MEASURING FERMENTATION OF YEAST UNDER VARIOUS CONDITIONS AND CELLULAR RESPIRATION OF MITOCHONDIRA IN LIMA BEANS
April 14, 2015
BSC 2010L Section: 904 Lab Parteners: Angelid Pabon, Taylor Taylor rig!t, rig!t, "iley Bla#e Abstract
T!e proble$ addressed in t!e %irst part o% t!e e&peri$ent is !o' do t!e $etabolic rates o% yeast co$pare (nder )arying conditions '!ic! incl(de %ood so(rce and te$perat(re* T!is proble$ 'as approac!ed setting (p nine di%%erent co$binations o% en)iron$ents %or t!e yeast+ s(crose, gl(cose and sat(rated starc! sol(tion along 'it! -º C, 25º C and 4º C* T!e res(lts s(pported t!e !ypot!esis 'it! gl(cose !a)ing on a)erage t!e %astest %er$entation rate %or %ood so(rce and -º C %or t!e te$perat(re* Introducton
Cell(lar "espiration re%ers to t!e biological pat!'ay by '!ic! cells release energy %ro$ c!e$ical bonds o% %ood $olec(les and pro)ide t!at energy %or essential processes o% li%e ./a)e, 2012* All li)ing cells $(st carry o(t cell(lar respiration 'et!er it be aerobic respiration .in t!e presence o% o&ygen or anaerobic respiration .'it!o(t o&ygen* Pro#aryotic cells carry o(t cell(lar respiration 'it!in t!e cytoplas$ or on t!e inner s(r%aces, '!ereas in e(#aryotic cells it occ(rs in t!e $itoc!ondria* T!e o(tco$e o% cell(lar respiration is t!e prod(ction o% ATP* ATP* ig(re 1 s!o's t!e e(ation %or cell(lar respiration*
Fi gur e1: Cel l ul arr es pi r at i oni nv ol v est heox i di z i ngofgl uc os et oy i el dc ar bondi o xi dewat erand36ATP
3nder anaerobic conditions, co nditions, %er$entation occ(rs* er$entation $a#es it possible %or ATP ATP to be contin(ally prod(ced in t!e absence o% o&ygen* as s!o'n in %ig(re 2 t!e e(ation %or %er$entation yields et!anol and carbon dio&ide * T!ere are t'o types o% %er$entation, alco!ol %er$entation and lactic acid %er$entation* Lactic acid %er$entation occ(rs in t!e $(scles o% ani$als '!en t!ey need energy %aster t!an t!e blood is can s(pply o& ygen* Alco!ol %er$entation %er$entation is per%or$ed by yeast (sed in bre'ing, 'ine $a#ing and ba#ing*
Fi gur e2: Su mma mm r i z ede qu at i o no ff e r me nt a t i on
T!e i$portance o% cell(lar respiration is it pro)ides t!e energy %or li)ing organis$s to per%or$ all o% t!e ot!er necessary %(nctions to $aintain li%e li%e and $ost singlecelled organis$s, s(c! as bacteria, do not re(ire $(c! energy and are able to s(r)i)e on glycolysis and %er$entation*
n ter$s o% %ood so(rces $onosacc!arides and disacc!arides represent on ly a s$all %raction o% t!e total a$o(nt o% carbo!ydrates in t!e nat(ral 'orld* T!e great b(l# o% t!e carbo! ydrates in nat(re are present as polysacc!arides, '!ic! !a)e relati)ely large $olec(lar 'eig!ts* T!e polysacc!arides ser)e t'o principal %(nctions* T!ey are (sed by bot! plants and ani$als to store store gl(cose as a so(rce o% %(t(re %ood energy and t!ey pro)ide so$e o% o % t!e $ec!anical str(ct(re o% cells* Plants store %ood energy as polysacc!arides #no'n as starc!* Cell(lar "espiration is a redo& reaction o&idi6ing %ood $olec(les, li#e gl(cose to carbon dio&ide and 'ater* A redo& reaction is a c!e$ical reaction in '!ic! a s(bstance loses electrons and anot!er gains electrons, t!ere is a trans%er o% electrons bet'een reactants* 7)erall it in)ol)es %o(r $ain s(bdi)isions, s!o'n in %ig(re + glycolysis is '!en gl(cose is bro#en do'n releasing !ydrogen $olec(les to %or$ pyr()ate+ t!e 8rebs cycle t!e carriers /A and A pic# (p electrons and !ydrogen ions to %or$ /A; and A;+ t!e electron transport c!ain and c!e$ios$osis also occ(r* occ (r*
Fi gur e3: I nput sandOut put sofCel l ul arRes pi r at i on
n t!e citric acid cycle, also #no'n # no'n as #rebs cycle, a critical steps occ(rs, e n6y$e cataly6ed con)ersion o% s(ccinate to %($arate t!ro(g! redo& reaction occ(rs* A depletes !ydrogen ions %ro$ s(ccinate and in t!e process %($arate is %or$ed, t!is is s!o'n in %ig(re 4* n t!is reaction PP acts as a electron acceptor t!at intercepts t!e ! ydrogen ions released %ro$ s(ccinate* T!e o&idation o% s(ccinate is re(ired %or t!e Citric Acid cycle to proceed*
Fi gur e4 : t heo xi dat i onofs uc c i nat eoc c ur st hr ought het r ans f erofh ydr ogeni onst oFAD.
n part one t!is e&peri$ent cell(lar respiration and alco!ol %er$entation 'ill be obser)ed* T!e goals is to %irst co$pare t!e %er$entation rates o% yeast (nder )ario(s conditions* T!ese conditions incl(ded gro'ing t!e yeast in one on e o% t!e t!ree carbo!ydrate sol(tions as a %ood so(rce .0*2 < s(crose, 0*2 < gl(cose or a sat(rated starc! sol(tion, at one o% t!e t!ree di%%erent te$perat(res .-º C, =25º C, or 4º C* And secondly $eas(re cell(lar respiration in $itoc!ondria t!at !a)e been isolated %ro$ li$a beans* east >east is #no'n %or brea#ing do'n co$ple& co $ple& carbo!ydrates to i$ply s(gars, so t!e gl(cose 'o(ld !a)e a %aster rate, %ollo'ing s(crose t!en t!e sat(rated starc! sol(tion* Mat!ra"s and M!t#ods
Fi gur e5: Fe r me nt a t i o nT u be Thear r owshowshow andwher eCO2gascol l ect edi n
n part t'o o% t!e e&peri$ent t!e spectrop!oto$eter 'as t(rned on and set to read t!e percentage o% trans$ittance o% @00 n$ o% lig!t* T!en si& c()ettes 'ere obtained labeling t!e tops B1, B2, 1 ,2 , , and 4* Si& s(ares o% para%il$ paper 'as t!en c(t to properly co)er co )er t!e tops o% t!e t(bes* T!e t(bes 'ere prepared according to a table si$ilar to %ig(re 5* B1 and B2 'ere (sed as blan#s* T!e blan#s 'ere prepared %irst t!en t!e c()ettes 'ere prepared* T!e s(ccinate 'as added last d(e to its %ast reaction ti$e to t!e sa$ple* 7nce t!e s(ccinate 'as ' as added eac! t(be 'as co)ered 'it! a piece p iece o% Para%il$ and 'as t!en )igoro(sly s!a#en %or t'o seconds to $i& t!e co$ponents*
Sa$%"! ID
Mtoc#ondra" Sus%!nson 'µ"(
DPIP 'µ"(
Bu&&!r '$"(
Succnat! 'µ"(
B1
2*15
0
150
200
B2
2*
0
0
200
1
2*2
150
150
0
2
2*1
150
150
100
2*0
150
150
200
4
2*15
150
0
200
F)ur! *+ Sc#!$! &or DPIP R!ducton R!actons 't#! $toc#ondra" sus%!nson ,as %r!-ous". %r!%ar!d and /!%t on c!(
Be%ore placing t!e c()ette in t!e spectrop!oto$eter, eac! t(be 'as 'iped do'n 'it! a 8i$'ipe to clear any s$(dges %ro$ inter%ering 'it! data* T!e Para%il$ %ro$ B1 'as re$o)ed and placed into t!e spectrop!oto$eter to blan# t!e $ac!ine* t 'as t!en re$o)ed and sa$ple id 1 'as inserted* "eadings at t!e 6ero ti$e point 'as ta#en* T!en sa$ple id 2 'as inserted and 6ero ti$e point 'as recorded, and t!is 'as repeated %or sa$ple id as 'ell* T!e spectrop!oto$eter 'as t!en blac# ed (sing B2, and t!e 6ero ti$e point %or sa$ple id 4 'as ta#en* or e)ery %i)e $in(tes t!ese steps 'ere repeated %or a total o% t!irty $in(tes*
R!su"ts
Fermentation Fermentation
n t!e %er$entation e&peri$ent, t!e a$o(nt o% g as prod(ced )aried a$ong t!e di%%erent carbo!ydrates and t!e di%%erent te$perat(res, ig(re - s!o's t!e res(lts o% t!e e&peri$ent* FoodSour ce Ti me ( mi n s )
37º C
~25º C
4º C
0 . 2M s u c r o s e
Cont r ol
0 . 2M s a t u r a t e d g l u c os e s t a r c h
H2O
5
0
0
0
0
10
0
2. 7
0. 2
0
15
0
5
0. 3
0
20
1
2. 4
0. 5
0
25
2. 6
3. 5
0. 7
0
30
4. 5
5
0. 7
0
35
6. 1
9( es t )
0. 8
0
40
7. 0
11( es t )
0. 8
0
5
0
0. 1
0. 1
0
10
0
0. 3
0. 1
0
15
0. 1
0. 7
0. 2
0
20
0. 4
0. 9
0. 4
0
25
0. 9
1. 0
0. 4
0
30
1. 5
1. 1
0. 4
0
35
1. 8
1. 8
0. 5
0
40
1. 9
2. 5
0. 5
0
5
1. 0
0. 3
0
0
10
1. 8
0. 9
0
0
15
2. 5
1. 5
0. 2
0
20
3. 1
1. 8
0. 3
0
25
3. 5
2. 3
1. 4
0
30
4. 0
2. 6
2. 5
0
35
4. 4
3. 1
2. 7
0
40
4. 7
3. 6
3. 1
0
Ti me( mi n s ) Samp mpl eI D 0
5
10
15
20
25
30
1
14. 0
14. 9
14. 6
14. 9
14. 5
13. 9
15. 0
2
16. 7
17. 1
18. 6
19. 5
20. 7
21. 6
22. 1
3
16. 9
18. 9
20. 4
21. 1
23. 2
24. 0
23. 9
4
9. 5
9. 1
9. 2
9. 7
10. 1
8. 9
9. 2
%T r a ns mi t t a nc eRe ad i n gso fRe du c edDPI P 3 0 . 0 2 4 . 0 1 8 . 0
%T r a ns mi mi t t a nc e 1
2
3
4
1 2 . 0 6. 0 0. 0
Ti me( mi ns)
Dscusson
T!e data !ig!ly s(pported t!e data* T!e di%%erence in $etabolic rates %or carbo!ydrates and te$perat(re di%%erence co(ld be acco(nted %or t!e rate o% alco!ol prod(ction increasing 'it! te$perat(re (p to a range o% 40º C* T!e cold te$perat(re 'o(ld slo' do'n t!e reaction* l(cose is a $onosacc!aride, s(crose is a disacc!aride, a nd starc! a polysacc!aride, t!is acco(nted %or t!e di%%ic(lty in yeast brea#ing do'n t!e s(gars, t!e $ore co$ple& t!e s(gar t!e slo'er t!e rate o% $etabolic reaction*
T!e reason %or sa$ple s!o'ing t!e s$all o% trans$ittance 'as d(e to t!e lac# o % $itoc!ondrial s(spension %or t!e PP to react on*
R!&!r!nc!s
/a)e, Carl "* .2012* Cellular Respiration* Respiration * "espiration* epart$ent o% P!ysics and an d Astrono$y, Astrono$y, eorgia State 3ni)ersity* 7nlineD* A)ailable: !ttp:EE!yperp!ysics*p!y astr*gs(*ed(E!baseEbiologyEcelres*!t$l** April @, 2015D* astr*gs(*ed(E!baseEbiologyEcelres*!t$l
Carbo!ydrates* .201* "etrie)ed April 14, 2015, %ro$ !ttp:EEc!e$ed*c!e$*p(rd(e*ed(Egenc!e$Etopicre)ie'EbpE1bioc!e$Ecarbo5*!t$l
