Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance

Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating online monitoring, pH control and feeding was developed and validated. Critical interference of the essential media component protocatechuic acid with pH measurement was revealed, but was effectively resolved by 80% concentration reduction without affecting biological performance. Microfluidic pH control and feeding (pulsed, constant and exponential) were successfully implemented: Whereas pH control improved performance only slightly, feeding revealed a much higher optimization potential. Exponential feeding with µ = 0.1 h−1 resulted in the highest product titers. In contrast, other performance indicators such as biomass-specific or volumetric productivity resulted in different optimal feeding regimes.

A Process Engineering Approach to Improve Production of P(3HB) byCupriavidus necatorfrom Used Cooking Oil

Different feeding strategies, namely, exponential feeding and DO-stat mode, were implemented for the production of poly(3-hydroxybutyrate), P(3HB), byCupriavidus necatorDSM 428 with used cooking oil (UCO) as the sole carbon source. With the exponential feeding strategy, a cell dry mass of 21.3 ± 0.9 g L−1was obtained, with a polymer content of 84.0 ± 4.5 wt.%, giving an overall volumetric productivity of 4.5 ± 0.2 g L−1 day−1. However, the highest P(3HB) volumetric productivity, 12.6 ± 0.8 g L−1 day−1, was obtained when the DO-stat mode was implemented together with the use of ammonium hydroxide for pH control, which served as an additional nitrogen source and allowed to reach higher cell dry mass (7.8 ± 0.6 g L−1). The P(3HB) obtained in all experiments had a high molecular mass, ranging from 0.6 × 105to 2.6 × 105 g mol−1, with low polydispersity indexes of 1.2-1.6. Melting and glass transition temperatures were also similar for the polymer produced with both cultivation strategy, 174°C and 3.0-4.0°C, respectively. The polymer exhibited a crystallinity ranging from 52 to 65%. The DO-stat strategy to feed oil containing substrates as the sole carbon sources was reported for the first time in this study, and the preliminary results obtained show that it is a promising strategy to improve P(3HB) production. Nevertheless, the process requires further optimization in order to make it economically viable.

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

ABSTRACT We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h−1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g � h), pyruvate formation rate (1.11 g/g � h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g � h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h−1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter � h and yield of 0.68 g/g.