Abstract
BackgroundThe physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed.ResultsWe report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW−1, respectively, at the lowest set dilution rate of 0.04 h−1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW−1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW−1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW−1 under phosphate-limiting conditions.ConclusionsSmall-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h−1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.
Highlights
Besides strain engineering, the establishment of a commercially viable bioprocess requires multiple screening procedures, process development, and optimizations as well as process validation and scale-up trials
The specific growth rate in fed-batch cultivations can be controlled by feeding a single growth-limiting nutrient at a desired rate. As both chemostat and fed-batch cultivations are capable of tightly maintaining the specific growth rate at a set value without the accumulation of residual substrate, key cultivation parameters obtained in chemostat and fed-batch cultivations can be compared [7]
Transferability of the physiological parameters measured in small‐scale chemostats to fed‐batch cultures in 1‐L bench‐top bioreactors under C‐ and P‐limiting conditions Since this study further aimed at assessing transferability and comparability of the physiological parameters measured under different cultivation conditions, the cultivation parameters obtained from the C- and P-limiting conditions determined in chemostat cultures were transferred to 1-L stirred bench-top reactors running in fed-batch mode
Summary
The establishment of a commercially viable bioprocess requires multiple screening procedures, process development, and optimizations as well as process validation and scale-up trials. Novel microbioreactor (MBR) systems, ranging from down-scaled stirred tank reactors to advanced shaken microtiter plate cultivation devices, have been developed to meet the increasing need for high throughput strain screening and testing of relevant cultivation conditions [1]. Efforts towards designing MBR systems are reflected in robust cultivation devices, such as the parallelized small-scale chemostat bioreactor system based on Hungate tubes [2] and the microtiter plate based system for high-throughput temperature optimization for microbial and enzymatic systems [3]. Chemostat cultivations provide defined and constant cultivation conditions, where single parameters such as temperature, pH, nutrient composition or concentration can be investigated in relation to the growth rate applied [4]. Fed-batch cultivations are characterized by feeding nutrients intermittently or continuously for microbial growth. The specific growth rate in fed-batch cultivations can be controlled by feeding a single growth-limiting nutrient at a desired rate. Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed
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