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Due to its characteristics, hydrogen is considered the energy carrier of the future. Its use as a fuel generates reduced pollution, as if burned it almost exclusively produces water vapor. Hydrogen can be produced from numerous sources, both of fossil and renewable origin, and with as many production processes, which can use renewable or non-renewable energy sources. To achieve carbon neutrality, the sources must necessarily be renewable, and the production processes themselves must use renewable energy sources. In this review article the main characteristics of the most used hydrogen production methods are summarized, mainly focusing on renewable feedstocks, furthermore a series of relevant articles published in the last year, are reviewed. The production methods are grouped according to the type of energy they use; and at the end of each section the strengths and limitations of the processes are highlighted. The conclusions compare the main characteristics of the production processes studied and contextualize their possible use.

HighlightsHydrogen-oxidising bacteria (HOBs) convert CO 2, hydrogen (H 2), and potentially nitrogen (N 2) into biomass via gas fermentation, promising to decouple food production from agriculture. Industrial-scale gas fermentation has been shown to be feasible across a variety of microbial species and gases. The development of engineered microbial strains for the precision fermentation of novel foods gains momentum due to maturing synthetic biology tools, pressing environmental concerns, and a consequential shift in consumer preferences. Combining industrial-scale gas fermentation capabilities with state-of-the-art precision fermentation technologies enables the development of gas precision fermentation processes. Product development and commercialisation would greatly benefit from focused generation of HOB tools and improved gas fermentation capacities in academia and industry.

Sustainable production of carbon-based products is urgently needed.A novel directed flow-through microbial electrosynthesis (MES) reactor was designed and characterized for carbon dioxide (CO2) conversion to C2–C6 carboxylates.Three-times denser biofilm, volumetric current density, and productivity were achieved compared with the state of the art.Biomass-specific production rates were maintained over more than 200 days, yet still an order of magnitude lower than that achieved by acetogens in syngas fermentation.Volumetric productivity in MES was comparable with that from syngas fermentation.Clostridium luticellarii and Eubacterium limosum were the dominant species. Carbon-based products are essential to society, yet producing them from fossil fuels is unsustainable. Microorganisms have the ability to take up electrons from solid electrodes and convert carbon dioxide (CO2) to valuable carbon-based chemicals. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative. Here, we show how a biofilm-based microbial porous cathode in a directed flow-through electrochemical system can continuously reduce CO2 to even-chain C2–C6 carboxylic acids over 248 days. We demonstrate a threefold higher biofilm concentration, volumetric current density, and productivity compared with the state of the art. Most notably, the volumetric productivity (VP) resembles those achieved in laboratory-scale and industrial syngas (CO-H2-CO2) fermentation and chain elongation fermentation. This work highlights key design parameters for efficient electricity-driven microbial CO2 reduction. There is need and room to improve the rates of electrode colonization and microbe-specific kinetics to scale up the technology. Graphical abstract [Display omitted] Microbial electrosynthesis (MES) of carboxylic acids from CO2 and electricity has been validated for over a decade, now reaching Technology Readiness Levels 3/4 in laboratory settings. However, process optimization is needed before demonstrating an industrial prototype. Key challenges for full-scale implementation include ensuring production stability. Critical areas to investigate and demonstrate are: (i) the impact of CO2 feed stream composition and properties; (ii) the short- and long-term effects of renewable electricity supply intermittency; and (iii) the flexibility of MES operations and the integrated process, including up- and downstream processes. Moreover, a comprehensive market analysis is required for each target product. For instance, hexanoic acid, which serves as a precursor for nylon, plasticizers, lubricants, pharmaceuticals, fragrances, fuels, and animal feed, necessitates the development of business models that consider complete supply chains and systems. Carbon-based products are crucial to society. However, their production from fossil-based carbon is unsustainable. We developed a scalable microbial electrosynthesis process that produces medium-chain carboxylic acids from CO2 and renewable electricity, using microorganisms as a catalyst. This represents a promising avenue for generating low CO2-footprint precursors for the chemical, fuel, feed, and food industries.

Living biological systems display a fascinating ability to self-organize their metabolism. This ability ultimately determines the metabolic robustness that is fundamental to controlling cellular behavior. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cultures exhibit rhythmic oscillatory behavior in high cell-density continuous cultures. Oscillatory behavior provides a unique opportunity for quantitating the robustness of metabolism, as cells respond to changes by inherently compromising metabolic efficiency. Here, we quantify the limits of metabolic robustness in self-oscillating autotrophic continuous cultures of the gas-fermenting acetogen Clostridium autoethanogenum. Online gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular byproducts synchronized with biomass levels. The data show initial growth on CO, followed by growth on CO and H₂. Growth on CO and H₂ results in an accelerated growth phase, after which a downcycle is observed in synchrony with a loss in H₂ uptake. Intriguingly, oscillations are not linked to translational control, as no differences were observed in protein expression during oscillations. Intracellular metabolomics analysis revealed decreasing levels of redox ratios in synchrony with the cycles. We then developed a thermodynamic metabolic flux analysis model to investigate whether regulation in acetogens is controlled at the thermodynamic level. We used endo- and exo-metabolomics data to show that the thermodynamic driving force of critical reactions collapsed as H₂ uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in acetogen gas fermentation are controlled at the thermodynamic level.

ABSTRACT Unabated mining and utilisation of petroleum and petroleum resources and their conversion to essential fuels and chemicals have drastic environmental consequences, contributing to global warming and climate change. In addition, fossil fuels are finite resources, with a fast-approaching shortage. Accordingly, research efforts are increasingly focusing on developing sustainable alternatives for chemicals and fuels production. In this context, bioprocesses, relying on microorganisms, have gained particular interest. For example, acetogens use the Wood-Ljungdahl pathway to grow on single carbon C1-gases (CO2 and CO) as their sole carbon source and produce valuable products such as acetate or ethanol. These autotrophs can, therefore, be exploited for large-scale fermentation processes to produce industrially relevant chemicals from abundant greenhouse gases. In addition, genetic tools have recently been developed to improve these chassis organisms through synthetic biology approaches. This review will focus on the challenges of genetically and metabolically modifying acetogens. It will first discuss the physical and biochemical obstacles complicating successful DNA transfer in these organisms. Current genetic tools developed for several acetogens, crucial for strain engineering to consolidate and expand their catalogue of products, will then be described. Recent tool applications for metabolic engineering purposes to allow redirection of metabolic fluxes or production of non-native compounds will lastly be covered. This review systematically discusses the challenges of genetically modifying acetogenic chassis, and the recent development of several genetic tools applied to engineer these industrially important microbes for sustainable production of fuels and chemicals from greenhouse gases using C1-gas fermentation.

Abstract The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures containing H2/CO2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol, the high productivity of continuous bioprocesses is needed. While the first industrial-scale gas fermentation facility operates continuously, the acetone–butanol–ethanol (ABE) fermentation is traditionally operated in batch mode. This review highlights the benefits of continuous bioprocessing for solvent production and underlines the progress made towards its establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current industrial bioproduction of solvents.

Nowadays, sustainable and renewable energy production is a global priority. Over the past decade, several Power-to-X (PtX) technologies have been proposed to store and convert the surplus of renewable energies into chemical bonds of chemicals produced by different processes. CO2 is a major contributor to climate change, yet it is also an undervalued source of carbon that could be recycled and represents an opportunity to generate renewable energy. In this context, PtX technologies would allow for CO2 valorization into renewable fuels while reducing greenhouse gas (GHG) emissions. With this work we want to provide an up-to-date overview of biomethanation as a PtX technology by considering the biological aspects and the main parameters affecting its application and scalability at an industrial level. Particular attention will be paid to the concept of CO2-streams valorization and to the integration of the process with renewable energies. Aspects related to new promising technologies such as in situ, ex situ, hybrid biomethanation and the concept of underground methanation will be discussed, also in connection with recent application cases. Furthermore, the technical and economic feasibility will be critically analyzed to highlight current options and limitations for implementing a sustainable process.

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.

Background Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential. Results In this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system. Initial efforts to retrofit the CRISPR/Cas9 system for C. autoethanogenum resulted in poor efficiency likely due to uncontrolled expression of Cas9. To address this, we constructed and screened a small library of tetracycline-inducible promoters that can also be used to fine-tune gene expression. With a new inducible promoter, the efficiency of CRISPR/Cas9-mediated desired gene deletion in C. autoethanogenum was improved to over 50 %, making it a viable tool for engineering C. autoethanogenum. Conclusions Addition of both an inducible promoter library and a scarless genome editing tool is an important expansion to the genetic tool box of industrial C. autoethanogenum strain.

Background Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator ; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator . The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO 2 . Results In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB − 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO 2 in the feed industry.