EVALUATION AND OPTIMIZATION OF MICROBIAL CELLULOSE(NATA) PRODUCTION USING PINEAPPLE WASTE AS SUBSTRACT

C,H.CLI 'NG & I , I . MUHAMADChelnical Engineering Deparlment, Facully ofChemical and Natural Resources Engineering,

University Technology Malaysja, 81310 Skuda;, Johor.

has seleral unique propeties such as high puriry and uhra-fine offiber nelwork, very hish hydrophific,good mechanical strcngth. and outslanding shape reienlion and high cfystallinity. This thesis Basundertaken !o evaluale rhe production of microbial ccllulose in stalic a.d shaken cullurc condilior usingpineappleNaste as tbe substmte lbf the tcrmentalion process. Shaken culiure techniqDe has been cariedoul at 80 rpin, 120 rpm and 160 $m. Optimum conditions $e.e fixed at pH 5.0 and tcmpcraturc of28"C for all the eapedmenl, in this.csc.trch project. frcm this resedch, mioobial cellulose ibrnationin shaken cullure with additional micro particles at 120 rpn is the nrosl suilable as culLure medium andthe nata producc is more thdn 70.230/0 by Neight compared !o slaric culture condition. Optimialionprocess for shaken culture lvith addiljonal micro pafticles (glass beads) is the bes! rnedium culture.Some wareFsoluble micro psticlcs were added inio medium to enhance rhe produciion olcellulose inshaken cullure condilion. With the presence of m;cfo particles. the nala wcight incrcase !o l5-l9o/ocompared to shaken culture condit;on. Statistical anal),sis oi erpe.inienlal design and numericalpfocedure is the tool in analyzing the yield. Yeast extrxct snd arc thc most si8nificant iactore alltctcellulose production. The best sclccted iomulalion lbr preparing tho medium cultufe are least extnct6g, suoose 209. bactopepton ] 49. KH:POIl.08g dan N,lgSOa 0.069 and nata yield tbr 441.175g,1.

Kelvords: nifiobial cellulose- Acetoblcto x)linum, pineapple waste, shaken culture coidition, nata

Microbial cellulose produc.d by lcetabactet rllthtun is a new lype olbiopolymer. It

Sclulosa mikrobial )og dihas;lkan oleh bakt tia Acetabttet xylimn merupakansejenis biololnner yang telbaru. Ia mempxnyai cifi-cni yang urik sepertijaringan fibef yang kua!,b€rupaya memegang air yang bany*, kckuatan nrekanikal dan pengekalan bentuk yang baik sertakebolehan penghabluran )ang tnrggi. Proiek penyelidikan ini bertujuan untuk ncmbandinskln kcsanpenghasilan seiulosa mikrobial dalam mcdium pcn8kuliuran stalik dar goncangan denganmenggunakan hasil sisa daripada nenas scbagsi srbsLrak dalam proses iermentxsi. Teknik pengkulturangoncangan irri diialankan pada 80 rp , 120 rpn dan 160 rpm. Keadaa| oplimun iailu nilai pll 5.0 dansuhu 28"C diterapkan dalam semua uikaji yang diialankan. Daripada pengkajian ini, penshasildselulosa mikrobial dalam nediurn perrgkulturan soncansan dergan penambahan mikro patikel pada120 l"m adalah paling sesuai dar jisin yang diperoleli adalah bertambah 70.230/".jika dibandingkandengan teknik pengkulturan staiik. Untuk menlcdiakan medium fer eitasi yang optimal. bebola kacascramik ditambahkan kc dalam medium pengkulxran goDcansan 120 rym untuk membantu dalampenghasilan selulosa mikrobial. Pcrtmbahan mikro padikel,.iisim nata beltambah sebrnyak 15.l9rojika dibddingkan dcngrn tcknik pengkulluran goncangan. Kacdah siatistik dalam relobenrukekspefimental dan kaeda| numedkal diSunakan daiam mcnganalisis hasil nala. Ekslrak !is dan Kll,POrmerupakan pembolehubah yang paling iiempensaruhi medium iemcnldsi. Fomulasi mcdium )!nedipilih ialah ekslrah yis 6g. sukrosa 209, baktopeplon 1.49, KHrPOrl.08g dan MSSOa 0.069 dan iisimnata yang diperolehi ialah 4,1l.l75el1.

1.0 INTRODUCTION

Nata is also known as microbial cellulose and it is the first commerciallyprodlrct in Philippiles. Nata derived from Latin word ralazc which means "to f]oat"from fementing coconut water or fermenting rotting ftuits. Nata can $owth rncoconut milk, an abundant domestic waste product or in a nutrient medium. Theculture characteristics that they identified as optimal were a temperature ofabout 28"C,pH 5 to 5.5, ammoni m salts as nitrogen source, and either glucose or sucrose ascarbon source. Tbe organism was identified as lcelol)acter rflinum. Tlle first study ofcellulose lormation in bacteria was reported by Adrian Brown in 1886. (Brown,

1986) noted that Acetobacter xyl[num c n produce cellulose at the surface ol themedium ("xyl jnuln" me4ning cotton).

Tlre bacterium that grows on the waste material is Acetobacter aceti. Se,,'eftlgenera that have shown lhe ability to s)nthesize cell$lose incfude Sarcinu,Agrobacterium, Rhizobium, and Acetobacler. HoNe\er, Acetobacter rylinum is theonly species known to be capable of producing cellulose. This organism and itsproduct were first identified and chaucterized over a century ago, although both werecommon place in the vinegar industry for quite a long time previous to identification.Acetobacter xyli um, is a Crum-negative, rod shaped bacteria and a prodigiousproducer ofcellulose, as pa|t ofit normal metabolic activit)'. The optimal temperaturefor growth is between 25"C to 30"C, and the pII optimum about 5.4 to 6.3 (Verschur€n,1999).

Pineapple waste is using in this research, because in lndonesia there's a hugeamount ofpineapples waste being thfown away every year. The pineapple prodlrctionat Nordl Jawa at 1994 reached 444,507 tonne (Tri Susanto et al., 2000). So now itstime to giving these pineapple wastes an added value to produce to a useful production.One ofthe alternative ways is extended jt into microbial cellulose product or known asnata de pina. From the view point of matorial recycling, microbial cellulose (nata)produced from pineapple wasie using cellulose-producing acetic acid bacteria withhigh microbial cellulose-producing ability in acjdic medium is attractive. Pineappiewaste can be stand for pineapple core, the peeling skin, or the pineapple cro\\'n.

Microbial cellulose is a folm of cellulose that is produced by an acetic acid-producing bacterium, lcetobdcter xllifinn. Bacteria from the species of Ae/obdctel,Acetobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotob.lcter,Pseudomonas, Rhizobium a d.lalclra synthesize cellulose. Only the Acetobacterspecies produce enough celldose tojustify commercial interest. Microbial cellulose rsdevoid oflignin and hemicelluloses. ll is extremely hydrophilic, and has an excellentshape and strength retention. lt can be ptoduced fron] many differcnt substmtes, andable to make to just about any shape or size in the reactor vessel. It has superiorproperties such as an ultra fine netlvork str[cture, high biodegradability and uniquemechanical strength as comparcd with green plant cellulose. Therefore, it is expectedto be a new biodcgradable biopolymer (Tsuchida T. and Yoshinaga F., 1997).

2.0 METHODOLOGY

2.1 Materials

The inoculums culture (stafer culture) used, was obtained f'rom MARDI, UPM.Pineapple waste was obtained from pineapple processing lactory at Johor state as a

substrate. Yeast extract, potassium dihydrogen phospbate, sucrose, magnesium sulfateand bactopepton were obtained from BDH Laboratory Supplies. AII chemicals usedwere of anahtical srade.

2.2 Experiment Method

For static culture it is study based on diflerent lermentation times on 4, 8, 10,dnd 13 days at 28"C. For shaken culture with and without micro padicles were studybased on different shaking speed on 80, 120, and 160 rpm (round per minute) in ashake incubator for 4 davs incubation at 280C.

Each medium was prcpared used the same amount ofchemical reagent. Juicef'rom pineapple waste is used as a substrate and has a concentration of (l;l) withdistilled water. All the ingredients were added into medium and the pH is adjusteduntil pH 5.0 using pH meter. The medium is plrt to autoclave at 121'C, 15 min. Aftercooled to room temperaturc, 10yo stafter culturc is transferred into medium withantiseptics technique. The solution is mixed apparently and put aside for feflnentationoccurs. The best selected medium culture is further with optimization process usingdesign expeft analyses with two level factodal designs.

2.J Delerminalion of Wcl \ eighl and Dn Weight Cellulose

Harvest the rnicrobial cellulose washed with NaOH solution 2% ( v) andagain washed with plenty of water and dried with tissue. Recording the clean wercellulose weight and dry in oven for 65 "C for one day. Cooled 1{) ambient temperatureand record the drv cellulose weisht.

2.4 Delermination of Clucose Conlenl of Microbisl Cellulosc

loml ofsample is taken and cenlrituged at 5000 rpm for 3 minutes to sedimentthe cellulose and others solutes. 3ml ofsupematants were carried out and added widl3m1 of 3,s-dinitocylicsilic (DNS) and centrifuged thoroughly. The mixing is placedinto water bath for 90'C at 15 minutes. After cooled to ambient temperature I.0ml ofpotassium natrilrm taftarate was pipette into the solution for color stabilizing. Cellcuvet fill with tbe mixing solution and rcad the optical density (OD) at 550nmwavelength.

3.0 RESULTS AI{D DISCUSSIONS

3.1 Static Culture Condition

The microbial cellulose produced is proportjonate to fermentation times infigure 1. As long as the medium is leave in a static condition, the cellulose willproduced more. lt can be concluding that cellulose weights will continuous to increasewhen the fermentation times is prolonged. The highest celllLlose weight produced is at13 days.

The differential value ofglucose absorb beforc and after putting in the sta erculture at 550 nm using spectrophotometer in figure 2.Ihe higher absorb value show *higher glucose concentration. Before inoculated the medium with lcetobacter xylinumhas a large amount of glucose content. Therefore, glucose concentmtion is high liomstarting. During exponential phase, bacteria used up the existing glucose as energy toproduce rnjoobial cellulose. Conseqlrently, glucose concentmtion decrcase drasticallyd ring exponential phase. By the way, bacteria also produced an enzyme to hydrolyzesucrose into glucose and fructose. On the days of 13, the glucose is found accumulatein the medium. lt is found that, the smaller the differential value, the higher quantityof microbial cellulose produced.

014

@

;>0.06

;0m

ec'hishu.$ 10 13 14 15 18

Figlre 1: Cellulose weight producedin different fermenlation duratiors

Time {days)

Figufe 2: Clucose analyses in diff'efentlennentation times at OD 550

3.2 Shaken Culture Condition

It is study based on the diflerent shaking speed showed by the bar charl offigure 3 for 4 days fermentation in incubator shaker at 28oC. The highest celluloseyield is at 1 20 rpm. When a medium culiurc is shaken or sti ed, lcebbacter. xylinumgrows morc rapidll,; however, the cellulose fibrils do not form a well-organizedpellicle like what produce under static condition. But it is folrnd that at cedain shakespeed it is suitable for bacteria grow and produce more cellulose. Cellulose mass willdecreased when the shake speed is increased or above 160 rpm. This may due toAcetobacter xylinum is difficult to adapt with harsh en\lironment. ln addition, theoriginal habitat of cellulose pioducer bacteria is on decaying fruits which is in stalrccondition.

sh.kins speed (l?nt

A dry wcighr ccil!lose 6 rvarer conieniK wer weieht ceUulosc

Figure 3: Cellulose produced in different shaking speed

3.3 Shaken Culture With Micro Particles

It is study based on different shaking speed with the presence of microparticles in the medium clrlture showed on figure 4. Microbial cellulose synthesisoccuned more rapidly when Ihe Acetatbacler ,,ftJ?rn bacteria cells are attached to thestatic palts of glass beads. Upon shaken in the presence of glass beads, therc will behigh dissolved oxygen in the mediurn that lead to an increase in cellulose fonnation.This water-insoluble glass beads can supply'mult iple adhesion sit€s' in the medium.Therefore, it is able to enhance cellulose synthesis with the result ofthe developmentofan oxygen-limiting biofilm arolrnd these padicles (Vandamme et. a1., 1997). Themicro pafiicles can be ranging frorn diatomaceous earth, silica gel, sea sand, sma..elass beads and loam.

r20Shakins Speod (rpm)

Xd.rcellulose wcight I \\ater !onlcnr E $et cellulose{,eight

Pigure 4: Cellulose produced in diflerent shal<ing speed with glass beads

3.4 ComDarison o{Medium Culture Condition

Microbial cellulose produced using different culture condition is shown rnfigure 5. Shaken culture (120 rpm) produces higher cellulose mass than static culturccondition bccause higher oxygen diffuses into medium at cefiain speed. The highestmicrobial cel lulose produced is 50.91 g. The value is 37.25 gram or 73.l7olo more thancellulose mass produce in static condition lbr the same composition nutfient medium.But after adding micro particles in shaken culture a1 I20 rpm, the microbial celluloseprodlrced is 60.03 g. This value is about 9.12 g or 15.19% more than microbialcellulose produce in shaken culture without micro particles.

s ass beads @12orpm

70

60

@120rpm sralcclrture

9qo!30

42f)

10

Figure 5: Comparison cellulose rveight in diff'erent cLrlture condition

3.5 ODtimization Process

T\ir'o level factorial designs are used to determine the optimum medium cultureconditions to ma,\imize the production ofmicrobial celluLose. Based on table l. thereare 5 variable involved, and th€se parameter are used in medium prcparation.

Table l: Variable for medium cultureFactor Factor Name Factor Level

veasl exoact 5s(- l). 6e(+l )B sucrose 4s( l ) , 20e(+1)C bactoDeDton 0.8gCl), 2.0s(+l)D KH,PO,I 0.6sCl), 1.08e(+l )E MeSO,r 0.05qG l) . 0. I qt+1 )

Based on tigure 6, normal plot gmph showed only most significant factorsused to analyzed in statistical analysis on design experiment. Yeast extract, potassiumdihydrogen phosphate (KHrPOr) and (interaction va.iable) consist of sucrose andbactopepton. Figure 7 showed that bactopepton and sucrose are totally not affectedtowards cellulose formation. The sugar content in pineapple waste is high; thereforethe cells used up the glucose content within the substrate before convelt the sucrcseinto glucose and fructose- So, sucrose not affects much in the cellulose production.There are two lactors affect the production of nata de pina during fermenlationprocess, they are yeast extract which contributes nitrogen source and potassiumdihydrogen phosphate also has the same function as yeast exhact. The factor A and Dshow a strong linear correlation with wet celllrlose weight. With the combination oforganic nitrogen (yeast extract) and inorganic nitrogen sources (potassium dihydrogenphosphate) will gave the highest yield ofmicrobial cellulose. (Budhiono et al., 1999).This trend in the eaperiment is agreed well in the theory.

HaliNorma plot

€. j

E. i

; l

!

"J&

,t i

ig

;

Figure 6: HalfNonnal Plot andEffect For Wet Cellulose Weight

Figure 7: Perturbation For WetCellulose Weight

The 3-D gaph showed in figure 8 will only analyses on variable of yeastextnct and potassium dihydrogen phosphate. This is due to these 2 variable are themost signiticant factor that effect towards cellulose production. From the g(aph, wbenthe amount of variable increase, the ceillrlose weight will goes up too. Thecombination oforganic and inorganic nitrogen source slrccessfully produced out morecellulose as high as 175.229 in 4 days t'ermentahon.

{- - .

Figure 8: Figure 4.17: 3-D Craph of Wet Cellulose Yield as Function ofKHrPOa andYeast Extract

The optimum medium culture to obtain the highest yield ofmicrobjal cellulosein producing Nata de Pina usingjuice from pineapple waste are shown in {igure 9. Theoptimum amount of each variable for medium clrlture are concluded as yeast extract6.09, sucrose 20.09, bactopepton 1.,199, KHTPOa 1.089 and MgSO+ 0.069 that caflproduced microbial cellulose as high as 176.479. Due to the design experiment, it rsshown that magnesium sulfate has no effect in the production of microbial cellulosc.Therefore, the variable ofmagnesium sulfate can ignore used in preparing the mediumculture.

5.00 5.00 4.00 20.00Yeast Extract = 6.00 Sucrose

- 20.00

0.80 2.OO 0.60tsactopepton = 1.49 KH2POa =

r .081.08

0.10 63.07 173.31gso4* (has no effect)

cettutose weight = 176.47gDesirabil itv = 0.962

Figxre 9: Ramp for Each Variable in Optimizalion Process

,1.0 Conclusion

As a conclusion, the juice pineapple waste is low in acid and high glucoseconcenhation this condition makes i1 suitable as a substrate for lbrmentation process.The shaken culture is better than static culture condition, but shaken culture withadditional glass beads promotes high dissolved oxygen in the medium that lead to anincrease in cellulose lbrmation. The cellulose weight produced in shaken culture withglass beads is 15.19% more cellulose compared to shaken culture without micropartjcles. Frcm optimization process, it is found that yeast exhact and KHTPOT is themost sjgnificant factors that can affect towards production ofNata de Pina usingjuicefrom pineapple waste. The best optimum medium culture that can optimize microbialcellulose yield is as below: yeast extract 6.09, sucrcse 20.09, bactopepton 1.499,potassium dihydrogen phosphate (KI-lrPOa) 1.089, and magnesium sulfate (MgSOa)0.06g with wet cellulose mass of 176.47g. From statistical analyses on designexperimenls it is found that Magnesium sulfate is not affecting toward celluloseformation. lt can bejust ignore to use in prepared medium culture for future.

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Budhiono, A., Rosidi, B., Taher, H., lgLtchi, M. (1999). "Kinetic Aspects ofBactefialCellulose Fomation Nata-de-Coco Culture System." Carbohydrate Polliners, Vol-40,pp.137-143.

Susanto, T., Adhjtia, R., Yunianta (2000). "Pembuatan Nata de Pina Dari Kulit NanasKajian Dari Sunrber Karbon dan Pengencemn Medium Fermentasi." JurnalTeknologiPeltanian, Vol.1. No.2 (2000).

Tsuchida, T. and Yoshinaga, F. (1997). "Production of Bacterial Cell lose byAgitation Cullure Systen". Pure & Appl. Chem., Vo1.69, No.ll, pp.2453-2458,(ree1).

Vandamme, E.J., DeBaets, S., Vanbaelan,"lmproved Production of bacterial CellliloseDegradation and Stability, Vol.59, pp.93-99.

Joris, K- ard DeWL'1f, P. (1997).Its Application Potential." Polymerand

Ve$churen, P.C., Cardon4 T.D., Robert Nout, M.J., DE Gooijer, K.D., and DenHuevel, J.V. (1999). "Location of Cellulose Production by Ace[obac[e/ XylinumEstablished liom Oxygen Profiles." Journal of Bioscienc€ & Bioengineering Vol.89,No.5.414-419. a2000)