To determine the most effective type of carbohydrate to produce the most ethanol through anaerobic yeast fermentation.

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To determine the most effective type of carbohydrate (sucrose, glucose, fructose and lactose) to produce the most ethanol through anaerobic yeast fermentation over a 24-hour period.

Introduction and theory:

Saccharomyces cerevisiae, also known as baker’s yeast, is an asexual, unicellular micro-organism belonging to the kingdom Fungi (Explore Yeast, 2013). It is found worldwide on plant surfaces and in soils, flower nectar and fruit, and is the type of yeast that is used to produce beer, bread and wine (Encyclopaedia Britannica, 2014) as has the ability to undergo fermentation (Schneiter, 2004). Yeast contains an enzyme called zymase that acts as a catalyst in the process of fermentation. This is because it has the ability to covert glucose to ethanol (AUS-e-TUTE, 2014). Yeast fermentation is an anaerobic process that begins with glycolysis[1] (Morton, 1980) using the hydrogen carrier, NADH, present in the yeast (see Figure 1). During this process, two ATP molecules are produced for each glucose molecule fermented (Morton, 1980).

Figure 1: Chemical Equation of the Reaction of Glucose to Pyruvic Acid (AUS-e-TUTE, 2014)

Zymase then coverts the pyruvic acid to ethanol and carbon dioxide using the hydrogen carrier NADH (see Figure 2).

Therefore when a carbohydrate such as glucose is converted into pyruvic acid by the enzyme zymase, ethanol, carbon-dioxide and adenosine triphosphate (ATP[2]) (Harris, 2009) are produced as by-products (see Figure 3). ATP is not gained in the fermentation process but rather produced during glycolysis (Morton, 1980).

Yeast fermentation rates can be affected by a number of variables such as pH, temperature, and nutrient type and availability (Encyclopaedia Britannica, 2014). The optimum temperature range for yeast fermentation is 25oC to 35oC (TransFermYield+, 2013) and the optimum pH level is 7.0 (Battcock & Azam-Ali, 2009). The type of carbohydrate present during the fermentation process can affect the rate of CO2 and ethanol production (McShaffrey, 2012). For signs of fermentation, yeast must be allowed at least 24 hours to ferment before measuring the concentration of ethanol and CO2 (McShaffrey, 2012).

Carbohydrates can be separated into two groups: monosaccharides and disaccharides. Both groups are composed of carbon; hydrogen and oxygen (refer to glucose in Figure 1, 2 and 3). Monosaccharides, such as glucose and sucrose, are simple sugars that are composed of single sugar units and cannot be converted into further simple sugars. Disaccharides, such as sucrose and lactose, are simple sugars composed of double sugar units and can be converted into further simple sugars (Kohn, 2013). Monosaccharides, particularly glucose, are easier for yeast to ferment than Disaccharides (Kohn, 2013). This is because each sugar has a different amount of carbon-hydrogen bonds and chemical composition; thus altering the pathway to glycolysis (see Figure 4).

Figure 4: Composition of Monosaccharides and Disaccharides (Bonner, 2010)

Yeasts are able to survive during fermentation because their nutritional requirements are much lower than those of complex, multicellular organisms and are able to metabolise carbohydrates very quickly; thus making it very useful to the biofuel industry (Marietta College, 2013). Biologists in the biofuel industry study the characteristics of yeasts to improve the rate and efficiency of ethanol production and fermentation processes to be more cost efficient and commercially competitive (Marietta College, 2013).

Ethanol, also referred to as a Biofuel, is commonly used as an alternative energy source to fossil fuels, such as Gasoline, which are non-renewable energy sources that greatly contribute to atmospheric pollution and greenhouse gas emissions (McShaffrey, 2012). Ethanol is also known to be used as an antiseptic and in paints and perfumes (Scottish Biofuel Programme, 2014). Gasoline is an unnatural, manufactured mixture that is produced from petroleum (Vermont Department of Health, 2014). Used worldwide as the main source of fuel for motor vehicles, gasoline is very harmful to the earth as it releases the exhaust pollutants carbon monoxide, nitrogen oxide and hydrocarbon into the environment (McShaffrey, 2012). Ethanol is often blended with gasoline to form ‘gasohol’ to create an environmentally-friendly alternative energy source. This has a number of environmental and health advantages as the presence of the oxygen atom in ethanol (refer to Figure 3) allows the gasohol to burn ‘cleaner’ than regular gasoline, leading to reduced emissions of exhaust pollutants. Ethanol is also ‘carbon neutral’, meaning that when CO2 is released, it is burned by the uptake of CO2 from the atmosphere by plants; thus addressing concerns about greenhouse gas emissions (McShaffrey, 2012).

Hypothesis:

As glucose is most efficiently utilised by during fermentation processes, glucose will produce the highest concentration of ethanol and CO2 over a 24-hour period.

Identification of Variables:

Independent

Carbohydrates (fructose, sucrose, glucose and lactose)

Dependant

Ethanol and CO2 (%)

Controlled

Quantity of yeast (5g), quantity of sugar (5g), water temperature (35o) and volume of water (200mL per conical flask)

Apparatus and Materials:

  • 1 x Kettle
  • 1 x Thermometer
  • 4 x Delivery Tubes with stoppers
  • 4 x 300mL Conical flasks
  • 5 x 200mL Beakers
  • 1 x 1L Beakers
  • 800mL boiled water + 300mL + 800 mL tap water (for beakers)
  • 1 x Hydrometer and plastic hydrometer cylinder
  • 5 x Spatula
  • 1 x Electronic Balance
  • 60g Yeast
  • 60g Sucrose
  • 60g Fructose
  • 60g Glucose
  • 60g Lactose
  • 14 x Plain white sticker label

Risk Assessment:

Hazard identified

Risks

Risk score[3]

Control measures

Person/s responsible

Glassware

  • Breaking conical flask, measuring cylinder or beaker, thus injuring self or others

5

  • Safety glasses
  • Lab coat
  • Enclosed shoes
  • Inform teacher or supervisor and dispose of in sharps bin
  • Take care when handling

Students

Boiling Water

  • Spilling or dropping kettle and burning self or others

5

  • Ensure kettle or glassware filled with water are handled with care and are kept away from table edges
  • Ensure safety goggles, gloves and lab coat are worn
  • Inform adult or supervisor and run injury under cold water

Students

Substances in eye or on skin

  • Yeast, sucrose, glucose, fructose and lactose entering eye or coming in contact with skin, causing irritation

3

  • Ensure safety goggles are worn
  • Avoid skin contact
  • If substances are in eyes, flush eyes with water

Students

Electrocution

  • Water from kettle spilling on electrical appliances, thus posing the threat of electrocution

6

  • Ensure electrical points are switched off
  • Where possible, ensure appliances are kept away from water
  • Handle electrical equipment with care
  • Inform teach or supervisor of electrocution

Students

Method:

  1. Using plain white sticker labels, label all conical flasks with the type of carbohydrate that will be placed into it, including sucrose, glucose, lactose and fructose.
  2. Label spatulas with type of material being measured by using sticker labels. These labels should include sucrose, fructose, glucose, lactose, and yeast. This will prevent cross-contamination of materials.
  3. Label all 200mL beakers with beaker number using sticker labels; for instance beaker 1, beaker 2, beaker 3, beaker 4 and beaker 5. Side aside beaker 5.
  4. Construct a data table to record results. This graph should include the type of carbohydrate, % of ethanol produced and trial number 1, 2 and 3.
  5. Fill beaker 1, 2 3 and 4 with 200mL of tap water. Set aside.
  6. Place conical flask labelled ‘Sucrose’ on electronic balance and tare. Measure 5g of yeast in the conical flask using the ‘yeast’ spatula. Tare electronic balance. Measure 5g of sucrose using the ‘sucrose’ spatula. Remove conical flask from electronic balance and set aside. Repeat this step for the three remaining conical flasks, ensuring the type of carbohydrate is changed for each. The end result should be 5g of yeast and 5g of carbohydrate in each conical flask.
  7. Boil 800mL of water in kettle. Once boiled, pour 500mL of water into a 1L beaker. Add 300mL of cold tap water to 1L beaker filled with 500mL of boiled water. Place the thermometer into beaker, ensuring the tip is submerged in water but is not touching the sides of the beaker. Continue to do this until water temperature reaches 35oC. If water temperate decreases, add remaining boiled water to increase temperature.
  8. Line all conical flasks in a straight line. Line beaker 1, 2, 3 and 4 directly behind each conical flask.
  9. Submerge the end of the delivery tube into water of beaker 1. Repeat this step for beaker 2, 3 and 4.
  10. Using beaker 5, measure 200mL of 35oC water and pour into ‘Sucrose’ conical flask. Qucikly place stopper of one delivery tube in conical flask. Ensure the stopper has been pushed firmly into the neck of the flask to prevent gasses from escaping or entering the flask. Repeat this step for ‘Glucose’, ‘Lactose’ and ‘Fructose’ conical flasks. Note time when this step has been completed.
  11. After 24 hours from the time noted after Step 9, remove delivery tube stopper and pour contents of ‘Sucrose’ conical flask into the plastic hydrometer cylinder.
  12. Place the hydrometer into the hydrometer cylinder, spinning it as it placed into the hydrometer, with the bulb end down. When the hydrometer is still and is not touching the sides of the hydrometer cylinder, record results into the data table. Ensure results are read at the meniscus and at eyelevel (see Appendix 1)
  13. Record results into a data table.
  14. Repeat steps 10, 11 and 12 with ‘Glucose’, ‘Lactose’ and ‘Fructose’ conical flasks.
  15. Repeat steps 1 to 14 two more times to obtain a total of three trials. This will ensure accuracy of results.

Diagram of Method:

Bibliography

AUS-e-TUTE, 2014. Fermentation. [Online] Available at: http://www.ausetute.com.au/fermentation.html [Accessed 06 06 2014].

Battcock, M. & Azam-Ali, S., 2009. Yeast Fermentations. [Online] Available at: http://www.fao.org/docrep/x0560e/x0560e08.htm [Accessed 04 06 2014].

Bonner, 2010. E105 Biology of Food. [Online] Available at: http://courses.bio.indiana.edu/L104-Bonner/F10/Part1.html [Accessed 06 06 2014].

Encyclopaedia Britannica, 2014. Yeast. [Online] Available at: http://www.britannica.com/EBchecked/topic/652395/yeast [Accessed 05 06 2014].

Explore Yeast, 2013. What is yeast?. [Online] Available at: http://www.exploreyeast.com/article/what-yeast [Accessed 1 06 2014].

Harris, K., 2009. Yeast Fermentation. [Online] Available at: http://www.hartnell.edu/faculty/kharris/bio10labmanual/labs/Yeast.pdf [Accessed 26 05 14].

Huxley, L., 2005. Biology: An Australian Perspective. 2nd ed. Sydney: Copyright Agency Limited.

Kohn, C., 2013. Fermentation. [Online] Available at: http://www2.waterforduhs.k12.wi.us/staffweb/ag/2011-2012 Courses/Biotech & Biofuels/7. Fermentation/Fermentation.pptx [Accessed 06 06 2014].

LUMCON, 2013. How to use a Hydrometer. [Online] Available at: http://www.lumcon.edu/education/K-12/studentdatabase/hydrometer.asp [Accessed 06 06 14].

Marietta College, 2013. Biol 105 Biofuel Lab Web Resource Page. [Online] Available at: http://www.marietta.edu/~biol/introlab/bfuelrsc.pdf [Accessed 06 06 2014].

McShaffrey, D., 2012. Yeast Fermentation. [Online] Available at: http://www.marietta.edu/~biol/introlab/BioFuel fermentation.pdf [Accessed 26 05 2014].

Morton, J. S., 1980. Glycolysis and Alcohol Fermentation. Acts & Facts, Volume 9, p. 12.

Schneiter, R., 2004. Genetics, Molecular and Cell Biology of Yeast. [Online] Available at: http://www.unifr.ch/biochem/assets/files/schneiter/cours/Yeast/YeastGenetics.pdf [Accessed 28 05 2014].

Science Buddies, 2013. http://www.sciencebuddies.org/science-fair-projects/project_ideas/Chem_p040.shtml#summary. [Online] Available at: http://www.sciencebuddies.org/science-fair-projects/project_ideas/Chem_p040.shtml#summary [Accessed 06 06 2014].

Scottish Biofuel Programme, 2014. Fermentation. [Online] Available at: http://biofuels-scotland.co.uk/technologies/fermentation [Accessed 02 06 2014].

TransFermYield+, 2013. Every Degree Counts. [Online] Available at: http://www.lallemandbds.com/transferm_yieldplus/images/LBDSMascoma_ThermostabilityDocument.pdf [Accessed 06 06 2014].

Vermont Department of Health, 2014. Gasoline. [Online] Available at: http://healthvermont.gov/emerg/gasoline.aspx [Accessed 04 06 2014].

Worthington, K., 2014. Catalase. [Online] Available at: http://www.worthington-biochem.com/ctl/default.html [Accessed 06 06 2014].

Appendix

Appendix 1: How to use a Hydrometer (LUMCON, 2013)

http://www.lumcon.edu/education/K-12/studentdatabase/Images/hydrometer.gifUsing a hydrometer is quite simple. The user must be careful, though, to not break the the hydrometer or the cylinder as they are made from glass. Follow the easy to follow directions below.

http://www.lumcon.edu/education/K-12/studentdatabase/Images/hydrometer_float.gif

  1. Fill the glass cylinder with sample water.
  2. Put the hydrometer with the bulb end down. It will bob up and down in the sample. Note that the sample may overflow from the cylinder.
  3. Assure that the hydrometer is not in contact with the sides of the cylinder and take the reading.

Reading the Hydrometer

Extreme care should be taken when reading the hydrometer; it is very easy to misinterpret the scale. Once the hydrometer has stopped bouncing up and down and the hydrometer is not touching the walls of the cylinder, a reading can be made. Note that a meniscus forms on the neck of the hydrometer. Just as reading the meniscus in a graduated cylinder, the user must take the reading where the plane of water is and not where the water clings up the neck of the hydrometer. See the image to the right. The correct reading of this hydrometer is about 0.982.

http://www.lumcon.edu/education/K-12/studentdatabase/Images/hydrometer_read.gif

Appendix 2: Risk Priority Chart

Risk Priority Chart

Likelihood

Consequences

Insignificant

1

Minor

2

Moderate

3

Major

4

Catastrophic

5

Almost certain 5

6

7

8

9

10

Likely 4

5

6

7

8

9

Possible 3

4

5

6

7

8

Unlikely 2

3

4

5

6

7

Rare 1

2

3

4

5

6

Risk level

9 – 10Extreme risk – STOP – Do something about the risk immediately

7 – 8High risk – Action plan required immediately, senior management attention urgently needed

5 – 6 Moderate risk - Specific monitoring or procedures required, management responsibility must be specified

2 – 4Low risk - Manage through routine procedures


[1] Glycolysis: The first stage in cellular respiration; occurs in cytoplasm and results in the formation of 2 ATP molecules and 2 pyruvic acid molecules.

[2] ATP: It is produced via cellular respiration and is stored in the glucose molecule of a cell to become a more transferrable form of chemical energy (Huxley, 2005).

[3] See Appendix 2

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