Coupling alkaline pre-extraction with alkaline-oxidative post

12 Pages·2014·1.38 MB·English

Liuetal.BiotechnologyforBiofuels2014,7:48
http://www.biotechnologyforbiofuels.com/content/7/1/48
RESEARCH Open Access
Coupling alkaline pre-extraction with
alkaline-oxidative post-treatment of corn
stover to enhance enzymatic hydrolysis
and fermentability
TongjunLiu1,2,DanielLWilliams1,3,SivakumarPattathil4,5,MuyangLi1,6,MichaelGHahn4,5,7andDavidBHodge1,3,6,8*
Abstract
Background: Atwo-stage chemical pretreatment ofcorn stoveris investigatedcomprising an NaOH pre-extraction
followed by analkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP
pretreatment can be improved using alkaline pre-extraction, which requires significantly less H O and NaOH.To
2 2
better understand the potential of this approach, this study investigates several componentsof this process
including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment,fermentation, and thecomposition
ofalkali extracts.
Results: Mild NaOH pre-extractionof corn stover uses less than 0.1g NaOH per gcorn stover at 80°C. The resulting
substrates were highly digestible bycellulolytic enzymes atrelatively low enzyme loadings and had a strong
susceptibility to drying-induced hydrolysis yieldlosses.Alkaline pre-extraction was highly selective for lignin removal
overxylanremoval;xylanremoval wasrelativelyminimal (~20%).Duringalkalinepre-extraction,upto0.10gofalkali
was consumed perg of corn stover. AHPpost-treatment atlow oxidant loading (25 mg H O perg pre-extracted
2 2
biomass) increased glucose hydrolysis yields by5%, which approachednear-theoretical yields. ELISA screening of
alkali pre-extraction liquors and theAHP post-treatment liquors demonstrated thatxyloglucan and β-glucans
likely remained tightly boundin thebiomass whereas themajorityof thesolublepolymeric xylans were glucurono
(arabino) xylans and potentiallyhomoxylans. Pectic polysaccharides were depleted inthe AHP post-treatment liquor
relative to the alkaline pre-extraction liquor.Because the already-low inhibitor content was further decreased inthe
alkaline pre-extraction,thehydrolysates generated by this two-stage pretreatment were highly fermentable by
Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation.
Conclusions: This work demonstrates that this two-stage pretreatment process is well suited for converting
lignocellulose to fermentable sugars and biofuels, such as ethanol. This approach achieved highenzymatic sugars
yields from pretreated corn stover using substantially lower oxidant loadings than have been reported previously
inthe literature. Thispretreatment approach allows for many possible process configurations involving novelalkali
recovery approaches and novel uses of alkaline pre-extraction liquors. Further work is required to identify the most
economical configuration, including process designs using techno-economic analysis and investigating processing
strategies that economize water use.
Keywords: Alkaline pretreatment,Oxidative delignification, Xylose fermentation
*Correspondence:[email protected]
1DOE-GreatLakesBioenergyResearchCenter,MichiganStateUniversity,East
Lansing,MI,USA
3DepartmentofChemicalEngineeringandMaterialsScience,MichiganState
University,48824EastLansing,MI,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Liuetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
unlessotherwisestated.
Liuetal.BiotechnologyforBiofuels2014,7:48 Page2of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
Introduction smelting black liquor recovery boiler, a fluidized bed
Relative to liquid transportation-fuels derived from boiler, or wet air oxidation [13,14], and alkali recovery
petroleum, biofuels derived from the pretreatment and through recausticization using a lime regeneration
enzymatic hydrolysis of lignocellulosic biomass offers cycle or autocausticization using Na-borate [15] or Fe O
2 3
many potential sustainability benefits. Many chemical [13] in the extraction liquor; (2) the use of gasification
pretreatments have been explored, covering a diverse ratherthancombustion,whichwouldenablethesynthesis
range of pH, solvents, and temperature. They have a ofFischer-Tropsch,mixedalcohol,ordimethyletherfuels
wide rangeofimpactsoncell-wallpolymers [1]. Alkaline from the sulfur-free syngas [16,17], or (3) recovery of
hydrogen peroxide (AHP) pretreatment significantly alkali-solubilizedligninand xylanbyultrafiltration[18,19]
improves the enzymatic digestibility of grasses (for or by acidification and filtration [20,21], which can pro-
example, corn stover and switchgrass) [2-4] because ducealow-sulfursolidfuelorafeedstockforfuels,chemi-
of several distinctive features of their cell walls [5]. cals,andpolymericmaterials.
Previous work has employed AHP as a single-step pre- Alkaline pre-extraction is similar to soda pulping of
treatment and required more than 100 mg H O per g graminaceous agricultural residues (for example, sugar-
2 2
biomass to improve digestibility over pretreatment with cane bagasse, wheat straw, and rice straw) in which
NaOH alone (unpublished data). The likely reason is that delignificationby NaOHaloneis followedbyanoxidative
asignificantportionoftheH O ispresumablyconsumed delignification or bleaching stage [22]. These commercial
2 2
by reacting with alkali-solubilized aromatics rather than pulping technologies are currently performed in China,
with the more recalcitrant lignin remaining in the cell India, South Africa, and Australia, among others [23,24];
walls. Additionally, catalytic amounts of transition metals however, there are different process objectives for a cellu-
in the biomass may contribute to the non-productive losic biofuels process. Grasses have both lower lignin
disproportionation of H O . For these process configu- contentanddifferencesintheircell-wallstructures;there-
2 2
rations, H O would be the primary cost input to the fore, alkali pulping is performed at considerably milder
2 2
process and must be decreased. At 100 mg H O per g conditions than for woody plants [25-27]. Atmospheric
2 2
biomass,thecostofH O is$1.50to$2.00pergalofetha- sodapulpingofsugarcanebagasseattemperatures<100°C
2 2
nol. New H O production technology improvements has been proposed to have positive economic advantages
2 2
may significantly decrease this cost; however to be eco- for small-scale operations because of the lower capital
nomical, new pretreatment process configurations must requirements[28].
beidentifiedthatusesignificantlylessH O . In the literature, there are many precedents for two-
2 2
At mild temperatures (< 100°C), treatment of grasses stage pretreatments employing an alkaline or oxidative
with NaOH induces significant solubilization of xylan post-treatment. As relevant examples, alkaline pretreat-
and lignin relative to dicots [6] and thus, has been pro- ment/pulping followedbyoxygendelignification hasbeen
posed as a standalone pretreatment for grasses including applied to softwoods [29] and hardwoods [30], lime
corn stover [7], wheat straw [8], sweet sorghum [9], and pretreatment followed by peracetic acid delignification
switchgrass[10].Inaddition,NaOHisproposedas adea- has been applied to kenaf (an herbaceous dicot) [31],
cetylation step prior to dilute acid pretreatment of corn AHP delignification has been applied as a post-treatment
stover [11]. The current work investigates an improved coupled to dilute-acid [32] and liquid hot water pretreat-
AHPprocessbyintroducinganNaOHpre-extractionstep ment [4] for grasses such as corn stover and switchgrass,
prior to subsequent AHP delignification. This approach and NaOH delignification has been coupled to the auto-
requires significantly less H O (and NaOH) by first hydrolysis of corn stover [33]. To our knowledge, mild
2 2
solubilizingandremovingeasilyextractedligninandxylan alkaline pretreatments of grasses coupled to oxidative
with alkali. Then, in a subsequent AHP step, an oxi- post-treatments-comparabletothecommercialpractices
dizing post-treatment removes the more recalcitrant ofalkalinepulpingandoxidativedelignificationorbleach-
lignin from the cell walls. Additional advantages of a ing of non-wood feedstocks - have not been explored
mild-temperature alkaline pre-extraction are that xylan as pretreatments. There is substantial need to improve
degradation to saccharinic acids through alkaline peeling knowledge of processing conditions that optimize hydro-
would notbe substantial. This alkali-solubilized xylan can lysis yields and minimize sugar degradation. With this in
be potentially recovered and used in other applications mind, the scope of the present workis toinvestigate con-
[6,12]. Further, soluble inhibitors of both enzymes and ditions for alkaline pre-extraction of corn stover coupled
microbes are removed using pre-extraction. This two- to an oxidative or alkali-only post-treatment. Specifically,
stage NaOH pretreatment approach provides many this work investigates: (1) the impact of NaOH loading
opportunities for integrating processes with the alkali and solids concentration on composition, biomass mass
pre-extraction liquor, including: (1) concentration in a yields, and alkali consumption during alkaline pre-extrac-
multiple-effect evaporator, combustion in a traditional tion; (2) improvement in glucose hydrolysis yield by
Liuetal.BiotechnologyforBiofuels2014,7:48 Page3of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
subsequent AHP or post-treatment with NaOH alone; (3) sugar hydrolysis yields). At relatively mild alkali con-
the comparison of alkali-solubilized glycans during alkali centrations,themaximumxylanremovalswereonly15to
pre-extraction and AHP post-treatment using an ELISA 24% (Figure 1C). Across all extraction conditions, the
screen for non-cellulosic cell-wall glycans, and (4) the average selectivity is 1.6 g lignin removed per g xylan
fermentability of the sugar hydrolysates generated by this removed. Earlier work for switchgrass demonstrated
two-stage pretreatment approach using Saccharomyces that under comparable extraction conditions with in-
cerevisiaestrainsmetabolicallyengineeredandevolvedfor creasing alkali far above the conditions used in the
xylosefermentation. present work, the xylanextractabilityreached a plateau at
70%removal[6].
Results and discussion Operating biomass conversion processes at high solids
NaOHpre-extraction concentrations minimizes process water-use and reduces
Treatment of graminaceous monocots such as corn costs for energy, capital equipment, and product recov-
stover with alkali at relatively modest concentrations and ery [34-36]. During pre-extraction, solids concentration
temperatures can solubilize up to 50% of the original is important because it impacts the pH for comparable
biomass, primarily extractives, hemicelluloses (xylans), alkali loadings. For example, an alkali loading of 0.10 g
andlignin[6].Thisabilitytosolubilizeplantcellwallscan NaOH per g biomass is an alkali concentration of only 5
beexploitedbypretreatmentsthatimprovetheenzymatic g/L at 5% (w/v) biomass solids concentration and 20 g/L
hydrolysis of cell-wall polysaccharides to fermentable at 20%(w/v)solids,resultinginsubstantial differences in
sugarsinbiofuelprocesses.Figure1presentstherelation- pH. In contrast, alkaline pulping using Kraft pulping for
ship between mass loss and compositional change in the woody biomass and soda pulping for grasses may use
biomass as a function of alkaline pre-extraction condi- alkali concentrations in the range of 150 to 180 g/L
tions. The obvious trend is that increasing alkali loading corresponding to alkali loadings of approximately 1.0 g
during the pre-extraction process increases solubilization NaOH per g biomass [14] and mass yields of app-
of hemicellulose (primarily xylan) and lignin. Glucan roximately 50%. As a function of solids concentration,
content exhibited a minor decrease (data not shown), Figure1Ashowsthatalkalinepre-extractionsat20%(w/v)
which likely results from removing glucan-containing solids concentration are slightly less effective than at 10%
hemicelluloses as well as sucrose and glucose in the (w/v)solids.Thisiscounter-intuitivebecausehighersolids
water-solubleextractives. concentrationsshouldyieldhigherpHvaluesforthesame
A relatively low alkali loading alkaline pre-extraction alkali loading and presumably result in more extraction.
allows for several advantageous potential process out- However, a problem with high-solids treatment is the
comes, including highly selective lignin removal versus difficulty ofpenetratingalkaliinto thebiomass becauseof
xylan. Further, it decreases alkali consumption and sub- limitations of laboratory mixing. Consistent with these
stantially decreases the required alkali recovery in the results is the concentration of solubilized cell-wall bio-
recausticization process, which decreases the capital polymers in the pre-extraction liquor (Figure 2). As the
requirements. Although lignin removal helps improve solids increase from 5 to 10% (w/v), extraction yields for
hydrolysis yields, xylan retention improves the overall hemicelluloses (primarily xylan) and lignin, increase and
sugar yields for the subsequent hydrolysis. In this sense, then decrease at 20% (w/v). Interestingly, the estimated
pre-extraction must balance lignin removal (to improve acetateyieldsfromthe5,10,and20%(w/v)solidsalkaline
theenzymatichydrolysis)withxylanretention(toimprove pre-extraction were 96, 96, and 67%, respectively. Again,
Figure1ImpactofNaOHandsolidsloading(w/v)duringalkalinepre-extractiononthesolubilizationofcell-wallpolymersandextractives.
Resultsareplottedfor(A)totalbiomasssolids,(B)Klasonlignin,and(C)hemicelluloses(Xyl+Gal+Man).Pre-extractionwasperformedat80°Cfor1h.
Liuetal.BiotechnologyforBiofuels2014,7:48 Page4of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
acylated to hemicelluloses and lignins such as acetyl,
p-coumaryl, and feruloyl esters) and potentially by
carboxylic acid degradation products of lignin and
polysaccharides (for example, formic acid, saccharinic
acids, hydroxy acids). Figure 3 plots alkali consumptions
for a range of pre-extraction conditions. At 80˚C and
higher solids concentrations (10%, w/v), alkali consump-
tion is nearly complete for the conditions tested in this
work,althoughslightlylessalkaliisconsumedat5%(w/v)
solids concentration. Alkali consumption profiles were
also generated for extractions at 30˚C over a wider range
ofNaOHloadings.UsingthiswiderrangeofNaOHload-
ings, alkali consumption approaches a maximum of 0.09
to 0.10 g NaOH per g biomass. Previously published re-
sults for mild-temperature NaOH pretreatment of wheat
Figure2Contentoflignin,acetate,andtotalpolymericand strawfoundcomparablealkaliconsumptions[8].
oligomericneutralpolysaccharidesinalkalinepre-extraction
liquorsasafunctionofsolidsloadingduringalkalinepre-
AHPandalkali-onlypost-treatment
extraction.Pre-extractionwasperformedat0.08gNaOHpergcorn
Alkali pre-extracted corn stover was generated for a
stover,at80°Cfor1handresultsshowamaximumextractionofcell
wallbiopolymersat10%(w/v)solids,whichislikelyaconsequence range of alkali loadings. First, corn stover was washed to
ofimperfectalkalipenetrationintothebiomassat20%(w/v)solids remove all of the solubles from the pre-extraction and
becauseofthelimitationsofthelaboratory-scalemixing. subjected to either an AHP post-treatment (25 mg H O
2 2
per g biomass loading, pH of 11.5, 30˚C, 24 h) or a sub-
thefailuretocompletelydeacetylatethebiomassindicates sequent alkali post-treatment (pH 11.5, NaOH only).
that alkali did not perfectly impregnate the biomass Following pH neutralization with concentrated H SO
2 4
because of poor mixing. Suitable mixing/impregnation with no additional solid–liquid separation or washing,
would likely eliminate this inefficiency. Previous pre- thesetreatedsamplesweresubjectedto24hof hydrolysis
extractions [8% (w/v) solids, 0.048 g/g NaOH loading, using a commercial cellulase cocktail. Figure 4 presents
70°C, 2 h] removed 30 to 45% of acetyl groups from the hydrolysis yields of both air-dried and never-dried
corn stover [11]. alkali pre-extracted corn stover. Several notable results
Alkali is consumed in both saponification reactions canbeobservedthatmeritcomment.
(for example, triglycerides and tannins and compounds The hydrolysis yields were high for short hydrolysis
times (24 h) and low enzyme loadings (5 to 15 mg
protein per g glucan). Although these enzyme loadings
are low compared to others reported for lignocellulose
hydrolysis, they are still an order of magnitude higher
than the amylase enzyme loadings required for starch
hydrolysisinthe corn ethanol industry [37].Only 24 his
employed for hydrolysis; therefore, longer hydrolysis
times would result in higher sugar yields, particularly for
low enzyme loadings. Another obvious result is the
substantial difference between the hydrolysis yields for
air-dried versus never-dried alkali pre-extracted corn
stover. Air-drying delignified corn stover decreases glu-
cose hydrolysis yields by 10 to 25%. This results from
drying-induced hornification or irreversible pore collapse
of cell walls at the nanometer scale and potentially the
collapse of the entire lumen at the whole-cell scale [38].
Pore collapse is a well-established outcome of drying
Figure3Alkaliconsumptionduringalkalinepre-extractionof delignifiedplantcellwalls;therefore,porepropertiesarea
cornstoverasafunctionofsolidsconcentration(w/v),NaOH strong function of biomass history (drying, pressing,
loading,andtemperature.Thealkaliconsumptionisshownto storage). This phenomena, which is well-known from
approachahorizontalasymptoteatbetween0.08and0.10g/g
literatureonpulpandpaper,decreaseswaterpenetrationin
NaOHloading.
cell-wall pores [38], which decreases cellulase penetration
Liuetal.BiotechnologyforBiofuels2014,7:48 Page5of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
Figure4Hydrolysisyieldsforalkali-extractedcornstoverasafunctionofalkalinepre-extractioncondition.Post-treatmentwas
performedand25mgHO /gbiomass,(A)15mgprotein/gglucanenzymeloading,(B)10mgprotein/gglucanenzymeloading,and(C)5mg
2 2
protein/gglucanenzymeloading.Extractionwasperformedat10%(w/v)solidsand80°Cfor1hwith100%displacementofpre-extractionliquor
withdistilledwaterfollowingpre-extraction.
andhydrolysis[39].Becausedelignifiedcellwallsarehighly treatment. Compared to the 24 h, 30˚C treatments, the
susceptible to drying-induced pore collapse, no drying yields were comparable or slightly higher (Figure 5), indi-
was performed between alkali pre-extraction, AHP post- catingthattheseconditionswouldbepreferable.
treatment,andhydrolysis.
Next, an additional post-treatment with either alkali ELISAscreeningofalkalipre-extractionandAHP
only or AHP at relatively modest H O loading (25 mg post-treatmentliquorsforsolubilizedcell-wallglycans
2 2
H O pergbiomass)wasperformedtoassesstheimpact In the analyses of major non-cellulosic plant glycans in
2 2
on enzymatic hydrolysis yields (Figure 4). The highest several bioenergy crops including corn stover, currently
achievable glucose hydrolysis yields were obtained for available collections of cell wall glycan-directed mono-
alkali pre-extracted corn stover (0.08 and 0.10 g NaOH/g clonal antibodies (mAbs) have been instrumental. Earlier
biomass) subjected to AHP post-treatment. Figure 4 studies employing ELISA screens of these mAbs against
shows that at an enzyme loading of 15 mg protein per g diverse structurally characterized plant cell-wall glycans
glucan, 24-h hydrolysis yields reached 95 to 96%. Mono- have categorized these mAbs into multiple groups based
meric xylose yields for hydrolysis only (for example, not on their specificity to distinct cell-wall glycans [40,41].
including xylan lost during prior treatments) were 50 to Taking advantage of this, ELISA screens with cell wall
60% for both AHP and alkali post-treated samples (data glycan-directed mAbs were performed to determine
not shown), although these should increase if hydrolysis the range and relative abundance of the non-cellulosic
times are extended. Although the glucose yields were
statistically higher than alkali-only post-treated materials
(P>0.999),identifyingthatthealkalipost-treatmentalone
canyieldamaterialthatishighlysusceptibletohydrolysis
isanimportantfinding.Thesealkali-onlypost-treatedma-
terials exhibited 24-h glucose hydrolysis yields that were,
on average, only 5% lower than the AHP post-treated
yields. Considering the cost of H O (approximately $700
2 2
pertonne),the5%increasedhydrolysisyieldscorresponds
to an increase in the estimated overall ethanol yield
(assuming 0.45 g/g and 0.30 g/g yields of ethanol from
glucose and xylose, respectively) from approximately 58
gal/tonne to 62 gal/tonne. This additional cost for H O
2 2
corresponds to $2.50 for each marginal gallon of EtOH
generated, or alternatively $0.17 per gallon of ethanol
overall.
Shorter time, higher temperature AHP post-treatments Figure5Impactofshortertime,highertemperaturepost-
were also tested (60˚C for 3 h) for some conditions that treatmentonglucosehydrolysisyieldsforarangeofalkali
pre-extractionconditions.
wouldbemorerealisticforaprocessemployingthispost-
Liuetal.BiotechnologyforBiofuels2014,7:48 Page6of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
polysaccharides that were solubilized during alkaline xyloglucans) is reasonably constant between the alkaline
pre-extraction, and how their distribution and relative pre-extraction liquor and the AHP post-treatment liquor,
abundance are altered in the AHP post-treatment liquor. indicating that both treatments solubilize similar pools of
Xylan epitopes represent the most abundant recognizable hemicellulosicglycans.
hemicellulose epitopes in both the pre-extraction liquor According to Figure 2, 7.6% of the original glucan in
(Figure6A)andtheAHPpost-treatmentliquor(Figure6B). the biomass is solubilized in the pre-extraction liquor
This is indicated by the significant binding of xylan-3, and can be hypothesized to be hemicelluloses (for ex-
xylan-4, and xylan-5 groups of mAbs (Additional file 1) ample, extracted β-glucans, xyloglucans, and glucoman-
that had been previously shown to be specific to either nans) and/or water-soluble sucrose, monomeric glucose,
unsubstituted (homoxylans) or highly substituted xylans and potentially even phenolic glycosides [42]. β-glucans
(glucurono(arabino)xylans) [40]. Other hemicellulosic epi- are known to be important components of the primary
topes, such as those for non-fucosylated xyloglucan and cell walls of graminaceous monocots and are hypo-
fucosylated xyloglucan, were present only in trace abun- thesized to play the role of xyloglucans in dicots [43].
dance, indicating that xyloglucans are not solubilized Interestingly, this work shows that β-glucans, which are
duringtheseprocessesandthatthemajorityofthesolubi- known to be present in cell walls of corn stover, are not
lized xyloseinFigure2arisesfromglucuronoxylansrather present in either the alkaline pre-extraction or the AHP
than from xyloglucans. Among these trace amounts of post-treatment liquors. It is important to note that ELISA
xyloglucans, the presence of non-fucosylated xyloglucan screens using mAbs conducted here allow the detection of
epitopeswereinrelativelyhigherproportionscomparedto relatively larger cell-wall glycans that effectively adsorb to
fucosylated xyloglucan epitopes. The relative distribution ELISA plates. Information on small glycan molecules (for
of solubilized hemicelluloses (xylans and trace amounts of example, oligomeric glycans, sucrose, and monosaccharides)
Figure6ELISAscreeningofglycanssolubilizedinpre-andpost-treatmentliquorsusingapanelofcellwallglycan-directedmonoclonal
antibodies(mAbs).TheseresultsplotthemAbbindingaffinityforliquorsof(A)alkalipre-extractedcornstoverat0.10g/gand(B)alkalinehydrogen
peroxide(AHP)post-treatmentofthispre-extractedcornstoverwiththemAbcategoriesdefinedinAdditionalfile1.
Liuetal.BiotechnologyforBiofuels2014,7:48 Page7of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
islostintheELISAscreenanalyses,becausethesesmallmol- onlypost-treatmentatpH11.5.Fromthesedata,theAHP
ecules do not adhere to the plates and thus cannot be de- post-treatmentclearlyimprovesthesubsequentenzymatic
tectedbymAbs[40]. hydrolysis for glucose, whereas the improvement realized
Other major glycan epitopes present in both alkali- for xylose hydrolysis yields are minimal. Additionally, the
solubilized and AHP post-treatment liquors were pectic results show only slightly lower hydrolysis yields than the
backbones (asindicated bythe bindingofHG backbone- thoroughly washed material at comparable treatment
1andRG-Ibackbone-1groupsofmAbs),pecticarabino- conditions (Figure 4A). This indicates that it is likely that
galactan (as indicated by the binding of RG-I/AG groups residualsolubles(forexample,xylanandsolublearomatics
of mAbs), and arabinogalactan (as indicated by the bind- from the alkaline pre-extraction) slightly inhibit the hy-
ing of AG-1 through AG-4 groups of mAbs) epitopes. drolysis, which has been clearly demonstrated in the past
These epitopes for pectic arabinogalactan polysaccha- [45,46]. This approach resulted in minimal xylan degrad-
ridesrelativetothetotalglycan abundanceintheextract ation, with only 5% of the total intial xylan unaccounted
is notably decreased in the AHP post-treatment liquor, for in a material balance across solid and liquid phases
because it is likely that a higher fraction of these more (data not shown). For the condition of 25mg H O per g
2 2
extractableglycansareremovedinthepre-extraction. biomass in Figure 7, alkaline pre-extraction removed 58%
of the lignin and the subsequent AHP post-treatment re-
Generationofhigh-sugarcornstoverhydrolysates sultedinatotalof73%ligninremoval(datanotshown).
Next, hydrolysates were generated for fermentation Using this approach with incomplete washing, hydroly-
using slightly different conditions than were used in the sates ofalkali-pre-extractedcornstoversubjectedtoAHP
preliminary screening of conditions for pre-extraction post-treatment weregenerated.Specifically,thehydrolysis
and post-treatment (Figure 4). Specifically, pre-extracted performed at two different solids concentrations during
corn stover (0.08 g NaOH/g biomass) was not washed, the pre-extraction, post-treatment, and hydrolysis gener-
but only subjected to dewatering before being subjected atesugarsatdifferentconcentrations.Table1summarizes
to AHP post-treatment. For these hydrolysates only 70% the conditions used togeneratethese hydrolysatesas well
of the pre-extraction liquor was removed and replaced as hydrolysate sugar and quantified compounds that are
with water up to the same solids content, resulting in a known to inhibit fermentation rates. To minimize capital
displacement ratio of 0.70. For this process, multistage costsandseparationcostsforethanolrecovery,highetha-
counter current washing schemes could be envisioned nol titers (requiring high sugar titers) and high ethanol
thatmakesefficientuseofwaterandresultinsubstantially productivities are necessary. As an example, corn ethanol
more alkali recoveryfrom the pre-extracted biomass [44]. fermentations often achieve ethanol concentrations in ex-
The 24-h hydrolysis yields for these incompletely washed cess of 18% (v/v) [37]. As such, it would be advantageous
materials are presented as a function of H O loading togeneratehighsugartitersinlignocellulosehydrolysates
2 2
(Figure 7) with 0 mg/g H O loading representing alkali- produced from high-solids enzymatic hydrolysis. A draw-
2 2
backto the fermentationof highsugar titer lignocellulose
hydrolysates is that inhibitors deriving from the de-
gradation and modification of cell-wall polymers during
pretreatment are typically present that are toxic to
fermentation.
Table1Conditionsusedtogeneratecornstover
hydrolysatesandthesugarandinhibitorconcentration
ofthesehydrolysates
Hydrolysate1 Hydrolysate2
SolidstoNaOHpre-extraction(w/v) 10.0% 20.0%
SolidstoAHPpost-treatment(w/v) 10.0% 23.5%
Enzymecocktail(proteinmassratio) CTec2+HTec2 CTec2+HTec2
(0.77:0.23) (0.77:0.23)
Enzymeloading(mg/gglucan) 15 15
Figure7EffectofH2O2loadingduringalkalinehydrogen Glc(g/L) 51.4 74.6
peroxide(AHP)post-treatmentonthehydrolysisyieldsof
Xyl(g/L) 20.9 37.9
alkalinepre-extracted,never-driedcornstover.Alkaline
pre-extractionwasperformedat10%(w/v)solidswitha70% Formate(g/L) 0 0
displacementofthepre-extractionliquorpriortoAHPpost-treatment
Acetate(g/L) 0.28 0.37
at23.5%(w/v)solids.Hydrolysiswasperformedat10%(w/v)solids.
AHP,alkalinehydrogenperoxide.
Liuetal.BiotechnologyforBiofuels2014,7:48 Page8of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
Our previouswork hasdemonstrated thathydrolysates Thetwo strainsinclude strain Y73,which wasengineered
generated from one-stage AHP pretreatment of corn to assimilate xylose using xylose reductase (XR)+xylitol
stover and switchgrass are already highly fermentable dehydrogenase (XDH) and strain Y128 which expresses a
without detoxification with xylose-fermenting Saccharo- bacterial xylose isomerase (XI) to facilitate xylose conver-
myces strains used in our laboratory [47]. For a process siontoxyluloseandsubsequentlytoethanol.Figure8pre-
using an alkaline pre-extraction, the alkali-solubilized sents the fermentation kinetics for these two hydrolysates
xylan, lignin, extractives, and alkali-saponifiable com- bythesetwostrains.Forthelow-sugar-concentrationcorn
pounds including acetate, p-coumarate, and ferulate as stover hydrolysate (Hydrolysate 1) complete conversion
well as inorganics (Na+) are removed prior to hydrolysis of both glucose (51 g/L) and xylose (21 g/L) to etha-
in the pre-extraction liquor. In particular, Na+ in these nol was realized within 100 h. For both strains, the
hydrolysates is expected to be at several-fold lower con- glucosewasrapidlyfermentedwithin18h,whereasxylose
centrations (approximately 100 to 200 mM) than those was fermented more rapidly in strain Y128 (Figure 8B).
presented in our previous work [47]. As a consequence, The high-sugar hydrolysate fermentations (Hydrolysate 2)
it is expected that hydrolysates generated using this resultedinincompletexyloseconsumptionafter120hfor
approach will be substantially less inhibitory to fermen- bothstrains (Figures 8Cand D) withethanol titersreach-
tation. This is comparable to the alkaline deacetylation ing more than 45 g/L for strain Y73. The average ethanol
pre-extraction performed at the National Renewable yields (YEtOH/Xyl) for each strain can be estimated by
Energy Laboratory (NREL) prior to dilute-acid pretreat- generating a regression for a plot of xylose consumption
ment, which generated hydrolysates substantially less versusethanolgeneration(Figure9)andthesewerefound
inhibitory to fermentation by metabolically engineered tobe0.31g/g forstrainY128and0.25g/g for strainY73.
Zymomonasmobilis[11]. Strain Y128, which performed better for the low-sugar
hydrolysate, showed slower xylose consumption for the
Hydrolysatefermentationbyxylose-fermentingyeaststrains high-sugarhydrolysate.
To demonstrate the fermentability of these hydrolysates, The fermentation results for strain Y73 are compar-
two hydrolysates were next subjected to fermentation by able to the performance on one-stage AHP-pretreated
evolved, metabolically engineered S. cerevisiae strains. corn stover hydrolysates of similar sugar concentrations,
Figure8Hydrolysatefermentationkinetics.Theserepresentfermentationofhydrolysatesofalkalipre-extracted,alkalinehydrogenperoxide
(AHP)post-treatedcornstoverbySaccharomycescerevisiaestrainsmetabolicallyengineeredforxylosefermentationusingeitherthexylosereductase
(XR)+xyitoldehydrogenase(XDH)pathway(strainY73)orthexyloseisomerase(XI)pathway(strainY128).Conditionsinclude(A)Y73inlow-sugar
hydrolysate,(B)Y128inlow-sugarhydrolysate,(C)Y73inhigh-sugarhydrolysate,(D)Y128inhigh-sugarhydrolysate.
Liuetal.BiotechnologyforBiofuels2014,7:48 Page9of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
the high 20% (w/v) solids concentration resulted in less
efficient treatment than at 10% (w/v) solids, presumably
because of limitations associated with laboratory-scale
mixing.TheapplicationoflowloadingsofH O (25mg/g)
2 2
asadelignifyingagentfollowingthealkalinepre-extraction
improved hydrolysis yields by 5% on average relative to
post-treatment with alkali alone. In future work, the
oxidant cost relative to the improved yield needs to be
evaluated by applying techno-economic modeling,
process design, and process optimization. Because water
use is an important environmental component of cellu-
losic biofuels processes, additional ongoing work is fo-
cused on identifying process options that can economize
water through minimization and recovery. This includes
the use of process liquors as backset while increasing the
solids content during processing and/or increasing the
washingefficiencytodecreasetheenergyloadtotheevap-
Figure9DeterminationofethanolyieldsinstrainsY73and
Y128.Plottingxyloseconsumptionversusethanolproductionfor oratorspriortochemicalrecovery.
bothcornstoverhydrolysates(Table1andFigure7)forstrainsY73
andY128resultsintheregressionoftheslopethatrepresentsthe Methods
averageethanolyieldonxylose,Y ,(g/g).Theaverageethanol
EtOH/Xyl Biomass
yieldonxyloseinthestrainutilizingthexyloseisomerasepathway
Corn stover (Zea mays L. Pioneer hybrid 36H56) was the
(Y128)wasslightlyhigherthanthatofthestrainutilizingthe
reductase/xylitoldehydrogenasepathway(Y73). same batch of material reported previously [5] and was
milled (Circ-U-Flow model 18-7-300, Schutte-Buffalo
Hammermill, LLC, Buffalo, NY, USA) to pass a 5-mm
which in our previously work [47] required nearly 240 h screen.Moisturecontent,structuralcarbohydratesinclud-
to completely consume all the sugars and generate more ing glucan, xylan+mannan+galactan, acid-soluble lignin
than 40 g/L ethanol. Rather than inhibition by com- and acid-insoluble lignin of corn stover before and after
pounds in the hydrolysate, this slower xylose consump- pretreatment as well as alkali-solubilized polysaccharides
tion is likely a consequence of combined stresses from and lignin weredetermined accordingto NREL standard-
the long fermentation time, which includes ethanol ized analytical procedures (NREL/TP-510-42618; NREL/
inhibition and nutrient depletion, because only min- TP-510-42619; NREL/TP-510-42623) with modifications
imal media components were added to the hydrolys- asreportedpreviously[5,6].
ate. Overall, these results provide positive validation
that these high-sugar hydrolysates are highly fermentable NaOHpre-extractionandAHPpost-treatment
to ethanol because of the low concentration of inhibitors Alkali pre-extractedcornstoverwaspreparedbysoaking
present. cornstoverinNaOHsolution at80°Cfor1h.The solids
loading of corn stover during alkaline pre-extraction was
Conclusions 5, 8, 10, or 20% (w/v) with NaOH loadings based on the
This work supports a number of notable conclusions that mass of corn stover. For some cases, after pre-extraction
have important implications for cellulosic biofuels the remaining insoluble solids were filtered and washed
processes that use NaOH pretreatment. Alkaline pre- with excess deionized water to remove all solubles. A
extraction coupled to alkaline or alkaline-oxidative sample of the wet biomass cake was taken to determine
post-treatment of corn stover can yield a biomass that moisture content after pre-extraction. For materials
is highly digestible at relatively low enzyme loadings using incomplete washing, after extraction the liquid
and was highly fermentable because soluble inhibitors was manually squeezed out of the biomass using a filtra-
to both enzymes (aromatics and xylan oligomers) and tion cloth (Miricloth, EMD Millipore, Billerica, MA,
microbes (p-hydroxycinnamic acids, acetate, and most USA). Subsequently, a known quantity of the removed
of the Na+) are removed during the pre-extraction. liquor was added back to the filter cake to yield a 70%
Minimalβ-glucanandxyloglucanwereidentifiedinanyof removal of the liquor. For AHP or alkali-only post-
theliquorsindicatingthatthesecell-wallglycansaremore treatment, the experiments were conducted at 30°C for
tightly bound into the cell walls and are not likely to be 24 h in 250-mL shake flasks, in which H O was mixed
2 2
strong contributors to the alkaline pre-extraction or AHP with 6 g alkali-extracted corn stover. The H O loading
2 2
post-treatment xylan or glucan content. Pre-extraction at was 25 mg H O per g biomass for AHP post-treatment,
2 2
Liuetal.BiotechnologyforBiofuels2014,7:48 Page10of12
http://www.biotechnologyforbiofuels.com/content/7/1/48
whereas no H O was included in post-treatment with sterilized (Millipore Stericup, Billerica, MA, USA) prior
2 2
alkali alone. The pH was periodically adjusted to 11.5 to inoculation. Fermentations were performed in 250-
using 5 M NaOH. Short-time, high-temperature AHP mL shake flasks capped with fermentation locks (Bac-
post-treatment was performed at 60°C for 3 h. Biomass chus & Barleycorn Ltd. Shawnee, KS, USA) with a work-
massyieldsafteralkalinepre-extractionweredetermined ing volume of 50 mL at 30°C and agitation in a rotary
gravimetrically by performing the pre-extraction on 1 g incubator at 180 rpm. Yeast seed cultures were prepared
corn stover, washing out solubles using several cycles of in yeast extract peptone dextrose media as described pre-
centrifugation, decanting, and resuspension in distilled viously [47]. A known volume of the seed culture at a
water. The washed pre-extracted biomass pellet was known optical density (OD) was centrifuged and resu-
600
oven-dried at 105°C to determine the mass yield. supended in hydrolysate to yield an initial OD of 1.0.
600
Consumption of NaOH was quantified by titrating di- Following inoculation and sampling, flasks were purged
luted pre-extraction liquors to their equivalence point withnitrogentomaintainanaerobicconditions.
using 0.1 M HCl (DMS Titrino716, Metrohm, Herisau,
Switzerland). Analysisofhydrolysatesandfermentations
The concentrations of glucose, xylose, and ethanol in
Enzymatichydrolysis hydrolysate and fermentation samples were determined
Hydrolysis was conducted at 50°C in a temperature- by high-performance liquid chromatography (HPLC)
controlled incubator with orbital shaking at 160 rpm. (Agilent 1100 Series, Agilent Technologies, Santa Clara,
Hydrolysiswasperformedat10%(w/v)solidswiththeex- CA, USA) using an Aminex HPX-87H column (Bio-Rad
ception of hydrolysates used for fermentation (Hydrolys- Laboratories, Inc., Hercules, CA, USA) operating at 65°C,
ate 1 and Hydrolysate 2,Table 1) which were hydrolyzed a mobile phase of 0.005 M H SO , a flow rate of 0.6 mL/
2 4
at the same solids as AHP post-treatment with dilution minute, and detection by refractive index. Cell densities
only for pH adjustment. The enzyme cocktails Cellic (OD )weredeterminedspectrophotometrically(Biomate
600
CTec2 and HTec2 were provided by Novozymes A/S 3, Thermo-Fisher Scientific, Waltham, MA, USA) follow-
(Bagsværd, Denmark) with the protein content deter- ing a 10-fold dilution in water. All plotted data points
mined by the Bradford assay (Sigma-Aldrich, St. Louis, represent averages of sample duplicates at a minimum,
MO, USA) using bovine serum albumin as standard. Na- whereaserrorbarsrepresentthedatarange.
citrate buffer (0.05 M, pH 5.2) was used for alkali pre-
extracted washed material, whereas incompletely washed
ELISAscreeningwithapanelofcellwall-directed
whole slurries of alkali pre-extracted or AHP post-treated
monoclonalantibodies
corn stover were neutralized to pH 5.2 [48] using 72%
To conduct the ELISA screens with a comprehensive
(w/w) H SO (approximately 3.3 mg H SO /g biomass).
2 4 2 2 collection of cell wall glycan-directed mAbs, each liquor
Cellic CTec2 and HTec2 were added at 15 mg total pro-
sample was loaded onto the ELISA plates (Corning 384-
tein content per g glucan in biomass. The protein mass
well clear flat-bottom polystyrene high-bind microplate,
ratio of CTec2:HTec2 was 0.77:0.23 based on the protein
product #3700) on an equal carbohydrate basis (15 μL
content according to the Bradford assay (Sigma-Aldrich,
per well from a solution of 20 μg/mL carbohydrate) for
St.Louis,MO,USA).Hydrolysisyieldsforsaccharification
conductingELISAscreensasdescribedpreviously[40,41].
were determined according to our previously outlined
Plant glycan-directed mAbs were from laboratory stocks
methodology[36]basedontheexperimentallydetermined
(CCRC, JIM and MAC series) at the Complex Carbohy-
compositionafterpre-extraction.
drate Research Center (available through CarboSource
Services; http://www.carbosource.net) or were obtained
Hydrolysatefermentation
from BioSupplies (Bundoora, Australia) (BG1, LAMP). A
S. cerevisiae strains engineered for xylose fermentation
description of the mAbs used in this study can be found
to ethanol using either XR+XDH (strain Y73) as repor-
inAdditionalfile1whichincludeslinkstoawebdatabase,
ted earlier [47] or the XI (strain Y128) were supplied by
WallMabDB (http://www.wallmabdb.net) that provides
Trey Sato (Great Lakes Bioenergy Research Center,
detailedinformationabouteachantibody.
University of Wisconsin, Madison, WI, USA). For fer-
mentation of the hydrolysates, the whole hydrolysis
Additional file
slurry was centrifuged (16,000×g) and the supernatant
was decanted. Yeast nitrogen base (YNB) without amino
Additionalfile1:Listingofplantcellwallglycan-directedmonoclonal
acids andammonium sulfateand urea wereadded tothe
antibodies(mAbs)usedforELISAscreening(Figure6).Thegroupings
supernatant to final concentrations of 1.67 g/L and 2.27 ofantibodiesarebasedonahierarchicalclusteringofELISAdata
g/L, respectively. The pH was next adjusted to pH 5.5 generatedfromascreenofallmonoclonalantibodies(mAbs)againsta
panelofplantpolysaccharidepreparationsthatgroupsthemAbs
using NaOH pellets and the hydrolysate was filter