The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC‐like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.
Objectives
With generation times of less than 10 min under optimal conditions, the halophilic Vibrio natriegens is the fastest growing non-pathogenic bacterium isolated so far. The availability of the full genome and genetic engineering tools and its ability to utilize a wide range of carbon sources make V. natriegens an attractive host for biotechnological production processes. However, high-cell-density cultivations, which are desired at industrial-scale have not been described so far.
Results
In this study we report fed-batch cultivations of V. natriegens in deep-well plates and lab-scale bioreactor cultivations at different temperatures in mineral salt medium (MSM). Upon switching from exponential glucose to constant glucose-feeding cell death was induced. Initial NaCl concentrations of 15–18 g L−1 and a temperature reduction from 37 to 30 °C had a positive effect on cell growth. The maximal growth rate in MSM with glucose was 1.36 h−1 with a specific oxygen uptake rate of 22 mmol gCDW−1 h−1. High biomass yields of up to 55 g L−1 after only 12 h were reached.
Conclusions
The shown fed-batch strategies demonstrate the potential of V. natriegens as a strong producer in industrial biotechnology.
Biobased and biodegradable polyhydroxyalkanoates (PHAs) are promising alternatives to common plastics. Due to their high production costs, only a minimal share of global plastic production is composed of PHA. A major contributor to the high costs minimizing the potential to occupy a larger market share is the downstream process. To obtain high recovery yields and pure products, most approaches rely on large amounts of solvents. While short-chain-length PHA (scl-PHA) is poorly soluble in nonhalogenated solvents, medium-chain-length PHA (mcl-PHA) was shown to be soluble in nonhalogenated solvents. In this study, an approach to recover poly(hydroxybutyrate-co-hydroxyhexanoate) with acetone and 2-propanol was scaled up 30-fold to 300 g of lyophilized cells per recovery cycle. High PHA purities of 90–100 % were reached from extractions at moderate temperatures from 30–58 °C. In two-stage extractions, up to 100 % PHA was recovered, while the molecular weight was not reduced. Solvents were recovered by distillation in a concentration step and after precipitation. Furthermore, the material properties were analyzed. PHA recovered from the distillation bottom had an increased HHx content compared to the first and second extractions using recovered solvents and was of low purity, indicating efficient and pure precipitation of the recovered PHA during the 2-stage extractions
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