Precultures Grown under Fed-Batch Conditions Increase the Reliability and Reproducibility of High-Throughput Screening Results.

One essential task in bioprocess development is strain selection. A common screening procedure consists of three steps: first, the picking of colonies; second, the execution of a batch preculture and main culture, e.g., in microtiter plates (MTPs); and third, the evaluation of product formation. Especially during the picking step, unintended variations occur due to undefined amounts and varying viability of transferred cells. The aim of this study is to demonstrate that the application of polymer-based controlled-release fed-batch MTPs during preculture eliminates these variations. The concept of equalizing growth through fed-batch conditions during preculture is theoretically discussed and then tested in a model system, namely, a cellulase-producing Escherichia coli clone bank containing 32 strains. Preculture is conducted once in the batch mode and once in the fed-batch mode. By applying the fed-batch mode, equalized growth is observed in the subsequent main culture. Furthermore, the standard deviation of cellulase activity is reduced compared to that observed in the conventional approach. Compared with the strains in the batch preculture process, the first-ranked strain in the fed-batch preculture process is the superior cellulase producer. These findings recommend the application of the fed-batch MTPs during preculture in high-throughput screening processes to achieve accurate and reliable results.

Modulation of collagen by addition of Hofmeister salts.

Collagen can be modified by addition of chaotropic or kosmotropic salts of the reversed Hofmeister series. Hence, telopeptide-poor collagen type I was suspended in H2SO4 (pH 2) and 0.05-0.5 M KCl and KNO3 (chaotropes), as well as KI and KSCN (kosmotropes). Rheological parameters, including storage and loss modulus, intrinsic viscosity, and critical overlap concentration, were assessed and the microstructure was characterized by applying confocal laser scanning microscopy and scanning electron microscopy. The addition of up to 0.1 M KCl and 0.05 M KNO3 increased the intrinsic viscosity from 1.22 to 1.51 L/g without salt to a maximal value of 1.74 L/g and decreased the critical overlap concentration from 0.66 to 0.82 g/L to a minimal value of 0.57 g/L. Higher salt concentrations increased the collagen-collagen interactions due to ions withdrawing the water from the collagen molecules. Hence, 0.1 M KSCN delivered the largest structures with the highest structure factor, area value and the highest critical overlap concentration with 17.6 L/g. Overall, 0.5 M salt led to salting out, with chaotropes forming fine precipitates and kosmotropes leading to elastic three-dimensional networks. The study demonstrated that collagen entanglement and microstructure depend strongly on the ionic strength and type of salt.