1. Pharmaceutical Biotechnology PHR 403
Chapter 2: Fermentation Technology

2. Fermentation Technology
In the context of Microbial biotechnology the term “fermentation” refers to the growth of large quantities of cells under anaerobic or aerobic conditions within a vessel called a fermenter or bioreactor
Similar vessels are used in processes involving cell-free and immobilized enzyme transformations

3. Bioprocess typically involves principle 3 steps(see figure)
Industrial Bioprocess – any large scale operation involving a transformation of a raw material into a product by moss or animal plant cells or enzymes or cell extracts
Product may be useable (eg. Insulin) or have commercial value (waste water treatment)
Bioprocess typically involves principle 3 steps(see figure)
Each step requires a combination of operating units.
Downstream processing may include-
extraction, ultra filtration, crystallization, chromatography, drying and packaging

4. Bioprocess stages and the commonly used operations in them
PROCESS STAGES OPERATIONS
Raw Material
Sorting
Sieving
Comminuting
Hydrolysis
Sterilization
PRETREATMENT
Biomass production
Metabolite biosynthesis
Biotransformations
BIOREACTION
Filtration
Centrifugation
Sedimentation
Flocculation
Cell Disruption
Precipitation
Evaporation
DOWNSTREAM PROCESSING
Product

5. Microbial Biotechnology
Use of micro-organisms for large scale industrial processes
Oldest form of microbiology and biotechnology which was used to make wine, beer, sake, bread with use of bacteria and yeasts without knowing scientific basis
Production of ethanol, lactic acid, butanol using microbes and Enzymes like amylase, protease, invertase were used during early 20th century
Pencillin was produced during WWII and other amino acids, nucleotides, enzymes were also produced later

6. MODERN
White Biotechnology
Now use in bioconversion/biotransformations of chemicals, genetic engineering of MO for non microbial products (insulin, interferons, HGH, vaccines)
Microbial Fermentations (organic acids, amino acids, vitamins, antibiotics, enzymes) and fermented foods (dairy, meat, plant, breads, alcoholic beverages)
Use of Microbes in increasing crop productivity
Icrobes as Biofertilizers and Biopesticides
Use
Microbes as food/feed: single cell protein
Bioenergy and Bioremediation
Mining and metallurgy

7. Isolation and culturing Micro-organisms
Sources
Soils, lakes, oceans, river, plant, animal, air, non living objects
Isolation methods
Serial dilution, spread plate, gradient pour plate, streak plate
Filtration, centrifugation
Importance of eliminating other organisms (antibiotics, heating)

8. Isolation and culturing Micro-organisms
Growth Media
MO require nutrients (C, N, P, Minerals), O2 requirement, temp, pH, salinity etc
Synthetic media
Semi synthetic media
Natural media
Media needs to be economical for large scale productions, consistent quality and available throughout. Raw material can be pre-treated if required
Cheap C and N2 sources can be used

9. Isolation and culturing Micro-organisms
Sources of nutrition
Carbon: sugarcane molasses, beet molasses, vegetable oil, starch, cereal grains, whey, glucose, sucrose, lactose, malt, hydrocarbons
Nitrogen: corn steep liquor, slaughter house waste, urea, ammonium salts, nitrate, peanut granules, soyabean meal, yeast extract etc
Growth factors: vitamins and amino acids are added when MO cannot synthesize them

10. Antifoams: sunflower oil, olive oil to prevent foaming
Isolation and culturing Micro-organisms
Sources of nutrition
Trace elements: Zn, Mo, Mn, Cu, Co required for metabolism or in metallo-enzymes or in proteins (Hb)
Inducers, precursors, repressors: for enzymes to function in metabolic processes inducers are required. Sometimes presence of presursors enhances production of a secondary metabolite or production an enzyme can be repressed due to repressors. Eg streptomycin is nduced by yeast extract, Sec metabolites can be repressed due to some cpds.
Antifoams: sunflower oil, olive oil to prevent foaming
Water: clean water of consistent composition, dissolved chemicals, pH is measured. Also required for cleaning, washing, rinsing, cooling, heating etc.

11. Culturing methods for Micro-organisms
Sterilization: devoid of MO (aseptic conditions)
Contamination free seed culture
Moist heat (121oC/15psi/20min), radiation, ultrasonic treatment, chemicals, mechanical, gases (ozone), filtration for sterilizing air
Sterilization of equipment, media and air
CULTURING
Avoidance of contamination can be achieved by
Use pure inoculum to start fermentation
Sterilize the media
Sterilize fermenter vessel
Sterilize all materials to be added to the fermentation during the process
Maintaining aseptic conditions during the fermentation

12. Control of environmental conditions for
Microbial growth
Temperature pH
Agitation
O2 conc
To be carefully monitored and maintained
Acidic pH: fungi and yeast
Psychrophiles, acidophiles etc

13. Sterilization:
Elimination of threads and welding of pipes and tubes to reduce contamination
Fermenters have pipes which flush steam into the system
Media along with fermenter is sterilized
Among the several factors that influence killing are-
temperature,
pH,
osmotic pressure,
shear,
mass transport, and
concentrations of extraneous substances that react with the killing agent.
These factors operate synergistically, and temperature plays roles other than simply affecting the kinetics of a reaction

14. Aeration and Mixing
Shake culture: flasks are kept on a shaker for required rotations
Fermenters: Stirrers for O2 mixing and baffles for increasing turbulence
V shaped notch
Increase turbulence
Increase efficiency of
O2 transfer
Improves growth of MO

15. Fermentation
Fervere: to boil
In Microbiology
Any process for the production of useful products through mass culture of MO
In Biochemistry
-The numerous Oxidation-Reduction reactions in which organic compounds used as carbon and energy act as acceptors and donors of H2 ion. The organic compound gives rise to various products of fermentation which accumulate in the growth medium
-Takes place in absence of O2
-Now term industrial fermentation for large scale cultivation of micro-organisms…most of them is aerobic

16. Fermenter and bioreactor
A biorector is a device in which the organisms are cultivated and motivated to form a desired product
Closed vessel or containment vessel to give a right environment for optimal growth and metabolic activity of the organism
Fermenter: for microbes/ Bioreactor : for eukaryotic cells
Size variable ranging from million litres or more.
Large scale production (10-100L to1000-million L capacity)
Helps to meet requirements of: pH
temperature
aeration
agitation
drain or overflow
control systems
sensors
cooling to achieve maximum microbial yield

17. What is fermentation technique?
Techniques for large-scale production of microbial products:
It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale.
They can be divided into surface (immersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors.
In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry

18. What is fermentation technique?
In the submersion processes, the microorganisms grow in a liquid medium.
Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium.
Most processes are aerobic, and for these the medium must be vigorously aerated.
All important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

19. Some important submerged fermentation products
Organism
Use
Ethanol
Saccharomyces cerevisiae
Industrial solvents, beverages
Glycerol
Production of explosives
Lactic acid
Lactobacillus bulgaricus
Food and pharmaceutical
Acetone and butanol
Clostridium acetobutylicum
Solvents
-amylase
Bacillus subtilis
Starch hydrolysis

20. General Aspects of Fermentation Processes

21. Fermenter The heart of the fermentation process is the fermenter.
In general:
Stirred vessel, H/D  3
Volume m3 (80 % filled)
Biomass up to 100 kg dry weight/m3
Product 10 mg/l –200 g/l

22. Component parts of a fermenter
Formulation of media to be used in culturing the organism during development of inoculum and in the production fermenter
Sterilization of the medium, fermenter and ancillary equipment
Production of an active, pure culture in sufficient quantity to inoculate the production vessel
The growth of the organism in the production fermenter under optimum conditions for product formation
The extraction of the product and its purification
Disposal of effluents produced by the process

23. Production fermenter Biomass Culture Cell fluid separation Cell free
DOWNSTREAM
PROCESSING
Culture
fluid
Cell
separation
Stock
culture
Shake
flask
Seed
fermenter
Cell free
supernatant
Medium STERILIZATION
Medium FORMULATION
Product
extraction
Medium raw material
Product
purification
Effluent treatment
Product packaging

24. Cross section of a fermenter for Penicillin production ( Copyright:  

25. Cross section of a fermenter for Penicillin production ( Copyright:

26. Flow sheet of a multipurpose fermenter and its auxiliary equipment

27. Batch fermentation refers to
Basic modes of operations of a fermenter
Batch culture
Batch fermentation refers to
a partially closed system in which most of the materials required are loaded onto the fermentor, decontaminated before the process starts and then, removed at the end.
The only material added and removed during the course of a batch fermentation is the gas exchange and pH control solutions.
In this mode of operation, conditions are continuously changing with time, and the fermentor is an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time.
The principal disadvantage of batch processing is the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermenter vessel, the cleaning, sterilization and re-start process

28. Basic modes of operations of a fermenter
Continuous culture
Continuous culture is a technique involving feeding the microorganism used for the fermentation with fresh nutrients and, at the same time, removing spent medium plus cells from the system
A unique feature of the continuous culture is that a time-independent steady-state can be attained which enables one to determine the relations between microbial behavior (genetic and phenotypic expression) and the environmental conditions.

29. Basic modes of operations of a fermenter
Fed-batch processes
The fed-batch technique was originally devised by yeast producers in the early 1900s to regulate the growth in batch culture of Saccharomyces.
Yeast producers observed that in the presence of high concentrations of malt, a by-product - ethanol - was produced, while in low concentrations of malt, the yeast growth was restricted. The problem was then solved by a controlled feeding regime, so that yeast growth remained substrate limited.
The concept was then extended to the production of other products, such as some enzymes, antibiotics, growth hormones, microbial cells, vitamins, amino acids and other organic acids.
An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion" 


30. By products may lead to cell death
Fed-batch processes
Basically, cells are grown under a batch regime for some time, usually until close to the end of the exponential growth phase.
At this point, the reactor is fed with a solution of substrates, without the removal of culture fluid.
This feed should be balanced enough to keep the growth of the microorganisms at a desired specific growth rate and reducing simultaneously the production of by-products (that can be growth or product production inhibitory and make the system not as effective).
By products may lead to cell death

31. A fed-batch is useful in achieving high concentration of products as a result of high concentration of cells for a relative large span of time.
Two cases can be considered:
the production of a growth associated product and the production of a non-growth associated product. In the first case, it is desirable to extend the growth phase as much as possible, minimizing the changes in the fermenter as far as specific growth rate, production of the product of interest and avoiding the production of by-products.
Growth associated products- These are those products formed simultaneously with microbial growth. The specific rate of product formation is proportional to the specific rate of growth. Example: production of constitutive enzyme.
Non-growth associated products- Products are formed during the stationary phase when the net growth rate is zero. The specific rate of product formation is constant. Example: Secondary metabolites like antibiotics (penicillin).
Mixed growth associated products- Here products are formed during the slow growth and stationary phases. Example: Production of lactic acid fermentation.

32. For non-growth associated products, the fed-batch would be having two phases: a growth phase in which the cells are grown to the required concentration and then a production phase in which carbon source and other requirements for production are fed to the fermenter.
This case is also of particular interest for recombinant inducible systems: the cells are grown to high concentrations and then induced to express the recombinant product.

33. Simple fermenters (batch and continuous) Fed batch fermenter
Types of Fermenter/Bioreactors
Simple fermenters (batch and continuous)
Fed batch fermenter
Air-lift or bubble fermenter
Cyclone column fermenter
Tower fermenter
Fluidized bed bioreactors
Packed bed bioreactor
photobioreactor
Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3 (industrial applications)

34. Outline of a fermentation process
Upstream processing
Raw Materials
Production microorganism
Fermentation
Downstream processing
Product purification
Product
Effluent waste

35. Fermentation Systems
We examine conventional fermenters used for microbial, plant and animal cell culture
Most fermentations use liquid media
Some are non stirred, non aerated and non aseptically operated (beer, wine) while others are stirred, aerated and aseptic
Also classified as to organization of biological phase – suspended growth or supported growth

36. Fermentations are performed in fermenters
Often with capacities of several thousand litres
Range from simple tanks (stirred or unstirred)
To complex integrated systems involving varying levels of computer controls
Fermenter and assoc pipe work must be made of stainless steel (for repeated sterilizationn and must not react with mos or metabolites

37. The essential components of a fermenter

38. The Fermentation Process

39. Operating Systems
Fermentations in liquid media can be carried out under batch, fed-batch or continuous culture conditions
Stirred batch fermentor : a closed system where all nutrients are present at start of fermentation within a fixed volume
Fed-batch fermentor: fresh medium is fed in throughout the fermentation and volume of batch increases with time
Continuous culture: fresh medium is fed into the vessel and spent medium and cells are removed (fixed volume)
In all systems, pH, temperature, aeration etc is monitored and adjusted

40. Batch Fermentor Medium added Fermentor sterilised Inoculum added
Fermentation followed to completion
Culture harvested
Glucose
Product
Cell biomass
Time

41. Fed-Batch Fermentors
Pump
Feedstock
vessel
(sterile)

42. Characteristics of Fed Batch Fermentors
Initial medium concentration is relatively low (no inhibition of culture growth)
Medium constituents (concentrated C and/or N feeds) are added continuously or in increments
Controlled feed results in higher biomass and product yields
Fermentation is still limited by accumulation of toxic end products
BATCH
FED-BATCH
Glucose
Glucose
Product
Product
Biomass
Biomass

43. Continuous Fermentor Flow rate1 = Flow rate2 Pump 1 Pump 2 Feedstock
vessel
(sterile)
Collection vessel

44. Fermentation Media
Media must satisfy all nutritional requirements of the organism and fulfil the objectives of the process
Generally must provide
a carbon source (for energy and C units for biosynthesis)
Sources of nitrogen, phosphorous ans sulfur
Minor and trace elements
Some require added vitamins e.g. biotinand riboflavin
Media generally contain buffers or pH controlled by adding acids / alkalis
Potential problems
Compounds that are rapidly metabolized may repress product formation
Certain compounds affect morphology

45. Factors affecting final choice of raw materials
Costs and availability
Ease of handling, transporting and storing
Sterilization requirements and denaturation problems
Formulation, mixing and viscosity characteristics
Concentration of product produced / rate of formation/ yield per gram of substrate
Levels and ranges of impurities which may produce undesirable by products
Health and safety considerations

46. Desirable Characteristics of Media
Should be inexpensive and available year round . Media may account for 60-80% of cost of process
Should ideally be stored at room T otherwise costs are high
Must be easily sterilized with minimal thermal damage
May influence aeration and cause foaming during process

47. Carbon sources Molasses Malt extract
Byproduct of cane sugar production
a dark viscous syrup containing 50% CHO (sucrose) with 2% nitrogen, vitamins and minerals
Malt extract
Use aqueous extracts of malted barley to produce C sources for cultivation of fungi and yeasts
Contain 90% CHO, 5% nitrogen and proteins, peptides and amino acids
Molasses : cant use pure glucose or sucrose (cost)

48. Carbon sources (2) Whey Alkanes and alcohols
Aqueous byproduct of dairy industry
Contains lactose and milk proteins
Difficult to store (refrigerate) so freeze dried
Many MO’s won’t metabolize lactose but whey is used in production of penecilluin, ethanol, SCP, xanthan gum etc
Alkanes and alcohols
C10 – C20 alkanes, metane and methanol used for vinegar and biomass production
Use is dependent on prevaling petroleum price

49. Nitrogen Sources Mo’s generally can use inorganic or organic N
Inorganic sources: ammonia, ammonium salts
Organic sources: amino acid, proteins and urea
Corn steep liquor
Yeast extract
Peptones
Soya bean meal