Fermentation

ABSTRACT The hypothesis which states that the simpler the nature of substrate, the faster the rate of cellular respiration of yeast was tested using the smith fermentation tube method. The experiment used six smith fermentation tubes, distilled water and sugar substrates. It composed of six set-ups which used 15ml of 10% yeast suspension, 15 ml distilled water and 15 ml of their assigned sugar substrate namely: starch, lactose, sucrose glucose and fructose respectively. Set-up six was the controlled set-up and did not contain any sugar substrate. The opening of the tube was covered with a cotton ball to prevent oxygen from entering. The set-ups were then observed every five minutes for thirty minutes. The volume and rate of carbon dioxide (CO2) evolved was calculated and recorded. Results showed that set-up 2 which contained the sucrose substrate yielded the highest rate of cellular respiration in yeast (0.46 ml/min.) followed by glucose (0.34 ml/min.), fructose (0.17 ml/min.) and starch (0 ml/min), lactose (0 ml/min.) and distilled water (0 ml/min.) respectively. The hypothesis was rejected but due to the sources of errors conducted by the researchers and the facts presented by the anaerobic respiration equation and other sources, it was accepted and concluded that the simpler the nature of substrate, the faster the rate of cellular respiration of yeast.

INTRODUCTION Living cells need transfusion of energy from outside sources to perform many tasks. A process called cellular respiration is done by living organisms to acquire this energy in the form of adenosine triphosphate or ATP. It is the process of breaking down nutrient molecules to from energy produced by photo synthesizers (Mader, 2010). It can also be defines as the transfer of energy from organic molecules into food and then converted to ATP (employee.heartland.edu). Cellular respiration can be classified as aerobic respiration in which oxygen is required as a reactant along with the organic fuel to produce energy and anaerobic respiration in which oxygen is not required to form ATP. Reece (2011) stated that aerobic respiration is similar to the principle of combustion of gasoline in an automobile engine in where oxygen is mixed with fuel. In this process, food serves as fuel for respiration and releases carbon dioxide (CO2)and water (H2O). This process can be summarized as : Organic Compounds + Oxygen ---------> carbon dioxide + water + energy The input-output equation for each reaction utilizing the use of glucose as a substrate may be written as: Aerobic:
C6H12O6 + 6H20 + 6O2 ------------> 6CO2 + 12H2O + energy (456,000 cal/mol) enzymes Anaerobic:
C6H12O6 ----------------------> 2CO2 + 2C2H5OH + energy ( 24, 000 cal/mol) enzymes C6H12O6 ----------------------> 2C3H6O3 + energy ( 36, 000 cal/mol) enzymes The breakdown of glucose in aerobic respiration is complete and the energy released is greater than that of anaerobic respiration as seen in the equations above (Duka et al., 2009). Anaerobic respiration can further be broken down into two types namely facultative and obligate anaerobes. Another term that is closely related to anaerobic respiration is fermentation, a process that produces small amounts of ATP molecules in the absence of oxygen (Mader, 2010). Fermentation is much the same like anaerobic respiration but without the net removal of electrons. In fermentation, the most common agent is yeast. Yeast uses substrates such as glucose to facilitate anaerobic respiration as seen from the equation above. These glucose substrates vary in their complex structures. Sugars also called saccharides come in three forms: monosaccharides, disaccharides and polysaccharides. Monosaccharides have the chemical formula C6H12O6 and are known to be the most simple sugars while disaccharides have the chemical formula of C12H22O12 and are composed of two monosaccharides joined together by glycocydic bonds (Reece,2011). However, many different configurations exist for each of the two kinds. These different configuration of atoms are called isomers. Isomers of sugars are important to life since organisms have evolved various enzymes to access the energy in each form. Some organisms are therefore better at getting at some forms of sugar than other forms because of the enzymes that they can use. Examples of monosaccharides are glucose, galactose and fructose while sucrose, lactose and maltose are examples of disaccharides. Polysaccharides such as starch and glycogen serves as storage form of energy in plants and animals respectively. If so, a hypothesis was derived which states that "the simpler the nature of substrate, the faster the rate of cellular respiration of yeast." This is due to the reason that monosaccharides are easily taken up during fermentation rather than disaccharides and polysaccharides. This study aimed to determine if the nature of substrates will have an effect on the rate of cellular respiration of yeast by anaerobic respiration under fermentation.
Specifically it aimed to:
1 To explain the effect of different nature of substrates on the rate of cellular respiration of yeast
2 To determine the substrate that would yield the highest rate of cellular respiration. This study was conducted at the Institute of Biology and Sciences (IBS), University of the Philippines Los Baños Campus, Laguna last September 16, 2013.

Materials and Methods To measure the rate of fermentation of yeast with different substrates, the smith fermentation tube method was used. This method involved the construction of six set-ups utilizing smith fermentation tubes, cotton plugs, 10 % yeast suspension, distilled water and five different sugars that differ in complexity, namely : starch, lactose, glucose, sucrose and fructose. In each set-up, 15 ml of their assigned sugar was poured inside the smith fermentation tube, all with a concentration of 10% to be use as substrates for the experiment. 15 ml of distilled water followed and lastly 15 ml of 10% yeast suspension. In Set-up 1, a polysaccharide with a chemical formula of (C6H10O5)n was used. This complex sugar is generally known as starch. In set-up 2, lactose (C12H22O11), a disaccharide made from glucose and galactose was used. Set-up 3 utilized another disaccharide in the presence of sucrose (C12H22O11). This sugar is made from the combination of glucose and fructose. The sugar used in set-up 4 is glucose, a monosaccharide with a chemical formula of C6H12O6. Fructose (C6H12O6), another common monosaccharide was used in set-up 5. Lastly, as the controlled set-up, set-up 6 did not contain any sugar substrate and used distilled water instead. The set-ups were then shaken to blend the mixture together. The opening of the tube was covered with the palm of one hand and was tilted horizontally so that no bubbles were trapped at the closed end of the tube. After it was ensured that no bubbles were formed, a cotton plug was placed at the opening of the tube to prevent oxygen from entering. The set-ups were then set-aside, secured with rubber bands in a upright position. This was done as to avoid having contact with the tube as the body emits energy in the form of heat that may also be a factor in the rate of fermentation. For an interval of five minutes for thirty minutes, the evolution of carbon dioxide (CO2), the height the gas occupied was measured, recorded and tabulated in Table 1. A graph showing the results in Table 1 was made as Figure 1.1 After collecting the data, the final height of the bubbles of each set-up after thirty minutes was used to compute for the volume of carbon dioxide (CO2) using the following equation: Volume of carbon dioxide (CO2) = V= pi r^2h
Where:
r = 4.5mm h = height of bubbles after thirty minutes. To calculate for the rate CO2 evolution in each set-up, the following formula was derived: Rate of carbon dioxide (CO2) production = Volume of CO2 time The calculated results on both computations can be seen in Table 2 and plotted in Figure 2.1 and 2.2 respectively.

Results and Discussion Table 1 shows the volume occupied by CO2 evolved every five minutes for a duration of thirty minutes. In set-up 1, the mixture containing starch, no evolution of gas was seen since the final height of CO2 gas evolved after thirty minutes was zero. This was also true for set-up 2 containing the mixture with lactose sugar. Set-ups 3 , 4 and 5, containing sugars of sucrose, glucose and galactose respectively showed an increase in the volume of CO2 gas evolved every 5 minutes for thirty minutes. In set-up 6, no evolution of CO2 gas was present during the span of time allotted for the experiment since no breakdown of glucose molecules occurred due to the absence of a substrate. The values in Table 1 was plotted using a line graph as seen in figure 1.1. The graph shows that sucrose had the highest rate for yeast metabolism (13.67 ml/min) for a span of thirty minutes. After sucrose is glucose (10.05 ml) and coming third is fructose (5.04 ml). Starch and lactose did not show any sign of having an effect on yeast metabolism within thirty minutes. The values of Table 2 was derived using the formula in V=pir^2h. Table 2 shows the volume and rate of CO2 produced after 30 minutes. In Figure 2.1 and 2.2, it can be observed that set-up 3 containing sucrose had the highest volume and rate of CO2 evolved with the value of 13.67 ml and 0.46 ml/min respectively. This was followed by set-up 4 containing glucose with values of 10.05 ml and 0.34 ml/min respectively. Set-up 5 containing fructose came in third with values of 5.04 ml and 0.17 ml/min respectively. Set-ups 1,2 and 6 containing starch, lactose an distilled water did not show any signs of CO2 evolution thus with values of zero. In Table 1.1, lactose and starch did not release any carbon dioxide (CO2) within thirty minutes because they are complex sugars. Yeast lacks enzymes to accesses these sugars and break their bonds. In set-up 6, yeast cannot produce CO2 since no substrate is present in the mixture which is needed for fermentation. The problem with the results in Table 1 and Figure 1.1 is that, glucose or fructose, a monosaccharide should perform the highest rate of yeast metabolism compared to sucrose, a disaccharide. This is due to the fact that they are simple sugar that are easily available in nature and can be broken down effortlessly rather than disaccharides and polysaccharides. Based on the results of the experiment, the hypothesis, which states that the simpler the nature of substrate , the higher the rate of cellular respiration in yeast, should be rejected but, based on the anaerobic respiration equation and from www.ukessays.com, simple sugars are used in the fermentation of yeast or it is used as starting compound for breakdown to produce CO2 and energy. Disaccharides are also great sources of food for yeast but before they are utilized, they are first transformed into simple sugars and thus begins fermentation. The only possible explanation for the outcome of the experiment is due to the errors conducted by the researchers . Some sources of errors may be the inaccurate amount of yeast suspension or substrates used, the exact time of intervals for recording measurements was not observed, negligence of the researcher while conducting the experiment, occurrence of spills, cotton balls not properly placed and oxygen entered the tube, not all bubbles were removed prior to observation, and constant touching of the set-up that may have resulted to contact with heat that may serve as a variable for the rate of respiration in yeast.

Summary and Conclusion To prove the hypothesis that the simpler the nature of substrate, the higher the rate of cellular respiration of yeast, an experiment using the Smith Fermentation Tube Method was performed. This involved the use of six smith fermentation tubes, 10% yeast solution, distilled water (H2O) and five different sugar substrates of 10% concentration namely: starch, lactose, sucrose, glucose and fructose.
Each set-up comprised of 15ml distilled H2O, 15 ml of 10% yeast solution and 15 ml of 10% glucose solution that is assigned to each set-up except in set-up 6, the controlled set-up, where the substrate used is distilled water. A cotton plug was placed at the opening of the tube after ensuring that no bubbles were formed after mixing the substances together. The set-ups were then set-aside for observation. The level of carbon dioxide gas evolved was recorded every five minutes for a duration of thirty minutes. The values were recorded in Table 1 and plotted in Figure 1.1.
After recording all the values, the volume and rate of carbon dioxide (CO2) after thirty minutes was computed and recorded in Table 2, and was graphed in Figure 2.1 and 2.2. Results showed that set-up 2 which contained the sucrose substrate yielded the highest rate of cellular respiration in yeast (0.46 ml/min.) This was followed by glucose (0.34 ml/min.) , fructose (0.17 ml/min.) and starch (0 ml/min), lactose (0 ml/min.) and distilled water (0 ml/min.) respectively. Upon the results of the experiments, the hypothesis was rejected but due to the facts present in the anaerobic equation for respiration, www.UKessays.com and possible sources of error committed by the researchers, the hypothesis is still accepted. Glucose, a monosaccharide performs the highest rate of respiration for yeast followed by fructose, sucrose, lactose, distilled water and starch respectively. The hypothesis was still accepted since it was evident that the researchers made errors during the experiment and thus the undesired outcome.
It can now be concluded based on the gathered facts that the simpler the nature of substrate, the faster the rate of cellular respiration of yeast. Furthermore, to reach desired results based on scientific claims, the experiment should be done with proper awareness and care.

LITERATURE CITED Duka, I.M.A., Diaz, M.G.Q., and Villa, N.P.O. 2009. Biology 1 Laboratory Manual: An Investigative Approach. 9th ed. Laguna. p.50. Mader, S.S. 2010. Biology. New York: McGraw-Hill Companies Inc. p. 134, 138. Recce J.B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Jackson, R.B. and Campbell, N.A., 2011, Cambell Biology. Ninth Edition. San Francisco: Pearson Education, Inc. p. 164, 70 Doherty, J. and I. Waldron. 2009. Cellular Respiration in Yeast. <http://employee.heartland.edu/hfei/Labs/CellularRespirationProtocol.pdf>. Accessed September 17, 2013. Fermentation . <http://www.ukessays.com/essays/science/fermentation.php>. Accessed September 17, 2013.

Cited: Duka, I.M.A., Diaz, M.G.Q., and Villa, N.P.O. 2009. Biology 1 Laboratory Manual: An Investigative Approach. 9th ed. Laguna. p.50.   Mader, S.S. 2010. Biology. New York: McGraw-Hill Companies Inc. p. 134, 138.   Recce J.B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Jackson, R.B. and Campbell, N.A., 2011, Cambell Biology. Ninth Edition. San Francisco: Pearson Education, Inc. p. 164, 70 Doherty, J. and I. Waldron. 2009. Cellular Respiration in Yeast. <http://employee.heartland.edu/hfei/Labs/CellularRespirationProtocol.pdf>. Accessed September 17, 2013.   Fermentation . <http://www.ukessays.com/essays/science/fermentation.php>. Accessed September 17, 2013.