Continuous feeding strategy for polyhydroxyalkanoate production from solid waste animal fat at laboratory- and pilot-scale.

Bioconversion of waste animal fat (WAF) to polyhydroxyalkanoates (PHAs) is an approach to lower the production costs of these plastic alternatives. However, the solid nature of WAF requires a tailor-made process development. In this study, a double-jacket feeding system was built to thermally liquefy the WAF to employ a continuous feeding strategy. During laboratory-scale cultivations with Ralstonia eutropha Re2058/pCB113, 70% more PHA (45 gPHA L-1 ) and a 75% higher space-time yield (0.63 gPHA L-1  h-1 ) were achieved compared to previously reported fermentations with solid WAF. During the development process, growth and PHA formation were monitored in real-time by in-line photon density wave spectroscopy. The process robustness was further evaluated during scale-down fermentations employing an oscillating aeration, which did not alter the PHA yield although cells encountered periods of oxygen limitation. Flow cytometry with propidium iodide staining showed that more than two-thirds of the cells were viable at the end of the cultivation and viability was even little higher in the scale-down cultivations. Application of this feeding system at 150-L pilot-scale cultivation yielded in 31.5 gPHA L-1 , which is a promising result for the further scale-up to industrial scale.

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population.

Tools for the determination of population heterogeneity caused by inhomogeneous cultivation conditions.

Population heterogeneity among a microbial culture can occur in various stages of a bioprocess. When a nutrient-limited fed-batch process is applied in the large scale, gradient formation (e.g. at the substrate concentration or the pH-value) can have a tremendous impact on the process performance. It might also have an impact on population heterogeneity, as the cells are opposed to stressful conditions that is an oscillating environment, when they pass the different zones evolved in the liquid phase. Nevertheless, the question whether these conditions support heterogeneity of a culture has not been clearly answered so far. Furthermore, if such a heterogeneity affects product formation, the usual analysis tools, which do not rely on a single-cell basis, certainly do not gain sufficiently suitable information for process optimization. This overview on the one hand provides information about the contribution of an oscillating environment to the formation of heterogeneities within a population and on the other hand a summary of tools, which can be used to investigate physiologic and morphologic heterogeneity during process development, scale up and production. If these techniques are considered, the identification of targets for accelerated process optimization on a single-cell basis becomes easier and faster.