A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer.

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in "Ca. Micrarchaeum harzensis A_DKE," and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated, and the micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described coculture, containing "Ca. Micrarchaeum harzensis A_DKE" and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of "Ca. Micrarchaeum harzensis A_DKE." Both organisms are clearly distinguishable by cell size, shape, and surface structure. IMPORTANCE Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member "Candidatus Micrarchaeum harzensis A_DKE," an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of "Ca. Micrarchaeum harzensis A_DKE" is an important step toward understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

Electron transfer of extremophiles in bioelectrochemical systems.

The interaction of bacteria and archaea with electrodes is a relatively new research field which spans from fundamental to applied research and influences interdisciplinary research in the fields of microbiology, biochemistry, biotechnology as well as process engineering. Although a substantial understanding of electron transfer processes between microbes and anodes and between microbes and cathodes has been achieved in mesophilic organisms, the mechanisms used by microbes under extremophilic conditions are still in the early stages of discovery. Here, we review our current knowledge on the biochemical solutions that evolved for the interaction of extremophilic organisms with electrodes. To this end, the available knowledge on pure cultures of extremophilic microorganisms has been compiled and the study has been extended with the help of bioinformatic analyses on the potential distribution of different electron transfer mechanisms in extremophilic microorganisms.

Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime.

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

Genetic engineering for enhanced productivity in bioelectrochemical systems.

A shift from petrochemical processes toward a bio-based economy is one of the most advocated developments for a sustainable future. To achieve this will require the biotechnological production of platform chemicals that can be further processed by chemical engineering. Bioelectrochemical systems (BESs) are a novel tool within the biotechnology field. In BESs, microbes serve as biocatalysts for the production of biofuels and value-added compounds, as well as for the production of electricity. Although the general feasibility of bioelectrochemical processes has been demonstrated in recent years, much research has been conducted to develop biocatalysts better suited to meet industrial demands. Initially, mainly natural exoelectrogenic organisms were investigated for their performance in BESs. Driven by possibilities of recent developments in genetic engineering and synthetic biology, the spectrum of microbial catalysts and their versatility (substrate and product range) have expanded significantly. Despite these developments, there is still a tremendous gap between currently achievable space-time yields and current densities on the one hand and the theoretical limits of BESs on the other. It will be necessary to move the performance of the biocatalysts closer to the theoretical possibilities in order to establish viable production routines. This review summarizes the status quo of engineering microbial biocatalysts for anode-applications with high space-time yields. Furthermore, we will address some of the theoretical limitations of these processes exemplarily and discuss which of the present strategies might be combined to achieve highly synergistic effects and, thus, meet industrial demands.

Extracellular reduction of solid electron acceptors by Shewanella oneidensis.

Shewanella oneidensis is the best understood model organism for the study of dissimilatory iron reduction. This review focuses on the current state of our knowledge regarding this extracellular respiratory process and highlights its physiologic, regulatory and biochemical requirements. It seems that we have widely understood how respiratory electrons can reach the cell surface and what the minimal set of electron transport proteins to the cell surface is. Nevertheless, even after decades of work in different research groups around the globe there are still several important questions that were not answered yet. In particular, the physiology of this organism, the possible evolutionary benefit of some responses to anoxic conditions, as well as the exact mechanism of electron transfer onto solid electron acceptors are yet to be addressed. The elucidation of these questions will be a great challenge for future work and important for the application of extracellular respiration in biotechnological processes.

Evaluation of productive biofilms for continuous lactic acid production.

In white biotechnology research, the putative superiority of productive biofilms to conventional biotransformation processes based on planktonic cultures has been increasingly discussed in recent years. In the present study, we chose lactic acid production as a model application to evaluate biofilm potential. A pure culture of Lactobacillus bacteria was grown in a tubular biofilm reactor. The biofilm system was cultivated monoseptically in a continuous mode for more than 3 weeks. The higher cell densities that could be obtained in the continuous biofilm system compared with the planktonic culture led to a significantly increased space-time yield. The productivity reached 80% of the maximum value 10 days after start-up and no subsequent decline was observed, confirming the suitability of the system for long-term fermentation. The analysis of biofilm performance revealed that productivity increases with the flow velocity. This is explained by the reduced retention time of the liquid phase in the reactor, and, thus, a minor pH drop caused by the released lactic acid. At low flow velocities, the pH drops to a value where growth and production are significantly inhibited. The biofilm was visualized by magnetic resonance imaging to analyze biofilm thickness. To deepen the understanding of the biofilm system, we used a simple model for cell growth and lactic acid production.

Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins.

This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

Biofilm systems as tools in biotechnological production.

The literature provides more and more examples of research projects that develop novel production processes based on microorganisms organized in the form of biofilms. Biofilms are aggregates of microorganisms that are attached to interfaces. These viscoelastic aggregates of cells are held together and are embedded in a matrix consisting of multiple carbohydrate polymers as well as proteins. Biofilms are characterized by a very high cell density and by a natural retentostat behavior. Both factors can contribute to high productivities and a facilitated separation of the desired end-product from the catalytic biomass. Within the biofilm matrix, stable gradients of substrates and products form, which can lead to a differentiation and adaptation of the microorganisms' physiology to the specific process conditions. Moreover, growth in a biofilm state is often accompanied by a higher resistance and resilience towards toxic or growth inhibiting substances and factors. In this short review, we summarize how biofilms can be studied and what most promising niches for their application can be. Moreover, we highlight future research directions that will accelerate the advent of productive biofilms in biology-based production processes.

Investigation on the anaerobic propionate degradation by Escherichia coli K12.

Propionate is an abundant carboxylic acid in nature. Microorganisms metabolize propionate aerobically via the 2-methylcitrate pathway. This pathway depends on a series of three reactions in the citric acid cycle that leads to the conversion of succinate to oxaloacetate. Interestingly, the γ-proteobacterium Escherichia coli can use propionate as a carbon and electron source under oxic but not under anoxic conditions. RT-PCR and transcriptomic analysis revealed a posttranscriptional regulation of the prpBCDE-gene cluster encoding the necessary enzymes for propionate metabolism. The polycistronic mRNA seems to be hydrolyzed in the 3'-5' direction under anoxic conditions. This regulatory strategy is highly constructive because the last gene of the operon encodes the first enzyme of the propionate metabolism. Further analysis revealed that RNase R is involved in the hydrolysis of the prp transcripts. Consequently, an rnr-deletion strain could metabolize propionate under anoxic conditions. To the best of our knowledge, this is the first study describing the influence of RNase R on the anaerobic metabolism of E. coli.

Chromate Resistance Mechanisms in Leucobacter chromiiresistens.

Chromate is one of the major anthropogenic contaminants on Earth. Leucobacter chromiiresistens is a highly chromate-resistant strain, tolerating chromate concentrations in LB medium of up to 400 mM. In response to chromate stress, L. chromiiresistens forms biofilms, which are held together via extracellular DNA. Inhibition of biofilm formation leads to drastically decreased chromate tolerance. Moreover, chromate is reduced intracellularly to the less-toxic Cr(III). The oxidation status and localization of chromium in cell aggregates were analyzed by energy-dispersive X-ray spectroscopy coupled to scanning transmission electron microscopy and X-ray absorption spectroscopy measurements. Most of the heavy metal is localized as Cr(III) at the cytoplasmic membrane. As a new cellular response to chromate stress, we observed an increased production of the carotenoid lutein. Carotenoid production could increase membrane stability and reduce the concentration of reactive oxygen species. Bioinformatic analysis of the L. chromiiresistens genome revealed several gene clusters that could enable heavy-metal resistance. The extreme chromate tolerance and the unique set of resistance factors suggest the use of L. chromiiresistens as a new model organism to study microbial chromate resistance.IMPORTANCE Chromate is a highly toxic oxyanion. Extensive industrial use and inadequate waste management has caused the toxic pollution of several field sites. Understanding the chromate resistance mechanisms that enable organisms to thrive under these conditions is fundamental to develop (micro)biological strategies and applications aiming at bioremediation of contaminated soils or waters. Potential detoxifying microorganisms are often not sufficient in their resistance characteristics to effectively perform, e.g., chromate reduction or biosorption. In this study, we describe the manifold strategies of L. chromiiresistens to establish an extremely high level of chromate resistance. The multitude of mechanisms conferring it make this organism suitable for consideration as a new model organism to study chromate resistance.

Kyrpidia spormannii sp. nov., a thermophilic, hydrogen-oxidizing, facultative autotroph, isolated from hydrothermal systems at São Miguel Island, and emended description of the genus Kyrpidia.

A Gram-stain-positive, rod-shaped, non-motile, spore-forming bacterium, strain EA-1T, was isolated from hydrothermal sediment samples from the Azores (São Miguel, Portugal). 16S rRNA gene sequence analysis of the isolated bacterium revealed a phylogenetic affiliation with the genus Kyrpidia. The sequence similarity of the five 16S rRNA gene copies to its closest relative, Kyrpidia tusciae, ranged from 97.79 to 97.85 %. The in silico estimate of DNA-DNA hybridization was 56.0 %. The dominant fatty acids of the novel isolate were anteiso-C17 : 0 (49.9 %), iso-C17 : 0 (23.0 %) and iso-C16 : 0 (13.3 %), while the quinone detected was menaquinone MK-7. Analysis of polar lipids identified phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and additional unidentified compounds comprising two glycolipids, two phospholipids and two lipids. The presence of meso-diaminopimelic acid in the peptidoglycan and mannose, arabinose and ribose in the cell wall of strain EA-1T were detected. The strain was able to grow heterotrophically as well as autotrophically with carbon dioxide as the sole carbon source and with hydrogen and oxygen as electron donor and acceptor, respectively. Based on its chemotaxonomic, physiological and genomic characteristics, the new strain is considered to represent a novel species within the genus Kyrpidia, for which the name Kyrpidiaspormannii sp. nov. is proposed. The type strain is strain EA-1T (=DSM 106492T=CCOS1194T).

Development of a production chain from vegetable biowaste to platform chemicals.

BACKGROUND: A future bioeconomy relies on the development of technologies to convert waste into valuable compounds. We present here an attempt to design a biotechnological cascade for the conversion of vegetable waste into acetoin and electrical energy. RESULTS: A vegetable waste dark fermentation effluent containing mainly acetate, butyrate and propionate was oxidized in a bioelectrochemical system. The achieved average current at a constant anode potential of 0 mV against standard hydrogen electrode was 177.5 ± 52.5 µA/cm2. During this step, acetate and butyrate were removed from the effluent while propionate was the major remaining component of the total organic carbon content comprising on average 75.6%. The key players with regard to carbon oxidation and electrode reduction were revealed using amplicon sequencing and metatranscriptomic analysis. Using nanofiltration, it was possible to concentrate the propionate in the effluent. The effluent was revealed to be a suitable medium for biotechnological production strains. As a proof of principle, the propionate in the effluent of the bioelectrochemical system was converted into the platform chemical acetoin with a carbon recovery of 86%. CONCLUSIONS: To the best of our knowledge this is the first report on a full biotechnological production chain leading from vegetable waste to the production of a single valuable platform chemical that integrates carbon elimination steps leading to the production of the valuable side product electrical energy.

Resilience, Dynamics, and Interactions within a Model Multispecies Exoelectrogenic-Biofilm Community.

Anode-associated multispecies exoelectrogenic biofilms are essential for the function of bioelectrochemical systems (BESs). The individual activities of anode-associated organisms and physiological responses resulting from coculturing are often hard to assess due to the high microbial diversity in these systems. Therefore, we developed a model multispecies biofilm comprising three exoelectrogenic proteobacteria, Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens, with the aim to study in detail the biofilm formation dynamics, the interactions between the organisms, and the overall activity of an exoelectrogenic biofilm as a consequence of the applied anode potential. The experiments revealed that the organisms build a stable biofilm on an electrode surface that is rather resilient to changes in the redox potential of the anode. The community operated at maximum electron transfer rates at electrode potentials that were higher than 0.04 V versus a normal hydrogen electrode. Current densities decreased gradually with lower potentials and reached half-maximal values at -0.08 V. Transcriptomic results point toward a positive interaction among the individual strains. S. oneidensis and G. sulfurreducens upregulated their central metabolisms as a response to cultivation under mixed-species conditions. G. sulfurreducens was detected in the planktonic phase of the bioelectrochemical reactors in mixed-culture experiments but not when it was grown in the absence of the other two organisms.IMPORTANCE In many cases, multispecies communities can convert organic substrates into electric power more efficiently than axenic cultures, a phenomenon that remains unresolved. In this study, we aimed to elucidate the potential mutual effects of multispecies communities in bioelectrochemical systems to understand how microbes interact in the coculture anodic network and to improve the community's conversion efficiency for organic substrates into electrical energy. The results reveal positive interactions that might lead to accelerated electron transfer in mixed-species anode communities. The observations made within this model biofilm might be applicable to a variety of nonaxenic systems in the field.

Acetoin production via unbalanced fermentation in Shewanella oneidensis.

This study describes the realization of an anoxic acetoin production process using the proteobacterium Shewanella oneidensis. Fermentative processes are of high biotechnological relevance since they offer high productivity and a low percentage of substrate consumption for anabolic processes. Nevertheless, the range of compounds that can be produced as sole end product of a fermentative process is limited, since the average oxidation state of substrate and products has to be identical in the absence of an external electron acceptor. This limitation could be overcome by the transfer of the surplus of electrons to a poised electrode surface, which of note is the only known anaerobic electron acceptor that cannot be depleted. In the first genetic engineering step, deletion mutants were developed that are devoid of either one, two, or all three prophages in their genome with the aim to construct a more stable chassis strain for microbe-electrode interaction, due to less prophage induced cell lysis (Gödeke et al., 2011). Current production in a bioelectrochemical system together with the analysis of cells on the anode surface were used as surrogate for the stability assessment. The λ-prophage deletion mutant produced overall 1.34fold more current (6.7 µA cm-2 ) than the wild type and all other constructed strains and showed with 1.1 × 1011 cells the highest cell density on the anode surface (2.3fold more than the wild type). The strain was further modified to contain codon optimized versions of acetolactate synthase and acetolactate decarboxylase derived from Bacillus subtilis. This allowed for the production of a mixture of acetoin and acetate from lactate in an almost 0.4:1 ratio. Further process improvement was reached by the deletion of the acetate kinase and phosphotransacetylase genes ackA/pta. The acetoin yield increased in this mutant from 40 to 86% of the theoretical maximum and acetoin was the only detectable end product. Biotechnol. Bioeng. 2017;114: 1283-1289. © 2017 Wiley Periodicals, Inc.

Arsenic-rich acid mine water with extreme arsenic concentration: mineralogy, geochemistry, microbiology, and environmental implications.

Extremely arsenic-rich acid mine waters have developed by weathering of native arsenic in a sulfide-poor environment on the 10th level of the Svornost mine in Jáchymov (Czech Republic). Arsenic rapidly oxidizes to arsenolite (As2O3), and there are droplets of liquid on the arsenolite crust with high As concentration (80,000-130,000 mg·L(-1)), pH close to 0, and density of 1.65 g·cm(-1). According to the X-ray absorption spectroscopy on the frozen droplets, most of the arsenic is As(III) and iron is fully oxidized to Fe(III). The EXAFS spectra on the As K edge can be interpreted in terms of arsenic polymerization in the aqueous solution. The secondary mineral that precipitates in the droplets is kaatialaite [Fe(3+)(H2AsO4)3·5H2O]. Other unusual minerals associated with the arsenic lens are behounekite [U(4+)(SO4)2·4H2O], stepite [U(4+)(AsO3OH)2·4H2O], vysokýite [U(4+)[AsO2(OH)2]4·4H2O], and an unnamed phase (H3O)(+)2(UO2)2(AsO4)2·nH2O. The extremely low cell densities and low microbial biomass have led to insufficient amounts of DNA for downstream polymerase chain reaction amplification and clone library construction. We were able to isolate microorganisms on oligotrophic media with pH ∼ 1.5 supplemented with up to 30 mM As(III). These microorganisms were adapted to highly oligotrophic conditions which disabled long-term culturing under laboratory conditions. The extreme conditions make this environment unfavorable for intensive microbial colonization, but our first results show that certain microorganisms can adapt even to these harsh conditions.

Biofilms for Production of Chemicals and Energy.

The twenty-first century will be the century of biology. This is not only because of breakthrough advances in molecular biology tools but also because we need to reinvent our economy based on the biological principles of energy efficiency and sustainability. Consequently, new tools for production routines must be developed to help produce platform chemicals and energy sources based on sustainable resources. In this context, biofilm-based processes have the potential to impact future production processes, because they can be carried out continuously and with robust stationary biocatalysts embedded in an extracellular matrix with different properties. We review productive biofilm systems used for heterotrophic and lithoautotrophic production and attempt to identify fundamental reasons why they may be particularly suitable as future production systems.

Beneficial applications of biofilms.

Many microorganisms live in the form of a biofilm. Although they are feared in the medical sector, biofilms that are composed of non-pathogenic organisms can be highly beneficial in many applications, including the production of bulk and fine chemicals. Biofilm systems are natural retentostats in which the biocatalysts can adapt and optimize their metabolism to different conditions over time. The adherent nature of biofilms allows them to be used in continuous systems in which the hydraulic retention time is much shorter than the doubling time of the biocatalysts. Moreover, the resilience of organisms growing in biofilms, together with the potential of uncoupling growth from catalytic activity, offers a wide range of opportunities. The ability to work with continuous systems using a potentially self-advancing whole-cell biocatalyst is attracting interest from a range of disciplines, from applied microbiology to materials science and from bioengineering to process engineering. The field of beneficial biofilms is rapidly evolving, with an increasing number of applications being explored, and the surge in demand for sustainable and biobased solutions and processes is accelerating advances in the field. This Review provides an overview of the research topics, challenges, applications and future directions in beneficial and applied biofilm research.

Elucidating the development of cooperative anode-biofilm-structures.

Microbial electrochemical systems are a highly versatile platform technology with a particular focus on the interplay of chemical and electrical energy conversion and offer immense potential for a sustainable bioeconomy. The industrial realization of this potential requires a critical focus on biofilm optimization if performance is to be controlled over a long period of time. Moreover, the aspect and influence of cooperativity has to be addressed as many applied anodic bioelectrochemical systems will most likely be operated with a diversity of interacting microbial species. Hence, the aim of this study was to analyze how interspecies dependence and cooperativity of a model community influence the development of anodic biofilms. To investigate biofilm activity in a spatially resolved manner, a microfluidic bioelectrochemical flow cell was developed that can be equipped with user-defined electrode materials and operates under laminar flow conditions. With this infrastructure, the development of single and co-culture biofilms of the two model organisms Shewanella oneidensis and Geobacter sulfurreducens on graphite electrodes was monitored by optical coherence tomography analysis. The interdependence in the co-culture biofilm was achieved by feeding the community with lactate, which is converted by S. oneidensis into acetate, which in turn serves as substrate for G. sulfurreducens. The results show that co-cultivation resulted in the formation of denser biofilms than in single culture. Moreover, we hypothesize that S. oneidensis in return utilizes the conductive biofilm matrix build by G. sulfurreducens for direct interspecies electron transfer (DIET) to the anode. FISH analysis revealed that the biofilms consisted of approximately two-thirds G. sulfurreducens cells, which most likely formed a conductive 3D network throughout the biofilm matrix, in which evenly distributed tubular S. oneidensis colonies were embedded without direct contact to the anode surface. Live/dead staining shows that the outermost biofilm contained almost exclusively dead cells (98 %), layers near the anode contained 45-56 % and the entire biofilm contained 82 % live cells. Our results exemplify how the architecture of the exoelectrogenic biofilm dynamically adapts to the respective process conditions.

Comparative analysis of the influence of BpfA and BpfG on biofilm development and current density in Shewanella oneidensis under oxic, fumarate- and anode-respiring conditions.

Biofilm formation by Shewanella oneidensis has been extensively studied under oxic conditions; however, relatively little is known about biofilm formation under anoxic conditions and how biofilm architecture and composition can positively influence current generation in bioelectrochemical systems. In this study, we utilized a recently developed microfluidic biofilm analysis setup with automated 3D imaging to investigate the effects of extracellular electron acceptors and synthetic modifications to the extracellular polymeric matrix on biofilm formation. Our results with the wild type strain demonstrate robust biofilm formation even under anoxic conditions when fumarate is used as the electron acceptor. However, this pattern shifts when a graphite electrode is employed as the electron acceptor, resulting in biofilm formation falling below the detection limit of the optical coherence tomography imaging system. To manipulate biofilm formation, we aimed to express BpfG with a single amino acid substitution in the catalytic center (C116S) and to overexpress bpfA. Our analyses indicate that, under oxic conditions, overarching mechanisms predominantly influence biofilm development, rather than the specific mutations we investigated. Under anoxic conditions, the bpfG mutation led to a quantitative increase in biofilm formation, but both strains exhibited significant qualitative changes in biofilm architecture compared to the controls. When an anode was used as the sole electron acceptor, both the bpfA and bpfG mutations positively impacted mean current density, yielding a 1.8-fold increase for each mutation.

Strain and model development for auto- and heterotrophic 2,3-butanediol production using Cupriavidus necator H16.

The production of platform chemicals from renewable energy sources is a crucial step towards a post-fossil economy. This study reports on the production of acetoin and 2,3-butanediol heterotrophically with fructose as substrate and autotrophically from CO2 as carbon source, H2 as electron donor and O2 as electron acceptor with Cupriavidus necator. In a previous study, the strain was developed for the production of acetoin with high carbon efficiency. Acetoin can serve as a precursor for the synthesis of 2,3-butanediol by the integration of a butanediol dehydrogenase. In this study, different plasmid backbones and butanediol dehydrogenases were evaluated regarding efficiency for CO2-based 2,3-butanediol production. The developed strain utilizes the pBBR1 plasmid bearing a 2,3-butanediol dehydrogenase from Enterobacter cloacae and is characterized by 2,3-butanediol as the main product and a heterotrophic total product yield of 88.11%, an autotrophic volumetric productivity of 39.45 mg L-1 h-1, a total product carbon yield of 81.6%, an H2 efficiency of 33.46%, and a specific productivity of 0.016 g product per gram of biomass per hour. In addition, a mathematical model was developed to simulate the processes under these conditions. With this model, it was possible to calculate productivities and substrate usage at distinct time points of the production processes and calculate productivities and substrate usage with high resolution which will be useful in future applications.