TKP4105/TKP4110
Hydrogen peroxide decomposition
by Baker’s yeast
Report
Audun F. Buene
audunfor@stud.ntnu.no
Elise Landsem
elisel@stud.ntnu.no
Group B19
Supervisor: Naresh Doni Jayavelu
Lab: K4-317
Date: 18th of September 2012
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TKP4105/TKP
Hydrogen peroxide decomposition
by Baker’s yeast
Report
Audun F. Buene
audunfor@stud.ntnu.no
Elise Landsem
elisel@stud.ntnu.no
Group B
Supervisor: Naresh Doni Jayavelu
Lab: K4-
Date: 18th of September 2012
Summary
In this experiment, the enzyme catalysed reaction of hydrogen peroxide decompo- sition was investigated. The parameter that was altered in order to investigate the enzymatic acticvity was the concentration of yeast. It was found that the initial rate of decomposition increased with increasing concentration of yeast, following the linear approximation for a volume of 30 mL: rH 2 O 2 (gyeast) = 5 · 10 −^4 · gyeast − 4 · 10 −^6.
1 Introduction
This experiment was conducted as a part of TKP4110 Chemical reaction tech- nique, at NTNU in the autumn of 2012. The main goal was to investigate the kinetic properties of Baker’s yeast in the process of decomposition of hydrogen peroxide to water and oxygen. This was done by measuring the volume of oxygen produced, which is directly related to the speed at which the hydrogen peroxide was decomposed. The variety of enzymes expressed in yeast makes this a suitable microfactory. In the experiment, the yeast consentration was altered in order to investigate its effect on the initial decomposition rate of H 2 O 2. From the experi- mental data, the initial reaction rates of the decomposition were calculated.
2 Theory
2.1 Decomposition process
Yeast is a eukaryote singlecellular microorganism. It has got a rich variety of enzymes, in order to get nutrition as well as to protect itself. Yeast is a lot less complicated than other eukaryotes, but all the more interesting because it has a lot of similar enzymes as its fellow and more complex eukaryotes. For instance the enzyme, catalase, that catalyse the reaction in which hydrogen peroxide is converted to water and oxygen.
2H 2 O 2 → 2H 2 O + O 2 (2.1)
The reason why the reaction is catalyzed by catalase is that the structure of the enzyme is made to match with the molecular structure of H 2 O 2. The active sites induce the breaking of chemical bonds, and promote the making of new ones. En- zymes lower the activation energy of chemical reactions by promoting a different reaction mechanism, and this is the reason for the increase in the initial reaction rates. These enzymes can not be consumed or altered in the reaction, which is the definition of a catalyst. [^3 ]
The initial reaction rate of this reaction will be the objective of this experiment, with respect to the consentration of yeast. There will be other factors to consider, conducting an experiment with living cells, for instance the effect of change in temperature, the concentraion of H 2 O 2 , pH of the mixture or other enzymes and reactions also occuring in the same batch. These effects are not to be investigated during this experiment.
The yeast is a living organism, so its activity will vary depending on a number of different factors. Therfore it is necessary to check the catalytic activity of the yeast that will be used. This test is explained in Section 3.2.
sa, is given by:
sa = sy ·
√ (^) n n · ∑n i=1(x^2 i^ )^ −^ (
∑n i=1 xi)^2
At last the error in the intersection of the linear estimate with the y-axis, sb, can be calculated from the following equation:
sb = sy ·
√ (^) ∑n i=1(x^2 i^ ) n · ∑n i=1(x^2 i )^ −^ (
∑n i=1 xi)^2
3 Method
The experiment was conducted as described in the document Hydrogen peroxide decomposition by Baker’s yeast[^1 ].
3.1 Apparatus
The reaction was run in a 50 mL round bottom flask. In order to measure the volume of gas developed during the reaction, a frictionless syringe was used, a glass syringe with a frictionless piston. It was connected to the reaction flask via a tube, just as the reaction was initiated.
Figure 3.1: Drawing of the experiment setup.
3.2 Preliminary test
A 250 mL sample of yeast suspension was produced in a volumetric flask. 3. grams of dry yeast was used, which amounts to 0.010432 g yeast/mL suspension. The yeast suspension was shaken untill the yeast was homogenously distributed. In order to get an idea about the activity of the yeast, a preliminary test of the
4 Results
The complete set of measurements are found in Appendix E.
4.1 Preliminary test
For the preliminary test it took 28.15 seconds for the reaction to produce 10 mL of O 2 -gas. This was a bit too fast, so all the volumes of yeast suspension were halved. This was done in order to get more accurate measurements.
4.2 Rate of decomposition
The initial rate of decomposition of H 2 O 2 as a function of the weight of yeast was found using a linear approximation from Figure 4.1. The data for the plot is shown in Appendix D. The initial decomposition rate for a 30 mL solution was found to be: rH 2 O 2 (gyeast) = 5 · 10 −^4 · gyeast − 4 · 10 −^6 (4.1)
Figure 4.1: All the initial reaction rates plotted as a function of the weight of yeast in the reaction mixture. A linear approximation to the curve has been added.
4.3 Statistical analysis
A statistical analysis was preformed on the measurements using the formulaes un- der Section 2.3. This gave the following errors for the inital reaction rate, sy, slope, sa and intersection with the y-axis, sb:
sy = 9. 9211 · 10 −^6 [mol H 2 O 2 /s]
sa = 1. 5568 · 10 −^4
[
mol H 2 O 2 /s gyeast
]
sb = 8. 3849 · 10 −^6 [mol H 2 O 2 /s]
from the system, corrupting the results. Also the connecting of the syringe to the reaction flask affected the volume, and made the syringe jump a tiny amount. During the experiment, as the initial reaction rate increased the size of the syringe was changed. This may have led to a difference in accuracy of the measurements.
6 Conclusion
A higher concentration of yeast gave a higher initial reaction rate. This is due to a higher number of active sites available. The relationship between the concentration of yeast and the initial reaction rate was found to be approximately linear, following the linear approximation for a 30 mL solution: rH 2 O 2 (gyeast) = 5 · 10 −^4 · gyeast − 4 · 10 −^6. It was also observed how efficient the yeast was in decomposing of the hydrogen peroxide. This experiment gave a good insight into how great some enzyme activities are, even when using living cells.
Trondheim, October 15, 2012
Elise Landsem Audun F. Buene
References
[1] Felleslab; Hydrogen peroxide decomposition by Baker’s yeast - Kinetic studies of a biocatalyst in action!, exercise description.
[2] Sigma-Aldrich; MSDS Hydrogen peroxide 3 wt. %, http://www. sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country= NO&language=no&productNumber=323381&brand=SIAL&PageToGoToURL= http%253A%252F%252Fwww.sigmaaldrich.com%252FMSDS%252FMSDS% 252FPleaseWaitMSDSPage.do%253Flanguage%253D%2526country%253D% 2526brand%253D%2526productNumber%253D323381%2526PageToGoToURL% 253D%252Fsafety-center.html.
[3] Elements of Chemical Reaction Engineering 4th Edition; H. Scott Fogler, Pear- son Education International, Massachusetts USA, 2010.
The total theoretical volume of O 2 is found by using the density of O 2 , ρO 2 =
- 309 · 10 −^3 g/m^3.
VO 2 = (^1). 3090 ·. 0564103 g/m 3 = 4. 309 · 10 −^5 m^3 = 43. 09 mL (A.6)
A.2 Calculation of initial reaction rate
As only the first measurements are of importance, the first 4-6 data points for the volume of produced oxygen gas were plotted against time. These plots can be found in Appendix D. From the linear function given by the computational program, the slope was read off. This slope indicates how fast the okxygen gas is produced. The volume of oxygen is then converted to moles of oxygen. Finally the number of moles of H 2 O 2 decomposed per unit of time can be found from the stoichiometry of the reaction.
To demonstrate the calculations, test number 6 will be used. For this test, 6 mL of yeast suspension was used, 4 mL 3 wt.% H 2 O 2 and 20 mL of water. The measurements were done with a 20 mL frictionless syringe and a stopwatch, and are shown in Table A.1.
Table A.1: Measurements from test no. 6. VO 2 is the collected volume of O 2 -gas.
Measurement no.
VO 2 [mL] Time [s]
1 1 14. (^2 2) 17. (^3 3) 19. (^4 4) 22.
These data are shown in Figure A.1, as well as the linear approximation made by the computational program.
Figure A.1: Values of volume of produced O 2 -gas plotted as a function of time.
From Figure A.1, the linear approximation made by the computational program is: y = 0. 3602 x − 4. 1381 (A.7)
This gives a slope of 0.3602 mL/s, which in turn gives the initial reaction rate:
dV (O 2 ) dt = 0. 3602 [mL/s] (A.8)
From the volume of oxygen, one can easily find the number of moles of oxygen produced, using the molar volume of an ideal gas.
dn(O 2 ) dt =^
- 3602 mL/s 22414 mL/mol O 2 = 1.^61 ·^10
− (^5) [mol O 2 /s] (A.9)
From the stoichiometry of the reaction, it is obvious that for each mole of O 2 -gas produced, two moles of H 2 O 2 have been decomposed. This gives:
dn(H 2 O 2 ) dt = (−2)^ ·^1.^61 ·^10
− (^5) [mol O 2 /s] = − 3 , 21 · 10 − (^5) [mol H 2 O 2 /s] (A.10)
which is the initial reaction rate of the reaction.
NTNU Norges teknisk- naturvitenskapelige universitet
MSDS
COMPOUND NAME Hydrogen peroxide solution, 3 wt. % FORMULA H 2 O 2 HEALTH RISKS Not particulary dangerous, but always contact a physician if in doubt.
PHYSICAL DATA
Molecular weight Relative density^ COMBUSTABILITY Not particulary dangerous, but can release O 2 in sertain reactions. 34.01 g/mol 1.000 g/cm^3
PRECAUSIONS Wear tightly fitting safety goggles. Handle with gloves.
HEALTH RISKS Breathing Ingestion Skin Eyes
May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if swallowed. May be harmful if absorbed through skin. Causes skin irritation. Causes eye burns
EXTINGUISHING Use water spray, alcohol-resistant foam, dry chemical or carbon dioxide
NOTES
FIRST AID MEASURES
EYES Rinse thoroughly with plenty of water for at least 15 minutes and consult a physician. SKIN Wash off with soap and plenty of water. Consult a physician.
INGESTION Never give anything by mouth to an unconscious person. Rinse mouth with water. Consult a physician. INHALATION If breathed in, move person into fresh air. If not breathing, give artificial respiration. Consult a physician.
SPECIAL NOTES R 5 - Heating may cause an explosion. R 8 - Contact with combustible material may cause fire. R20/22 - Harmful by inhalation and if swallowed. O - Oxidising R35 - Causes severe burns. SPILLAGE/ LEFT-OVERS To be collected and disposed of properly. STORAGE Store in a cool, well-ventilated place. Light sensitive.
C Risk assessment and chemical data sheets
Data necessary to fill out the chemical data sheets was found from Sigma-Aldrich [ 2 ]
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av
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Nummer
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HMS-avd.
HMSRV
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Side
Erstatter
HMS Unit:
Kjemisk prosessteknologi
Line manager:
Øyvind Gregersen
NDJ
Hazardous activity identification process
Date:
Participants in the identification process (including their function): Short description of the main activity/main process:
ID no.
Activity/process
Responsible person
Laws, regulations etc.
Existing documentation
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measures
Comment
Yeast fermentation
Hydrogen peroxide decomposition by Baker’s yeast
Safety goggles and lab coat