Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

Laboratory 3 Fermentation and Photosynthesis

Purpose
This laboratory is an introduction to the metabolic processes of fermentation and photosynthesis. In the fermentation experiment, you will examine the substrates required and products generated during fermentation, as well as determine the effect of substrate structure on the rate of fermentation. In the photosynthesis experiment, you will investigate the relationship between the color of a plant and the wavelengths of light it absorbs. Then you will determine what colors of visible light result in the highest rates of photosynthesis.

Part I. The effect of sugar structure on fermentation rate in yeast

Introduction (modified from K. Kearns, Biology ept., Boston !niversity" In respiration, electrons are transferred from a high free energy food molecule (such as glucose or other sugars" to a low free energy electron acceptor (such as oxygen, iron(III" or other molecules", releasing energy in the process. Fermentation is a process for producing energy from sugars (or other high energy food molecules" in the absence of oxygen or any other external electron acceptor. Instead of an external acceptor, the cell uses a low energy organic molecule produced from internal metabolic processes. In yeast, for example, the byproduct of glucose brea#down (called glycolysis" is acetaldehyde. This molecule serves as an electron acceptor for the electrons stripped from the original glucose molecule. The product of electron transfer to acetaldehyde (reduction" is ethanol, which the yeast cannot use for anything further and must excrete. Thus you can thin# of ethanol as yeast pee$pee. %any different microorganisms are capable of fermentation, including people, E. coli and several other #inds of bacteria. In these organisms, fermentation can also produce other waste products such as lactic acid, propanol, and butanol. &lthough fermentation has the benefit of wor#ing under difficult conditions (e.g., no oxygen", it produces far less &T' than respiration (( &T')glucose via fermentation, *+, &T')glucose via respiration" because there is a much smaller free energy drop between the organic electron donors and acceptors used. In yeast, fermentation uses the glycolytic pathway to produce pyruvate- pyruvate is converted into acetaldehyde, which is then reduced to ethanol. 3lucose (4 carbon" ( pyruvate (+ carbon" ( acetaldehyde5 ( 67( (( carbon 5 8 carbon" ( ethanol (( carbon"

( &T'

/ermentation is a process used extensively in food microbiology for the production of many foods, including wine, pic#les and bread. 0ine$ma#ers are interested in the production of alcohol by fermentation. Bread ma#ers are interested in the production of carbon dioxide gas by fermentation to ma#e bread rise. The rate of fermentation depends on the quantity and quality of sugar present. 1ugars that are easily shunted into the glycolytic pathway are rapidly and easily fermented. 1ugars that must be modified before entering glycolysis, such as disaccharides, will slow down the fermentation process. If the organism does not have the necessary modification en2ymes, the sugar cannot be fermented at all. Today, you will determine the relative quality of three different sugars for yeast fermentation by measuring the amount of gas produced over time. This experiment should elucidate the substrates necessary for and the products resulting from fermentation. 0hile the fermentation experiment is incubating, you may proceed with the photosynthesis experiments.

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

Procedures 8. 7btain four pairs of fermentation tubes. 9ach pair should have one small and one large tube. %ar# the bottom of the smaller tubes as follows. 6 : control 3 : glucose (a monosaccharide" % : maltose (a disaccharide of glucose plus glucose" ; : lactose (a disaccharide of glucose plus galactose" (. To each of the smaller tubes, add 8, ml of the appropriate sugar. The control tube should receive 8, ml of water. +. %ix the yeast (premeasured to 8, g" with 8,, ml of warm water. !se a stirrer to mix the solution thoroughly. <. &dd the yeast mixture to each sugar solution to fill the smaller tube. =. 'lace your thumb over the opening of the smaller tube and sha#e the contents vigorously. 4. 'ut the larger tube over the smaller one, inverted (i.e. the smaller tube will be right$side up and the larger one will be upside down over the smaller one". >. Invert the tubes so that the larger tube is now on the bottom as shown in /igure 8. Figure 1. /ermentation experimental design and mixing protocol.

?. !sing a metric ruler, measure the distance from the top of the yeast solution to the top of each small tube. @ecord these values in Table 8 as your Time , data. A. 'lace all four tubes in the +> B6 water bath. 3as from fermentation will be produced during this time. The gas will accumulate in the top of the smaller tube, pushing it up over time. 8,. %easure the distance from the top of the yeast solution to the top of each small tube every (, minutes for 4, minutes. Cou will probably need to remove the tubes from the water bath to do this. @ecord these data in Table 8. Table 1. %easurement of gas produced by yeast fermentation on different sugars. Time (min) Control (mm) Glucose (mm) Maltose (mm) Lactose (mm) ,

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

(, <, 4, 88. 0hen you are done, pour the contents of the tubes down the sin#. @inse out your tubes thoroughly with water and allow them to dry on a paper towel at your bench.

Part II. Photosynthesis

Introduction (modified from 9. 3odric# ((,,8" Principles o Biology !!. Biology !niversity" epartment, Boston

'hotosynthesis is the process by which external energy (derived from the sun" is made available to the living world through a two component process. the light reactions and the dar# reactions (/igure (". In the light reactions, light energy stri#ing pigments in the chloroplast is transformed first to electrical energy (excited electrons" and then to chemical energy bonds in the molecules &T' and D& 'E (. In the dar# reactions (also called the 6alvin 6ycle", some of these bonds are subsequently bro#en down and in the process energy is released. This energy is used to drive the en2ymatic reactions which change atmospheric carbon dioxide, a low energy molecule, into sugars. In this exercise you will be dealing with the energy$capturing reactions which occur only in the presence of light. &lthough photosynthesis is restricted to chlorophyll$containing organisms (plants and some protists" and some bacteria, the sugars they produce can be used by all living organisms, via glycolysis and respiration, to provide chemical energy for living processes. Figure ! The ;ight @eactions of 'hotosynthesis

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

"! The "bsorption #pectrum o$ a Chloroplast #uspension In the first experiment examining photosynthesis, you will determine what wavelengths of visible light (<,,$>,, nm" are absorbed by a plant of your choice. 'lants available will include spinach and red cabbage. The color of the plant should give you a hint about the absorption spectrum of the plant because the color you perceive is the color that is reflected and all other colors are absorbed by the chloroplasts. D7T9. uring the photosynthesis experiments, the lights must be turned off in the laboratory, the doors closed, and curtains drawn. Cou will have only the light from the experimental lamps to wor# from. 'repare your materials and plan your experiments accordingly.

Procedures 'reparation of 6hloroplasts by the Teaching /ellow 8. In the cold room, remove the mid$vein from one leaf and use only the leafy portion. (. In a blender, add the leaf (8g" to 8,, ml phosphate buffer. +. Blend +,$<, quic# pulses in blender followed by a +, second blend. 'ieces should be about 8mm in diameter suspended in deeply colored buffer. <. /ilter the mixture through +$< layers of cheesecloth. 1quee2e cloth to retrieve buffer. Cou should be able to retain A,F of original volume. iscard the residue. =. Test the activity of preparation. 1et the spectrophotometer with the red filter at 4,, nm. Gero the spectrophotometer with =., ml of the phosphate buffer. Insert another cuvette containing =., ml of the newly blended solution. Ideally, you should get a reading of ,.+,,$,.<,,. 4. @etain the filtrate on ice and protect it from light throughout the experiment by covering it with foil. etermination of &bsorbance 1pectrum by the students >. 7btain two cuvettes. Into one cuvette, place = ml of phosphate buffer. Into the other cuvette, place = ml of chloroplast solution. ?. 1tarting with the spectrophotometer with blue tape (blue filter" on it, set the wavelength to <,, nm. A. %a#e sure your spectrophotometer is calibrated. &bsorbance:infinity, Transmission:,F with no tube in the cuvette holder. 8,. Dow set the absorbance Transmission:8,,F". to 2ero using the phosphate buffer (&bsorbance:,,

88. %easure the absorbance of the chloroplast solution every +, nm starting at <,, nm (<,, nm, <+, nm, <4, nm, etc.". Cou %!1T re$2ero the machine with phosphate buffer 9H9@C TI%9 you change the wavelength of the spectrophotometer. 7nly go up to =?, nm on the blue filter spectrophotometer. @ecord you results in Table (. o not #eep your chloroplast solution in the spectrophotometer too long or you will bleach the chloroplasts.

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

8(. Dow go to the spectrophotometer with red tape (red filter" on it. 1et the wavelength to 4,, nm. 6alibrate the machine by setting the &bsorbance:infinity, Transmission:,F with no tube in the cuvette holder. 1et &bsorbance:,, Transmission:8,,F with phosphate buffer. D7T9. If you are using a digital spectrophotometer, you do not need to switch between machines. Eowever, you do need to ma#e sure that the switch at the bottom, left$hand side corresponds to the wavelength range you are measuring. 8+. %easure the absorbance of the chloroplast solution every (, nm starting at 4,, nm (4,, nm, 4(,, 4<,, etc.". Cou %!1T re$2ero the machine with phosphate buffer 9H9@C TI%9 you change the wavelength. 1top at >,, nm. Table . &bsorbances of chloroplast suspension at specific wavelengths. &a'e ()) nm (3) nm (*) nm (+) nm , ) nm ,,) nm Length "bsorbance &a'e Length "bsorbance

,-) nm

%lue

*)) nm

* ) nm

*() nm

**) nm

*-) nm

/)) nm

.ed

%! The 0$$ect o$ &a'elength on Photosynthesis .ate 'lants experience environments with different light qualities. /or example, tall canopy plants in a tropical rain forest have access to the broad spectrum of light. Eowever, plants living at the forest floor are limited in the wavelengths of light available, mostly in the green wavelength range. 1imilarly, water absorbs all colors of light except blue, giving oceans and la#es a blue appearance. This means that mostly blue light is available for photosynthesis in aquatic environments. Dow that you #now which wavelengths of light are best absorbed by your plant of choice, you need to #now how much photosynthetic activity occurs in these various wavelengths of light. 1pecifically, you will compare photosynthesis rate for chloroplasts exposed to blue, green, red, and full spectrum light. /ormulate a hypothesis regarding the possible effects of light quality on the energy$capturing reactions of photosynthesis. !se /igure + and results from your analysis of the absorption spectrum of chloroplasts to help you in writing this hypothesis. Figure 3. 0avelength range and relative amount of energy in visible light. Hiolet Blue 3reen Cellow 7range @ed &a'elengths <,,$<=,nm <=,$=,,nm =,,$==,nm ==,$4,,nm 4,,$4=,nm 4=,$>,,nm

energy

Cou will measure photosynthesis activity by determining the extent of color loss of the dye dichlorophenolindophenol ( 6'I'". This dye intercepts the flow of electrons in the photosynthesis electron transport chain. 0hen it accepts electrons, it becomes reduced and changes color, from blue (oxidi2ed form" to colorless (reduced form" (/ig. <". This color change can be measured as a reduction in

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

absorbance readings on a spectrophotometer. The amount of color lost is proportional to the number of electrons activated during photosynthesis, which is proportional to photosynthesis activity. Figure (. Interception of electron flow in photosystem II by 6'I'.

Procedure 8. etermine the position of each of the four color boxes that gives a light intensity of <, footcandles inside the box. !se the diagram in /igure = to set up your experiment properly. Figure ,. 9xperimental set$up for measuring photosynthetic rate using 6'I'.

;ight

6hromatography Tan#

1amples

(. 9ach group will examine the photosynthetic reaction for all four colors. Therefore, prepare four sets of two tubes. one tube will contain = ml of chloroplast suspension and the other tube will contain = ml of chloroplast suspension 5 <, Il 6'I'. +. Invert the tubes to mix. <. 1et the wavelength of your spectrophotometer with red tape (red filter" on it to 4(, nm. 1et the absorbance of your spectrophotometer to 2ero using a tube containing = ml of phosphate buffer. %easure the absorbances of the other tubes. This is your t:, reading. @ecord these values in Table + on the next page. =. Incubate one chloroplast only tube and one chloroplast5 6'I' tube in each color box for 8, minutes. %a#e sure the tubes are lined up along the length of the acetate sheet. 9ach tube should be equally exposed to the light source. 4. Ta#e a final absorbance measurement at the end of 8, minute incubation period, using the phosphate$only tube to set the absorbance of the spectrophotometer to 2ero. This is your t:8, reading.

Calculations

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

8. 1ubtract the ending absorbance (&8," from the beginning absorbance (&," for each tube. Biochemically spea#ing, the absorbance should decrease in tubes with 6'I' over time as the 6'I' goes from blue to colorless as it accepts electrons from photosynthesis. There should be little change in the Jchloroplast onlyK tube. (. 1ubtract tube L( (chloroplast only" $rom tube L8 (chloroplast5 6'I'". In biochemical terms, we have now subtracted out any absorbance change in the chloroplast itself and the resulting value only represents changes in 6'I' absorbance. Table 3. &bsorbance change for chloroplasts incubated in different colors Color Tube L8 6hloroplast5 6'I' L( 6hloroplast only L8 6hloroplast5 6'I' L( 6hloroplast only L8 6hloroplast5 6'I' L( 6hloroplast only L8 6hloroplast5 6'I' L( 6hloroplast only "bs at T) "bs at T1) Change in "bs (T) 1 T1)) Change in 2CPIP "bsorbance (Tube 31 4 Tube 3

@ed 3reen Blue Broad 1pectrum

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

Dame. MMMMMMMMMMMMMMMMMMMMMMM B! I . MMMMMMMMMMMMMMMMMMMMMMM Teaching /ellow name and section number. MMMMMMMMMMMMMMMMMMMMMM

Laboratory 3 Fermentation and Photosynthesis

Pre1lab 5uestions
8. Eow will fermentation rate be measured in our lab experimentN Eow does this measurement relate to the metabolic process of fermentationN

(. 0hich sugar should give higher fermentation rate, glucose, maltose or lactoseN 0hyN

+. 0hich color should spinach absorb the mostN 0hyN 0hich color should red cabbage absorb the mostN 0hyN

<. Eow will

6'I' be used as an indicator of photosynthetic activity in our lab experimentN

=. 0here does 6'I' intercept electrons chemically (in relation to molecules" and physically (in relation to the cell" during photosynthesisN

Laboratory 3: Fermentation and Photosynthesis BE 209 Spring 2007

Dame. MMMMMMMMMMMMMMMMMMMMMMM B! I . MMMMMMMMMMMMMMMMMMMMMMM Teaching /ellow name and section number. MMMMMMMMMMMMMMMMMMMMMM

Laboratory 3 Fermentation and Photosynthesis

Post1lab "ssignment
8. !sing 9xcel ma#e one graph showing height of gas column versus time for each substrate according to the data you collected in Table 8. The graph should have < lines on it, one for each substrate. (. /ind the slope of each line. The fermentation rate of yeast with any sugar is the change in gas column height over the change in time, which is equivalent to the slope of the line. 9xcel will easily calculate slopes for you- as# your T/ if you need help with this function. &ttach the graph with fermentation rate to this sheet. +. !sing the data of fermentation rate of yeast with each substrate, ma#e one graph showing fermentation rate (change in gas column height per unit time" versus substrate. !se the column graph function in 9xcel. Be sure your figure axes are labeled properly with units and given a descriptive title. &ttach the graph to this sheet. <. escribe (but do not explain" what you see in the figure by comparing characteristics among the substrates. o 0hich substrate results in the best fermentation rateN o 0hich substrate results in the worst fermentation rateN o Eow does the fermentation rate correlate with the complexity of the sugarN o If the results are not what you have expected, what might have happenedN

=. !sing 9xcel graph the relationship between absorbance of chloroplasts and wavelength of visible light. &ttach the graph to this sheet. 4. &ccording to the photosynthetic rates for chloroplasts exposed to different light source, what is your hypothesis regarding the possible effects of light quality on the energy$capturing reactions of photosynthesisN

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