Activity Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
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Measuring Yeast Fermentation Kinetics with a Homemade Water Displacement Volumetric Gasometer Richard B. Weinberg* Departments of Internal Medicine and Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, United States S Supporting Information *
ABSTRACT: A simple homemade water displacement volumetric gasometer constructed from plastic water bottles, plastic tubing, and a graduated cylinder was used to measure the evolution of carbon dioxide by the fermentation of sugars by baker’s yeast. The apparatus provides reproducible, quantitative data that can be graphed to display phenomena such as induction lag time, steady-state growth, and nutrient depletion, and can be used to demonstrate the effect of a variety of conditions (such as carbohydrate type and concentration, inhibitors, pH, and temperature) on yeast fermentation kinetics. Advanced students can use the gasometer to calculate kinetic parameters such as the Michaelis constant, maximal velocity, and activation energy. The gasometer can be a versatile tool for exploring the principles of fermentation and enzyme kinetics in science laboratories ranging from elementary school to high school advanced placement chemistry and first-year college chemistry. KEYWORDS: Elementary/Middle School Science, High School/Introductory Chemistry, First-Year Undergraduate/General, Biochemistry, Laboratory Instruction, Collaborative/Cooperative Learning, Inquiry-Based/Discovery Learning, Consumer Chemistry, Kinetics, Metabolism
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INTRODUCTION Although humans have been familiar with fermentation since Neolithic times,1 the underlying biological basis of fermentation was not firmly established until 1857 when Louis Pasteur reported that alcoholic fermentation required living yeast organisms.2 Three decades later in 1897, Nobel laureate Eduard Buchner showed that a cell-free yeast filtrate could initiate fermentation,3 thereby inaugurating the field of enzymology, and laying the foundation of modern biochemistry. Soon thereafter in 1905, Harden and Young elucidated the roles of cofactors such as inorganic phosphate (Pi), ADP, and ATP in the overall alcoholic fermentation reaction:4
kinetics of fermentation may be monitored by measuring the disappearance of glucose or the appearance of ethanol, the most expeditious method is to measure the volume of carbon dioxide (CO2) produced by the reaction. Many methods for measuring the amount of evolved CO2 have been reported, including the following: balloon inflation;5 counting gas bubbles;6 liquid displacement from inverted graduated cylinders,7 pipettes,8 test tubes,9,10 capillary tubes,11 or microfuge tubes;12 and use of electronic sensors.13,14 However, each of these approaches has practical limitations: some yield only semiquantitative data, whereas others have limited accuracy or require expensive equipment. Herein is described a water displacement volumetric gasometer constructed from inexpensive, easily available materials that can accurately measure the volume of CO2 produced by the fermentation of sugar by baker’s yeast,
C6H12O6 + 2ADP + 2Pi → 2CH3CH 2OH + 2CO2 + 2ATP
(1)
Given the simplicity of its biochemistry and the easy availability of required materials, yeast fermentation has long been a popular classroom demonstration and the basis of laboratory exercises suitable for a broad age range. Although the © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: October 4, 2017 Revised: March 1, 2018
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DOI: 10.1021/acs.jchemed.7b00043 J. Chem. Educ. XXXX, XXX, XXX−XXX
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STANDARD EXPERIMENTAL PROCEDURE The free end of the long tube is placed into the graduated cylinder, and the distance between the bottle and cylinder is adjusted so the tip just hangs over the top edge (see Figure 1). If the tubing extends too far down into the graduated cylinder, it will siphon water from the reservoir and produce spurious data. One teaspoon of rapid-acting dry yeast, 2 teaspoons of table sugar (sucrose), and any desired additives (see below) are added to the reaction bottle using a small funnel. Then, 6 ounces of 110 °F tap water is poured into the bottle, and the timer is started. The reaction bottle is gently swirled for 30 s to mix the ingredients, and then the cap is screwed on tightly by turning the bottle counterclockwise onto it from beneath. It is important not to move or agitate the reaction bottle thereafter during an experiment, as doing so will release dissolved CO2 and give spurious data. Initially, bubbling is seen in the reservoir bottle as the reaction bottle cools down slightly and creates a partial vacuum. Then, after a lag period of 6−8 min (during which no CO2 is released as the yeast becomes rehydrated and begins to synthesize the enzymes needed for fermentation), the solution begins to foam and evolve CO2 as the yeast ferments the sugar, and the increased pressure in the reaction bottle volumetrically displaces water from the reservoir into the graduated cylinder. At this point, the volume of water displaced is recorded at 1 min intervals. When the volume in the cylinder reaches 100 mL, the water is quickly poured out into an empty container, the tube is replaced in the cylinder, and recording the displaced water volume is resumed. Alternatively, the tube can be transferred to a second empty graduated cylinder. At the end of the experiment, the caps are loosened to release pressure in the bottles, the displaced water is poured back into the reservoir bottle, and the reaction bottle is rinsed out with tap water in prepared for the next experiment.
Saccharomyces cerevisiae. This device yields reproducible, quantitative data that can be used to explore the principles of fermentation and enzyme kinetics in classroom and science laboratory settings from elementary school through high school advanced placement (AP) chemistry and first-year college chemistry.
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Activity
APPARATUS AND MATERIALS
Gasometer Construction
The gasometer was constructed from two 12 ounce polyethylene (recycle code 1) plastic bottles with screw-on caps (such as used for bottled water), 3/16 in. o.d. 1/8 in. i.d. vinyl tubing (Ace Hardware, item 46132), and a 100 mL plastic graduated cylinder.15 Two lengths of tubing, one 10 in. long and the other 15 in. long, were cut on an acute diagonal using sharp scissors. A hole slightly smaller than the outer diameter of the tubing was punched through the center of one bottle cap using a nail and hammer; two holes, each slightly off-center, were punched in the other cap. One end of the 10 in. tube was threaded through the top of the 1-hole cap so that about 1/2 in. protruded inside; the other end was similarly threaded through one hole in the 2-hole cap. One end of the 15 in. tube was threaded through the remaining hole so that about half of its length extended below the cap. The ends of both tubes were then cut off square. A small dab of silicone caulk was placed on top of both caps to ensure an airtight seal. One bottle (“the reaction chamber”) was screwed onto the one-hole cap. The other bottle (“the displacement reservoir”) was filled with 10 ounces of water to which several drops of blue food coloring were added (to improve readability) and screwed onto the twohole cap. The position of the long tube was adjusted so that it just reached the bottom of the bottle. The assembled gasometer is shown in Figure 1.
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HAZARDS AND PRECAUTIONS Students should wear eye protection and be instructed on proper laboratory techniques. The use of materials of low and high pH carries the risk of skin or eye irritation or chemical burns if they are carelessly handled. Solid plant food can cause severe intestinal distress if ingested. Experiments using water temperatures above 120 °F carry the risk of thermal burns if hot water is spilled.
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THE ACTIVITY The gasometer was used in a series of laboratory exercises in three fifth-grade science classes, all under the direction of a single teacher. Class size was 18−24 students. At the beginning of the first lab, the teacher briefly discussed the basic principles of yeast fermentation with the class, and then described how the gasometer worked and demonstrated the setup procedure. The class was divided into groups of three students, and each group was assigned a station at a desk on top of which a gasometer, a graduated cylinder, reaction materials, a digital timer, and a data recording sheet (see the Supporting Information) had been placed. Each group chose a member responsible for keeping time, reading the volume of water displaced into the graduated cylinder, and recording the data; all group members participated in the initial setup.17 At the end of the experiment, students graphed their volume versus time data on the worksheets, and determined the length of the lag period and the fermentation rate from the slope of the linear
Figure 1. Water displacement volumetric gasometer in use during an experiment.
Materials
The following materials were obtained from local supermarkets and health food stores: rapid-acting dry yeast,16 sugar (sucrose), salt, baking soda, white vinegar, washing soda (Na2CO3), canned beef broth, solid plant food (Miracle Grow), Lactaid enzyme pills, kitchen measuring spoons, an 8 ounce liquid measuring cup, a small plastic funnel, a digital count-up timer, and a digital probe-style kitchen thermometer. B
DOI: 10.1021/acs.jchemed.7b00043 J. Chem. Educ. XXXX, XXX, XXX−XXX
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growth phase (Figure 2). Data from different groups were compared using an overhead projector, and the teacher led
Figure 3. Substrate specificity of baker’s yeast. Reactions were performed containing 1 teaspoon of lactose or 6 ounces of milk diluted 1:1 with warm water with and without two crushed Lactaid tablets, and 1 teaspoon of cornstarch boiled in 6 ounces of water and cooled to 120 °F with and without 2 teaspoons of freshly collected human saliva.
Figure 2. Fermentation of sucrose by baker’s yeast. Cumulative CO2 production (mL) vs time (min) was recorded for different amounts of sucrose added to the initial fermentation reaction mixture.
discussion of possible explanations for why some of the values differed among groups (e.g., spills, inaccurate measurements or readings). Finally, the students were invited to brainstorm ideas for different substrates and reaction conditions, and predict their impact on length of the lag period, the reaction rate, and the amount of CO2 generated. The following are conditions that students suggested and evaluated in subsequent laboratory sessions.
Although lacking lactase and amylase, baker’s yeast possesses enzymes that enable it to ferment many different types of carbohydrate, including monosaccharides (e.g., glucose, fructose, galactose), disaccharides (e.g., sucrose, maltose), and oligosaccharides (e.g., maltotriose, raffinose).18 By substituting these fermentable substrate sugars (available from specialty grocery stores, health food stores, and Internet venders) for sucrose in the standard reaction mixture, students in high school or college biochemistry laboratories could measure and compare their fermentation efficiency.
Amount of Sucrose
Fermentation Inhibitors
Students ran reactions containing different amounts of sucrose. They observed that reactions containing less than 1 teaspoon of sugar displayed slower CO2 evolution; reactions with 1 or 2 teaspoons exhibited a plateau (possibly due to depletion of required nutrients or end-product inhibition), and reactions containing 3 teaspoons showed faster CO2 evolution and generated a larger volume of CO2 (Figure 2). They also observed that the amount of sugar in the reaction had little effect on the length of the lag phase. These observations anchored discussions about the effect of substrate concentration on the rate of yeast growth, and how yeast growth slows down when the supply of nutrients needed to support enzyme activity has been depleted.
Fermentation by baker’s yeast is inhibited by many organic and inorganic compounds, including sodium chloride.19 Students added increasing amounts of salt (NaCl) to the standard reaction mixture and observed the inhibition of yeast growth at high salt concentrations (Figure 4). These data led to a
Substrates
Students used the gasometer to evaluate how effectively baker’s yeast fermented other foodstuffs, such as apple juice, honey, milk, and starch. They were surprised to observe that yeast produced no CO2 when it was incubated with either warm milk or a cornstarch solution. These observations led to discussions about enzyme substrate specificity, in which students learned that baker’s yeast does not make lactase (the enzyme that digests the disaccharide lactose into its component monosaccharide sugars glucose and galactose), or α-amylase, the enzyme that digests starch, a polysaccharide composed of linear chains of glucose molecules. However, when students predigested milk by addition of lactase tablets to the reaction mixture, or predigested the cornstarch solution using the αamylase present in fresh saliva, they observed brisk fermentation of the liberated monosaccharides (Figure 3).
Figure 4. Inhibition of yeast fermentation by sodium chloride. Different amounts of NaCl were added to the standard reaction mixture before adding water. Inset: Plot of CO2 production rate, calculated from the slope of the linear portions of the curves, versus NaCl concentration. C
DOI: 10.1021/acs.jchemed.7b00043 J. Chem. Educ. XXXX, XXX, XXX−XXX
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discussion of the use of salt as a preservative. End-product inhibition of fermentation by ethanol20 or the effect of specific fermentation inhibitors21 could be similarly examined by advanced students. Nutrients
The optimum growth of baker’s yeast requires factors besides a carbohydrate energy source, such as amino acids, inorganic phosphate, magnesium, and trace minerals.22 Students explored the impact of nutrients by adding canned beef broth and solid plant food to the standard reaction (Figure 5). They observed
Figure 6. Effect of pH on yeast fermentation. Reactions were run over a pH range of pH 3−9 using acetate (pH 3−7) and carbonate (pH 8− 9) buffers. Initial reaction pH was measured with an electronic pH probe. Fermentation lag phase and reaction rate were determined from plots of CO2 volume versus time at each pH, as described above.
their data and compare results among their peers. The fermentation laboratory exercises proved to be so popular that the students in each class requested to use the gasometer for additional laboratory sessions, on a monthly basis, throughout the remainder of the school year. However, the simplicity of gasometer and the safety of the required materials could also make it suitable for use as a demonstration in elementary school settings. The youngest students could simply watch the apparatus in action, with the yeast mixture bubbling in the reaction bottle and water spilling out of the reservoir into the graduated cylinder. Older students could work in groups to compare different brands or types (regular vs rapid-acting) of yeast, or different substrates (e.g., honey, maple syrup, fruit juices). To simplify data collection, students could record the time it takes to displace a set volume of water or, alternatively, the volume of water displaced after a set elapsed time. On the other hand, the quantitative data generated by the gasometer readily lends it to use in high school AP chemistry or college biochemistry laboratory exercises. In these settings, students could use precisely measured amounts of solids and liquids to achieve greater accuracy and enable calculations in SI units, and also use the ideal gas equation to transform the volume of displaced CO2 into moles of product generated and substrate consumed. Students could evaluate end-product inhibition by adding increasing amounts of ethanol to the reaction mixture, compare the potency of various chemical inhibitors, test different commercial growth media and enzyme cofactors, measure the effect of substrate concentration on reaction rate to calculate the reaction Michaelis constant (Km) and maximal velocity (vmax), and examine the impact of temperature on fermentation rate to calculate fermentation activation energy.23 Examples of these advanced applications and calculations are provided in the Supporting Information. In summary, the water displacement volumetric gasometer is an inexpensive homemade apparatus that can accurately measure the volume of CO2 produced by the fermentation of sugar by baker’s yeast. This versatile device is easy to use in the classroom; uses nontoxic, readily obtained materials; quickly engages student interest and participation; and yields quantitative data that can be used to explore the principles of
Figure 5. Effect of supplemental nutrients on yeast fermentation. Reactions were conducted with 110 °F water containing no additives; a 1:1 dilution of canned beef broth in water, warmed to 110 °F; 1/8 teaspoon of solid plant food; or both additives.
that the additives increased the reaction rate and total CO2 production, but not the lag phase. They also found that more than 1/8 teaspoon of plant food inhibited fermentation, due to its high salt concentration. Calculated CO2 production rates for these experiments follow: no additives, 9.3 mL/min; solid plant food, 13.9 mL/min; broth, 15.6 mL/min; and both additives, 18.6 mL/min. pH Range
Students investigated the impact of pH on yeast fermentation by adding common materials of known pH [such as vinegar (pH ∼ 3), baking soda (NaHCO3, pH 8.2), and washing soda (Na2CO3, pH ∼ 11)] to the standard reaction mixture. They measured the initial reaction pH using pH test strips. As above, they calculated the impact of these additives on the fermentation lag phase and rate. They observed that baker’s yeast was most active near neutral pH, and that its growth was inhibited at both low and high pH (data not shown). More advanced students in high school and AP courses could examine the effects of pH over a continuous range by using standard biological buffers (Figure 6; note that these fermentation reactions were conducted later by the author outside of the classroom).
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DISCUSSION In a middle school setting, the gasometer apparatus proved to be an effective, intuitive tool that enabled students to explore the basic principles of yeast fermentation and enzyme action, brainstorm and evaluate their own hypotheses and experimental conditions, and apply simple graphical techniques to analyze D
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(12) Grammer, R. T. Quantitation and Case-Study-Driven Inquiry To Enhance Yeast Fermentation Studies. Am. Biol. Teach 2012, 74, 414−420. (13) Heinzerling, P.; Schrader, F.; Schanze, S. Measurement of Enzyme Kinetics by Use of a Blood Glucometer: Hydrolysis of Sucrose and Lactose. J. Chem. Educ. 2012, 89, 1582−1586. (14) Vernier Software & Technology. Respiration of Sugars by Yeast. https://www.vernier.com/experiments/bwv/12a/respiration_of_ sugars_by_yeast/ (accessed Feb 2018). Vernier Software & Technology. Comparing Fermentation Rates of Various Sugars. http://www.vernier.com/products/sensors/eth-bta/#section5 (accessed Feb 2018). (15) Plastic graduated cylinders are available from many Internet venders. Cylinders made from clear polymethylpentene yield a flat liquid meniscus that is easier to accurately read, especially for younger students. (16) Red Star Quick Rise Yeast was used to generate the data presented in this paper. Regular yeast can also be used, but because it has a longer lag phase (∼10−14 min), students may become frustrated waiting for the onset of gas production. (17) In a classroom laboratory session, it is expedient to fill a large picnic thermos or insulated water container with very hot water (∼140 °F). Students can draw hot water into measuring cups and cool it down to the desired starting temperature by adding cold water while monitoring it with a digital probe-style kitchen thermometer. (18) Lagunas, R. Sugar Transport in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 1993, 104, 229−242. (19) Branham, S. E. The Effects of Certain Chemical Compounds upon the Course of Gas Production by Baker’s Yeast. J. Bacteriol. 1929, 18, 247−264. (20) D’Amore, T.; Panchal, C. J.; Russell, I.; Stewart, G. G. A Study of Ethanol Tolerance in Yeast. Crit. Rev. Biotechnol. 1989, 9, 287−304. (21) Dai, Y.; Normand, M. D.; Weiss, J.; Peleg, M. Modeling the Efficacy of Triplet Antimicrobial Combinations: Yeast Suppression by Lauric Arginate, Cinnamic Acid, and Sodium Benzoate or Potassium Sorbate as a Case Study. J. Food Prot. 2010, 73, 515−523. (22) Ishtar Snoek, I. S.; Yde Steensma, H. Factors Involved in Anaerobic Growth of Saccharomyces cerevisiae. Yeast 2007, 24, 1−10. (23) Ortiz-Muniz, B.; Carvajal-Zarrabal, O.; Torrestiana-Sanchez, B.; Aguilar-Uscanga, M. G. Kinetic Study on Ethanol Production Using Saccharomyces cerevisiae ITV-01 Yeast Isolated from Sugar Cane Molasses. J. Chem. Technol. Biotechnol. 2010, 85, 1361−1367.
fermentation and enzyme kinetics in classroom and laboratory settings ranging from elementary school through high school AP chemistry and college chemistry.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00043. Calculation of fermentation kinetic parameters and activation energy (PDF, DOC) Student information handout (PDF, DOC) Data collection/graphing sheet for middle school students (PDF, DOCX) Instructor application notes (PDF, DOCX) Data collection sheet for advanced students (PDF, DOCX)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Richard B. Weinberg: 0000-0002-9081-7668 Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS The author thanks Ms. Millicent Foreman and the students in her fifth grade science classes at the Summit School, Winston Salem, NC, for field testing the water-bottle gasometer and providing helpful suggestions for improving its use in the classroom.
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REFERENCES
(1) Sicard, D.; Legras, J. L. Bread, Beer, and Wine: Yeast Domestication in the Saccharomyces sensu stricto Complex. C. R. Biol. 2011, 334, 229−236. (2) Pasteur, L. Memoire sur la Fermentation Alcoolique. Compt. Rend. 1857, 45, 1032−1036. (3) Buchner, E.; Rapp, R. Alkoholische Gährung ohne Hefezellen (vorläufige Mitteilung). Ber. Dtsch. Chem. Ges. 1897, 30, 2668−2678. (4) Harden, A.; Young, W. J. The Influence of Phosphates on the Fermentation of Glucose by Yeast-Juice: Preliminary Communication. Proc. Chem. Soc. (London) 1905, 21, 189−191. (5) Slaa, J.; Gnode, M.; Else, H. Yeast and Fermentation: The Optimal Temperature. J. Org. Chem. Dut. Aspects 2009, 134, a−c. (6) Thiel, P. Bubbly Yeast. Science in the Real World: Microbes in Action; University of Missouri, St Louis, MO, 1999. http://www.umsl. edu/~webdev/microbes/Classroom%20Activities/fungus1.pdf (accessed Feb 2018). (7) Tatina, R. Apparatus and Experimental Design for Measuring Fermentation Rates in Yeast. Am. Biol. Teach. 1989, 51, 35−39. (8) Yurkiewicz, W. J.; Ostrovsky, D. S.; Knickerbocker, C. B. A Simple Demonstration of Fermentation. Am. Biol. Teach 1989, 51, 168−169. (9) Reinking, L. R.; Reinking, J. L.; Miller, K. Fermentation, Respiration and Enzyme Specificity: A Simple Device and Key Experiments with Yeast. Am. Biol. Teach 1994, 56, 164−168. (10) Collins, L. T.; Bell, R. P. How To Generate Understanding of the Scientific Process in Introductory Biology. Am. Biol. Teach 2004, 66, 51−53. (11) Knabb, M. T.; Misquith, G. Assessing Inquiry Process Skills in the Lab Using a Fast, Simple, Inexpensive Fermentation Model. Am. Biol. Teach 2006, 68, 25−28. E
DOI: 10.1021/acs.jchemed.7b00043 J. Chem. Educ. XXXX, XXX, XXX−XXX
