Biotechnology Fermentation Types PDF

Biotechnology (PMP-404) Level 4 Pharm D Lecture 2 Dr. Enas Yasser Sultan Lecturer of Microbiology and Immunology  Identify the basic parts of fermenters.  Describe the methods to control the conditions inside the fermenters.  Discriminate between types of fermentation...

Biotechnology (PMP-404) Level 4 Pharm D Lecture 2 Dr. Enas Yasser Sultan Lecturer of Microbiology and Immunology  Identify the basic parts of fermenters.  Describe the methods to control the conditions inside the fermenters.  Discriminate between types of fermentation processes.  Fermenter Specifications Requirements 1) Minimize liquid loss from the vessel 2) Provide operation free from contamination 3) Provide adequate mixing and aeration 4) Maintain a specific temperature 5) Control the pH of the culture 6) Facilitate the growth of a wide range of organisms 7) Allow feeding of nutrient solutions and reagents Biotechnology Biotechnology Biotechnology Biotechnology  1- Submersion technique Fermenter may be: 1- Shake flask fermentations (Laboratory fermentations): conical flasks that are kept on a shaker for required rotations and aeration. - These vessels are normally plugged with cotton wool or a Styrofoam bung to prevent microbial contamination, but this can lead to restricted exchange of gases. Biotechnology  1- Submersion (Liquid-state fermentation) technique 2- Fermenters (industrial processes): fermenters with capacities up to several hundred-thousand liters are used. Biotechnology  Control of Chemical and Physical Conditions 1) Agitation Agitation of suspended cell fermentations is performed in order to mix the three phases within a fermenter. a- liquid phase contains dissolved nutrients and metabolites, b- gaseous phase is predominantly oxygen and carbon dioxide, c- solid phase is made up of the cells and any solid substrates that may be present. Mixing should produce homogeneous conditions and promote nutrient, gas and heat transfer. Biotechnology Fermenter agitation requires a substantial input of energy, including 3 mechanisms: Biotechnology  a- Stirred Tank Reactors (STRs) Contain mechanically moving agitators or impellers within a baffled cylindrical vessel. Baffles are usually flat vertical plates. About 4–6 baffle plates are fitted to the inside vessel walls to aid mixing and mass transfer. Within each vessel, the impeller is connected to an external motor, which drives the stirrer system. Biotechnology  b- Airlift fermenter, ALF (Pneumatic systems) No moving parts and use the expansion of compressed gas for mixing. Liquid movement is initiated by the injection of compressed air at the bottom of the internal or external-loop ALF and the air bubbles expand in the riser causing the upward movement of liquid and initiating its cycling within the fermenter. Liquid circulation in airlift reactors results from the density difference between the riser and downcomer regions. Biotechnology b- Airlift fermenters, ALF (Pneumatic systems) Biotechnology Biotechnology  b- Airlift fermenter, ALF (Pneumatic systems) Advantages: avoid excess heat produced during mechanical agitation, so elimination of corrosion effects generally encountered in mechanical agitated reactors. Biotechnology  c- Deep-jet fermenter (Hydrodynamic mechanisms) Use liquid kinetic energy to mix the fermenter contents, which is achieved by using an external liquid pump for external circulation and reinjection. Biotechnology  Control of chemical and physical conditions 2) Heat transfer Heat is generated in the fermentation due to metabolic activity of microorganisms and mechanical agitation processes. In fermenter design, efficient heat transfer is important in controlling the temperature during the fermentation run. Biotechnology  Most fermentations: heat needs to be dissipated by cooling. Conversely, fermentations that operate above ambient temperature, such as those involving thermophilic organisms, there needs to be an input of heat. Biotechnology  Control of chemical and physical conditions 2) Heat transfer How to achieve it??? Heat transfer is primarily achieved using an outer jacket surrounding the internal phase or via internal coils. Biotechnology  Control of chemical and physical conditions 3) Aeration Some fermentations operate anaerobically, but the majority are aerobic and require the provision of large quantities of normally sterile air or oxygen that must be dispersed throughout the fermenter. Biotechnology  Sterile filtered air or oxygen normally enters the fermenter through a sparger system. To promote aeration in stirred tanks, the sparger is usually located directly below the agitator. Biotechnology  Submersion Operating Modes 1. Batch Closed systems where there are no additions following inoculation, apart from acid/base, air and antifoam. In batch fermentations there is a definite beginning and end to the process. The product is then harvested and the fermenter must be cleaned before restarting the cycle (down-time). Alcoholic beverages, most amino acids, enzymes, organic acids. Biotechnology Advantages: Chance of contamination of culture is minimum. Disadvantage: Slower growth rate because nutrient levels decline with time. Increased non-productive down-time Batch-to-batch variability of the product Less efficient as the fermenter is not in operation all the time. Biotechnology Biotechnology  2. Fed-batch It is a semi- closed system in which extra nutrients are added as the fermentation progresses, which increases the fermentation volume and the product(s) remain in the bioreactor until the end of the run. A fresh aliquot of the medium is continuously added without the removal of the culture. eg: Baker’s yeast and penicillin. Phenyl acetic acid is fed into the fermenter continuously but at low rate??? Biotechnology Advantages: This method is useful where a substrate causes viscosity problems or is toxic at high concentrations. Disadvantage: Chance of contamination of culture is higher. Biotechnology  3. Continuous Open system where fresh medium is continuously added and culture broth (containing cells and metabolites) is simultaneously removed at the same rate, resulting in a constant working volume. The system has the property of reaching a steady state in which the concentration of limiting nutrient and the cell number do not vary with time. Biotechnology Advantages: Higher growth rate as nutrients are continuously added to the fermentation tank Disadvantages: High level of contamination occurs, huge volumes of product may be lost. Biotechnology Biotechnology Biotechnology  2- Solid state fermentation, SSF It involves the growth of microorganisms on solid support in the absence or near absence of water. The support used include: cereal grains (rice, wheat, barley, and corn), legume seeds, straws, sawdust (wood shavings). Products include: food (cheeses and mushrooms), fuel, pharmaceutical preparations (antibiotics), enzymes, organic acids and ethanol. Biotechnology Biotechnology  Fungi do not form spores in LSF, but sporulation is often accomplished in SSF. Fungi perform better than bacteria, because of its low moisture requirement. Lack the sophisticated control mechanisms that are usually associated with submerged fermentations. Biotechnology SSF may be employed in the form of: 1.Monocultures: mushroom production, e.g. Agaricus bisporus; 2.Dual cultures: straw bioconversion using Chaetomium cellulilyticum and Candida tropicalis 3.Mixed cultures: as used in composting and the preparation of silage Biotechnology SSF is a multistep process: 1. Pretreatment of a substrate that often requires mechanical, chemical or biological processing to enhance the availability of the bound nutrients and also to reduce the size of the components, 2. Hydrolysis of primarily polymeric substrates (ex.: polysaccharides and proteins) 3. Utilization of hydrolysis products (Fermentation) 4. Separation and purification of end-products. Biotechnology  Advantages of Solid State Fermentation (SSF) It produces a minimum amount of waste and liquid effluent so not very damaging to the environment. Solid substrate fermentation employs simple natural solids as the media. Low technology. No need for sterilization. The yield of the products is high. Bioreactor design are quite simple. Many industrial and agricultural wastes can be fruitfully used in SSF. Biotechnology Course Title 38 General Microbiology  Biotechnology (PMP-404) Level 4 Pharm D Lecture 3 Dr. Enas Yasser Sultan Lecturer of Microbiology and Immunology  Identify the process of solid-state fermentation.  Describe the methods to control the conditions inside the fermenters.  Discriminate between types of the used fermenters.  2- Solid state fermentation, SSF It involves the growth of microorganisms on solid support in the absence or near absence of water. The support used include: cereal grains (rice, wheat, barley, and corn), legume seeds, straws, sawdust (wood shavings). Products include: food (cheeses and mushrooms), fuel, pharmaceutical preparations (antibiotics), enzymes, organic acids and ethanol.Biotechnology  Fungi do not form spores in LSF, but sporulation is often accomplished in SSF. Fungi perform better than bacteria, because of its low moisture requirement. Lack the sophisticated control mechanisms that are usually associated with submerged fermentations. Biotechnology Bioreactors used for SSF Most solid-substrate fermentations are batch processes, although attempts are being made to develop fed-batch and continuous systems. Bioreactors used for SSF Excellent SSF processes, resulting in 1. Effective oxygen transfer, 2. Efficient heat removal, 3. Excellent water distribution and 4. Good substrate mixing with minimal mycelia damage.  1- Tray bioreactor The top of the tray is opened and bottom& sides may be perforated for aeration. Substrates are spread onto each tray to a depth of only a few centimeters (5-15 cm) and then stacked in a chamber through which humidified air is circulated.  Temp. is regulated by circulating worm/cold water as needed. Humidity is controlled b passing dry air around the bed. Scale up is achieved by increasing the area & no. of trays. Uses: production of most fermented foods and enzymes.  2- Bed systems Consisting of a bed of substrate up to 1 m deep, through which humidified air is continuously forced from below.  2.a- Packed bed reactor, PBR Tubular types of reactors which are packed with immobilized microbial cells as biocatalysts and fed with nutrients either from top or from bottom. Operated under conditions of forced aeration (humidified air is continuously forced from below). Heat removal, column may cover with heat jacket OR used heat transfer plates inserted into the beds Uses: engineering of waste water management. 2.b- Fluidized bed reactor, FBR  The mixture of solid particles and gas will behave like a liquid (gas-solid fluidized-bed reactors). Forced aeration is applied at the bottom chamber at sufficient speed to fluidize the solid substrate particles and cause mixing.  Provide continuous agitation with forced air to prevent adhesion and aggregation of substrate particles. Clump breaker. Headspace. Substrate properties is important or NOT? Uses: biomass production for animal feed.  3- Rotating drum bioreactor Cylindrical drum that is semi-filled with a bed of substrate and air passes around the bed. The drum rotate around central axis to mix the beds and mounted on its side onto rollers that both support and rotate the vessel.  It is necessary to limit the substrate bed to achieve good O2 and CO2 (high O2 transfer). might include use of baffles, why????? Uses: enzyme and microbial biomass production. Disadvantage: drum is filled to only 30% capacity, otherwise mixing is inefficient.  Stages of Fermentation process Industrial fermentations comprise both upstream processing (USP) and downstream processing (DSP) stages. I) USP: involves all factors and processes leading to the fermentation and consists of three main areas. a) The producer microorganism: including: 1. The strategy for initially obtaining a suitable microorganism, 2. Industrial strain improvement to enhance productivity and yield, 3. Maintenance of strain purity. b) The fermentation medium: selection of suitable cost-effective carbon and energy sources, along with other essential nutrients. This media optimization is a vital aspect of process development to ensure maximization of yield and profit (the basis of industrial media are waste products from other industrial processes) C) The fermentation process: which is usually performed under controlled conditions, developed to optimize the growth of the organism or the production of a target microbial product. II) DSP: encompasses all processes following the fermentation as (cell harvesting, cell disruption, product purification, finishing process ). It has the primary aim of efficiently and safely recovering the target product to the required specifications (biological activity, purity, etc.), while maximizing recovery yield and minimizing costs.  Products of Fermentation process I) Microbial cells (biomass): Baker’s yeast, Mushroom II) Microbial metabolites: -Primary metabolites -Secondary metabolites III) Microbial enzymes: catalase, amylase, protease, lipase, etc….  Microbial metabolites Primary metabolites: Essential for growth, development, and reproduction of the organism. It is a key component in maintaining normal physiological processes thus, it is often (central metabolite). Produced by all Microorganisms. Primary metabolites are produced during active cell growth (lag, log and stationary). 1ry metabolites are produced in large quantities, and their extraction is easy. Ex. alcohols such as ethanol, lactic acid, citric acids, vitamins, amino acids, proteins, enzymes, nucleic acids, carbohydrates and lipids. Secondary metabolites: Do not play a role in growth, development, and reproduction like primary metabolites do. Not central metabolites. Observed in some Microorganisms. Ecological function, including defense mechanism, by serving as antibiotics, by producing pigments, alkaloids, essential oil and glycosides (steroids and phenolic). 2ry metabolites are produced in small quantities, and their extraction is difficult.  Produced when the cell is not operating under optimum conditions (when primary nutrient source is depleted). Formed during the end or near the stationary phase of growth. Phases of metabolites production in batch culture 1- Trophophase - Culture is nutrient sufficient - Exponential Growth - No Product Formation 2- Idiophase - Carbon limitation - Growth slowing or stopped - Product formation& harvested At the end of this phase. 3- Senescence - Product formation has been stopped. - Degeneration/lysis of mycelium (Fungi, Actinomycetes). - Product degraded. Course Title 42 General Microbiology