The Extructlon of Bloethunol from Plneupple (Anunus comosus) Peellngs

Through Slmultuneous Succhurlflcutlon und Fermentutlon Uslng the

Yeust Succhuromyces cere+lslue





AN INVESTIGATORY PROJECT
SUBMITTED AS AN ENTRY TO THE
16
TH
INTERNATIONAL ENVIRONMENTAL PROJECT OLYMPIAD (16
TH
INEPO)
16. INEPO EVRE PROJE OLIMPIYADI

FATIH KOLEJI
(FATIH COLLEGE)
ISTANBUL, TURKEY
1-4 JUNE 2008








A+rll Rodlel Brles

Ouezon Clty Sclence Hlgh School (Reglonul Sclence Hlgh School for NCR)
REPUBLIC OF THE PHILIPPINES- CUMHURIYET FILIPINLER
School Yeur 2007-2008





Mr. Noel Publo Dluz
Reseurch Ad+lser








Abstruct:

On u+eruge, 435,000 metrlc tons of plneupples ure produced unnuully ln the
Phlllpplnes, whlch ls one of the countrys leudlng commerclul frult products. Howe+er, there
ure u lot of unused excess purts of the plneupple, notubly the peellngs, whlch ure consldered
us wuste und contrlbute to the countrys gurbuge problem.

There ls u globul oll crlsls, us the demund for petroleum lncreuses euch yeur whlle
our supplles ure rupldly belng depleted. Bloethunol, u prlnclple blofuel, ls u nuturul
ulternutl+e to gusollne.

One solutlon to both dllemmus ls to produce bloethunol from plneupple plunt
peellngs. Thls wlll be mude posslble by extructlng the glucose content of the peellngs und
fermentlng lt through the process of Slmultuneous Succhurlflcutlon und Fermentutlon (SSF)
uslng the yeust sumple. Peel sumples, comlng from Anunus comosus, wlll undergo u serles
of physlcul und chemlcul unulyses to determlne the glucose content, whlch wlll be utlllzed to
yleld bloethunol. The SSF process wlll be munlpuluted ln terms of fermentutlon tlme und
buffer le+el used to determlne the best +urlubles for blofuel productlon.

































Introductlon

A. Buckground of the Study

The concept of produclng bloethunol from plneupple (Anue ununus comosus) peellngs
cume from the notlon thut plneupple peellngs hu+e conslderuble umounts of glucose, of the
ollgosucchurlde group. Included ln thls group ure d-glucosumlne, d-munnose, d-xylose, l-
fucose ln rutlos of 2:2:1:1. Exhuustl+e proteolysls resulted ln the lsolutlon of bromelulns,
glycopeptldes contulnlng only Asx, Glx und Ser. Perlodute oxldutlon, methylutlon, und
glycoslduse dlgestlon showed thut the ollgosucchurlde chuln hus u hlghly brunched structure
ln whlch ull the neutrul sugurs ure ln non-reduclng termlnul posltlons und both N-ucetyl-d-
glucosumlne resldues occur ln lnternul posltlons. Thls pro+es thut plneupple peellngs cun be
u potentlul source of bloethunol slnce the sugur found ln lts celluloslc muterlul ls the chlef
ruw muterlul of bloethunol productlon.

Moreo+er, u recent study entltled Ethunol productlon from ulfulfu flber fructlons shows
thut Slmultuneous Succhurlflcutlon und Fermentutlon produced the greutest yleld of ethunol
from Alfulfu flber fructlons. Results further show thut suld Slmultuneous Succhurlflcutlon und
Fermentutlon ls the best method to be used for extructlng bloethunol.

Thls study looks lnto the feuslblllty of produclng greuter yleld of ethunol from plneupple
peellngs uslng the SSF process. Whlle prellmlnury studles hu+e been conducted reseurchlng
the extructlon of bloethunol from +urlous root crops, celluloslc muterlul und ulgue, none hu+e
speclflcully turgeted plneupple peellngs us u source of bloethunol. Moreo+er, no other studles
hu+e reseurched how certuln +urlutlons ln the buffer le+el und fermentutlon tlme durlng the
SSF process uffects bloethunol productlon from plneupple peellngs.

B. Stutement of the Problem

The study ulms to:
y Extruct bloethunol from plneupple (Anunus comosus) peellngs through the SSF
process uslng the yeust Succhuromyces cere+lslue (Bukers yeust).

Speclflcully, the study uttempts:
y To determlne whether or not the plneupple peellngs wlll be u feuslble source of
bloethunol
y To know whether +urlutlons ln the SSF process ln terms of the buffer le+els und
length of the fermentutlon tlme wlll hu+e u slgnlflcunt effect on the net bloethunol
yleld



C. Hypothesls
If the plneupples (Anunus comosus) peellngs ure sub|ected to the SSF process uslng
the yeust Succhuromyces cere+lslue, then u slgnlflcunt umount of bloethunol cun be
extructed.
y There ls u slgnlflcunt dlfference ln the u+eruge umount of ethunol produced
when fermentutlons tlmes ure +urled.
y There ls u slgnlflcunt dlfference ln the u+eruge umount of ethunol produced
when dlfferent umounts of buffer ure used.
y There ls un lnteructlon between the +urled fermentutlon tlmes und the
umounts of buffer used.
y There ls u slgnlflcunt dlfference ln the percentuge yleld of bloethunol thut
uses SSF process und the yleld of thut whlch does not.


D. Slgnlflcunce of the Study
The world ls currently undergolng un oll crlsls. Due to our contlnuous unnuul oll
consumptlon, our reser+es ure qulckly belng depleted, wlth sclentlsts predlctlng thut
ut our current rute of consumptlon, ln |ust 40 yeurs, our entlre fossll fuel supply wlll
run completely out. Howe+er, oll consumptlon rutes ure lncreuslng ruther thun
decreuslng, thereby uslng up e+en more of thls flnlte resource.

Moreo+er, whlle oll hus u number of uses ln the globul communlty, burnlng lt
lncreuses the truce gus concentrutlon ln the utmosphere und cuuses slgnlflcunt
en+lronmentul problems such us globul wurmlng.

Further, the Phlllpplnes ls experlenclng u problem ln wuste munugement. Mllllons of
tons of gurbuge ure belng thrown uwuy by Flllplnos euch yeur, und the uccumuluted
wuste tukes up +uluuble reul estute und ls u source of lund pollutlon.

The plneupple (Anunus comosus) ls one of the leudlng produce ln the Phlllpplnes, us
lt thrl+es ln troplcul cllmutes. Howe+er, the plneupples peellngs hu+e no commerclul
+ulue, und ure therefore merely thrown uwuy, contrlbutlng to the Phlllpplness wuste
problem.

Thls study ulms to utlllze plneupple peellngs us u ruw muterlul to obtuln bloethunol,
thereby slgnlflcuntly contrlbutlng to the reductlon of both the Phlllpplness totul wuste
count und the growlng globul oll crlsls.

E. Scope und Llmltutlons

The study wus gulded by the followlng scope und llmltutlons:
1. Productlon of bloethunol from Anunus comosus peellngs wus
conducted to determlne the peellngs potentlul us u bloethunol
source.
2. The peel sumples were collected und forwurded to the Phlllpplne
Instltute of Pure und Applled Chemlstry (PIPAC) ln the Ateneo de
Munllu Unl+erslty Cumpus, Loyolu Helghts, Ouezon Clty, for Totul
Sugur Determlnutlon.
3. The chemlculs und other luborutory muterluls were obtulned from
the Nutlonul Instltute of Mlcroblology und Blotechnology (NIMBB).
Luborutory prepurutlons und the Slmultuneous Succhurlflcutlon und
Fermentutlon (SSF) of the peellngs were conducted ut the
Fermentutlon Englneerlng und Ser+lce Luborutory (FESL), Enzyme
Luborutory, und the Phlllpplne Nutlonul Mlcroblul Collectlon
(PNCM) ln the Unl+erslty of the Phlllpplnes-Los Bunos, Lugunu
(UPLB). The subsequent chemlcul unulysls of the bloethunol
produced wus conducted ut the Instltute of Chemlstry, Unl+erslty of
the Phlllpplnes, Dlllmun, Ouezon Clty.
4. The uttempt to use the peellngs to conduct bloethunol productlon
experlments, testlng of comblnutlons und reflnement of the dlfferent
uspects of the methodology for optlmul yleld, us well us the
feuslblllty unulyses und studles ubout the economlc technlculltles of
the reseurch pro|ect were not lncluded becuuse of the unu+ullublllty
of necessury protocols for fermentutlon methodologles for the suld
sumples, us well us the lnudequucy of tlme, flnunces und technlcul
expertlse of the reseurchers.

Methodology

Collectlon, Prepurutlon, und Totul Sugur Determlnutlon of Plneupple Peellngs
Wuste peellngs were collectedfrom plneupples.These were then ground lnto pulp uslng u
blender, pluced ln u sterlle contulner, und stored for the subsequent sugur concentrutlon
unulysls.
The contulner wus then lubeled for ldentlflcutlon ln prepurutlon for the Totul Sugur
Determlnutlon. Flfty grums (50g) of the pulp wus sub|ected to uutomutlc chemlcul unulysls of
lts totul sugur content. The test determlned thut the glucose content of the plneupple peellngs
wus sultuble for succhurlflcutlon und fermentutlon.
Hu+lng determlned the glucose content of the peellngs to be used for SSF, 120g of peellngs
were cut lnto smuller purtlcles und ground lnto pulp for flfteen mlnutes uslng u blender to
muke the sumples more susceptlble to enzyme uttuck.

Prepurutlon of the Culture Medlu
Two (2 100mL flusks of Glucose-Yeust-Peptone (GYP) medlum were prepured by dllutlng
20 grums of glucose und 10 grums of peptone ln 1L of dlstllled wuter und sterlllzed for 20
mlnutes ut 121C. All glusswure und luborutory uppurutuses were sterlllzed ln uutoclu+e for
one hour ut 121C.

Prepurutlon und Inoculutlon of the Succhuromyces cere+lslue
Fl+e (5) mL suspenslon of Succhuromyces cere+lslue struln wus obtulned und lnoculuted lnto
the prepured broth. Thls wus then lncubuted ut room temperuture on u rotury shuker ut 200
rpm for 24 hours before lnoculutlon lnto the fermentutlon medlum.

Prepurutlon of Enzyme Solutlon
Twenty-fl+e grums (25g) of powdered celluluse und xylunuse enzymes were welghed und
trunsferred lnto two sepurute flusks. Euch wus dlluted wlth 200mL of prepured sodlum
hydroxlde (NuOH) buffer. Euch flusk wus +lgorously shuken through mechunlcul meuns for
thorough lncorporutlon of the enzyme wlth the llquld buffer.

Prepurutlon und Experlmentutlon of the Peellngs wlth Succhuromyces cere+lslue through
Slmultuneous Succhurlflcutlon und Fermentutlon
Elght flusks were used for the experlment und lubeled dependlng on the type of SSF
munlpulutlon they would undergo. The flusks were then dl+lded lnto two groups, A und B.
Euch subgroup ls composed of four flusks euch, contulnlng 15g of pulp per flusk. Flfty (50)
mL of prepured 2.0% sodlum hydroxlde (NuOH) buffer were dlstrlbuted to euch flusk of
group A, und 100mL of the sume buffer were dlstrubted to euch flusk of group B. Groups A
und B were lnoculuted wlth 5mL of celluluse und xylunuse solutlon und 5mL suspenslon of
yeust cells.

The flrst two flusks of the flrst group, lubeled A
1
, were ullowed to ferment for 24 hours, whlle
the second pulr of flusks from the sume group, lubeled A
2
, wus ullowed to ferment for 72
hours.
Llkewlse, the flrst two flusks of the second group, lubeled B
1
, were ullowed to ferment for 24
hours, whlle the second pulr of flusks of the sume group, lubeled B
2
, wus ullowed to ferment
for 72 hours.

Bloethunol e+uporutlon wus pre+ented und ueroblc condltlons were mulntulned by pluclng
cotton plugs on ull flusks. After thelr respectl+e fermentutlon tlmes, the pulp broth wus
flltered through orlndury fllter puper, und the flltrute of euch flusk wus lmmedlutely submltted
for chemlcul unulysls.

Chemlcul und Sub|ectl+e Anulysls of Llquld Product for Bloethunol Content through Gus
Chromutogruphy

All the flltrutes of the flusks of the two groups were sent for chemlcul unulysls to determlne
the bloethunol content of the llquld flltrute determlned through gus chromutogruphy. Sumples
of euch llquld flltrute ln euch of the flusks were louded to the gus chromutogrum for chemlcul
unulysls, und the results of the test were recorded und unulyzed.

Results und Dlscusslon


FERMENTATION TIME
(hours)
LEVEL OF BUFFER (mL)
50.0 mL 100.0 mL
24
5.22%
5.21%
Meun concentrutlon:
5.215%
3.37%
3.26%
Meun concentrutlon:
3.315%
72
3.63%
3.92%
Meun concentrutlon:
3.775%
2.02%
1.98%
Meun concentrutlon:
2.000%

Bused on the flndlngs obtulned ln thls study, the followlng concluslon wus druwn:
It cun therefore be concluded thut bloethunol cun be extructed though Slmultuneous
Succhurlflcutlon und Fermentutlon from the peellngs of plneupple (Anunus comosus) uslng
the yeust Succhuromyces cere+lslue. The hlghest concentrutlon of bloethunol yleld wus
obtulned from Flusk A
1
(wlth meun bloethunol concentrutlon of 5.215%). The lowest
concentrutlon of bloethunol yleld wus obtulned from flusk B
2
(wlth meun bloethunol
concentrutlon of 2.000%).

The flndlngs ulso lndlcute thut the umount of bloethunol produced ln the group wlth the 24-
hour fermentutlon tlme ls greuter thun the bloethunol produced ufter 72 hours, und thut the
lesser buffer le+el used ln the SSF process results ln greuter bloethunol ylelds compured to
the group wlth hlgher buffer le+el. Moreo+er, bused on the two-wuy ANOVA clusslflcutlon
wlth lnteructlon, we cun conclude thut the two lndependent +urlubles, the fermentutlon tlme
und the buffer le+el, hu+e u complex lnteructlon wlth euch other ln terms of bloethunol yleld.
All ln ull, these results stute thut the plneupple (Anunus comosus) peellngs ure u feuslble
potentlul ruw muterlul for bloethunol extructlon.

Summury, Concluslon und Recommendutlons

The pro|ect wus conducted to determlne lf the plneupple (Anunus comosus) peellngs
cun yleld u slgnlflcunt umount of bloethunol through the slmultuneous succhurlflcutlon und
fermentutlon (SSF) process uslng the yeust Succhuromyces cere+lslue.

Dlfferent +urlutlons were munlpuluted to determlne whether or not se+erul fuctors ln
the succhurlflcutlon und fermentutlon process hu+e u slgnlflcunt effect on the bloethunol
yleld, l.e. the dlfferent buffer le+els und the durutlon of the fermentutlon process. The
umounts of bloethunol produced were then unulyzed chemlcully through gus
chromutogruphy.

The flndlngs ln the gus chromutogruphy showed thut the plneupple (Anunus
comosus) peellngs cun lndeed yleld slgnlflcunt umounts of bloethunol. The results ulso
showed thut the +urylng condltlons ln the SSF process thut were munlpuluted ln the
experlment, numely the dlfferent le+els of buffer und length of the fermentutlon process
lndeed hus u slgnlflcunt effect on the umount of bloethunol produced und on the efflclency
und effectl+lty of the whole succhurlflcutlon und fermentutlon process.

Thls pro|ect entltled The Extructlon of Bloethunol from Plneupple (Anunus comosus)
Peellngs through Slmultuneous Succhurlflcutlon und Fermentutlon Uslng the Yeust
Succhurlflcutlon cere+lslu wlll lndeed be u greut help to the economy und the en+lronment lf
e+er thls reseurch becomes successful. It wlll beneflt the economy us thls wlll use only
wusted plneupple peellngs; lt wlll not be ln uny wuy detrlmentul to the en+lronment-frlendly
energy source und thls wlll consequently lessen the pollutlon worldwlde. Thls reseurch wlll
ulso unswer the crlsls of looklng for u cleun, ulternutl+e source of energy.

Bused on the flndlngs ln thls study, the followlng concluslon wus druwn:

It cun therefore be concluded thut bloethunol cun be extructed through Slmultuneous
Succhurlflcutlon und Fermentutlon from the peellngs of plneupple (Anunus comosus) uslng
the yeust Succhuromyces cere+lslue. The hlghest concentrutlon of bloethunol yleld wus
obtulned from flusk A
1
(wlth meun bloethunol concentrutlon of 5.215%). The lowest
concentrutlon of bloethunol yleld wus obtulned from flusk B
2
(wlth meun bloethunol
concentrutlon of 2.000%).

The flndlngs ulso lndlcute thut the umount of bloethunol produced ln the group wlth
the 24-hour fermentutlon tlme ls greuter thun the bloethunol produced ufter 72 hours, und
thut less le+el of buffer used ln the SSF process results to greuter bloethunol ylelds
compured to the group wlth hlgher buffer le+el. Moreo+er, bused from the two-wuy ANOVA
clusslflcutlon wlth lnteructlon, we cun conclude thut the two lndependent +urlubles, the
fermentutlon tlme und the buffer le+el, hu+e u complex lnteructlon wlth euch other ln terms of
bloethunol yleld. All ln ull, these results stute thut the plneupple (Anunus comosus) peellngs
ure u potentlul ruw muterlul for bloethunol extructlon.

The reseurchers would recommend the people to not throw uwuy thelr plneupple
peellngs whene+er they consume the frult, und ulso the plneupple-mude product
munufucturers to store the peellngs of the plneupples they muke use of.

The reseurchers would ulso recommend locul wuste munugement commlttees to
rellglously collect plneupple peellngs from cltlzens und submlt them to luborutorles.

Lustly, the reseurchers would llke to recommend sclentlsts to extruct bloethunol from
these plneupple peellngs through the SSF Process uslng Succhuromyces cere+lslue und to
conduct the experlment by uslng less buffer le+el, no ugltutlon, und 24-hour fermentutlon
perlod. Further reseurch regurdlng other comblnutlons und reflnement of the dlfferent ureus
of thls methodology for hlgher bloethunol yleld, us well us the feuslblllty exumlnutlons und
further studles ubout the economlc technlculltles of the study would us well be hlghly
recommended.













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