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

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.

Mass transfer resistance of sterile plugs in shaking bioreactors.

One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (k(O(2))), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D(CO(2))) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.

Device for sterile online measurement of the oxygen transfer rate in shaking flasks.

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.