The different properties of acceptor-doped (hard) and donor-doped (soft) lead zirconate titanate (PZT) ceramics are often attributed to different amounts of oxygen vacancies introduced by the dopant. Acceptor doping is believed to cause high oxygen vacancy concentrations, while donors are expected to strongly suppress their amount. In this study, La³⁺ donor-doped, Fe³⁺ acceptor-doped and La³⁺/Fe³⁺-co-doped PZT samples were investigated by oxygen tracer exchange and electrochemical impedance spectroscopy in order to analyse the effect of doping on oxygen vacancy concentrations. Relative changes in the tracer diffusion coefficients for different doping and quantitative relations between defect concentrations allowed estimates of oxygen vacancy concentrations. Donor doping does not completely suppress the formation of oxygen vacancies; rather, it concentrates them in the grain boundary region. Acceptor doping enhances the amount of oxygen vacancies but estimates suggest that bulk concentrations are still in the ppm range, even for 1% acceptor doping. Trapped holes might thus considerably contribute to the charge balancing of the acceptor dopants. This could also be of relevance in understanding the properties of hard and soft PZT.

The Gram-negative bacterium E. coli is the host of choice for a multitude of used recombinant proteins. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures using isopropyl β-d-1 thiogalactopyranoside as inducer are often referred to cause stress reactions, leading to a phenomenon known as “metabolic” or “product burden”. These high expressions of recombinant proteins mainly result in decreased growth rates and cell lysis at elevated induction times. Therefore, approaches tend to use “soft” or “tunable” induction with lactose and reduce the stress level of the production host. The usage of glucose as energy source in combination with lactose as induction reagent causes catabolite repression effects on lactose uptake kinetics and as a consequence reduced product titer. Glycerol—as an alternative carbon source—is already known to have positive impact on product formation when coupled with glucose and lactose in auto-induction systems, and has been referred to show no signs of repression when cultivated with lactose concomitantly. In recent research activities, the impact of different products on the lactose uptake using glucose as carbon source was highlighted, and a mechanistic model for glucose-lactose induction systems showed correlations between specific substrate uptake rate for glucose or glycerol (q_s,C) and the maximum specific lactose uptake rate (q_s,lac,max). In this study, we investigated the mechanistic of glycerol uptake when using the inducer lactose. We were able to show that a product-producing strain has significantly higher inducer uptake rates when being compared to a non-producer strain. Additionally, it was shown that glycerol has beneficial effects on viability of cells and on productivity of the recombinant protein compared to glucose.

The bacterium Escherichia coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body–based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.

The bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes. Classical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein. Time dependent monitoring of IB QA—titer, purity, IB bead size—showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate—time dependently—quality attributes in upstream processing to enable robust downstream processing.

Continuous cultivation with E. coli has several benefits compared to classical fed-batch cultivation. The economic benefits would be a stable process, which leads to time independent quality of the product, and hence ease the downstream process. However, continuous biomanufacturing with E. coli is known to exhibit a drop of productivity after about four to five days of cultivation depending on dilution rate. These cultivations are generally performed on glucose, being the favorite carbon source for E. coli and used in combination with IPTG for induction. In recent works harsh induction with IPTG was changed to softer induction using lactose for the T7-based plasmids, with the result of reducing the metabolic stress and tunability of productivity. These mixed feed systems based on glucose and lactose result in high amounts of correctly folded protein. In this study we used different mixed feed systems with glucose/lactose and glycerol/lactose to investigate productivity of E. coli based chemostats. We tested three strains producing different model proteins, with the final aim of a stable long-time protein expression. While glucose fed chemostats showed the well-known drop in productivity after a certain process time, glycerol fed cultivations recovered productivity after about 200 h of induction, which corresponds to around 30 generation times. We want to further highlight that also the cellular response upon galactose utilization in BL21 might be a key parameter in process design to achieve stable productivity soon. This “Lazarus” phenomenon has not been described in literature before and may enable a stabilization of continuous cultivation with E. coli using different carbon sources.