Introduction to bioprocess Engineering

1. Introduction to Bioprocess Engineering
Harinath Reddy A
Department of Life sciences
biohari14@gmail.com).
Bangalore

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3.  U. Sathyanarayana. Biotechnology. Books and Allied
(P) Ltd, Kolkota: 2008.
 P. F. Stanbury. A. Whitaker and S.J. Hall. Principles of
Fermentation Technology. 2nd ed, Edinburgh:
Butterworth Heinemann Press, 2003.
 M. D. Pauline. Bioprocess Engineering Principles. 2nd
ed, London: Academic Press, 2000

4.  A bioprocess is a specific process that uses complete living cells or
their components (e.g., bacteria, enzymes, chloroplasts) to obtain
desired products.
 Bioprocess Engineering is a specialization of Biological
Engineering or Biotechnology.
 It deals with the shape or style and development of equipment
and processes for the creation of products such as
pharmaceuticals, nutrients, food, feed, chemicals, and many
polymers.
Definition of a Bioprocess:

5. Over view of bioprocesses:
 When our early ancestors made alcoholic liquid beverages, they
used a bioprocess method the combination of live yeast cells and
nutrients (like cereal grains) create a fermentation system.
 In fermentation system organisms used the nutrients for their own
growth and produced several by-products (as alcohol and
carbon dioxide gas) that helped to produce the liquid beverage.

6.  It also deals with studying different biotechnological processes for
large scale manufacturing of biological product and increasing
the quality of end product.
 Bioprocess Engineers to “Bring Engineering to Life” through the
conversion of biological materials into other forms needed by
mankind.

7.  In the first half of the 20th century, the first biggest scale fermentation
processes, namely penicillin and citric acid, were realized.
 The process of recombinant DNA technology or genetic engineering
then lead to a substantial increase in the number of bioprocesses.
 Test example is insulin, the first product produced with recombinant
DNA technology.

8.  The bioprocessing has highly visible significance in the area of
human health care, with product such as human insulin,
interferons, erythropoietin, and monoclonal antibody, Hepatitis B
vaccine are bioprocess products.
 Thus the field of bioprocessing has become a very significant
part of biotechnology.

9. Current bioprocess technology and products:
 Biopharmaceuticals: Mainly includes therapeutic proteins, antibiotics,
antibodies, polysaccharides, hormones, vaccines, and diagnostic agents.
 Industrial chemicals and Energy: Mainly includes alcohols, organic acids,
fuels and other chemicals.
 Food Industries: Mainly includes alcoholic beverages and dairy products,
several novel foods (starch, amino acids and vitamins) and food additives.

10.  Agriculture: Mainly includes veterinary vaccine and antibiotics,
use of nitrogen fixing bacteria, production of transgenic plants and
genetic modification of foods.
 Environmental-management: Mainly includes municipal
wastewater treatment plants to provide clean and safe drinking
water.
 The recent attention to the environment has focused some
bioprocessing technology on the transformation of xenobiotics to
biodegradable forms.

11. Major products of bioprocessing:
Fermentation products Organisms used
Organics:
Alcohol
Acetone/butanol
Yeast
Clostridium acetobutylicum
Organic acids:
Citric acid
Gluconic acid
Lactic acid
Aspergillus niger
Aspergillus niger
Lactobacillus sp.,
Amino acids:
L-glutamic acid
L-Arginine
Corneybacterium glutamicum
Brevibacterium flavum
Antibiotics:
Penicillin
Tetracyclines
Penicillium chrysogenum
Streptomyces aureofaciens

12. Fermentation products Microorganisms used
Enzymes:
Proteases
Alpha amylases
Pectinases
Bacillus spp.
Bacillus Stearothermophilus,
Bacillus licheniformis
Aspergillus niger.
Vitamins:
Vit B12 Pseudomonas denitrificans
Therapeutic proteins:
Insulin
Growth hormones
Interferon alpha 2
Erythropoietin
Recombinant E. coli cells
Recombinant E. coli cells
Recombinant E. coli cells
Recombinant mammalian cell
culture

13. Various components of Bioprocess:
The entire bioprocess can be divided in three stages.
 Stage I : Upstream processing
 Stage II: Fermentation
 Stage III: Downstream processing

14. Stage I : Upstream processing:
 Stage I : Upstream processing which involves preparation of
liquid medium.
 Separation of particulate and inhibitory chemicals from the
medium, sterilization, air purification etc.
 Upstream processes include selection of a microbial strain
characterized by the ability to synthesize a specific product
having the desired commercial value.


15.  Usually, waste products from other industrial agriculture processes,
such as molasses, plant biomass, rice husks and corn steep after
modifying with the incorporation of additional nutrients, are used as
the substrate for many industrial fermentations.

16. 
Sterilisation is essential for preventing the contamination with any
undesired microorganisms.
 Air is sterilised by membrane filtration while the medium is
usually heat sterilised.
 Any nutrient component which is heat labile is filter-sterilised and
later added to the sterilised medium.
 The fermenter may be sterilised together with the medium or
separately.

17.  Fermentation process itself which usually is carried out in large
tanks known as fermenters or bioreactors.
 In addition to mechanical parts which provide proper conditions
inside the tank such as aeration, cooling, agitation, etc.,
 The tank is usually also equipped with complex sets of monitors
and control devices in order to run the microbial growth and
product synthesis under optimized conditions.


18.  The processing of the fermentation reactions inside the fermenter
can be done using many modifications of engineering technologies.
 One of the most commonly used fermenter types is the stirred-tank
fermenter which utilizes mechanical agitation principles, mainly
using radial-flow impellers, during the fermentation process.

19. Stage II: Fermentation:
 The fermentation metaboilc process involves the propagation of
the microorganism and production of the desired product.
 The fermentation process can be categorised depending on various
parameters.
 It can be either aerobic fermentation, carried out in the presence
of oxygen or anaerobic fermentation, carried out in the absence of
oxygen.
 Many industrial fermentation are carried out under aerobic
conditions where a few processes such as ethanol production by
yeast require strictly anaerobic environments.

20.  The fermentation process can also be divided into three basic
systems, namely depending on the feeding strategy of the culture
and the medium into the fermenter.
 Batch fermentation
 Continuous fermentation
 Fed-batch fermentation.


21.  The process can also be categorised as solid state fermentation
(SSF) or submerged fermentation (SmF), depending on
the amount of free water in the medium.
 In a Solid state fermentation, the medium contains no free flowing
water. The organisms are grown in a solid substrate.
 Generally used for the production of amylase and protease by
Aspergillus oryzae.
 Submerged fermentation is in which microorganisms grow in a liquid
medium where free water is abundant. This is the method of choice for
many industrial operations.
 Generally used for the production of recombinant vaccines and
probiotics production.

22. Step 3: Downstream Processing:
 Downstream processing which involves separation of cells from
the fermentation broth, purification and concentration of
desired product and waste disposal or recycle.


23. The different stages in downstream processing:
(1) Solid-Liquid Separation
(2) Release of Intracellular Products
(3) Purification by Chromatography
(4) Formulation.
Step 3: Downstream Processing:

24. Stage 1. Solid-Liquid Separation:
 The first step in product recovery is the separation of whole cells
(cell biomass) and other insoluble ingredients from the culture
broth.
 Several methods are in use for solid-liquid separation.
 Filtration and Centrifugation

25. Filtration:
 Filtration is the most commonly used technique for separating the
biomass and fermented broth or culture broth.
 Several filters such as Depth filters, Membrane filters and
Rotary drum vacuum filters are in use.
t

26. Centrifugation:
 The technique of centrifugation is based on the principle of
density differences between the particles to be separated and the
medium.
 In recent years, continuous flow industrial centrifuges have been
developed.
 .
A. Tubular bowl centrifuge
B. Disc centrifuge

27. Stage 2: Release of Intracellular Products:
 Several biotechnological products (protiens, vitamins, enzymes)
which are located within the cells.
 The microorganisms or other cells can be disintegrated or
disrupted by physical, chemical or enzymatic methods.

28. Stage 3: Purification by Chromatography:
 The biological products of fermentation (proteins, pharmaceuticals,
diagnostic compounds and research materials) are very effectively
purified by chromatography.
 Chromatography usually consists of a stationary phase and
mobile phase.

29. The different types of chromatography techniques used for
separation of desired product(mainly proteins):
Chromatography Principle
Ion exchange chromatography Net charge
Gel filtration chromatography Size and shape
Affinity chromatography Net charge

30. Ion-exchange chromatography:
 Ion-exchange chromatography depends on the ionic bonding of
proteins to an inert matrix material.
 Two of the most commonly employed ion-exchange resins (inert
matrix material) are:
 Diethylaminoethyl cellulose: (+) (binds to negative charged
molecules: Anion exchanger)(DEAE) and
 Carboxymethyl cellulose (-) (CM): Cation exchanger.

31. Column

32.  The resin is packed into a column, and the protein solution is
allowed through the column in a buffer whose composition
promotes the binding of some or all of the proteins to the resin.
 Proteins are bound to the resin reversibly and can be displaced
by increasing or changing the ionic strength (or pH) of the
buffer. (which adds small ions to compete with the charged
groups of the macromolecules for sites on the resin).
 Proteins are eluted from the column in order from the least
strongly bound to the most strongly bound.

33. Gel Filtration Chromatography or size exclusion
chromatography :
 Gel filtration separates proteins (or nucleic acids) primarily on the
basis of their effective size.
 Like ion-exchange chromatography, the separation material consists
of gel beads that are packed into a column through which the
protein solution slowly passes.
 The materials used in gel filtration are composed of cross-linked
polysaccharides (agarose or Sephadex G-150 beads) of different
porosity, which allow proteins to diffuse in and out of the beads.

34. Gel Filtration Chromatography:
For example if a solution consists of three different proteins such as 75 kDa and 25
kDa and 120 kDa.,
Agarose
or
Sephadex
G-150
beads are
generally
used in
chromato
grphy
column

35.  For example if a solution consists of three different proteins such as
120 kDa, 75 kDa and 25 kDa.
 To purify 120 kDa protein form mixture, the sample pass through a
column of Sephadex G-150 beads.
 When the protein mixture passes through the column bed, the 120
kDa protein is unable to enter the beads and remains dissolved in the
moving solvent phase.
 The gel beads allows only the entry of proteins that are less than
about 100 kDa size.

36.  As a result, the 120 kDa protein is eluted as soon as the preexisting
solvent in the column (the bed volume) has dripped out.
 In contrast, the other two proteins can diffuse into the interstices
within the beads and are retarded in their passage through the
column.
 As more and more solvent moves through the column, these proteins
move down its length and out the bottom, but they do so at different
rates.
 Among those proteins that enter the beads, smaller species are
retarded to a greater extent than larger ones.
 Consequently, the 120-kDa protein is eluted in a purified state, while
the 75-kDa and 25 kDa protein remains in the column.

37. Gel-filtration chromatography:
 This is also referred to as size-exclusion
chromatography.
 In this technique, the separation of molecules is
based on the size, shape and molecular weight.
 The sponge-like gel beads with pores serve as
molecular sieves for separation of smaller and
bigger molecules.
 A solution mixture containing molecules of different
sizes (e.g. different proteins) is applied to the
column and eluted.
 The smaller molecules enter the gel beads
through their pores and get trapped.
 On the other hand, the larger molecules cannot pass
through the pores and therefore come out first with
the mobile liquid.
 At the industrial scale, gel-filtration is particularly
useful to remove salts and low molecular weight
compounds from high molecular weight products.

38. Ion-exchange chromatography:

39.  It involves the separation of molecules based on their surface charges.
 Ion-exchangers are of two types (cation- exchangers which have
negatively charged groups like carboxymethyl and sulfonate, and anion-
exchangers with positively charged groups like diethylaminoethyl
(DEAE).
 In ion-exchange chromatography, the pH of the medium is very crucial.
 The ionic bound molecules can be eluted from the matrix by changing the
pH of the elutant buffer.
 Ion-exchange chromatography is useful for the purification of
antibiotics, besides the purification of proteins.

40. Affinity chromatography:

41. Affinity chromatography:
 Affinity chromatography is based on an interaction of a protein
with an immobilized ligand.
 The ligand can be a specific antibody, substrate, or an inhibitor.
 The protein bound to the ligand can be eluted by reducing their
interaction. This can be achieved by changing the pH of the buffer.

42. Stage 4: Formulation:
 Formulation broadly refers to the maintenance of activity and
stability of a biological products during storage and distribution.
 For certain small molecules like (antibiotics, citric acid),
formulation can be done by crystallization.

43.  Proteins may be formulated in the form of solutions, or dry
powders.
 The sugars (sucrose, lactose), salts (sodium chloride, ammonium
sulfate), polyhydric alcohols (glycerol) used as stabilizers for
protein formulation.

44.  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.

45. Bioprocess operation & their global impact/ Role of bioprocess
engineering.

46. History:
 More than 8,000 years ago, it was used to make leavened bread.
 The malting of barley and fermentation of beer was used in Egypt
in 2500 BC.
 Louis Pasteur proved in 1857 that yeast is a living cell that
ferments sugar to alcohol; in 1877.
 In 1928, Alexander Fleming showed that growing colonies of
Penicillium notatum inhibit Staphylococcus cultures.

47. Role of bioprocess engineering:
 Biopharmaceuticals: Mainly includes therapeutic proteins, antibiotics,
antibodies, polysaccharides, hormones, vaccines, and diagnostic agents.
 Industrial chemicals and Energy: Mainly includes alcohols, organic acids,
fuels and other chemicals.
 Food Industries: Mainly includes alcoholic beverages and dairy products,
several novel foods (starch, amino acids and vitamins) and food additives.

48.  Agriculture: Mainly includes veterinary vaccine and antibiotics, use of
nitrogen fixing bacteria, production of transgenic plants and genetic
modification of foods.
 Environmental-management: Mainly includes municipal wastewater
treatment plants to provide clean and safe drinking water.
 The recent attention to the environment has focused some bioprocessing
technology on the transformation of xenobiotics to biodegradable forms.
 Mining: Natural microorganisms have been used for mineral leaching
and metal concentration.

49. Proteins from Recombinant Microorganisms:
 Most of the products manufactured today are made either in
recombinant E. coli or in animal cells.
 E. coli is the microbial system of choice for the expression of
heterologous proteins. No other microorganism is used to produce
so large a number of products at high level.
 Typical levels of foreign protein expressed represent 10–30% of
total cellular protein.

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