Simultaneous saccharification and fermentation of dextrin£zed cassava starch to glucose and

alcohol, respectively, were carried out by co-immobilized Zymomonas mobilis and immobilz'zed

glucoamylase (IG). Calcium alginate-entrapped cells and IG (4 : 1 ratio) gave an alcohol productivity

of 0.30 glgww cellslh in a batchfermentation process. For continuous fermentation, 54.3 gil alcohol

was produced at a dilution rate of 0.3Ih. 60% of the initial activity was lost within 3 days and, thereafter,

the system entered a slowly decreasing phase ofalcohol production

Ho, Y. C.

Mohd Ghazali, Hasanah

Universiti Putra Malaysia Institutional Repository

Pertanika 9(2), 235 - 240 (1986)Alcohol Production from Cassava Starch byCo-immobilized Zymomonas mobilis andImmobilized GlucoamylaseY.C. HO and H.M. GHAZALI IDepartment of Food Science,Faculty of Food Science and Technology,Universiti Pertanian Malaysia,43400 Serdang, Selangor, Malays£a.Key words: Alcohol; Zymomonas mobilis; glucoamylase; co-immobilized system.ABSTRAKPensakaridaan dan penapaian serentak kanji ubi kayu terdekstrin kepada glukosa dan alkohol,masing-masing, dilakukan oleh Zymomonas mobilis dan glukoam£lase tersekat-gerak yang tersekat-gerak bersama. Sel-sel dan glukoamilase tersekat-gerak yang terperangkap di dalam kalsium alg£nat(nisbah 4 : 1) memberi pro.duktiviti alkohol sebanyak 0.30 gig berat basahljam untuk prosespenapaian sekumpul. Bagi penapaian selanjar, 54.3 gil alkohol dihasilkan pada kadar pencairan0.3Ih. 60% darzpada aktiviti awal terhapus dalam masa 3 hari dan selepas itu, sistem tersebutmemasukifasa penghasz'lan alkohol yang menurun dengan perlahan.ABSTRACTSimultaneous saccharification and fermentation of dextrin£zed cassava starch to glucose andalcohol, respectively, were carried out by co-immobilized Zymomonas mobilis and immobilz'zedglucoamylase (IG). Calcium alginate-entrapped cells and IG (4 : 1 ratio) gave an alcohol productivityof 0.30 glgww cellslh in a batchfermentation process. For continuous fermentation, 54.3 gil alcoholwas produced at a dilution rate of 0.3Ih. 60% of the initial activity was lost within 3 days and, there-after, the system entered a slowly decreasing phase ofalcohol production.INTRODUCTIONCo-immobilizing microbial cells and anenzyme together in the same support in order tocarry out a certain biochemical reaction hassome merits. Besides supplementing the array ofintracellular enzymes of the cells, the productfrom the enzyme reaction can be channelleddirectly to the cells for further conversion ormodification. This is especially advantageous iffurther conversion or modification of theproduct requires a sequential reaction. It may bepossible to reduce production costs with such anarrangement since both reactions are carried outin a single reactor.I To whom requests for off-prints should be addressedFew reports have been published on co-immobilized enzymes and cells. One of theearliest reports on such a system concerned theco-immobilization of glucoamylase and Sac-charomyces cerevisiae (Hough and Lyons, 1972).TiCl 4 was used to immobilize the enzyme on thesurface of the yeast cells. Hirano et al. (1979)and Karube et al. (1977) studied systems whereaminoacylase and glucose oxidase, respectively,were immobilized on the natural mycelial pelletsof Aspergillus ochraceus. Glutaraldehyde wasused as the coupling agent and albumin wasfound to stabilize the pellets by preventing theleakage of the enzymes. Ghazali and Cheetham(1983) pursued the approach of co-immobiliza-Y.C. HO AND H.M. GHAZALItion by using a system where an enzyme that hadalready been immobilized was further immo-bilized with a microorganism in a commonsupport. They demonstrated that continuousbioconversion of solubilized com starch intoalcohol was possible when using a co-immo-bilized system comprising immobilized gluco-amylase (IG) and Saccharomyces uvarum. Boththe enzyme and the yeast were immobilized incalcium alginate pellets. However, the stabilityof the system in continuous fermentation was lowowing to cell inactivation in the presence of highalcohol concentrations.Zymomonas mobilis has been shown to be abetter alcohol producer than yeast (Rogers et al.,1980) and has also been reported to be able totolerate high concentrations of alcohol (Lavers etal., 1980). The bacterium, however, can onlyferment glucose, fructose and sucrose intoalcohol and CO 2' but it cannot utilize othercarbon sources such as maltose and starch(Swings and De Ley, 1979). The inability toutilize starch, which is a cheap substrate com-pared with sugars, can be overcome by sac-charifying the starch first.This paper reports on the use of a co-immobilized system, comprising Z. mobilisATCC 2304 and immobilized glucoamylase, tocarry out the simultaneous saccharification andfermentation of dextrinized cassava (Manihotesculenta, L) starch into alcohol.MATERIALS AND METHODSMaintenance and Preparation of z. mobilisZ. mobilis ATCC 2304 was maintained in agrowth medium containing glucose (100 gil),yeast extract (10 gil), ammonium sulphate (1gil), potassium dihydrogen phosphate (1 gil)and magnesium sulphate heptahydrate (0.5 gil)(Rogers et al., 1979). The culture was grown at30°C for 24 h and stored at 4°C between suc-cessive transfers of 3 to 4 days.For immobilization, a total of 2 1 of theabove medium in shake-flasks was inoculatedwith Z. mobilis and incubated with shaking (70rpm) for 24 h at 30°C. The bacterial cells werethen harvested by centrifugation at 13000 g for 15minutes at 4°C. The weight of the cells used forimmobilization was expressed in gram wetweight (gww) cells.Preparation ofStarch SubstrateThe cassava starch was dextrinized accord-ing to the method described by Subhi andGhazali (1986). The dextrinized starch used forthe fermentation process has a pH of 5.5 and aglucose content of 3 to 5%. Cassava starch wasbought locally.Immo bilization ofGlucoamylaseThe method used for the immobilization ofglucoamylase was that described by Daniels andFarmer (1981) and Subhi and Ghazali (1986).The Extent ofSaccharification ofSubstrateby Alginate-entrapped IGA 5% (w/v) suspension of IG (supportparticle size of 355 - 600 11m) in 5% sodiumalginate (BDH Chemicals Co. Ltd, Poole,England) solution was prepared by mixing in a10 ml hypodermic syringe. The slurry formedwas extruded from a height of 20 cm into abeaker containing a continuously stirred 0.1 MCaCl 2 solution. The calcium alginate pelletsformed had a diameter of 0.4 - 0.5 cm and wereleft in the liquor for 1 h. The pellets wereremoved and rinsed with distilled water bydecantation. They were then added to 100 ml ofsubstrate and incubated with shaking (100 rpm)at 55°C. Samples were withdrawn at regularintervals and analysed for glucose (Barton,1966). A similar set-up was used for the controlusing non-entrapped IG.Co-immob1:lization of Z. mobilis and IGA 5% cell load was prepared by mixing 0.5g of freshly harvested Z. mobz'lz"s and IG in 5%sodium alginate solution in a 10 ml syringe. Theweight (gww) of IG was varied to give the desiredcell-to-enzyme ratio. The slurry formed was thenextruded into a continuously stirred 0.1M CaCl2solution containing 15 % glucose to obtain beads236 PERTANIKA VOL. 9 NO.2, 1986ALCOHOL PRODUCTION BY CO-IMMOBILIZED BIOCATALYSTSContinuous Fermentatz."on ofSubstrate 200of calcium alginate pellets. The pellets were leftin the liquor for 1 hr and were then rinsed bydecantation with distilled water before use. 240Pellets containing a 4 : 1 ratio of Z. mobilis-to-IG were packed into a jacketed glass column,37 em X 1 em internal diameter. 4 g (ww) ofcells were used for the column. The temperatureof the column and the flow rate of the substratewere maintained at 30°C and 0.3/h, respec-tively. The substrate solution was fed upwardinto the column so as to facilitate the removal ofCO 2 formed during fermentation. 10 ml ofeluate was collected at the time of analysis andassayed for glucose and alcohol (Amerine andOugh, 1974).160120HO40o 2 3 4 5 6 7RESULTS AND DISCUSSIONThe effect of enzyme carrier particle size onthe activity of the IG was studied in order toobtain the best enzyme preparation for co-immobilization with Z. mob£lis. As shown inFigure 1, decreasing the carrier particle sizeincreased the saccharification activity of IG. Theincrease in activity could be attributed to greateramounts of glucoamylase being bound to thecarrier: the smaller the particle size, the largerthe specific surface area. However, decreasingthe particle size further did not give an enzymepreparation of greater activity (Ghazali, 1981).IG bound to charcoal of less than 300 J1 m indiameter was found to have very low activity.In subsequent studies, glucoamylase im-mobilized on charcoal of particle size between355 and 600 J.1 m was used. Furthermore, it wasfound that it was easier to extrude the alginate-Z. mobilis-IG mixture from the syringe into theCael bath to form even sized, spherical pellets2that contained the IG evenly dispersed through-out the gel matrix, when using particles in thissize range.Immobilizing the IG in calcium alginatepellets caused a decrease in the amount ofglucose produced, but the rate of glucose pro-duction after an initial lag period was similar toFig. 1: The effect of support particle size on theefficiency of immobilization of glucoamylase.2 g of IG were used to sacchanfy 100 mlsubstrate..355-600 Jim • >600-710 m• > 710-1000 J1mthat with free IG (Figure 2). The lag periodobserved can be ascribed to establishing a diffu-sion .gradient and to the diffusion limitations ofthe higher molecular weight components of thedextrinized cassava starch. Kierstan (1982)reported that diffusion characteristics of lowermolecular weight substances were unaffected byalginate and ion concentrations; however, diffu-sion of higher molecular weight components wasaffected by alginate concentration. That the rateof glucose production was similar in both casesindicated that immobilizing IG in alginate geldid not affect its activity significantly. There-fore, in a co-immobilized system, the glucosethat was produced by the gel-entrapped IGwithin the gel would be in contact with thebacteria at all tImes and could be utilizedimmediately for fermentation.The effect of varying the amount of IG(gww) in calcium alginate pellets containing afixed amount of Z. mobz'lis (gww) on glucose andalcohol production is shown in Figure 3. ThePERTANIKA VOL. 9 NO.2. 1986Y.C. HO AND H.M. GHAZALIF£g. 4: Tt'me course on the stability ofca-immabz"lt"zed Z. mobilis and Ie at acell-to-enzyme ratio of 4 : 1.Time (days)A similar trend for alcohol production wasobserved by Ghazali and Cheetham (1982) whenusing co-immobilized S. uvarum and IG. Withthe yeast system, 50% of the initial alcoholproducing activity was lost within 8 days ofoperation. The major limiting factor in theoverall stability of the system was the yeast cells,since in sz"tu regeneration of the cells with growthmedium could restore the activity of the system.Continuous fermentation by co-immobiliz-ed Z. mobz"lis and IG (4 : 1 cells-to-IG ratio) wascarried out to study the stability of the systemwith respect to time (Fz"gure 4). During the firstthree days of operation, the concentration ofalcohol produced dropped by about 60 % of theinitial value of 54.3 gil and thereafter the systementered a gradually 'decreasing phase of alcoholproduction. A corresponding initial increase wasobserved for glucose but eventually the amountproduced gradually decreased with time. Thedecrease in the production of both alcohol andglucose can be attributed to the decline in theactivities of the bacteria and the IG in thealginate pellets.where the availability of glucose was neither "inexcess nor deficient, based on the glucose toalcohol ratio (Figure 3). At this ratio, the con-centration of alcohol produced was 36 gil,which corresponds to a productivity of 0.3glgww cells/h.~~ 100e..i 508"8 1~40 80"G ~"-bO 30 60 ~e..ic: e..ie c:"0 e.c: 20 ItO §e:;;:"G10 20Cells-to-IG ratioNFig. 3: The effect of z. mobilis-to-Ie ratio onsacchanfication and fermentation. Theprocesses were carried out for 24 hrs.study was carried out by batch fermentation,whereby pellets of known cells-to-IG ratio wereadded to 100 ml of substrate and incubated withshaking (100 rpm) at 30°C for 24 h. The resultsobtained indicated that. the ratio of cells-to-IG of10 : 1 appeared to be best in terms of glucoseutilization (based on the amount present afterfermentation) and a ratio of 1 : 1 for alcoholproduction. However, at the ratio at whichglucose was best utilized, the concentration ofalcohol produced was low (28.6 g/i) and, at theratio of 1 : 1, glucose was produced in excess(33.5 gil) where it remained unutilized. Theoptimal cells-to-IG ratio was taken to be 4 : 1,Time (h)Fig. 2: The effect of gel entrapment on Ie; 1 g ofIe was used for the saccharification process.o free Ie • alginate entrapped Ie160"- 120tel~M 80~400 2 -3 4 5 6 770602.0- 100~'i':>O 1.6 ~ I fk).c:'-" 0~40 1.2 il 60c:e 30"0.c: 0.8 §. 40e:;;: 2010 0.4 :.to0 0 0238 PERTANIKA VOL. 9 NO.2, 1986ALCOHOL PRODUCTION BY CO-IMMOBILIZED BIOCATALYSTSThe low operational stability of the Z. mobz"lis-IG system may be overcome by intermittentsupply of growth medium or by continuoussupply of diluted growth medium so that a'growing immobilized cells' system is obtained.Alternatively, strains of Z. mob£lis with greatertolerance to higher concentrations of glucosewhen maintained in a non-growing state can besought.Visual observations showed that CO 2 evolu-tion during the first day of fermentation was veryvigorous indicating that the immobilized bac-teria were very active. Although most of the gasevolved escaped from the column through theeluate outlet at the top end of the columnreactor, some CO 2was retained as bubbles in thecolumn. The escaping gas caused the pellets tobe more closely packed at the top of the column.New pockets of CO 2 were formed as fermenta-tion continued and these replaced the CO 2bubbles that eventually escaped from thecolumn. The installation of a multiple-disk shaft(McGhee et al., 1982) in the column may help toeliminate gas hold-up and uneven distribution ofthe alginate pellets.ACKNOWLEDGEMENISThe authors are grateful to UniversitiPertanian Malaysia for making available theUniversity Research Grants for this project.REFERENCESAMERINE, M.A. and OUGH, C. (1974): Wine and MustAnalysis. New York. John Wiley and Sons. Inc.BARTON, R.R. (1966): A specific method for quanti-tative detennination of glucose. Anal. Biochem.14: 258 - 260.DANIELS, M.J. and FARMER, D.M. (1981): UK PatentGBO 022 B.GHAZALI, H.M. (1981): Production of ethanol by co-immobilized Saccharomyces uvarum and amylo-glucosidas. M.Sc. Thesis, Reading University,England.GHAZALI, H.M. and CHEETHAM, P.S.]. (1982):Ethanol Production by Co-immobilized Sac-sharomyces uvarum and glucoamylase. In 'Pro-ceeding of the Regional Workshop on Immo-bz'lt"zed MicTobial Enzymes and Cells'. Flegel,T.W., Meevootisom, V., Bhumisituna, A., andMatangkasombut, P. (eds). Bangkok. p. 137-144.HARANO, K., KARUBE, I. and SUZUKI, S.S. (1979):Aminoacylase pellets. Biotechnol. Bioeng. 19:311- 321.HOUGH, J.S. and LYONS, T.P. (1972): Coupling ofenzymes onto micro-organims. Nature 235: 389.KARUBE, I., HIRANO, K. and SUZUKI, S.S. (1977):Glucose oxidase pellets. Biotechnol. Bioeng. 19.:1233 -1238.CONCLUSIONCassava starch, an important agriculturalby-product in this region, can be further KIERSTAN, M. (1982): Studies on the characteristics ofmodified to produce value-added products such alginate gels in relation to their use in separationand immobilization applications. Biotechnol.as glucose syrup, high fructose-glucose syrups Bioeng. 24: 1507 - 1517.and alcohol. The work described in this paperI f h b d KLEIN,J., STOCK,J. and VORLOP, Z.K.D. (1983): Poreshows an examp e 0 a system t at can e use to size and properties of spherical calcium alginateproduce alcohol. Zymomonas mob£lis, a bacte- biocatalysts. Eur. J. Appl. Microbiol. Biotechnol.rium capable of producing and tolerating high 18: 86 - 91.oncentrations of alcohol but incapable of utilis- -:: LAVERS, B.H., PANG; P., MACKENZIE, C.R.,ing starch as a substrate, can be made to LAWFORD, G.R., PIK, J.R. and LOWFORD, H.G.produce alcohol from starch by supplementing (1981): In: Advances z~ Biotechnol. Vol. II. Moo-its cellular enzyme system externally with gluco- Young, M. and Robmson, C.W. (Eds). Perga-h h d I . H mon Press, Toronto, p. 195.amylase, a starc y ro ySlng enzyme. owever, • .it is important to evaluate the potential of this MCGHEE, J.E., ST. JULIAN, G., DETROY, R.W. and.. BOTHAST, R.J. (1982): Ethanol Production bywork for larger scale operations smce the I b'l' d S h . . S. . mmo llZe acc aromyces cerevzszae, ac-demand for alcohol, espeCially as fuel, IS depen- chaTomyces uvarum and Zymomonas mob£lis.dent on current market price for petroleum. Biotechnol. Bioeng. 24: 1155 -1163.PERTANIKA VOL. 9 NO.2, 1986 239Y.C. HO AND H.M. GHAZALIOSBORN, J.A., JANNELI, R.M., WIECK, M.J., DECKER,T.F., GORDON, B.L. and YACYNYCH, A.M.(1982): Use of chemically modified activatedcarbon as a support for immobilized enzymes.Biotechnol. Bioeng. 24: 1653 -1669.ROGERS, P.L., LEE, K.J. and TRIBE, D.E. (1979):Kinetics of alcohol production by Zymomonasmobilis at high sugar concentrations. Biotechnol.Letts. I: 165 - 170.ROGERS, P.L., LEE, K.J. and TRIBE, D.E. (l979):'Highproductivity ethanol fermentation with Zymo-monas mobilis. Process Biochem.15(6): 7 - 11.SUBHI. B. and GHAZALI, H.M. (1986): Production ofcassava syrup with glucoamylase immobilized. toacid-washed charcoal. Pertanika 9(1): 125 -129.SWINGS, J.J. and DE LEY, J. (1979): The biology ofzymomonas. Bacterial Rev. 41: 1-46.(Received 24 February, 1986)240 PERTANIKA VOL. 9 NO.2, 1986

Alcohol Production from Cassava Starch by Co-immobilized Zymomonas mobilis and Immobilized Glucoamylase

http://psasir.upm.edu.my/2326/1/Alcohol_Production_from_Cassava_Starch_by.pdf

Alcohol Production from Cassava Starch by Co-immobilized Zymomonas mobilis and Immobilized Glucoamylase

Abstract

Similar works

Full text

Available Versions

Universiti Putra Malaysia Institutional Repository