Fermentation and Respiration - Laboratory Study 3 | HBIO 111 | Lab Reports Biology

General Biology Laboratory BIO 111 Morehouse College

Laboratory Study 3

Fermentation and Cellular Respiration

Objectives

1. Demonstrate the production of carbon dioxide by anaerobic fermentation

2. Evaluate the rate of fermentation as a function of yeast cell concentration

3. Evaluate the inhibitory effects of kitchen spices on fermentation

Introduction

At the cellular level, respiration is the oxidation of food materials to carbon

dioxide and water, which required an input of atmospheric oxygen. This process is the

major source of energy, in the form of ATP, for cellular processes in aerobic organisms.

Respiration of Glucose

Glucose (C6H12O6) is the most common, directly metabolized, respiratory

substrate. In the process of cellular respiration, glucose is broken-down, oxidized (it

gives-up electrons) in multiple chemical steps that ultimately yield carbon dioxide and

water (in the presence of oxygen), and chemical energy stored in the covalent bonds of

ATP. There are three sequential groups of chemical processes that constitute cellular

respiration: glycolysis, citric acid cycle, and the electron transport chain (or oxidative

phosphorylation). Glycolysis occurs in the cytoplasm of all cells, but the citric acid cycle

and electron transport chain reactions occur exclusively in the mitochondria of eukaryotic

cells. Review the processes of glycolysis, citric acid cycle and electron transport in your

textbook and recall that glycolysis alone results in only partial oxidation of glucose, a

relatively small energy yield, and no direct requirement for atmospheric oxygen. The

complete oxidation of energy containing substrates occurs in the citric acid cycle

occurring in the matrix of mitochondria, and the majority of ATP production is in the

electron transport chain embedded in the inner membranes (cristae) of mitochondria.

Atmospheric oxygen is the final electron acceptor at the end of the electron transport

chain where two electrons (and two protons) are transferred to an oxygen atom yielding

one water molecule. Oxygen provides a means of dumping electrons and thereby

permitting the recycling (oxidation) of the electron carrier molecules NADH and FADH2

which are reduced in glycolysis (NAD >> NADH) and in the citric acid cycle (NAD >>

NADH and FAD >> FADH2).

Fermentation

Louis Pasteur defined fermentation as “la conséquence de vie sans oxygène libre”

(the process of life without oxygen gas) in the 1870’s. What would happen to the

respiration of glucose when a cell is deprived of oxygen gas? The only reaction directly

affected is the very last step in the electron transport chain where electrons are transferred

to oxygen and water is formed. Yet, the inhibition of this dumping of electrons results in

and accumulation of reduced electron carriers (both NADH and FADH2) so all reactions

in the electron transport chain and the citric acid cycle would stop. In the absence of

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Laboratory Study 3

Fermentation and Cellular Respiration

Objectives

Demonstrate the production of carbon dioxide by anaerobic fermentation
Evaluate the rate of fermentation as a function of yeast cell concentration
Evaluate the inhibitory effects of kitchen spices on fermentation Introduction At the cellular level, respiration is the oxidation of food materials to carbon dioxide and water, which required an input of atmospheric oxygen. This process is the major source of energy, in the form of ATP, for cellular processes in aerobic organisms. Respiration of Glucose Glucose (C 6 H 12 O 6 ) is the most common, directly metabolized, respiratory substrate. In the process of cellular respiration, glucose is broken-down, oxidized (it gives-up electrons) in multiple chemical steps that ultimately yield carbon dioxide and water (in the presence of oxygen), and chemical energy stored in the covalent bonds of ATP. There are three sequential groups of chemical processes that constitute cellular respiration: glycolysis, citric acid cycle, and the electron transport chain (or oxidative phosphorylation). Glycolysis occurs in the cytoplasm of all cells, but the citric acid cycle and electron transport chain reactions occur exclusively in the mitochondria of eukaryotic cells. Review the processes of glycolysis, citric acid cycle and electron transport in your textbook and recall that glycolysis alone results in only partial oxidation of glucose, a relatively small energy yield, and no direct requirement for atmospheric oxygen. The complete oxidation of energy containing substrates occurs in the citric acid cycle occurring in the matrix of mitochondria, and the majority of ATP production is in the electron transport chain embedded in the inner membranes (cristae) of mitochondria. Atmospheric oxygen is the final electron acceptor at the end of the electron transport chain where two electrons (and two protons) are transferred to an oxygen atom yielding one water molecule. Oxygen provides a means of dumping electrons and thereby permitting the recycling (oxidation) of the electron carrier molecules NADH and FADH 2 which are reduced in glycolysis (NAD >> NADH) and in the citric acid cycle (NAD >> NADH and FAD >> FADH 2 ). Fermentation Louis Pasteur defined fermentation as “la conséquence de vie sans oxygène libre” (the process of life without oxygen gas) in the 1870’s. What would happen to the respiration of glucose when a cell is deprived of oxygen gas? The only reaction directly affected is the very last step in the electron transport chain where electrons are transferred to oxygen and water is formed. Yet, the inhibition of this dumping of electrons results in and accumulation of reduced electron carriers (both NADH and FADH 2 ) so all reactions in the electron transport chain and the citric acid cycle would stop. In the absence of

oxygen (anaerobic environments), cells are limited to glycolysis alone, but must use alternative means of recycling NAD. This recycling is performed in one of two alternative processes in which the end product of glycolysis, pyruvate is used as an electron acceptor. These anaerobic processes are lactic fermentation and alcohol fermentation. Lactic fermentation occurs in animal muscle cells (including human skeletal muscles) during repeated muscle contraction. Many plant and fungal cells can perform both types of fermentation, but alcohol fermentation is particularly common in yeasts. Some bacteria use lactic fermentation under anaerobic conditions. Although the ATP yield from either fermentation pathway is a fraction of that possible under aerobic conditions, the ATP from fermentation can be used to sustain life processes in some cells. Many bacteria are obligate anaerobes and conduct all their life processes in the absence of oxygen often using fermentation metabolism. There are other anaerobic processes performed only by Bacteria and Archaea which we will not consider at this time. Some cells are obligate aerobes that either lack the enzymes for fermentation or have energy demands too great to tolerate the low energy conditions of fermentation (for example, brain cells). There are many facultative anaerobes that can survive periods of low oxygen conditions by means of fermentation. Anaerobic conditions are very common in liquid (water) environments in which the diffusion of oxygen is often much slower than in the air. In this study, you will be evaluating the rates of alcohol fermentation by live yeast cells. Materials and Methods Fermentation by Yeast Each group will conduct their study in the water bath at your bench. Start by conducting the fermentation experiment in which the main variable is yeast concentration in the reaction tube. After you conduct the initial study, you should conduct a second study in which 1% solutions of commonly used spices (naturally occurring materials that, in the days before refrigeration, were used to preserve foods and prevent spoilage) will be introduced to the reaction mixture. Your objective will be to determine which spices inhibit fermentation and therefore would be the best preservatives against yeasts. In this study, the rate of carbon dioxide production by fermenting yeast, Saccharomyces cerevisiae , will be used to measure the rate of fermentation. Carbon dioxide will be measured in a respirometer apparatus (Figure 1) in which the increasing volume of gas in the graduated pipette will be recorded during a 20 minute period. You will be able to evaluate a number of variables that might influence the rate of fermentation, concentration of yeast, and the presence of spices. What hypotheses will you be testing when you conduct this experiment with different yeast concentrations? What are the predicted results for each hypothesis? What hypotheses will you be testing when you conduct this experiment with different spices? What are the predicted results of each hypothesis?

Graph your results in Excel and give your graph a descriptive title and prose legend. What is the dependent variable (plotted on the y-axis)? What is the independent variable (plotted on the x-axis)? What were the fermentation rates for each treatment? Post your results on the chalkboard.
Which treatment had the fastest fermentation rate? Which had the slowest rate of fermentation? Which of your original hypotheses about the effect of yeast concentration on fermentation rate can you reject? Procedure: Effects of Spices on Fermentation Set-up a control and three different concentrations of a given spice by replacing deionized water with the spice solution in each treatment (see Table 1). Collect data in the same manner as you did for the yeast concentration experiment and record data for a 20 minute period. How do your results compare to those you obtained in the first study? Which spices inhibited fermentation? Are some spices stimulators of fermentation? Assignment Prepare a Results Summary of the findings of your entire class. Literature Cited Morgan, J.G. and M.E.B. Carter (2002) Investigating Biology , 4th^ edition, Benjamin Cummings, NY, NY. Acknowledgements Parts of this study were reprinted with revision from H. Ikuma, P. Harley and C. Yocum (1985) Respiration and Photosynthesis, in Biology 105 Course Pack, Division of Biological Sciences, University of Michigan, Ann Arbor. The experimental design is based on Morgan, J.G. and M.E.B. Carter (2002) Investigating Biology , 4th^ edition, Benjamin Cummings, NY. Revisions made 10/2004 by L. Blumer.

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Laboratory Study 3

Fermentation and Cellular Respiration