Simultaneous saccharification and co-fermentation for bioethanol production using corncobs at lab, PDU and demo scales.

0.45 MB·English

by  Koppram, Rakesh

Kopprametal.BiotechnologyforBiofuels2013,6:2
http://www.biotechnologyforbiofuels.com/content/6/1/2
RESEARCH Open Access
Simultaneous saccharification and co-fermentation
for bioethanol production using corncobs at lab,
PDU and demo scales
Rakesh Koppram1, Fredrik Nielsen2, Eva Albers1,3, Annika Lambert4, Sune Wännström4, Lars Welin3,
Guido Zacchi2 and Lisbeth Olsson1*
Abstract
Background: While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising
process for bioconversion of lignocellulosic materials to ethanol, thereare still relatively littledemo-plant data and
operating experiences reported in theliterature.In thecurrentwork, we designed a SSCF process and scaledup
from lab to demo scale reaching 4% (w/v) ethanolusing xylose rich corncobs.
Results: Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluatedfor their
fermentation performance inhydrolysates of steam pretreatedcorncobs. Twostrains, RHD-15 and KE6-12 with
highest ethanol yield and lowest xylitol yield, respectively were further screenedin SSCF using thewhole slurry
from pretreatment.Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the
preferred strain since it produced 26%lower xylitol from consumed xylosecompared to RHD-15.Model SSCF
experiments withglucose or hydrolysate feed incombination withprefermentation resulted in79% of xylose
consumption and more than 75% of thetheoretical ethanol yield on available glucose and xylose in lab and PDU
scales. The results suggestthatfor an efficient xylose conversion to ethanol controlled release of glucose from
enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout theSSCF. Fed-batch
SSCF inPDU withadditionof enzymes atthree different time points facilitated controlled release ofglucose and
hence co-consumptionof glucose and xylose was observed yielding 76% of thetheoretical ethanol yield on
available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF incombination with
prefermentationand a feedof substrate and enzymes 47 and 40 g l-1 ofethanolcorresponding to 68% and 58%of
thetheoretical ethanol yield on available glucose and xylose were produced at10.5% WIS inPDU and demo scale,
respectively. The strain KE6-12 was able to completely consume xylose within 76 hduring thefermentationof
hydrolysate ina 10 m3demo scale bioreactor.
Conclusions: The potentialof SSCF is improved in combinationwith prefermentation and a feed of substrate and
enzymes. It was possibleto successfully reproduce the fed-batch SSCF atdemo scale producing 4% (w/v) ethanol
which is the minimum economical requirement for efficient lignocellulosic bioethanol production process.
Keywords: S.cerevisiae,SSCF, Prefermentation, Xylose
*Correspondence:[email protected]
1DepartmentofChemicalandBiologicalEngineering,Industrial
Biotechnology,ChalmersUniversityofTechnology,GöteborgSE-41296,
Sweden
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Kopprametal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background processreferredtoassimultaneoussaccharificationandco-
The global CO emissions in 2010 from fossil energy use fermentation (SSCF). Besides reduced capital cost [16],
2
grew at the fastest rate since 1969. The year 2010 also SSCF process offers several advantages which include con-
witnessed that the global oil production did not match the tinuous removal of end-products of enzymatic hydrolysis
rapidgrowthinconsumption[1].Theserecentdatafurther that inhibit cellulases or β-glucosidases [17] and higher
intensify worldwide concerns about greenhouse gas emis- ethanolproductivityand yieldthanseparatehydrolysisand
sions and energy security for a sustained economic deve- fermentation [18,19]. It is required to operate a SSCF
lopment. For a reduced dependence on oil from fossil process at high content of water-insoluble-solids (WIS) to
reserves, use of biofuels such as bioethanol from abun- achieve high concentrations of ethanol. However, it has
dantly available lignocellulosic biomass is of great interest been shown that at high WIS content ethanol yield was
nowadays because they will count towards meeting the decreased due to increased mass transfer resistance and
mandateof10%bindingtargetforbiofuelsfromrenewable inhibitors concentration [20]. Operating SSCF in a fed-
sourcesinthetransportforallEuropeanmemberstatesby batch mode at high WIS content not only assists ease of
2020 [2]. Along with this interest comes increased interest mixing and produces high ethanol concentrations [21] but
incommercializingethanolproductiontechnologyfromin- also offers a possibility to maintain glucose at low levels
expensive lignocellulosic feedstocks which includes wood allowing efficient co-fermentation of glucose and xylose
biomass, agricultural and forestry residues, biodegradable [22].Loweringofglucoseconcentrationcanbeachievedby
fraction of industrial and municipal wastes. Irrespective of initially fermenting free hexoses before adding enzymes to
type,thebasicstructuralcompositionoflignocellulosicbio- a SSCF process in a concept referred as prefermentation
mass consists of cellulose, hemicellulose and lignin. The enhanced xylose uptake irrespective of batch or fed-
cellulose and hemicellulose that form the polysaccharide batch SSCF [23]. These flexibilities offered by a SSCF
fraction are embedded in a recalcitrant and inaccessible processmakesitapromisingprocessoptionforbioethanol
arrangement[3]andthereforerequiresapretreatmentstep productionfromlignocellulosicmaterials.
to disrupt the structure and make it accessible for subse- The heterogeneity of raw materials together with a
quent steps. Since lignocellulosic materials are very com- variety of pretreatment methods, lack of detailed under-
plex, not one pretreatment method can apply for all the standingofdynamicchangesofsubstrateduringenzymatic
materials.Severalmethodsthatareclassifiedintophysical, hydrolysis and unavailability of microorganisms that can
physico-chemical, chemical and biological pretreatment ferment a wide range of carbohydrates and can tolerate
have been investigated and an elaborate review on each of highconcentrationsofinhibitorsproducedfrompretreated
these methods has been presented by Taherzadeh and biomass makes SSCFa highly researched area yet to reach
Karimi [4]. One of the most commonly used pretreatment the commercial status. There come additional technical
methodsissteamexplosion,withtheadditionofH SO or challenges when operating at larger scales which include
2 4
SO ,whichremovesmostofthehemicellulose,followedby longertimestoaddmaterialintothereactor,longermixing
2
enzymatichydrolysistoconvertcellulosetoglucose[5,6]. times and therefore concentration gradients are inevitable.
The release of hexose and pentose sugars during pre- On-site propagation of yeast in large volumes is needed
treatment and enzymatic hydrolysis is often accompanied whichalsoincreasestheprobabilityofcontaminationsince
by liberation of compounds such as furans, weak organic lignocellulosic ethanol plants will not employ aseptic ope-
acids and phenolics compounds [7] that inhibits growth, rating conditions. Moving cellulosic ethanol technology
ethanol yield and productivity of fermenting microorgan- from the laboratory to a commercial scale biorefinery
ism, Saccharomyces cerevisiae [8-10]. Traditionally and in- is an expensive proposition and requires process data
dustriallyrelevantmicroorganismforethanolfermentation at sufficient scale to obtain engineering and process
is S. cerevisiae, but its inability to consume pentose sugars guarantees. Some prominent players that are working
like xylose and arabinose has led to intensive research on on this proposition include Chemtex, Inbicon, DuPont
metabolic and evolutionary engineering to develop strains cellulosic ethanol, POET-DSM advanced biofuels,
that can tolerate high concentration of inhibitors and fer- Iogen, Abengoa Bioenergy, Mascoma and SEKAB. A
ment xylose and arabinose [11-15]. However, it has been category of feedstock that is of considerable interest
shown that recombinant S. cerevisiae strain utilizing pen- is corn derived residues due to that it is inexpensive
tose sugar may lose its xylose consuming ability in a long and available in abundance. Corncob is an agricultural
term evolutionary engineering for inhibitor tolerance [15]. residue and a byproduct of corn production. Currently,
Consequently, to ensure that all properties are retained 12.1 billion tons and 120 million tons of corn are being
during evolutionary engineering requires careful design of produced in the US and China, respectively. About 70
theselectionpressure. million metric tons of corncobs are available annually
The enzymatic hydrolysis can be performed simulta- accounting only from the US and China markets [21,24].
neouslywiththeco-fermentationofglucoseandxyloseina Removal of corncobs from the agricultural grounds does
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not contribute to decreased soil organic matter since
corncobsarelowinnutrients.
Inthiswork,axylosefermentingS.cerevisiaestrainwas
usedinSSCFofpretreatedcorncobs withthe objectiveof A: 47 %
determining suitable conditions for co-consumption of
B: 0.18
glucose and xylose. Fed-batch mode of SSCF in combi-
C: 46 %
nationwithprefermentationwasinvestigatedathighWIS
D: 14.5
content. To validate the designed SSF process and verify
A: 19 %
the reproducibility at different scales, the process was AD2-10
scaledupfromlabconditionstoprocessdevelopmentunit B: 0.17
(PDU)(30liters)andfurthertodemoscale(10m3). C: 30 %
D: 9.8 A: 46 %
Results and discussion
KE4-22 B: 0.13
The SSCF concept is one of the interesting process
C: 46 %
options and the potential of such process for biological
D: 14.5
conversion of lignocellulosic raw materials to bioethanol
in large scales has to the best of our knowledge not been
KE6-12
reportedpreviously.Apromising xylose consumingstrain
ofS.cerevisiaewasselectedfromscreeningsevendifferent
recombinant S. cerevisiae strains. The glucose influence
on xylose consumption of the selected strain was investi-
A: 22 %
gated by model SSCF with glucose or hydrolysate feed.
The potential of fed-batch SSCF process in combination B: 0.13
with prefermentation was finally demonstrated in 10 m3 C: 36 %
demoscalebioreactors. D: 10.7
RHA-15
ScreeningandselectionofS.cerevisiaestrain
Anaerobicfermentationofcorncobhydrolysate
The seven different S. cerevisiae strains (Table 1) were
evaluated on their fermentation performance in corncobs A: 47 %
A: 19 %
hydrolysate in shake flasks equipped with glycerol loops. B: 0.26
B: 0.17
Since,xyloseconstitutesasignificantproportionofmono- C: 51 %
C: 30 %
saccharides in corncobs hydrolysate xylose consumption
D: 14.4
and xylitol yield together with ethanol yield were deter- D: 9.8
RHC-15
mined (Figure 1) and used as parameters for strain selec-
AD1-13
tion. The strains, AD2-10, KE6-12 and RHC-15, RHD-15
displayed similar ethanol concentration, ethanol yield and
performed better than their respective parental strains A: 55 %
with regard to xylose consumption. The strain RHD-15 B: 0.21
displayed the highest ethanol yield and xylose consumed. C: 53 %
The strain KE6-12 stands alone among other strains in
D: 15.8
xylitol yield producing the lowest amount of xylitol from
RHD-15
consumedxylose.Eventhoughthescreeningrevealedsig-
nificant differences in fermentation of hydrolysate, it is Figure1ScreeningofS.cerevisiaestrainsincorncobs
important to evaluate the microbial performance in the hydrolysate.Xyloseconsumption,xylitolandethanolyields,ethanol
concentrationincorncobshydrolysateafter96hoffermentationin
Table1Scerevisiaestrainsusedinthisstudy anaerobicshakeflasks.KE4-22istheparentalstrainofAD2-10and
KE6-12.AD1-13istheparentalstrainofRHA-15,RHC-15andRHD-15.
Parentalstrain Evolvedstrain
A:xyloseconsumed(%),B:xylitolyieldonconsumedxylose
KE4-22 AD2-10 (gg-1),C:ethanolyield(%,basedonmaximumtheoreticalethanol
yield on available glucose and xylose), D: ethanol concentration
KE6-12
(g l-1) at the end of 96 h.
AD1-13 RHA-15
RHC-15
RHD-15
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whole slurry in a SSCF process. The strains RHD-15 and KE6-12consumedmarginallyloweramountofxylosebut,
KE6-12, due to their high ethanol and low xylitol produced 56% less xylitol. Since, bioconversion of xylose
yields, were therefore, selected as the preferred strains to ethanol is one of the predominant requirements for an
for subsequent investigations in the SSCF process. economical lignocellulosic bioethanol production, further
fermentationandSSCFexperimentswerecarriedoutwith
SSCFofcorncobswholeslurry the strain KE6-12, unless otherwise stated. In screening
To assess the fermentation performance, the strains RHD- experiments using corncobs hydrolysate, the strain
15andKE6-12wereevaluatedinabasecasebatchSSCFof RHD-15 performed relatively better than KE6-12,
corncobswholeslurryat7.5%WISforethanolproduction. however, in SSCF using corncobs whole slurry both
During the SSCF process, the glucose concentration was the strains resulted in similar ethanol yields and
quickly reduced to less than 1 g l-1 within 10 h and there- RHD-15 was clearly outperformed by KE6-12 in lower
after,itwasmaintainedatthislevelthroughouttheprocess xylitol yields. The differences in results from the two
(Figure 2a & 2b). Immediately after inoculation, both the screening experimental systems could be attributed to the
strainsstartedtoconsumexyloseforaperiodof72hafter differencesinexperimentalconditions.TheeffectofpHon
which the xylose concentration in the reactor started to xylose consumption by a recombinant xylose utilizing S.
level off. After 96 h, the strain KE6-12 had consumed 37% cerevisiae has been previously shown that increasing the
oftheavailablexyloseand30%oftheconsumedxylosewas pH from 5.0 to 5.5 resulted in 46% increase in xylose
converted to xylitol (2.8 g l-1) whereas, the strain RHD-15 consumptionrate[25].Screeningusingcorncobshydrolys-
had consumed 42% of the available xylose and 66% of the atein shakeflaskswereperformed at an initial pH6.0and
consumed xylose was converted to xylitol (6.4 g l-1). An 30°C which clearly resulted in higher xylose consumption
ethanol concentration of 21.9 and 21.5 g l-1 were achieved compared to screening in SSCF where the pH was con-
corresponding to a yield of 0.28 g g-1 and 0.27 g g-1 based trolled at 5.0 and sub-optimal temperature of 35°C. It
on total available sugars for the strains KE6-12 and shouldbenotedthatoftenstrainengineeringand develop-
RHD-15, respectively. In comparison to RHD-15, strain ment results in a numerous strains and a high throughput
screening of these strains in SSCF process in shake flasks
could be impractical due to difficulties in mixing at high
7 28 WIScontent.Thedifferenceintwoscreeningsystemsillus-
a
trate the importance of choice of experimental setup and
6 24
-1ylitol, g l 45 1260 1-hanol, g l cuosenddiitniotnhsefaocrtusaclreeexnpienrgimteonbtse. as close as possible to that
se, x 3 12 e, Et MInoodredleSrStCoFuansdaertsotaonldtothdeeseifgfenctthoefSgSluCcFosperoocnesxsylosecon-
o s
uc 2 8 ylo sumption and to optimally design the SSCF process with
Gl X effectivexyloseconsumptionamodelSSF[26]withprefer-
1 4
mentation [23] was performed. A model SSCF is a SSCF
0 208 processwithouttheadditionofenzymesbutfedwithpure
b glucose solution or hydrolysate to the reactor mimicking
6 24
thereleaseofglucoseduringenzymatichydrolysisofcellu-
-1ol, g l 45 1260 -1ol, g l lforesee.gPlurceofesremweanstafteiromneinsteadcboenfcoerpetswtahrteirnegitnhietiafelleyda.vailable
xylit 3 12 han Labscale
Glucose, 12 48 Xylose, Et MdstraoonldytesrlaatSteeS.wCAiFthginlaucfleaoebsdesofcefael1de00cwoagrsrle-p1spegrolfunocdrmoinseegdstooinluthtcieoonranmactoobausnctohnoy--f
glucosefrom7.5%WISwasstartedafter2hofinoculation
0 0
0 20 40 60 80 100 andterminatedat96h.Duringtheprefermentationperiod
Time, h of2h,theglucoseconcentrationwasreducedtonearly0g
Figure2ScreeningofS.cerevisiaestrainsincorncobswhole l-1 and maintained at this level until 72 h (Figure 3a).
slurry.Glucose(diamonds)andxylose(squares)consumption, Immediately after prefermentation, xylose was rapidly con-
ethanol(circles)andxylitol(crosses)productioninSSCFat7.5%WIS sumed until 48 h and thereafter, the concentration started
content,5gl-1ofyeastloadingand5FPUgWIS-1ofenzyme
to level off. After 96 h, 79% of xylose was consumed and
loadingusingKE6-12(a)andRHD-15(b).
37%ofconsumedxylosewasconvertedtoxylitol(6.4gl-1).
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observedimmediatelyaftertheadditionofenzymesolution
45
indicating the hydrolysis of xylan. Thereafter, the xylose
a
40 wasconsumedquicklyfor10h,however,whentheglucose
-1g l 3305 wticaasllcyosmlopwleetdeldyocwonn.sTumhiesdintdhiecaxtyelsotsheactothnesucmonpstiuomnpdtrioamnao-f
on, 25 glucose with maintained low concentration of glucose is
ati beneficial for efficient xylose consumption. Previous study
entr 20 onfed-batchSSCFusing xyloserichwheat strawhashigh-
c 15
n lighted thatmaintaininglow levelsofglucose consequently
o
C 10 increased consumed xylose twice as compared to a batch
5 SSCF [26]. It also has been discussed that presence of glu-
450 cose at high concentrations may inhibit xylose uptake due
b to competition for transporters [27,28]. Feeding of the
40
liquid fraction from enzymatic hydrolysis was started after
-1g l 35 24hofprefermentationandwasmaintainedfor24hcorre-
n, 30 sponding to a final WIS content of 7.5%. The glucose
o
ati 25 concentration gradually increased during the 24 h feeding
ntr 20 phase until 48 h and thereafter was completely consumed.
e
nc 15 The xylose started to accumulate when glucose concentra-
o
C tionreachedapeakof10gl-1andthereafternoxylosewas
10
consumed and no change in ethanol concentration was
5
observed indicating the end of fermentation. The increase
0
in xylose concentration after 50 h could be due to enzy-
0 20 40 60 80 100
matichydrolysisofxylan.After96h,anethanolconcentra-
Time, h
tion of 32 g l-1 was produced corresponding to 77% of the
Figure3ModelSSCF.Glucose(diamonds)andxylose(squares)
consumption,ethanol(circles)andxylitol(crosses)productionina theoretical yield based on available sugars. This ethanol
modelSSCFincorncobshydrolysatewith5gl-1ofKE6-12atlab yield is well in accordance with ethanol yields of model
scaleusingafeedofglucosesolution(a)andatPDUusingafeed SSCF in lab scale. Evidences from model SSCF with prefe-
ofliquidfractionafterenzymatichydrolysis(b).Amountofglucose rmentation clearly suggest that fermentation of initial free
fediscorrespondingto7.5%WIScontent.
glucoseandthereafter,maintenanceofglucoseatlowlevels
arecrucialfactorsforefficientxyloseconsumption.
An ethanol concentration of 31.2 g l-1 was achieved corre-
sponding to a yield of 0.38 g g-1 based on total available Fed-batchSSCF
sugars (75% of the theoretical yield). Higher ethanol PDUscale
concentration in model SSCF compared to batch SSCF It was also possible to achieve similar ethanol yields in a
maypossiblybeduetohigherxyloseconsumptionandalso fed-batch SSCF as that in the model SSCF. Fed-batch
points to a direction that cellulose fibers were not com- SSCF in PDU was carried out using the whole slurry
pletely hydrolyzed in batch SSCF to yield similar ethanol with a total WIS of 7.9%. Initially, prefermentation was
concentrationsasthatobtainedinmodelSSCF. carried out for 2 h by adding 6 g dry cell weight l-1 of
yeast from cell suspension. To maintain glucose concen-
PDUscale trations at a minimum level in the reactor and thereby
A model SSCF in PDU scale similar to lab scale model facilitate effective xylose consumption, a strategy to add
SSCF was performed with a feed of hydrolysate from en- enzyme solution at multiple time points to ensure con-
zymatic hydrolysis. In order to completely hydrolyze cellu- trolled release of glucose was investigated. An enzyme
lose fibers, enzymatic hydrolysis of solid fraction of solution corresponding to 3 FPU gWIS-1 was added at 2
corncobs slurry was carried out at 50°C with enzyme loa- h, 24 h, and 48 h. The glucose concentration was main-
ding of 6 FPU gWIS-1. The liquid fraction remaining after tained around 5 g l-1 until 72 h before it was completely
filtering the enzymatically hydrolyzed solid fraction was consumed at 96 h (Figure 4). A steady co-consumption
used as a feed. Prefermentation in corncobs hydrolysate of glucose and xylose was observed throughout the
wasinitiatedbyaddingyeastandanenzymesolutioncorre- SSCF. After 96 h, 50% of the available xylose was con-
sponding to 3 FPU gWIS-1. The glucose was rapidly con- sumed producing xylitol with aconcentration ofonly 1.5
sumed during the initial 10 h of prefermentation, reduced g l-1. An ethanol concentration of 32 g l-1 was achieved
to near 0 g l-1 and maintained at this level until 24 h corresponding to 76% of the theoretical ethanol yield
(Figure 3b). A sharp increase in xylose concentration was basedonavailablesugars.
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40 50
a
35
40
1
-g l 30 -1g l 30
ation, 25 ntration, 20
tnr 20 once
ec C 10
n 15
o
C
500
10
b
5 40
00 20 40 60 80 100 -1n, g l 30
Figure4Fed-batchSSCFwithpTreimfeer,m hentationandsplit entratio 20
c
additionofenzymeatPDU.Glucose (diamonds) and xylose n
o
(squares) co-consumption, ethanol (circles) and xylitol (crosses) C 10
productionusingcorncobswholeslurryat7.9%WIS,6gl-1ofKE6-12
with3FPUgWIS-1ofenzymeloadingateachtimepointsof2h,24h 0
and48h. 0 20 40 60 80 100 120 140 160 180
Time, h
Figure5SSCFwithprefermentationandfed-batchadditionof
In a commercial bioethanol production process it is
substrateandenzyme.Glucose(diamonds)andxylose(squares)
desirable that the substrate load is higher than 7% WIS co-consumption,ethanol(circles)andxylitol(crosses)production
toachieve4%(w/v)ethanolconcentrationtoyieldasub- usingcorncobswholeslurryat10.5%WIS,5gl-1ofKE6-12and15
sequent economical distillation process [29]. It has been FPUgWIS-1ofenzymeloading.Splitadditionofsubstrateat0h,5h,
27h,49handenzymesolutionat2h,24h,48h,72h,96hinPDU
shown that working at high WIS content increases the
(a).Fed-batchadditionofsubstratefor48handsplitadditionof
concentration of inhibitors and results in inhibition of
enzymesolutionat2h,24h,48h,72h,96hindemoscale(b).
yeast and lower ethanol yields [26]. Therefore, along
with split addition of enzymes, fed-batch SSCF at higher
WIS was investigated with split addition of substrate Demoscale
resultinginloweramountofinhibitorsforeachaddition. Xylosefermentationinhydrolysate
Fed-batch SSCF experiment was performed with a corn- A time span of 24 to 48 h was used to pump a substrate
cobs slurry addition at 0 h, 5 h, 27 h and 49 h to a total intothedemoscalereactorof10m3.Inordertoaddress
final WIS of 10%. Enzyme solution was added at multiple thepotentialofthestrainKE6-12onxyloseconsumption,
time points of 2 h, 24 h, 48 h, 72 h and 96 h to a total of fed-batch fermentation of corncobs hydrolysate corre-
15 FPU gWIS-1. During the first 2 h of prefermentation, sponding to a WIS content of 6% was evaluated. The
the glucose concentration was reduced to nearly 0 g l-1 corncobs hydrolysate was fed into the reactor for 24 h.
and reached around 5 g l-1 after the first addition of The glucose concentration was reduced to nearly 0 g l-1
enzyme (Figure 5a). The glucose concentration was then within5handallxylosewasconsumedin76h(Figure6).
maintained below 5 g l-1 throughout the SSCF process. Only10.6%oftheconsumedxylosewasconvertedtoxyli-
Thexylosewasco-consumedalongwithglucoseformore tol (2.0 g l-1). An ethanol concentration of 10.9 g l-1 was
than100h.Attheendoffed-batchSSCF,55%oftheavai- achieved corresponding to a yield of 0.46 g g-1 on total
lable xylose was consumed and 11% of the consumed availablesugars(90%ofthetheoreticalyield).
xylose was converted to xylitol (3.4 g l-1). An ethanol
concentration of47g l-1 wasachieved correspondingtoa Fed-batchSSCF
yield of 0.35 g g-1 based on total available sugars (69% of Afed-batchSSCFwithsubstrateandenzymefeedandpre-
the theoretical yield). Higher xylose consumption and fermentation for 2 h similar tothe one performed at PDU
ethanol yield at 10% WIS clearly suggest that the combi- scale was carried out in the demo scale. The corncobs
nation of prefermentation and a feed of enzymes and slurry was fed into the reactor for 48 h resulting in a total
substratesasoneofthepossibleSSCFstrategiesfordemo WISof10.5%.Enzymesolutionwasaddedatfivedifferent
scaleexecution. timepoints, 2h,24h,48h,72and 96h corresponding to
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SSCFprocess.Thepotentialofthefed-batchSSCFprocess
12
is more vivid and we demonstrated that with prefermenta-
tionandsubstrateandenzymefeeditispossibletoproduce
10 ethanol from corncobs as high as 40 g l-1 and more, with
relatively high WIS content at both 30 l (PDU scale)
1 and 10 m3 (demo scale). Using such a strategy it was
-g l 8 possible to maintain low levels of glucose concentra-
n, tion, which facilitated co-consumption of glucose and
o
ti 6 xylose. We also confirmed that the results of fed-batch
a
r
t SSCF were similar at PDU and demo scales and the
n
cne 4 experimental system was reproducible at both the scales.
o However, at higher WIS content an optimal feeding
C
strategy is required to ferment all xylose and avoid
2 glucose accumulation.
Materials and methods
0
0 20 40 60 80 Saccharomycescerevisiaestrains
Time, h ThesevenS.cerevisiaestrainsusedinthisstudy(Table1)
were developed by a combination of different evolution-
Figure6Fed-batchfermentationincorncobshydrolysateat
ary engineering strategies and random mutagenesis
demoscale.Glucose(diamonds)andxylose(squares)consumption,
ethanol(circles)andxylitol(crosses)productionusing5gl-1ofKE6- (Albers et al.,manuscriptinpreparation) onS.cerevisiae
12.Corncobshydrolysatecorrespondingto6%WIScontentwasfed TMB 3400 [30] that harbours the xylose reductase gene
for24h. and xylitol dehydrogenase from Scheffersomyces stipitis
(formerly known as Pichia stipitis) and endogenous
a total of 15 FPU gWIS-1. The glucose concentration was xylulokinase overexpressed. All the strains were stored
quicklyreducedtonearly0gl-1within10handthereafter, at −80°C in culture aliquots containing 20% sterile gly-
it was maintained at low concentration throughout the cerol. Volumes of 100 μl from the vials were used to
process(Figure5b).Co-consumptionofxyloseandglucose inoculateprecultures.
wasobservedformorethan100hsimilartothefed-batch
SSCFatPDUscale.After150h,65%oftheavailablexylose Media
wasconsumedand24.7%oftheconsumedxylosewascon- Corncobsslurry
verted to xylitol (9.3 g l-1). Surprisingly, in comparison to Corncobs slurry with a water-insoluble-solids (WIS) con-
fed-batchSSCFatPDUscale,higheramountofxylosewas tent of 15% was received from SEKAB-E-Technology AB
consumed in demo scale, however, also higher amount of (Örnsköldsvik, Sweden) and was stored at −20°C. The
xylitol was produced. An ethanol concentration of corncobs were pretreated at 185°C for 5 min with 0.6%
39.8 g l-1 was achieved corresponding to a yield of 0.29 g dilute sulfurous acid (SO in water). Two batches were
2
g-1 based on total available sugars (58% of the theoretical pretreated and the composition of which are pre-
yield).Morecontrolledconditionsoftemperature,pHand sented in the Table 2. Batch 1 was used for screening
homogenous mixing in PDU scale resulted in higher final and selection experiments. Batch 2 was used in the
ethanol concentration and yield compared to demo scale
conditionswithhighermasstransferlimitations.
Table2Compositionofthepretreatedcorncobs
Contentinsolidfraction(%ofWIS) Contentinliquidfraction(gl-1)
Conclusion
The performance of recombinant xylose utilizing S. Batch1 Batch2 Batch1 Batch2
cerevisiae strainsvariedintwodifferentscreeningexperi- Glucan 66.9 61.4 Glucose* 17.0 15.0
ments, which highlights the importance of experimental Mannan 0 0 Mannose* 0 0
setup and conditions for screening of strains to be highly Galactan 0 2.9 Galactose* 6.5 0
similar to that of the actual experiments. The choice of Xylan 5.8 8.2 Xylose* 79.4 74.4
the strain KE6-12 seems well justified when xylose was
Arabinan 1.0 1.4 Arabinose* 11.8 14.0
completely consumedat demo scale duringthe fermenta-
Lignin 27.6 28.9 HMF 2.0 1.9
tion of hydrolysate with 90% of the theoretical ethanol
yield.Differentfeedingprofilesofglucoseanditsinfluence Furfural 3.8 4.0
on xylose consumption was studied using model SSCF Aceticacid 10.4 8.3
and it proved to be a valuable tool to optimally design a *Bothmonomericandoligomericformisincluded.
Kopprametal.BiotechnologyforBiofuels2013,6:2 Page8of10
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demoscaleexperiments.Thecorncobshydrolysate(liquid followed by an aerobic fed batch on corncobs hydrolysate
fractionofcorncobsslurry),pHadjustedto5.0with10M and molasses. In the demo scale molasses was used as the
NaOH,wasusedinyeastcellcultivationswhenrequired. medium in aerobic batch and fed batch cultivation. The
yeast strain was inoculated in to 50 ml (lab scale), 150 ml
Molasses (PDU) of minimal medium contained in a 150 ml
Molasses was obtained from SEKAB-E-Technology AB (lab scale) and 300 ml (PDU) shake flasks, respectively;
(Örnsköldsvik,Sweden)andwaseitherusedaloneormixed incubated at 30°C on an orbital shaker at 180 rpm for 24
with liquid fraction of corncobs slurry for cultivating yeast h. Aerobic batch cultivation was performed in 50 g l-1
cellsthatwasthenusedforSSCFexperiments. molassessupplementedwith23.5gl-1(NH ) SO ,3.0gl-1
42 4
KH PO , 2.25 g l-1 MgSO ·7H O, 33 μg l-1 biotin, 125
2 4 4 2
Minimalmedium ppm vitahop (Betatech Gmbh, Schwabach, Germany) (to
The initial inoculum for screening yeast strains and suppress bacterial growth) and 0.5 ml l-1 antifoam. The
SSCF experiments were cultivated in minimal medium yeastcultivationwascarriedoutin3.6l InforsHT-Labfors
containing 20 g l-1 glucose and xylose, respectively and bioreactor (lab scale), 30 l bioreactor (PDU) and 10 m3
enriched with salts, two folds of vitamins and trace bioreactor(demoscale).Thecultivationwasinitiatedinthe
elements according to Verduyn et al. [31]. The pH of bioreactorsbyadding50mlor150mlofminimalmedium
the medium was set to 6.0 with 1 M NaOH for all shake culture to a working volume of 500 ml (lab scale) or 1.5 l
flaskcultivations. (PDU) of molasses medium, respectively. The cultivation
was carried out until all sugars are consumed which was
Cultivationofyeast indicated by CO evolution in the gas-out and dissolved
2
In order to improve inhibitor tolerance by adaptation, oxygen concentration in the culture. Upon depletion of
yeast cells were grown briefly in presence of corncobs sugars in batch phase, a feed solution containing corncobs
hydrolysate duringthe cultivation for screeningand SSCF hydrolysateandmolasseswasfedlinearlyfor20htoafinal
experiments (as described below). It has been previously volumeof1.5l(labscale)or4.5l(PDU).Theconcentration
shown that the cultivation procedure of yeast signifi- of corncobs hydrolysate in the feed solution was same as
cantly influences the performance in SSF and small-scale thatofconcentrationofcorncobsslurryintheSSCFexperi-
fermentationsofhydrolysateliquor[32]. ments. Molasses concentration was 100 g l-1 in the feed
The precultures for screening S. cerevisiae strains for solution. The stirrer speed during the batch phase in
ethanol productionwere cultivatedin 150mlshake flasks lab scale was 700 rpm and increased linearly to 1000
with50mlofminimalmedium.Thecultureswereinocu- rpm during the fed batch phase; whereas, the stirrer
latedtoaninitialOD of0.005,incubatedat30°Conan speed was maintained at 700 rpm throughoutthe cultiva-
650
orbital shakerat 180 rpm.After 18h ofincubation, corn- tion in PDU scale; aeration rate was maintained at 1 vvm
cobshydrolysatesupplementedwith23.5gl-1(NH ) SO , and the pH was maintained at 5.0 by automatic addition
4 2 4
3.0 g l-1 KH PO and 2.25 g l-1 MgSO ·7H O was added of2MNaOH.
2 4 4 2
to the preculture cultivation flask to a final volume of After the cultivation, cells were harvested by centrifu-
35% (v/v) and incubated for another 24 h. gation for 8 min at 4°C, 1800 g and the cell pellet was
The yeast cells for SSCF experiments in lab and PDU resuspended in 0.9 % sterile NaCl solution to yield a cell
scales were cultivated in aerobic batch on molasses, suspension withadryweightof80gl-1.
Table3BrieflistofSSCFexperimentscarriedoutinlab,PDUanddemoscales
Modeofoperation_Scale InitialPre-fermentationtime,h Amountof Strain Totalcell Totalenzymeamount,FPUgWIS-1
solids,%WIS amount,gl-1
BatchSSCF_Lab None 7.5 RHD-15 5 5
BatchSSCF_Lab None 7.5 KE6-12 5 5
Fed-batchModelSSCF_Lab1 2 7.5 KE6-12 5 None
Fed-batchModelSSCF_PDU2 24 7.5 KE6-12 5 None*
Fed-batchSSCF_PDU 2 7.9 KE6-12 6 9
Fed-batchSSCF_PDU 2 10 KE6-12 5 15
Fed-batchSSCF_Demo 2 10.5 KE6-12 5 15
1ModelSSCFinlabscalewithafeedofglucosesolutionwithglucoseamountscorrespondingto7.5%WIS.
2ModelSSCFinPDUscalewithafeedoffilteredhydrolysatefromenzymatichydrolysisofwholeslurryat7.5%WIS.
*NoenzymeaddedduringthemodelSSCF.However,filteredhydrolysatefromenzymatichydrolysisofwholeslurryusinganenzymesolutionof6FPUgWIS-1
wasusedasafeedsolution.
Kopprametal.BiotechnologyforBiofuels2013,6:2 Page9of10
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Anaerobicfermentationinshakeflasks flasks and SSCF experiments were analyzed by high per-
The pH of corncobs hydrolysate was set to 6.0, supple- formance anion exchange chromatography using 4 × 250
mented with 0.5 g l-1 (NH ) HPO , 125 ppm vitahop mmDionexCarboPacPA1columnwith4×50mmguard
4 2 4
and filter sterilized using 0.45 μm cellulose acetate filter. column maintained at 30°C. Eluent A: 300 mM NaOH,
This fermentation medium was inoculated using the cell eluent B: 100 mM NaOH+85 mM sodium acetate were
suspension to reach a yeast concentration of 3 g dry cell used for elution at a flow rate of 1 ml min-1. Monosac-
weight l-1. The fermentations were carried out in 50 ml charides including arabinose, galactose, glucose, xylose
working volume in 100 ml shake flasks fitted with gly- and mannose were detected using pulsed amperometric
cerol loops providing anaerobic condition. The flasks detector.Optical density (OD)was used as an estimate of
were incubated at 30°C on an orbital shaker at 180 rpm cell concentration in shake flask experiments. OD was
for 96 h and samples were withdrawn for OD mea- measured at 650 nm using the cell free medium at the
650
surement and extracellular metabolite analysis. Possible pointofsamplingasbackground.
contamination during the shake flask fermentation was
checkedbyocular inspectioninmicroscope. Yieldcalculations
Ethanolyield(%ofmaximumtheoreticalyield)
SSCF Thesumofavailablefermentablesugarsincludingglucose,
The SSCF experiments were carried out in lab scale mannose, galactose, and xylose in liquid fraction and glu-
(3.6 l Infors HT-Labfors), PDU scale (30 l), and demo canandxylanfibersintheWISwascalculated.Duetothe
scale (10 m3) bioreactors with a total working weight addition of water during hydrolysis, the theoretical weight
of 1.5 kg, 20 kg and 4000 kg, respectively. In the lab of glucose and xylose released are 1.11 and 1.13 times the
and PDU scale experiments the corncobs slurry was weight of glucan and xylan, respectively. By using the
pH adjusted to 5.0 with 10 M NaOH and supplemented maximum theoretical ethanol yield of 0.51 g g-1 sugar, the
with0.5 g l-1(NH ) HPO . Inthedemo scale the pHwas maximum ethanol that can be produced from total avail-
42 4
adjusted using NH solution and supplemented with 0.25 able sugars was calculated. The percentage of the theoret-
3
g l-1 H PO . To avoid possible contamination and foam icalethanolyieldisdefinedasY =100*producedamount
3 4 SE
formation 125 ppm of Vitahop solution and 0.5 ml l-1 ofethanol(g)/maximumtheoreticalamountofethanol(g).
antifoam,respectivelywereaddedtothemedium.Inorder
to obtain the desired WIS content the supplemented Xyloseconsumed(%)
mediumwasdilutedwithwaterandusedforSSCFexperi- The percentage xylose consumed=100*amount of xylose
ments. Unless otherwise stated, all the experiments were consumed (g)/total amount of available xylose in liquid
initiatedbyadding5gdrycellweightl-1ofyeastfromcell andWISfraction(g).
suspension. An enzyme preparation, Cellic Ctec-2 from
Novozymes A/S, Denmark with filter paper activity of 95 Xylitolyield(%)
FPU g-1 enzyme, β-glucosidase activity of 590 IU g-1 The percentage xylitol yield=100*amount of xylitol
enzymewasaddedtoSSCFexperimentscorrespondingto produced (g)/amount of xylose consumed (g).
the desired cellulase activity. All SSCF experiments were
carried out at 35°C; pH was maintained at 5.0 by auto- Abbreviations
SSCF:Simultaneoussaccharificationandco-fermentation;SSF:Simultaneous
matic addition of 3 M NaOH and the stirrer speed was
saccharificationandfermentation;WIS:Waterinsolublesolids;PDU:Process
maintainedat400rpminlabandPDUscales,respectively. developmentunit;FPU:Filterpaperunit;OD:Opticaldensity;HPLC:High
A brief summary of all SSCF experiments carried out is performanceliquidchromatography;HPAEC:Highperformanceanion
exchangechromatography.
listed in theTable 3. All SSCF experiments performed in
duplicates in lab scale and one of them is represented in Competinginterests
theresultsanddiscussionsection. SW,ALwereemployedbySEKABE-Technologyduringthetimeofthiswork.
EAwasemployedbyTaurusEnergyABduringthetimeofthiswork.LWis
employedbyTaurusEnergyABandLOdoesconsultancyforTaurusEnergyAB.
Analysisofmetabolites
Samples for extracellular metabolites were analyzed by Authors’contribution
RK,EA,AL,SW,LW,GZandLOparticipatedintheconceptionanddesignof
high performance liquid chromatography using Aminex
thestudy.RK,ALandFNperformedtheexperimentalwork.RKwrotethe
HPX-87H column with 30 × 4.6 mm Cation-H Biorad manuscript.Alltheauthorscommentedonthemanuscript,readand
micro-guard column maintained at 45°C. 5 mM H SO approvedthefinalmanuscript.
2 4
was used as an eluent at a flow rate of 0.6 ml min-1.
Acknowledgements
Ethanol, xylitol, and acetic acid were detected using RI Thisworkwaspartoftheproject‘Industrialverificationofpentose
detector maintained at 35°C and HMF, furfural and lactic fermentingyeasts’financedbyEnergimyndigheten,SEKABE-Technologyand
TaurusEnergyAB,Sweden,grantnumber:30805–1.Wewouldliketothank
acid were detected using UV detector at 210 nm. The
membersofIndustrialBiotechnologygroupatChalmersUniversityof
sugars in corncobs hydrolysate and samples from shake Technologyforreadingandcommentingonthemanuscript.
Kopprametal.BiotechnologyforBiofuels2013,6:2 Page10of10
http://www.biotechnologyforbiofuels.com/content/6/1/2
Authordetails 19. AlfaniF,GallifuocoA,SaporosiA,SperaA,CantarellaM:ComparisonofSHF
1DepartmentofChemicalandBiologicalEngineering,Industrial andSSFprocessesforthebioconversionofsteam-explodedwheat
Biotechnology,ChalmersUniversityofTechnology,GöteborgSE-41296, straw.JIndMicrobiolBiotechnol2000,25:184–192.
Sweden.2DepartmentofChemicalEngineering,LundUniversity,P.O.Box 20. HoyerK,GalbeM,ZacchiG:Productionoffuelethanolfromsoftwoodby
124,LundSE-22100,Sweden.3TaurusEnergyAB,Ideon,OleRömersväg12, simultaneoussaccharificationandfermentationathighdrymatter
LundSE-22370,Sweden.4SEKABE-TechnologyAB,P.O.Box286, content.JChemTechnolBiotechnol2009,84:570–577.
ÖrnsköldsvikSE-89126,Sweden. 21. ZhangMJ,WangF,SuRX,QiW,HeZM:Ethanolproductionfromhighdry
mattercorncobusingfed-batchsimultaneoussaccharificationand
Received:26October2012Accepted:7January2013 fermentationaftercombinedpretreatment.BioresourTechnol2010,
Published:14January2013 101:4959–4964.
22. ÖhgrenK,BengtssonO,Gorwa-GrauslundMF,GalbeM,Hahn-HägerdalB,
ZacchiG:Simultaneoussaccharificationandco-fermentationofglucose
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