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Synthetic materials are integral components of consumable and durable goods and are indispensable in the modern world. Polyesters are the most versatile bulk- and specialty-polymers but their production is not sustainable and their fate at end-of-life is of great concern. Bioplastics are highly regarded alternatives but often fall behind conventional synthetic plastics due to shortcomings in material properties and commercial competitiveness. This has limited the success of sustainable replacements at global market scale. Enabling production of bioplastics with superior properties from waste-derived feedstocks could change that. To this end, we created a synthetic entry into the metabolic pathway of bio-polyester synthesis of Cupriavidus necator H16 by means of heterologous hydroxyacyl-CoA transferase and mutant PHA synthase. The resulting microbial cell factories produced a range of aliphatic and aromatic bio-polyesters and enabled co-polymerization of a range of hydroxy carboxylates, including a hydroxyphenylic and a hydroxyfuranoic acid, for the first time incorporating aromatic rings in the backbone of biological polyesters. These diverse polymers were then characterized in terms of their physical properties. The resulting polymers were structurally analogous to synthetic polyesters like PET, PEF and other polyarylates. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters from in-situ generated O2, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility of de-novo production of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of various high-performance thermoplastics and thermosets.

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.

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.

Synthetic materials are integral components of consumable and durable goods and are indispensable in the modern world. Polyesters are the most versatile bulk- and specialty-polymers but their production is not sustainable and their fate at end-of-life is of great concern. Bioplastics are highly regarded alternatives but often fall behind conventional synthetic plastics due to shortcomings in material properties and commercial competitiveness. This has limited the success of sustainable replacements at global market scale. Enabling production of bioplastics with superior properties from waste-derived feedstocks could change that. To this end, we created a synthetic entry into the metabolic pathway of bio-polyester synthesis ofCupriavidus necatorH16 by means of heterologous hydroxyacyl-CoA transferase and mutant PHA synthase. The resulting microbial cell factories produced a range of aliphatic and aromatic biopolyesters and enabled co-polymerization of a range of hydroxy carboxylates, including a hydroxyphenylic and a hydroxyfuranoic acid, for the first time incorporating aromatic rings in the backbone of biological polyesters. These diverse polymers were then characterized in terms of their physical properties. The resulting polymers were structurally analogous to synthetic polyesters like PET, PEF and other polyarylates. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters fromin-situgenerated O2, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility ofde novoproduction of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of various high-performance thermoplastics and thermosets.Significance statementBiomaterials can facilitate the transition of chemical industry to a carbon-neutral and circular economy and prevent the accumulation of greenhouse gases and plastic waste in the natural environment by developing bio-replacements for existing fossil carbon-based plastics along with end-of-life strategies. Accomplished via the genetic engineering of a microbial cell factory that assimilates carbon dioxide, this work demonstrates the first biocatalytic polymerization of aromatic building blocks and their incorporation into the backbone of a bio-polyester. Employing a bio-electrochemical system for cultivation of the microbes, oxyhydrogen is formed and consumedin-situ, thus avoiding explosive gasmixtures. The obtained aromatic polyesters are structural analogs to synthetic bulk- and high-performance polymers such as PET (polyethylene terephthalate) and PEF (polyethylene furanoate).Highlights-First demonstration of bio-polyarylates – microbial polyesters with aromatic rings in the backbone-Production of novel PHAs from CO2and H2+O2achievedin-situin a bio-electrochemical system-Expression-level of PHA synthase and molecular weight of polyesters is inversely correlated-In silicodesign and pathway analysis of bio-polyesters from low-cost carbon-feedstocks

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.

CO

A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2-C4 carboxylic acids and alcohols from carbon dioxide. Each cathode chamber provides ideal pH conditions to favor acetogenesis/carbon chain elongation (pH = 6.9), and solventogenesis (pH = 4.9), respectively, without the requirement of external acid/base dosing.

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.

Amphiphilic polycations are an alternative to biocides but also toxic to mammalian cells. Antimicrobially active hydrophilic polycations based on 1,4-dibromo-2-butene and tetramethyl-1,3-propanediamine named PBI are not hemotoxic for porcine red blood cells with a hemocytotoxicity (HC50) of more than 40,000 μg · mL(-1). They are quickly killing bacterial cells at their MIC (minimal inhibitory concentration). The highest found selectivity HC50 /MIC is more than 20,000 for S. epidermidis. Investigations on sequentially prepared PBIs with defined molecular weight Mn and tailored end groups revealed that there is a dependence of antimicrobial activity and selectivity on Mn and nature of the end groups.

More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering.

Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]3+) were tested for their toxicity and electro-fermentations were performed in 1L bioreactors covering 38 biotic and 8 abiotic runs. When using C. kluyveri and mediators, maximum absolute current densities higher than the abiotic controls were detected for all runs. At the same time, no significant impact on the cell metabolism (product formation, carbon recovery, growth rate) was found. From this observation, we deduce general potential limitations of electro-fermentations with C. kluyveri and discuss strategies to successfully overcome them.

Microbial electrosynthesis (MES) and electro-fermentation are novel approaches to increase microbial production by stimulating the metabolism of the cells electrically. The technique is still in its infancy and while already hyped as promising method to convert CO2 or cheap carbon sources and electrical energy into valuable chemicals and fuels, little is known about its true potential. This project uses a combination of in silico and in vivo approaches to gather novel information about the benefits and limitations of microbial electrosynthesis as well as its fundamental mechanisms. Understanding microbial electron transport mechanisms is the key to optimization of any bioelectrochemical technology. Therefore, this work analyses the different electron transport chains that nature offers for organisms such as metal respiring bacteria and acetogens, but also standard biotechnological organisms currently used in bioproduction. Special focus lies on the essential connection of redox and energy metabolism, which is often ignored when studying bioelectrochemical systems. The possibility of extracellular electron exchange at different points in each organism is discussed regarding required redox potentials and effect on cellular redox and energy levels. Key compounds such as electron carriers (e.g. cytochromes, ferredoxins, quinones, flavins) are identified and analysed regarding their possible role in electrode-microbe-interactions. Based on these findings a stoichiometric network analysis is performed analysing the effect of electrical stimulation on the microbial metabolism theoretically. Escherichia coli is used as model organism for production with the aim to identify target processes for MES. For the first time, 20 different valuable products were screened for their potential to show increased yields during anaerobic electrically enhanced fermentation. Surprisingly it was found that an increase in product formation by electrical enhancement is not necessarily dependent on the degree of reduction of the product but rather the metabolic pathway it is derived from. Contrary to the usual assumption a reduced product would always require electron supply by a cathode this study shows that a complex metabolic analysis is needed to identify the overall redox state of each process. A variety of beneficial processes is presented with product yield increases of maximal +36% in reductive and +84% in oxidative fermentations and final theoretical product yields up to 100%. This includes compounds that are already produced at industrial scale such as succinic acid, lysine and diaminopentane as well as potential novel bio-commodities such as isoprene, para-hydroxybenzoic acid and para-aminobenzoic acid. Furthermore, it is shown that the way of electron transport has major impact on achievable biomass and product yields. The coupling of electron transport to energy conservation could be identified as crucial for most processes. The in vivo approach of this project introduces the development of a standardised reactor platform suitable for reproducible, fully controlled electrically enhanced fermentations. Different organisms that are used in industrial bio-production were screened for their ability of electron uptake: E. coli, Propionibacterium (P.) acidipropionici, P. freudenreichii, Citrobacter werkmanii and Clostridium (C.) autoethanogenum. A growth-related current consumption was monitored for all tested strains, however in most cases the electron uptake was too small compared to substrate uptake to result in significant metabolic changes. An exception is presented by the GRAM-positive acetogen C. autoethanogenum, which shows a considerable metabolic shift from acetate towards lactate and 2,3 butanediol caused by extracellular electron supply. This is the first report of electro-activity of the close relative to C. ljungdahlii. In heterotrophic fermentations, extracellular electron supply cut acetate production by more than half while production of lactate and 2,3-butanediol increased thirty-five fold and three fold, respectively. Interestingly this metabolic response crucially depends on the redox potential at which the electrons are supplied, which is proven by the use of different mediator molecules. In agreement with the findings from the in silico study, this again is an indication for the importance of the metabolic pathway by which electrons enter the cellular metabolism for the success of microbial electrosynthesis. This work applies a powerful metabolic modelling tool and studies electron transport mechanisms in the industrial alcohol producing Clostridium autoethanogenum. As such it adds an important piece of fundamental understanding of microbial electron transport possibilities to the research community and will help to optimize and advance bio-electrochemical techniques beyond the level of lab-scale studies.

The replacement of petrochemical aromatics with bio-based molecules is a key area of current biotechnology research. To date, a small number of aromatics have been produced by recombinant bacteria in laboratory scale while industrial production still requires further strain development. While each study includes some distinct analytical methodology to quantify certain aromatics, a method that can reliably quantify a great number of aromatic products and relevant pathway intermediates is needed to accelerate strain development. In this study, we developed a robust reverse phase high performance liquid chromatography method to quantify a wide range of aromatic metabolites present in host microorganisms using the shikimate pathway, which is the major metabolic pathway for biosynthesis of aromatics. Twenty-three metabolites can be quantified precisely with the optimized method using standard HPLC equipment and UV detection, with the mobile phase used for chromatography also compatible with mass spectrometry (MS). The limit of quantification/detection is as low as 10 to 10 mol, respectively, which makes this method feasible for quantification of intracellular metabolites. This method covers most metabolic routes for aromatics biosynthesis, it is inexpensive, robust, simple, precise and sensitive, and has been demonstrated on cell extracts from S. cerevisiae genetically engineered to overproduce aromatics.

Background Microbial electrosynthesis is a novel approach that aims at shifting the cellular metabolism towards electron-dense target products by extracellular electron supply. Many organisms including several acetogenic bacteria have been shown to be able to consume electrical current. However, suitable hosts for relevant industrial processes are yet to be discovered, and major knowledge gaps about the underlying fundamental processes still remain. Results In this paper, we present the first report of electron uptake by the Gram-positive, ethanol-producing acetogen, Clostridium autoethanogenum. Under heterotrophic conditions, extracellular electron supply induced a significant metabolic shift away from acetate. In electrically enhanced fermentations on fructose, acetate production was cut by more than half, while production of lactate and 2,3-butanediol increased by 35-fold and threefold, respectively. The use of mediators with different redox potential revealed a direct dependency of the metabolic effect on the redox potential at which electrons are supplied. Only electrons delivered at a redox potential low enough to reduce ferredoxin caused the reported effect. Conclusions Production in acetogenic organisms is usually challenged by cellular energy limitations if the target product does not lead to a net energy gain as in the case of acetate. The presented results demonstrate a significant shift of carbon fluxes away from acetate towards the products, lactate and 2,3-butanediol, induced by small electricity input (~0.09 mol of electrons per mol of substrate). This presents a simple and attractive method to optimize acetogenic fermentations for production of chemicals and fuels using electrochemical techniques. The relationship between metabolic shift and redox potential of electron feed gives an indication of possible electron-transfer mechanisms and helps to prioritize further research efforts. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0663-2) contains supplementary material, which is available to authorized users.

Improving the performance of cathodes for the electrochemical CO2 reduction reaction (CO2RR) will benefit from the discovery of new materials as well as the introduction of new paradigms for the development of integrated systems. This poster presents work on the synthesis and CO2 reduction activity of a novel quasi-2D Pd/Pt bimetallic ‘nanoclam’ catalyst synthesized on carbon cloth electrodes as well as the integration of these novel nanostructured cathodes with microbial communities. The integration of microbial communities with these nanostructured Pd/Pt catalysts has the potential to combine the best-of-both-worlds from electrochemical and biological systems to achieve a regenerative catalytic system with high-selectivity, high activity, and low overpotential.

Amphiphilic polycations are an alternative to biocides but also toxic to mammalian cells. Antimicrobially active hydrophilic polycations based on 1,4-dibromo-2-butene and tetramethyl-1,3-propanediamine named PBI are not hemotoxic for porcine red blood cells with a hemocytotoxicity (HC50) of more than 40,000 μg · mL(-1). They are quickly killing bacterial cells at their MIC (minimal inhibitory concentration). The highest found selectivity HC50 /MIC is more than 20,000 for S. epidermidis. Investigations on sequentially prepared PBIs with defined molecular weight Mn and tailored end groups revealed that there is a dependence of antimicrobial activity and selectivity on Mn and nature of the end groups.

Background Microbial electrosynthesis and electro fermentation are techniques that aim to optimize microbial production of chemicals and fuels by regulating the cellular redox balance via interaction with electrodes. While the concept is known for decades major knowledge gaps remain, which make it hard to evaluate its biotechnological potential. Here we present an in silico approach to identify beneficial production processes for electro fermentation by elementary mode analysis. Since the fundamentals of electron transport between electrodes and microbes have not been fully uncovered yet, we propose different options and discuss their impact on biomass and product yields. Results For the first time 20 different valuable products were screened for their potential to show increased yields during anaerobic electrically enhanced fermentation. Surprisingly we found that an increase in product formation by electrical enhancement is not necessarily dependent on the degree of reduction of the product but rather the metabolic pathway it is derived from. We present a variety of beneficial processes with product yield increases of maximal 36% in reductive and 84% in oxidative fermentations and final theoretical product yields up to 100%. This includes compounds that are already produced at industrial scale such as succinic acid, lysine and diaminopentane as well as potential novel bio-commodities such as isoprene, para-hydroxybenzoic acid and para-aminobenzoic acid. Furthermore, it is shown that the way of electron transport has major impact on achievable biomass and product yields. The coupling of electron transport to energy conservation could be identified as crucial for most processes. Conclusions This study introduces a powerful tool to determine beneficial substrate and product combinations for electro-fermentation. It also highlights that the maximal yield achievable by bio electrochemical techniques depends strongly on the actual electron transport mechanisms. Therefore it is of great importance to reveal the involved fundamental processes to be able to optimize and advance electro fermentations beyond the level of lab-scale studies. Electronic supplementary material The online version of this article (doi:10.1186/s12859-014-0410-2) contains supplementary material, which is available to authorized users.

The production of fuels and chemicals by electricity-driven bio-production (i.e., using electric energy to drive biosynthesis) holds great promises. However, this electrification of white biotechnology is particularly challenging to achieve because of the different optimal operating conditions of electrochemical and biochemical reactions. In this article, we address the technical parameters and obstacles to be taken into account when engineering microbial bioelectrochemical systems (BES) for bio-production. In addition, BES-based bio-production processes reported in the literature are compared against industrial needs showing that a still large gap has to be closed. Finally, the feasibility of BES bio-production is analysed based on bulk electricity prices. Using the example of lysine production from sucrose, we demonstrate that there is a realistic market potential as cost savings of 8.4 % (in EU) and 18.0 % (in US) could be anticipated, if the necessary yields can be obtained.