152 Upstream Processing
barium chloride and sulfuric acid at varying quorum sensing A phenomenon by which bacterial cells
concentrations. communicate with each other by the release of signaling
media Solid or liquid substances that support the growth molecules, which aids their survival in varying
of microorganisms or used for collection and environmental conditions.
transportation of organisms to ensure their viability. single cell protein A mixture of proteins extracted from
protoplast fusion A type of genetic modification in plants pure or mixed cultures of algae, yeasts, fungi, or bacteria
where two distinct protoplasts (from different species of and used as a substitute for protein-rich foods, in human
plants) are fused, resulting in the formation of a new and animal feeds.
hybrid plant possessing the characteristic of both plants; it vaccine An immunobiological substance designed to
is also called as somatic fusion. produce specific protection against a given disease.
2.13.1 Introduction
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 is prepared and optimized before the fermentation process commences. The inoculum needs to be
optimized for better performance, which can be done on the basis of various parameters, including mutation (DNA recombination,
radiation, and chemical addition). In medical terminology, an inoculum can be defined as a population of bacteria or viruses,
which is introduced in the body to cause or to increase immunity to a specific disease or condition, or into the medium for different
medical microbiological procedures.
During the first stage of inoculum development, the inoculum is taken from the working stock culture 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
influences the final product. Bacterial vegetative cells and spores are suspended in sterile tap water or sterile saline, which is then
added to the broth. In case of nonsporulating fungi and actinomycetes, hyphae are used for inoculum, and then transferred to the
final solution. Inoculum development is done in sequential steps to increase the volume of the initial inoculum to the desired
level. At each step, the inoculum used is 0.5–5% of the medium volume, which constitutes a 20–200-fold increase in the inoculum
volume at each step from its original size. An active growth stage is required in the final production stage of fermentation processes.
Many fermentation processes have several stages of development and scale-up of inocular microbial growth. The objective is
usually to achieve a high level of viable biomass in a suitable physiological form and a growth stage for use as an inoculum for the
next stage where small cell protein (SCP) for enzymes, proteins, or metabolite production, or viable biomass is the required end
product.
In microbiological techniques, inoculation of samples and microbiological cultures in the relevant media is crucial to obtain a
proper yield for diagnosis and antimicrobial sensitivity, both for bacterial and for fungal cultures. Correct preparation of inoculum
for cultures is integral to many microbiological procedures. One main component is standardization of size of cultures, which has
implications mostly in antimicrobial susceptibility testing. Counting methods of bacteria also require standardization of bacterial
inoculum for the same. In case of viral cultures, inoculation requires meticulous preparation of viral stock to ensure maximum viral
yield. Animal cells from tissues are cultivated followed by expansion of the cells for mammalian cell-culture techniques for
industrial and medical use.
This article briefly describes the criteria, process, and monitoring of inoculum development for fermentation biotechnology
followed by inoculum preparation in reference to various microbiological and industrial techniques.
2.13.2 Criteria for Inoculum Preparation for Fermentation Process
For proper inoculum development, certain criteria are considered as detailed below.
[Link] Physiology and Morphology
Optimized fermentation is often associated with particular physiological and morphological forms. For instance, in strepto
mycin-producing strains, mycelia fragmentation, loss of conidia formation, and progressive change to noncardial-type
morphologies are associated with culture degeneration and antibiotic producing capacity. The production of some microbial
products is associated with spore formation, whereas the synthesis of other products is inhibited by spore formation.
Spore formation, in turn, can be regulated by media design. In general, low levels of complex nitrogen induce
spore formation.
Certain medium-related conditions have been shown to affect the morphology. These include pH, viscosity, divalent cations,
chelating agents, anionic polymers, surface-active agents, and the presence of solids in medium.
Inoculum Preparation 153
[Link] Optimum Size
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 fermentors. 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
fermentor 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. Studies have demonstrated that inoculum size has a significant influence on the growth
of culture under extreme conditions. Studies have also demonstrated that, for identical combination of sodium chloride and
pH, a difference was observed between medium- and low-level inoculua, with medium inoculum exhibiting more growth at
more extreme conditions, indicating that inoculum size plays a very important role in the growth and, eventually, the yield.
[Link] Contamination
The risk of contamination is always present in inoculum development. Contamination can result in lower productivity by killing
the microbes used for inoculum preparation, or by competition by the contaminating organisms. Therefore, every effort must be
made to detect and prevent contamination. This can be done by various sterilization methods and offline and online monitoring.
The kinetics of culture media sterilization describes the rate at which microorganisms are destroyed using a first-order reaction-
rate model [16].
dN
− ¼ Kd N
dt
where N is the number of viable organisms present in the culture media, t is the treatment time or sterilization time, and Kd is the
reaction rate constant.
In different industrial processes, different contaminating organisms pose problems. In the canning industry, Clostridium
butylicum, an obligate anaerobe, may grow in sealed cans and produce heat-resistant spores and deadly toxins. However, it is not a
problem for sauces, which are too acidic, jams and jellies because of their very high sugar content, and milk as it is stored at low
temperature.
Organisms such as Lactobacillus are common contaminants in wine production. Similarly, in the antibiotic industry, there are
many potential contaminants such as molds, yeast, and many bacteria, including Bacillus. The most dreaded contaminants in the
fermentation industry include phages for which the only effective protection is to develop resistant strains.
[Link] Retaining Productivity
The culture should retain its essential product-forming capabilities. A culture usually loses its productivity due to depletion of
media, degeneration of culture, accumulation of toxins, contamination, 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
have to 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 postexponential 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.