Introduction to fermentation and Bioreactor Design

Maulik P. Suthar Ganpat University

Introduction
Pharmaceutical proteins produced via fermentation in transgenic microbes or mammalian cell culture systems provide economical systems for production of therapeutic proteins. These include antibodies, vaccines, blood proteins, etc. Biopharmaceuticals are medical drugs (see pharmacology) produced using biotechnology. They are proteins (including antibodies), nucleic acids (DNA, RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes, 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 microbes have been approved for therapeutic use in the USA. Hundreds of additional biotech drug candidates are in various stages of research or clinical trials. Fermentation systems can be scaled up to produce quantities of pharmaceuticals that are difficult or impossible to produce via traditional methods. Pharmaceutical quality may also be improved. For example, pharmaceuticals produced from blood must be carefully purified to ensure no transmission of viruses as accidental contaminants in the pharmaceutical product. Microbial systems that do not allow human viruses to replicate enable pharmaceutical production with little or no risk of virus contamination.

Introduction
Fermentation technology is the oldest of all biotechnological processes. The term is derived from the Latin verb fevere, to boil--the appearance of fruit extracts or malted grain acted upon by yeast, during the production of alcohol. Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat. Microbiologists consider fermentation as 'any process for the production of 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 produced without the participation of oxygen or other inorganic electron acceptors.

Commercially important Fermentation

Microbial cells or Biomass as the product: Eg. Bakers Yeast, 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

Metabolite
Ethanol - Saccharomyces cerevisiae alcoholic beverages - Kluyveromyces fragilis Citric acid - Aspergillus niger food industry Acetone and Clostridium butanol acetobutyricum solvents Lysine Corynebacterium nutritional additive Glutamic acid glutamacium flavour enhancer Riboflavin Ashbya gossipii nutritional Eremothecium ashbyi Vitamin B12 Pseudomonas denitrificans nutritional Propionibacterium shermanii Dextran Leuconostoc mesenteroides industrial Xanthan gum Xanthomonas campestris industrial

Secondary metabolites
Penicillin Penicillium chrysogenum antibiotic Erythromycin Streptomyces erythreus antibiotic Streptomycin Streptomyces griseus antibiotic Cephalosporin Cephalosporium acrimonium antibiotic Griseofulvin Penicillium griseofulvin antifungal antibiotic Cyclosporin A Tolypocladium inflatum immunosuppressant Gibberellin Gibberella fujikuroi plant growth regulator Secondary metabolism may be repressed in certain cases. Glucose represses the production of actinomycin, penicillin, neomycin and streptomycin; phosphate represses streptomycin and tetracyclin production. Hence, the culture medium for secondary metabolite production should be carefully chosen.

PRODUCTION ENZYMES
Aspergillus oryzae Amylases Aspergillus niger Glucamylase Trichoderma reesii Cellulase Saccharomyces cerevisiea Invertase Kluyveromyces fragilis Lactase Saccharomycopsis lipolytica Lipase Aspergillus species -Pectinases and proteases Bacillus species Proteases Mucor pusillus Microbial rennet Mucor meihei Microbial rennet

RECOMBINANT PRODUCTS
Therapeutics Proteins mAbs Enzymes Hormones DNA for gene therapy

BIOTRANSFORMATION
Production of a structurally similar compound from a particular one, during the fermentation process is transformation, or biotransformation, or bioconversion. The oldest instance of this process is the production of acetic acid from ethanol. Immobilised plant cells may be used for biotransformation. Using alginate as the immobilising polymer, digitoxin from Digitalis lanata was converted into digoxin, which is a therapeutic agent in great demand. Similarly, codeinone was converted into codeine and tyrosine from Mucuna pruriens was converted into DOPA.

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

Design of Fermenter

Design of Fermenter
Factors to consider when designing a fermenter Aseptic and regulatory capability, longterm reliability Adequate aeration and agitation Low power consumption Temperature and pH controls Sampling facilities

FERMENTERS AND BIOREACTORS DESIGN

There are many requirements that need to be met in the design of a large production scale fermentation facility. Aspects of design to be considered include design yield basis, operating schedule, media sterilization, fermenter and ancillary vessel design, piping systems, CIP/SIP and CGMP compliance. To be successful, a well thought-out and well-designed sanitary/sterile envelope is therefore crucial to the fermentation/biotech facility. A fermenter is the set up to carry out the process of fermentation. The fermenters vary from laboratory experimental models of one or two litres capacity, to industrial models of several hundred litres capacity, which refers to the volume of the main fermenting vessel. A bioreactor differs from a fermenter in that the former is used for the mass culture of plant or animal cells, instead of micro-organisms. The chemical compounds synthesised 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 and bioreactors are identical. With the involvement of micro-organisms as elicitors in some situations, the distinction between the two concepts is being gradually obliterated.

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

Economy of scale

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

Typical Bioprocessing

Three Myths of Scale-Up

We can just make it bigger. Technology is already there. It can be done very quickly.

Scale-up is the key for the commercial 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

Penicillin Production

Approaches for Scale-Up

Kinetics
Cell Kinetics Enzyme Kinetics

Bioreactor (Fermenter) Design Operation and Control Separations Engineering Aspects

Transport Phenomena Unit Operations

Cost Estimations

Guidelines for Fermenter Design and Operation

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

Instrumentation and Control

The success of a fermentation process is highly dependent on environmental factors The fermenter needs to be able to control such factors as temperature, pH, and dissolved oxygen levels

Aeration and Agitation

Most industrial fermentations are aerobic processes meaning that the production microbe requires oxygen to grow The oxygen demand is met by sparging air through the fermentation vessel and using an agitator increase the amount of dissolved oxygen

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 and oxygen

Bioreactor Specifications

Di
1.3 m

HL*= m

5.5 Dt
1.50 m

HL**= 6.06m

Total volume = 11.8 m3

Final Design Specifications

Initial Reactor Volume Max Oxygen Demand Target maximum kLa Tank Diameter Impeller Diameter Tank Height (HL) HL* HL** Rpm Qg Ni Corrected Power (Pg) Pg/V Design kLa Pm Geometry Correction Factor ( ) 8500L 8mmole/gh 2088h-1 1.5m 1.3m 4.81m 5.51m 6.06m 45 0.036m3/s 2
6789W

776.21W/m3 1998h-1 8.06W/kg 0.13

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 (minus medium)

Novonordisk Bioreactor

Steps
1. Organism selection, with regard to:
substrate versatility byproduct formation characteristics robustness of the organism, e.g., to process upsets viability with regard to cell recycling physiological characteristics (maximum growth rate, aeration requirements, etc.) genetic accessibility

2. Metabolic and cellular engineering: improve existing properties of the organism introduce novel functions, for example, by simplify- ing product recovery, expanding substrate and product ranges, and enabling fermentation to occur under nonstandard conditions

3. Fermentation process development: culture and media optimization (from complex to defined minimal media) optimization of cultivation parameters that take into account product recovery and purification (minimize byproduct formation, minimize chemical inputs, and develop high-cell-density cultivation) incorporation of cell retention/recycling

Roadmap for Integrated Process Development

Analyze of economic and process constraints based on preliminary process design Identify opportunities for improvement, e.g., reduced waste streams, energy use, impurity levels and raw material use Put together a wish list of physiological characteristics and downstream separation performance Evaluate feasibility of achieving the wish list based on technical difficulty and economics Define the best strategy for addressing each opportunity by taking into account both downstream and fermentation capabilities, such as high cell density, extractive fermentation, simplify broth, etc. Integrated fermentation and downstream process development

Types of Fermenter
1. 2. 3. 4. 5. 6. 7. 8. 9. Activated sludge Fermenter Air Lift Fermenter Bubble cap Fermenter Loop Fermenter Mist Fermenter Packed Bed Fermenter Rotating Drug Fermenter Tower Fermenter Trickling Film Fermenter

Classification of Fermenters

1. Activated sludge Fermenter

2. Air-lift Fermenters

2. Air-lift Fermenters

3. Bubble cap Fermenter

4. Loop Fermenter

5. Mist Fermenter

6. Packed Bed Fermenter

7. Rotating Drum Fermenter

8. Tower/Column Fermenters

9. Trickling Film Fermenter

References
Stanbury, P.F., A. Whitaker, and S. J. Hall, Principles of Fermentation Technology, 2nd ed., Butterworth Heinemann, Oxford, 2000.

References

References

References