Engineering in Life Sciences最新文献

Bioelectrochemical systems (BESs) offer a sustainable method for chemical production, including the enhanced production of succinic acid. By combining fermentation with BES, it could be possible to achieve sustainable succinic acid production and CO₂ fixation using Actinobacillus succinogenes. In literature, the potential application of BES is commonly associated with increased succinate yields, as it is expected to enhance the availability of NADH, thereby influencing the intracellular nicotinamide adenine dinucleotide (NADH/NAD⁺) balance. However, it remains unclear whether BES can improve NADH regeneration and achieve higher NADH/NAD⁺ ratios across all growth phases of A. succinogenes. This study investigates the impact of an applied electrical potential on the intracellular NADH/NAD⁺ ratio during an electrochemical-assisted fermentation process. Using an adapted high-performance liquid chromatography method with a Supelcosil LC-18-T column, it was demonstrated that NADH availability in BES, particularly during the stationary growth phase, improved by up to 1.98-fold compared to the control. This enhancement in reducing power led to a succinate yield of 0.747 ± 0.01 g g⁻¹, representing a 15.65% increase compared to a fermentation without electrochemical assistance. These findings support the expectation that the use of BES could enhance the competitiveness of bio-based succinate production.

Raman spectroscopy, a robust and non-invasive analytical method, has demonstrated significant potential for monitoring biopharmaceutical production processes. Its ability to provide detailed information about molecular vibrations makes it ideal for the detection and quantification of therapeutic proteins and critical control parameters in complex biopharmaceutical mixtures. However, its application in Saccharomyces cerevisiae fermentations has been hindered by the inherent strong fluorescence background from the cells. This fluorescence interferes with Raman signals, compromising spectral data accuracy. In this study, we present an approach that mitigates this issue by deploying Raman spectroscopy on cell-free media samples, combined with advanced chemometric modeling. This method enables accurate prediction of protein concentration and key process parameters, fundamental for the control and optimization of biopharmaceutical fermentation processes. Utilizing variable importance in projection (VIP) further enhances model robustness, leading to lower relative root mean squared error of prediction (RMSEP) values across the six targets studied. Our findings highlight the potential of Raman spectroscopy for real-time, on-line monitoring and control of complex microbial fermentations, thereby significantly enhancing the efficiency and quality of S. cerevisiae-based biopharmaceutical production.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology and biomedical research, but there is still a need to shorten the operation process and reduce the cost of antibodies. This study aims to assess a novel IHC technique that incorporates mechanical microvibration (MMV) to expedite the process, reduce antibody consumption, and enhance staining quality. MMV was generated using coin vibration motors attached to glass slides mounted with consecutive tissue sections. Multiple antibodies targeting various antigens were used to stain cancerous and normal tissues, with and without microvibration. Various parameters were tested, including incubation durations, temperatures, and antibody dilutions. The novel method showed the potential to achieve comparable or superior outcomes in significantly less time, utilizing over 10 times less antibody than controls. MMV improved specific staining quality, yielding stronger, and better-defined positive reactions. This was validated through a multicenter double-blind assessment and quantitative image analysis. The possible mechanisms were also investigated. MMV shortens immunohistochemical staining duration, reduces antibody usage, and enhances staining specificity, likely by accelerating antibody movement and diffusion. These improvements translate to time and cost savings, offering clinical and financial value for diagnostic pathology and biomedical research.

Ethyl acetate is at present exclusively produced from fossil resources. Microbial synthesis of this ester from sugar-rich waste as an alternative is an aerobic process. Ethyl acetate is highly volatile and therefore stripped with the exhaust gas from the bioreactor which enables in situ product recovery. Previous research on microbial formation of ethyl acetate has focused on the kinetics of ester synthesis and in part on the ester stripping, while the separation of the ester from the exhaust gas has hardly been investigated. A mixed matrix membrane was developed consisting of Silikalite-1 embedded in polydimethylsiloxane which was installed in a radial–symmetrical membrane module. Evaluation of the separation of ethyl acetate was based on the analysis of the composition of the feed and retentate gas by mass spectrometry. The separation efficiency of the membrane was first tested with varied flows of artificial exhaust gas, containing defined amounts of ethyl acetate. A model for describing the separation process was parametrized by the measured data and used to design a real separation experiment. Ethyl acetate produced from delactosed whey permeate by Kluyveromyces marxianus DSM 5422 in a stirred bioreactor gassed with 0.5 vvm air was successfully separated from the exhaust gas by membranes; 93.6% of the stripped ester was separated. Liquid ethyl acetate was recovered by cooling the permeate gas to ‒78°C, whereby 99.75% of the condensed organic compounds were ethyl acetate. This study demonstrates for the first time that microbially produced and stripped ethyl acetate can be effectively separated from the exhaust gas of bioreactors by membrane technology to obtain the ester in high yield and purity.

In this work, the degradation of mono- and polyfluorinated phenolic compounds was demonstrated by a series of crude plant peroxidases, including horseradish root (HRP) and six members of the Cucurbita genus. Highly active samples were identified using a library screening approach in which more than 50 crude plant samples were initially evaluated for defluorination activity toward 4-fluorophenol. The highest concentrations were observed in the HRP, pumpkin skin (PKS), and butternut squash skin (BNS), which consistently gave the highest intrinsic rates of decomposition for all the substrates tested. Although HRP exhibited a significant decrease in activity with increased fluorination of the phenolic substrate, PKS showed only minor reductions. Furthermore, in silico studies indicated that the active site of HRP poorly accommodates the steric bulk of additional fluorines, causing the substrate to dock farther from the catalytic heme and thus slowing the catalysis rate. We propose that the PKS active site might be larger, allowing closer access to the perfluorinated substrate, and therefore maintaining higher activity compared to the HRP enzyme. However, detailed kinetic characterization studies of the peroxidases are recommended. Conclusively, the high catalytic activity of PKS and its high yield per gram of tissue make it an excellent candidate for developing environmentally friendly biocatalytic methods for degrading fluorinated aromatics. Finally, the success of the library approach in identifying highly active samples for polyfluorinated aromatic compound (PFAC) degradation suggests the method may find utility in the quest for other advanced catalysts for PFAS degradation.

The L-tryptophan–derived purple pigment violacein (VIO) is produced in recombinant bacteria and studied for its versatile applications. Microbial synthetic co-cultures are gaining more importance as efficient factories for synthesizing high-value compounds. In this work, a mutualistic and cross-feeding Escherichia coli co-culture is metabolically engineered to produce VIO. The strains are genetically modified by auxotrophies in the tryptophan (TRP) pathway to enable a metabolic division of labor. Therein, one strain produces anthranilate (ANT) and the other transforms it into TRP and further to VIO. Population dynamics and stability depend on the choice of carbon source, impacting the presence and thus exchange of metabolites as well as overall VIO productivity. Four carbon sources (D-glucose, glycerol, D-galactose, and D-xylose) were compared. D-Xylose led to co-cultures which showed stable growth and VIO production, ANT-TRP exchange, and enhanced VIO production. Best titers were ∼126 mg L^–1 in shake flasks. The study demonstrates the importance and advantages of a mutualistic approach in VIO synthesis and highlights the carbon source's role in co-culture stability and productivity. Transferring this knowledge into an up-scaled bioreactor system has great potential in improving the overall VIO production.