Bio6–Fermentation&CellularRespirationLab

INTRODUCTION

Thecellsofalllivingorganismsrequireenergytokeepthemselvesaliveandfulfillingtheirroles.Where does this energy come from? The answer is energy released frommolecules of thenucleotideadenosinetriphosphateorATP.

Asyoucanseefromthediagramabove,thehydrolysisofATPtoADP(adenosinediphosphate)andinorganicphosphate(Pi)isexergonicandthusreleasesenergywhichcellscanusetodoanynumberof things. Oncehydrolyzed,ATP canbe regenerated fromADPandPi, though this isendergonicandthusrequiresenergy. TheenergyneededtoregenerateATP isobtainedfrom“food”,whateverthatmaybe.Thefoodweeatisfirstdigestedbyenzymesasyoulearnedinthepreviouslab.Oncethepolymersinyour food(e.g.,polysaccharides, triglycerides,protein)havebeenbrokendownbyenzymesintomonomers(e.g.,monosaccharidessuchasglucose,fattyacids,aminoacids),theyentertheblood circulation and are delivered to the cells of the body. Within cells, the processes offermentation and cellular respiration will further catabolize (break down) these molecules,harvestingtheenergytheycontainforthesynthesisofATP.Letusnowtakeabrief lookatfermentationandcellularrespiration toseehoweachprocessproducesATPusingenergyreleasedfrommoleculesofglucose.Keepinmindthat,althoughwearefocusingonglucose,othermoleculessuchasfattyacidscanbeusedforthesamepurpose,thoughinslightlydifferentways.

171

Part1:FERMENTATIONToproduceATP fromglucose,whetherby fermentationorcellular respiration,cellsmust firstpartiallybreakitdownbyglycolysis(“sugar”“separation”).Theenzymesinvolvedinglycolysisarelocatedinthecellcytoplasmandsequentiallybreakdowneach6-carbonmoleculeofglucosetotwo3-carbonmoleculesofpyruvate.Intheprocess,enoughenergyisextractedtoproduce2moleculesofATP.

glucose (6-carbons) ààààà 2 pyruvate (3-carbons)

2 ADP + 2 Pi 2 ATP Inconjunctionwithglycolysis,cellswillcarryoutfermentationifthereisnooxygen(O2)available.When you overexert yourself for example, your muscles do not receive enough oxygen andtemporarilyfermentglucose.Inanotherfamiliarexample,yeastwillfermentwhenplacedinanenclosedenvironmentwithasourceofcarbohydratesuchasgrapes(formakingwine)orhopsandbarley(formakingbeer).Interestingly,fermentationdoesnotproduceanyadditionalATP.Whatitdoesdoisregenerateanimportantmoleculeneededforaparticularstepinglycolysis. ThismoleculeistheelectroncarrierNAD+,whichifdepletedwillbringahalttoglycolysisandATPproduction,resultingincelldeath. FermentationthereforecontributestoATPproduction indirectlybyallowingglycolysis,andtheproductionof2ATPperglucose,tocontinueunhindered.

2 NAD+ 2 NADH 2 NAD+

glucose ààààà 2 pyruvate 2 fermentation products

2 ADP + 2 Pi 2 ATP

Glycolysis Fermentation

Asshownabove,NAD+,anemptyelectroncarrier,isconvertedtoNADH,afullelectroncarrier(the electrons being “carried” are associated with the hydrogen atom) during glycolysis.FermentationissimplyoneormorebiochemicalstepsthattransfertheHinNADHandanextraelectrontoamoleculeofpyruvate.Asaresult,NADHisrestoredtoNAD+,whichisneededforglycolysis,andpyruvateisconvertedtoa“fermentationproduct”whichcanbeavarietyofthingsdependingontheorganism.Animals,includinghumanbeings,producelacticacidwhentheircellsferment.Inorganismsfromotherkingdomsthefermentationproductscanbequitedifferent.Somebacterialspeciesproduceaceticacid(vinegar)whentheyferment,whereasothersproduceacetone(themainingredientinnail polish) or other organic molecules. In the Kingdom Fungi, single-celled yeasts when

energy

2H- 2H-

172

fermentingwillproduceCO2andethanolinstead.Thisprocess,knownasalcoholfermentation,isthebasisforbeerandwineproduction.Regardlessofthefermentationproducts,thepurposeof fermentation is always the same – to regenerate NAD+ so that glycolysis can continue toproduce2ATPperglucosewithoutinterruption.In the following exercise you will investigate alcohol fermentation in yeast under differentconditionsandmeasuretheproductionofonefermentationproduct–CO2.Exercise1–ObservingandMeasuringAlcoholFermentationinYeast

1. On your worksheet, write a hypothesis addressing theeffect of increasing concentrations of yeast on CO2production.

2. To test your hypothesis, plan an experiment combiningvariousamountsofwater,yeastsolutionandcornsyrup(asourceofsugar).Youwillplan3reactionsthateachtotal12mlinwhichtheindependentvariableistheconcentrationof yeast. You should also have a control reaction. Noreactionshouldcontainmorethan4mlofyeastsolutionorcornsyrup.

3. Assemble each reaction in small beakers, and once all 3

reactionsarecomplete,transfereachmixturetoalabeledsaccharometer(asshownontheright).Simultaneouslytiltall3saccharometers(gently)untilnoairistrappedinsidethecolumn.Atthispointyouwillbeginthetimingofyourexperiment(recordstarttimeonyourworksheet).

4. At5minuteintervals,usethegraduationsonthecolumntoestimatethevolumeofgastrappedin

thecolumninmlforatotalof30minutes.

5. Recordthedataonyourworksheet,graphthedata,andanswertheassociatedquestions.

Part2:CELLULARRESPIRATION

While2ATPperglucosemoleculeisclearlybetterthannothing,itisnotnearlyenoughtomeetthe energy needs of complexmulticellular organisms such as plants and animals. To get themaximumATPyieldfrommoleculesofglucoserequirescellularrespiration,whichandproduceupto36ATPperglucosemolecule.Inaerobicorganisms,cellularrespirationrequiresO2(whichiswhywebreathe!),hencethetermaerobicrespiration.

173

Theoverallprocessofcellularrespirationcanbesummarizedinthefollowingequation:

glucose oxygen carbondioxide waterC6H12O6+6O2ààààà6CO2+6H2O

36ADP+36Pi36ATP

Ineukaryoticcells,cellularrespirationbeginswithglycolysisinthecytoplasmandcontinuesinthemitochondriaasoutlinedbelow:

TheCitricAcidCycle–ThisisabiochemicalpathwayinvolvedinbreakingpyruvatedowntoCO2.Intheprocess,energyrichelectronsinhydrogenatomsaretransferredtoNAD+andFADproducingNADHandFADH2. Inaddition, 2ATPperoriginalglucosearealsoproduced.

OxidativePhosphorylation–Thisistheprocessbywhichtheremaining32ATPmoleculesareproducedinvolvingtwodistinctstages:

• ElectronTransport-electronsgatheredbyNADHandFADH2duringglycolysisand

thecitricacidcycleareusedtoproduceanH+gradientwithinmitochondriainaprocessthatrequiresO2

• Chemiosmosis–theH+gradientproducedbyelectrontransportprovidesenergy

forATPsynthasetomake32ATPperoriginalglucose

TheimportanceofO2forcellularrespirationcannotbeoveremphasized.O2isthefinalelectronacceptorintheelectrontransportchain.WithoutO2electrontransportdoesnotoccur,bringingcellularrespirationtoahalt,andtheonlyoptionforATPproductionisfermentation.Thismeans2ATPperglucoseinsteadof36.ThecelldiagrambelowsummarizesfermentationandcellularrespirationinrelationtoO2andwhereeachprocessoccursineukaryoticcells,andthenumberofATPmoleculesproduced.

Glycolysis

FERMENTATION

CELLULAR

RESPIRATION CitricAcidCycle OxidativePhosphorylation

2 ATP (per glucose)

32 ATP (per glucose)

2 ATP (per glucose) no O2

O2 present

energy

174

Inthenextexerciseyouwilldetecttheoxidationofsuccinate,ametabolicintermediateintheCitricAcidCycle,asevidenceofcellularrespiration.Succinatedehydrogenase(SDH)isanenzymein the Citric Acid Cycle which catalyzes the removal of 2 hydrogens from succinate (i.e., theoxidationofsuccinate)whicharetransferedtotheelectroncarrierFAD.ThisyieldstheproductsfumarateandFADH2asshownbelow:

FAD + succinate FADH2 + fumarate

FADH2inturnwilldonatetheelectronsfromthese2hydrogenstocoenzymeQintheelectrontransport chain. The compoundDCPIP (di-chlorophenol-indophenol) is not normally found incells, however when added to mitochondria it will substitute for coenzyme Q and receiveelectronsfromFADH2.Beforereceivingtheelectrons(initsoxidizedstate)DCPIPisabluecolor,howeverafterreceivingtheelectrons(beingreducedbyFADH2)DCPIPiscolorless. Becauseofthiscolorchange,DCPIPisagoodindicatorofrespirationasillustratedbelow.

InthenextexerciseyouwilladdDCPIPtoamitochondrialsuspensionmadefromlimabeans(yes,plantscarryoutcellularrespirationtoo!)anddetectthecitricacidcyclestepillustratedabovebythelossofbluecolorinDCPIP.

succinate dehydrogenase

fumarate

succinate

DCPIP DCPIP (colorless)

SDH

175

Exercise2–Detectingcellularrespirationinamitochondrialsuspension

1. Review the experiment below, write your hypothesis on your worksheet and identify theindependentanddependentvariablesaswellasthecontrol.

2. Label3testtubesandaddthecomponentsindicatedinthechartbelow,inorder:

Tube#1 Tube#2 Tube#3PhosphateBufferSolution 4.0ml 3.7ml 3.4ml

DCPIP 0.5ml 0.5ml 0.5mlSuccinateSolution 0ml 0.3ml 0.6mlLimaBeanExtract 0.5ml 0.5ml 0.5ml

TOTAL 5.0ml 5.0ml 5.0ml

3. Makesureeachtubeismixedandscorethecolorofeachtubeevery5minutesforatotalof30minutesusingthescaleshownbelow:

0 1 2 3 4 5

4. Graphcolorscorevstimeforeachtubeandanswertheassociatedquestions.

Part3:DESIGNINGANEXPERIMENT

Having investigated alcohol fermentation in yeast and cellular respiration in a mitochondrialsuspension,youandyourgroupwilldesignandcarryoutanewexperimenttoexpandonwhatyouhavealreadylearned.Exercise3–Designanexperiment

1. Decideasagroupto further investigateyeast fermentationorcellular respiration in limabeanmitochondrialsuspension.

2. Identifyanindependentvariableyouhavenotalreadyinvestigated(e.g.,amountofcornsyrupormitochondrialsuspension)andcomeupwithahypothesiswithregardtothisvariable.Writethehypothesisonyourworksheet.

3. Design an experiment to test this hypothesis. On your worksheet, briefly describe your

experimentalplan,andidentifytheindependentvariable,dependentvariableandcontrol.

4. Carry out your experiment, record and graph the results on your worksheet, and write yourconclusion.

176

Fermentation&CellularRespirationLabWorksheetName:____________________________Group:________ Date:_______________Exercise1–Yeastfermentation Starttime:_________Stateyourhypothesisbelowandidentifytheindicatedcomponentsofthisexperiment:Ø Hypothesis:Ø Independentvariable: Ø Dependentvariable: Ø Control:Results:

0min 5min 10min 15min 20min 25min 30min

Tube1

Tube2

Tube3

Onthegridbelow,graphtheresultsforeachtubebyplottingtheamountofgasproducedvstime.

Ø Didtheseresultssupportyourhypothesis?Explain.

177

Exercise2–Cellularrespiration Starttime:_________EndTime:________Indicatetherolesofeachofthefollowingcomponentsinyourexperiment:

Limabeanextract: Succinate:

DCPIP: Buffer:

Stateyourhypothesisbelowandidentifytheindicatedcomponentsofthisexperiment:Ø Hypothesis:Ø Independentvariable: Ø Dependentvariable: Ø Control:Results:

0min 5min 10min 15min 20min 25min 30min

Tube1

Tube2

Tube3

Onthegridbelow,graphtheresultsforeachtubebyplottingthecolorscorevstime.

178

Ø Didtheseresultssupportyourhypothesis?Explain.Ø Whywas it important for this and thepreviousexperiment to keep the total volumeofeach tube

constant?Exercise3–DesignanexperimentØ Stateyourhypothesis:Ø Providethedetailsofyourexperimentbelow:Identifytheindicatedcomponentsofyourexperiment:Ø Independentvariable: Ø Dependentvariable: Ø Control:Drawachartortableandrecordtheresultsofyourexperimentbelow:

179

Graphyourresultsonthegridbelow:

Ø Didtheseresultssupportyourhypothesis?Explain.

180