TKP4105/TKP4110Yeast fermentation
Workplan
Audun F. [email protected]
Elise [email protected]
Group B19Supervisor: Naresh Doni Jayavelu
Laboratory: K4-212To be conducted on: 18th of September 2012
Contents
1 Introduction 1
2 Theory 22.1 Decomposition process . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4 Various information . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 Method 43.1 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2 Preliminary test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.3 Experiment 1: Dependence of the rate of the yeast consentration . . 53.4 Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
A Calculations 7
B Risk assessment and chemical data sheets [2] 8
C Measurements from Preliminary test 11
D Measurements from Experiment 1: Dependence of the rate of theyeast consentration 11
1 Introduction
In this experiment, the main goal is to investigate the kinetic properties of Baker’syeast in the process of decomposition of hydrogen peroxide to water and oxygen.This is done by measuring the volume of oxygen, and can be related to the speedat which the hydrogen peroxide is decomposed. The variety of enzymes expressedin yeast makes this the perfect microorganism for such reactions. For part one ofthe experiment it is the yeast consentration that is varied in order to investigateits effect on the decomposition rate of H2O2.
1
2 Theory
2.1 Decomposition process
Yeast is a eukaryote singlecellular microorganism. It has got a rich variety ofenzymes, in order to get nutrition as well as to protect itself. Yeast is a lot lesscomplicated than other eukaryotes, but all the more interesting because it has alot of similar enzymes as its fellow and more complex eukaryotes. For instance theenzymes that catalyse the reaction where hydrogen peroxide is converted to waterand oxygen.
2H2O2 → 2H2O+O2 (2.1)
The reaction rate of this reaction will be the objective of this experiment, withrespect to the consentration of yeast. There will be other factors to consider,conducting an experiment with living cells, for instance the effect of change intemperature, the concentraion of H2O2, pH of the solution or other enzymes andreactions also occuring in the same batch. These effects are not to be investigatedduring this experiment.
The yeast is a living organism, so its activity will vary depending on a numberof different factors. Therfore it is necessary to check the catalytic activity of theyeast that will be used. This test is explained in section 3.2.
2.2 Statistical analysis
In order to estimate the standard errors in the slope and the intersection of thereaction rate curves as a function of yeast consentration, statistical analysis isused. The method used will be the method of least squares. This is usually donewith computers directly from the plots of the measurements.
2.3 Data analysis
Because this experiment is run as if in a batch reactor, we’re only interested inthe initial reaction rate, because there is no easy way of measuring the H2O2-
2
concentration. The data gathered is then converted and plotted, to find the re-action rate as afunction of the concentraion of yeast. This is possible because foreach parallel, the yeast concentration is constant.
There are two different ways to estimate the initial reaction rate. The first is byestimating the tangent to the curve of the first measuring point. Secondly one canestimate the slope from a straight line through the 3-4 first measuring points.
2.4 Various information
• Q: Oxygen absorption in water, explain
– A: If the water volume is kept constant, the amount of oxygen absorbedwill also be constant for all the parallels. This means all the results are’equally’ shifted.
• Q: Measuing of volume intervalls rather than time intervalls
– A: The resolution of the watch is greater than the resolution on thesyringe. This will be the most accurate way of measuring.
• Q: Reaction delay
– A: Water will absorb the first oxygen gas formed
– A: The use of yeast as a microfactory in stead of pure enzymes willdelay the process, because of the diffusion in and out of the cell. Otherenzymes present in the cell may also cause the reaction to delay.
– A: The mixing of the H2O2 and the yeast suspension may be slow be-cause of little or no strirring, until O2-gas bubbles are formed and con-tribute to the mixing of the solution.
• Q: Error of measured point due to human factor
– A: This will not affect the relative initial reaction rates because theerrors will affect all the points equally.
3
3 Method
The experiment is described in the document Hydrogen peroxide decomposition byBaker’s yeast [1].
3.1 Apparatus
The reaction is run in a 50 mL round bottom flask. In order to measure the volumeof gas developed during the reaction, a syringe is used. This is a cylinder witha frictionless piston, and is connected to the reaction flask just as the reaction isinitiated.
3.2 Preliminary test
In order to get an idea about the activity of the yeast, there is to be conducteda preliminary test of the enzyme activity of the yeast. The time it takes for thereaction to produce a gas volume of 10 mL is expected to be ranging from 80 to120 seconds.
1. Prepare a 100 mL sample of yeast suspension in a 100-150 mL beaker. De-pending on what kind of yeast is used the concentraition of the suspensionsample vary (12g/L for dry yeast while 50 g/L for fresh yeast). Fresh yeastshould first be crumbled.
2. Prepare the set-up before starting any part of the experiment.
3. Add 8.0 mL of homogenized suspension and 18 mL of distilled water to theround bottom reaction flask.
4. Add 4.0 mL 3 wt% H2O2 to and close the reaction flask. START the STOP-WATCH and observe the position of the piston!
5. Monitor the reaction volume increase, and make notes of how far the pis-ton has moved at a certain time interval. CHECK TIME INTERVAL withNahres.
6. The syringes are very delicate, so make sure they don’t break.
4
3.3 Experiment 1: Dependence of the rate of the yeast con-
sentration
The experiment is run almost identically to the preliminary test, using a constantvolume of 4 mL of 3 wt% H2O2 for each of the parallels. The total volume of thereaction mix is to be a constant 30 mL. The yeast suspension is made in a 250 mLvolumetric flask.
Table 3.1: List of how to prepare the different solutions of reaction mixtures.
Seriesno.
Yeast suspen-sion, [mL]
Hydrogen peroxidesolution, [mL]
Distilled water,[mL]
1 2.0 4.0 24.02 4.0 4.0 22.03 6.0 4.0 20.04 8.0 4.0 18.05 10.0 4.0 16.06 12.0 4.0 14.07 14.0 4.0 12.08 16.0 4.0 10.09 18.0 4.0 8.010 20.0 4.0 6.0
3.4 Tips
• Add the peroxide as the last component of the reaction mix!
• Don’t break the syringe!
• In making of yeast suspension, only fill half way before shaking!
• Always homogenize the suspension before use.
• Remember to close the system immediatly after adding of the peroxide.
5
Trondheim, September 14, 2012
Elise Landsem Audun F. Buene
References
[1] Felleslab; Hydrogen peroxide decomposition by Baker’s yeast - Kinetic studiesof 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.
6
A Calculations
The following assumptions have been made for this calculation:
• Room temperature in reaction flask
• No biproducts of the decomposition
• Oxygen as an ideal gas
• Amount of O2 absorbed in the water of the solution is negligible.
The overall reaction is:2H2O2 → 2H2O+O2 (A.1)
4 mL of a 3 wt.% solution of hydrogen peroxide is used. This gives a total weightof H2O2 in the reaction mix:
mH2O2= 4 · 10−3kg · 0.03 = 0.12 gram (A.2)
This molecular weight of H2O2 is 34.015 g/mol, so this gives the total number ofmoles of H2O2:
nH2O2=
mH2O2
MmH2O2
=0.12 gram
34.015 g/mol= 3.528 · 10−3 mol (A.3)
From the stoichiometry of the reaction, the number of moles of oxygen gas isobtained:
nO2=
1
2· nH2O2
= 1.764 · 10−3 mol (A.4)
The mass of O2 is obtained by:
mO2=MmO2
· nO2= 32 g/mol · 1.764 · 10−3 mol = 0.0564 grams (A.5)
The total theoretical volume of O2 is found by using the density of O2, ρO2=
1.309 · 10−3 g/m3.
VO2=
0.0564
1.309 · 103 g/m3 = 4.309 · 10−5m3 = 43.09 mL (A.6)
7
NTNUNorges teknisk-
naturvitenskapelige universitet
MSDS
COMPOUND NAMEHydrogen peroxide solution, 3 wt. % FORMULAH2O2 HEALTH RISKSNot particulary dangerous, but always contact a physician if in doubt.PHYSICAL DATA Molecular weight Relative density COMBUSTABILITYNot particulary dangerous, but can release O2 in sertain reactions.
34.01 g/mol 1.000 g/cm3
PRECAUSIONS Wear tightly fitting safety goggles. Handle with gloves. HEALTH RISKSBreathingIngestionSkinEyes
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
EXTINGUISHINGUse water spray, alcohol-resistant foam, dry chemical or carbon dioxideNOTES
FIRST AID MEASURESEYESRinse thoroughly with plenty of water for at least 15 minutes and consult a physician.SKINWash off with soap and plenty of water. Consult a physician.
INGESTIONNever give anything by mouth to an unconscious person. Rinse mouth with water. Consult a physician.INHALATIONIf breathed in, move person into fresh air. If not breathing, give artificial respiration. Consult a physician.SPECIAL NOTESR 5 - Heating may cause an explosion.R 8 - Contact with combustible material may cause fire.R20/22 - Harmful by inhalation and if swallowed.O - OxidisingR35 - Causes severe burns.SPILLAGE/LEFT-OVERS To be collected and disposed of properly.STORAGE Store in a cool, well-ventilated place. Light sensitive.
B Risk assessment and chemical data sheets [2]
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C Measurements from Preliminary test
Table C.1: Measurements from Preliminary test. Time is the time it takes for thesystem to reach 10 mL of produced oxygen gas.
Seriesno.
Vyeast susp.
[mL]VH2O2(aq)
[mL]VDistilledH2O
[mL] Time [s]
123
D Measurements from Experiment 1: Dependence
of the rate of the yeast consentration
Table D.1: Measurements from Experiment 1: Dependence of the rate of the yeastconsentration. Series X, parallel Y.
Measurementno.
VO2 (g) [mL] Time [s]
12345678
11
