Enhanced ethanol production at commercial scale from molasses using high gravity technology by mutant S. cerevisiae

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Enhanced

ethanol

production

at

commercial

scale

from

molasses

using

high

gravity

technology

by

mutant

S. cerevisiae

Muhammad

Arshad

a

_,

_Tariq

_Hussain

a

_,

_Munawar

_Iqbal

b,∗

_,

_Mazhar

_Abbas

c

a_Department_of_Basic_Sciences,_Biochemistry_Section,_College_of_Veterinary_and_Animal_Sciences,_Jhang_Campus_35200,_Pakistan b_The_University_of_Lahore,_Department_of_Chemistry,_Lahore,_Pakistan

c_The_University_of_Lahore,_Institute_of_Molecular_Biology_and_{Biotechnology,}_Lahore,_Pakistan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5June2015

Accepted29November2015

Availableonline16February2017

AssociateEditor:SolangeInes

Mussatto

Keywords:

Veryhighgravitytechnology

Ethanol Molasses Aeration Brix

a

b

s

t

r

a

c

t

Veryhighgravity(VHG)technologywasemployedonindustrialscaletoproduceethanol

frommolasses(fermented)aswellasby-productsformationestimation.Theeffectof

dif-ferentBrix◦ (32,36and40)air-ﬂowrates(0.00,0.20,0.40,and0.60vvm)wasstudiedon

ethanolproduction.Themaximumethanolproductionwasrecordedtobe12.2%(v/v)at40

Brix◦with0.2vvmair-ﬂowrate.Atoptimumlevelaerationand40Brix◦VHG,theresidual

sugarlevelwasrecordedintherangeof12.5–18.5g/L,whereastheviablecellcountremained

constantupto50hoffermentationanddrymatterproductionincreasedwithfermentation

time.Bothwaterandsteamconsumptionreducedsigniﬁcantlyunderoptimumconditions

ofBrix◦andaerationratewithcompromisingtheethanolproduction.ResultsrevealedVHG

withcontinuousairﬂowisviabletechniquetoreducetheethanolproductioncostform

molassesatcommercialscale.

Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Due to higher petroleum product prices and air pollution

issues,researchersarefocusingtoexplorealternative

renew-ablesources.Inthisconnection,ethanolhasbeenemergedas

apotentialalternativecandidateasitiseco-friendlyand

use-ableasblendingwithgasolineincurrentcombustionengine

systems.1_Sugar_cane_molasses_(high_fermentable_sugars)_is

themajorfeedstockfordistilleriesinPakistanforethanol

pro-duction.Theindustrialprocessiswellestablishedwith7–8%

∗ _{Corresponding}_author.

E-mail:bosalvee@yahoo.com(M.Iqbal).

(v/v)ethanol productionfrom dilutedmolasses(15–16%).2,3

However,duringethanolproduction,hugeamountof

efﬂu-ent (∼12liter efﬂuent/literabsolute alcohol)withvery high

biologicaloxygendemandBOD(45,000–60,000mg/L)andCOD

(80,000–120,000mg/L) isproduced,whichisdischargedinto

the environment without proper treatment.4 _So _for, _the

ethanol production using current distilleries is a potential

source ofenvironmentalpollution in Pakistanand ethanol

productionprocessneedstobeimprovedandoptimizedforan

environmentfriendly,fastandcheapethanolproduction.The

sugarcanemolasses(sugarproductionby-product)isagood

http://dx.doi.org/10.1016/j.bjm.2017.02.003

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optiontoproduceethanol.5,6_Biomasses_are_efﬁcient_source_of

energyandareeco-friendlyinnature.7–11

The application of VHG fermentation technology (the

preparationandfermentationofmashescontaining27gor

more dissolved solids per 100g mash) has been proposed

forefﬁcient ethanol productionand to reducestillage

vol-ume.Amongdifferentethanolproductionmethods,theVHG

fermentationtechnologyisveryefﬁcientatindustrialscale

sinceit offershigher yield (12–15%), low waste generation

and can beoperated atlow cost. Higher ethanol levelcan

reduce the distillationcost as compared tothe traditional

fermentation,wheremaximum7–8%ethanolisproduced.2,12

Theethanolpercentageproductioncanbeenhancedthrough

VHGtechnologyusingstrainstolerabletohighsugarcontents.

Yeasts are known to tolerate higher sugar contents (up

to40%), however, athigher sugar concentration, the yeast

growthisaffected,whichgrowsveryslowly.13 _Yeast_growth

isregulatedbymetabolitesgeneratedduringethanol

produc-tion. Generally,industrial ethanol production isperformed

atlowcarbohydrate concentrationinfermentablesugar.2,14

Previous reports indicated that yeast can grow up to 50%

sucroseinfermentingmedia15_and_batch_fermentation_with

23.8% (v/v) ethanol at laboratory scale was achieved in a

simultaneoussacchariﬁcationandfermentationmode.16

Lab-scalemultistagecontinuouscascade,chemostatfermentation

system have been constructed to investigate its

applica-bility toward ethanol production under increased gravity

conditions.17_Ethanol_production_using_VHG_technology_can_be

enhancedapplyingvariousstrategies,i.e.,vitaminfeeding,12

nutrient feeding,13 _media_{supplementations,}18 _temperature

optimization19andaerationrate12havebeenstudiedwellto

improvethe ethanolpercentageyield.Amongthese

strate-gies,aerationimprovedtheviabilityofyeastduringhigh-level

ethanolproduction.20

The VHG fermentation technology in alcohol industry

(operatedinPakistan)isofgreatinterest,asitsaves

consider-ableamountofwaterwithhigheralcoholyield,whichneeds

lowenergyinput,lesscapitalcost,moreefﬁcientandisalso

freefrombacterialcontamination.21_Therefore,_this_study_was

aimedtoevaluatetheethanolproductionusingSaccharomyces

cerevisiae(mutantstrain).Moreover,theeffectsofaerationrate

andBrix’sonethanolpercentageyieldalongwithby-products

estimationinfed-batchmodefermentationatindustrialscale

(usingcurrentdistilleriessysteminPakistan)wasalso

inves-tigated.

Materials

and

methods

Microorganismandculturemedia

TheSaccharomycescerevisiaeparentalstrainwasobtainedfrom

Shakarganjdistillery(Instant-France).Themutantstrain of

Saccharomyces cerevisiae UAF-1 was used for fermentation (S. cerevisiae SAF-INSTANT strain were exposed to gamma

radiation(500KradusingCo-60gammaradiationsource)and

resultantly,1135mutantstrainswererecorded.Allsurvivors

were tested for sugar tolerance and ethanol production.

The survivor (GAMMA-11) furnished higher tolerance to

sugar aswell as ethanolproduction, whichwas named as

SaccharomycescerevisiaeUAF-1andusedforethanol

produc-tion).Theselectedstrainwasculturedinmediumcontaining

(g/L):sucrose(10.0), yeastextract(3.0), (NH4)2SO4 (2.0), and

MgSO4(0.5).WefoundthatgenecreAdelta4wassigniﬁcantly

mutatedandlevelsofmRNAofCreAproteininthewildstrain

were moreinquantitythan thoseofthemutantderivative

(creA delta4 geneisuseful inenhancing theproductionof

extracellularinvertase(protein)levelinthemediumaswell

astakepartincarboncataboliterepression.TheSko1gene

hasroleinactivationandrepressionofproteintranscription

involvedinosmoticandoxidativestressandalsosuppression

offkinaseoverexpressioniscontrolledbySko1gene).

More-over,copynumberofsko1geneinthemutantderivativewas

sufﬁciently decreased to cause repression of transcription

ofCreAmotifasdoneinwildstrain.Goodqualitymolasses

having 89 ◦Bx, total reducing sugars (TRS) (50% w/v) with

additionofnitrogenandphosphoruscontentswasusedfor

the inoculate preparationand very high gravity technique

wasusedforfermentationofmolasses.

Molassespre-treatment

For molasses pretreatment, diluted molasses was settled

in tanks of conical bottom type, especially designed for

the removalofsludge,ashcontentsand otherparticulates.

SodiumhexametaphosphatewasaddedandpHwasadjusted

at4.0–4.5withcommercialsulfuricacidwhichconvertedCa2+

intocalciumsulfate.22_Brix◦_of_the_substrate_was_adjusted_at

32,36and40andsugarcontentswere21%,24%,and27%(w/v)

inpre-treatmentmolasses,respectively.

Inoculumpreparation

Initially,thepropagationofyeastwasstartedin1m3_vessel

andcellcountreachedto3.00×106_CUF/mL;_it_was_transferred

to60m3_vessel_to_maintain_appropriate_cell_count2_and_this

inoculumwasusedforfermentationofmolasses.

Fermentation

Fed-batch experiments were performed in fermenters of

300m3_capacity._The_temperature_was_maintained_at₃₂_±₁◦_C

and stirred by circulation of mash with the help of

elec-tric pumpat 300rpm. To controlthe foaming, silica based

antifoamingagentwasused.Airwassuppliedbytheair

blow-ers.Theaerationratesusedwere0.0,0.1,0.3,0.6and0.9(vvm).

Thefermenterwasﬁlledin16hafterthetransferof

inocu-lum(25%)withappropriatelydilutedmolasses.Levelriseof

thefermenterwaskeptat5%/h(15m3_molasses_were_fed_to

fermenter/h(Fed-batchmode),whichis5%oftotalfermenter

molassesvolumeusedforfermentation).Aftertransferringof

inoculum(100m3_),_the_substrate₍₂₀₀_m3₎_was_supplied

contin-uously.Fivefermenterswererunforeachtreatmentinorder

tocheckreproducibility.

Distillation

Fermented mash was distilled in double effect

distilla-tion plant (Firlli, Italy) having 40,000 liters processing

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columnandsteamwasappliedfrombottominsuchaway

thattemperaturemaintainedinthe rangeof78–80◦C.

Vac-uumwasappliedthroughcondensers.Vaporstraveledfrom

columntocondenserswerecondensedandcooled.The

resul-tantliquidwasre-boiledindepurationcolumnforremoving

impuritiesand ﬁnally,fedtorectiﬁcationcolumn.

Rectiﬁca-tionofethanolwasdoneintherangeof96.0–96.4%andthis

columnwasoperatedat1.5kgm/s2_pressure.

Determinationofcellcount/viability

Forcellcountdeterminationhematocytometerwasused,

fer-menterbrothsamplewasseriallydilutedwithasterilesaline

solution(0.89%(w/v)NaCl)toapointwherereasonable

num-ber ofcells could be counted (3.00×106_CUF/mL). _The _cell

viabilitywasdeterminedbytheMethyleneBluetechnique.12

Brix,reducingsugars,ethanolandby-products determination

Concentration of TRS in diluted molasses and fermented

mash(after sucroseinversionusingHCl) wasmeasured by

Fehling-Soxhletmethod.23_Brix_was_measured_with_the_help_of

ATAGOdensimeter(model2312;ATAGOCo.Ltd.,Tokyo,Japan).

Ethanolandby-products infermentedsampleswere

deter-minedusingGC(ShimadzuGC-17A,3.0)equippedwithﬂame

ionization detector(FID)as reportedearlier.2 _The_aldehyde

contentwasestimatedthroughASTM-D-1363method.

Results

TheVHGtechnologywithandwithoutaerationwasevaluated

onindustrialscaletoimprovethetraditionalfermentationfor

ethanolproduction.VHGtechnologyresultedinhigheralcohol

production(11–12%,v/v)ascompareto7–8%(v/v)in

conven-tionalprocessandoverall,variationamongreplicateswasin

the range of±2–3% throughoutthe experimentation. High

gravitymolassesmediacontainingdifferentconcentrationof

dissolvedsolid,rangingfrom32to40◦Brixwerepreparedand

fermentedfor60hin300m3 _vessel._Maximum _ethanol_was

produced(12.2%)from27%TRSat0.2vvmaerationrate.The

highersugarcontentresultsinhigherethanolproductionand

theseﬁndingsareinlinewithreportedﬁnding,i.e.,pilot-scale

ethanolproductionfromricestrawhydrolysatesusing

xylose-fermentedwithPichiastipiteswasstudied24_and_inﬂuence_of

aerationonbioethanolproductionfromozonizedwheatstraw

hydrolysatesusingPichiastipitis.25

Viablecellcount,ethanolformation,residualsugar

con-tentanddrymattergenerationweredeterminedduringthe

fermentationperiod.By-products,i.e.,methanol,aceticacid,

higheralcohols(1-propanol,1-butanol,2-butanol)production

andaldehydeappearancetimewererecordedandresultsare

depictedinTable2.Ethanol(%,v/v)andresidualsugar(g/L)

at32◦BrixwithdifferentaerationratesareshowninFig.1A

andB,respectively.Residualsugarshowedincreasingtrend

uptothecompletionofbatch.Withoutaeration,theethanol

productionwas8.9%,with35.41g/Lresidualsugarcontents.

Fig. 1C shows the viablecell countat 32◦ Brix with

differ-entaerationrates.Maximumviablecellcountremained417

Table1–Fermentationkineticparametersatdifferent Brix◦.

Parameters 32◦ 36◦ 40◦

Fermentationtime(h) 60 60 60

Residualsugars(g/L)max 35.4 39.7 60.4

Residualsugars(g/L)min 12.5 14.5 18.7

max(h−1) 0.38 0.34 0.35 min(h−1) 0.31 0.42 0.43 Xmax(g/L) 20.3 22.2 22.7 Xmin(g/L) 16.1 18.4 19.0 Ethanolmax(g/L) 8.2 8.9 9.6 Ethanol(ming/L) 7.18 7.30 7.50

million/mL at0.60vvm.Highoxygenconcentration exerted

stronginfluenceonyeastgrowthduringfermentationandthis trendwasinlinewithpreviousfindingsthatoxygen supple-menthassignificanteffectonethanolproductionaswellas cellviability.26_The_viable_cell_count_increased_to_16%,_20%_and

22% at0.2,0.4and0.6vvm,respectively. Theﬁnalbiomass

(Fig.1D)andethanolproductionwasrecorded considerably

athigher sugar level, which indicates thatincreasing level

ofsugar,the yeastcopetheosmoticstressdueinresponse

ofethanolproductioninfermenter.Thestudiedparameters

(Table1)duringethanolfermentationclearlyshowedthatthe

aerationaffectedtheprocessathighersugarlevel.

Ethanolproduction(%)wasrecordedtobe9.3%(v/v)at36◦

Brixwithoutaerationandreachedto11.3%(v/v)withaeration

(Fig.2A).Aerationratesof0.20,0.40and0.60vvmincreasedthe

ethanolproductionby20%,16%and14%overthenon-aerated

fermentation, respectively.Theresidualsugar increasedup

to 30h offermentationand then,decreased sharplyup to

60h(Fig.2B).Maximumviablecellcountwasenhancedwith

increasing sugar level and 499million/mL cell count was

recordedat36◦Brix(Fig.2C).Similarly,thebiomassincreasing

trendwasobservedasthetimeoffermentationisincreasedat

differentVHGlevels(Fig.2D).Thereductionisresidualsugar

and productionof biomassisa good indicationofethanol

productionand Brix levelsigniﬁcantly affectedthe ethanol

production.Themaximumethanolproductionof12.2%(v/v)

wasrecordedat40◦Brixwith0.2vvmaerationrate(Fig.3A).

Theresidualsugar levelswere18.7g/L, 24.8g/Land21.2g/L

at0.2,0.4and0.6vvm,respectively(Fig.3B),whereasin

non-aerated fermenter, the residual sugar was recorded to be

60.4g/L,whichindicatesthataerationalongwithBrix◦ level

isalsoimportantfactorinethanolproduction.Thecellcount

wasrecordedtobe514million/mLat40◦Brix(Fig.3C),which

wasmaximum cellcountascomparedtoother Brixlevels.

Thebiomassproductiontrendat40◦Brixwasrecorded

sim-ilartootherBrixlevels,whichincreasedwiththepassageof

time.

Differentby-products, i.e.,methanol, acetic acid, higher

alcohols (1-propanol, 1-butanol, 2-butanol) were recorded

andby-productsproductionwasrecordedtobeminimumat

0.2vvmairﬂow-rate(Table2)ascomparedtocontrol(0.00vvm

aerationrate).Thealdehydeappearedafterlongertimeof

fer-mentationwhenaerationratewas0.2vvmversusallother

airﬂow-rates.Higheralcohols,i.e.,1-propanol,1-butanol,

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60 50 40 30 20 10 4 5 6 7 8 9 10 11 Ethanol, % Time (h)

A

60 50 40 30 20 10 0 0 20 40 60 80 100 120 140 Residual sugar (g/L) Time (h) 0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

B

C

D

60 50 40 30 20 10 0 280 300 320 340 360 380 400 420 Cell count (x 10 6 /mL) Time (h) 60 50 40 30 20 10 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Dry matter (g) Time (h)

Fig.1–(A)Ethanolproduction,(B)residualsugarcontents,(C)cellcountand(D)drymatterat32◦Brixatdifferentveryhigh

gravitylevels.

Table2–Effectofaerationonby-productsformationsatdifferentBrix◦levels.

Brix◦ _Air_level _Methanol_(g/h_L) _Acetic_acid_(g/h_L) _Higher_alcohols_(g/h_L) _APT*_(min)

32◦ 0.00vvm 2.3 3.4 1.2 28 0.20vvm 1.8 2.1 1.3 40 0.40vvm 3.1 4.1 2.2 24 0.60vvm 3.5 5.0 2.4 16 36◦ None 3.7 3.5 1.3 9 0.20vvm 2.7 2.1 1.3 30 0.40vvm 4.7 4.1 2.6 18 0.60vvm 5.2 5.0 2.8 12 40◦ None 4.4 5.2 1.5 10 0.20vvm 3.2 3.1 1.7 26 0.40vvm 5.6 6.1 3.3 19 0.60vvm 6.2 7.4 3.6 14

∗ _Aldehydes_appearance_time_and_higher_alcohols_include_1-propanol,_1-butanol,_2-butanol.

inhighaerated(0.6vvm)fermenterincomparisonwith

non-aerationmedium(1.2,1.3and1.5g/hL),respectively. Itwas

observedthatincreasedethanolproductioninhibitedthecell

growth.Cellcountviabilitydeclinedupto25%innon-aerated

cultureat32◦Brix.Overall,atBrix◦ threelevelsused,

resid-ualsugarswereveryhighinnon-aeratedfermenters.Aeration

improvedtheethanolproductionby18%ataerationlevelof

0.2vvm,however,theethanolproductiondecreasedathigher

aeration rates.Sugarfermentation byS.cerevisiaeis

gener-ally inhibitedinthe presenceofoxygen,butsmall amount

ofdissolvedoxygenenhancesethanolproductioncompared

tohighlyaerobiccondition.27 _Residual_sugar _recorded_to_be

39.7%innon-aeratedfermenterandonly14.5%inaerated

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60 50 40 30 20 10 4 5 6 7 8 9 10 11 12 Ethanol, % Time (h)

A

60 50 40 30 20 10 0 0 20 40 60 80 100 120 140 Residual sugar (g/L) Time (h)

B

60 50 40 30 20 10 0 280 300 320 340 360 380 400 420 440 460 480 500 Cell count (x 10 6 /mL) Time (h)

C

60 50 40 30 20 10 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Dry matter (g) Time (h)

D

0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

gravitylevels.

to130.5g/L.Theviabilityofcellsmaintained80%evenafter

48hoffermentationascomparedtocontrol(60%).Higher

alco-holsproductionalongwithotherby-productswashigherin

aeratedfermentationandaciditywaslowat0.2vvmas

com-paredtonon-aeratedprocessandhighaeratedculturesatall

Brix◦ levels.Theformationofby-productstookplaceunder

certainunfavorablefermentingconditions.Therefore,

signif-icantreductioninthe by-productswasobservedat0.2vvm

controlledaerationatallhighgravityfermentingmedium.The

production of oxidized metabolites (acetaldehyde, acetate,

andacetoin)isalwaysfavored,2_however,_under_aerobic

con-ditionsandaceticacidproductionwasrecordedtobehigh.

Discussion

Theinhibitory effect of by-products in ethanol production

ismainfactorinethanolproduction.28_It_was_observed_that

underaeratedconditions,declineincellcountviabilitywas

muchlowerascomparedtonon-aeratedcultureatallsugar

levels.Residualsugarundernonaeratedconditionand

aer-ated process as well as the viability of cells after 48h of

fermentationclearlyindicatesthattheyeastcellsneedsome

aerationin order toovercome the osmotic stress atinitial

stageandethanolinducedoxidativestressattheendof

fer-mentation.Theseﬁndingsareinlinewith.29_Authors_reported

thataeratedfedbatchprocessonglucosemediumisusefulin

ethanolproduction.Similarly,Maemura30_also_found_increase

inviablecellcountwiththeaerationlevel. Thetotal

num-berofcellsreachedmaximumlevelafterincubationforabout

24handcellgrowthcultivationwasfoundtobedependent

ontheaeration.Moreaerationenhancedthedissolved

oxy-genandathigherBrix◦,theviablecellcountdecreasedand

thisisinagreementwithobservationsreportedpreviously.31

Ithasbeen reportedthatthecellviabilitydecreasedasthe

concentrationofethanolincreasedusingS.cerevisiaeduring

fermentation.16_In_present_{investigation,}_ethanol_production

was maximum at the lowest aeration rate and Seo32 _also

observed similar trend. Moreover, they reported that the

growthandethanolproductionmaydecreaseatlaterstages

of fermentation, when the ethanol concentration reached

∼100g/L.Overall,ethanolproductionwashigherinthe

aer-atedfermentersandtheseﬁndingsareinlinewithAlfenore

etal.12_and_in_another_study,_production_of_higher_alcohols_was

markedlyenhancedunderoxidativeconditionsmaintainedby

agitationorspargingwithair.2_So_for,_based_on_current

ﬁnd-ing,it isconcludedthat theethanolproductionusingVHG

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60 50 40 30 20 10 0 4 5 6 7 8 9 10 11 12 13 Ethanol , % Time (h)

A

60 50 40 30 20 10 0 0 20 40 60 80 100 120 140 Re s idu al s ug ar ( g /L ) Time (h)

B

60 50 40 30 20 10 0 300 320 340 360 380 400 420 440 460 480 500 520 Cell count (x 10 6 /mL) Time (h) 0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

C

60 50 40 30 20 10 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Dry matter (g) Time (h)

D

gravitylevels.

optimizedconditionasigniﬁcantlyhigherethanolproduction

wasachievedalongwithminimumby-productsproduction.

Conclusions

Ethanolproductionalong withotherby-products was

eval-uated at different aeration rates and Brix levels. Aeration

increased the ethanol production up to 12.0% (v/v) in

highgravitymediumversus7–9%(conventionallyproduced

ethanol). Since ethanol production is the major objectives

infermentationusingeasilycontrolleddistillationprocesses

atreducedcosts.Therefore,theuseofhighgravitymedium

(40◦Brix)underaeratedconditionisusefulinreducingwater

consumption up to 35%, which would ultimately decrease

theefﬂuentsgeneration.Moreover,witheaseindistillation

process,thismethodalsohelpincuttingdownthesteam

con-sumption,resultinginlowerdistillationcostsandimproved

ethanolproductionatindustrialscale.

Conﬂict

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

TheJhangsugarmillAuthoritiesarehighlyacknowledgedfor

providingresearchfacilitiesatJhangsugarmilllaboratories

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