The bio-based production of aromatics is experiencing a renaissance with systems and synthetic biology approaches promising to deliver bio-catalysts that will reach yields, rates, and titers ...comparable to already existing bulk bio-processes for the production of amino acids for instance. However, aromatic building blocks derived from petrochemical routes have a huge economic advantage, they are cheap, and very cheap in fact. In this article, we are trying to shed light on an important aspect of biocatalyst development that is frequently overlooked when working on strain development: economic and environmental impact of the production process. We estimate the production cost and environmental impact of a microbial fermentation process depending on culture pH, carbon source and process scale. As a model molecule we use
para
-hydroxybenzoic acid (pHBA), but the results are readily transferrable to other shikimate derived aromatics with similar carbon yields and production rates.
Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. ...Production of other organic acids reduces propionic acid yield and productivity, pointing to by‐products gene‐knockout strategies as a logical solution to increase yield. However, removing by‐product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well‐conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.
Traditionally derived from oil‐based resources, biological production of propionic acid (PA) using Propionibacterium has gained interest in recent years. In this study, genome shuffling on two P. acidipropionici strains was used to increase growth rate and PA production. Sequencing of the new strains suggests that recombination events, in well‐conserved regions of the genome, are responsible for the phenotype improvement. This work provides base resolution insight on the underlying mechanisms leading to a higher PA production using genome shuffling.
Hyper-performing whole-cell catalysts are required for the renewable and sustainable production of petrochemical replacements. Chassis cells—self-replicating minimal machines that can be tailored for ...the production of specific chemicals—will provide the starting point for designing these hyper-performing 'turbo cells'.
The NASA InSight Lander on Mars includes the Heat Flow and Physical Properties Package HP
3
to measure the surface heat flow of the planet. The package uses temperature sensors that would have been ...brought to the target depth of 3–5 m by a small penetrator, nicknamed the mole. The mole requiring friction on its hull to balance remaining recoil from its hammer mechanism did not penetrate to the targeted depth. Instead, by precessing about a point midway along its hull, it carved a 7 cm deep and 5–6 cm wide pit and reached a depth of initially 31 cm. The root cause of the failure – as was determined through an extensive, almost two years long campaign – was a lack of friction in an unexpectedly thick cohesive duricrust. During the campaign – described in detail in this paper – the mole penetrated further aided by friction applied using the scoop at the end of the robotic Instrument Deployment Arm and by direct support by the latter. The mole tip finally reached a depth of about 37 cm, bringing the mole back-end 1–2 cm below the surface. It reversed its downward motion twice during attempts to provide friction through pressure on the regolith instead of directly with the scoop to the mole hull. The penetration record of the mole was used to infer mechanical soil parameters such as the penetration resistance of the duricrust of 0.3–0.7 MPa and a penetration resistance of a deeper layer (
>
30
cm
depth) of
4.9
±
0.4
MPa
. Using the mole’s thermal sensors, thermal conductivity and diffusivity were measured. Applying cone penetration theory, the resistance of the duricrust was used to estimate a cohesion of the latter of 2–15 kPa depending on the internal friction angle of the duricrust. Pushing the scoop with its blade into the surface and chopping off a piece of duricrust provided another estimate of the cohesion of 5.8 kPa. The hammerings of the mole were recorded by the seismometer SEIS and the signals were used to derive P-wave and S-wave velocities representative of the topmost tens of cm of the regolith. Together with the density provided by a thermal conductivity and diffusivity measurement using the mole’s thermal sensors, the elastic moduli were calculated from the seismic velocities. Using empirical correlations from terrestrial soil studies between the shear modulus and cohesion, the previous cohesion estimates were found to be consistent with the elastic moduli. The combined data were used to derive a model of the regolith that has an about 20 cm thick duricrust underneath a 1 cm thick unconsolidated layer of sand mixed with dust and above another 10 cm of unconsolidated sand. Underneath the latter, a layer more resistant to penetration and possibly containing debris from a small impact crater is inferred. The thermal conductivity increases from 14 mW/m K to 34 mW/m K through the 1 cm sand/dust layer, keeps the latter value in the duricrust and the sand layer underneath and then increases to 64 mW/m K in the sand/gravel layer below.
Due to their excellent energy resolution, the intrinsically fast signal rise time, the huge energy dynamic range, and the almost ideally linear detector response, metallic magnetic calorimeters ...(MMC)s are very well suited for a variety of applications in physics. In particular, the ECHo experiment aims to utilize large-scale MMC-based detector arrays to investigate the mass of the electron neutrino. Reading out such arrays is a challenging task which can be tackled using microwave SQUID multiplexing. Here, the detector signals are transduced into frequency shifts of superconducting microwave resonators, which can be deduced using a high-end software-defined radio (SDR) system. The ECHo SDR system is a custom-made modular electronics, which provides 400 channels equally distributed in a 4 to 8 GHz frequency band. The system consists of a superheterodyne RF frequency converter with two successive mixers, a modular conversion, and an FPGA board. For channelization, a novel heterogeneous approach, utilizing the integrated digital down conversion (DDC) of the ADC, a polyphase channelizer, and another DDC for demodulation, is proposed. This approach has excellent channelization properties while being resource-efficient at the same time. After signal demodulation, on-FPGA flux-ramp demodulation processes the signals before streaming it to the data processing and storage backend.
To our present best knowledge, microwave SQUID multiplexing (
μ
MUXing) is the most suitable technique for reading out large-scale low-temperature microcalorimeter arrays that consist of hundreds or ...thousands of individual pixels which require a large readout bandwidth per pixel. For this reason, the present readout strategy for metallic magnetic calorimeter (MMC) arrays combining an intrinsic fast signal rise time, an excellent energy resolution, a large energy dynamic range, a quantum efficiency close to
100
%
as well as a highly linear detector response is based on
μ
MUXing. Within this paper, we summarize the state of the art in MMC
μ
MUXing and discuss the most recent results. This particularly includes the discussion of the performance of a 64-pixel detector array with integrated, on-chip microwave SQUID multiplexer, the progress in flux ramp modulation of MMCs as well as the status of the development of a software-defined radio-based room-temperature electronics which is specifically optimized for MMC readout.
Providing an anodic potential in a bio‐electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, ...the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro‐fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio‐electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro‐fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio‐electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro‐fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet‐to‐be‐identified oxygen‐dependent regulatory mechanism.
Providing an anodic potential in a bio‐electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances to achieve high yield of products. However, it is largely unclear how P. putida uptake the carbon into cytosol under electro‐fermentative conditions, while the cells have three native transporting systems for glucose differing in energy and redox demand. By creating three gene deletion mutants of P. putida KT2440 that force the cells to exclusively utilize one of the three routes, our results showed the gene deletion did not alter significantly the secreted acetate concentrations and thus suggested the stoichiometry of the sugar uptake routes was not a limiting factor during electro‐fermentation.
Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO
. It has been a longstanding challenge to engineer an obligate ...aerobe organism, such as
, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for
under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of
in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of
and possibly also other obligate aerobes.
Escherichia coli is currently used by many research institutions and companies around the world as a platform organism for the development of bio-based production processes for bulk biochemicals. A ...given bulk biochemical bioprocess must be economically competitive with current production routes. Ideally the viability of each bioprocess should be evaluated prior to commencing research, both by metabolic network analysis (to determine the maximum theoretical yield of a given biocatalyst) and by techno-economic analysis (TEA; to determine the conditions required to make the bioprocess cost-competitive). However, these steps are rarely performed. Here we examine theoretical yields and review available TEA for bulk biochemical production in E. coli. In addition, we examine fermentation feedstocks and review recent strain engineering approaches to achieve industrially-relevant production, using examples for which TEA has been performed: ethanol, poly-3-hydroxybutyrate, and 1,3-propanediol.
Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most ...heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23–55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.
•Phototrophic production of trans-4-hydroxy-L-prolin.•pH dependency of product accumulation in Synechocystis PCC6803.•Comparative analysis of land use efficiency in phototrophs & heterotrophs.
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