AND OPTIMIZATION
OF MICROBIAL
EVALUATION
CELLULOSE
(NATA) PRODUCTION USING PINEAPPLE WASTE AS SUBSTRACT
& I,I. MUHAMAD
C,H.CLI'NG
FacullyofChemicalandNaturalResources
Engineering,
ChelnicalEngineeringDeparlment,
UniversityTechnologyMalaysja,81310Skuda;,Johor.
Microbialcelluloseproduc.dby lcetabactetrllthtun is a new lype olbiopolymer.It
hasseleral uniquepropetiessuchas high puriryand uhra-fineoffiber nelwork,very hish hydrophific,
good mechanicalstrcngth.and outslandingshapereienlionand high cfystallinity.This thesisBas
undertaken
!o evalualerheproductionof microbialccllulosein stalica.d shakencullurccondilior using
pineappleNaste
astbe substmtelbf the tcrmentalionprocess.ShakenculiuretechniqDe
hasbeencaried
oul at 80 rpin, 120 rpm and 160 $m. Optimumconditions$e.e fixed at pH 5.0 and tcmpcraturcof
28"C for all the eapedmenl,in this.csc.trchproject.frcm this resedch,mioobial celluloseibrnation
in shakencullurewith additionalmicro particlesat 120rpn is the nroslsuilableas culLuremediumand
the nata produccis more thdn 70.230/0
by Neight compared!o slaricculturecondition.Optimialion
processfor shakenculture lvith addiljonalmicro pafticles(glassbeads)is the bes! rnediumculture.
micro psticlcs wereaddedinio mediumto enhancerhe produciionolcellulose in
SomewareFsoluble
shakencullure condilion. With the presenceof m;cfo particles.the nala wcight incrcase!o l5-l9o/o
comparedto shakenculture condit;on.Statisticalanal),sisoi erpe.inienlal design and numerical
pfocedureis the tool in analyzingthe yield. Yeastextrxctsnd arc thc most si8nificantiactorealltct
celluloseproduction.The bestsclcctediomulalion lbr preparingtho mediumcultufeare least extnct
] 49. KH:POIl.08g danN,lgSOa
0.069and natayield tbr 441.175g,1.
6g, suoose209.bactopepton
Kelvords: nifiobial cellulose-Acetoblcto x)linum, pineapplewaste,shakenculturecoidition, nata
Sclulosamikrobial )og dihas;lkanoleh bakt tia Acetabttet xylimn merupakan
sejenisbiololnner yang telbaru.Ia mempxnyaicifi-cni yang urik sepertijaringanfibef yang kua!,
b€rupayamemegangair yang bany*, kckuatannrekanikaldan pengekalanbentuk yang baik serta
kebolehanpenghabluran
)ang tnrggi.Proiekpenyelidikanini bertujuanuntuk ncmbandinskln kcsan
penghasilan seiulosa mikrobial dalam mcdium pcn8kuliuran stalik dar goncangan dengan
menggunakan
hasilsisadaripadanenasscbagsisrbsLrakdalamprosesiermentxsi.Teknik pengkulturan
goncanganirri diialankanpada80 rp , 120 rpn dan 160rpm. Keadaa|oplimun iailu nilai pll 5.0 dan
suhu 28"C diterapkandalam semua uikaji yang diialankan.Daripadapengkajianini, penshasild
selulosamikrobial dalam nediurn perrgkulturan
mikro patikel pada
soncansandergan penambahan
120 l"m adalahpaling sesuaidar jisin yangdiperoleli adalahbertambah70.230/".jika
dibandingkan
denganteknik pengkulturan
staiik.Untuk menlcdiakanmediumfer eitasi yang optimal.bebolakaca
scramikditambahkankc dalam medium pengkulxrangoDcansan120 rym untuk membantudalam
penghasilan
selulosamikrobial.Pcrtmbahanmikro padikel,.iisim natabeltambahsebrnyak15.l9ro
jika dibddingkan dcngrn tcknik pengkullurangoncangan.Kacdah siatistik dalam relobenruk
ekspefimental
dan kaeda|numedkaldiSunakan
daiammcnganalisis
hasilnala.Ekslrak!is dan Kll,POr
yang paling iiempensaruhimedium iemcnldsi. Fomulasi mcdium )!ne
merupakanpembolehubah
dipilih ialahekslrahyis 6g. sukrosa209,baktopeplon1.49,KHrPOrl.08gdan MSSOa0.069dan iisim
natayangdiperolehiialah4,1l.l75el1.
1.0INTRODUCTION
Nata is also known as microbial celluloseand it is the first commercially
prodlrctin Philippiles. Nata derivedfrom Latin word ralazc which means"to f]oat"
from fementing coconut water or fermentingrotting ftuits. Nata can $owth rn
coconutmilk, an abundantdomesticwasteproductor in a nutrient medium.The
thatthey identifiedasoptimalwerea temperature
culturecharacteristics
ofabout 28"C,
pH 5 to 5.5, ammoni m salts as nitrogensource,and either glucoseor sucroseas
carbonsource.Tbe organismwas identifiedaslcelol)acterrflinum. Tlle first studyof
celluloselormation in bacteriawas reportedby Adrian Brown in 1886. (Brown,
1986)noted that Acetobacterxyl[num c n producecelluloseat the surfaceol the
medium("xyljnuln" me4ningcotton).
Tlre bacteriumthat grows on the wastematerialis Acetobacteraceti.Se,,'eftl
genera that have shown lhe ability to s)nthesize cell$lose incfude Sarcinu,
Agrobacterium,Rhizobium,and Acetobacler.HoNe\er, Acetobacterrylinum is the
only speciesknown to be capableof producingcellulose.This organismand its
productwerefirst identifiedand chaucterizedovera centuryago,althoughbothwere
commonplacein the vinegarindustryfor quitea long time previousto identification.
Acetobacterxyli um, is a Crum-negative,rod shapedbacteriaand a prodigious
producerofcellulose,as pa|t ofit normalmetabolicactivit)'.The optimaltemperature
for growthis between25"Cto 30"C,andthe pII optimumabout5.4to 6.3 (Verschur€n,
1999).
Pineapplewasteis using in this research,
becausein lndonesiathere'sa huge
amountofpineappleswastebeingthfownawayeveryyear.The pineappleprodlrction
at Nordl Jawaat 1994reached444,507tonne(Tri Susantoet al., 2000).So now its
time to givingthesepineapplewastesan addedvalueto produceto a usefulproduction.
Oneofthe alternativewaysis extendedjt into microbialcelluloseproductor knownas
nata de pina. From the view point of matorialrecycling,microbialcellulose(nata)
producedfrom pineapplewasie using cellulose-producing
aceticacid bacteriawith
high microbial cellulose-producing
ability in acjdic medium is attractive.Pineappie
wastecanbe standfor pineapplecore,the peelingskin,or the pineapplecro\\'n.
Microbial celluloseis a folm of cellulosethat is producedby an aceticacidproducing bacterium, lcetobdcter xllifinn. Bacteria from the speciesof Ae/obdctel,
Acetobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotob.lcter,
Pseudomonas,
Rhizobiuma d.lalclra synthesizecellulose.Only the Acetobacter
speciesproduceenoughcelldosetojustify commercialinterest.Microbialcellulosers
devoidoflignin and hemicelluloses.
ll is extremelyhydrophilic,and hasan excellent
shapeand strengthretention.lt can be ptoducedfron] many differcntsubstmtes,
and
able to make to just aboutany shapeor size in the reactorvessel.It has superior
propertiessuchas an ultra fine netlvorkstr[cture,high biodegradability
and unique
mechanicalstrengthas comparcdwith greenplantcellulose.Therefore,it is expected
to be a new biodcgradable
biopolymer(TsuchidaT. andYoshinagaF., 1997).
2.0METHODOLOGY
2.1 Materials
The inoculumsculture(stafer culture)used,wasobtainedf'romMARDI, UPM.
Pineapplewastewas obtainedfrom pineappleprocessinglactory at Johorstateas a
substrate.
Yeastextract,potassiumdihydrogenphospbate,
sucrose,magnesiumsulfate
and bactopepton
were obtainedfrom BDH LaboratorySupplies.AII chemicalsused
wereof anahticalsrade.
2.2 Experiment Method
For staticcultureit is studybasedon diflerentlermentationtimes on 4, 8, 10,
dnd 13 daysat 28"C.For shakenculturewith and without micro padicleswerestudy
basedon differentshakingspeedon 80, 120,and 160 rpm (round per minute)in a
shakeincubatorfor 4 davsincubationat 280C.
Eachmediumwas prcparedusedthe sameamountofchemical reagent.Juice
f'rom pineapplewaste is used as a substrateand has a concentrationof (l;l) with
distilledwater.All the ingredientswere addedinto mediumand the pH is adjusted
until pH 5.0 usingpH meter.The mediumis plrtto autoclaveat 121'C, 15 min. After
cooledto room temperaturc,10yostafterculturc is transferredinto medium with
antiseptics
technique.The solutionis mixed apparentlyandput asidefor feflnentation
occurs.The best selectedmediumcultureis furtherwith optimizationprocessusing
designexpeftanalyseswith two levelfactodaldesigns.
2.JDelerminalionof Wcl \ eighland Dn WeightCellulose
Harvestthe rnicrobialcellulosewashedwith NaOH solution 2% ( v) and
againwashedwith plenty of water and dried with tissue.Recordingthe cleanwer
celluloseweightanddry in ovenfor 65"C for oneday.Cooled1{)ambienttemperature
andrecordthe drv celluloseweisht.
2.4Delermination
of ClucoseConlenlof MicrobislCellulosc
loml ofsampleis takenandcenlritugedat 5000rpm for 3 minutesto sediment
the celluloseand otherssolutes.3ml ofsupematantswere carriedout and addedwidl
3m1of 3,s-dinitocylicsilic(DNS) and centrifugedthoroughly.The mixing is placed
into waterbathfor 90'C at 15 minutes.After cooledto ambienttemperature
I.0ml of
potassiumnatrilrmtaftaratewas pipetteinto the solutionfor color stabilizing.Cell
cuvet fill with tbe mixing solution and rcad the optical density (OD) at 550nm
wavelength.
3.0 RESULTS AI{D DISCUSSIONS
3.1 StaticCulture Condition
The microbial celluloseproducedis proportjonateto fermentationtimes in
figure 1. As long as the medium is leave in a static condition,the cellulosewill
producedmore.lt canbe concludingthat celluloseweightswill continuousto increase
whenthe fermentationtimesis prolonged.The highestcelllLlose
weightproducedis at
13days.
The differentialvalueofglucoseabsorbbeforcand after putting in the sta er
cultureat 550 nm usingspectrophotometer
in figure2.Ihe higherabsorbvalueshow*
higherglucoseconcentration.
Beforeinoculatedthe mediumwith lcetobacterxylinum
hasa largeamountof glucosecontent.Therefore,glucoseconcentmtionis high liom
starting.During exponentialphase,bacteriausedup the existingglucoseas energyto
producernjoobial cellulose.Conseqlrently,
glucoseconcentmtiondecrcase
drastically
d ring exponentialphase.By the way,bacteriaalsoproducedan enzymeto hydrolyze
sucroseinto glucoseand fructose.On the daysof 13,the glucoseis foundaccumulate
in the medium.lt is foundthat,the smallerthe differentialvalue,the higherquantity
of microbialcelluloseproduced.
014
@
;
>0.06
;0 m
ec'hishu.$
10 13 14 15 18
Time
{days)
Figlre 1: Celluloseweightproduced
in differentfermenlationduratiors
Figufe2: Clucoseanalysesin diff'efent
lennentation
timesat OD 550
3.2 ShakenCulture Condition
It is study basedon the diflerentshakingspeedshowedby the bar charl of
figure 3 for 4 days fermentationin incubatorshakerat 28oC.The highestcellulose
yield is at 120 rpm. Whena mediumculiurcis shakenor sti ed,lcebbacter. xylinum
grows morc rapidll,; however,the cellulosefibrils do not form a well-organized
pellicle like what produceunderstaticcondition.But it is folrndthat at cedainshake
speedit is suitablefor bacteriagrow and producemore cellulose.Cellulosemasswill
decreased
when the shakespeedis increasedor above 160 rpm. This may due to
Acetobacterxylinum is difficult to adaptwith harshen\lironment.ln addition,the
originalhabitatof cellulosepioducerbacteriais on decayingfruits which is in stalrc
condition.
sh.kins speed (l?nt
A dry wcighr ccil!lose
K wer weieht ceUulosc
6 rvarer conieni
Figure3: Celluloseproducedin differentshakingspeed
3.3 ShakenCulture With Micro Particles
It is study basedon different shakingspeedwith the presenceof micro
particlesin the medium clrltureshowedon figure 4. Microbial cellulosesynthesis
occunedmore rapidlywhenIheAcetatbacler
,,ftJ?rn bacteriacellsare attachedto the
staticpalts of glassbeads.Upon shakenin the presenceof glassbeads,thercwill be
high dissolvedoxygenin the mediurnthat leadto an increasein cellulosefonnation.
glassbeadscansupply'multiple
This water-insoluble
adhesion
sit€s'in the medium.
Therefore,it is ableto enhancecellulosesynthesiswith the resultofthe development
ofan oxygen-limitingbiofilm arolrndthesepadicles(Vandammeet. a1.,1997).The
micro pafiiclescan be rangingfrorn diatomaceous
earth,silica gel, seasand,sma..
elassbeadsand loam.
r20
(rpm)
Shakins
Speod
Xd.rcellulose
wcight I \\ater!onlcnr E $et cellulose{,eight
Pigure4: Celluloseproducedin diflerentshal<ing
speedwith glassbeads
3.4 ComDarisono{Medium Culture Condition
Microbial celluloseproducedusing different culture condition is shown rn
figure 5. Shakenculture(120 rpm) produceshighercellulosemassthan staticculturc
conditionbccausehigheroxygendiffusesinto mediumat cefiain speed.The highest
produced
microbial
cellulose
is 50.91g. Thevalueis 37.25gramor 73.l7olo
morethan
cellulosemassproducein staticconditionlbr the samecompositionnutfientmedium.
But after addingmicro particlesin shakenculturea1 I20 rpm, the microbialcellulose
prodlrced
is 60.03g. This valueis about9.12 g or 15.19%more than microbial
celluloseproducein shakenculturewithoutmicro particles.
s ass beads @12orpm
70
60
9qo
!30
42f)
10
@120rpm
sralcclrture
Figure5: Comparison
celluloserveightin diff'erentcLrlturecondition
3.5 ODtimizationProcess
T\ir'olevel factorialdesignsareusedto determinethe optimummediumculture
conditionsto ma,\imizethe productionofmicrobial celluLose.
Basedon table l. there
are5 variableinvolved,andth€separameter
areusedin mediumprcparation.
Tablel: Variablefor mediumculture
Factor FactorName
FactorLevel
veaslexoact
5s(-l). 6e(+l)
B
sucrose
C
bactoDeDton
D
E
KH,PO,I
MeSO,r
4s( l) , 20e( + 1)
0.8gCl),
2.0s(+l)
0.6sCl),1.08e(+l
)
0.05qG
l). 0 . I q t + 1)
Basedon tigure 6, normal plot gmph showedonly most significantfactors
usedto analyzedin statisticalanalysison designexperiment.Yeastextract,potassium
dihydrogenphosphate(KHrPOr) and (interactionva.iable) consistof sucroseand
bactopepton.
Figure 7 showedthat bactopepton
and sucroseare totally not affected
towardscelluloseformation.The sugarcontentin pineapplewasteis high; therefore
the cells usedup the glucosecontentwithin the substratebeforeconveltthe sucrcse
into glucoseand fructose-So, sucrosenot affectsmuch in the celluloseproduction.
There are two lactors affect the productionof nata de pina during fermenlation
process,they are yeast extract which contributesnitrogen sourceand potassium
dihydrogenphosphate
alsohasthe samefunctionas yeastexhact.The factorA and D
show a stronglinearcorrelationwith wet celllrloseweight.With the combinationof
organicnitrogen(yeastextract)and inorganicnitrogensources(potassiumdihydrogen
phosphate)
will gavethe highestyield ofmicrobial cellulose.(Budhionoet al., 1999).
This trendin the eaperimentis agreedwell in the theory.
HaliNormaplot
,ti
i
€. j
g
;
E. i
;l
!
"J&
Figure6: HalfNonnal Plotand
EffectFor Wet CelluloseWeight
Figure7: Perturbation
For Wet
CelluloseWeight
The 3-D gaph showedin figure 8 will only analyseson variableof yeast
extnct and potassiumdihydrogenphosphate.
This is due to these2 variableare the
mostsigniticantfactorthat effecttowardscelluloseproduction.From the g(aph,wben
the amount of variable increase,the ceillrlose weight will goes up too. The
combinationoforganic and inorganicnitrogensourceslrccessfully
producedout more
celluloseashigh as 175.229in 4 dayst'ermentahon.
{--.
Figure8: Figure4.17:3-D Craphof Wet CelluloseYield asFunctionofKHrPOaand
YeastExtract
The optimummediumcultureto obtainthe highestyield ofmicrobjal cellulose
producing
in
Natade Pinausingjuicefrom pineapplewasteareshownin {igure9. The
optimumamountof eachvariablefor mediumclrltureare concludedas yeastextract
6.09, sucrose20.09, bactopepton1.,199,KHTPOa1.089and MgSO+0.069that cafl
producedmicrobialcelluloseas high as 176.479.Due to the designexperiment,it rs
shownthat magnesiumsulfatehasno effectin the productionof microbialcellulosc.
Therefore,the variableofmagnesiumsulfatecan ignoreusedin preparingthe medium
culture.
5.00
5.00
YeastExtract= 6.00
0.80
2.OO
= 1.49
tsactopepton
4.00
20.00
Sucrose 20.00
0.60
r .0 8
KH2POa= 1.08
0.10
63.07
173.31
gso4* (hasno effect)
cettutoseweight = 176.47g
= 0.962
Desirabilitv
Figxre9: Rampfor EachVariablein OptimizalionProcess
,1.0Conclusion
As a conclusion,the juice pineapplewasteis low in acid and high glucose
this conditionmakesi1 suitableas a substratefor lbrmentationprocess.
concenhation
The shakenculture is betterthan static culture condition,but shakenculturewith
additionalglassbeadspromoteshigh dissolvedoxygenin the mediumthat leadto an
increasein celluloselbrmation.The celluloseweightproducedin shakenculturewith
glass beadsis 15.19%more cellulosecomparedto shakenculture without micro
partjcles.Frcm optimizationprocess,it is foundthat yeastexhactand KHTPOTis the
mostsjgnificantfactorsthat canaffecttowardsproductionofNata de Pinausingjuice
from pineapplewaste.The bestoptimummediumculturethat can optimizemicrobial
celluloseyield is as below: yeastextract6.09, sucrcse20.09, bactopepton1.499,
potassiumdihydrogenphosphate(KI-lrPOa)1.089,and magnesiumsulfate(MgSOa)
0.06g with wet cellulose mass of 176.47g.From statisticalanalyseson design
experimenlsit is found that Magnesiumsulfate is not affectingtoward cellulose
formation.lt canbejust ignoreto usein prepared
mediumculturefor future.
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