This document provides an overview of fermentation and bioprocess technology. It begins with definitions of fermentation and discusses the basic requirements for microbial growth. It then covers topics like batch, fed-batch, and continuous fermentation processes. Different types of fermenters and components like spargers and impellers are described. The document discusses strain selection and improvement methods. It also provides examples of industrial fermentation processes like ethanol production and antibiotic production. Finally, it gives an overview of downstream processing techniques used to purify products from fermentation broth, such as centrifugation, filtration, and extraction.

1. DR. DARSHAN MARJADI
SRKI, SURAT
Bioprocess & Fermentation
Technology
GSBTM N-BT-CBC e-CRASH
WORKSHOP JUNE2020

2. 2

3. What is Fermentation?
3

4. F E R M E N T A T I O N
The term Fermentation is derived from the Latin
verb "fevere" which means "to boil".
Pasteur’s definition: “life without air”, anaerobe
redox reactions in organisms
New definition: the chemical breakdown of a
substance by bacteria, yeasts, or other
microorganisms, typically involving
effervescence and the giving off of heat.
4

5. F E R M E N T A T I O N
φ the process of deriving energy from the oxidation of organic
compounds, such as carbohydrates, using an endogenous
electron acceptor, which is usually an organic compound.
φ Any Microbe requires Water, Oxygen, Energy source, Carbon
source, Nitrogen source and Micronutrients for the growth.
φ Carbon & Energy source + Nitrogen source + O2 + other
requirements → Biomass + Product + byproducts + CO2 + H2O
+ heat
5

6. 6

7. Bioprocess Technology:
Bio-Life
Technology- Practical application of knowledge; adapting
ourselves to physical world
Definition:
A bioprocess is any process that uses complete living
cells or their components (e.g., bacteria, enzymes,
chloroplasts) to obtain desired products.

8.  Components are…..
• First part is concerned with obtaining the best
biological catalyst for a specific function or
process,
• Second part creates (by construction and
technical operation) the best possible
environment for the catalyst to perform, and
• Third part (downstream processing) is
concerned with the separation and
purification of an essential product or
products from a fermentation process.

9. Difference between
FERMENTER AND BIOREACTOR

10. 10
Parameter Fermenter/ Microbial
Bioreactor
Mammalian Cell Bioreactor
Use for Growth and maintenance of
bacterial and fungal cell.
Growth and maintenance
of Mammalian cell.
Size Large Small
Growth Rate Few days to week Several weeks
pH Addition of acids and bases Addition of gases (CO2
Cylinder)
rpm 800 or more 150 or less
Foam Antifoam agents, Sensor,
Peristaltic pump
Not common
Condition Aerobic and Anaerobic Aerobic only

11. Outline of Fermentation Technology
11

12. . 12

13. 13

14. Basic design of fermenter 14

15. 15
Parts of a fermentor
• Aeration & agitation system
• Impeller
• Sparger
• Baffles
• Load cells
• Inlet & exit gas analyser
• ph meter
• Flow cell
• Steam line
For thorough mixing of medium and
inoculum the part of fermentor useful is
a. Shaft
b. Headspace
c. Impeller
d. Sparger

16. Bioreactor - Baffles
Baffle
Baffles obstruct the flow in the
vessel to help mix the contents.

17. 17
Antifoam agent is
a. Silicon compounds b. Corn oil c. Soyabean oil d. All of these

18. Bioreactor – Load Cell
Compact
Compression
Load Cells
Spacing of
Load Cells
Large vessel containing all
the parts and condition
necessary for the growth of
desired microorganisms is
called
a. Bio reactor
b. Auto reactor
c. Impeller
d. Sparger

19. Selection of Microbial
strain for the fermenter
19

20. Isolation- Seperation

21. Screening
 Screening may be defined as use of highly selective
procedure to allow the detection & isolation/ sorting of
only those interested microorganisms from large
microbial population.

22. Types of Screening
 Primary screening:
The process which involve detection and isolation of
microorganisms that posses potential industrial application.

23. Sample Medium
Soil
Sample
Sterile
Nutrient agar
plate
(3%sucrose+
1%CaCo3 )
Isolation and Screening of
Organic acid producing Bacteria

24. Secondary Screening
 Secondary screening is a specialized screening
technique which involve checking capabilities of
microbes or nature of the products produced by
microbial isolates is carried out carefully.
Industrially important Antibiotic producing organisms shall be isolated
by
a. Disk plate method
b. Direct plate method
c. Serial dilution method
d. Crowded plate method

25. Eg. Of Secondary
screening
 Selective pressure:
 Media contain Bile salts inhibit G+ve but allow G-ve
bacteria to grow.
 Mannitol salt agar is a medium selected for growth of
halotolerant bacteria.
To differentiate lactose and non- lactose
fermentors the medium used is
a. Mac Conkey’s medium
b. Stuart’s medium
c. Sugar medium
d. Citrate medium

26. What is Strain Improvement?
 A strain of bacteria is basically same species of
bacteria but with different functions. Ex. two bacteria,
both are the same species, however, one is pathogenic
(causes disease) while the other isn't.
• Improve means to make something better

27. Methods of manipulating the genetic apparatus of
industrial organisms
A. Methods not involving foreign DNA
1. Conventional mutation
B. Methods involving DNA foreign to the organism (i.e.
recombination)
2. Transduction
3. Conjugation
4. Transformation
5. Heterokaryosis
6. Protoplast fusion
7. Genetic engineering
8. Metabolic engineering
9. Site-directed mutation
Protoplasts can be prepared from
a. Gram positive bacteria
b. Gram negative bacteria
c. Both a & b
d. None of these

28. 28
• The term protoplast refers to the
spherical shape assumed by Gram-
positive bacteria.
• Spheroplast refers to the spherical
shape assumed by Gram-negative
bacteria.
• The difference is essentially the
presence of a single membrane, in
the case of the protoplast, and the
two membranes (inner and outer) of
the Gram-negative spheroplasts.
• It is also possible to generate a
gram-negative protoplast by the
removal of the outer membrane.
• Thus, in essence, protoplast refers
to a bacterial sphere that is
bounded by a single membrane and
spheroplast refers to a sphere that
is bounded by two membranes

29. Selection of mutants
 Auxotrophs: Are the organisms which require complete
nutrients for their growth.
 They lack certain enzymes to manufacture certain required
nutrients & Consequently such nutrients must therefore be
added to growth medium.
 (Auxotrophy is the inability of an organism to synthesize a
particular organic compound required for its growth).

30.  Prototrophs: Are the organisms which able to grow
in less supply of nutrients.
 They posses all the enzymes needed to synthesize
all growth requirements.

31. Formulation
-Its fundamental meaning is the putting together of
components in appropriate relationships or structures,
according to a formula.
MEDIA
- Medium- Support
- Here Growth components
- A growth medium or culture medium is a liquid or gel
designed to support the growth of microorganisms.

32. Media
Carbon
Nitrogen
Phosphorus
Buffers,
Growth
factors
Antifoaming agent
Trace elements
Inducers
Precursors
Chelators
Requirements for the growth & fermentation of the strain

33. 33

34. Fermentation Media 34

35. 35

36. 36

37. •Oxygen is always provided in water.
•Some organisms require molecular oxygen as
terminal oxidizing agents to fulfill their energetic
needs through aerobic respiration.
•These organisms are obligatorily aerobic. For
obligate anaerobes molecular 02, is a toxic
substance.
•Some organisms are facultative anaerobes and
can grow with or without molecular 02.
Oxygen Sources

38. Fermentation
Aerobic
Anaerobic
38

39. Aerobic fermentation
 Adequate aeration
 Bioreactors- adequate supply of sterile air
 In addition, these fermenters may have a
mechanism for stirring and mixing of the
medium and cells
 Antibiotics, enzymes, vitamins.
39

40. Anaerobic fermentation
 In anaerobic fermentation, a provision for aeration
is usually not needed.
 Lactic acid, ethanol, wine
40

41. Types of Fermentation
 Batch: Media and cells are added to the reactor and it is run until a
predetermined set point (i.e. time, concentration). The bioreactor has a
constant volume (the initial volume).
 Fed-Batch: The bioreactor is a batch process in the beginning and after a
certain point a feed input is introduced and the volume of the vessel
increases.
 Continuous: The bioreactor starts with an initial volume and media is
constantly introduced and product is constantly taken out. The inputs and
outputs are at the same rate, so the volume always remains the same.

42. Fermentation
 Fermentation could be:
 Batch mode
 Fed batch mode (continuous)
42

43. TYPES OF FERMENTATION
BATCH TYPES FERMENTATION
FED BATCH FERMENTATION
CONTINUOUS FERMENTATION
SOLID STATE FERMENTATION

44.  Considered to be a closed system.
 The sterilized media in the fermenter is
inoculated with the microorganism.
 Incubation is allowed under the optimum
conditions (aeration, agitation,
temperature).
 During entire fermentation nothing is
added except air, antifoam and acid/base.
44Batch fermentation

45. Batch fermenter system 45

46. Fed-Batch fermentation
 It is enhancement of batch fermentation.
 Continue adding the nutrients (feeding) in a
small doses during the fermentation.
 The method in controlling nutrients feeding
process is by measuring methods.
 The main advantage of fed-batch
fermentation is the elimination of catabolite
repression (feed-back inhibition).
46

47. CONTINUOUS FERMENTATION
It is a continuous process where the nutrient is
continuously added to the fermented at a fixed
rate.
The organisms are continuously maintained at
logarithmic stage. the products are recovered
continuously.
The fomenters in this type are called “flow
through’’ fermentation.
Example: Primary metabolites

48. Microbial growth rate 48
Primary
metabolites
Secondary
metabolites

49. Continuous fermenter system 49

50. SOLID STATE FERMENTATION
The growth of microorganisms on moist solid substrate
particles in the absence or mere absence of visible liquid
water between the particles.
The moisture content of solid substrate ranges between 12-
80%.

51.  Ethanol fermentation (performed by yeast and
some types of bacteria) breaks the pyruvate
down into ethanol and carbon dioxide. It is
important in bread-making, brewing, ethanol fuel
and wine-making.
51

52.  is a biological process in which sugars such as
glucose, fructose, and sucrose are converted into
cellular energy and thereby produce ethanol and
carbon dioxide as metabolic waste products.
 Because yeasts perform this process in the absence
of oxygen, ethanol fermentation is classified as
anaerobic.
52
Industrial alcohol will be produced by using starter
culture
a. Top yeast
b. Middle yeast
c. Bottom yeast
d. Feeder yeast

53. Types of Yeast
 Top-cropping yeasts are so called because they
form a foam at the top of the wort during
fermentation. An example of a top-cropping yeast
is Saccharomyces cerevisiae,
 Bottom-cropping yeasts are typically used to
produce lager-type beers, An example of bottom-
cropping yeast is Saccharomyces pastorianus
53

54. 54
In alchoholic fermentation, CO2 is evolved during
a. Decarboxylatin of pyruvic acid
b. Formation of acetaldehyde
c. None
d. Both a and b

55. Major Industrial Products for
the Health Industry
Antibiotics: Isolation and
Characterization

56. The industrial production of antibiotics begins with
screening for antibiotic producers
56

57. 57

58. • Once new producers are identified,
purification and chemical analyses of the
antimicrobial agent are performed.

59. 59

60. Downstream Processes

61. Introduction to Purification
Many biological processes require a
purification scheme to reduce the
fermentation broth to its pure final
product.
By Extracting Intra cellular or Extra
cellular components

62. 62

63. Bacteria
Cell membrane
Cell wall
Peptidoglycan
Cell membrane
Periplasm
Lipopolysaccharides +
proteins
•Sub-micron to 2 microns in size
•Have thick cell walls, 0.02-0.04
microns, peptidoglycan +
polysaccharide+ teichoic acid
•Phospholipid cell membrane
present
•Sub-micron to 1 micron in size
•Cell capsule present
•Peptidoglycan layer is thin
•Periplasmic space present
•Mechanically less robust than gm+
bacteria
•Chemically more resistant than
gm+ bacteria
G+Ve G-Ve

64. Cell disruption methods
Physical methods
• Disruption in bead mill
• Disruption using a colloid mill
• Disruption using French press
• Disruption using ultrasonic vibrations
Chemical and physicochemical methods
• Disruption using detergents
• Disruption using enzymes e.g. lysozyme
• Disruption using solvents
• Disruption using osmotic shock

65. Types of Purification Efforts
A purification scheme can be made up any
combination of the following isolation efforts
Coagulation and Flocculation
Extraction
Precipitation
Centrifugation
Filtration

66. Coagulation aim 66

67. Flocculation aids 67

68. Floc formation with polymers
6/15/2020
watertreatment
68

69. Liquid-liquid extraction is a useful method to
separate components (compounds) of a mixture
Extraction

70. Let's see an example.
Suppose that you have a mixture of sugar in vegetable
oil (it tastes sweet!) and you want to separate the
sugar from the oil.
You observe that the sugar particles are too tiny to
filter and you suspect that the sugar is partially
dissolved in the vegetable oil.

71. How about shaking the mixture
with water
Will it separate the sugar from
the oil? Sugar is much more
soluble in water than in
vegetable oil, and, as you know,
water is immiscible (=not
soluble) with oil.
Did you see the result? The
water phase is the bottom layer
and the oil phase is the top
layer, because water is denser
than oil.
*You have not shaken the mixture
yet, so sugar is still in the oil
phase.

72. By shaking the layers (phases) well, you
increase the contact area between the two
phases.
The sugar will move to the phase in which it
is most soluble: the water layer
Now the water phase tastes
sweet, because the sugar is moved to the
water phase upon shaking.**You extracted
sugar from the oil with water.**
In this example, water was the extraction
solvent ;the original oil-sugar mixture was
the solution to be extracted; and sugar
was the compound extracted from one
phase to another.
Separating the two layers accomplishes the
separation of the sugar from the vegetable
oil

73. 73

74. Diffusion
Osmosis & RO

75. Filtration
 0.1- 10
µm
 Pore
 Diamete
r- 1- 100
A
 1000-
1,000,00
0 dalton

76. 76
MWCO generally represents
the
a. Lowest molecular weight
b.Highest molecular weight
c. Average molecular weight
d.Smallest pore size

77. The rotary vacuum drum filter

78. 78
Basket Centrifuge

79. Primary and Secondary
Metabolites
Primary metabolites are
produced during active cell
growth, and
Secondary metabolites are
produced near the onset of
stationary phase.

83. STRUCTURE OF INSULIN
 Insulin
Polypeptide hormone
51 amino acids
Two chains
A chain 21 a.a.
B chain 30 a.a.
 Held by interchange disulfide bridges

85. GENETICS OF INSULIN SYNTHESIS
The proinsulin precursor of
insulin is encoded by the INS
gene.
Located on Short arm of
Chromosome -11

86. Recombinant DNA Technology in
the Synthesis of Human Insulin
 Since 1921: Treatement with
insulin derived from animals
 Bovine & porcine insulin
slightly different from human
insulin
 Sometimes inflammation at
injection sites
 Fear: long term complications
 Solution: Inserting insulin gene
into E.coli to produce
identical human insulin using
Recombinant DNA
Technology

87. THANK YOU
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