BIOPROCESS

PRINCIPLES
 Biotechnology
• Field of applied biology that involves the use of living organisms and
bioprocesses in Engineering, technology, medicine and other fields
requiring bioproducts.
• Biotechnology also utilizes these products for manufacturing purpose.
• Modern biotechnology includes genetic engineering and cell/tissue culture
technologies.
• It covers a range of methods to modify living organisms for human needs,
including: Domestication of animals, Cultivation of plants, Breeding
programs using artificial selection and hybridization.
 Bioprocess Engineering
• Bioprocess Engineering is a specialization in biotechnology, chemical engineering, and
agricultural engineering.
• It focuses on designing and developing equipment and processes for manufacturing products
like food, feed, pharmaceuticals, chemicals, polymers, and paper using biological materials.
• Bioprocees engineering is a combination of mathematics, biology and industrial design, and
consists of various spectrums like designing of Fermentors, study of fermentors (mode of
operations etc.).
• It also deals with studying various biotechnological processes used in industries for large
scale production of biological product for optimization of yield in the end product and the
quality of end product.
• Bio process engineering may include the work of mechanical, electrical and industrial
engineers to apply their principles to processes using living cells or their components.
• Bio processing is an essential part of many food, chemical and
pharmaceutical industries.
• It uses microbial, animal, and plant cells or their components (e.g.,
enzymes) to Create new products and Eliminate harmful waste.
• The practice of using microorganisms for fermented food production
dates back to ancient times.
• Modern bioprocesses produce a wide range of products, including:
Common products: Industrial alcohol, organic solvents.
Specialty products: Antibiotics, therapeutic proteins, vaccines.
• Commercial bioproducts include industrial enzymes and living cells like
baker’s and brewer’s yeast.
• Advances in microbiology, biochemistry, and cell physiology boost
bioprocessing.
• Tools like recombinant DNA and tissue culture enable new products or
improve Bio processing methods.
• Innovations include medicines, cultured tissues, biochips, eco-friendly
pesticides, and pollution-degrading microbes.
• Biological systems are complex but follow chemical and physical laws,
enabling engineering analysis.
Engineering in bioprocessing involves:
• Designing and operating bioreactors, sterilizers, and product-recovery
equipment.
• Developing systems for automation and control.
• Ensuring efficient and safe layouts for fermentation factories.
Steps in Bioprocess
Development
Step-1: Genetic Manipulation of the host organism where a gene from
animal DNA is cloned into Escherichia coil.
Tools: Petri dishes, micropipettes, micro centrifuges, nano-or microgram
quantities of restriction enzymes, and electrophoresis gels for DNA and
proteifractionation.
• In terms of bioprocess development, parameters of major importance are
Stability of strains and product expression levels.
• Step-2 :
• After cloning, the growth and production characteristics of the cells must
be measured as a function of culture environment .
• Small-scale cultures (250 mL–1 L) are used to test growth and production.
• Optimal conditions (medium, pH, temperature) are determined.
• Calculated parameters such as cell growth rate, specific productivity and
product yield are used to describe performance of the organism
Step-3 : Scale-up of the process
• Use a 1–2 L bench-top bioreactor equipped with instruments for
measuring and adjusting temperature, pH, dissolved-oxygen
concentration, stirrer speed and other process variables.
• Bioreactors allow better monitoring and control compared to shake
flasks.
• Gather data on oxygen needs, shear sensitivity, and foaming
characteristics and other parameters.
• Analyze cell activity to assess the economic feasibility of the process
for commercial use.
Step 4 :scaled up again to a Pilot-Scale Bioreactor
• Scale up to a 100–1000 L vessel based on bench-scale
specifications.
• Bioprocess engineers oversee pilot-scale operations.
• Even with similar designs (e.g., reactor geometry, aeration,
mixing), performance variations can occur.
• Loss in productivity may impact economic projections and
require reassessment.
Step 5 : Design of the industrial-scale operation
1. This part of process development is clearly in the territory of bioprocess engineering.
2. Bioprocess engineers design the reactor and auxiliary systems, including:
• Air supply and sterilization.
• Steam generation and supply.
• Medium preparation and cooling-water systems.
• Process control networks.
3. Emphasis on aseptic fermentation, containment, and safety requirements.
Step 6 : Product Recovery (downstream processing)
• After fermentation, raw broth undergoes treatment to produce the final product.
• Purification is costly, especially for recombinant DNA products (up to 80-90% of processing cost).
• Recovery methods depend on the product and broth, including:
 Filtration, centrifugation, and flotation to separate cells from liquid.
 Mechanical disruption for intracellular products.
 Solvent extraction, chromatography, membrane filtration, adsorption, crystallization, and drying.
• Experts in chemistry, biochemistry, and chemical engineering design recovery and purification systems.
Step 7 : Packing and Marketing
• Clinical trials (animal and human) are needed for new
pharmaceuticals like recombinant human growth hormone or
insulin to test effectiveness and safety.
• After successful trials, the product can be released for healthcare
use.
• Food products also undergo testing before release.
• Manufacturing standards must be met, especially for recombinant
products, which require stricter safety measures.
 Unit operations and Downstream processing.
• Bioprocesses treat raw materials to produce useful products.
• Unit operations are individual steps that change or separate components in the process.
• Common unit operations in bioprocessing include:
Centrifugation, chromatography, cooling, crystallization, dialysis.
Distillation, drying, evaporation, filtration, heating, humidification.
Membrane separation, milling, mixing, precipitation, solids handling, solvent extraction.
• In fermentation, raw materials are altered mainly by reactions in the fermenter.
• Physical changes before and after fermentation prepare substrates and help extract/purify
the product.
• Fermentation broths are complex mixtures with products in dilute form.
• Downstream processing involves treating the broth after fermentation to concentrate and
purify the product, typically requiring only physical modifications.
 Steps of downstream processing
a. Cell removal:
• The first step in product recovery is removing cells from the fermentation liquor.
• This is needed if the biomass (e.g., baker’s yeast) or intracellular product is the
desired product.
• Removal of cells can also assist recovery of product from the liquid phase.
• Filtration and centrifugation are common methods for cell removal.
b. Primary isolation:
• Techniques are used to isolate fermentation products from cells or cell-free broth.
• The choice of method depends on the physical and chemical properties of the
product.
• Primary isolation processes treat large volumes and are typically non-selective, but
can significantly improve product concentration and quality.
• Methods like adsorption, liquid extraction, and precipitation are commonly used for
primary isolation.
c. Purification:

• Purification processes are highly selective, separating the product from similar
impurities.Common unit operations include chromatography, ultrafiltration, and fractional
precipitation.

• d. Final isolation:

• The final required purity depends on the product's intended use.

• For high-quality products like pharmaceuticals, typical operations include crystallization,

followed by centrifugation, filtration, and drying.
 Process Flow Diagrams

• Large-scale manufacturing processes are complex, requiring specialized methods to

communicate information.

• Flow diagrams (or flow sheets) simplify processes and present key information and data.

• Flow sheets can range from simple block diagrams to detailed schematic drawings,
showing main and auxiliary equipment like pipes, valves, pumps, and bypass loops
Production of the antibiotic, bacitracin
 Discovery of Penicillin

• Penicillin was discovered by accident when Alexander Fleming left a dish of

staphylococcus bacteria uncovered.
• He found bacterial growth everywhere except in an area where Penicillium
notatum mould was growing.
• The mould produced a substance called penicillin, which stopped bacterial
growth.
• It wasn’t until World War II that Howard Florey and Ernst Chain isolated
penicillin for use.
• Fleming, Florey, and Chain won the Nobel Prize in 1945 for their discovery,
which changed medicine and led to life-saving antibiotics.
 Manufacture of Penicillin
• The exact composition of industrial penicillin production media is often
considered a trade secret or patented information.
• Common ingredients in the media include:
Corn steep liquor solids, lactose, glucose, calcium carbonate, KH2PO4,
edible oil, and a penicillin precursor.
• A typical medium, as outlined by Jackson (1958), includes:
1. pH maintained between 5.5 to 6.0 after sterilization.
2. A 4-5% lactose content with vigorous aeration and agitation in the
fermentor.
3. Lactose and precursor are included in the production media but not in
the inoculum media.
Penicillin Production
Penicillin Production Process
1. Filtration: The fermented culture is filtered using a rotary vacuum
filter to remove mycelium and other solid residues.
2. pH Adjustment: The pH of the clear broth is adjusted to 2-2.5
using phosphoric or sulfuric acid, converting penicillin to its
anionic form.
3. Extraction: The broth is extracted using a countercurrent solvent
extractor and an organic solvent (e.g., amyl acetate, butyl
acetate, or methyl isobutyl ketone).
4. Back Extraction: Penicillin is back extracted into an aqueous
medium by adding KOH or NaOH, forming the corresponding
potassium or sodium salt of penicillin.
5. Re-extraction: The aqueous solution (containing the penicillin
salt) is acidified and re-extracted using the solvent methyl isobutyl
ketone.
6. Back Extraction: The solvent extract is back-extracted with
aqueous NaOH several times until penicillin is fully extracted. This
combines the aqueous extractions, leading to crystallization of
penicillin as sodium or potassium penicillin.
7. Crystallization and Drying: The crystalline penicillin is washed,
dried under vacuum, and tested to meet the required specifications
for the final product.
 Ethanol Production
• Ethanol has been produced since ancient times
through the fermentation of sugars.
• Beverage ethanol and over half of industrial
ethanol are still produced this way.
• The raw material for fermentation is simple sugars.
• The enzyme zymase from yeast converts the sugars
into ethanol and carbon dioxide.
• The fermentation reaction can be represented as:

Glucose → Ethanol+ Carbon Dioxide

• The fermentation process is complex and impure yeast cultures
produce varying amounts of glycerine and organic acids.
• In beverage production (e.g., whiskey and brandy), these impurities
contribute to the flavor.
• Starches from plants like potatoes, corn, wheat, and others can also
be used for ethanol production, but they must first be converted into
simple sugars.
• The enzyme diastase, released by germinating barley during malting,
converts starches into sugars.
• Germination of barley called Malting, is the first step in brewing beer
from starchy plants such as corn and wheat.
• The ethanol concentration from fermentation typically ranges
from a few percent to about 14%.
• Above 14% ethanol, the zymase enzyme is destroyed, halting the
fermentation process.
• Distillation is used to concentrate ethanol, but the vapor from
aqueous ethanol contains 96% ethanol and 4% water.
• Pure ethanol cannot be obtained by distillation alone.
• Commercial ethanol typically contains 95% ethanol and 5% water.
• To produce absolute ethanol, dehydrating agents are used to
remove the remaining water.
 Manufacture of Lactic Acid
• Lactic acid was first discovered in 1780 as a sour
component of milk.
• It has since found applications in the food,
pharmaceutical, and cosmetic industries.
• Lactic acid bacteria are microorganisms that
degrade carbohydrates and produce lactic acid.
• Key genera of lactic acid bacteria include
Streptococcus, Lactobacillus, Lactococcus, and
Leuconostoc.
 Enzyme Production

• Sources of Industrial Enzymes can be

a. Plant
b. Animals
c. Microorganism
• Factors for Selecting Industrial Enzymes
a. specificity
b. pH
c. Thermo stability
d. activation or inhibition
e. availability and cost
Enzymes and their producer microorganisms
Industrial Enzyme Production:
• Most Enzymes are Produced through batch processes, with few via
continuous fermentation.

• Fermenter Capacity:

Bulk enzymes: Produced in fermenters up to 100m³.

Fine enzymes: Produced in smaller fermenters, usually a few hundred liters

or less.

• Fermenter Type: Most fermenters are stirred tank reactors that are Operated
under aseptic conditions and use low-cost, undefined complex media.
 Enzyme production processes

1. Search for a suitable organism to produce the enzyme.

2. Screen microorganisms to select based on enzyme properties like optimum pH, heat
resistance, and ability to secrete the target enzyme.
3. Determine the fermentation system and media, using inexpensive carbon and
nitrogen sources for maximum enzyme production per biomass.
4. Downstream processing involves separating, purifying, stabilizing, and preserving
the enzyme.
5. Enzyme stability must be tested as it affects the timing and processes used in
downstream processing.
6. The level of purification varies based on whether the enzyme is intracellular or
extracellular, and its intended use.
Generalized manufacture of enzymes
Processing of Biological material
 Filtration
• Filtration separates solid particles from a fluid-solid mixture by forcing
the fluid through a filter medium, which retains the solids.
• As the solids accumulate or collected, they form a filter cake, which
increases resistance to filtration.
• Filtration can be done using vacuum or positive pressure equipment,
and the pressure difference across the filter is called the filtration
pressure drop.
• Fermentation broths can be challenging to filter due to the small,
gelatinous cells and viscous, non-Newtonian behavior of the broth.
• Microbial filter cakes are often compressible, meaning the porosity
decreases as pressure increases, causing reduced filtration rates and
potential loss of product.
 Filter Aids
• Filter aids like diatomaceous earth are widely used in
the fermentation industry to improve filtration efficiency.

• Pre-coating: Filter aid is applied as a layer on the filter

medium to prevent blockages or "blinding" by solids,
which could otherwise clog the filter pores.

• Adding to the broth: Filter aid is also mixed into the

fermentation broth to increase the porosity of the cake
formed during filtration, making it easier to filter.
 Centrifugation
• Centrifugation separates materials based on their density using a force
greater than gravity.
• In bioprocessing, it is used to:
 Remove cells from fermentation broth
 Eliminate cell debris
 Collect precipitates
 Clarify fermentation media (e.g., molasses or wort for brewing).
• Centrifugation works best when:
 Particles are large
 Liquid viscosity is low
 There is a significant density difference between particles and the fluid.
• Effectiveness is enhanced by: A large centrifuge radius and High rotational
speed.
• In biological solid centrifugation, the particles (e.g., cells) are small, the
medium is viscous, and particle density is close to the fluid density.
 Cell Disruption

• Cell disruption is necessary for releasing products like

enzymes and recombinant proteins that remain in the
biomass.
• Mechanical methods include grinding with abrasives, high-
speed agitation, high pressure pumping and ultrasound.
• Non-mechanical methods such as osmotic shock, freezing
and thawing, enzymatic digestion of cell walls, and
treatment with solvents and detergents can also be applied.
• The high-pressure pump forces cells through an adjustable
valve with a restricted orifice, reaching pressures up to 550
atm.
• The homogeniser is widely used for cell disruption but may
block when working with highly filamentous organisms..
 Aqueous Two-Phase Liquid Extraction
• Liquid extraction is used to recover fermentation
products by transferring dissolved components into a
suitable solvent.
• Examples include:
Penicillin extraction using solvents like butyl acetate,
amyl acetate, or methyl isobutyl ketone.
Erythromycin isolation using pentyl or amyl acetate.
Steroid recovery, vitamin B12 purification, and alkaloid
isolation (e.g., morphine and codeine) from plant
materials.
• Aqueous two-phase extraction uses solvents that form
two distinct phases, providing conditions to separate
proteins, cell fragments, and organelles while preserving
their biological activity
 Chromatography
• Chromatography is a technique for separating mixtures
and isolating components based on differential
migration.
• Principle: Solute molecules move at different speeds
through a column due to varying affinities for the
stationary phase (resin particles).
• Separation occurs as solutes are retained (trapped)
differently and elute at different times, creating a
chromatogram (pattern of solute peaks).
• The mobile phase (fluid) carries solutes through the
column, while the stationary phase (material inside the
column) causes the separation.
• Chromatography is ideal for high-purity recovery of therapeutics and
pharmaceuticals due to its high resolution.
• Chromatographic methods available for purification of proteins,
peptides, amino acids, nucleic acids, alkaloids, vitamins, steroids and
many other biological materials include adsorption chromatography,
partition chromatography, ion-exchange chromatography, gel
chromatography and affinity chromatography.
• These methods differ in how they retard the movement of molecules
through the column.
• Distillation
Distillation is a process where a mixture of
liquids or vapors of two or more substance
is separated into its parts based on their
purity, using heat to boil and then cool to
condense them.
• Evaporation
 Crystallization Drying
• In process industries, products often contain • Drying refers to an operation in which
impurities that need to be removed for purity. the moisture of a substance is
• Crystallization is used to recover a solid product
from a solution, by making the solution
removed by thermal means the
supersaturated. removal of relatively small amount of
• Supersaturation is achieved by methods such as: water or other liquids from the solid
 Cooling the solution. material.
 Evaporation of solvent.
• Drying is one of the oldest methods
 Adiabatic cooling, which combines evaporation and
cooling without adding heat.
of preserving food.
• If the solubility of a substance increases with • Primitive societies practiced the
temperature, cooling the solution will make it drying of meat and fish in the sun long
supersaturated and cause crystallization. before recorded history.
• Sometimes, a solid mixture is purified by dissolving
the desired component in a solvent and then
crystallizing it.
• The solubility of the substance is key in the
crystallization process