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Unprecedented increase in atmospheric CO2 levels calls for efficient, sustainable, and cost-effective technologies for CO2 removal, including both capture and conversion approaches. Current CO2 abatement is largely based on energy-intensive thermal processes with a high degree of inflexibility. In this Perspective, it is argued that future CO2 technologies will follow the general societal trend towards electrified systems. This transition is largely promoted by decreasing electricity prices, continuous expansion of renewable energy infrastructure, and breakthroughs in carbon electrotechnologies, such as electrochemically modulated amine regeneration, redox-active quinones and other species, and microbial electrosynthesis. In addition, new initiatives make electrochemical carbon capture an integrated part of Power-to-X applications, for example, by linking it to H2 production. Selected electrochemical technologies crucial for a future sustainable society are reviewed. However, significant further development of these technologies within the next decade is needed, to meet the ambitious climate goals.

Our understanding ofin situmicrobial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for novel taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of novelNegativicutesandMethanobacteriafrom stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.

Next-generation electro-bioreactors will require development of novel reactor-tailored components to improve reactor productivity while maintaining high energy efficiency and biocompatible reactor conditions.

Microbial electrosynthesis (MES) of acetate is a process using electrical energy to reduce CO2to acetic acid in an integrated bioelectrochemical system. MES powered by excess renewable electricity produces carbon-neutral acetate while benefitting from inexpensive but intermittent energy sources. Interruptions in electricity supply also cause energy limitation and starvation of the microbial cells performing MES. Here, we studied the effect of intermittent electricity supply on the performance of hydrogen-mediated MES of acetate. Thermoanaerobacter kivui produced acetic acid for more than 4 months from intermittent electricity supplied in 12 h on-off cycles in a semicontinuously-fed MES system. After current interruptions, hydrogen utilization and acetate synthesis rates were severely diminished. They did not recover to the steady-state rates of continuous MES within the 12 h current-on period under most conditions. Accumulating high product (acetate) concentration exacerbated this effect and prolonged recovery. However, supply of a low background current of 1-5% of the maximum current during "off-times" reduced the impact of current interruptions on subsequent MES performance. This study presents sustained MES at a rate of up to 2 mM h-1acetate at an average concentration of 60-90 mM by a pure thermophilic microbial culture powered by intermittent electricity. We identified product inhibition of accumulating acetic acid as a key challenge to improving the efficiency of intermittently powered MES.

SummaryProtists in the gut microbiota influence intestinal immunity and epithelial composition, yet the mechanisms used by these protists to shape the gut environment remain poorly understood. Here, we identify a new protist species, Tritrichomonas casperi, and show that metabolic differences between closely related commensal protists modulate their effects on host immunity and determine their ecological niche within the murine microbiota. Genomic and metabolomic analysis of commensal tritrichomonads reveal species-level differences in excretion of the tuft cell activating metabolite succinate, leading to differential induction of Th1, Th2, and Th17 cells in the small intestine. Using defined diets and in vitro growth assays, we show that different tritrichomonad species preferentially rely on dietary polysaccharides or mucus glycans, which leads to trans-kingdom competition with specific bacteria in the microbiota. Our findings elucidate differences in commensal tritrichomonad metabolism and suggest how dietary interventions could regulate the impact of these protists on gut health.

While enzymes catalyze reactions with high selectivity and specificity at ambient temperature and pressure, electroactive enzymes retain such remarkable catalytic properties for catalyzing redox re...

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin‐dependent transport‐coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO₂ reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H₂ consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.

Algal-bacterial interactions provide clues to algal physiology, but mutualistic interactions are complicated by dynamic exchange. We characterized the response of Chlamydomonas reinhardtii to the presence of a putative alga-benefitting commensal bacterium (Arthrobacter strain ‘P2b’). Co-cultivation promoted chlorophyll content, biomass, average cell size, and number of dividing cells, relative to axenic cultures. Addition of bacterial spent medium (whole, size-fractionated and heat-treated) had similar effects, indicating P2b does not require algal interaction to promote growth. Nutrients and pH were excluded as putative effectors, collectively indicating a commensal interaction mediated by Arthrobacter-released small exometabolite(s). Proteogenomic comparison revealed similar response to co-cultivation and spent media, including differential cell cycle regulation, extensive downregulation of flagellar genes and histones, carbonic anhydrase and RubisCO downregulation, upregulation of some chlorophyll, amino acid and carbohydrate biosynthesis genes, and changes to redox and Fe homeostasis. Further, Arthrobacter protein expression indicated some highly expressed putative secondary metabolites. Together, these results revealed that low molecular weight bacterial metabolites can elicit major physiological changes in algal cell cycle regulation, perhaps through a more productive G1 phase, that lead to substantial increases in photosynthetically-produced biomass. This work illustrates that model commensal interactions can be used to shed light on algal response to stimulating bacteria.

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

High-temperature tolerant enzymes offer multiple advantages over enzymes from mesophilic organisms for the industrial production of sustainable chemicals due to high specific activities and stabilities towards fluctuations in pH, heat, and organic solvents. The production of molecular hydrogen (H2) is of particular interest because of the multiple uses of hydrogen in energy and chemicals applications, and the ability of hydrogenase enzymes to reduce protons to H2 at a cathode. We examined the activity of Hydrogen-Dependent CO2 Reductase (HDCR) from the thermophilic bacterium Thermoanaerobacter kivui when immobilized in a redox polymer, cobaltocene-functionalized polyallylamine (Cc-PAA), on a cathode for enzyme-mediated H2 formation from electricity. The presence of Cc-PAA increased reductive current density 340-fold when used on an electrode with HDCR at 40 °C, reaching unprecedented current densities of up to 3 mA·cm−2 with minimal overpotential and high faradaic efficiency. In contrast to other hydrogenases, T. kivui HDCR showed substantial reversibility of CO-dependent inactivation, revealing an opportunity for usage in gas mixtures containing CO, such as syngas. This study highlights the important potential of combining redox polymers with novel enzymes from thermophiles for enhanced electrosynthesis.

The efficient delivery of electrochemically in situ produced H2 can be a key advantage of microbial electrosynthesis over traditional gas fermentation. However, the technical details of how to supply large amounts of electric current per volume in a biocompatible manner remain unresolved. Here, we explored for the first time the flexibility of complex 3D-printed custom electrodes to fine tune H2 delivery during microbial electrosynthesis. Using a model system for H2-mediated electromethanogenesis comprised of 3D fabricated carbon aerogel cathodes plated with nickel-molybdenum and Methanococcus maripaludis, we showed that novel 3D-printed cathodes facilitated sustained and efficient electromethanogenesis from electricity and CO2 at an unprecedented volumetric production rate of 2.2 LCH4 /Lcatholyte/day and at a coulombic efficiency of 99%. Importantly, our experiments revealed that the efficiency of this process strongly depends on the current density. At identical total current supplied, larger surface area cathodes enabled higher methane production and minimized escape of H2. Specifically, low current density (2) enabled by high surface area cathodes was found to be critical for fast start-up times of the microbial culture, stable steady state performance, and high coulombic efficiencies. Our data demonstrate that 3D-printing of electrodes presents a promising design tool to mitigate effects of bubble formation and local pH gradients within the boundary layer and, thus, resolve key critical limitations for in situ electron delivery in microbial electrosynthesis.

Bioelectrochemical power-to-gas presents a promising technology for long-term storage of excess renewable energy in the form of methane. The transition of the technology from laboratory to applied scale is currently challenged by low volumetric production rates, energy losses at the cathode, as well as the unknown physiology of the microbes in an electrochemical reactor. Here, we introduce a stable electromethanogenesis system based on efficient in situ hydrogen production by non-precious-metal catalysts and effective hydrogen uptake by the methanogenic microorganisms. Using NiMo-cathodes and pure cultures of hydrogenotrophic Methanococcus maripaludis, our system achieved an unprecedented volumetric methane production rate from CO2 of 1.4 L methane per L per day. The system performed stably for over 4 weeks with columbic efficiencies steadily above 90%. A physiological analysis of cells in the electromethanogenic reactor revealed robustly-growing cells with nearly identical protein expression patterns to gas-fed controls. Local pH fluctuations at the surface of cathode and cation exchange membrane resulted in a small but noticeable fraction of cell lysis. Our data collectively indicate that physiologically uncompromised cells of a pure methanogenic culture can perform methanogenesis robustly at high specific rate in a biocompatible electromethanogenic reactor using inexpensive, earth-abundant cathode materials.

A microbial battery (MB) breaks down waste organics and stores the liberated electrons in a solid-state cathode. Here, we demonstrate that these electrons can be utilized, along with the products (e.g., carbon dioxide, CO2) of the MB, to electrochemically generate higher-value products from waste. We utilize a series-stacked MB with mediated enzymatic electrosynthesis (EES) to produce either hydrogen (H2) from protons (H+) or formate (HCOO–) from CO2 with the electrons and carbon directly derived from waste organics. We also demonstrate that a reduced solid-state cathode can be used as a solid-state anode as an electron source for EES. This work shows that higher-value products can be electrochemically generated at high faradaic efficiencies (up to 91% for hydrogenase and 72% for formate dehydrogenase) using the energy, electrons, and carbon contained in the waste organic input without the need for continuous oxygen, potentiostatic control, or supplemental power.

CO2 reduction by combined electro- and bio-catalytic reactions is a promising technology platform for sustainable production of chemicals from CO2 and electricity. While heterogeneous electrocatalysts can reduce CO2 to a variety of organic compounds at relatively high reaction rates, these catalysts have limitations achieving high selectivity for any single product beyond CO. Conversely, microbial CO2 reduction pathways proceed at high selectivity; however, the rates at bio-cathodes using direct electron supply via electricity are commonly limiting. Here we demonstrate the use of non-precious metal cathodes that produce hydrogen in situ to support microbial CO2 reduction to C1 and C2 compounds. CoP, MoS2 and NiMo cathodes perform durable hydrogen evolution under biologically relevant conditions, and the integrated system achieves coulombic efficiencies close to 100% without accumulating hydrogen. Moreover, the one-reactor hybrid platform is successfully used for efficient acetate production from electricity and CO2 by microbes previously reported to be inactive in bioelectrochemical systems. The reduction of CO2 by electro- and biocatalysis offers a promising route to the sustainable production of chemicals and fuels. Here, the integration of methanogenic or homoacetogenic microbes with earth-abundant electrodes allows robust, rapid, and selective CO2 reduction with coulombic efficiencies of up to 100%.

CO

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h(−1), the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h(−1)). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh., A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

Quantitative molecular diagnostic methods can effectively detect pathogen-specific nucleic acid sequences, but costs associated with multi-pathogen panels hinder their widespread use in research trials. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Amplification efficiencies ranged from 88 to 98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Due to nanofluidic volumes, detection limits were 1-2 orders of magnitude less sensitive for nL-qPCR than an enteric TaqMan Array Card (TAC). However, higher detection limits do not hinder detection of diarrhea-causing pathogen concentrations. Compared to TAC, nL-qPCR displayed 99% (95% CI 0.98, 0.99) negative percent agreement and 62% (95% CI 0.59, 0.65) overall positive percent agreement for presence of pathogens across diarrheal and non-diarrheal fecal samples. Positive percent agreement was 89% among samples with concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log10 copies/gram of stool [IQR -0.40, -0.28] compared with TAC. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.

Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero-valent iron (Fe0 ) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe0 -corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate-starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO4 2- reduction. The insensitivity of Fe0 corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe0 corrosion.

Molecular hydrogen is a major high-energy carrier for future energy technologies, if produced from renewable electrical energy. Hydrogenase enzymes offer a pathway for bioelectrochemically producing hydrogen that is advantageous over traditional platforms for hydrogen production because of low overpotentials and ambient operating temperature and pressure. However, electron delivery from the electrode surface to the enzyme's active site is often rate-limiting. Here, it is shown that three different hydrogenases from Clostridium pasteurianum and Methanococcus maripaludis, when immobilized at a cathode in a cobaltocene-functionalized polyallylamine (Cc-PAA) redox polymer, mediate rapid and efficient hydrogen evolution. Furthermore, it is shown that Cc-PAA-mediated hydrogenases can operate at high faradaic efficiency (80-100 %) and low apparent overpotential (-0.578 to -0.593 V vs. SHE). Specific activities of these hydrogenases in the electrosynthetic Cc-PAA assay were comparable to their respective activities in traditional methyl viologen assays, indicating that Cc-PAA mediates electron transfer at high rates, to most of the embedded enzymes.

Electrosynthesis of formate is a promising technology to convert CO2 and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H2 by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H2 and formate formation in electrochemical reactors (-800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO2 reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO2 to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate.

CO2 emissions can be transformed into high-added-value commodities through CO2 electrocatalysis; however, efficient low-cost electrocatalysts are needed for global scale-up. Inspired by other emerging technologies, the authors report the development of a gas diffusion electrode containing highly dispersed Ag sites in a low-cost Zn matrix. This catalyst shows unprecedented Ag mass activity for CO production: -614 mA cm-2 at 0.17 mg of Ag. Subsequent electrolyte engineering demonstrates that halide anions can further improve stability and activity of the Zn-Ag catalyst, outperforming pure Ag and Au. Membrane electrode assemblies are constructed and coupled to a microbial process that converts the CO to acetate and ethanol. Combined, these concepts present pathways to design catalysts and systems for CO2 conversion toward sought-after products.

Correction for ‘In situ electrochemical H2 production for efficient and stable power-to-gas electromethanogenesis’ by Frauke Kracke et al., Green Chem., 2020, 22, 6194–6203, DOI: 10.1039/D0GC01894E.

Objectives The selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model. Methods Pharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7. Results Meropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone. Conclusions Simulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

BackgroundA fundamental clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity.MethodsCholera was chosen as a model disease to investigate this important question. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection, as well as exposure to the first-line antibiotic azithromycin detected by mass spectrometry.ResultsAmong diarrheal samples positive by nanoliter quantitative PCR for Vibrio cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively.ConclusionsEstimations of cholera burden may improve by accommodating for the negative effect of antimicrobial exposure on diagnostic positivity. Furthermore, the findings herein challenge our current approach to interpreting and developing bacterial diagnostics given variable rates of lytic bacteriophage and antibiotic exposure.

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that ‘everything is everywhere’. With the discovery of lateral budding in ‘Kolteria novifilia’ and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how ‘microbial dark matter’ can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection., This work was funded by the Deutsche Forschungsgemeinschaft (grant no. JO 893/4-1) and the Volkswagen foundation (experiment no. 89256). M.Y.G. was funded by the NIH IRP at the US National Library of Medicine. Work in the Mascher lab was supported by the Deutsche Forschungsgemeinschaft (grant no. MA2837/2-2) and the Bundeministerium für Bildung und Forschung in the framework of the ERAnet Synthetic Biology (project: ERASynBio2-ECFexpress). R.A. and A.M. were funded by the Max Planck Society.

Quantitative molecular diagnostic methods, such as qPCR, can effectively detect pathogen-specific nucleic acid sequences. However, costs associated with multi-pathogen quantitative molecular diagnostics hinder their widespread use. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Assay efficiencies ranged from 88-98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to complex fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Detection limits were 1-2 orders of magnitude higher for nL-qPCR than an existing enteric TaqMan Array Card (TAC), due to nanofluidic volumes. Compared to the TAC, nL-qPCR displayed 97% (95% CI 0.96, 0.98) negative percent agreement and 63% (95% CI 0.60, 0.66) overall positive percent agreement. Positive percent agreement was 90% for target concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log10copies/gram of stool [IQR -0.41, -0.28] compared with the enteric TAC. Higher detection limits, inherent to nL-qPCR, do not hinder detection of clinically relevant pathogen concentrations. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip described here is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.

Mathematical modeling holds great potential for quantitatively describing biofilm growth in presence or absence of chemical agents used to limit or promote biofilm growth. In this paper, we describe a general mathematical/statistical framework that allows for the characterization of complex data in terms of few parameters and the capability to (i) compare different experiments and exposures to different agents, (ii) test different hypotheses regarding biofilm growth and interaction with different agents, and (iii) simulate arbitrary administrations of agents. The mathematical framework is divided to submodels characterizing biofilm, including new models characterizing live biofilm growth and dead cell accumulation; the interaction with agents inhibiting or stimulating growth; the kinetics of the agents. The statistical framework can take into account measurement and interexperiment variation. We demonstrate the application of (some of) the models using confocal microscopy data obtained using the computer program COMSTAT.

Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides population. Although both vinyl chloride and 1,2-dichloroethane could be simultaneously transformed to ethene, prolonged exposure to 1,2-dichloroethane diminished the vinyl chloride transforming capacity of the culture. Kinetic tests revealed that dechlorination of 1,2-dichloroethane by the consortium was strongly inhibited by cis-dichloroethene but not vinyl chloride. Native polyacrylamide gel electrophoresis and mass spectrometry revealed that a trichloroethene reductive dehalogenase (TceA) homologue was the most consistently expressed of four detectable reductive dehalogenases during 1,2-dichloroethane exposure, suggesting that it catalyzes the reductive dihaloelimination of 1,2-dichloroethane to ethene.

Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1–0.14 μmol cm−2 h−1 at −400 mV vs standard hydrogen electrode and 0.6–0.9 μmol cm−2 h−1 at −500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21–0.23 μmol cm−2 h−1 at −400 mV and 0.57–0.74 μmol cm−2 h−1 at −500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.

Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al 2 O 3 (1 −1 0 2) and α-Fe 2 O 3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO 3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (⩾10 −5 M), more than 99% of these ions partitioned into the biofilms at S. oneidensis /α-Al 2 O 3 (1 −1 0 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Thus, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (⩽10 −6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al 2 O 3 (1 −1 0 2) substantially (∼52% to ∼13% at 10 −7 M, and ∼23% to ∼5% at 10 −6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al 2 O 3 (1 −1 0 2) surfaces (∼15% to ∼28% at 10 −7 M, and ∼1% to ∼7% at 10 −6 M). The higher observed partitioning of Zn(II) (∼28%) at the α-Al 2 O 3 (1 −1 0 2) surfaces compared to Pb(II) (∼13%) in the mixed-metal-ion systems at the lowest concentration (10 −7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al 2 O 3 (1 −1 0 2) surfaces under our experimental conditions. Competitive sorption of Pb(II) and Zn(II) at S. oneidensis /α-Fe 2 O 3 (0 0 0 1)/water interfaces at equi-molar metal-ion concentrations of ⩽10 −6 M showed that the presence of Pb(II) ions decreased Zn(II) partitioning onto α-Fe 2 O 3 (0 0 0 1) significantly (∼45% to −7 M, and ∼41% to 3% at 10 −6 M), whereas adding Zn(II) caused only small changes in Pb(II) partitioning (∼59% to ∼47% at 10 −7 M, and ∼26% to ∼23% at 10 −6 M), suggesting that Pb(II) strongly outcompetes Zn(II) for sorption sites on S. oneidensis -coated α-Fe 2 O 3 (0 0 0 1) surfaces. Our study implies that caution should be taken when applying results obtained from partitioning studies of single-metal-ion systems to mixed-metal-ion systems at complex biofilm/mineral interfaces.

Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) binds preferentially to the α-Al2O3 (1–102) and α-Fe2O3 (0 0 0 1) surfaces at low Pb concentration ([Pb] = 10−7 M) and then increasingly partitions into the biofilm coatings at higher concentrations (10−6 to 10−4 M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10−7 to 10−4 M). In comparison, the α-Al2O3 (0 0 0 1) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0 0 0 1) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1–102) and α-Fe2O3 (0 0 0 1). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1–102) and α-Fe2O3 (0 0 0 1) at [Me(II)] of 10−7 M; at 10−5 M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20–24 h), both Pb(II) and Zn(II) increasingly partition to the metal-oxide surfaces at [Me(II)] = 10−5 M and pH 6.0, indicating possible reaction/diffusion-controlled sorption processes. Pb LIII-edge and Zn K-edge grazing-incidence extended X-ray absorption fine structure (GI-EXAFS) measurements suggest that both Pb(II) and Zn(II) ions may be complexed by carboxyl groups in S. oneidensis biofilms after 3-h exposure at pH 6.0 and [Me(II)] = 10−5 M. In contrast with Burkholderia cepacia, which was used in our previous studies of monolayer biofilm-coated metal-oxide surfaces (Templeton et al., 2001), S. oneidensis MR-1 forms relatively thick biofilm coatings (6–20 μm) that are rich in reactive functional groups and are expected to dominate metal-ion adsorption. Our results show that even thick and highly reactive biofilms like S. oneidensis do not cause much change in the intrinsic chemical reactivities of the underlying metal-oxide surfaces with respect to aqueous Pb(II) and Zn(II) and don’t block reactive sites on the metal-oxide surfaces; instead they reduce the rate of Pb(II) and Zn(II) sorption onto these surfaces.

Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO2 to formylmethanofuran with H2-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu. We have identified and purified three Hdr complexes of M. maripaludis, where homodimeric Hdr complexes containing (Vhu)2 or (Fdh)2 were found, in addition to a heterocomplex that contains both Vhu and Fdh. Formate was found in in vitro assays using the purified Hdr complex to act directly as the electron donor for FBEB via the associated Fdh. Furthermore, while ferredoxin was slowly reduced to 30% [-360 mV vs the standard hydrogen electrode (SHE)] by H2 and formate (0.8 atm and 30 mM, according to thermodynamics), the addition of CoB-S-S-CoM as the high-potential electron acceptor ( E°' = -140 mV vs SHE; to induce FBEB) resulted in the rapid and more complete reduction of Fd to 94% (-455 mV vs SHE).

Methanogenic archaea can be integrated into a sustainable, carbon-neutral cycle for producing organic chemicals from C1 compounds if the rate, yield, and titer of product synthesis can be improved using metabolic engineering. However, metabolic engineering techniques are limited in methanogens by insufficient methods for controlling cellular protein levels. We conducted a systematic approach to tune protein levels in Methanosarcina acetivorans C2A, a model methanogen, by regulating transcription and translation initiation. Rationally designed core promoter and ribosome binding site mutations in M. acetivorans C2A resulted in a predicable change in protein levels over a 60 fold range. The overall range of protein levels was increased an additional 3 fold by introducing the 5′ untranslated region of the mcrB transcript. This work demonstrates a wide range of precisely controlled protein levels in M. acetivorans C2A, which will help facilitate systematic metabolic engineering efforts in methanogens.

The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi., IMPORTANCE The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi. Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides.

Methane is the primary end product from cathodic current in microbial electrolysis cells (MECs) in the absence of methanogenic inhibitors, but little is known about the archaeal communities that develop in these systems. MECs containing cathodes made from different materials (carbon brushes, or plain graphite blocks or blocks coated with carbon black and platinum, stainless steel, nickel, ferrihydrite, magnetite, iron sulfide, or molybdenum disulfide) were inoculated with anaerobic digester sludge and acclimated at a set potential of −600 mV (versus a standard hydrogen electrode). The archaeal communities on all cathodes, except those coated with platinum, were predominated by Methanobacterium (median 97% of archaea). Cathodes with platinum contained mainly archaea most similar to Methanobrevibacter. Neither of these methanogens were abundant (

It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods. Here, we report that trichloroacetic acid increases the MS signal of tobramycin. To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed. Aliquots of 25 μL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid. The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode. ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification. The calibration curve concentration range was 50–25000 ng/mL. Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold). The method is simple, fast, and reliable. Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.

The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi . In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi . 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi . We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi . IMPORTANCE The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi . In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi . Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides .

A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe–S cluster–containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe–S cluster cofactors and discuss physiological implications.