UPSTREAM PROCESSING
Primary processing or pre-manufacturing: All the factors and processes leading to fermentation
o Obtaining a suitable microorganism
o Strain improvement to enhance productivity and yield
o Maintenance of strain purity
o Preparation of a suitable inoculum and the continuing development of selected strains to increase
the economic efficiency of the process.
o Selection of suitable cost-effective carbon and energy source along with other essential nutrients.
o Media optimization
o Cheap substrates: cane molasses, beet molasses, cereal grains, starch, glucose, sucrose and lactose
as carbon sources, ammonium salts, urea, nitrates, corn, soya bean meal, slaughter-house waste and
fermentation residues as nitrogen sources
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Criteria for the selection of a medium at industrial scale
o Produce the maximum yield of product or biomass (per gram of substrate used)
o Maximum biomass formation rate, maximum specific growth rate
o Minimum yield of undesired products
o Consistent quality and be readily available throughout year
o Minimal problems during media making and sterilization
o Minimal problems in aeration & agitation, extraction, purification and waste treatment.
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Calculation of Kinetic Parameters
Kinetic Parameter Method of Calculation
Maximum biomass formation rate
Xmax (g L-1 d-1)
Maximum specific growth rate
µmax (d-1)
Doubling time
td (day)
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Process from the laboratory to the pilot scale and industrial scale
o The scale-up process performed at three scales.
1. LAB SCALE (100mL – 1L): Conditions are optimized (Temperature, pH, media and gas requirements)
e.g. Temperature ranging from 250C-450C is used in synthesis of lactic acid by Lactobacillus bulgaricus. The one
with better result is selected for future use. Likewise, pH and media are also optimized.
2. PILOT SCALE (1 – 12L): A pilot scale is a small-scale preliminary study conducted in order to
evaluate feasibility, time, cost, adverse events, and effect size (statistical variability). If the results
are satisfactory the process is practiced on industrial scale, otherwise not.
3. INDUSTRIAL SCALE (100 – 10,000 L): Production of desired product in higher amount (equipment
design, technology and time)
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Focus areas of research of upstream processing
o Studying the physiology of microorganisms under extreme conditions
o Development of microbioreactors as a screening tool to shorten the process development time
o Development of low-cost fermenters, alternative novel reactor concepts, advanced simulation tools
for modelling fermentation processes on different scales
o Combination of energy production and bioprocesses (utilization of waste for biogas plants)
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DOWNSTREAM PROCESSING
o Series of steps used to purify and isolate a product created through bioprocessing
o When they reach the desired density, they are harvested and moved to the downstream section
o Recovery and purification of biosynthetic products
o Recycling and the proper treatment and disposal of waste
o Examples of industrial products: antibiotics, hormones (insulin and human growth hormone),
antibodies and vaccines; antibodies and enzymes used in diagnostics; industrial enzymes; natural
fragrance and flavor compounds.
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Steps of Downstream Processing
1. Disruption of the Cell
i. PHYSICAL METHOD
o Mechanical Disruption: rotating, blender, mixer etc.
o Mortar and Pestle:
o Sonication: pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores and finely
chopped tissue. Sonication is best suited for volumes <100 ml.
ii. CHEMICAL METHOD
o Organic solvents like methanol, ethanol and acetone for this purpose. Surfactants like soap, detergents etc.
are also used.
iii. BIOLOGICAL METHOD
o Enzymes --- disrupting cells to get intracellular products (e.g Lysozyme and cellulase for gram negative
bacterial lysis & glycoprotein, mannanase, glucanase for yeast/fungi)
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2. Removal of Insolubles
o FILTRATION: separation of solids from fluids by interposing a medium through which only the fluid can pass.
➢ Dead End Filtration : permeate flux drops fast to dead end filtration.
➢ Cross Flow Filtration : filter cake is completely washed away during this process and permeate flux does not drop as fast
when compared to dead end filtration e.g. extraction of soluble antibiotics from fermentation liquors.
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o CENTRIFUGATION: use of the centrifugal force for the sedimentation of mixtures with a centrifuge
➢ Decantation: separation of mixtures, achieved by carefully pouring a solution from a container in order to
leave the precipitate in the bottom of the container
➢ Pipetting: supernatant part of the mixture is carefully removed with the help of a pipette
o FLOCCULATION: process of contact and adhesion whereby the dispersed particles form larger-size
clusters. It is a process wherein colloids come out of suspension in the form of flakes by the addition
of a clarifying agent.
➢ The commonly used flocculating agents are aluminum sulphate, calcium hydride, sodium aluminate and
sodium silicate
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3. Product Isolation
o SOLVENT EXTRACTION: method to separate compounds based on their relative
solubilities in two different unmixable liquids, usually water and an organic
solvent like n-hexane (used in production of fine organic compounds, the
processing of perfumes, the production of vegetable oils and biodiesel etc.).
o ULTRAFILTRATION: membrane filtration in which hydrostatic pressure forces a
liquid against a semi permeable membrane. Pore size used is 0.1-0.001 um.
Suspended solids and solutes of high molecular weight are retained and is called
retentate, while water and low molecular weight solutes pass through the
membrane and is called permeate. Both dead-end and cross flow methods can
be used for ultrafiltration.
o PRECIPITATION: formation of a solid in a solution or inside another solid during a
chemical reaction or by diffusion in a solid. Precipitation can also be obtained by
making a super saturated solution through heating. The heat is then removed
and the excess amount of solutes gets settle in the bottom resulting in
precipitation.
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4. Product Purification
Recovery of the product in a highly purified state. The most expensive
step of downstream processing
o AFFINITY CHROMATOGRAPHY: method of separating biochemical
mixtures based on properties such as antigen and antibody or enzyme
and substrate. The target molecule is trapped on a solid or stationary
phase (gel matrix, agarose) while other molecules in solution will not
become trapped as they do not possess this property.
o REVERSE PHASE CHROMATOGRAPHY: uses a non-polar stationary
phase and polar compounds are removed while non-polar compounds
are retained, also called "reversed phase" or “hydrophobic
chromatography”. It is similar to ion exchange chromatography.
Hydrophilic proteins will flow through the column, while lipophilic
proteins will remain in the column.
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Some other Chromatographic Techniques
o Adsorption: mixture of gas or liquid
gets separated on the adsorbent bed
that adsorbs different compounds at
different rates
o Ion Exchange: separation of charged
biomolecules (proteins, peptides,
amino acids, or nucleotides)
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o Gel Filtration/Size Exclusion: molecules in
solution are separated by their size, and in
some cases molecular weight. e.g proteins
and industrial polymers etc.
o Partition Chromatography: separating and
identifying mixtures that are colored,
especially pigments. The least soluble
particles do not move further on the filter
paper, but the most soluble particles move
further
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o HPLC: High Performance Liquid Chromatography is a
process of separating components in a liquid mixture.
A liquid sample is injected into a stream of solvent
(mobile phase) flowing through a column packed with
a separation medium (stationary phase).
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5. Product Drying & Polishing
The product is brought to a commercial form for consumer usage.
o Vacuum Drying: using vacuum pump
o Spray Drying: forming a dry powder from a liquid or slurry by rapidly
drying with a hot gas
o Freeze Drying: completely frozen sample is placed under a vacuum in
order to remove water or other solvents from the sample, allowing
the ice to change directly from a solid to a vapor without passing
through a liquid phase
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o CRYSTALLIZATION: If a saturated hot solution is allowed to cool,
the solute is no longer soluble in the solvent and forms crystals of
pure compound. Impurities are excluded from the growing
crystals and the pure solid crystals can be separated from the
dissolved impurities by filtration.
o USE OF ANTI-SOLVENT: it reduces the solubility of the solute in
the solvent, causing it to precipitate out. This process is useful in
pharmaceuticals, separation of chemicals and recovery of
valuable materials from waste streams
o DESICCATION: absorbs water content leaving pure product &
increases the shelf life. Drying agent or desiccant (hygroscopic
substance --- dry silica gel or anhydrous sodium hydroxide)
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6. Packing of the Product
Following precaution should be practiced while packing a product.
o Protection from mechanical shock, vibration, electrostatic discharge, compression, temperature, etc.
o Desiccants or Oxygen absorbers --- to help extend shelf life
o Liquids, powders, and granular materials need containment
o Packages and labels should depict how to use, transport, recycle, or dispose of the package or product
o The packaging and labels could be used by marketers to encourage potential buyers to purchase the
product
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7. Advertisement
o Communication used to convince consumers to buy the product
o Traditional media
o Newspaper
o Magazines
o Television commercial
o Radio advertisement
o Social media
o Websites etc.
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Focus areas of research of downstream processing
o Computer-aided design systems
o Cost-effective toolbox of generic technique
o Minimizing energy and water input as well as waste streams
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A summary of major
steps in upstream and
downstream processing
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