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FL3_REQUIREMENTS FOR BIOPROCESSING MSBTW-Bioprocess Technology (Dr. Kiani)

Brief discussion about the requirements:
The overarching goal of biotech is to benefit society through the creation of pharmaceutical, diagnostic,
agricultural, environmental, and other products that utilize living cells and cellular materials. These products
necessitate the development of novel bioprocessing techniques as well as sophisticated equipment that can
support each stage in the bioprocessing flow from product formulation, hydration, and cell culture (upstream) to
harvest and finally separation and purification, finish, bulk storage, and filling (downstream).
Successful development of a microbial fermentation process requires the multidisciplinary contributions,
particularly biochemistry, chemistry, genetics and molecular biology, chemical & process engineering, and
mathematics for statistical modeling and computer technology.
A typical process engages both upstream (USP) and downstream (DSP) processing stages. The upstream
process is associated with all factors that are essential for the fermentation. This mainly includes:
1. Types of media used
2. Types of organisms used
1. TYPES OF MEDIA USED
The media used for the growth of microorganisms in industrial fermentation must contain all the elements in a
suitable form for the synthesis of cellular substances as well as the metabolic products. While designing a
medium, several factors must be taken into consideration. The most important among them is the ultimate
product desired in the fermentation.
There are two uses of fermentation media: 1. Growth media 2. Fermentation media
1. Growth media: Growth medium contains low amounts of nutrients. It is useful in creating raw material
for further fermentation processes.
2. Fermentation media: Fermentation media contains high amounts of nutrients. It is used in creating
final products using fermentation.
For example, growth of yeast requires 1% carbon. But during fermentation of alcohol, yeast requires 12
to 13 % carbon in the medium.
Optimization of the media components:
Optimization of the media components is a vital aspect to ensure maximization of yield and economics.
For growth-linked products (primary metabolites e.g. ethanol, citric acid), the product formation is directly
dependent on the growth of the organisms, hence the medium should be such that it supports good growth.
On the other hand, for products which are not directly linked to the growth (secondary metabolites e.g.
antibiotics, alkaloids, gibberellins), the substrate requirements for product formation must also be considered.
Primary metabolites: Some microbial products are primary metabolites, produced during
active vegetative growth (growth phase or trophophase). For example; amino acids, organic
acids, vitamins and industrial solvents such as alcohols and acetone.
Secondary metabolites: Many valuable industrial products are secondary metabolites, they are
not necessary for microbial growth. For example: antibiotics and alkaloids, usually achieved
after optimum biomass production (stationary phase growth or the idiophase).
In the laboratory fermentation, pure defined chemicals may be used for culturing microorganisms. However,
for industrial fermentations, undefined and complex substrates are frequently used for economic reasons.
Cheaper substrates are advantageous since they minimize the production cost of the fermented products. Wastes
from agriculture, and by-products of other industries are generally preferred, although they are highly variable
in composition.
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Raw materials used in fermentation largely depend on their cost at a particular time, since there are seasonal
variations. In many of the industrial media, the basic raw materials are waste products from other industrial
processes, particularly sugar processing wastes, corn steep liquor, lignocellulosic wastes, cheese and whey.
Choice of the medium:
The choice of the medium is very critical for successful product formation. For industrial fermentation, the
microorganisms, in general, utilize a luxury metabolism. Therefore, good production yields are expected with
an abundant supply of carbon and nitrogen sources, besides requisite growth factors. The media used in
fermentation processes may be synthetic or crude.
What is the role of Fermentation Media?
During the fermentation process, media contains high amounts of nutrients, micro-organism
and optimum conditions. When these micro-organisms are incubated at the desired optimum
conditions, they enjoy luxurious metabolism. Here, the fermentation organisms become
hyperactive due to the presence of high quantities of nutrients, thus it results in consumption
of excess nutrients and partial degradation of fermentation media. The waste effluents
excreted by microbes could be the desired output product of the fermentation process.
Types of fermentation media used in industries
There are two types of fermentation media used in industries.
1. Synthetic media 2. Crude media
1. SYNTHETIC MEDIA:
Media with all the requisite constituents in a pure form in the desired proportion represents synthetic media. Use
of this type of media in fermentations is not practicable.
Synthetic media is useful in the field of research as each component is chemically known, and the exact
composition of nutrients is predetermined. So, in case of synthetic media, variation in levels and concentration
of nutrients can be controlled. Here, by experimentation with synthetic media, the effect of nutrients on growth
and yield of product can be analyzed. We can redesign the synthetic media as per our needs.
Advantages of a well-designed synthetic media:
1. Synthetic media lacks sources of protein and peptides; hence, there is no foam formation
2. Chances of contamination are very less
3. Product recovery is easier because synthetic media contains pure components.
2. CRUDE MEDIA:
Crude media is generally used on an industrial scale for fermentation process. Crude media contains a rough
composition of media required for fermentation. It gives high yield of product and contains undefined sources
of ingredients. Crude media contains high level of nutrients, vitamins, proteins, growth factors, anti-foaming
agents and precursors.
It is important to ensure that crude media should not contain toxic substances that could affect the growth of
bacteria and yield of product.
The non-synthetic media with naturally available sources are better suited for fermentation. In practice, crude
media with an addition of required synthetic constituents is ideal for good product yield in fermentation.
The most frequently used substrates for industrial fermentation with special reference to the supply of carbon
and nitrogen sources and growth factors are briefly described below.
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INGREDIENTS OF CRUDE MEDIA:

1) Inorganic nutrients: Crude media contains inorganic salts containing cations and anion along with a carbon
source. Sometimes, fermentation micro-organisms have a specific requirement of ions like magnesium ions,
phosphates or sulphates. These requirements are fulfilled by addition of these ions to balance the crude media.
2) Carbon source: Simple to complex carbohydrates can be added to media as a source of carbon. The
selection of carbon source depends upon the availability as well as the cost of raw material. In most of the
fermentation media, crude source of carbon is added.
1. Simple carbohydrates – simple sugars are semi purified polysaccharides. Sources of simple
carbohydrates are Black strap molasses, Corn molasses, Beet molasses, sulphite waste liquor, Hydrol
(corn sugar molasses), etc.
2. Complex carbohydrates – Source of complex carbohydrates are Starch, Corn, Rice, Rye, Milo,
wheat potatoes etc. Source of starch cellulose are corn cobs, straws, wood waste, saw meal etc.
3) Nitrogen source: Salts of urea, ammonia, and nitrate can be used as a nitrogen source. When fermentation
organisms are non-proteolytic in nature, pure form of urea, ammonia and nitrate are used as a source of
nitrogen. When fermentation organisms are proteolytic in nature, animal and plant raw materials are used; like
distillery dried solubles, Casein, Cereal grains, peptones, yeast extract, hydrolysate, and soybean meal etc.
4) Growth factors: Crude media constituents provide enough amount of growth factors so no extra addition of
growth factor is required. If there is a lack of any kind of vitamins or nutrients, growth factors can be added to
media. Examples are yeast extract, and beef extract.
5) Precursors: Precursors are generally present in the media as crude constituents. Precursors are added in the
fermentation media at time of fermentation as it gets incorporated in the molecules of product without bringing
any kind of change to the final product. This helps in improving yield and quality of product. Sometimes,
precursors are added in pure form depending upon the need of product. For example, Cobalt chloride is added
less than 10 ppm in fermentation of vitamin B12.
6) Buffers: Buffers are used to control drastic changes of pH. Sometimes, media components may act as
buffers. For example, protein, peptides, amino-acids act as good buffers at neutral pH. Sometimes inorganic
buffers like K2HPO4, KH2PO4, and CaCO3 etc, can be added as required. Generally, during fermentation
process, pH changes to acidic or alkaline pH. The cheapest and easily available buffer is CaCO3

2. TYPES OF ORGANISMS USED

The key factor that is responsible for the product fermentation is the microorganism itself. To achieve efficient
microorganism, following strategies are included:
 Initial screening of suitable industrial microorganism
 Strain improvement to enhance productivity and yield
 Pure culture maintenance of the strain
 Preparation of a suitable inoculum and fermentation conditions to improve the economic
efficiency of the process.
For example, the production of stable mutant strains that greatly overproduce the expected metabolite is
often crucial.
CRITERIA USED FOR CHOICE OF ORGANISMS
1. The nutritional characteristics of the organism: Organism should be capable to utilize the ingredients
present in the medium to produce interested product.
2. The optimum temperature of the organisms: For instance, the use of an organism having an
optimistic temperature above 40oC considerably reduces the cooling costs of a large-scale fermentation.
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3. Genetic stability: The stability of the organism and its amenability to genetic manipulation.
4. High yielding strain: Ability to convert maximum substrate into product and give a high yield of
product per unit time.
5. Easy product recovery: The easy product recovery from the cultures during downstream processing.
6. It should not produce undesirable substances
7. It should be easily cultivated on a large scale.
8. Selective pressure may be used in the isolation of organism that will grow on particular substrates in the
presence of certain compounds or under agricultural conditions adverse in their types

FL4_TEMPERATURE & pH REQUIREMENTS FOR GROWTH OF DIFFERENT ORGANISMS

Q: What is considered a critical process parameter?
Ans: In the context of a bioprocess, a Critical Process Parameter (CPP) is a measurable and controllable key
variable that directly shapes the outcome and efficiency of a biological process. These parameters play a crucial
role in determining:
o How well the process operates.
o Broad spectrum spans, encompassing physical, chemical, and biological factors.
Specific parameters that require monitoring can vary based on the unique characteristics and goals of different
bioprocesses. Nonetheless, certain parameters consistently stand out as top priorities in most fermentations.
TEMPERATURE REQUIREMENTS:
 Temperature, a central parameter in bioprocessing, profoundly influences microbial activities and
enzymatic reactions. The right temperature serves as a catalyst for optimal cell growth, metabolism, and
the production of target compounds, while deviations may compromise these results.
 Both excessively high and low temperatures can impede cellular activities, resulting in decreased
productivity or undesirable by-products. Higher temperature deviations can have a dramatic effect on
cell viability, while lower temperatures slow down cell metabolism.
 Temperature variations also impact the solubility of gases crucial for certain processes. Therefore,
continuous monitoring and adjustment of temperature, facilitated by sophisticated heating or cooling
systems, are fundamental to consistently achieving high-quality outcomes in bioprocessing.
 Most bioprocess temperatures are within the range of 30°C to 37°C. The temperature must be tightly
controlled to within about 1°C.
 During the bioprocess, cell density increases, and consequently the metabolic activity increases, which
generates a substantial amount of heat. The generated heat must be removed before the temperature in
the vessel increases. Thus, efficient heat transfer to operate and maintain the system at optimal growth
temperature for the working organism is needed.
 The temperature in a bioreactor can be managed by removing excess heat using an external jacket or
coil, through which cooling water is circulated. Or in the case of adding heat, steam is used for
circulation. Alternatively, the equipment can be located internally, such as helical or baffle coils.
 An additional option for maintaining a set temperature is to pump liquid from the reactor through a
separate heat exchange unit.
TEMPERATURE REQUIREMENTS FOR BIOREACTORS:
Bioreactors are mainly used for biological processes such as fermentation and cell culture, where temperature
control is crucial as it directly affects the metabolic activity, growth rate, product formation, and structural
stability of microorganisms or cells. The specific requirements are as follows:
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1. Physiological suitability range: The temperature inside the bioreactor should be set within the optimal
growth temperature range of the cultivated microorganisms or cells. For example:
i. For bacteria, this range may be between 30°C and 37°C;
ii. For yeast and certain fungi, it may be 25°C to 30°C;
iii. For mammalian cell culture, it is usually around 37°C.
Maintaining a suitable temperature helps maintain high activity, high yield, and good cellular health.
2. Stability and uniformity: The temperature not only needs to be maintained at a specific value, but also
should be highly uniform throughout the entire reaction volume to avoid cell death or metabolic
abnormalities caused by local overheating or undercooling.
This usually requires an efficient mixing system and good heat transfer design, as well as precise
temperature control systems.
3. Process control: Based on specific biological processes and research needs, temperature may need to be
dynamically adjusted according to preset procedures. For example, in certain fermentation processes, it
may be necessary to induce specific gene expression or regulate metabolic pathways by changing
temperature.
4. Accuracy and precision: Accuracy of temperature control systems is usually required within ± 1°C, or
even higher, to ensure the repeatability of experiments or production and the reliability of data.

PH REQUIREMENTS FOR GROWTH OF DIFFERENT ORGANISMS

pH, a measure of the acidity or alkalinity of a solution, is a fundamental parameter in bioprocessing. Its
significance lies in its profound influence on various biological and chemical reactions, including microbial
growth and enzyme activity, making it a critical factor for the success of bioprocesses.
The pH homeostasis in metabolism is critical for several reasons:
1. First, because structure/function of biological macromolecules, especially proteins, depends on pH.
2. Second, because pH, as any other cellular metabolite concentration, may affects the kinetic and
thermodynamic force of chemical reactions involving protons as metabolites.
3. Third, because pH changes severely affect the energetic metabolism, provided that the proton motive
force use to be the main source of electrochemical potential for ATP synthesis.
Different organisms thrive in specific pH ranges, and deviations can lead to inhibited growth or undesirable
metabolic shifts. Microbes can grow in different environments by adapting to the pH of their habitat. Most
bacteria grow over a pH range of 3 units.
The precise control of pH in bioprocesses ensures the creation of an environment favorable to produce desired
compounds. Such control is achieved through methods such as chemical indicators, electrodes, or spectroscopy.
Automated pH controllers and dedicated equipment facilitate real-time assessment, allowing for immediate
adjustments to maintain the ideal pH conditions.
Based on pH, microorganisms can be divided into three main groups:
1. Acidophiles are organisms that thrive under highly acidic conditions (less than pH 5.5). Some examples
are Leptospirillum ferriphilum and Sulfolobus acidocaldarius.
2. Neutrophiles live and thrive in an environment with a relatively neutral pH (pH 5.5 to 9). Some of the
most common include Escherichia coli, Staphylococcus aureus, and Lactobacillus acidophilus.
3. Alkaliphiles are microorganisms that grow well at pH values exceeding pH 9. An example of this group
is Halorhodospira halophila.