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Introduction to fermentation and
Bioreactor Design
Maulik P. Suthar
Ganpat University
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Introduction
Pharmaceutical proteins produced via fermentation in transgenic microbes ormammalian cell culture systems provide economical systems for production oftherapeutic proteins. These include antibodies, vaccines, blood proteins, etc.
Biopharmaceuticals are medical drugs (see pharmacology) produced usingbiotechnology. They are proteins (including antibodies), nucleic acids (DNA,
RNA orantisense oligonucleotides) used for therapeutic orin vivo diagnosticpurposes, and are produced by means other than direct extraction from a native(non-engineered) biological source
Dozens of new pharmaceuticals produced via fermentation in transgenic microbeshave been approved for therapeutic use in the USA. Hundreds of additional biotechdrug candidates are in various stages of research or clinical trials. Fermentationsystems can be scaled up to produce quantities of pharmaceuticals that are difficult
or impossible to produce via traditional methods. Pharmaceutical quality may also beimproved. For example, pharmaceuticals produced from blood must be carefullypurified to ensure no transmission of viruses as accidental contaminants in thepharmaceutical product. Microbial systems that do not allow human viruses toreplicate enable pharmaceutical production with little or no risk of virus contamination.
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Introduction
Fermentation technology is the oldest of all biotechnological processes. Theterm is derived from the Latin verb fevere, to boil--the appearance of fruitextracts or malted grain acted upon by yeast, during the production ofalcohol.
Fermentation is a process of chemical change caused by organisms ortheir products, usually producing effervescence and heat.
Microbiologists consider fermentation as 'any process for the productionof a product by means of mass culture of micro-organisms'.
Biochemists consider fermentation as 'an energy-generating process in
which organic compounds act both as electron donors and acceptors';hence fermentation is an anaerobic process where energy is producedwithout the participation of oxygen or other inorganic electronacceptors.
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Commercially important Fermentation
Microbial cells or Biomass as the product: Eg. BakersYeast, Lactic acid bacillus, Bacillus sp.
Microbial Enzymes: Catalase, Amylase, Protease,Pectinase, Glucose isomerase, Cellulase, Hemicellulase,
Lipase, Lactase, Streptokinase etc. Microbial metabolites : Primary metabolites Ethanol, Citric acid, Glutamic acid,
Lysine, Vitamins, Polysaccharides etc. Secondary metabolites: All antibiotic fermentation
Recombinant products : Insulin, HBV, Interferon,GCSF, Streptokinase Biotransformations: Eg. Phenyl acetyl carbinol,Steroid
Biotransformation
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Metabolite
Ethanol - Saccharomycescerevisiae alcoholic beverages - Kluyveromycesfragilis Citric acid - Aspergillus nigerfood industry Acetone and Clostridium
butanol acetobutyricum solvents Lysine Corynebacterium nutritional additive Glutamic acid glutamacium flavour enhancer RiboflavinAshbyagossipiinutritional Eremothecium ashbyi Vitamin B12 Pseudomonas denitrificansnutritional
Propionibacterium shermanii Dextran Leuconostocmesenteroides industrial Xanthan gum Xanthomonascampestris industrial
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Secondary metabolites
Penicillin Penicillium chrysogenum antibiotic Erythromycin Streptomyceserythreus antibiotic Streptomycin Streptomycesgriseus antibiotic Cephalosporin Cephalosporium acrimonium antibiotic
Griseofulvin Penicillium griseofulvin antifungal antibiotic Cyclosporin A Tolypocladium inflatum immunosuppressant Gibberellin Gibberellafujikuroiplant growth regulator Secondary metabolism may be repressed in certain cases. Glucose
represses the production of actinomycin, penicillin, neomycin andstreptomycin; phosphate represses streptomycin and tetracyclinproduction. Hence, the culture medium for secondary metaboliteproduction should be carefully chosen.
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PRODUCTION ENZYMES
Aspergillus oryzae Amylases Aspergillus niger Glucamylase Trichodermareesii Cellulase
Saccharomycescerevisiea Invertase Kluyveromycesfragilis Lactase Saccharomycopsis lipolytica Lipase Aspergillus species -Pectinases and proteases
Bacillus species Proteases Mucorpusillus Microbial rennet Mucormeihei Microbial rennet
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RECOMBINANT PRODUCTS
Therapeutics
Proteins mAbs
Enzymes
Hormones DNA for gene therapy
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BIOTRANSFORMATION
Production of a structurally similar compound from aparticular one, during the fermentation process istransformation, orbiotransformation, orbioconversion. The oldest instance of this process is
the production of acetic acid from ethanol.
Immobilised plant cells may be used forbiotransformation. Using alginate as the immobilisingpolymer, digitoxin from Digitalis lanata was convertedinto digoxin, which is a therapeutic agent in greatdemand. Similarly, codeinone was converted intocodeine and tyrosine from Mucunapruriens wasconverted into DOPA.
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Vitamins
Figure 11.13, Vitamin B12
Used as supplements for human food and animal feeds
Nearly $1B/year
Synthesized chemically, but some by biocatalysis
Selected high-yield strains for B12 produce up to 60 mg/L
For riboflavin, up to 7 g/L
Amino Acids
L-Glutamate (MSG) flavor enhancer
Aspartame (phe + asp) sweetener
L-Lysine nutritive additive
DL- Methionine nutritive additive
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Design of Fermenter
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Design of Fermenter
Factors to consider when designing afermenter
Aseptic and regulatory capability, long-term reliability
Adequate aeration and agitation
Low power consumption Temperature and pH controls
Sampling facilities
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FERMENTERS AND BIOREACTORSDESIGN
There are many requirements that need to be met in the design of a large productionscale fermentation facility. Aspects of design to be considered include design yieldbasis, operating schedule, media sterilization, fermenter and ancillary vessel design,piping systems, CIP/SIP and CGMP compliance. To be successful, a well thought-outand well-designed sanitary/sterile envelope is therefore crucial to thefermentation/biotech facility.
A fermenter is the set up to carry out the process of fermentation. The fermentersvary from laboratory experimental models of one or two litres capacity, to industrialmodels of several hundred litres capacity, which refers to the volume of the mainfermenting vessel.
A bioreactordiffers from a fermenter in that the former is used for the mass cultureof plant or animal cells, instead of micro-organisms. The chemical compoundssynthesised by these cultured cells, such as therapeutic agents, can be extracted
easily from the cell biomass.
The design engineering and operational parameters of both fermenters andbioreactors are identical. With the involvement of micro-organisms as elicitors insome situations, the distinction between the two concepts is being graduallyobliterated.
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Ideal requirements of fermenter
1) Provide operation free from contamination;
2) Maintain a specific temperature;
3) Provide adequate mixing and aeration;
4) Control the pH of the culture;5) Allow monitoring and/or control of dissolved oxygen;
6) Allow feeding of nutrient solutions and reagents;
7) Provide access points for inoculation and sampling;
8) Minimize liquid loss from the vessel;
9) Facilitate the growth of a wide range of organisms
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Economy of scale
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Scale Up
Definition
Adaptation of biological methods of
production to large-scale industrial use Objectives
Obtain the best biological catalyst
Create the best possible environment Purify the products in the most economical
ways
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Typical Bioprocessing
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Three Myths of Scale-Up
We can just make it bigger.
Technology is already there.
It can be done very quickly.
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Scale-up is the key for thecommercial success
Penicillin
Process developed in England
Scaled up in the US
Commercial success by the US
High Fructose Corn Syrup
Process developed in the US Scaled up in Denmark
Commercial success by Denmark
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Penicillin Production
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Approaches for Scale-Up
Kinetics Cell Kinetics
Enzyme Kinetics
Bioreactor (Fermenter) Design Operation and Control
Separations
Engineering Aspects Transport Phenomena
Unit Operations
Cost Estimations
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Guidelines for Fermenter Design andOperation
Material: Stainless steel (Type 316) Height to diameter ratio of the vessel: 2 to 1 or 3 to 1 Impeller Two or three disk turbine impellers Diameter: 0.3 to 0.4 of tank diameter Agitation speed: 50 200 rpm Impeller shaft enters either from the top or bottom. Baffle - Four equally spaced to prevent vortex formation Width: one tenth of the tank diameter Sparger -Ring sparger (Single orifice for a small fermenter)
Heating or cooling coil For sterilization or to control the temperature
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Instrumentation and Control
The success of a fermentation process ishighly dependent on environmental factors
The fermenter needs to be able to controlsuch factors as temperature, pH, anddissolved oxygen levels
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Aeration and Agitation
Most industrial fermentations are aerobicprocesses meaning that the productionmicrobe requires oxygen to grow
The oxygen demand is met by sparging airthrough the fermentation vessel and usingan agitator increase the amount ofdissolved oxygen
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How Unit Operates
Substrate feed
Glucose, ammonia, mineral salts
Cellular metabolism of substrate Extracellular production of insulin
Air sparging for oxygen delivery
Impellers for mixing of nutrients andoxygen
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Bioreactor Specifications
HL*
= 5.5m
HL
**=
6.06m
Di1.3
m
Dt1.50
m
Total volume = 11.8 m3
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Final Design Specifications
Initial Reactor Volume 8500LMax Oxygen Demand 8mmole/gh
Target maximum kLa 2088h-1
Tank Diameter 1.5m
Impeller Diameter 1.3m
Tank Height (HL) 4.81mHL* 5.51m
HL** 6.06m
Rpm 45
Qg 0.036m3/s
Ni 2
Corrected Power (Pg) 6789W
Pg/V 776.21W/m3
Design kLa 1998h-1
Pm 8.06W/kg
Geometry Correction
Factor ()
0.13
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Companies produce Insulin
Sanofi Pasteur
Novonordisk
Eli Lilly Average cost = $40/100 I.U. or ~$800/kg
Final production cost ~$12/kg precursor
Includes capital and operating costs (minusmedium)
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Novonordisk Bioreactor
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Steps
1. Organism selection, with regard to:
substrate versatility
byproduct formation characteristics robustness of the organism, e.g.,to processupsets
viability with regard to cell recycling
physiological characteristics (maximumgrowth rate, aeration requirements, etc.)
genetic accessibility
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2. Metabolic and cellular engineering:
improve existing properties of the
organism introduce novel functions, forexample, by simplify- ing product recovery,expanding substrate and product ranges,and enabling fermentation to occur under
nonstandard conditions
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3. Fermentation process development:
culture and media optimization (fromcomplex to defined minimal media)
optimization of cultivation parameters thattake into account product recovery andpurification (minimize byproduct formation,
minimize chemical inputs, and develophigh-cell-density cultivation)
incorporation of cell retention/recycling
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Roadmap for Integrated ProcessDevelopment
Analyze of economic and process constraints based on preliminary processdesign
Identify opportunities for improvement, e.g., reduced waste streams, energyuse, impurity levels and raw material use
Put together a wish list of physiological characteristics and downstreamseparation performance
Evaluate feasibility of achieving the wish list based on technical difficultyand economics
Define the best strategy for addressing each opportunity by taking intoaccount both downstream and fermentation capabilities, such as high celldensity, extractive fermentation, simplify broth, etc.
Integrated fermentation and downstream process development
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Types of Fermenter
1. Activated sludge Fermenter2. Air Lift Fermenter3. Bubble cap Fermenter
4. Loop Fermenter5. Mist Fermenter6. Packed Bed Fermenter7. Rotating Drug Fermenter
8. Tower Fermenter9. Trickling Film Fermenter
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Classification of Fermenters
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1. Activated sludge Fermenter
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2. Air-lift Fermenters
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2. Air-lift Fermenters
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3. Bubble cap Fermenter
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4. Loop Fermenter
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5. Mist Fermenter
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6. Packed Bed Fermenter
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7. Rotating Drum Fermenter
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8. Tower/Column Fermenters
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9. Trickling Film Fermenter
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References
Stanbury, P.F., A. Whitaker, and S. J. Hall,Principles of Fermentation Technology, 2nd
ed., Butterworth Heinemann, Oxford,2000.
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References
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References
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References
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