About upstream and down stream processing in fermentation

1. REACCREDITED WITH A+ GRADE WITH A CGPA OF 3.9 IN THE THIRD CYCLE OF NAAC
AFFILIATED TO MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI
Post Graduate & Research Centre – Department of Microbiology
(GOVERNMENT AIDED)
UNIT-1 :TYPESOF PROCESSING- UPSTREAMANDOWNSTREAM
SUBMITTED BY : SUBMITTED TO :
K.JANAKI SUJITHA GUIDE: DR. S.VISWANATHAN, PH.D,
REG NO – 20211232516111 ASSISTANT PROFESSOR AND HEAD,
II MSC MICROBIOLOGY SPKC, ALWARKURICHI.

2. Introduction
A bioprocessing is a particular procedure that utilizes complete
living cells or their segment.
Industrial fermentation involves two process
• Upstream processing
• Downstream processing

4. Upstream
processing
 It is an entire process from which the initial stage of cell
isolation and cultivation until the final harvest.
 Includes formulation of the fermentation medium,
sterilization of air, fermentation medium, inoculum
preparation and inoculation of the medium.
 selection of a microbial strain characterized by the ability to
synthesize a specific product having the desired commercial
value.This strain then is subjected to improvement
protocols to maximize the ability of the strain to synthesize
economical amounts of the product.

6. Upstream processing normally deals with three important points.
1.The first relates to fermentation media, especially the selection of suitable cost effective carbon
and energy sources, along with other essential nutrients. The media optimization is a vital aspect
of process development to ensure maximization of yield and profit.
2.The second aspect involves aspects associated with the producer microorganism. They include
the strategy for initially obtaining a suitable microorganism, industrial stain improvement to
enhance productivity and yield, maintenance of strain purity, preparation of a suitable inoculums
and continuing development of selected strains to increase the economic efficiency of the
process.
3.The third component relates to the fermentation which is usually performed under rigorously
controlled conditions developed to optimize the growth of the organism or the production of a
target microbial product.

7. The first step in developing a producer strain is the isolation of concerned microorganisms from their
natural habitats. The success of an industrial fermentation process chiefly depends on the microbial strain
used. The strain should have the following characteristics:
1. It should be able to grow on relatively cheaper substrates.
2. It should grow well in an ambient temperature preferably at 30-40°C. This reduces the cooling costs.
3. It should yield high quantity of the end product.
4. It should possess minimum reaction time with the equipment used in a fermentation process.
5. It should possess stable biochemical characteristics.
6. It should yield only the desired substance without producing undesirable substances.
7. It should possess optimum growth rate so that it can be easily cultivated on a large scale.
ISOLATION OF MICROORGANISMS

8. The economics of a fermentation process largely depends upon the type of
microorganism used. If fermentation process is to yield a product at a cheaper price
the chosen microorganism should give the desired product in a predictable and
economically adequate quantity. The microorganism with a desired characters is
generally isolated from natural substrates like soil etc. Such an organism is generally
called as a producer strain.
Screening is divided into two broad categories.
They are:
1. Primary screening
2. Secondary screening.
SCREENING

9. 1. Primary Screening of Microorganisms:
Primary screening may be defined as detection and isolation of the desired
microorganism based on its qualitative ability to produce the desired product like antibiotic or
amino acid or an enzyme etc. In this process desired microorganism is generally isolated from a
natural environment like soil, which contains several different species. Sometimes the desired
microorganism has to be isolated from a large population of different species of
microorganisms.
2. Secondary Screening of Microorganisms:
Secondary screening can be used for determining the quantitative and
qualitative information about the cells, desired organisms or strains. In the case of qualitative
screening, it can help to determine the yield potential and antibiotics of a product. However, in
the case of quantitative screening, it can help to determine the product’s quantity that is
obtained by various types of fermentation media. secondary screening also helps to study and
determine the biological, chemical and physical properties of a selected product.

10. Strain improvement is the process by which we can improve the strain for novel
quantities. Hence it is the process of altering the property of an organism.
Strain improvement is based on three approaches:
1. Mutant selection: In the early stages, selection of spontaneous mutants may be helpful but induced
mutations are the most common source of improvements. Many mutations bring about marked changes
in the biochemical character of practical interest; these are called major mutations.
2. Recombination: It may be defined as formation of new gene combinations among those present in
different strains. Recombination may be used based on a) protoplast fusion and b) sexual reproduction.
3. Recombinant DNA technology: Involves the isolation and cloning genes of interest for the production of
the necessary gene constructs using appropriate enzymes were made followed by the transfer and
expression of these genes into an appropriate host organism. The technique used to achieve two broad
objectives:
a) Production of recombinant proteins
b) Modification of the organisms metabolic pattern for production of new, modified or
more quantity of metabolites.
STRAIN IMPROVEMENT

11. Isolation of strains from natural sources
↓
Primary and secondary screening for
bioactive strains
↓
Select different types of bioactive strains
↓
Mutagen treatment (UV, X-rays, 60CO, NTG
etc.).
↓
Check the activity
↓
Select higher yielding strains
↓
Large-scale testing
↓
Highest yielding strain
↓

12. MEDIA FORMULATION
Growth medium must have essential nutrients for microbial growth for successful fermentation
process.There are two kinds of media
1. Inoculum media: For growth and development. It plays a crucial role in the
production of desired product. Usually inoculum is added 5 - 10 % .
2. Production media:Addition of precursor.
A medium which is used for large scale fermentation should have the following characteristics:
It should cheap and easily available.
It should maximize the growth of the microorganisms, productivity and the rate of
formation of the desired product.
It should minimize the formation of undesired products.

13. STERILIZATION
Sterilization is essential for preventing the contamination with any
undesired microorganisms. This may be achieved by
1. Heating
2. Irradiation
3. Chemicals
4. Filtration
In general, the industrial fermentations are carried out under vigorous and
continuous aeration. For an effective fermentation, the air should be completely sterile, and
free from all microorganisms and suspended particles. There is a wide variation in the
quantity of suspended particles and microbes in the atmospheric outdoor air.
Air is usually sterilized by membrane filtration while the medium is usually heat
sterilized. Aerobic fermentation requires a very high rate of air supply . Air contains both
fungal spores and bacteria, which are removed either by depth filter or screen filter.

14. Downstream
processing
 The extraction and purification of a biotechnological product
from fermentation is referred to as Downstream processing
(DSP) or product recovery.
 DSP is as complex and important as fermentation process. It
often requires expertise and technical skills.
 The methodology adopted for downstream processing depends
on the nature of the end product, its concentration stability and
the degree of purification required.

15. The desired products for isolation by DSP are most frequently metabolites which can
present as follows.
1. Intracellular metabolites:These products are located within the cells e.g. vitamins,
enzymes.
2. Extracellular metabolites:They are the products outside the cells e.g. most
antibiotics (penicillin, streptomycin), amino acids, alcohol.
3. Both intracellular and extracellular: e.g. vitamin B12, flavomycin.
 Sometimes, the microorganism itself be the desired end product e.g.
single cell protein.

16. STAGES IN
DOWNSTREAM
PROCESSING
SEPARATION OF PARTICLES
 CELL DISRUPTION
 EXTRACTION
 CONCENTRATION
 PURIFICATION
 DRYING

18. SEPARATIONOF PARTICLES
 Primary recovery operation.
 It is the first step pf DSP and usually involve the separation of solid substances,
from the liquid media.
 Separate whole cells from culture broth, removal of cell debris, collection etc.
Separation of particles can be done by,
1. Filtration
2. Centrifugation
3. Flocculation
4.Floatation

19. Filtration
Filtration is the most commonly used technique for separating the biomass and culture filtrate. The
efficiency of filtration depends on many factors the size of the organism, presence of other organisms,
viscosity of the medium, and temperature.
Several filters such as depth filters, absolute filters, rotary drum vacuum filters and membrane filters
are in use.
Depth filters : They are composed of a filamentous matrix such as glass wool, asbestos or filter paper.
The particles are trapped within the matrix and the fluid passes out. Filamentous fungi can be removed
by using depth filters.
Absolute filters : These filters are with specific pore sizes that are smaller than the particles to be
removed. Bacteria from culture medium can be removed by absolute filters.

20. Rotary drum vacuum filters : These filters are frequently used for separation of broth containing 10-
40% solids (by volume) and particles in the size of 0.5-10um. Rotary drum vacuum filters have been
successfully used for filtration of yeast cells and filamentous fungi. The equipment is simple with low
power consumption and is easy to operate. The filtration unit consists of a rotating drum partially
immersed in a tank of broth. As the drum rotates, it picks up the biomass which gets deposited as a
cake on the drum surface. This filter cake can be easily removed.

21. Membrane filters : In this type of filtration, membranes with specific pore sizes can be used. However,
clogging of filters is a major limitation. There are two types of membrane filtrations—static filtration
and cross-flow filtration. In cross-flow filtration, the culture broth is pumped in a crosswise fashion
across the membrane. This reduces the clogging process and hence better than the static filtration.

22. There are 3 major types of filtrations based on the particle
sizes and other characters. These are microfiltration, ultrafiltration and
reverse osmosis.

23. It can be used for bacteria, usually protein precipitates. The technique of centrifugation is based on the
principle of density differences between the particles to be separated and the medium.
Tubular bowl centrifuge : This is a simple and a small centrifuge, commonly used in pilot plants. Tubular
bowl centrifuge can be operated at a high centrifugal speed, and can be run in both batch or continuous
mode. The solids are removed manually.
Disc centrifuge : It consists of several discs that separate the bowl into settling zones. The feed/slurry is fed
through a central tube. The clarified fluid moves upwards while the solids settle at the lower surface.
Multi-chamber centrifuge : This is basically a modification of tubular bowl type of centrifuge. It consists of
several chambers connected in such a way that the feed flows in a zigzag fashion. There is a variation in the
centrifugal force in different chambers. The force is much higher in the periphery chambers, as a result
smallest particles settle down in the outermost chamber.
Centrifugation

24. Scroll centrifuge or decanter : It is composed of a rotating horizontal bowl tapered at one end. The
decanter is generally used to concentrate fluids with high solid concentration (biomass content 5-80%).
The solids are deposited on the wall of the bowl which can be scrapped and removed from the narrow
end.

25. Flotation:
When a gas is introduced into the liquid broth, it forms bubbles. The cells and other solid
particles get adsorbed on gas bubbles. These bubbles rise to the foam layer which can be collected and
removed. The presence of certain substances, referred to as collector substances, facilitates stable foam
formation e.g., long chain fatty acids, amines.
Flocculation:
In flocculation, the cells (or cell debris) form large aggregates to settle down for easy removal. The
process of flocculation depends on the nature of cells and the ionic constituents of the medium. Addition
of flocculating agents (inorganic salt, organic polyelectrolyte, mineral hydrocolloid) is often necessary to
achieve appropriate flocculation.

27. CELL DISRUPTION
1. Physical methods of cell disruption
Certain physical procedures can disturb bacteria or cells to liberate intracellular products.
i. Ultra sonication
•In the laboratory, ultrasonic disintegration is widely utilized. However, due to its high cost, it is not
appropriate for industrial application on a broad scale.
ii. Osmotic shock
•This technique involves suspending cells (devoid of growth media) in 20% buffered sucrose.
•The cells are subsequently transferred to roughly 4°C water.
•Gram-negative bacteria utilize osmotic shock to release hydrolytic enzymes and binding proteins
iii. Heat shock (thermolysis)
•By subjecting cells to heat, their destruction is relatively simple and inexpensive.
•However, this method can only be applied to a small number of heat-stable intracellular substances.
iv. High pressure homogenization
•This method involves forcing a high-pressure cell suspension through a very narrow orifice to atmospheric
pressure.
•This abrupt release of high pressure generates a liquid shear capable of rupturing the cells.

28. v. Grinding with glass beads
•In a reaction vessel, cells combined with glass beads are treated to a very high speed.
•As the beads press the cells against the vessel wall, the cells rupture. Size and number of the glass beads,
concentration and age of the cells, temperature and agitator speed all influence cell lysis.
•Under optimal conditions, approximately 80% of the cells may be destroyed..
•It has a cylindrical body with an inlet, an outlet, and a motor-driven Centre shaft. Attached to this shaft are radial
agitators.
•The cylinder incorporates glass beads. The cell suspension is introduced by the inlet, while the disrupted cells exit
via the outlet.
•During operation, the body of the cell disruptor is kept at a cold temperature.

29. B. Chemical methods of cell disruption
i. Alkalies
•Some bacterial proteins have been extracted using an alkaline solution. However, the alkali stability of the desired
product is critical to the success of this procedure; for instance, recombinant growth hormone can be effectively
extracted from E. coli by treating the bacteria with sodium hydroxide at a pH of 11.
ii. Organic solvents
•Several water-miscible organic solvents, such as methanol, ethanol, isopropanol, and butanol, can be employed to
disturb the cells.
•These compounds are combustible, necessitating the use of specialized fire safety equipment. Toluene, an organic
solvent, is often utilized.
•Toluene is thought to breakdown membrane phospholipids and form membrane holes for intracellular content
release.
iii. Detergents
•Ionic detergents, such as cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl sulphate, have the
ability to denature membrane proteins and lyse cells.
•Non-ionic detergents, such as Triton X-100 or Tween, are also utilized to some extent, despite being less reactive
than ionic detergents.
•The difficulty with using detergents is that they interfere with purifying processes, namely salt precipitation.
•Purification by ultrafiltration or ion-exchange chromatography can circumvent this constraint.

30. C. Enzymatic methods of cell disruption
•Cell lysis happens under mild conditions and in a selective manner when cells are disrupted by enzymatic
techniques.
•This is really beneficial for product recovery. Lysozyme is the most widely utilized and commercially available
enzyme (produced from hen egg white).
•It hydrolyzes β-1, 4-glycosidic linkages in bacterial cell wall mucopeptides. Gram-positive bacteria (those with a high
concentration of cell wall mucopeptides) are more vulnerable to lysozyme’s activity.
•In conjunction with EDTA, lysozyme can kill Gram-negative bacteria by destroying their cells. As lysozyme digests
the cell wall, the osmotic effects rupture the periplasmic membrane, releasing the intracellular contents.
•Additionally, but less frequently, additional enzymes are utilized for cell disruption. In conjunction with proteases,
glucanase and mannanase are used to lyse yeast cell walls.
D. Combination of methods
•A combination of physical, chemical, and enzymatic processes are utilized to increase the efficiency of cell
disintegration in a cost-effective manner.

31. •Typically, 80-98% of the filtrate that is free of suspended particles (cells, cell debris, etc.) is water.
•The desired substance is a minor component. Water must be extracted to get the desired
product concentration.
•These are the most typical ways for concentrating biological products:
• evaporation.
• liquid-liquid extraction.
• membrane filtration.
• precipitation.
• adsorption.
•Actual process depends on the nature of the desired product (quality and quantity to be
preserved to the greatest extent possible) and the cost element.
CONCENTRATION

32. Evaporation:
Water in the broth filtrate can be removed by a simple evaporation process. The evaporators, in
general, have a heating device for supply of steam, and unit for the separation of concentrated
product and vapour, a condenser for condensing vapour, accessories and control equipment.
Some of the important types of evaporators in common used are
Plate evaporators
Falling film evaporators
Forced film evaporators
Centrifugal forced film evaporators

33. Liquid-Liquid Extraction:
The concentration of biological products can be achieved by transferring the desired product
(solute) from one liquid phase to another liquid phase, a phenomenon referred to as liquid-liquid extraction.
Besides concentration, this technique is also useful for partial purification of a product.
Membrane Filtration:
Membrane filtration has become a common separation technique in industrial biotechnology.
It can be conveniently used for the separation of biomolecules and particles, and for the concentration of
fluids. The membrane filtration technique basically involves the use of a semipermeable membrane that
selectively retains the particles/molecules that are bigger than the pore size while the smaller molecules
pass through the membrane pores.
Membranes used in filtration are made up of polymeric materials such as polyether sulfone and
polyvinyl di-fluoride.

34. Precipitation:
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides. Further, precipitation technique can also be
employed for the removal of certain unwanted byproducts e.g. nucleic acids, pigments.
Adsorption:
The biological products of fermentation can be concentrated by using solid adsorbent particles. In
the early days, activated charcoal was used as the adsorbent material. In recent years, cellulose-based
adsorbents are employed for protein concentration.
And for concentration of low molecular weight compounds (vitamins, antibiotics, peptides)
polystyrene, methacrylate and acrylate based matrices are used. The process of adsorption can be carried
out by making a bed of adsorbent column and passing the culture broth through it. The desired product,
held by the adsorbent, can be eluted.

35. The journey of a drug to market begins with a molecule which is showing potential to
treat, cure or prevent disease. There are many steps along this journey; one of the most
important steps is the need to separate and purify the molecule from a complex feed
stream.
It aims at recovery of the product in a highly purified state.
Purification is achieved by the following procedures:
•Crystallization: This is used for the low molecular mass compound like antibiotics.
•Chromatographic methods: Chromatography is the
answer to getting high-purity products, especially
with proteins. Chromatography is still the major tool
on all levels of the DSP from the first capture to the
final polishing step.
PURIFICATION

36. There are many methods of executing the chromatography step. These are the most used
methods:
•Ion exchange chromatography (IEX) – is the most popular method for the purification of
proteins and other charged molecules. Positively charged molecules are attracted to negatively
charged solid molecules.
•Hydrophobic interaction chromatography (HIC) – separates protein molecules using the
properties of hydrophobicity. In this method, proteins containing both hydrophilic and
hydrophobic regions are applied to a HIC column under high salt buffer conditions. Due to these
salt buffer conditions; proteins of the hydrophobic area can precipitate out of the solution.
•Affinity chromatography (AC) – can be used to purify and concentrate a substance from a
mixture to a buffering solution, or it can be used to reduce the number of unwanted substances
and to identify the biological compound of a particular substance.
•Gel filtration chromatography (GFC) – separates proteins and peptides based on size.

37. Formulation broadly refers to the maintenance of activity and stability of a
biotechnological products during storage and distribution. The formulation of low
molecular weight products (solvents, organic acids) can be achieved by
concentrating them with removal of most of the water. For certain small molecules,
(antibiotics, citric acid), formulation can be done by crystallization by adding salts.
Proteins are highly susceptible for loss of biological activity; hence their formulation
requires special care. Certain stabilizing additives are added to prolong the shelf life
of protein. The stabilizers of protein formulation include sugars (sucrose, lactose),
salts (sodium chloride, ammonium sulfate), polymers (polyethylene glycol) and
polyhydric alcohols (glycerol). Proteins may be formulated in the form of solutions,
suspensions or dry powders.
FORMULATION

38. Drying is an essential component of product formulation. It basically involves the transfer of
heat to a wet product for removal of moisture. Most of the biological products of fermentation
are sensitive to heat, and therefore require gentle drying methods. Based on the method of
heat transfer, drying devices may be categorized as contact, convection, radiation dryers.
Freeze-drying:
Freeze-drying or lyophilization is the most preferred method for drying and formulation of a
wide-range of products—pharmaceuticals, foodstuffs, diagnostics, bacteria, viruses. This is
mainly because freeze-drying usually does not cause loss of biological activity of the desired
product.
Lyophilization is based on the principle of sublimation of a liquid from a frozen state. In the
actual technique, the liquid containing the product is frozen and then dried in a freeze-dryer
under vacuum. The vacuum can now be released and the product containing vials can be
sealed e.g., penicillin can be freeze dried directly in ampules.
DRYING

39. Spray drying:
Spray drying is used for drying large volumes of liquids. In spray drying, small droplets of liquid
containing the product are passed through a nozzle directing it over a stream of hot gas. The
water evaporates and the solid particles are left behind.

40. REFERENCES
AN INTRODUCTION TO INDUSTRIAL MICROBIOLOGY by Dr. P.K. SIVAKUMAR, Dr. M.M. JOE,
Dr. K. SUKESH
INDUSTRIAL MICROBIOLOGY BY L.E. CASIDA
http://ecoursesonline.iasri.res.in/mod/page/view.php?id=5158
https://www.biotechnologynotes.com/microorganisms/screening/screening-of-microorganisms-
primary-and-secondary-techniques-industrial-biotechnology/13697

41. Skills gained by seminar
 Gained subject knowledge
 Increased confidence level
 Gained communication skills
 Searching ability
 Time management