Ethol Production From Potatoe

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Indian Journal of Science and Technology Vol. 3 No. 7 (July 2010) ISSN: 0974- 6846

Research communication Ethanol production Rani et al.Indian Society for Education and Environment (iSee) http://www.indjst.org Indian J.Sci.Techno

Ethanol production from potato flour by

accharomyces cerevisiae

P.Rani,S.Sharma,F.C.Garg,KushalRajandLeelaWati

DepartmentofMicrobiology,CCSHaryanaAgriculturalUniversity,Hisar-125004,India.

[email protected]

Abstract

Flour prepared from potato tubers (Solanum tuberosum) after cooking and drying at 70C was used for ethanoproduction.Homogenousslurryofpotatoflourwaspreparedinwateratsolidliquidratio1:4.Liquefactionofpotatoflou

slurrywith-amylase(2.05DUNU/gstarch)at80Cfor30minfollowedbysaccharificationwithglucoamylase(20.5GAU/gstarch)at60Cfor2hgenerated15.2%totalreducingsugarsinthehydrolysate.FermentationofhydrolysatewithSaccharomycescerevisiaeHAU-1at30Cfor48hresultedinproductionof56.8gl

-1ethanol.Supplementationo

nitrogen sources to potato flour did not contribute significantly to ethanol yield. Simultaneous saccharification andfermentationofhydrolysatewasaseffectiveasseparatehydrolysisandfermentation.

Keywords Ethanol,fermentation,liquefaction,potatoflour,saccharification,S.cerevisiae.Introduction

Bioethanol as an alternative source of energy hasreceivedspecialattentionworldoverduetodepletionoffossil fuels. According to United States department ofenergy, for every unit of energy put towards ethanolproduction 1.3 units are returned (Hill et al., 2006). InIndia sugarcane molasses is the main raw material forethanol production but now the short supply andincreased cost is themain hindrance for its use. Thereareabout342distilleriesinthecountrywithaninstalledcapacityofover3billionlitresofethanolannually(Narde,2009)which isshort ofrequirement and ismet throughimports. The efficiency of ethanol production largelydepends on the availability of suitable substrate, yeaststrainandmethodemployed. The sugary substrates available are comparativelyexpensive than molasses but can be easily used forethanolproductionwithsomemodificationintheprocess.Onthe otherhand cellulosicmaterials are cheaper andavailableinplentybuttheirconversiontoethanolinvolvesmanystepsandisexpensive.Thestarchysubstratesarepromisingdueto theireconomicviabilityandavailability.Starchy crops like corn, barley, wheat, rice and tubercropsviz.potato,sweetpotatoarebeingexploitedfortheproduction of bioethanol worldwide (Szambelan et al.,

2004;Shigechietal.,2004).Theworldoverproductionofpotatoesin2007was325.3milliontoneswhileinIndiaitwas approximately 26 million tons (Rani et al., 2009)showing this asa promising crop but isbeingused forproductionofethanolinsomecountries(Kobayashietal.,1998). Moreover, potatoes are rich in starch, whichmakes ita cheapsubstrate for ethanol production.Theproblemsassociatedwithitsprocessingwillalsobelessthaninothergrains.Itisalsosemi-perishablefoodwhichcan be stored for considerable period without spoilage.Goodqualityalcoholcanbeproducedfrompotatowhich

can be used for both fuel as well as potable purposeTherefore there is a need to explore the possibility oethanolproduction frompotatoafter suitableprocessingand this study was planned to develop a suitabletechnologyforconversionofpotatostarchintoethanol.

Materials and methods

Substrate: Potato tubers were procured from universityfarmandanalyzedfordifferentcomponentsbystandardmethods (AOAC, 1990). Thoroughly washed unpeeledpotatoes(1kg)werecookedinapressurecookerinonelitre water containing 0.5% potassium metabisulphiteBoiled potatoes were mashed, dried overnight at 70Candgroundtofinepowder(210m).Enzymeforliquefactionandsaccharification:Commercia

-amylase(Termamyl-100,specificactivity30DUNU/mlandamyloglucosidase (Amylo300,Specificactivity400GAU/ml)werereceivedfromMs.JagatjeetIndustryLtd.,Jalandhar(Punjab).Preparationofpotatoflourslurry:Slurrieswerepreparedfrompotatoflourmixedwithwateratdifferentsolidliquidratio(1:1-1:10)andtreatedwithliquefyingenzyme(1000l/100ml)at80Cfor30minundershakingconditions.

Theslurrypreparedbymixing20gflourin80mlwate(1:4)beinghomogenous,loose,easytohandlewasusedforfurtherexperiments.Liquefactionof potato flour (100mlslurry)wascarriedoutat80Cinashakingwaterbathusing varying concentrationof enzyme (100-2000l)adifferent incubation temperatures (60-90C) for differentime intervals (10-40 min). The progressof liquefactionwasmonitoredbyemployingstarch-iodine(1gofIodine&2gKIin100mlwater)reaction. Saccharificationof liquefiedstarchwascarriedouta60Cfordifferenttimeintervalsusingvarying


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concentration (100-1000 l/100 ml) ofAmylo-300 containingamyloglucosidase. The reaction wasmonitoredby theyield of total reducingsugars estimated by dinitrosalicyclicacidmethod(Miller,1959).

Yeastculture:AfastfermentingstrainofSaccharomyces cerevisiaeHAU-1 wasobtainedfromculturecollectionDept.ofMicrobiology,CCSHaryanaAgriculturalUniversity, Hisar (Haryana) andmaintained on yeast extract peptone dextrose (YEPD)agar medium containing yeast extract (1%), peptone(2%),dextrose (2%)andagar (2%).Dextrose inoculummedium (IM) used for inoculum preparation containeddextrose (6%), peptone (0.5%), yeast extract (0.5%).Yeastcellspregrownininoculummediumfor18hunder

shaking condition (100 rpm) werecentrifugedat8000rpmfor15minandinoculated into hydrolysate at aconcentrationof 0.5% (w/v)wetweighbasis.Flaskswereincubatedat302Cunder stationaryconditionsandethano

contentwasmeasuredatanintervalof24 h by gas liquid chromatography(Systronics, GC 8200) using capillarycolumnandflameionizationdetector.

Results and discussion

Potatotuberscontained80.3%moisture,20%starch2.19% proteins and 0.41% total sugars while therespective value for potato flourwere 8.12,73.0,10.86and0.91%(Table1).

Table1.Compositionofpotatoandpotatoflour

ParametersComposition(%)

PotatoPotatoflour

Moisture 80.28 8.12

Starch 20.0 73.0Totalprotein 2.19 10.86

Crudefiber 0.85 1.65

Ashcontent 0.65 2.15

Totalsugars 0.41 0.91

Totallipids 0.12 1.00

Fig.1.EffectofAmylo300conc.onsaccharificationofpotatoflourat60C

Fig.2.Ethanolproductionfrompotatoflourbysimultaneoussaccharificationandfermentation

Fig.3.Effectoftemperatureonethanolproductionfrompotatoflour

Fig.4.Ethanolproductionfrompotatoflourafterremovalofresidue


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Hydrolysis of potato flour: 100 ml slurry of potato flour(20%)wasliquefiedwith1000lTermamyl-100at80Cwithin30min.Theliquefiedpotatofloursaccharifiedwithdifferent doses of Amylo 300 (100-1000 l) at 60Creleased 15.2% total reducing sugars at enzymeconcentrationof750lafter2h,howeverfurtherincrease

in reaction time and temperature and enzymeconcentrationdidnothaveappreciableeffect(Fig.1).Ethanolproduction fromhydrolysateof potato flour:Thehydrolysate from potato flour on inoculation withSaccharomyces cerevisiaeat 0.5% concentration (w/v)generated 56.8 gl

-1ethanol after 48 h fermentation at

302C. On supplementation ofvarious nitrogen sources viz.urea, ammonium sulphate,peptoneandyeastextractat0.1-0.5% (w/v) maximum 59.9 gl

-1

ethanol was obtained with

supplementation of peptone(0.2%) or yeast extract (0.2%)under similar conditions (Table2). A maximum of 51.2 gl

-1

ethanolwasproducedin48hascompared to56.8gl

-1 ethanol in

48 h when potato starch wassaccharified simultaneouslyduring fermentation suggestingthat simultaneoussaccharificationand fermentationis equally effective as separatehydrolysisandfermentation(Fig.

2).Effect of temperature on ethanol production: Duringsummerseasonthereisnostrictcontrolovertemperatureoffermentationvatswhichevergoesto40Caffectingtherate of ethanol production (Cazetta et al ., 2007).Fermentationof20%slurryofpotatoflourwascarriedoutat different temperatures (30, 35, 37 & 40C) understationary conditions up to 96 h. A maximum ethanolcontent of 56.8 gl

-1 was recorded after 48 h of

fermentationat30C(Fig.3).Howeverattemperature35,37and40C, thecorrespondingvalueswere53.6,50.0and 46.0 gl

-1 respectively showing a decline with

increasedtemperatureoffermentation.Ethanol production after removal of residue fromhydrolysate: The residual suspended matter afterliquefactionandsaccharificationofstarchinpotatoslurrymayinterfereinthehandlingofslurryduringlargescalefermentation. Thus the sediment was removed bycentrifugation at 4000 rpm and the supernatant withsoluble sugars was used for production of ethanol.Ethanol level of 53.6 gl

-1 was produced in supernatant

after24hwhichfurtherincreasedto56.8gl-1after48h

(Fig. 4). This suggests that the supernatant afteliquefaction and saccharification of starch present inpotatoflourcanbeusedaseffectivelyasthepotatoflour.

Discussion

Potatoescanbeanappropriatealternatesubstratefo

bioethanolproductionduetotheirplentyavailability,lowcost and easy processing. For better penetration oenzymeintosubstratesizereductionismust.Potatoflourpreparedbycooking,mashingandgrindingwasusedfoethanol production by Saccharomyces cerevisiaeSmooth slurry is prerequisite for fermentation that waspreparedby liquefaction ofpotato flourwithamylase a

80C for30minand it took 2hfor saccharification of liquefiedslurrybyglucoamylaseat60C. Addition of nutrients such asammoniumsulphate,urea,yeasextract and peptone have been

reported to play a vital role inboosting ethanol productionandits rate (Fundora et al ., 2000)Wang et al. (2007) found thaaddition of 1.5% peptone in themedium increased the finaethanoltitrefrom14.2%-17.0%in48 h. The response of addednutrients inpotato flourwas lowforethanolproductionsuggestingthat potato as such may be agood substrate for fermentationwas an interesting observationBecauseofthelongperiod(2h)

needed toachieve thesaccharification itwas necessaryto explore the possibilities to carryout simultaneoussaccharificationandfermentation(SSF)afterliquefactionthat was foundequally effective as separate hydrolysisand fermentation.Chenetal.(2007) found thatethanoconcentration in solid state SSF using temperaturecycling(10hat37Cwith15minat42C)was2timesmoreeffectivethanusingconstant37C.MontesinosandNavaroo (2000) also reported reduction in fermentationtimeinSSFofwheatflourgivingethanolconcentrationof67gl

-1.

Conclusion

Potato flour prepared by cooking, mashing andgrinding was used for ethanol fermentation bySaccharomyces cerevisiae. Homogenised (1:4) slurrywasobtainedontreatmentwith-amylase(2.05DUNU/gstarch) at80C for 30min whichwas saccharified withglucoamylase(20.5GAU/gstarch)at60Cfor2h.Theaddition of nitrogen sources did not affect ethanoproduction.Theotherconditionswerealsostandardizedfor optimization of ethanol production. The interestingobservationwasthefeasibilityofethanolproductionwith

Table2.Ethanolproductionfrompotatoflouraftersupplementationwithvariousnitrogensources

Nitrogensourceconc.(%w/v)

Ethanol(gl-)

24h 48h 72h

Ammoniumsulphate

0.1 56.0 56.8 56.4

0.2 56.8 58.3 58.3

0.3 55.2 56.0 56.0Peptone 0.1 56.8 57.5 57.5

0.2 58.3 59.9 59.1

0.3 57.5 57.5 56.8

Urea 0.1 56.0 56.0 55.2

0.2 57.5 59.1 56.8

0.3 55.2 56.0 55.2Yeastextract

0.1 56.0 58.3 58.3

0.2 58.3 59.9 59.1

0.3 56.0 57.5 57.5

ControlNo

nitrogensource

54.4 56.8 55.2


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simultaneous saccharification and fermentation (SSF)whichisbeingscaledupatpilotscale.

References

1. AOAC (1990)Officialmethods of analysis. 15th edn.

Assoc.ofofficialanalyticalchemists.WashingtonDC,

USA.2. CazettaML,CelligoiMA,BuzatoJBandSearminoIS

(2007) Fermentation of molasses by Zymomonasmobilis: Effects of temperature and sugarconcentrationonethanolproduction. Biores.Technol.98,2824-2828.

3. ChenHZ,XuJandLiZH(2007)Temperaturecyclingto improve the ethanol production with solid statesimultaneous saccharification and fermentation.PriklBiokhimMikrobiol.43:65-68.

4. FundoraN,VldesI,GarciaR,HernandezLM,PortoOand GonzalezMD(2000) Influence ofnitrogensaltson alcohol production. Revista ICIDCA Sobre Los

DerivadosDeLa.Cana.DecAzucar,34,2.5. HillJ,NelsonE,TilmanD,PolaskySandTiffancyD(2006)Environmental, economicandenergeticcostsandbenefitsofbiodieselandethanolbiofuels.Proc.oftheNat.AcademyofSci.USA,103,11206-11210.

6. Kobayashi F,Sawada T, Nakamuraa Y, Ohnaga M,GodlivingM andUshiyamaT (1998)Saccharificationandalcoholfermentationinstarchofsteamexplodedpotato.Appl.Biochem.Biotechnol.69,177-189.

7. MillerGL(1959)Useofdinitrosalicylicacidreagentfordeterminationofreducingsugar.Anal.Chem.31,426-428.

8. Montesinos T and Navarro J (2000) Production ofalcoholfromrawwheatflourbyamyloglucosidaseand

Saccharomycescerevisiae.Enz.Microb.Technol.27,362.

9. Narde A (2009) Distilleries in India. In: sugar Indiayear book09 (ed. Ajit Narde) Anekant Prakashan,Kolhapur.pp:401-432.

10.Rani P, Garg FC, Sharma SS and Wati L (2009)Ethanol production from potato starch. Ind. FoodPacker.63(4),63-68.

11.Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y,Ueda M, Satoh E and Kondo A (2004) Directproduction of ethanol from raw corn starch viafermentation by use of a novel surface- engineeredyeast strain co-displaying glucoamylase and alpha-

amylase.Appl.Environ.Microbiol.70,5037-5040.12.SzambelanK,NowakJandCzarneckiZ(2004)UseofZymomonas mobilis and Saccharomyces cerevisiaemixed with Kluyveromyces fragilis for improvedethanol production from Jerusalemartichoke tubers.Biotechnol.Lett.26,845-848.

13.Wang FQ, Gao CJ, Yang CY and Xu P (2007)Optimizationofanethanolproductionmediuminveryhigh gravity fermentation. Biotechnol. Lett. 29, 233-236.

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Ethol Production From Potatoe