InBt121 Bioprocess I - Batch Fermentation 2024 PDF

InBt121 Bioprocess I Pretest: Type Your Answers in the VSUEE Instructions: TRUE or FALSE. Identify the statement if it is TRUE or FALSE if the statement is not correct. 1. Continuous culture is carried out in a closed culture system which contains an initial amount of nutrients. 2. During the s...

InBt121 Bioprocess I Pretest: Type Your Answers in the VSUEE Instructions: TRUE or FALSE. Identify the statement if it is TRUE or FALSE if the statement is not correct. 1. Continuous culture is carried out in a closed culture system which contains an initial amount of nutrients. 2. During the stationary phase, the microorganism appears to have no growth but there is only minimal increase in cell density 3. In batch culture, the log phase or exponential phase is when the microorganism is growing at its maximum specific growth rate. 4. Fed-batch culture is similar to the batch fermentation process but there is an extension of the batch culture by feeding periodically with medium with no removal from vessel. 5. In continuous culture, the nutrient feed should be equal to the unused nutrient of the vessel. Review: Bioreactor A vessel or container in which a biochemical process is carried out containing organisms or biochemically active substances derived from such microorganisms in an aerobic or anaerobic process.  It is used to convert any starting material ( or raw material or substrate into some product. The conversion occurs through the action of a biocatalyst- enzymes, microorganisms, cells of animals and plants, or subcellular structures such as chloroplasts and mitochondria.  The starting substrate may be a simple organic chemical (e. g. sugar and penicillin), an inorganic chemical such as carbon dioxide, or a poorly defined complex material such as meat and animal manure. Different bioreactor designs are needed to accommodate the great diversity of substrates, products and biocatalysts and the different requirements of the different bioconversion processes. Parts and Functions of Bioreactor: 1. Cooling Jacket The fermenter is fitted externally with a cooling jacket through which steam (for sterilization) or cooling water (for cooling) is run. Cooling jacket is necessary because sterilization of the nutrient medium and removal of the heat generated are obligatory for successful completion of the fermentation in the fermenter.  For very large fermenters, insufficient heat transfer takes place through the jacket and therefore, internal coils are provided through which either steam or cooling water is run.  2. Aeration System: Sparger and Impeller. It is one of the most critical part of a fermentor. In a fermenter with a high microbial population density, there is a tremendous oxygen demand by the culture, but oxygen being poorly soluble in water hardly transfers rapidly throughout the growth medium.  The sparger is typically just a series of holes in a metal ring or a nozzle through which filter-sterilized air (or oxygen-enriched air) passes into the fermenter under high pressure. The air enters the fermenter as a series of tiny bubbles from which the oxygen passes by diffusion into the liquid culture medium. 2. Aeration System The impeller (also called agitator) is an agitating device necessary for stirring of the fermenter. The stirring accomplishes two things: (i) It mixes the gas bubbles through the liquid culture medium and (ii) It mixes the microbial cells through the liquid culture medium. In this way, the stirring ensures uniform access of microbial cells to the nutrients.  The size and position of the impeller in the fermenter depends upon the size of the fermenter. In tall fermenters, more than one impeller is needed if adequate aeration and agitation is to be obtained. Ideally, the impeller should be 1/3 of the fermenter's diameter fitted above the base of the fermenter. 3. Baffles The baffles are normally incorporated into fermenters of all sizes to prevent a vortex and to improve aeration efficiency. They are metal strips roughly one-tenth of the fermenters diameter and attached radially to the walls. 4. Controlling Devices for Environmental Factors In any microbial fermentation, it is necessary not only to measure growth and product formation but also to control the process by altering environmental parameters as the process proceeds. For this purpose, various devices are used in a fermentor.  Environmental factors that are frequently controlled includes temperature, oxygen concentration, pH, cells mass, levels of key nutrients, and product concentration. Use of Computer in Fermenter Computer technology has produced a remarkable impact in fermentation work in recent years and the computers are used to model fermentation processes in industrial fermentors. Integration of computers into fermentation systems is based on the computers capacity for process monitoring, data acquisition, data storage, and error- detection.  Some typical, on-line data analysis functions include the acquisition measurements, verification of data, filtering, unit conversion, calculations of indirect measurements, differential integration calculations of estimated variables, data reduction, tabulation of results, graphical presentation of results, process stimulation and storage of data.  Module 2: Microbial Growth Kinetics of Culture Fermentation Lesson 2.1: Batch Culture Process Lesson 2.2: Fed-batch Culture Process Lesson 2.3: Continuous Culture Lesson 2.1 Batch Culture Process Learning Outcomes 1. Define the batch culture fermentation 2. Understand the microbial growth kinetics 3. Familiarize the advantage and disadvantage of batch culture fermentation Batch Culture A type of culture operation in a closed culture system which contains an initial limited amount of nutrient. As the inoculated culture or inoculum cells grow, it will undergo several phases in the growth curve. The bioreactor is first charged with medium, inoculated with cells, and the cells achieve the required density or optimum product concentrations.  The bioreactor contents are discharged, and the bioreactor is prepared for a fresh charge of medium. The Stages of Batch Fermentation Shake Flask Seed Fermenter Production Fermenter  All nutrients are provided at the beginning of the cultivation and products are removed at the end. Nutrients, cells, reagents-----------------→PRODUCT Advantages Short duration Less chance of contamination as nutrients are added at the beginning Separation of batch material for traceability Easier to manage Disadvantages Product is mixed in with nutrients , reagents , cell debris, and toxins. Shorter productive time. Productivity is low.  Microbial Growth Phases Associated with Batch Fermentation Lag Phase Exponential Phase Stationary Phase Death Phase https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002/section002/black/page001.html Operation is thus characterized by three periods of time Filling period, Cell growth and cell production period Final emptying period Phases of Batch Culture Process: 1. Lag Phase It is the first major point of microbial growth in batch culture process. After the inoculation of microorganisms into the fresh medium, it will undergo a period that may be suspected that there is no growth taking place. Still, there is only a minimal increase in cell density.  1. Lag Phase It is a period of adapting or adjusting to the cells’ new environment. In some fermentation, it is not taking place.  On a commercial scale, the lag phase duration should be shortened as much as possible, and it can be resolved by using a suitable inoculum. dX/dt and dS/dt are essentially zero. Yield Coefficient and Specific Growth Rate  The lag phase is an adaptation period, where the bacteria are adjusting to their new conditions. The length of the lag phase can vary considerably, based on how different the conditions are from the conditions that the bacteria came from, as well as the condition of the bacterial cells themselves.  Actively growing cells transferred from one type of media into the same type of media, with the same environmental conditions, will have the shortest lag period. Damaged cells will have a long lag period, since they must repair themselves before they can engage in reproduction.  This process could include the repair of macromolecular damage that accumulated during stationary phase and the synthesis of cellular components necessary for growth. 2. Log or Exponential Phase Growth rate of the cells gradually increases, Cells grow at a constant, maximum rate. Nutrients are in excess, and the organism is growing at its maximum specific growth rate, µmax, for the optimal conditions. 2. Log or Exponential Phase The exponential growth of a single-celled organism is replicated through binary fission. The growth of microorganisms will continue indefinitely in the culture. However, when the growth of microorganism and the excretion of the microbial product depends on the consumption of nutrients dramatically affects the organism‘s growth.  Hence, after a particular time, the growth rate of the culture decreases until growth stops. The termination of growth is due to the exhaustion of some essential nutrients in the medium, toxin accumulation (toxin limitation), or the combination of the two. 2. Log or Exponential Phase Cells have adjusted to their new environment The cells are dividing at a constant rate resulting in an exponential increase in the number of cells present. This is known as the specific growth rate and is represented mathematically by first order kinetics as the following: 𝑑𝑥 =(μ-kd)X 𝑑𝑡 where X is the cell concentration, μ is the cell growth rate, and kd is the cell death rate.  The term μ– kd can be referred to as μnet. The cell death rate is sometimes neglected if it is considerably smaller than the cell growth rate.  There are other models used to determine cell growth rate that depend upon inhibition Substrate Inhibition Product Inhibition Toxic Compounds Inhibition The type of inhibition causes mathematical changes in the previously presented Monod equation for batch kinetics Substrate Inhibition In batch fermentation, this can occur during the initial growth phases while substrate concentrations are high If this is a major problem, continuous or fed-batch fermentation methods should be considered Product Inhibition In batch fermentation, this can occur after induction of the recombinant gene 3. Stationary Phase It is the third major phase microbial growth in a batch fermentation process. The growth rate has declined zero but may still metabolically active and produce secondary metabolites. It occurs when the number of cells dividing and dying is in an equilibrium state. It can result in depletion of one or more essential growth nutrients, accumulation of toxin by product.  Occurs when the number of cells dividing and dying is in equilibrium and can be the result of the following: Depletion of one or more essential growth nutrients Accumulation of toxic growth associated by- products Stress associated with the induction of a recombinant gene  Primary metabolite, or growth associated, production stops Secondary metabolite, or non-growth associated, production may continue  4. Death Phase It is the last major phase of microbial growth in a batch fermentation process, which is also known as the decline phase. The rate of cells dying is greater than the rate of cells dividing. The decrease in growth rate and the cessation of growth, due to the depletion of the substrate, may be described by the relationship between µ and the residual growth-limiting substrate. 4. Death Phase Similar to exponential phase, it is represented mathematically by first order kinetics as the following: 𝑑𝑋 =-kdX 𝑑𝑡 Determination of Microbial Growth Curve There are a two main methods primarily used to establish a growth curve. Both of which are represented on the previously shown growth curve. Viable Cell Count Initially lower curve representing the number of cells that are actually viable Determined by plating a sample from the culture Optical Density Initially higher curve representing the number of cells that are both viable and non-viable Determined by taking an optical measurement using a spectrophotometer Optical Density Measuring the optical density with a spectrophotometer is a quick and easy way to develop a growth curve. One takes a sample of the fermentation broth and measures the absorbance at a particular wavelength in the spectrophotometer. For E. coli cells in a typically LB medium, the wavelength used in 600 nm.  The measured value can be compared to previous measurements made in conjunction with cell plating or cell counting. The negative side of using and non-viable cells absorb this wavelength. As a result, the values taken are not representative of only viable cells. Learning Tasks Using a block flow diagram, present the general fermentation process in batch culture with short explanation in your OWN words of each block. Cite references used for the explanation. (10pts) Lesson 2: Fed-Batch Culture REVIEW Learning Outcomes At the end of the lesson, you will be able to: 1. Understand the concepts and principles of fed-batch culture 2. Familiarize the uses, advantages, and disadvantages of the fed-batch culture fermentation. Characteristics of Fed-Batch Culture It is similar to the batch fermentation process However, there is an extension of the batch culture by feeding periodically with medium with no removal from the vessel. A volume of medium is inoculated with the organism and allowed to grow in a period of time.  Subsequently, a fed is initiated into the fermenter when a ‘quasi-steady state is attained in which the growth limiting substrate has exhausted. The supply of growth-limiting substrate or medium will sustain the microorganisms’ growth in fed-batch reactor.  In terms of medium, the initial concentration is relatively low. Then the reactor will then continually be fed with medium concentrated with carbon and nitrogen in increments.  Pump 1 Separator Pump 2 (Biomass X) Feedstock vessel (sterile) Collection vessel Comparison of Batch vs. Fed-batch Process Advantages of Fed-batch culture than batch culture The maintenance of the fed-batch culture is in the low substrate level. The most problem of fermentation is when substrates are fed at a high concentration, repression occurs due to high concentration of the substrate. The productive phase of the fermentation will be extended.  In the cycle fed-batch system, a periodic shift of the growth rate enhances secondary metabolite production as many secondary metabolites are not growth-associated. Kojic acid production is an example of a secondary metabolite produced by Aspergillus oryzae. Advantages of Fed-batch culture than batch culture The product formation in this system depends on the specific growth rate of microorganisms. Nutrients are fed exponentially to obtain optimum desired products by monitoring the specific growth rate of the microorganism. Example: antibiotic production, which is mostly secondary metabolites.  There is a drastic reduction of oxygen transfer and heat because of high viscosity culture broth. Using fed-batch culture, the fed of nutrients is controlled pulse wise followed by the output of a certain volume from the fermenter.  This technique reduces the high viscosity effect of the culture. Biopolymer production usually encounters this problem and fed-batch culture is suited in the fermentation.  Advantages of Fed-batch culture than Batch Culture In extracellular enzyme production, it is controlled by catabolite repression. Enzyme synthesis is inhibited by the presence of readily utilized carbon source or inhibitory carbon source. Using fed-batch culture technique, the inhibitory carbon source is fed gradually to overcome the inhibitory effect for the production of extracellular enzymes. Disadvantages Provides another point of ingress for contamination May produce high cell density numbers and product yields which are difficult to deal within downstream, creating bottlenecks in the whole process. Classification of Fed-Batch Culture Fix volume Fed-Batch Culture Variable volume fed-batch Culture Fix volume fed-batch culture In this type of fed-batch, the limiting substrate is fed without diluting the culture. The culture volume can also be kept constant by feeding the growth limiting substrate in undiluted form, such as a very concentrated liquid. Additionally, the feed is provided at constant rate.  The production of mass of biomass per mass of substrate is constant during the fermentation time. Variable volume fed-batch culture Volume changes with fermentation time due to the substrate feed. The substrate is provided in a manner that maximizes the specific growth rate. The substrate is added exponentially for biomass production to maintain a constant growth rate in a culture growing exponentially.  Meanwhile, this culture considers the limitation of one substrate concentration. The conditions of variable fed-batch culture are; (a) the input of substrate is equal to the consumption of cell, and (b) the input of substrate depends on the specific growth rate and total cell in the culture. Questions Lesson 3: Continuous Culture Learning Outcomes 1. To understand the concepts and principles of continuous culture 2. To determine the microbial growth kinetics of continuous culture 3. To explain the uses, advantages, and disadvantages of continuous culture Continuous Culture Fresh fermentation media is continuously added to the reactor while fermenter broth containing biomass, products and unused nutrient are continuously removed. Exponential growth in batch culture may be prolonged by the addition of fresh medium to the vessel. Growth can be maintained for long duration (medium has been designed such that growth is substrate limited and not toxin limited). Continuous Growth Kinetics The added medium displaced an equal volume of culture from the vessel. If medium is fed continuously to such a culture at a suitable rate, a steady state is achieved eventually Formation new biomass by the culture is balanced by the loss of cells from the vessel. Under this steady state, the µ is controlled by the dilution rate (→ [S]).  Flow rate1 = Flow rate2 Pump 1 Pump 2 Feedstock vessel (sterile) Collection vessel Culture System in Continuous Process Chemostat- Growth of cells is controlled by the availability of the growth chemical component of the medium. Turbidostat- Concentration of cells in the culture is kept constant by controlling the flow of medium such that the turbidity of the culture is kept within certain, narrow limits.  Kinetics of Continuous Culture The flow of medium into the vessel is related to volume of vessel by the term of dilution rate D, defined as; D = F/V ……………………………….1 Where; F: flow rate (dm3/h-1) V: volume (dm3) D :Dilution rate (h-1) The net change in cell concentration over a time period may be expressed as; dx/dt = growth – output ………………………. 2 dx/dt = μx-Dx ……………………………….. 3 During steady state, the rate of cell growth is equal to the rate of cell being displaced or wash out (dx/dt = 0) μx = Dx ……………………………………….4 μ = D …………………………………………5 Under steady state condition specific growth rate (μ) is controlled by dilution rate (D). Continuous culture only operate below maximum specific growth rate (D Growth rate culture washes out Dilution rate < Growth rate culture overgrows Dilution rate = Growth rate steady state culture  Product is harvested from the outflow stream Stable chemostat cultures can operate continuously for weeks or months.  [Substrate] determine the dilution rate. If [Substrate] depleted below level that support growth rate, the following events take place: The growth rate of the cells will be less than the dilution rate and they will be washed out of the vessel at a rate greater than they are being produced, resulting in a decrease in biomass concentration. The substrate concentration in the vessel will rise because fewer cells are left in the vessel to consume it.  The increased substrate concentration in the vessel will result in the cells growing at a rate greater than the dilution rate and biomass concentration will increase. The steady state will be re-established Theoretical Advantage of Continuous Culture Constant condition at steady state, thus productivity will be constant and always maximum. Can be operated for very long time so unproductive time would be minimal Process control is constant allowing process automation Metabolite productivity Disadvantages of Chemostat Strain lost or strain degeneration Industrial strain contain recombinant plasmid or mutant Industrial strain grows inefficiently with low max and normally have high Ks value Mutated strain change to wild type in order to have better growth capacity Wild type will overgrow the culture and industrial strain will be wash out Disadvantages of Chemostat Operational problems Culture mixing is not homogenous Growth on the wall of the fermenter made the problem worse Contamination Problem of equipment reliability Application of Continuous Culture Biomass production Growth associated product or primary metabolite – ex, ethanol, citric acid Not suitable for non-growth associated or secondary metabolite – ex antibiotic Fermentation Conclusion Now that the fermentation process is over, the fermentation broth containing the cells and the extracellular media is removed from the production fermentor. This is called harvesting and that completes the upstream process of fermentation.  After the cells are harvested, the recombinant protein needs to be separated from the cells that produced them. This is accomplished through the downstream process of purification. Learning Tasks 1. Give other fermentation products that are produced by continuous culture fermentation. (5pts) Posttest Instructions: TRUE or FALSE. Identify the statement if it is TRUE or FALSE if the statement is not correct. 1. During the stationary phase, the microorganism appears to have no growth but there is only minimal increase in cell density 2. In batch culture, the log phase or exponential phase is when the microorganism is growing at its maximum specific growth rate. 3. In continuous culture, the nutrient feed should be equal to the unused nutrient of the vessel. 4. Continuous culture is carried out in a closed culture system which contains an initial amount of nutrients. 5. Fed-batch culture is similar to the batch fermentation process but there is an extension of the batch culture by feeding periodically with medium with no removal from vessel. References: https://aasnig.com/fermentor-bioreactor-design-and- its-functions/ https://projects.ncsu.edu/project/actionagenda/copr otein/media/Fermentation.pdf  Thank you and God bless! If any of you lacks wisdom, you should ask God, who gives generously to all without finding fault, and it will be given to you. James 1:5