Labster Virtual Lab Manual Fermentation.pdf

Virtual Lab Manual Fermentation: Optimize bio-ethanol production Synopsis You will experiment with the growth conditions of the yeast Saccharomyces cerevisiae in order to produce as much bio-ethanol as quickly as possible. T...

Virtual Lab Manual Fermentation: Optimize bio-ethanol production Synopsis You will experiment with the growth conditions of the yeast Saccharomyces cerevisiae in order to produce as much bio-ethanol as quickly as possible. The production of ethanol through fermentation by the yeast is widely used to produce alcoholic drinks such as beer and wine. It can also be harnessed to sustainably produce bio-ethanol that could be used as fuel. Setting up your experiment Your mission will be to optimize bio-ethanol production on a pilot scale. After setting up the bench-top fermenter or bioreactor, you will add a yeast inoculum and run a fermentation experiment. Dr. One will guide you through the first experiment, including how to work aseptically and the first set of culture conditions. Once you are familiar with the equipment you can experiment freely with the fermentor. Pilot scale fermentation Because you are working in the virtual lab, you will be able to gather data very quickly. This allows you to test many different growth parameters and see the effects of different combinations of temperature, gas composition, level of stirring, and pH in a matter of minutes. 1 Copyright Labster ApS 2020 All Rights Reserved Qualitative data analysis Once you have performed a number of fermentation experiments, you will compare the results. Dr. One will guide you through qualitative analysis of key parameters of the fermentation process. Can you decide together which culture conditions should be used for optimal bio-ethanol production on an industrial scale. Learning Objectives At the end of this simulation, you will be able to… Simulate yeast batch fermentation Outline the principles of fermentation and its applications Summarize the principal components of a fermentor and their function Experiment with the effect of temperature, pH, gas, and agitation on fermentation Analyze growth curves qualitatively to identify optimal growth parameters Techniques in Lab Aseptic Technique Bioreactor or fermentor Theory Ethanol fermentation Fermentation is an anaerobic pathway of producing energy for the cells from sugars. Lactic acid or alcohol are produced as byproducts. Fermentation can be harnessed to produce dairy products such as yogurt using specific strains of bacteria or alcoholic drinks using yeast such as Saccharomyces cerevisiae. The carbon dioxide that is also produced is used in breadmaking. In alcohol fermentation, yeast converts pyruvate into ethanol (ethyl alcohol), releasing carbon dioxide in the process. There are two steps involved in pyruvate conversion: Release of carbon dioxide from pyruvate, which is converted to the two-carbon compound acetaldehyde Acetaldehyde reduction by NADH to ethanol. These steps provide a continuous supply of NAD⁺ for ATP generating glycolysis. 2 Copyright Labster ApS 2020 All Rights Reserved Figure 1: Alcohol fermentation pathway. Bioreactor or fermentor A bioreactor is a vessel in which microorganisms can act upon a given substrate in a controlled environment. The substrate is biochemically processed by the microorganism, and the resulting product can be harnessed. Sometimes, the word fermentor is used instead of bioreactor, because the vessel can be used for fermentation. Figure 2: Fermentor diagram. 3 Copyright Labster ApS 2020 All Rights Reserved Fermentation preparation Inoculum To start a fermentation, you need a preculture with a certain number of healthy cells as an inoculum. A culture used to inoculate (inoculum) for fermentation should meet the following criteria: Healthy and in an active state to minimize lag phase time Free of contamination Suitable morphological form Available in sufficient volume Retain its product-forming capabilities Avoid contamination One of the worst things that can happen during fermentation experiments is to detect contaminants. Contaminants are unwanted microorganisms that compete with the original strain over nutrients, and disrupt the experiment. Contaminants can be detected microscopically, or from the dataset. A contaminant will often grow rapidly, thereby giving rise to very high CO₂-levels, and a high biomass concentration relative to the product. It is extremely important to prevent contamination; therefore, the bioreactor must be sterilized before starting with the fermentation. Fermentors or bioreactors can be sterilized with or without the medium. Bioreactors are normally sterilized by heating the jacket or coil of the bioreactor using steam in the vessel. For sterilization, steam pressure inside the vessel is 15 psi and held for 20 min. After sterilization is complete, it is important to maintain positive pressure inside the vessel; otherwise, a vacuum may develop and unsterile air be drawn into the vessel. Fermentation process The key for an effective fermentation is to control and maintain the microbial growth and metabolite production inside the fermentor or bioreactor. To achieve sustainable microbial growth and metabolite production, certain parameters must be controlled. Those parameters are as follows: Sterilization of media and vessel Sufficient substrate and growth factors (e.g., vitamins) in media Agitation (stirring) Temperature control pH control Anti-foaming (During the fermentation foam is formed, which can disrupt the measuring equipment) Control overflow (used when media are added continuously) Aeration (oxygen supply for aerobic processes) 4 Copyright Labster ApS 2020 All Rights Reserved It is very important to perform fermentation using sterile technique. Contaminants compete with the production organism for nutrients and lead to a decreased product output. Batch culture growth A batch culture is a closed-culture system which contains an initial, limited amount of nutrients, hence the exponential growth is limited to a few generations. Figure 3: Yeast growth phases. The phase of microbial growth in a batch culture is generally divided into four phases; the lag phase, log phase or exponential phase, stationary phase and death phase. During the lag phase, microbes are growing and adapting to the new environment so the biomass does not increase significantly. During the exponential phase, the cells are at their most active and consume large amounts of nutrients, hence maximum biomass is achieved. The limited amount of nutrients will eventually lead to nutrient depletion and growth rates will decrease and become zero. When the number of dying cells is greater than the number of cells generated, the biomass will decrease. This is the death phase. Saccharomyces cerevisiae grown with excess sugar will show two distinct exponential phases with different growth rates. This phenomenon is called the diauxic shift. 5 Copyright Labster ApS 2020 All Rights Reserved