h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Biotechnology
and
Industrial
Microbiology
Ethanol
production
from
sweet
sorghum
by
Saccharomyces
cerevisiae
DBKKUY-53
immobilized
on
alginate-loofah
matrices
Sunan
Nuanpeng
a,
Sudarat
Thanonkeo
b,
Preekamol
Klanrit
a,c,
Pornthap
Thanonkeo
a,c,∗aKhonKaenUniversity,FacultyofTechnology,DepartmentofBiotechnology,KhonKaen,Thailand bMahasarakhamUniversity,WalaiRukhavejBotanicalResearchInstitute,Mahasarakham,Thailand
cKhonKaenUniversity,FermentationResearchCenterforValueAddedAgriculturalProducts(FerVAAPs),KhonKaen,Thailand
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27November2016
Accepted14December2017
Availableonline13March2018
AssociateEditor:AdalbertoPessoa
Keywords: Alginate-loofah-matrix Ethanolproduction Saccharomycescerevisiae Sweetsorghum Thermotolerantyeast
a
b
s
t
r
a
c
t
Ethanolproductionfromsweetsorghumjuice(SSJ)usingthethermotolerantSaccharomyces
cerevisiaestrainDBKKUY-53immobilizedinanalginate-loofahmatrix(ALM)was
success-fullydeveloped.Asfoundinthis study,an ALMwithdimensions of20×20×5mm3 is
effectiveforcellimmobilizationduetoitscompactstructureandlong-termstability.The
ALM-immobilizedcellsystemexhibitedgreaterethanolproductionefficiencythanthefreely
suspendedcellsystem.Byusingacentralcompositedesign(CCD),theoptimum
condi-tionsforethanolproductionfromSSJbyALM-immobilizedcellsweredetermined. The
maximumethanolconcentrationandvolumetricethanolproductivityobtainedusing
ALM-immobilizedcellsundertheoptimalconditionswere97.54g/Land1.36g/Lh,respectively.
TheuseoftheALM-immobilizedcellswassuccessfulforatleastsixconsecutivebatches
(360h)withoutanylossofethanolproductionefficiency,suggestingtheirpotential
applica-tioninindustrialethanolproduction.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Theuse of alternative energy resources has been
increas-ingly replacing the use of petroleum-based fossil fuels
due to a continuously decreasing fossil fuel reservoir and
∗ Correspondingauthorat:KhonKaenUniversity,FacultyofTechnology,DepartmentofBiotechnology,KhonKaen,Thailand.
E-mail:portha@kku.ac.th(P.Thanonkeo).
environmental problems caused by the growing
consump-tion of oil and its derivatives.1,2 Bioethanol is one of
the high-potential alternative fuel energy sources, since
it isclean, renewable, carbon-neutral and environmentally
friendly.3–5 Bioethanolcanbeproducedfromvarious
renew-ableresources,suchassugar-basedmaterials(e.g.,sugarcane
https://doi.org/10.1016/j.bjm.2017.12.011
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
molasses,beetmolasses), starch-based materials(e.g.,
cas-sava,corn,potato),andlignocellulosic-basedmaterials(e.g.,
rice straw,sugarcane bagasse, corn stalk, grass, pineapple
peel). Apart from sugarcane and cassava, sweet sorghum
[(Sorghumbicolor(L.) Moench)]isoneofthemostpromising
alternativeenergycropsforindustrialbioethanolproduction
inThailand.6–8Itisconsideredahigh-potentialfeedstockfor
ethanol fuel production because it has high levels of
fer-mentablesugars,suchassucrose,glucoseandfructose,and
ahighyieldofgreenbiomass.9Sweetsorghumisalso
con-sideredahigh-efficiencyenergycropbecauseitrequiresless
fertilizerandwaterusage,hasawideadaptabilityfor
cultiva-tion,andhasashortgrowingperiodof3–4months.10,11
Conventional industrial bioethanol production is
gener-allycarriedoutusingafree-cellsystem.Thefree-cellsystem
hasseveraldisadvantages,suchasahighoperatingcostand
a low ethanol productivity.12 To improve ethanol
produc-tion efficiency,cell-immobilization hasbeen proposed.The
immobilized-cellsystem hasseveral advantages,suchas a
reducedriskofmicrobialcontaminationduetohighcell
densi-tiesandfermentationactivity,increasedsubstrateuptakerate,
increasedethanolproductivityandyield,prolongedactivity
andstability ofthecells,the abilitytorecyclethe
biocata-lysts,increasedtolerancetoahighsubstrateconcentration,
reduced inhibition ofend products, protectionof the cells
frominhibitors,easyproductrecovery,andminimal
produc-tion costs.1,13–17 Severaltechniques forcell immobilization,
suchasadsorption,entrapment,cross-linking,covalent
bond-ing,andencapsulation, havebeenreported.16Amongthese
techniques,entrapmentincalciumalginatebeadsisthemost
widelyusedbecause it iseasily prepared,inexpensive and
non-toxic.18 However,it also hassome disadvantages,e.g.,
gel degradation,lowmechanical strength and severe mass
transferrestriction.14,17Inaddition,thecomplexand
sophis-ticatedequipmentrequiredforthelarge-scalepreparationof
calciumalginatebeadscanleadtohighproductioncosts.19
Loofahsponge,alignocellulosicmaterialfromthegourdLuffa
cylindricacomposedmainlyofcellulose(60%),hemicellulose
(30%)and lignin (10%),20 isconsidered as oneof the most
promisingnaturalcarriersforcellimmobilizationinindustrial
ethanolproduction.Loofah spongehasseveral advantages,
suchaslowcost,abundance,chemicalstability,high
poros-ity, high surface area and non-toxicity.17 Ganguly et al.21
reported that the structure and shape of loofah sponges
remained unchanged under various pH conditions (1.1–14)
andremainedstableinhightemperaturesdespiterepeated
autoclavingat121◦Cfor20–40min.Thespongeissuitablefor
celladhesionbecauseitiscomposedofhighlyporousrandom
latticesofsmallcrosssections.15
Inrecentyears,statisticalexperimentaldesignshavebeen
widelyusedtooptimizeethanolproductionconditions.22–25
Thesetechniqueshaveseveraladvantages,suchasa
reduc-tionintimeconsumptionandareductioninoperatingcosts
due to fewer experimentalunits. The interaction between
independent variables can also be evaluated. In addition,
thesecondorderpolynomialequationcanbeusedto
deter-minetheoptimumconditions.26,27Manyfactors,suchasthe
incubationtemperature, the initialyeast cellconcentration
and the initial sugar concentration, affect the growth and
ethanolproduction offreeand immobilizedyeast cells.1,17
Althoughthereareanumberofstudiesonethanol
produc-tion using immobilizedcells,15,17,28,29 little isknown about
ethanolproductionusingyeastcellsimmobilizedspecifically
withinthealginate-loofahmatrix(ALM).Therefore,
optimiza-tion ofethanol productionfrom sweet sorghum juice(SSJ)
using yeast cells entrapped inALMwas performed in this
studyusingacentralcompositedesign(CCD).Theethanol
pro-ductionefficiencyduringrepeatedbatchfermentationusing
ALM-immobilizedcellswasalsoexamined.
Materials
and
methods
Yeaststrain,cellpreparationandrawmaterials
Saccharomyces cerevisiae DBKKUY-53, a high-yield
ethanol-producing thermotolerant yeast strain,7 was used in this
study. Itwas cultured ina yeastextract malt extract (YM)
medium(0.3%yeastextract,0.3%maltextract,0.5%peptone
and1%glucose)at30◦Cfor2daysandthenstoredat4◦Casa
stockculture.Forinoculumpreparation,aloopfulofthestock
culturewastransferredto100mLoftheYMbrothwithan
initialpHof5.0.Thepreculturewasgrowninacontrolled
tem-peratureincubatorshakerat30◦Candshakenat150rpmfor
18h.Then,thepreculturewastransferredintoSSJcontaining
sugarconcentrationof100g/Landsubsequentlyincubatedat
30◦Cwithshakingat150rpmfor18h.Thecellswerecollected
bycentrifugationat5000rpmfor10min,washedtwicewith
0.85%(w/v)NaClsolution,andthenresuspendedinthesame
solution.Theresultingcellswereusedasastarterculturefor
cellimmobilizationandethanolproduction.
ConcentratedSSJ(75◦Bx)obtainedfromtheDepartmentof
Agronomy,FacultyofAgriculture,KhonKaenUniversity,
Thai-land,wasusedasarawmaterialforethanolproductioninthis
study.Thejuicewasstoredat−18◦Cuntiluse.
Cellimmobilization
Loofahspongesusedinthisworkwerepurchasedfromalocal
market,KhonKaen,Thailand.Theywerecutintosmallthin
squarepieceswithdifferentdimensionsof10×10×5mm3,
15×15×5mm3 and 20×20×5mm3. S. cerevisiae
DBKKUY-53 was immobilizedon the alginate-loofah matrices using
theentrapmentmethod.Thestarterculturewasinoculated
intoasterile2%(w/v)sodiumalginatesolutionwithan
ini-tial cell concentration of 2×109cells/mL. Sterilized loofah
cubicspongeswithdifferentdimensionswereimmersedin
thealginate-cellsolution.Then,thealginate-loofahsponges
weredroppedintoa0.1MCaCl2solutionandgentlyagitated
for15min.Theresultingalginate-loofahmatrixorALMwas
washedwithasteriledistilled watertoremoveexcessCa2+
ionsandunentrappedcellsbeforebeingusedforethanol
pro-duction.
Ethanolproductionbyfreeandimmobilizedcellsusing
varioussizesofALM
SSJ containing sugar concentration of 200g/L was added
to a500-mL air-lockedErlenmeyer flaskwith afinal
Table1–KineticparametersofethanolproductionfromsweetsorghumjuicebythefreeandALM-immobilizedcellsofS. cerevisiaeDBKKUY-53at37◦C.
Conditions Kineticparameters
P(g/L) Qp(g/Lh) S(g/L) Yp/s(g/g) T(h)
Freecell 73.64±2.50a 1.53±0.05a 31.69±1.53a 0.44±0.01ab 48
10mm×10mmimmobilizedcell 75.14±2.74a 1.57±0.06a 13.94±0.83b 0.45±0.03a 48
15mm×15mmimmobilizedcell 76.75±1.11a 1.60±0.02a 14.82±1.94b 0.39±0.01bc 48
20mm×20mmimmobilizedcell 76.46±1.63a 1.59±0.03a 12.67±0.28b 0.38±0.01c 48
P,ethanolconcentration;Qp,ethanolproductivity;S,residualsugarconcentration;Yp/s,ethanolyield;andt,fermentationtime.Meanvalues±SD withdifferentlettersinthesamecolumnaresignificantlydifferentatp<0.05basedonDMRTanalysis.
Ethanol production was carried out by transferring the free cells and the cells immobilized in loofah sponges of variousdimensionsinto sterileSSJwithaninitialcell con-centration of 1×107cells/mL.30 The flasks were statically
incubatedat37◦Cinacontrolledtemperatureincubator.
Dur-ingethanolfermentation,sampleswerewithdrawnatcertain
timeintervals,and cells,sugar andethanolconcentrations
weredetermined.
Optimizationofethanolproductionconditionsduring
fermentationbyALM-immobilizedcellsusingacentral
compositedesign(CCD)
Basedonareviewoftheliterature,6,7,14,18threefactors,namely
the inoculum size, the initial sugar concentration and the
incubationtemperature,wereselectedasparametersforthe
optimizationofethanolfermentationusingaCCD.Thecodes
andactualvaluesoftheindependentfactorsfortheCCDare
showninadditionalinformationsection,Table1.The
relation-shipbetweenthecodeandactualvaluesisdescribedbythe
followingequation:
Xi=(XiX−X0), i=1,2,...,k (1)
whereXiisacodedvalueofthevariable;Xiistheactualvalue
ofthevariable;X0istheactualvalueofthevariablesatthe
centerpoint;andXisthestepchangeofthevariable.
Theexperimentaldatawereexplainedbyasecondorder
polynomialfunctionasfollows:
Y=ˇ0+
ˇ iXi+ ˇiiX2i + ˇijXiXj, i=1,2,...,k (2)where Y is the predicted response; Xi and Xj represent
the independent variables; ˇ0 is a constant term; ˇi
rep-resents the linear coefficients; ˇii represents the squared
coefficients; and ˇij represents the cross product
coeffi-cients.
The Design-Expert 7.0 Demo version (STAT EASE
Inc., Minneapolis, USA) was used for the experimental
designs and regression analysis. An analysis of variance
(ANOVA) was used to estimate the statistically significant
parameters. The quality of the quadratic model equation
was expressed as the coefficient of determination (R2).
The validation experiment was conducted using
opti-mized conditions determined from the response surface
plots.
Ethanolproductionbyrepeatedbatchfermentation
Repeatedbatchfermentationwasperformedina500-mL
air-lockedErlenmeyerflaskwithafinalworkingvolumeof300mL.
The fermentation conditions were based on the
optimiza-tionresultsobtainedfromtheCCD,aspreviouslydescribed.
Theflaskswerestaticallyincubatedfor60h,thefermentation
brothwas withdrawnat50% oftheworkingvolumeatthe
endofeachbatch,andthecells,sugarandethanol
concen-trationswereanalyzed.Freshfermentationmedium(150mL)
wasaddedtotheflasksforsubsequentbatchfermentation.8
Analyticalmethods
Theviableyeastcell numberswere determinedbyadirect
counting methodusing ahaemacytometerwithmethylene
blue staining technique.31 For immobilized cells, 10g of
wetALM-immobilizedcellsweredissolvedin0.05Msodium
citratebufferasdescribedbyBangraketal.,17Aftertheloofah
sponge was removed, viable cells were determined using
methylenebluestainingasmentionedearlier.31The
fermen-tation brothwas centrifuged at 12,000rpmfor 10min, and
the supernatantwasthen analyzedfortotalsugar
concen-trationsusingthephenol-sulphuricacidmethod.32Thetotal
solublesolidsofthefermentationbrothwereestimatedusing
hand-heldrefractometer.31Theethanolconcentration(P,g/L)
was analyzed by gas chromatography (Shimadzu GC-14B,
Japan)usingapolyethyleneglycol(PEG-20M)-packedcolumn
with aflame ionizationdetector. Thetemperatures forthe
injector,detectorand columnovenwere 180◦C,250◦C and
150◦C,respectively.Nitrogenwasusedasacarriergas,and
2-propanolwasusedasaninternalstandard.6Thepressure
of nitrogen gas was controlled at 200kPa. The volumetric
ethanolproductivity(Qp,g/Lh)wascalculatedbythe
follow-ing equation:Qp=P/t,wherePisthe ethanolconcentration
(g/L),andtisthefermentationtime(h),givingthegreatest
ethanolconcentration.Theethanolyield(Yp/s)wascalculated
astheactualethanolproducedand wasexpressedasgram
ethanolpergramsugarutilized(g/g).Theethanol
fermenta-tionefficiency(Ey,%)wascalculatedbythefollowingequation:
Ey=((Yp/s)/0.511)×100,whereYp/sistheethanolyield(g/g),and
0.511isthe theoreticalmaximum ethanolyieldperunitof
glucosefromglycolyticfermentation(g/g).
TheScanningElectronMicroscope(SEM,Hitachimodel
S-3000N,Tokyo, Japan)wasusedtovisualizecharacteristicof
theimmobilizedcells.Theimmobilizedcellswerefrozenin
Table2–Thecentralcompositedesign(CCD)matrixforethanolproductionfromsweetsorghumjuicebythe
ALM-immobilizedcellsofS.cerevisiaeDBKKUY-53.
Runs Inoculum(%) Sugarconcentration(g/L) Temperature(◦C) P(g/L) Qp(g/Lh) Y
p/s(g/g) 1 10.0 260 37.5 83.13 1.73 0.42 2 10.0 260 37.5 79.71 1.66 0.44 3 10.0 201 37.5 84.03 1.75 0.49 4 7.0 295 33.0 96.64 1.61 0.45 5 13.0 295 42.0 44.85 0.75 0.46 6 10.0 260 37.5 77.01 1.60 0.43 7 7.0 225 42.0 50.15 1.04 0.43 8 10.0 260 37.5 81.35 1.69 0.44 9 13.0 225 33.0 96.43 1.61 0.48 10 5.0 260 37.5 81.58 1.36 0.43 11 7.0 295 42.0 25.71 0.54 0.39 12 10.0 319 37.5 78.04 1.30 0.41 13 10.0 260 45.0 24.36 0.51 0.39 14 13.0 295 33.0 99.70 1.66 0.45 15 10.0 260 30.0 93.84 1.30 0.43 16 7.0 225 33.0 95.39 1.59 0.45 17 10.0 260 37.5 85.98 1.79 0.45 18 13.0 225 42.0 49.86 1.04 0.42 19 15.0 260 37.5 86.06 1.79 0.42
P,ethanolconcentration;Qp,ethanolproductivity;Yp/s,ethanolyield;Ey,ethanolfermentationefficiency.
weresputteredwithgold(EMITECHmodelK500X,Kent,UK) andphotographed.
Results
Ethanolproductionusingfree-andALM-immobilizedcell systems
Theresultsoftheethanolproductionatrelativelyhigh tem-perature (37◦C) from SSJ containing 200g/Lof sugar using freecellsandcellsimmobilizedinloofahspongesofdifferent dimensionsaresummarizedinTable1.Theethanol
concen-trationsand volumetricethanolproductivitiesproduced by
boththefreeand theALM-immobilized cellswere not
sig-nificantly different. Thismight bedueto the factthat the
initiallivingcellconcentrationsofbothfreelysuspendedcells
andimmobilizedcellsweresimilar.Itshouldbenotedfrom
thecurrentstudythatcellsintheALM-immobilizedsystem
couldutilizehigheramountofsugarsthanthatinthe
free-cellsystem,resultinginlessresidualsugarconcentrationin
the fermentation broth. As a result, the ALM-immobilized
cells exhibited slightly higher ethanol concentrations and
volumetric ethanol productivities than the free cells. The
highestethanolconcentration(76.46–76.75g/L)and
volumet-ricethanolproductivity(1.59–1.60g/Lh)wereachievedbythe
ALM-immobilizedcellswithadimensionof15×15×5mm3
and 20×20×5mm3, which were approximately 3.87% and
4.08%greaterthanthoseofthefreecells,respectively.With
respecttothe ethanolyield, thefree-cell systemexhibited
slightlyhigherethanolyieldthantheALM-immobilized
sys-tem. The cell concentrations during ethanol fermentation
weredetermined.Asobservedinthisstudy,theinitialliving
cellconcentration offreelysuspendedcells inthe
fermen-tationbrothwas1.68×107cells/mL,thenitwasenumerated
and reachedthe concentrationof1.37×108cells/mL atthe
end of fermentation (48h). On the other hand, the initial
living cell concentrations of ALM-immobilized cells in
dif-ferent dimensions ofloofah sponges were similar, ranging
from 1×107 to1.16×107cells/mL.Itshould benotedfrom
this study that the freely suspended cells were observed
apart from the immobilized cells in the ALM-immobilized
cellssystem,resultinginthetotallivingcellconcentrations
of8.85×107to9.38×107cells/mLafter48hoffermentation,
which was slightly lower than that of the free cells
sys-tem (Additionalinformationsection,Table2). Accordingto
the results demonstrated in Table 1 and additional
infor-mation section, Table 2, it was plausible that both freely
suspendedcellsandimmobilizedcellscontributedinthe
con-version of sugar into ethanol in the ALM-immobilized cell
system.
It should be noted that the ethanol
concentra-tions and volumetric ethanol productivities obtained
from the ALM-immobilized cells in 15×15×5mm3 and
20×20×5mm3loofahspongeswerenotsignificantly
differ-ent(Table1).BecausetheALM-immobilizedcellsystemusing
20×20×5mm3 loofah sponges exhibited more compact
structure and its preparationwas alsoeasier than the cell
system using the 15×15×5mm3 loofah sponges, it was
selectedasthesystemforfurtherexperiments.
Optimizationofethanolproductionconditionsduring
fermentationbyALM-immobilizedcellsusingacentral
compositedesign(CCD)
Theoptimumlevels ofthe inoculumsize, the initialsugar
concentrationandtheincubationtemperatureduringethanol
productionfromSSJbyALM-immobilizedcellswereevaluated
usingaCCDviatheDesignexpertsoftware.The
experimen-taldesignmatricesalongwiththeresponsevariables(ethanol
concentrationand ethanolproductivity)aresummarizedin
experi-Table3–KineticparametersofrepeatedbatchethanolproductionfromsweetsorghumjuicebytheALM-immobilized
cellsofS.cerevisiaeDBKKUY-53.
Batch P(g/L) Qp(g/Lh) Yp/s(g/g) X0(cells/cm3) 1 82.54±0.60 1.38±0.02 0.40±0.01 1.40×107 2 84.75±0.87 1.41±0.01 0.44±0.01 6.04×107 3 82.29±1.08 1.37±0.02 0.42±0.02 5.68×107 4 81.50±1.16 1.36±0.06 0.40±0.01 4.84×107 5 80.63±1.15 1.34±0.10 0.42±0.02 3.06×107 6 82.80±1.27 1.38±0.12 0.43±0.00 2.87×107
P,ethanolconcentration;Qp,ethanolproductivity;Yp/s,ethanolyield;X0,initiallivingcellconcentrationineachbatch.
mentsrangedfrom24.36to99.70g/Lwiththeethanolyields ranging from 0.39 to 0.49g/g. The predicted values of the ethanolconcentrationsinthisstudywererangedfrom15.71to 100.31g/L(datanotshown).Aregressionanalysiswasapplied totheexperimentaldatashowninTable2,andthe
second-orderpolynomialequation (3)fortheprediction ofethanol
concentration(P,g/L)asafunctionoftheprocessparameters
includingtheinoculumsize(A,%),initialsugarconcentration
(B,g/L),andincubationtemperature(C,◦C),wasasfollows:
P(g/L)=+82.39+2.22A−2.61B−24.37C+3.08AB+
2.71AC−4.29BC−0.34A2−1.35B2−9.08C2 (3)
Therelativelyhighcoefficientofdetermination(R2)ofthe
regressionoftheabovemodel(0.9698)indicatesthat96.98%
oftheethanolconcentrationcouldbeexplainedbythe
estab-lishedmodel.Thep-valueofthelack-of-fittestwasabove0.05
(0.0891),suggestingthatthemodelisreliable.Thestatistical
significanceoftheregressionmodelofethanolconcentration
wasevaluated byANOVA, andthe resultsare summarized
inadditionalinformationsection,Table3.Inthisstudy,the
establishedmodelwasfoundtobehighlysignificantbecause
the p-value of the experiment was less than 0.05. These
resultsindicatedthattheincubationtemperature(C)andthe
quadratictermsoftheincubationtemperature(C2)had
sig-nificanteffectsontheethanolconcentrationcomparedwith
otherprocessparameters.
The3-Dresponsesurfaceplots(Fig.1A)showingtheeffects
oftheprocessparametersonethanolconcentrationwere
gen-eratedusingthedatainEq.(3).Generally,theresponsesurface
plotscanbeusedtoexplaintheinteractionbetweentwo
pro-cess parameters when another process parameter is fixed
atacentrallevel. Basedon theresponsesurface plots,the
optimumlevelsoftheprocessparametersneededtoobtain
themaximumethanolconcentrationcanbedetermined.9As
showninFig.1A,increasingtheinoculumsizeincreasesthe
ethanolconcentration.Ontheotherhand,theethanol
con-centrationdecreases when theinitial sugarconcentrations
andtheincubationtemperaturesareincreased.
Theregression and the response surfaceanalyses were
alsoevaluatedtostudy theeffects ofinoculum size,initial
sugarconcentrationandincubationtemperatureonethanol
productivity.As shown in Table 2, the actual ethanol
pro-ductivitiesmeasuredin19experimentsrangedfrom0.51to
1.79g/Lh,whicharereliablyclosetothepredictedvaluesof
0.32–1.76g/Lh.Todevelopaquadraticpolynomialregression
modelandasecond-orderpolynomialequation(4)forthe
pre-dictionofthefinalethanolproductivity(Qp,g/Lh)asafunction
oftheprocessparameters,includingtheinoculumsize(A,%),
theinitialsugarconcentration(B,g/L),andincubation
tem-perature(C,◦C),theethanolproductivitiesprovidedinTable2
wereused,andthepredictionequationwasasfollows:
Qp(g/Lh)=+1.71+0.073A−0.011B−0.32C+0.030AB+
0.017AC−0.11BC−0.065A2−0.085B2−0.30C2 (4)
TheresultsoftheANOVAshowninadditionalinformation
section,Table4revealedthatthismodelwashighly
signifi-cant.Inaddition,thelineartermsofthesugarconcentration
(B),incubationtemperature(C),theinteractionbetweensugar
concentrationandincubationtemperature(BC),thequadratic
termsofthesugarconcentration(B2)andincubation
temper-ature(C2)werealsosignificant.TheR2valueoftheregression
(0.9512)suggeststhat95.12%ofthevariabilityintheresponse
couldbeexplainedbythismodel.Thep-valueof“lack-of-fit
tests”(0.0644)wasnotsignificant.Theseresultsclearly
indi-catethatthemodelisreliable.
The 3-D response surface plots showing the effects of
various parameters on ethanol productivity are illustrated
in Fig. 1B. The results indicate that ethanol productivity
increaseswhentheinoculumsizeisincreased.Ontheother
hand, the ethanol productivity decreases when the initial
sugar concentrations and the incubation temperatures are
increased.
Repeatedexperimentswereperformedtoverifythe
pre-dicted optimum values. The final optimum values of the
parametersfromtherepeatedexperimentswereasfollows:
an inoculum size of11.05%, an initialsugar concentration
of 277.06g/L, and anincubation temperature of33.55◦C.A
maximum ethanolconcentration and avolumetricethanol
productivity of 97.54g/L and 1.36g/Lh, respectively, were
obtainedfromtheALM-immobilizedcellsystemunderthese
optimumconditions(Fig.2).Theactualethanolconcentration
obtainedinthisstudyisreliablyclosetothepredictedvalue
(97.76g/L),whereas thatofavolumetricethanol
productiv-ityisslightlydifferentfromthepredictedvalue(1.76g/Lh).It
shouldbenotedfromthecurrentstudythathighlevelsof
sug-arsandtotalsolublesolidremainedinthefermentationbroth.
Thismightbeduetothenegativeeffectsofhigh
concentra-tionsofsugarandethanolongrowthandmetabolicprocesses
inyeastcells.15Bytheway,theresultsofthisstudy
87 1.8 1.675 1.55 1.425 1.3 12.97 11.49 10.00 8.51 7.03 1.9 1.55 1.2 0.85 0.5 33.04 35.27 37.50 39.73 41.96 7.03 8.51 99 80.25 61.5 42.75 24 295.68 277.84 260.00 242.16 224.3241.96 39.73 37.50 35.27 33.04 10.00 11.49 12.97 24 42.5 61 79.5 98 12.97 11.49 10.00 8.51 1.9 1.55 1.2 0.85 0.5 295.68 277.84 260.00 242.16 224.32 41.96 39.73 37.50 35.27 33.07 7.0341.96 39.73 37.50 35.27 33.04 295.68 277.84 260.00 242.16 224.32 83.25 79.5 75.75 72 12.97 11.49 10.00 8.51 7.03 295.68 277.84 260.00 242.16 224.32 B: sugar concentration B: sugar concentration B: sugar concentration productivity productivity productivity ethanol concentr ation ethanol concentr ation ethanol concentr ation B: sugar concentration A: Inoculum size A: Inoculum size C: temperature C: temperature C: temperature C: temperature A: Inoculum size A: Inoculum size
A
B
Fig.1–The3-Dresponsesurfaceplotsshowingtheeffectsofvariousparametersonethanolconcentration(A)andethanol productivity(B)bytheALM-immobilizedcellsofS.cerevisiaeDBKKUY-53.
Table4–Comparisonofrepeated-batchethanolproductionbytheALM-immobilizedcellsofthermotolerantyeastS.
cerevisiaeDBKKUY-53andotherimmobilizedcellsystems.
Strain C-source Carrier P(g/L)a Q
p(g/Lh)a Yp/s(g/g)a References
S.cerevisiae Glucoseand sucrose
Sorghumbagasse 96(13batches) 100(21batches)
NA 0.48 Yuetal.35
S.cerevisiaeNP01 Sweetsorghum juice
Freshsorghum stalk
99.28(8batches) 1.36 0.47 Ariyajaroenwong
etal.34
S.cerevisiaeM30 Palmsugar Thin-shellsilk cocoon
81.9(5batches) 1.71 0.45 Rattanapanetal.1
S.cerevisiaeIR2 Sugarbeetjuice Loofahsponge 76(3batches) NA 0.39 Ogbonnaetal.28
S.cerevisiaeM30 Canemolasses Alginate-loofah-matrix (ALM)
81.4(3batches) NA 0.43 Phisalaphongetal.15
S.cerevisiae Glucose Sucrose Chitosan-cover calciumalginate 30.7(8batches) 31.8(8batches) NA NA 0.31 0.34 Duarteetal.36 S.cerevisiae DBKKUY-53 Sweetsorghum juice Alginate-loofah-matrix (ALM)
82.4(6batches) 1.37 0.42 Thisstudy
S0,initialsugarconcentration;P,ethanolconcentration;Qp,ethanolproductivity;Yp/s,ethanolyield;NA,notavailable.
Time (h) 0 12 24 36 48 60 72 84 96 R e si d u al su ga r c o n c e n tr a ti o n ( g L -1) 0 50 100 150 200 250 300 T o ta l solu b le solid ( B x) 0 5 10 15 20 25 30 E th a no l c o nc e n tr a ti o n ( g L -1) 0 20 40 60 80 100 120 pH 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
Fig.2–VerificationexperimentsofethanolproductionfromsweetsorghumjuiceusingtheALM-immobilizedcellsofS.
cerevisiaeDBKKUY-53.ThefermentationconditionsusedinthisexperimentwerebasedontheCCDresults,asfollows:an
inoculumsizeof11.05%,aninitialsugarconcentrationof277.06g/Landanincubationtemperatureof33.55◦C.䊉:ethanol, :totalsolublesolids,:totalresidualsugar,:pH.
modelisapowerfultooltodeterminetheoptimumvaluesof
theindividualvariablesandthemaximumresponsevaluesas
alsodescribedbyDongetal.33
Ethanolproductionbyrepeatedbatchfermentation
Ethanol production from SSJ by repeated batch
fermenta-tion for sixcycles usingthe ALM-immobilized cells ofthe
thermotolerantS.cerevisiaestrainDBKKUY-53wasevaluated.
Thetimeprofileofrepeatedbatchethanolproductionbythe
ALM-immobilizedcellsisshowninFig.3,andthemain
fer-mentationparameters are summarized inTable 3. Overall,
the almost constant ethanol concentrations and
volumet-ric ethanol productivities, ranging from 80.63 to 84.75g/L
and from 1.34 to 1.41g/Lh, respectively, suggest that the
ALM-immobilizedcellsarestablethroughouttheentire
oper-ation. Although a longer period of fermentation may be
needed to determine the long-term stability of the
ALM-immobilized cell system, repeated fermentation had to be
ceasedaftersixcyclesduetoalimitedamountofSSJ.A
previ-ousstudybyOgbonnaetal.19reportedthatcellsimmobilized
onloofahspongeswerestableaftermorethan35 cyclesof
repeatedbatchfermentationandmorethan500hof
contin-uousethanolproductionusingeithersucroseormolassesas
rawmaterials.
Fig.4showstheyeastcellsadsorbedontheinnerandouter
surfacesandinthemicro-porousstructureoftheALM.The
numberofyeastcellsattheendoftherepeatedbatch
fer-mentation(360h)increasedascomparedtotheinitialtimeof
fermentation.
Acomparativeanalysis betweenrepeated batchethanol
fermentationbyALM-immobilizedcells ofthe
thermotoler-antyeastS.cerevisiaeDBKKUY-53andotherimmobilizedcell
systemsreported inthe literaturewas illustrated (Table 4).
Theethanolconcentration,volumetricethanolproductivity
andethanolyieldproducedbytheALM-immobilizedcellsof
S.cerevisiaeDBKKUY-53werecomparabletothosereportedby
Rattanapanetal.,1Phisalaphongetal.15andOgbonnaetal.28
However,theethanolconcentration,volumetricethanol
pro-ductivityandethanolyieldobtainedinthisstudywerelower
thanthosereportedbyAriyajaroenwongetal.34andYuetal.35
Thismightbeduetothedifferencesinyeaststrainsand
car-riersusedforcellimmobilization.Ontheotherhand,lower
levels of ethanol concentrations and ethanol yields using
immobilizedcellssystemhavealsobeenreported.For
exam-ple, Duarte et al.36 reported the ethanol concentration of
30.7g/Lwiththeethanolyieldof0.31g/gfromglucose and
31.8g/Lwiththeethanolyieldof0.34g/gfromsucroseusingS.
cerevisiaeimmobilizedinchitosan-coveredcalcium alginate.
Inthissystem, chitosanactedasabarriertosubstrate and
products, resulting in the low substrate consumption and
ethanolproductionrate.
Discussion
Cell-immobilization has been proposed as one of the
fer-mentation approaches to improve the ethanol production
efficiencysinceitprovidesseveraladvantages,suchas
pro-longedactivityandstabilityofthecells,theabilitytorecycle
thebiocatalysts,increasedtolerancetoahighsubstrate
con-centration, and easy product recovery.1,13–17 In this study,
S.cerevisiaeDBKKUY-53wassuccessfullyimmobilizedinthe
ALM,oneofthemostpromisingnaturalcarriersforcell
immo-bilization, and its ethanolfermentationactivities usingSSJ
as a raw material were determined. Although the ethanol
production efficiencies in terms of ethanol concentrations
and volumetric ethanol productivities between the
ALM-immobilized cells and the free cells were not significantly
different,theethanolconcentrationsandvolumetricethanol
productivitiesproducedbytheALM-immobilizedcells were
slightlyhigherthanthoseofthefreecells.Theresidualsugar
concentrationsinthefermentationbrothoftheimmobilized
cell cultures were alsoless than those inthe broth ofthe
Time (h) 0 24 48 72 96 120144168192216240264288312336360384 pH 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 Tota l s o lu b le so lid ( B x) 0 5 10 15 20 25 30 35 R e s id u a l s u g a r co n c en tr a tio n ( g L -1) 0 50 100 150 200 250 300 E th a n o l c o n c en tr a tio n ( g L -1) 0 20 40 60 80 100
Fig.3–Timeprofileofrepeatedbatchethanolproduction fromsweetsorghumjuicebytheALM-immobilizedcellsof
S.cerevisiaeDBKKUY-53.䊉:ethanolconcentration,:
residualsugarconcentration,:totalsolublesolid,:pH.
study,theALM-immobilizedcellsexhibitedslightlylowercell
numberthanthefreecells.Thesefindingsdemonstratedthat
the immobilizedcell cultures exhibitedhigher ethanol
fer-mentationactivitythanthefreecellcultures.Onepossibilityis
thatthematrixofcarriersmayprotectyeastcellsfromstress
conditionssuchashightemperatureorhighethanol
concen-trationduringfermentation,allowingthemtoperformbetter
fermentationactivitythanthefreecells.15Withrespecttothe
ethanolyieldinthecurrentstudy,theALM-immobilizedcells
exhibitedslightlylowerethanolyieldthanthefreecells.This
mightbeduetothefactthattheimmobilizedcellcultures
utilized sugarsnotonlyforethanolproductionbut alsofor
synthesisofothermetabolizessuchassaturatedfattyacids,
glycerolortrehalose,whichareinvolvedintheethanol
tol-eranceofyeastcellsunderstressconditions.35Toclarifythis
hypothesis,furtherinvestigationisneeded.
Itshouldbenotedthattheutilizationofsugarsbythe
ALM-immobilizedcellswasnotrestrictedwiththecarriersystem,
suggestedthatthe diffusionofthesubstrates wasnot
pre-ventedbythecarriers,whichwerehighlyporousandthus,
facilitatedthemasstransferofthesystem.Theresultsofthe
currentstudywereingoodagreementwiththosereportedby
Phisalaphongetal.,15Pachecoetal.37andLeetal.38
Thereareseveralfactorsinfluencingtheethanol
produc-tionefficiency6,7,14,18;however,inthisstudy,onlythreemajor
factors including the inoculum size, the initialsugar
con-centrationandtheincubationtemperaturewerefocused.As
foundinthisstudy,increasingtheinoculumsizeresultedin
theincreasesoftheethanolconcentration,whichwas
sim-ilarwiththatreportedbyNuanpengetal.7Onthecontrary,
theethanolconcentrationandethanolproductivitydecrease
whentheinitialsugarconcentrationsandtheincubation
tem-peratures are increased (Fig. 1). This might be due to the
negativeeffectsofhighsugarconcentrationsandhigh
incu-bation temperaturesongrowth,cell viabilityandmetabolic
processesinyeastcells.Baietal.39andOzmichiandKargi40
reported that high sugar concentrations caused negative
effectsoncellviabilityandmorphologyduetoanincreasein
theosmoticpressure,leadingtoareductioninthecellbiomass
andethanolproductionefficiency.Likewise,ithasalsobeen
reportedthatrelativelyhightemperatureconditionscausea
modificationofplasmamembranefluidityandareductionin
theeffectivenessoftheplasmamembrane,resultinginthe
leakage ofessential cofactorsand coenzymes requiredfor
theactivityofenzymesinvolvedinglucosemetabolismand
ethanolproduction.41Relativelyhightemperatureconditions
havealsobeenreportedtocauseadenaturationofcellular
pro-teins,leadingtothereductionofcellgrowthandfermentation
activity.42
ItshouldbenotedfromtheexperimentalusingCCDmodel
that the maximum ethanol concentration and volumetric
ethanolproductivitywerederivedfromdifferentfermentation
conditions.Inthisstudy,amaximumethanolconcentration
wasachievedfromrun14withtheconditionsasfollows;13%
inoculumsize,295g/Lsugarconcentrationand33◦C,whilea
maximumvolumetricethanolproductivitywasachievedfrom
run 19 with the conditions asfollows; 15% inoculum size,
260g/Lsugarconcentrationand37.5◦C.Thereisacorrelation
betweentheinoculumsize,theinitialsugarconcentrationand
produc-A
B
C
D
E
D
Fig.4–SEMimageoftheALM-immobilizedcellsinloofahspongeswithadimensionof20mm×20mm.(A)Pureloofah sponge;(B)alginate-loofahsponge(withoutyeastcells);(C)yeastcellsincorporatedontheoutersurfaceoftheALM;(D) yeastcellsincorporatedontheinnersurfaceoftheALM;(E)ALM-immobilizedcellsat0h,and(F)ALM-immobilizedcellsat 360hafterfermentation.Thewhiteandblackarrowheadsindicatetheyeastcellsandloofahsponge,respectively.
tivity.Itclearlyindicatedfromthisstudythatincreasingthe
inoculumsizeand incubationtemperaturesresultedinthe
increasesofthevolumetricethanolproductivity.Ontheother
hand,thevolumetricethanolproductivitydecreaseswhenthe
initialsugarconcentrationsincreased.
Based on the CCD model, the optimal conditions for
ethanol production from SSJ using the ALM-immobilized
cells were obtained in the current study. In order to
ver-ifythe predicted optimalvaluesoftheindividual variables
from CCD results, repeated ethanolfermentation was
per-formed(Fig. 2). Althoughthe actual ethanolconcentration
(97.54g/L)obtainedinthisexperimentwasrelativelycloseto
thepredictedvalue(97.76g/L),theactualvolumetricethanol
productivity (1.36g/Lh)was slightly lower than that ofthe
predictedvalue(1.76g/Lh).Itwas plausiblethathighsugar
concentration (277.06g/L) may cause prolongation of
com-pletesugarutilization,resultinginlowervolumetricethanol
productivity.7,39,40
Oneoftheadvantagesofthecell-immobilizationsystemis
theabilitytorecyclethebiocatalysts.Inthisstudy,theethanol
productionfromSSJbyrepeatedbatchfermentationusingthe
ALM-immobilized cells ofS. cerevisiaeDBKKUY-53 was
per-formed. Asfoundinthis study,the ALM-immobilized cells
couldbeusedforatleastsixsuccessivecycleswithoutanyloss
ofethanolproductionefficiency(Fig.3).Ithasbeenreported
that highconcentrationsofethanolinhibit thegrowth and
metabolicprocessesofyeastcells.15Theenhancedcell
stabil-ityoftheimmobilizedcellsasobservedinthepresentstudy
suggests that the ALMcarriersmay protectthe yeast cells
fromsevereconditionsduringthefermentationprocess.The
increasedcellstabilityandcell productivityofthe
immobi-lizedsystemdemonstratedinthisstudyareconsistentwith
the reportsbyRattanapanetal.,1 Phisalaphongetal.15 and
Ariyajaroenwongetal.34Inaddition,anincreaseinthe
num-berofyeastcellsadsorbedontheinner andoutersurfaces
and inthe micro-porous structureofthe matrix based on
SEM observationatthe end ofthe repeatedbatch
fermen-tation(360h)ascomparedwiththatattheinitialtime(0h)
wasobserved(Fig.4),suggestingthathighethanolandsugar
yeastgrowth.Wepropose,fromthesefindings,thatthe
regen-erationandprotectionofimmobilizedcellsbytheALMarethe
mainfactorsthatworksynergisticallytopreventcellactivity.
S.cerevisiaeDBKKUY-53hasbeenreportedasoneofthe
goodcandidatesforethanolproductionathightemperature
conditions.7 Although, in this work, the predicted optimal
temperaturebased on the CCD resultsforethanol
produc-tionfromSSJbytheALM-immobilizedcellsofthisstrainwas
33.55◦C,ithasanabilitytoproducerelativelyhighlevelsof
ethanol concentrations and volumetric ethanol
productivi-ties atrelatively high temperature ofup to 40◦C.Previous
studybyNuanpengetal.,7demonstratedthatthemaximum
ethanolconcentrationsandvolumetricethanolproductivities
producedundertheoptimalconditionsusingthefree-cell
sys-temofS.cerevisiaeDBKKUY-53were106.82g/Land2.23g/Lhat
37◦C,and85.01g/Land2.83g/Lhat40◦C,respectively,using
SSJcontainingsugarconcentrationof250g/Lasaraw
mate-rial.ThesefindingssuggeststhatS.cerevisiaeDBKKUY-53has
ahigh potentialforhigh-temperatureethanolfermentation
(HTEF),whichprovidesseveraladvantages,suchasincreasing
therateoffermentation,decreasingariskofcontamination,
andreducing the costofthecoolingsystem andoperating
processes.43,44 Itshould be notedthat the level ofethanol
concentrationandvolumetricethanolproductivityobtained
at37◦Cusingthefree-cellsysteminthisstudy(Table1)was
lowerthanthatofNuanpengetal.7Thismightbeduetothe
differencesinthefermentationconditions.Inthisstudy,the
sugarconcentrationusedintheethanolfermentation
exper-imentusingthe free-cellsystem was200g/L,whilethat of
Nuanpengetal.7was250g/L.Furthermore,theconditionfor
ethanolproductionusing the free-cellsystem inthis work
wasnotyetoptimizedascomparedwiththatofNuanpeng
etal.7
Conclusion
ALM-immobilized cells were successfully developed, and
their ethanol fermentation activities using SSJ as a raw
material were evaluated. The ethanol concentration and
volumetricethanol productivity produced bythe free cells
andthecellsimmobilizedindifferentdimensionsofloofah
spongeswerenotsignificantlydifferent.Basedonthestability
and mass transfercharacteristics, the ALM with a
dimen-sion of 20×20×5mm3 was shown to be effective for cell
immobilization. The optimum conditions for ethanol
pro-ductionbythe ALM-immobilizedcells were asfollows:the
inoculum sizeof11.05%, the initialsugar concentration of
277.06g/Landtheincubationtemperatureof33.55◦C.Under
theseoptimum conditions, the maximum ethanol
concen-trationandthe volumetricethanolproductivity of97.54g/L
and 1.36g/Lh, respectively, were achieved. The ability of
ALM-immobilized cells to be used in repeated batch
fer-mentation for at least six successive cycles without any
loss of ethanol production efficiency indicates that
ALM-immobilized cells can be potentially used for industrial
ethanolproduction.Furtherstudiestoimproveethanol
pro-ductivity and yield such as testing ethanol production by
ALM-immobilizedcellsduringcontinuousfermentation,need
tobeperformed.
Conflict
of
interest
Theauthorsdeclarethattheyhavenoconflictofinterest.
Acknowledgments
Thisresearch wasfinancially supported bytheEnergy
Pol-icyandPlanningOffice,MinistryofEnergy,Thailand,andby
theResearchFundforSupportingaLecturertoAdmita
High-PotentialStudenttoStudyandPerformResearchonHisExpert
ProgramYear2009,bytheGraduateSchool,KhonKaen
Univer-sity,Thailand,bytheFermentationResearchCenterforValue
AddedAgriculturalProducts(FerVAAP),andbytheKhonKaen
University.WewouldliketothankAssociateProf.Dr.Prasit
Jaisil,FacultyofAgriculture,KhonKaenUniversity,Thailand
forprovidingsweetsorghumjuice.
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound,
intheonlineversion,atdoi:10.1016/j.bjm.2017.12.011.
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