Yeast Fermentation in an anabolic Environment
In the event that there is an oxygen deficit within the muscles either from intensive or sustained exercise, they will acquire a large percentage of their energy from Lactic Acid Fermentation. This is an anaerobic form of energy production, meaning that oxygen is not present within the process. Yeast obtains its energy in a nearly identical way, Alcoholic Fermentation. In Lactic Acid Fermentation, the substance Glucose is broken down to form Lactic Acid (Jean Sloat Morton, 1980). The key difference with alcoholic fermentation on the other hand is that glucose is broken down to form a substance acetaldehyde, which breaks down further to form ethanol. A carbon molecule is given off as carbon dioxide also at this stage, and is used to make bread rise. The alcoholic fermentation process has also been used for centuries in the production of alcohol (Portal, 2005).
What Is ATP?
Adenosine Tri Phosphate (ATP) is the main source of energy for cells in all living organisms, whether plant or animal. ATP consists of one molecule of adenosine bonded with three molecules of phosphate. When a cell requires energy it breaks off a phosphate from this chain. The energy released from the breaking of the bond is harnessed by the cells, and phosphate is used in the process. The ATP becomes Adenosine Di Phosphate (ADP). This is when lactic acid fermentation or alcoholic fermentation commences. These processes replace the phosphate in the ADP, to form another ATP for the cells to utilise. (Nave, 2001)
Lactic acid fermentation and alcoholic fermentation are both anaerobic methods of energy production that use the sugar glucose as their primary fuel source. Lactic acid fermentation uses a total of 11 enzymes to turn glucose into lactic acid. Alcoholic fermentation makes use of the same enzymatic pathway as lactic acid fermentation. In alcoholic fermentation however, there is an extra step that turns pyruvic acid into carbon dioxide and ethanol. Instead of the enzyme lactate dehydrogenase which is the final enzyme in lactic acid fermentation that turns pyruvic acid into lactic acid, two different enzymes pyruvate decarboxylase and alcoholic dehydrogenase turn pyruvic acid into carbon dioxide and ethanol. This is the major difference between alcoholic fermentation and anaerobic respiration.
All cells require energy to perform their given tasks. This is an issue for cells, as they are constantly using up their stored energy. For this reason, blood contains a certain level of glucose so as that it is readily available for cells that have depleted their ATP. (Ophart, 2003)
The Chemical reaction for Glycolysis can be displayed by -
C6H12O6 + 2 NAD+ + 2 ADP + 2 P -----> 2 pyruvic acid, (CH3(C=O)COOH + 2 ATP + 2 NADH + 2 H+
The first step of glycolysis is to break down the six carbon molecules that come from glucose into three carbon molecules. This requires two ATP, which then become ADP (Adenosine Di Phosphate). The three carbon molecules then become pyruvate. This creates a net gain of four ATP Molecules. In the process, two NADH’s are produced. (Unknown, 2005)
The Citric Acid Cycle (Krebs Cycle)
The end goal of the Citric Acid Cycle is to get the pyruvate and put it into the Mitochondria so that an NADH and FAD2 are produced.
As the Pyruvate enters the Krebs Cycle, two NADH’s are created, (one per Pyruvate) along with two Co2 molecules (one Per Pyruvate). The Pyruvate has now been converted to Acetyl CoA, or Co-enzyme A. The rest of the Krebs Cycle can be seen in Fig. 1.1.
Electron Transport Chain
The final step in anaerobic respiration is the Electron Transport Chain. The goal of the electron transport chain is to break down the NADH and FAD2 so as that H+ can be forced to the outer of the mitochondria. ATP is produced in this cycle, as hydrogen molecules move through a special enzyme called ATP Synthase. This is possible through a concentration gradient, which can be referred to in Fig. 1.2
The main reason for identifying which substrate produces the most Co2 is for reasons of alcohol production. The alcohol and beverage industry is a multimillion dollar industry, and by finding out which substrate is the most efficient in the fermentation process would significantly benefit the production and time costs of alcoholic beverages.
The hypothesis for this experiment is that there will be a difference in the rate of Co2 production between four different substrates (Glucose, Fructose, Sucrose and Lactose). Glucose will produce the most amount of Co2 as it is the main sugar used by most cells for respiration and fermentation in the first stage of energy production which is glycolysis.
The aim of the experiment is to measure the differences in Co2 production by yeast when given one of four substrates (Glucose, Fructose, Lactose and Sucrose).
It was decided from the beginning of the experiment that the variable being measured was the Co2 Production. This was measured because it provides a clear indication as to how well the yeast is metabolising the substrate that it is immersed in. An uncontrolled variable was the PH of the water the yeast was contained in. This variable was left because most tap water would be at a PH of 7.
An apparatus for measuring production of Co2 via a conical flask and stopper was first set up. Two were set up in unison using a burette clamp. Two 100ml, 10% sugar solutions were made up in a 150 ml beaker at a temperature of 36 degrees Celsius. These were added to the 1 gram of yeast in the flask and let to sit for 5 minutes. The stopper was then placed over the conical flask and Co2 production was measured for a 15 minute time period. The temperature was taken of each solution. The experiment then repeated with the other two sugar substrates.
The results that were acquired were analysed in a number of different ways. Firstly, the mean of the raw data was acquired for each different type of substrate, so as that a more accurate measurement for each individual substrate could be acquired. This was using the formula all scores added/the number of scores. This gave averages for the raw data and provided empirical information and the general idea of what was occurring in the experiment.
The amount of Co2 produced was measured in Mls. This is because it is the only measurement on the gas tube and therefore it is the most accurate way to measure the data.
As can be seen in the graph, the glucose finished at the 15 minute period with a total of 33.8 mls, sucrose was 31.8 mls, fructose 28.4 mls and lactose at 2.4 mls.
Fig.2 shows that lactose finished with the least amount of Co2 produced while glucose produced the highest amount of Co2.
Standard deviation gives scientists a general idea as to how spread data is from the mean. The standard deviations for the substrates are as follows;
Glucose: mean – 33.8 mls / Standard Dev. 2.241 mls
Fructose: mean – 28.4 mls / Standard Dev. 1.545 mls
Sucrose: mean – 31.8 mls / Standard Dev. 1.621 mls
Lactose: Mean – 2.4 mls / Standard Dev. 0.917 mls
Rate of Change (Mls/Min)
Glucose – 2.3 mls
Fructose – 1.9 mls
Sucrose – 2.12 mls
Lactose – 0.16 mls
The results show glucose to be the most promising substrate in terms of Co2 production with a total production of 33.8 mls after 15 minutes. Lactose on the other hand was found to be the least efficient at producing Co2 after 15 minutes, with a total production of 2.4 mls. This raises the question as to why Lactose is such an inefficient substrate in the alcoholic fermentation process? The reason for this is, for yeast to be able to break down lactose into glucose and galactose, they must produce a special type of enzyme for the job. This enzyme is Beta-Galactosidase, or lactase. Yeast also doesn’t have carrier proteins for lactose, so it is unable to be used in the normal enzymatic pathway. This means that the yeast will simply die, as there is no energy source for it to be able to metabolise and produce energy.
A way for yeast to be able to metabolise lactose would be to breed a strain that produces the enzyme Lactase. This means that the yeast could break down the lactose into glucose and galactose, thus enabling it to use the Glucose as its energy source. (ROGOSA, 1948)
Glucose as the most efficient substrate
Glucose was found to be the most efficient source of energy for the yeast, producing the highest yield of Co2 of the three other substrates. Glucose is a Monosaccharide which means it is only made up of one type of sugar (Glucose). Sucrose and lactose are Disaccharides meaning they are made up of two sugars. Disaccharides require the most energy to be broken down, as the bonds between the two simple sugars need to be broken before anything can be metabolised. Glucose is able to be metabolised by the yeast very efficiently due to the fact that it is already in its simplest form, and ready to be metabolised by the yeast. Using the fermentation of fructose as a substrate, yeast must break it down into sucrose and glucose, and then break the sucrose into glucose. This requires energy on behalf of the yeast, which is why it was the third most inefficient with a production rate of 28.4mls. For the simple fact that Glucose is the only sugar used in Glycolysis, the first stage of anaerobic respiration, it was able to be utilised by the yeast the fastest. (Unknown, What are the differences between Monosaccharides and Disaccharides?, 2009)
The main variable that was left uncontrolled was the temperature of the yeast/sugar solution during the alcoholic fermentation process. This means that if one day had a colder room temperature than another day, the yeast/sugar solution may reach a temperature where alcoholic fermentation ceases. To control this, aluminium foil can be placed around the 150ml flask while the experiment is being performed. This would insulate the solution, and sustain the temperature so the yeast can survive for the duration of the experiment.
Another variable that could the controlled is the amount of sunlight and UV rays that the yeast/sugar solution receives. When yeast is exposed to UV radiation, mutation or even cell death can occur (J Svihla, 1960). This cell death and mutation could affect the amount of Co2 that is produced as there is less yeast to ferment the available substrate. To control this, the experiment could be performed in a room where there was no natural sunlight to ensure that there is the maximum amount of yeast in the solution.
The tests conducted and the information and analysis provided in this report demonstrate that the source of energy producing the highest amount of Co2 in yeast fermentation is glucose; producing 33.8 mls of Co2 in a 15 minute time period. Lactose is not a viable option for any type of fermenting process involving yeast and should be avoided. The implications of these results are that glucose is the most efficient source of energy for organisms and should be the main substrate used in brewing and baking.
The results confirmed and supported the original hypothesis, and demonstrated that Glucose is the most efficient energy source for Alcoholic Fermentation.
References and further reading
J Svihla, F. S. (1960). Effects of irradiation on yeast cells. Retrieved from Jstor: http://www.jstor.org/discover/10.2307/3570864?uid=3737536&uid=2129&uid=2&uid=70&uid=4&sid=21102157262621
Jean Sloat Morton, P. (1980). Glycolysis and Alcoholic Fermentation . Retrieved from Institute for creation research.
Nave, R. (2001). ATP. Retrieved from Hyper Physics: http://hyperphysics.phy-astr.gsu.edu/hbase/biology/atp.html
Ophart, C. E. (2003). Glycolysis, A summary. Retrieved from Virtual Chembook.
Portal, E. (2005). Lactic Acid & Alcoholic Fermentation: Comparison, Contrast & Examples. Retrieved from Education Portal.
ROGOSA, M. (1948). Mechanism of the fermentation of Lactose By Yeast. UNKNOWN: Bureau of Dairy Industry US.
Unknown. (2005). Glycolysis, Krebs Cycle, and other Energy-Releasing Pathways. Retrieved from uic.edu: http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect12.htm
Unknown. (2009). What are the differences between Monosaccharides and Disaccharides? Retrieved from WiseGeek: http://www.wisegeek.com/what-are-the-differences-between-monosaccharides-and-disaccharides.htm