Fermentation Technology (Bio-Processing) Learning Outcome 1. Trace the capability of biological systems as tools for technology 2. Evaluate living cells and enzymes as biological catalysts 3. Knowledge of both the structure and function of industrial biological systems 4. Compare and contrast between different fermentation processes 5. Recognize how to screen for, preserve and propagate important genotypes 6. Awareness of fermentation technology as a tool of drug production Fermentation Technology (Bio-Processing) Alcoholic fermentation: Conversion of glucose to ethanol (anaerobic respiration) Fermentation (Bio-Processing): Manufacturing of desired microbial products Commercially important Fermentations: 1. Microbial cells or biomass as the product: e.g. Bakers yeast 2. Microbial enzymes: e.g. Amylase, protease, glucose isomerase 3. Microbial metabolites: A. Primary metabolites: e.g. Ethanol, citric acid, amino acids, vitamins B. Secondary metabolites: All antibiotic fermentations 4. Recombinant products: e.g. Insulin, interferon 5. Bio-transformations: e.g. Steroid transformation 2 Microbial Cell Fermentation Over half of bio-pharmaceuticals are produced by microorganisms Microbial biocatalysts may be wild type or genetically modified strains Hosts used for the production of recombinant pharmaceuticals are approved by: FDA (Food and Drug Administration) or EMEA (European Medicines Agency) Other bio-pharmaceuticals are produced using animal or plant cell cultures Cell cultures share many common principles with microbial fermentation systems Cell Banking Systems Bio-pharmaceutical production cell lines can be preserved for indefinite periods: 1. Small culture volumes mixed with glycerol in Eppendorf tubes are frozen at -80°C 2. Aliquots in small ampoules are immersed in liquid nitrogen 3. Dormant spores in small vials are stored in the refrigerator (spore formers only) 3 Microbial Growth 1. Main Constituents of Growth Media Water Nitrogen sources: nitrogen-containing compounds such as proteins Carbon & energy sources: such as carbohydrates, lipids, and proteins Mineral salts: phosphates, sulfates, potassium, magnesium, manganese & iron Growth factors: may include vitamins, amino acids & nucleotides 2. Examples of Media Categories A. Nutrient Broth: suitable for growth of many bacterial species B. Selective Media: favor the growth of certain microorganisms and prevent others C. Differential Media: distinguish different growing microorganisms 4 3. Natural Raw Materials for Biotechnology: • Growth medium can be formulated based on natural raw materials • These may be: 1. Wastes: unwanted or unusable materials 2. By-products: a secondary product deriving from a manufacturing process • They are mainly of agricultural, industrial or domestic origins • They could be economic nutrient sources for industrial fermentation • Getting benefits from a waste turns it into a by-product (it became utilizable) • Their utilization eliminates a major source of environmental pollution • May require chemical or enzymatic pretreatments to become utilizable • Many of these substrates were successfully converted to: Fuels (e.g. ethanol, methane) Feeds (e.g. single cell protein) Enzymes (e.g. proteases, amylases) Fertilizers (compost) • Examples of natural raw substrates: Nutrient Compound Raw material Origin Carbon Sucrose • Sugarcane Agriculture • Molasses Industry Lactose • Milk whey Industry Fats • Vegetable oil Industry Hydrocarbons • Petroleum fractions Industry Starch & Cellulose • Straw Agriculture • Maize cobs Nitrogen Protein • Soybean meal Industry • Wheat bran • Milk whey 3. Phases of Microbial Growth Normally, growth follows a typical curve with the following phases The shape of the curve varies according to species and medium used Log Number of Cells Time A. Lag phase: the period of adaptation (metabolic regulations) B. Log (exponential) phase: a steadily increase of growth rate C. Stationary phase: biomass remains constant due to: 1. decreasing concentrations of nutrients 2. increasing concentrations of toxins D. Death (decline) phase: accumulated chemicals lyse the cells 7 5. Monitoring Microbial Growth By measuring dry mass (also known as biomass or dry weight) By direct counting of bacterial cells (e.g. by Haemocytometer) By measurement of turbidity (absorbance or optical density) Total Cell Count on a Measuring Bacterial Haemocytometer Grid Growth by Turbidimetry 8 6. Fermentation Parameters Some quantitative parameters are vital in the design of fermentation processes They can easily be derived from experimental data They include: 1. Kinetics of substrate consumption Substrate uptake rate: e.g. in g substrate/l/h 2. Yield which specifies the ratio of amount produced per substrate consumed Product yield: e.g. in g product/g substrate 3. Productivity which specifies the rate of product formation Productivity: e.g. in g product/l/h Specific productivity: e.g. in g product/g cells/h Bioreactors Definition: A device or system in which a biological reaction takes place Could be: flask, bottle, tank or vessel (Fermenter) Supports growth of prokaryotic or eukaryotic cells Fermenter sizes: > I liter up to thousands of liters A typical fermenter consists of three major parts: The culture vessel Supply systems Measurement & control systems Important features of a typical fermenter: 1. Ease of installation 5. Computer controlled operation 2. Ease of sterilization 6. Foam control facility 3. Convenient aeration and agitation 7. Integrated piping system 4. Heating and cooling facilities 8. Multiple entry and exit ports 10 Types of Bioreactors: Packed Bed Reactor Stirred Tank Reactor Fluidized Bed Reactor Types of Bioreactors (continued): 1. Packed Bed Bioreactor: Cells are immobilized on large particles which do not move with the liquid Simple to construct and operate but May suffer from blockages and from poor oxygen transfer 2. Stirred Tank Reactor: A cylindrical vessel with motor driven central agitators Allow efficient distribution of the culture components including gas 3. Fluidized Bed Reactor: A combination of the two most common, packed-bed and stirred tank The substrate is forced upward through the immobilized biocatalyst 4. Photo-bioreactor: A bioreactor which incorporates some type of light source Used to grow small phototrophs such as cyanobacteria and algae Types of Liquid culture systems A. Batch (closed) systems • All the nutrient components are added at the beginning • Involves nutrient consumption and/or toxin accumulation • Leads to decline of the growth rate • Metabolism of the growing organism is always in a changing state B. Fed-batch (semicontinuous) systems • It is a modification of the batch process • Volumes of nutrient may be added during the fermentation • The system remains closed since there is no continuous outflow C. Continuous (open) systems It is possible to achieved a steady microbial metabolic state • Example: A prolonged log phase of growth • Tool: A continuous flow fermenter system • Involves: A continuous input of fresh nutrient media An output of biomass and other products Advantages of batch and fed batch cultures: 1. Easy 2. Fermenter could be used for alternative purposes 3. In case of contamination, one batch is lost Advantages of continuous cultures: 1. Smaller vessels are needed 2. More economic Disadvantages of continuous cultures: 1. Difficult to monitor all environmental factors 2. Foaming 3. Blocking connections by biomass Solid State Fermentation: Definition: Growth of microorganisms (mainly fungi) on moist solid materials (in the absence of free-flowing water) Substrates traditionally used include: a variety of agricultural products such as: Rice, wheat, grains, beans, corn and soybeans Working of A Bioreactor A. Sterilization (1) In situ sterilization: • Bioreactor is filled with the medium • Pressurized steam is injected into the jacket surrounding the reaction vessel • The whole system is heated to about 1200°C for about 20 minutes • This method may destroy or precipitate some medium components (2) Continuous heat sterilization: • Empty bioreactor is first sterilized by injecting pressurized steam • The medium is rapidly heated to 1400°C for a short period B. Aeration: • The upper part of the bioreactor (about 20% of it’s volume) is a vacant space • Compressed oxygen is introduced at the bottom of the bioreactor • The gases released during fermentation pass out through an air outlet at the top C. Inoculation and sampling Involves a gradual scaling up of the culture volume Inoculum → Flask → Bench-top fermenter → Mass production fermenter D. Control systems: Specified sensors allow automated monitoring of various factors including: Temperature, pH, dissolved oxygen, foam formation, etc Setting up an industrial process: Major Steps of a fermentation process involves: 1. Screening 3. Scaling up 2. Optimization 4. Downstream processing 1. Screening: • Bioreactor: Small laboratory glassware (e.g. conical flasks & Petri dishes) • Goal: searching for a suitable biocatalyst • Example: Isolation of a cellulase producer bacterium Selective medium: A formula that contains cellulose as a sole carbon source Cost-effective: Replacement of pure cellulose by cellulose rich raw material 2. Optimization: maximizing the desired property • Includes: 1. Detecting the best concentration of each medium component 2. Optimization of other factors such as pH and temperature 3. Genetic modification of the biocatalyst if necessary 3. Scaling up: • Involves: 1. Cultivation & optimization in a pilot scale fermenter (e.g. 5 liters) 2. Fermentation in a plant scale fermenter (thousands of liters) 4. Downstream processing: • It is the extraction and purification of the desired end product • Needs the skills of biochemists and chemical engineers • It involves: 1. Initial separation of broth into a liquid phase and a solid phase 2. Concentration and purification of the product e.g. by distillation, centrifuging, precipitation & filtration 3. Introducing the product in a stable form e.g. by immobilization or drying Keywords: 1. Fermentation 17. Specific productivity 2. Bio-Processing 18. Bioreactor 3. FDA 19. Fermenter 4. EMEA 20. Packed Bed Reactor 5. Growth factor 21. Stirred Tank Reactor 6. Selective Medium 22. Fluidized Bed Reactor 7. Differential Medium 23. Photo-bioreactor 8. Waste 24. Batch (closed) culture 9. By-product 25. Fed-batch (semicontinuous) culture 10. Lag phase 26. Continuous (open) culture 11. Log (exponential) phase 27. Solid State Fermentation 12. Stationary phase 28. In situ sterilization 13. Death (decline) phase 29. Bench-top fermenter 14. Haemocytometer 30. Pilot scale 15. Yield 31. Plant scale 16. Productivity 32. Optimization Training Exercises: A) List the disadvantages of continuous cultures B) Illustrate diagrammatically a bioreactor (name the type) that can be used in aerobic wastewater treatment. C) Suggest an appropriate scientific term for each of the following statements: 1. Searching for a suitable biological catalyst 2. Maximizing the desired property 3. Waste materials that can be utilized in other applications 4. The use of animal, plant, or microbial cells or enzymes to synthesize, breakdown, or transform materials 5. A laboratory scale bioreactor 6. A fermentation culture during which a substrate is added several times during the run D) Put the mark ( ) or ( x ): 1. All acids or alkalis added to adjust the pH of a medium must be sterilized. 2. Temperature sensitive amino acids can be sterilized by UV radiation. 3. Every primary metabolite is formed by many microorganisms. 4. All naturally produced organic compounds can be used as carbon and energy sources by at least some microorganisms. 5. Solid fermentations could be carried out by batch or continuous methods.