Ethanol fermentation from raw starch of sweet sorghum grains

8/18/2019 Ethanol fermentation from raw starch of sweet sorghum grains

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Ethanol fermentation from raw starch of sweet sorghum grains

Najiah Nadir 1, Maizirwan Mel2, Mohd Ismail Abd Karim3, osli Mohd !unus"123#io$rocess and Molecular Engineering esearch %nit, &acult' of Engineering, International Islamic %ni(ersit'

Mala'sia, )*+* #o 1-, .-/20, Kuala um$ur, Mala'sia "e$artment of hemical Engineering, &acult' of hemical and Natural esources Engineering, %ni(ersiti Mala'sia)ahang, ME it', 243-- 5ambang, Kuantan, )ahang

orres$onding Email6 najdir7'ahoo*com

Abst ra ct— Ethanol can be readily produced by

fermentation of simple sugars that are converted from

either starchy crops or cellulosic materials. The

hydrolysis and fermentation were used to produce ethanol

from raw starch of sweet sorghum grains by utilizing

commercially available α-amylase and glucoamylase.

Ethanol production from hydrolyzed sweet sorghum was

analyzed under different fermentation conditions using

Saccharomyces cerevisiae in batch fermentation. Theconcentration of inoculum, urea, NP !nitrogen,

phosphorus, and potassium", temperature, initial p#, and

agitation were investigated simultaneously through two-

level factorial design. The ma$imum ethanol

concentration !%%.&' g()" were obtained at concentration

of inoculum of '.*+ !w(w", urea of '.%'+ !w(w", NP of

'.'+ !w(w", temperature of *', initial p# of %.', and

agitation of /'' rpm. 0s shown in the analysis of variance

!0N120" result, the concentration of inoculum, urea,

initial p#, and agitation have contributed more

significant effect on fermentation of hydrolyzed sweet

sorghum. The ma3or factors for fermentation wereoptimized by the central composite design !4" under

the response surface method !567". 0fter further

optimization using 4, the optimum fermentation

conditions for ma$imum ethanol production of &.89 g()

were predicted at '.8*+ !w(w" of inoculum concentration,

.%: of initial p# of fermentation media, and ' rpm of

agitation speed.

eywords-ethanol; sweet sorghum; hydrolysis; fermentation

I* IN8+%8I+N

Ethanol or eth'l alcohol is the most utilized li9uid

biofuel either as a fuel or as a gasoline enhancer :1;*8here has been a $romotion in new a$$lications andmarecondl', thecentral com$osite design ?@ was em$lo'ed to obtain

the o$timum le(el of the significant factors b' de(elo$inga model followed b' other statistical tests such as anal'sisof (ariance ?AN+FA@, coefficient of determination, 2contour and 3 surface $lots for the maimum ethanol*


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II* MA8EIA> AN ME8+>

A. Substrates

>weet sorghum grains were obtained from Indonesian#ioenerg' &oundation*

B. Microorganisms

riedform industrial Saccharomyces cerevisiae

'east was used in this research*C. Enzymes

#oth Cam'lase from Bacillus subtilis andglucoam'lase from Aspergillus niger were obtained fromenz'me industr' in iau, Indonesia* 8he acti(ities of thetwo enz'mes were identified to be 2.,--- %Gm and13-,--- %Gm, res$ecti(el'*

D. Substrate Preparation

>weet sorghum grains were blended into small sizeof a$$roimatel' 2- Hm to enhance the h'drol'sis

$rocess* 8he microstructure of the starch granules were(iewed with a field emission scanning electron

microsco$e ?&E>EM@* E. Hyrolysis

'drol'sis was $erformed based on $re(iouso$timization stud' :22;* 8he shaam$les for glucose and ethanol determination werecentrifuged at .--- r$m for 3- minutes to remo(e the

substrates and cells* 8he su$ernatant was filtered througha -*". m membrane and anal'zed b' high $erformanceli9uid chromatogra$h' ?)@ e9ui$$ed with a refracti(einde detector* 8he $recolumn and column used for

se$aration were 4*- .- mm >1-11) and /*0 1.- mmI)a


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where 0 is the de$endent (ariable ?ethanol@B 1 %, 1 and 1 2are the inde$endent (ariable ?inoculum, initial $, andagitation@B 3 - is the regression coefficient at centre $ointB

3 %, 3 and 3 2 are the linear coefficientsB 3 %%, 3 and 3 22 arethe 9uadratic coefficientsB and 3 %, 3 %2 and 3 2 are the

second order interaction coefficients*8A#E II* INE)ENEN8 FAIA#E> IN 8E EN8A

+M)+>I8E E)EIMEN8A E>I5N

2ariables 6ymboloded levels

-2 -1 0 +1 +2

Inoculum, J?wGw@

1 -*1 -*2 -*3 -*" -*.

Initial $ 2 .*. 4*- 4*. /*- /*.Agitation, r$m 3 2. .- /. 1-- 12.

8he de(elo$ed regression model was e(aluated b'

anal'zing the (alues of regression coefficients, anal'sis of (ariance ?AN+FA@, p) and !)(alues* 8he 9ualit' of fit of the model e9uation was e$ressed b' the coefficient of determination, 4* 8he statistical software $actat Ease Inc*, Minnea$olis, %>A@was used to identif' the e$erimental design as well as toestablish a regression model to $redict the o$timumcombinations considering the effects of linear, 9uadraticand interaction on the amount of ethanol $roduced*

III* E>%8> AN I>%>>I+N

A. Screening o5 Signi5icant !ermentation Parameters

!or Ethanol Prouction 6sing '(o)*evel !actorial

Design&ig* 1 shows the &E>EM images of raw sweetsorghum starch granules* 8he surfaces of granules were9uite smooth, but there is slight e(idence of fissures,indentations or $ores*

>i fermentation $arameters were screened b' thetwole(el factorial design, which showed eighte$erimental runs for ethanol $roduction ?8able III@*%sing the results of the e$eriments, the regressionmodel relating the ethanol ?!@ with the inde$endent(ariables, the inoculum ?A@, urea ?#@, initial $ ?E@, and

agitation ?&@ is as follows6

?3@

where ! is the de$endent (ariable ?ethanol 'ield@B A, #,E, and & are the inde$endent (ariable ?amount of

inoculum, urea, $, and agitation, res$ecti(el'@*

&igure 1* &E>EM images ?1---@ for sweet sorghum ?scale barO1- m@*

8he main effect of each $arameter on ethanol 'ieldwas estimated as the difference between the a(erage of the measurements made at the low ?1@ and high le(el ?2@of the factors* 8he main effects of each fermentation

$arameter are shown in &ig* 2*

8able III shows the achie(ed ethanol concentrationsafter 2" h of fermentation as the maimum ethanol wasobtained at this moment of time* &rom 8able III, out of eight runs, un 2 ga(e the lowest ethanol ?41*4- gG@while un 3 ga(e the highest ethanol ?44*/-@* Also, thereis onl' small difference between the obser(ed and

$redicted ethanol (alues* 8he $redicted ethanol 'ieldcalculated using ?3@ was gi(en in 8able III along with theobser(ed (alue* In this stud', the determinationcoefficient, 2 O -*=3=1 indicates a high correlation

between the e$erimentall' obser(ed and $redicted(alues*

8A#E III* 8E +#>EFE AN )EI8E E>%8> &+ E8AN+ !IE

5un


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8he correlation between the obser(ed and $redicted(alues is better when the (alue of 4 is closer to 1* In thise$eriment, the (alue of 4 was -*=3=1* 8his (alueindicates a high degree of correlation between theobser(ed and $redicted (alues* 8he (alue of 4 indicatesthat =3*=1J of the (ariables6 inoculum, urea, initial $,

and agitation $la' an im$ortant role to the res$onse* 8he(alue of 4 is also a measure of fit of the model and it can be mentioned that onl' about 4*-=J of the total (ariationswere not e$lained b' the ethanol $roduction :23;*

8he results were anal'zed using the anal'sis of (ariance ?AN+FA@ as a$$ro$riate to the e$erimentaldesign used as shown in 8able IF* 8he com$uted !)(alue?11*./@ indicates that the model was highl' significant athigh confidence le(el* 8he $robabilit' p(alue was alsorelati(el' low ? p(alue P ! O -*-342@ which indicates thesignificance of the model* 8he &isher (ariance ratio, the

! (alue is a statisticall' (alid measure of how well thefactors describe the (ariation in the mean of data* 8he

greater the ! (alue indicates that the factors e$lainade9uatel' the (ariation in the data about its mean, andthe estimated factor effects are real* Also, the ! (alue isin(ersel' $ro$ortional to p(alue P ! * igher ! (alue willresult to lower p(alue P ! *

8A#E IF* ANA!>I> +& FAIANE ?AN+FA@ &+ >EENIN5

6ource 6um of 6=uares ! -value "-value > !

Model 2.*12 11*./ -*-342A =*1/ 14*=- -*-241# 1*34 2*.- -*2122E =*3. 1/*23 -*-2."

& .*2" =*4. -*-.3-

&or the inde$endent (ariables, the p)(alue for initial $is the lowest ?-*-2."@, followed b' inoculum ?-*-241@,agitation ?-*-.3-@, and lastl' urea ?-*2122@* 8he p(alueswere used to chec< the significance of each coefficient*8he lower the p)(alue indicates the more significantcorrelation of coefficients ? p)(alue Q -*-. indicate themodel terms are significantB p)(alue Q -*-1 indicate themodel terms are highl' significant@*

Lhen the factor is highl' significant, the small changein the factor ?either increase or decrease@ will gi(e bigim$act on the res$onse* )ositi(e effect means increasing

the factor will result to an increase in the res$onse whilenegati(e effect means reducing the factor will result to anincrease in the res$onse* inear and 9uadratic effects of

$arameters were significant, meaning that the' can act aslimiting factor and little (ariation in their (alue wouldchange either the growth rate or the $roduct formationrate or both to a considerable etent :2";*

&rom the $lot in &ig* 2, three factors, which are N)K,tem$erature, and initial $ ga(e $ositi(e effect to theres$onse* +n the other hand, the inoculum, urea, andagitation ga(e negati(e effect to the res$onse* 8he resultsshowed that the highest (alue re$resents the mostsignificant factor, b' considering the absolute (alue onl'

?neglect the $ositi(e and negati(e sign@* 8he initial $ of the media ga(e highest im$act on ethanol $roduction,

followed b' amount of inoculum, agitation, urea,fermentation tem$erature, and lastl' amount of N)K* )ositi(elinear coefficient means $ositi(e effect and (ice (ersa*8herefore, the most im$ortant factors that affect theh'drol'sis $rocess of sweet sorghum are the initial $ themedia ?E@, amount of inoculums ?A@, and agitation ?&@*

In this stud', the growth of S. cerevisiae was not monitored b' the method of o$tical absorbance because the mediumcontained $reci$itate from the sweet sorghum and also thecolor of the h'drol'sate (er' dar


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of the fermentation time :20;* 8he reason might be thaturea together with N)K ecessi(el' stimulated the growthof the 'east and decreased its ethanol bios'nthesis :2;*

S. cerevisiae is re$orted to grow well within thetem$erature range 20"-R :2=;* &or fermentationtem$erature, it has slightl' $ositi(e effect on the

$roduction of ethanol* igher ethanol was achie(ed at3.R* &rom :3-;, it is mentioned that the maimum $ointfor ethanol $roduction from Korean food waste leachatewas at tem$erature of 30R* Meanwhile, :31; found thatethanol 'ield from simultaneous saccharification andfermentation of citrus $eel waste was highest when thefermentation tem$erature at 3/R* #esides, :32; statedthat higher ethanol $roducti(it' from the


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initial $ ?12@, inoculum and agitation ?13@, andinitial $ and agitation ? 23@ shown in the AN+FAanal'sis were significant*

8A#E FI* ANA!>I> +& FAIANE ?AN+FA@ &+ +)8IMISA8I+N

6ource 6um of 6=uares ! -value "-value > ! Model .1*31 40*.- -*---1

1 2"*22 2=-*=/ Q -*---12 -*4. /*/0 -*-30.3 -*/1 0*"/ -*-33"12 1-*02 13-*-3 Q -*---122 -*.4 4*/. -*-"0"32 -*-" -*"= -*.1"3

12 2*-1 2"*13 -*--""13 3*20 3=*30 -*--1.23 -*43 /*43 -*-3=0

8he 2 contour $lots and 3 res$onse surface are thegra$hical re$resentation of the regression model used todetermine the o$timum (alues of the $arameters withinthe considered ranges :3/;* 8he 2 and 3 $lots for theinteraction between two (ariables among three the(ariables are shown in &ig* 3 to &ig* .* 8he $ur$ose of

res$onse surface is to determine the o$timum (alues of the (ariables, which mean the res$onse is at maimum(alue :3/;* 8he contour $lot re$resents an infiniti(enumber of combinations of the two test (ariables whilethe other (ariable maintained at zero le(el ?centre@* 8hemaimum $redicted (alue is obtained from the surfaceconfined in the smallest elli$se in the contour $lot*Elli$tical contours are obtained when there is a $erfectinteraction between the two inde$endent (ariables :30;*Meanwhile, the 3 surface $lot shows whether the elli$sein the contour $lot is at maimum or minimum*

&igure 3* 2 contour $lot and 3 res$onse surface show the effect of amount of inoculum ?J ?wGw@@ and initial $ on the ethanol $roduction ?gG@?agitation was /. r$m@*

&igure "* 2 contour $lot and 3 res$onse surface show the effect of amount of inoculum ?J ?wGw@@ and agitation ?r$m@ on the ethanol $roduction?gG@ ?$ was 4*.-@*


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&igure .* 2 contour $lot and 3 res$onse surface show the effect of initial $ and agitation ?r$m@ on the ethanol $roduction ?gG@ ?amount of inoculum was -*3-J ?wGw@@*

&ig* 3 illustrates the res$onse surface of ethanol $roduction from the interaction of inoculumconcentration and initial $ of the media* 8he

$redicted ethanol 'ield increases as the amount of inoculum increases and initial $ decreases* 8hemaimum ethanol obtained of about /"*"0 gG was

$redicted at inoculum concentration and init ial $around -*.J ?wGw@ and .*. while agitation s$eed was/. r$m*

A res$onse surface in &ig* " shows the (ariation of ethanol 'ield as a function of inoculum concentrationand agitation b' maustain* Energ' e(*, (ol* =, no*4, $$* .3...., ecember 2--.*

:"; #* * >olomon, V* * #arnes, and K* E* al(orsen, X5rain andcellulosic ethanol6 istor', economics, and energ' $olic',Y #iomass#ioenerg', (ol* 31, no* 4, $$* "14"2., Vune 2--/*


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:.; L* 8* 8sai, * &* an, and * 8* in, XAn anal'sis of bioethanolutilized as renewable energ' in the trans$ortation sector in8aiwan,Y enew* >ustain* Energ' e(*, (ol* 12, no* ., $$* 134"1302, Vune 2--0*

:4; M* * Ngu'en, and * 5* * )rince, XA sim$le rule for bioenerg'con(ersion $lant size o$timisation6 #ioethanol from sugar cane andsweet sorghum,Y #iomass #ioenerg', (ol* 1-, no* .4, $$* 34134.,1==4*

:/; &* * Vohnson, and E* Matsiustain* e(*, (ol* 1-, no* 1, $$* "2.3, March 2--4*

:0; A* >* #ennett, and * )* Ane, X)roduction, trans$ortation andmilling costs of sweet sorghum as a feedstoc< for centralized bioethanol $roduction in the u$$er Midwest,Y #ioresour* 8echnol*,(ol* 1--, no* ", $$* 1.=.14-/, &ebruar' 2--=*

:=; * Amutha, and )* 5unase* 8anahen, XIm$acts of main factors on bioethanolfermentation from stal< juice of sweet sorghum b' immobilizedSaccharomyces cerevisiae ?I 13-0@,Y #ioresour* 8echnol*, (ol*==, no* ", $$* 0"/0.", March 2--0*

:1"; +* +* +la'in* Nieib, V* >* Mcaren, * * Madl,M* 8uinstra, M* * enz, and * Lang, X&actors im$acting ethanol $roduction from grain sorghum in the dr'grind $rocess,Y erealhem*, (ol* 0", no* 2, $$* 13-134, MarchGA$ril 2--/*

:2-; V* Loods, X8he $otential for energ' $roduction using sweetsorghum in southern Africa,Y Energ' >ustain* e(*, (ol* ., no* 1, $$* 3130, March 2--1*

:21; A* emirbas, X#iofuels sources, biofuel $olic', biofuel econom'and global biofuel $rojections,Y Energ' on(ers* Manage*, (ol* "=,no* 0, $$* 21-42114, August 2--0*

:22; N* Nadir, M* Mel, M* I* A* Karim, and * M* !unus, X+$timisationof h'drol'sis conditions for ethanol $roduction from sorghumstarch,Y V* Inst* Eng* Mala's*, (ol* /1, no* 3, $$* 243", >e$tember 2-1-*

:23; M* S* Alam, )* Vamal, and M* M* Nadzir, X#iocon(ersion of $almoil mill effluent for citric acid $roduction6 statistical o$timizationof fermentation media and time b' central com$osite design,YLorld V* Microbiol* #iotechnol*, (ol* 2", no* /, $$* 11//110.,2--0*

:2"; >* #* Imandi, F* F* * #andaru, >* * >omalan* * #andaru,and * * 5ara$ati, XA$$lication of statistical e$erimentaldesigns for the o$timization of medium constituents for the $roduction of citric acid from $inea$$le waste,Y #ioresour*8echnol*, (ol* ==, no* 1-, $$* """."".-, Vul' 2--0*

:2.; * aluce, V* +* 8ognolli, K* &* de +li(eira, * >* >ouza, and M* *Morais, X+$timization of tem$erature, sugar concentration, andinoculum size to maimize ethanol $roduction without significantdecrease in 'east cell (iabilit',Y A$$l* Microbiol* #iotechnol*, (ol* 03,no* ", $$* 42/43/, Vune 2--=*

:24; >* * )rescott, and * 5* unn, Industrial Microbiolog', "th ed*, New!or* Siaulussnain,

Xom$arati(e stud' on two commercial strains of Saccharomycescerevisiae for o$timum ethanol $roduction on industrial scale,Y V*#iomed* #iotechnol*, (ol* 2-1-, $$* 1., A$ril 2-1-*

:20; )* #afrnco(W, * [mogro(i\o(W, I* >lW(ici*@, (ol* "2, $$* 20.2=3, 2--0*

:3.; * A* ardona, `* V* >Wnchez, and * &* 5uti^rrez, XMicroorganisms for ethanol $roduction,Y in )rocess s'nthesis for fuel ethanol $roduction,#oca aton6 )ress, 2-1-, $$* 1311.3*

:34; N* >harma, K* * Kalra, * >* +beroi, and >* #ansal, X+$timization of fermentation $arameters for $roduction of ethanol from hah, and M* I*ajo

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Ethanol fermentation from raw starch of sweet sorghum grains