Exploring small-scale chemostats to scale up microbial processes: 3-hydroxypropionic acid production in S. cerevisiae

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.

Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae

“…Chemostats are strict nutrient-limited over the range of dilution rates with constant biomass yield [ 36 ]. For this study, we also chose to increase the dilution rate above this threshold and approaching the maximum growth rate,and wash out situation.…”

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

“…Furthermore, higher biomass concentrations and thus higher volumetric titers can be achieved in fed-batch and repetitive fed-batch mode. As biomass yield is decreasing upon production of recombinant proteins, this can potentially lead to washout (Lis et al, 2019). Hence, trying to achieve "fed-batch like" biomass concentrations in chemostat cultivation is highly difficult.…”

Adaptation of Central Metabolite Pools to Variations in Growth Rate and Cultivation Conditions in Saccharomyces Cerevisiae

“…Chemostats are strict nutrient-limited over the range of dilution rates with constant biomass yield 27 .…”