27 FEB 19 MICROBIAL PHYSIOLOGY PRACTICALS 1 & 2 EXPERIMENT 1 : FACTORS AFFECTING ETHANOL PRODUCTIVITY INTRODUCTION: Ethanol is a commercially valuable product. It can be made by using microbial cells to convert the sugars in a substrate into ethanol in the process of fermentation. Hence to understand the rate at which the ethanol is being produced, or its productivity, the factors that affect growth of the microbes and the metabolic pathways they use must be taken into consideration. Fermentation is an anaerobic process, i.e. A process which occurs in the absence of oxygen. It is a catabolic reaction used by many organisms to produce ATP and is exploited in industry to produce a wide variety commercially valuable products, such as ethanol, from inexpensive starting materials. Microorganisms are cultured in vessels containing growth medium which acts as an inexpensive source of carbon and energy. The microorganisms are kept under controlled conditions. Altering the organism, initial source of carbohydrates and/or growth conditions results in altered products and amounts of those products. The metabolic pathways used in each microorganism to produce the same product may be different. In the case of this experiment, the yeast Saccharomyces Cerevisiae is used and it produces ethanol through the following reaction: C6H1206 -> 2C2H5OH+2CO2 The sugars are broken down into pyruvate during glycolysis (produces 2 ATP) which is then degraded to ethanol in a two-step process: Glucose -> 2 Pyruvate -> 2 Acetaldehyde -> 2 Ethanol + 2CO2 Pyruvate is decarboxylated to acetaldehyde by pyruvates decarboxylase and acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase. To maximise the ethanol production, the growth rate of the microbes must be maximised and oxygen be removed before the start of the death phases of a batch fermentation. This ensures that the maximum number of microorganisms is acting on the substrate to convert as much of it as possible to ethanol. The aim of this experiment is to investigate the factors that affect ethanol production. Before the start of the experiment, standard samples with varying percentages of ethanol were prepared and their CG area counts were measured. This will be used to prepare a standard curve to determine the ethanol content of the other solutions in the experiment. Table 1: Values of % ethanol and Area count for Standard curve % Ethanol 10 5 12.5 2 4.8 1 2.5 0 GC Area Count (x 106) 25 0
27 FEB 19 1.1 EFFECT OF AERATION ON ETHANOL PRODUCTION The production of ethanol using Saccharomyces Cerevisiae is not strictly an anaerobic process. There is an optimum level of oxygen concentration for this yeast to product ethanol. In this part of the experiment, flasks are filled in volumetric flasks at different volumes of media so that the different surface areas in each flask in contact with air will mean each sample will have a different level of aeration. The 100% volume flask will have a smaller surface area in contact with the oxygen in the air and hence have the