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October 15, 2015 (Vol. 35, No. 18)

Rating the BactoVessel Single-Use System

Evaluating Performance against a Standard Glass/Steel Fermentation Technology

  • The decision to use single-use systems for manufacturing pharmaceuticals depends on many factors. However the major driving force is a desire to increase throughput.

    Single-use fermenters (SUFs) eliminate cross contamination, reduce water consumption, and eliminate time-consuming sterilization of conventional glass/steel stirred tank reactors (STRs).

    This tutorial compares process parameters between conventional STRs and CerCell’s latest generation SUF and examines the growth data for a wild-type E. coli bacterium. The volumetric oxygen mass transfer coefficient (kLa) value for different bioreactor sizes with three different turbine designs and four different agitation speeds was determined.

    Two kLa high-speed optical DO sensors—Presens’ OpTrode and Hamilton’s VisiFerm—were used. The  values were compared relative to the different sizes of bioreactors, turbines, stirring speed, and sensors. Finally, the kLa values of conventional STRs are compared with kLa values for the SUF.

  • Fermentation Process

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    Fermentation processes, commonly used in the production of many foods, beverages, and pharmaceuticals, include chemical reactions such as oxidation, reduction, hydrolysis and biosynthesis. Some processes may be aerobic and others anaerobic. The rate of the fermentation process depends on the concentration of microorganisms, cellular components, and enzymes, as well as temperature, pH, and oxygen level.

    For the tests performed, the STR and SUF temperatures were kept constant as controlled by the Biostat PCS at 27.5°C. The 3 L BactoVessel SUF has an internal diameter (ID) of 124 mm and the 5.7 L SUF ID 144 mm (Table 1).

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    The kLa, the most important parameter in a fermentation process, is defined as the reciprocal of time covering the transfer of oxygen from the gas phase to liquid. The kLa describes the efficiency with which oxygen can be supplied to a fermentation process for a given set of operating conditions.

    Dissolved oxygen (DO) is often the limiting substrate in the fermentation. For bacteria and yeast cultures oxygen concentration is usually 10–50% of air saturation. For optimum growth, it is important to maintain the DO level above this critical value by aerating (sparging) the bioprocesses with air or pure oxygen. The oxygen mass transfer rate to the liquid fermentation liquid should be equal to or exceed the rate at which growing cells take up oxygen.

    In aerobic fermentation, oxygen molecules pass several mass transfer steps engaged in the transport of oxygen from the interior of the gas bubbles before facilitating intracellular reactions.

  • Discussion

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    Figure 1. Cell density measurements of seven different SUF/STRs relative to agitation speeds of 1,000 rpm and time in hours according to Table 1.

    The kLa value increased linearly with agitation speed until 1,500 RPM. The best kLa value was obtained in the 5.7 liter SUF with Bakker turbine at 1,500 rpm and 212 h-1. At higher agitation speed (2,000 rpm) the kLa value decreases (Table 2).

    Growth data show no difference for conventional STR and BactoVessel SUF. Such a SUF delivers the same results as conventional STR fermenters under equal conditions. Data for the growth of E. coli showed a cell densitry (CD) max about 30, a specific growth rate of 0.3 h–1, and a maximum of about 0.028 dry weight (DW) g/L for all bioreactor types (Figure 1).

    The kLa values for a 5 L STR with Rushton turbine measured to 142 h–1 at 1,000 rpm, and the kLa value of 5.7 liter SUF with Rushton turbine is 144 h–1.

    In conclusion, tray turbines have good kLa values: Bakker at 212 h–1, Smith turbine at 187 h–1, and Rushton at 182 h–1 (Table 2). Bakker tray turbines exhibit the highest gas collection level relative to Smith tray and Rushton flat blade. Further the oxygen mass transfer rate is linked to power take up and RPM, i.e., higher RPM increase kLa. Visual inspection of mixing gradients shows flat blade Rushton being the least effective in gradient free mixing. Smith is significantly better and Bakker even slightly better.

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