Inoculation development for production of recombinant microbial products

Recombinant DNA technology:
Recombinant DNA technology introduces foreign genes into plants, animals and microorganisms in order to express new characteristics. This technique has been used in production of livestock, food products, biopharmaceuticals, antibiotics, and industrial chemicals. Recombinant microorganisms are also used for small scale and large-scale fermentations.
Inoculum development:
An inoculum can be defined as the population of microorganisms or cells that is introduced in the fermentation medium or any other suitable medium. It needs to be prepared and optimized before any production process involving use of microorganisms is commenced. However, in some cases the products are the microorganisms themselves (example – SCPs or yeast). The reason for inoculum optimization is better performance in terms of quality and/or quantity of the end product. Microorganisms contribute to production of a variety of commercial and industrial products. This text will particularly focus on inoculation development for recombinant microbial products with respect to fermentation processes. Certain criteria for inoculum development are mentioned which can be applied to recombinant as well as nonrecombinant microorganisms and, safety guidelines for developing inoculum by recombinant microorganisms are also mentioned in this text.
Inocula are usually prepared in several stages of increasing volume and it usually consists of 5-20% of the total product. During the first stage of inoculum development, the inoculum is taken from the working stock culture in order to initiate the growth in a suitable liquid medium. Growth at this stage is influenced by the adaptation of inoculum to the new environment, which further affects the final product. Bacterial vegetative cells and spores added to the broth post suspension in sterile tap water or sterile saline. In case of nonsporulating fungi and actinomycetes, hyphae are used for inoculation process and then transferred to the final solution. As mentioned earlier, inoculum development is carried out in sequential steps to increase the volume of the initial inoculum to the desired level.
Criteria for inoculum development:
Cells to be used must be actively growing, young and must therefore be in the phase of logarithmic growth thereby minimizing the length of the lag phase by taking less time to adapt to the environmental conditions. This will only be possible with healthy and actively growing cells. This can be achieved by providing the microorganisms with proper medium, necessary conditions, and a good monitoring system.
The risk of contamination in inoculum development is always present. Hence, it is necessary to detect and prevent contamination. This can be achieved by various sterilization methods as well as offline and online monitoring. Contamination can result in lower productivity by killing of the microorganisms used in inoculum development by the contaminating microorganisms or they could also compete with the cells in inoculum for nutrients and survival. The most atrocious contaminants in the fermentation industry include phages for which the only effective measure is to employ resistant strains.
A suitable morphological form should also be ensured as optimized fermentation is often associated with physiological and morphological forms. Certain medium related conditions have been shown to affect the morphology. These conditions include pH, viscosity, divalent cations, chelating agents, anionic polymers, surface active agents, and the presence of solids in the medium.
The culture must also retain its product forming capabilities. A culture may usually lose its productivity due to depletion of media, degeneration of culture, accumulation of toxins, contaminants, etc. A common example is antibiotic production by fermentation where the reversion of high-yielding strains is rare. The retention of productivity depends on the likelihood of stability of culture, which, in turn, depends on the medium conditions. If the product is growth associated, medium conditions must satisfy cell growth and product formation in an optimal manner. In case of enzymes and antibiotics, the optimum rate of product formation is often observed during the post exponential phase. Thus, the main objective of controlled media and other environmental conditions is to extend the exponential phase as long as possible, that is, to retain productivity for as long as possible.
The inoculum must be available in sufficient quantities to provide an optimum volume. Starting from a slant or stock, the inoculum is usually built up in two or three stages in the laboratory followed by one or more stages in conventional fermenters. During this process, the cells may undergo 20–50 generations or more. At each step, the inoculum is used at 0.5–5% of the medium volume, which allows a 200-fold increase in inoculum volume at every step. Mostly, the inoculum used for the production stage is approximately 5% of the medium volume, or it can be adjusted to obtain maximum fermenter productivity. Low inoculum levels result in long fermentation cycles, lowered productivity, and increased likelihood of contamination. Taking into consideration another perspective of inoculum size, at high cell densities, a phenomenon called inoculum effect has been observed with some organisms, which is attributed to several mechanisms. Prominent among the proposed mechanisms is quorum sensing.