature growth or attachment to solid surfaces, respectively. Overall, the data presented herein provides a framework for examining the ecology and evolution of theAcetobacteriumgenus and highlights the potential of these species as a source of fuels and chemicals from CO₂-feedstocks.

The price for renewable electricity is rapidly decreasing, and the availability of such energy is expected to increase in the coming years. This is a welcomed outcome considering that mitigation of climate disruption due to the use of fossil carbon is reaching a critical stage. However, the economy will remain dependent on carbon-based chemicals and the problem of electricity storage persists. Therefore, the development of electrosynthetic processes that convert electricity and CO₂ into chemicals and energy dense fuels, perhaps even food, would be desirable. Electrochemistry has been applied to the manufacture of many valuable products and at a large industrial scale, but it is difficult to produce multicarbon chemicals from CO₂ by chemistry alone. Being that the biological world possesses expertise at the construction of C–C bonds, it is being examined in conjunction with electrochemistry to discover new ways of synthesizing chemicals from electricity and CO₂. One approach is microbial electrosynthesis. This Account describes the development of a microbial electrosynthesis system by the authors. A biocathode consisting of a carbon-based electrode and a microbial community produced short chain fatty acids, primarily acetate. The device works by electrolysis of water, but microbes facilitate electron transfer from the cathode while reducing CO₂ by the Wood–Ljungdahl pathway possessed by an Acetobacterium sp. While this acetogenic microorganism dominates the microbiome growing on the cathode surface, 13 total species of microbes overall were ecologically selected on the cathode and genomes for each have been assembled. The combined species may contribute to the stability of the microbiome, a common feature of naturally selected microbial communities. The microbial electrosynthesis system was demonstrated to operate continuously at a cathode for more than 2 years and could also be used with intermittent power, thus demonstrating the stability of the microbiome living at the cathode. In addition to the description of reactor design and startup procedures, the possible mechanisms of electron transfer are described in this Account. While mysteries remain to be solved, much evidence indicates that the microbiome may facilitate electron transfer by supplying catalyst(s) external to the bacterial cells and onto the cathode surface. This may be in the form of a hydrogen-producing catalyst that enhances hydrogen generation by an inert carbon-based electrode. Through the enrichment of the electrosynthetic microbiome along with several modifications in reactor design and operation, the productivity and efficiency were improved. In addition to the intrinsic value of the current products, coupling the process with a secondary stage might be used to produce more valuable products from the acetic acid stream such as lipids, biocrude oil, or higher value food supplements. Alternatively, additional work on the mechanism of electron transfer, reactor design/operation, and modification of the microbes through synthetic biology, particularly to enhance carbon efficiency into higher value chemicals, are the needed next steps to advance microbial electrosynthesis so that it may be used to transform renewable electrons and CO₂ directly into products and help solve the problem of climate disruption.

It was hypothesized that a lack of acetogenic biomass (biocatalyst) at the cathode of a microbial electrosynthesis system, due to electron and nutrient limitations, has prevented further improvement in acetate productivity and efficiency. In order to increase the biomass at the cathode and thereby performance, a bioelectrochemical system with this acetogenic community was operated under galvanostatic control and continuous media flow through a reticulated vitreous carbon (RVC) foam cathode. The combination of galvanostatic control and the high surface area cathode reduced the electron limitation and the continuous flow overcame the nutrient limitation while avoiding the accumulation of products and potential inhibitors. These conditions were set with the intention of operating the biocathode through the production of H2. Biofilm growth occurred on and within the unmodified RVC foam regardless of vigorous H2 generation on the cathode surface. A maximum volumetric rate or space time yield for acetate production of 0.78 g/Lcatholyte/hour was achieved with 8 A/Lcatholyte (83.3 A/m2projected surface area of cathode) supplied to the continuous flow/culture bioelectrochemical reactors. The total Coulombic efficiency in H2 and acetate ranged from approximately 80% to 100%, with a maximum of 35% in acetate. The overall energy efficiency ranged from approximately 35% to 42% with a maximum to acetate of 12%.

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).

A microbial community originating from brewery waste produced methane, acetate, and hydrogen when selected on a granular graphite cathode poised at −590 mV versus the standard hydrogen electrode (SHE) with CO ₂ as the only carbon source. This is the first report on the simultaneous electrosynthesis of these commodity chemicals and the first description of electroacetogenesis by a microbial community. Deep sequencing of the active community 16S rRNA revealed a dynamic microbial community composed of an invariant Archaea population of Methanobacterium spp. and a shifting Bacteria population. Acetobacterium spp. were the most abundant Bacteria on the cathode when acetogenesis dominated. Methane was generally the dominant product with rates increasing from 4 mM day ⁻¹ (accumulated to 28.5 mM over 12 days), and methanogenesis ceased following the addition of 2-bromoethanesulfonic acid. Traces of hydrogen accumulated during initial selection and subsequently accelerated to >11 mM day ⁻¹ (versus 0.045 mM day ⁻¹ abiotic production). The hypothesis of electrosynthetic biocatalysis occurring at the microbe-electrode interface was supported by a catalytic wave (midpoint potential of −460 mV versus SHE) in cyclic voltammetry scans of the biocathode, the lack of redox active components in the medium, and the generation of comparatively high amounts of products (even after medium exchange). In addition, the volumetric production rates of these three commodity chemicals are marked improvements for electrosynthesis, advancing the process toward economic feasibility.

Microbial fuel cells (MFCs) are bioelectrochemical devices capable of converting chemical energy to electrical energy using bacteria as the catalysts. Mechanisms of microbial electron transfer to solid electrode surfaces are not well defined in most electrochemically-active microorganisms, particularly for Gram-positive bacteria. In this study, we investigated the electrochemical characteristics of the Gram-positive, thermophilic bacterium Thermincola ferriacetica strain Z-0001. This organism was capable of transferring electrons from acetate to the anode of an MFC to generate an electric current. T. ferriacetica exhibited rapid recovery of current following medium exchanges, recovering to near-maximum current output in less than three hours. The recovery of electrons from acetate was 97% in air-cathode MFCs inoculated with T. ferriacetica. Further insights into the anode reduction by these biofilms were gained through cyclic voltammetry (CV). A continuous steady-state current was reached above −0.1 V vs.Ag/AgCl reference electrode in CV scans of an established T. ferriacetica biofilm. A catalytic wave with a midpoint potential consistently near −0.28 V indicated a continuous electron-transporting interface between the attached microbial biofilm and the electrode surface. Additionally, no significant peaks were observed when scanning cell-free spent medium from active MFCs. These data suggest that T. ferriacetica directly transfers electrons to an electrode through a mechanism that is tightly associated with the biofilm that forms on the electrode. This is the first mechanistic insight into how Gram-positive extracellular electron transfer might occur without the addition of soluble electron shuttling mediators. These mechanistic evaluations will be essential for the improvement and application of such biocatalysts in microbial fuel cells and other bioelectrochemical systems.