A fluorescent reporter system for anaerobic thermophiles

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli—Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes

Physiological characterization and promoter engineering of Acetobacterium wieringae for acetone production via gas fermentation

The pressing need to mitigate environmental concerns has driven research into sustainable energy and chemical production methods that reduce carbon emissions. Gas fermentation offers a promising avenue for low-carbon fuel and chemical synthesis. Acetobacterium wieringae, particularly strain A. wieringae JM, has emerged as an attractive host for gas-based biorefineries due to its unique abilities, including growth in diverse gas compositions and pH ranges, and efficient growth on carbon monoxide without co-substrates. This study focuses on enhancing the potential of A. wieringae for acetone production through genetic modification. A transformation protocol was developed, and the acetone production operon from Clostridium acetobutylicum was introduced. Novel promoters were explored to widen gene expression possibilities in A. wieringae. The stability of the plasmid backbone pMTL83151 carrying replicon pCB102 was assessed. Additionally, the tolerance of A. wieringae to gas synthesis derived from biogenic residue gasification was evaluated for potential industrial application. Gas composition significantly influenced acetone production by A. wieringae, with distinct physiological effects observed between strain A. wieringae DSM 1911 and A. wieringae JM. Four constitutive promoters from A. wieringae JM and four from C. autoethanogenum were successfully expressed, exhibiting stronger activity than the reference Pthl promoter from C. acetobutylicum. Notably, A. wieringae JM demonstrated robust growth in synthesis gas from biomass gasification, though with physiological variations. This study unveils the intricate relationship between gas composition, physiological attributes, and acetone production in A. wieringae. The expanded promoter repertoire enhances genetic manipulation potential, propelling the strain's capacity for versatile gene expression. Moreover, the resilience of A. wieringae JM to gasification-derived gas synthesis highlights its viability for industrial implementation. These findings contribute to advancing the development of gas-based biorefineries, paving the way for sustainable chemical production with reduced environmental impact.info:eu-repo/semantics/publishedVersio

Structure and ion dynamics of mechanosynthesized oxides and fluorides

In many cases, limitations in conventional synthesis routes hamper the accessibility to materials with properties that have been predicted by theory. For instance, metastable compounds with local non-equilibrium structures can hardly be accessed by solid-state preparation techniques often requiring high synthesis temperatures. Also other ways of preparation lead to the thermodynamically stable rather than metastable products. Fortunately, such hurdles can be overcome by mechanochemical synthesis. Mechanical treatment of two or three starting materials in high-energy ball mills enables the synthesis of not only new, metastable compounds but also of nanocrystalline materials with unusual or enhanced properties such as ion transport. In this short review we report about local structures and ion transport of oxides and fluorides mechanochemically prepared by high-energy ball-milling

Microbial Upgrading of Acetate into Value-Added Products—Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches

Ecological concerns have recently led to the increasing trend to upgrade carbon contained in waste streams into valuable chemicals. One of these components is acetate. Its microbial upgrading is possible in various species, with Escherichia coli being the best-studied. Several chemicals derived from acetate have already been successfully produced in E. coli on a laboratory scale, including acetone, itaconic acid, mevalonate, and tyrosine. As acetate is a carbon source with a low energy content compared to glucose or glycerol, energy- and redox-balancing plays an important role in acetate-based growth and production. In addition to the energetic challenges, acetate has an inhibitory effect on microorganisms, reducing growth rates, and limiting product concentrations. Moreover, extensive metabolic engineering is necessary to obtain a broad range of acetate-based products. In this review, we illustrate some of the necessary energetic considerations to establish robust production processes by presenting calculations of maximum theoretical product and carbon yields. Moreover, different strategies to deal with energetic and metabolic challenges are presented. Finally, we summarize ways to alleviate acetate toxicity and give an overview of process engineering measures that enable sustainable acetate-based production of value-added chemicals

Optimized Operating Conditions for a Biological Treatment Process of Industrial Residual Process Brine Using a Halophilic Mixed Culture

Residual process brine is a sustainable raw material for chlor-alkali electrolysis processes. This study investigates the influence of critical process parameters on the performance of a continuous treatment process for residual process brine using halophilic microorganisms. The goal of the bioprocess is an efficient degradation of the organic impurities formate, aniline, phenol, and 4,4′-methylenedianline from this residual stream. It was shown that formate could be degraded with high efficiencies (89–98%) during the treatment process. It was observed that formate degradation was influenced by the co-substrate glycerol. The lowest residual formate concentrations were achieved with specific glycerol uptake rates of 8.0–16.0 × 10−3 g L−1 h−1 OD600−1. Moreover, a triple-nutrient limitation for glycerol, ammonium, and phosphate was successfully applied for continuous cultivations. Furthermore, it was shown that all aromatic impurities were degraded with an efficiency of 100%. Ultimately, this study proposed optimized operating conditions, allowing the efficient degradation of organics in the residual process brine under various process conditions. Future optimization steps will require a strategy to prevent the accumulation of potential intermediate degradation products formed at high aniline feed concentrations and increase the liquid dilution rates of the system to achieve a higher throughput of brines

Optimized Operating Conditions for a Biological Treatment Process of Industrial Residual Process Brine Using a Halophilic Mixed Culture

Residual process brine is a sustainable raw material for chlor-alkali electrolysis processes. This study investigates the influence of critical process parameters on the performance of a continuous treatment process for residual process brine using halophilic microorganisms. The goal of the bioprocess is an efficient degradation of the organic impurities formate, aniline, phenol, and 4,4′-methylenedianline from this residual stream. It was shown that formate could be degraded with high efficiencies (89–98%) during the treatment process. It was observed that formate degradation was influenced by the co-substrate glycerol. The lowest residual formate concentrations were achieved with specific glycerol uptake rates of 8.0–16.0 × 10−3 g L−1 h−1 OD600−1. Moreover, a triple-nutrient limitation for glycerol, ammonium, and phosphate was successfully applied for continuous cultivations. Furthermore, it was shown that all aromatic impurities were degraded with an efficiency of 100%. Ultimately, this study proposed optimized operating conditions, allowing the efficient degradation of organics in the residual process brine under various process conditions. Future optimization steps will require a strategy to prevent the accumulation of potential intermediate degradation products formed at high aniline feed concentrations and increase the liquid dilution rates of the system to achieve a higher throughput of brines

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

Abstract Background Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production. Results The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l−1 h−1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l−1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l−1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals. Conclusion A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol

Photosynthetic poly-β-hydroxybutyrate accumulation in unicellular cyanobacterium Synechocystis sp. PCC 6714

Abstract Poly-β-hydroxybutyrate (PHB) production from CO2 has the potential to reduce the production cost of this biodegradable polyesters, and also to make the material more sustainable compared to utilization of sugar feedstocks. In this study the unicellular cyanobacterium, Synechocystis sp. PCC 6714 has been identified as an unexplored potential organism for production of PHB. Synechocystis sp. PCC 6714 was studied under various cultivation conditions and nutritional limitations. Combined effects of nitrogen and phosphorus deficiency led to highest PHB accumulation under photoautotrophic conditions. Multivariate experimental design and quantitative bioprocess development methodologies were used to identify the key cultivation parameters for PHB accumulation. Biomass growth and PHB accumulation were studied under controlled defined conditions in a lab-scale photobioreactor. Specific growth rates were fourfold higher in photobioreactor experiments when cultivation conditions were controlled. After 14 days of cultivation in nitrogen and phosphorus, limited media intracellular PHB levels reached up to 16.4% from CO2. The highest volumetric production rate of PHB was 59 ± 6 mg L−1 day−1. Scanning electron microscopy of isolated PHB granules of Synechocystis sp. PCC 6714 cultivated under nitrogen and phosphorus limitations showed an average diameter of 0.7 µm. The results of this study might contribute towards a better understanding of photoautotrophic PHB production from cyanobacteria

Characterizing the effect of expression of an acetyl-CoA synthetase insensitive to acetylation on co-utilization of glucose and acetate in batch and continuous cultures of E. coli W

Abstract Background Due to its high stress tolerance and low acetate secretion, Escherichia coli W is reported to be a good production host for several metabolites and recombinant proteins. However, simultaneous co-utilization of glucose and other substrates such as acetate remains a challenge. The activity of acetyl-CoA-synthetase, one of the key enzymes involved in acetate assimilation is tightly regulated on a transcriptional and post-translational level. The aim of this study was to engineer E. coli W for overexpression of an acetylation insensitive acetyl-CoA-synthetase and to characterize this strain in batch and continuous cultures using glucose, acetate and during co-utilization of both substrates. Results Escherichia coli W engineered to overexpress an acetylation-insensitive acetyl-CoA synthetase showed a 2.7-fold increase in acetate uptake in a batch process containing glucose and high concentrations of acetate compared to a control strain, indicating more efficient co-consumption of glucose and acetate. When acetate was used as the carbon source, batch duration could significantly be decreased in the overexpression strain, possibly due to alleviation of acetate toxicity. Chemostat cultivations with different dilution rates using glucose revealed only minor differences between the overexpression and control strain. Accelerostat cultivations using dilution rates between 0.20 and 0.70 h−1 indicated that E. coli W is naturally capable of efficiently co-utilizing glucose and acetate over a broad range of specific growth rates. Expression of acetyl-CoA synthetase resulted in acetate and glucose accumulation at lower dilution rates compared to the control strain. This observation can possibly be attributed to a higher ratio between acs and pta-ackA in the overexpression strain as revealed by gene expression analysis. This would result in enhanced energy dissipation caused by an imbalance in the Pta-AckA-Acs cycle. Furthermore, yjcH and actP, genes co-transcribed with acetyl-CoA synthetase showed significant down-regulation at elevated dilution rates. Conclusions Escherichia coli W expressing an acetylation-insensitive acetyl-CoA synthetase was shown to be a promising candidate for mixed feed processes using glucose and acetate. Comparison between batch and continuous cultures revealed distinct differences in glucose-acetate co-utilization behavior, requiring additional investigations such as multi-omics analysis and further engineering towards even more efficient co-utilization strains of E. coli W

Separating bulk from grain boundary Li ion conductivity in the sol–gel prepared solid electrolyte Li1.5Al0.5Ti1.5(PO4)3

Lithium aluminium titanium phosphate (LATP) belongs to one of the most promising solid electrolytes. Besides sufficiently high electrochemical stability, its use in lithium-based all-solid-state batteries crucially depends on the ionic transport properties. While many impedance studies can be found in literature that report on overall ion conductivities, a discrimination of bulk and grain boundary electrical responses via conductivity spectroscopy has rarely been reported so far. Here, we took advantage of impedance measurements that were carried out at low temperatures to separate bulk contributions from the grain boundary responses. It turned out that bulk ion conductivity is by at least three orders of magnitude higher than ion transport across the grain boundary regions. At temperatures well below ambient long-range Li ion dynamics is governed by activation energies ranging from 0.26 to 0.29 eV depending on the sintering conditions. As an example, at temperatures as low as 173 K, the bulk ion conductivity, measured in N2 inert gas atmosphere, is in the order of 8.1 × 10−6 S cm−1. Extrapolating this value to room temperature yields ca. 3.4 × 10−3 S cm−1 at 293 K. Interestingly, exposing the dense pellets to air atmosphere over a long period of time causes a significant decrease of bulk ion transport. This process can be reversed if the phosphate is calcined at elevated temperatures again