Spreading Knowledge is a great Legacy. MMG

1. Dr. Imran Sajid

2. Raw materials Microbial strain
Fermentation
Product purification
ProductEffluent wastes
Upstream processing
Downstream processing

3. Contents
• Fermentation and types of fermentation
• Fermenter design and construction
• Control of physical and chemical parameters
• Stages in a fermentation process
• Solid substrate fermentation

4. Fermentation Systems
Fermentation two different contexts
• Physiological point of view
• Industrial point of view
• Culturing of microbial cells in large vessels under
aerobic or anaerobic conditions
• For Microbial cell mass + different products +
bioconversion
The large vessels ????? (Fermenters, Bioreactors)

5. But remember ???????
Recombinant DNA technology
• Setting new trends
• Expression of required genes in plants and animals
Examples
• Monoclonal antibodies from ascitic fluid of rodents
• Vaccines in sheep milk
• Malaria vaccine in banana fruit
• Expression in different other agricultural plants

6. Types of Fermentation
Based on media composition/condition
• Submerged fermentation
• Solid substrate fermentation/Surface
fermentation

7. Types of Fermentation
Based on operating modes
• Batch cultures
• Fed batch fermentation
• Continuous fermentation
Batch cultures
• Most of the fermentation processes are batch
cultures
• Closed type of systems are used
• There are no additions except acid/base and air
• There are definite start and end points
• Fermenter is loaded, sterilized, and inoculated,
• The product is harvested and fermenter is cleaned

8. Operating modes
• The duration between two batches is called down
time
Batch cultures are used for the production of
• Secondary metabolites, antibiotics
• Traditional products alcoholic beverage, amino acids,
enzymes, organic acids etc
Advantages
• Initial capital expenditure is lower
• In case of contamination batch can be terminated
Disadvantages
• Less effective for the production of cell mass &
primary metabolites

9. Operating modes
• Batch to batch variation of product quality
• Increased non productive down time
• Increased frequency of sterilization may cause
damage to the instrument and probes
• Running cost of stock cultures
• More labour is required in batch fermentation
Fed batch fermentation
• Extra nutrients are added as fermentation progresses
• Additions are made at the end of rapid growth phase
• Additions are made continuously, intermittently, or
as a single supplementation

10. Operating modes
Fed batch fermentation is used for
• Producing backer’s yeast
• Some time for penicillin
• Some ethanol fermentations
• Waste water treatment
Advantages
• Can extend the product formation phase
• May overcome the problem of suppressive or rapidly
metabolized substrate
• Is also useful where substrate causes viscosity
problems

11. Operating mode
Continuous fermentation
• Open type of systems
• Fresh medium is continuously added
• Also culture is simultaneously removed
• The process is maintained at a constant working
volume
• Cell grow at exponential rate for extended period of
time
Continuous fermentation is used for
• Biomass and primary metabolites production
• Production of biofuels/ ethanol
• Effluent treatment

12. Operating modes
Advantages
• More productive than batch cultures
• Reduced down time
• Low operating costs
Disadvantages
• Higher initial capital expenditure
• Maintenance of sterility during longer operation (20-50 days)
• Continuous supply of constant composition medium
• Any genetic instability may lead to low yielding mutants etc
• However the problems can be overcome by GMP and good
microbiological practices

13. Fermenter design and construction

14. Fermenter design and construction
What may be the main function of a fermenter ????
“To provide a suitable environment in which an organism can
efficiently produce a target product”
• Cell biomass
• A metabolite
• A bioconversion product
Fermenters are designed to ensure above function
Performance parameters
• Rate of agitation, oxygen transfer
• pH, temperature, and foam production

15. Fermenter design and construction
Laboratory fermentations
• Can be performed in
• bottles, conical flasks etc
• Covered with plugs or Styrofoam bungs etc
However even on laboratory scale small vessels are designed
Design depends on the following factors
• Producing organism
• Optimal operating conditions
• Product value & Scale of production
• Reliability and minimum capital investment

16. Fermenter design and construction
Structure of a fermenter & specifications
Traditional fermenters
• An open cylindrical or rectangular vessel
• Made up of wood or stone
• Utilization in food and beverage fermentations
Modern fermenters
• Closed vessels (designed to exclude contamination)
• Must with stand repeated sterilization and cleaning
• Should be constructed with non toxic & corrosion resistant
material

17. Fermenter design and construction
Construction material
Small fermenters (few liter capacity)
• Glass or stain less steel
Pilot scale fermenters
• Stainless steel with polished internal surfaces
Production fermenters
• Mild steel lines with glass or plastics
In case of aseptic fermentation
• All the pipelines must be sterilize able
• Most fermenters have an external jacket

18. Fermenter design and construction
Aseptic fermentations needs special arrangements
• There should be no direct contact between sterile and non
sterile sections
• All the connections must be suitable for steam sterilization
• System must have facility for aseptic
• Inoculation
• Sampling
• Harvesting
• There should be no horizontal pipes or unnecessary joints

19. Fermenter design and construction
Additional features
• Pressure gauges
• Safety valves
• Structure of safety valves (metal foil discs)
• Pumps should be avoided as
• May cause contamination
• May create shear forces
• Alternative methods for liquid transfer are
• Gravity feeding or vessel pressurization etc
• Fermenters are operated under positive pressure

20. Fermenter design and construction

21. Fermenter design and construction

22. Fermenter design and construction

23. Fermenter design and construction

24. Fermenter design and construction

29. Types of Fermenters
There are three principal types of fermenters
1- Stirred tank reactors (STRs)
• Mechanically moving agitators or impellors with in a baffled
fermenter or vessel
• Baffles--- flat vertical plates, width is about one tenth of
vessel diameter, number varies from 4-6 in a vessel
• Functions – aid in mixing, increase turbulence, prevent vortex

30. Types of fermenters
• STRs are commonly used reactors
• Impeller is connected to external motor
• Seals on the shaft may cause
contamination, two to three seals
• Effectiveness of agitation depends on
• Design of impellor blades
• Speed of agitation, depth of liquid
• The height to diameter ratio 3:1 or 4:1
• STRs must create high turbulence but it
creates high shear force
• Some time animal and plant cells are
sensitive to shear force

31. Types of Fermenters
2- Pneumatic systems (airlift fermenters)
• No moving parts
• Use expansion of compressed gas for mixing
• Lower energy requirements
• Creates less shear force
• Gas is pumped from bottom
• Air bubbles move upward
• It cause the upward movement of liquid
• Internal cooling coils are usually not required
• Jacket can provide sufficient heat transfer

32. Types of Fermenters

33. Types of Fermenters
3- Hydrodynamic mechanism (deep-jet fermenters)
• Use the liquid kinetic energy for mixing
• Mixing of nutrients and gases is complex
• It is influenced by
• Medium density
• Rheology
• Size and geometry of vessel
• Amount of power used in the system

34. Control of chemical and physical conditions
The basic concept of fermenter
“ to separate internal fermentation environment from
the external environment”
Two types of properties
Intensive properties (cannot be balanced)
• Temperature, concentration, pressure and specific
heat
Extrinsive properties (can be balanced)
• Mass, volume, entropy and energy
• e.g. 10g of water + 35 g of water at 30° C = 45 g of
water but temperature remains 30° C not 60° C

35. Fermenter control and monitoring
Control of physical and chemical conditions
• By sensors (electrodes)
• By sampling
• Data logging
Internal & external sensors
• Sensor for pH (monitor the level of acid and base)
• For oxygen and CO2 ( monitors the level of O2 CO2)
• For foam etc
• Pressure gauges
• Sensors should be sterilize able
• External sensors do not need sterilization

36. Fermenter control and monitoring
• sensors are attached to central control system
• Central control system automatically monitors and adjust
parameters according to the program
In process control
• Collection of samples at specific intervals
• Analysis of samples for different parameters e.g.
• Product concentration
• Nutrients consumption
• Cell number or biomass etc
Data logging
Permanent record of data (results obtained) in soft and hard

37. Control of chemical and physical conditions
1- Agitation
Why agitation is necessary????
• To mix the three phases in fermenter
• The liquid phase (dissolved nutrients and metabolites)
• The gaseous phase (oxygen + carbon dioxide)
• The solid phase (cell or any solid substrate)
Mixing should produce
• Homogeneous conditions
• Promote heat and gas transfer
• It prolong retention of air bubbles, reduce bubble size, hence
increase the surface for oxygen transfer

38. Control of chemical and physical conditions
Agitation and internal conditions of the fermenter
• STRs have agitators with multiple impellors
• In larger fermenters internal conditions are usually non
uniform
• Flow pattern may be laminar or turbulent as a function of the
Reynolds number (Re) of the impellor
Re =
High Re value = laminar flow
Low Re value = turbulent flow There are no units of Re
Inertial force
Viscous force

39. Control of chemical and physical conditions
Rheological behavior of the fluids
• Rheology is the study of flow and deformation of materials
under the applied forces
• Have major impact on the mixing and mass transfer
• There are three types of fluids
Newtonian fluids ( obey Newton’s law of viscosity)
• Their viscosity does not vary with agitation
Non Newtonian fluids
• Viscosity varies with agitation or shear force
• E.g. pseudo plastic fluids (decreasing viscosity)
• Dilatant fluids (increasing viscosity)
Viscoelastic fluids
• do not observe liquid state properties

41. Control of chemical and physical conditions
2- Heat Transfer: Temperature Control
Efficient heat transfer is necessary
• 1- to control the temperature during sterilization process
• 2- to maintain the required temperature during fermentation
Heat is generated in the fermenter due to
• Metabolic activity of the organism
• Mechanical agitation process
Temperature Control (Sensors)
In most fermentations heat needs to be dissipated by cooling
Heat transfer is achieved by an outer jacket, internal coils etc.

47. Control of chemical and physical conditions
3- Mass transfer: Nutrients and Aeration
• Transfer of nutrients into cells is straight forward
• However transfer of oxygen is complex
Aeration
• Some fermentations operate anaerobically
• Most require oxygen (aerobic fermentation)
Air serve in two ways
• Supply oxygen for aerobic respiration
• for purging volatile metabolic products, oils etc
The air provided must be Sterile
• Air should be passed through sterile filters

48. Control of chemical and physical conditions
• Filters should also be present on outlets
• Air enters to the fermenter by a sparger system
• Air transfer rate 0.5-1.0 volumes/ volume of medium/min
• Sparger is fitted at bottom directly below the agitator
• Structure of sparger can effect the oxygen transfer
• Sparger with small pores are preferred (small bubbles)
• Solubility of oxygen in aqueous solutions
• Decreases with increase in temperature
• This makes aeration into the inner areas of fermenter more
difficult
• Oxygen transfer is complex as it involves a phase change

49. Control of chemical and physical conditions
• Gaseous phase to liquid phase
Factors affecting oxygen transfer
Physical conditions,
• temperature, pressure, surface area
• Composition of medium,
• volume of gas/unit volume of reactor
• Type of sparger system,
• Speed of agitation or combination of all
Oxygen mass balance can be controlled by
Oxygen transfer rate OTR
Oxygen utilization rate (critical oxygen demand COD)

55. Stages of a fermentation process
Overall scheme for fermentation consists of four stages
• Inoculum preservation
• Inoculum build-up
• Fermenter pre-culture
• Production fermenter
1- Inoculum preservation
• Preservation of organism along with its activity is necessary
• Working strains are derived from master strains
• Different methods of storage
• Storage at low temperature (2-6o C)
• Frozen storage (-18 or -80o C in freezers or at -196 in liquid N2)
• Lyophilization (freeze drying)

56. Stages of a fermentation process
2- Inoculum build-up (growth of the Inoculum)
• Preserved culture is revised as
• Shaking culture
• On solid medium (if spores are required)
Growth times can be expected as follows
Lyophilized cultures 5-10 days
Frozen cultures Bacteria 4-48 hours
Actinomycetes 1-5 days
Fungi 1-7 days
Refrigerated cultures Bacteria 4-24 hours
Actinomycetes 1-3 days
Fungi 1-5 days

57. Stages of a fermentation process
3- Fermenter pre-culture
• Pre-cultures are made for having enough inoculum for a large
size fermenter
• Small size inoculum in a production fermenter may delay
growth and lower overall yield
• Optimal concentration of inoculum is required
• Bacteria 0.1-3.0%
• Actinomycetes 5-10%
• Fungi 5-10%
• Spore suspension 1-5. 105/L
• Usually production culture medium is used for pre-cultures

58. Stages of a fermentation process
4- Production fermentation
• Different sizes of fermenters are used for large or commercial
fermentation, 10-20 -------100,000 ---- 450,000 L
Different parameters are optimized before the process
• 1- composition of medium
• 2- order of solution,
• 3- pH
• 4- changes in sterilized nutrients
• Inoculation depends on the size of the fermenter
Important parameters during the process
Temperature , aeration, pressure, stirring

59. Stages of a fermentation process
Temperature
Mesophylic range 20-45o C
Thermophylic range above 45o C
Some time temperature differs for tropho and idiophases
Aeration
In most cases 0.25-1.0 vvm (air volume/liquid volume. min)
Pressure
In order to avoid contamination 0.2-0.5 bar
Stirring
Stirring rate with disc impellers
0.02 250-450
0.2 250-350
1-20 120-180
40-150 120-150
450 60-120

60. • Optimization of different parameters
• Scale up
• Scale down

61. Solid substrate fermentation
Introduction
• Various conventional food fermentations in Asia e.g
• Oriental temph and sufu, cheeses and mushrooms
etc
• In addition enzymes, organic acids and even ethanol
particularly in areas where modern equipment is not
available
• Rarely used in Europe and America
• Growth of microorganism on solid
• Usually organic materials in the absence of free
water e.g
• Straw, wood chippings etc

62. Solid substrate fermentation
Processing
• Lack the sophisticated control mechanisms
• Intrinsic kinetics of microorganisms are usually
unknown
• Control inside the fermenter is difficult to achieve
• Some time useful process e.g. formation of fungal
spores
Multistep process
1- pretreatment of substrate
Mechanical , chemical, or biological treatment
2-hydrolysis of primary polymeric substrate
3- utilization of hydrolysis product
4-separation and purification of end product

63. Solid substrate fermentation
Microorganisms involved in Solid substrate
fermentation
• Those that tolerate lower water activity
• Aw values of around 0.7
They may be employed as
1- Monocultures e.g. mushroom production (Agaricus
bisporus)
2- dual cultures e.g. straw bioconversion
(Chaetomium cellulilyticum and Candida tropicalus)
3- mixed cultures e.g. composting and silage
preparation
Indigenous or inoculants

64. Solid substrate fermentation
Environmental parameters influencing SSF
Water activity Aw
• Water is lost by evaporation and metabolic activity
• Replaced by humidification and addition of water
• Too low water level --- substrate is less accessable
• Too high ------ reduction in porosity of substrate
• Reducing the gaseous exchange
Temperature
• heat generation is more problematic--- effect on
humidity
• Temperature is controlled by aeration/ agitation

65. Solid substrate fermentation
Aeration
• Process is usually aerobic
• Oxygen requirements depends on microorganism &
• Specific process
• Kinetics of oxygen transfer are poorly under stood
• Rate of oxygen transfer is influenced by substrate
used
Bioreactors used for solid substrate fermentation
• Usually batch processes
• Some processes simply involve spreading the
substrate onto a suitable floor

66. Solid substrate fermentation
There are five different types of
bioreactors used for SSF
1- Rotating drum fermenters
• Cylindrical vessel of about 100 L
• Mounted on its sides onto rollers
• Used in enzyme and microbial biomass
production
Disadvantages
• Drum is filled only up to 30% capacity

70. 2- Tray fermenters
• Used extensively for the production
of oriental foods
• Substrate is spread on trays which
are stacked in a chamber which is
humified, large volume --- 150 m3
capacity

73. Solid substrate fermentation
3- Bed systems
• As used in commercial koji production
• Bed substrate up to one meter deep
• Humified air is continuously forced from
bottom

77. 4- Column bioreactors
• Glass or plastic column
• Solid substrate is loosely packed
• Surrounded by a jacket (helps in
temperature control)

81. 5- Fluidized bed reactors
• Provide continuous agitation by
forced air (prevent aggregation)
• Used for biomass production for
animal feed