Inoculum propagation

Inoculum propagation and small-scale culture systems... 

Industrial fermentation plants consist of three main sections preparation, fermentation, and product recovery. The preparation section usually contains operations such us medium preparation and sterilization and inoculum propagation. The fermentation section is the heart of the plant where the transformation of raw material into products takes place. The product recovery section encompasses the downstream operations needed to obtain the product of interest with the required purity (Reisman 1988). In the design of fed-batch and batch fermentation plants, one faces the problem of figuring out the adequate combination of the number and size of fermenters to be used to meet the desired production schedule. In principle, the problem has an infinite number of solutions because for any given fermenter size, a number of units of that size will do the work. Nevertheless, not all solutions are equal from an economic standpoint (Simpson et al. 2005). 

Specific procedures exist for storing (16,17) and propagating microorganisms to obtain reproducible fermentations. The stock culture is stored frozen (<—80°C) or freeze-dried. To prepare the inoculum (seed) mixture, an aUquot is taken and grown in consecutive soHd or Hquid cultures of increasing volume. The volume of the last step, the seed fermentor, is typically 4—12% of the main fermentor volume. 

Adherent insect cell release from solid surfaces generally does not require trypsinization, unlike anchorage-dependent mammalian cells. Insect cells, like mammalian cells, need rigorous aseptic manipulation during cell transfer, inoculation, and propagation in bioreactors. A minimal inoculum density is required for both cell types. Typically, insect and mammalian cell cultures are initiated with inocula of 1-2 X 105 cells per milliliter of liquid medium (Agathos, 1991). Table 2.3 summarizes the major differences and similarities between these cells. 

After eliminating the cryoprotector (generally DMSO - dimethyl sulfoxide), cells are suspended in fresh medium at a concentration between 0.1 and 0.4 million cells per-mL, depending on the cell line and the culture medium, to be used subsequently for incubation and propagation. This range of inoculum concentration is used as a standard for the inoculation of animal cell cultures, since lower concentrations result in long lag phases and in a delay in cell growth, which is detrimental to process time and productivity. 

All the procedures carried out on a small scale are labor-intensive and require skilled operators. Manipulation of the cultures is carried out in laminar-flow cabinets, to maintain asepsis. During this stage of cell propagation/inoculum development, activities are carried out by direct operator manipulation, so that the process is considered to be open . 

Figures 9.2 and 9.3 show schemes that illustrate inoculum development from the cryotubes to production scale for suspension and adherent cells, respectively. In these hypothetical process schemes, the expression production bioreactor is used arbitrarily for any of the types of bioreactor presented in the next section of this chapter. In general, different flasks and several intermediate bioreactors are used for cell propagation to reach the quantity of cells necessary to inoculate the production bioreactor. The number of propagation steps is a function of the final scale of the production bioreactor.

Cryotube Expansion in stationary flasks Propagation in stirred flasks Inoculum bioreactor Production bioreactor... 

Lastly, the cellular sources subsection of the methods of manufacture section must include a detailed description of the process of inoculation, cell growth, and harvesting. The stages of cell growth should be described carefully, including the selection of the inoculum, scale-up for propagation, and established and proposed (if different) production batch size. 

It is important to emphasize that for moderate- to large-scale fermentation plants, bank deposits of suitable yeast strains are necessary to guard against contamination. The capability of propagating active yeast strains adapted to the particular feedstock and conditions used in the process should be maintained to make sufficient inoculum available for fermenter startup after plant upsets and scheduled shutdowns. Also, if yeast cells are separated for recycling as inoculum for fresh feed, the most active cell strata should be selected. 

To this effect, culture the cells over several (e.g. 10-20) generations in the presence of the additive in static cultures, and check if they can overcome the inhibitory effect. If they do, then propagate the cells (when preparing the culture inoculum) in the presence of the additive. 

Cell Culture Section. The activities in this section include media sterilization, inoculum preparation, cell growth, and virus propagation (Figure 10.1-8). Serum-free (SF) medium is sterilized by 0.2- rm filtration and used for both inoculum preparation and cell growth/virus propagation. The culture is assumed to take... 

The total equipment purchase cost was estimated to be around 2.8 million. The most expensive piece of equipment is the bioreactor used for cell culture and virus propagation, priced at 506,000. The cost of unlisted equipment (including the equipment used in the inoculum preparation section) was assumed to represent 20% of the total equipment cost. 

Fig. 13.3 Overlaid MALDI-TOF mass spectra of initial Staphylococcus bacteriophage 53 inoculum (2 X10 PFU mL ), grown in N-labeled medium—green trace, and after its propagation for 2 h in S. aureus culture (6.7 x 10 CPU mL ) blue trace. The downward shift in mass of the observed biomarkeis before and after amplification is due to replacement of the isotope of the...

The following steps are always run in fermentors. Volume of the end production step is decisive for the number of previous cultivation steps for propagation of necessary amount of inoculum. During the inoculum preparation it can be also advantageously manipulated to evoke an optimal state of the... 

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