Fed-batch mode in shake flasks by slow-release technique

Abstract

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.

Microfluidic biolector—microfluidic bioprocess control in microtiter plates

“…Scheidle et al (2009) and Stöckmann et al (2009) proved the thesis discussed above by demonstrating that the screening result markedly depends on the cultivation strategy used. The authors performed screenings of hundreds of Hansenula polymorpha clones under fed-batch conditions, feeding glucose by applying a polymer-based controlled-release system (Jeude et al, 2006) in 96-well deepwell MTP. By comparing these screenings to conventional screenings in batch mode, they clearly showed that fed-batch screenings yielded completely different results with respect to the best-producing clones.…”

Control of specific carbon dioxide production in a fed-batch culture producing recombinant protein using a soft sensor

“…This could not be met with previous soft sensors used by us (Warth et al, 2010;Gustavsson and Mandenius., 2013) where delay times of up to 20 min seriously hampered the controllability of the system. An alternative setup would have been to extend the gas analyser with on-line measurement of O 2 , hence allowing monitoring of oxygen uptake rate (OUR) as well as respiratory quotient (RQ) by combining CO 2 and O 2 measurements (Levisauskas et al, 1996;Jeude et al, 2006). However, the on-line measurability of CO 2 with existing CO 2 sensors seems better than with available O 2 analyser alternatives.…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…Use of hydrogels has been reported for controlled delivery of growth factors to animal cells 64 . Continuous delivery of glucose and sodium carbonate using silicone elastomer discs has been reported for in situ release to microbial systems over a period of one or two days to achieve nutrient feeding and pH control respectively 65,66 . Unlike microbial cultures which run for short durations of 24-48 h, animal cells in fed batch are cultured for 7-15 days.…”