Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses.

1.7 MB·English

by  Wang, ZJ

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Lignosulfonate and elevated pH can enhance
enzymatic saccharification of lignocelluloses
Wang et al.
Wangetal.BiotechnologyforBiofuels2013,6:9
http://www.biotechnologyforbiofuels.com/content/6/1/9
Wangetal.BiotechnologyforBiofuels2013,6:9
http://www.biotechnologyforbiofuels.com/content/6/1/9
RESEARCH Open Access
Lignosulfonate and elevated pH can enhance
enzymatic saccharification of lignocelluloses
ZJ Wang1,2, TQ Lan2,3 and JY Zhu2*
Abstract
Background: Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key
barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass.Sulfite
Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL)is a relatively new process, but demonstrated
robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields
and energy efficiencies.This study demonstrated therole of lignin sulfonation inenhancing enzymatic
saccharification of lignocelluloses –lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses,
contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding ofcellulase.
Results: The study found thatlignosulfonate from SPORL pretreatment and from a commercial source inhibits
enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding ofcellulase.
Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered
as the concentrations of thesetwo lignosulfonates increased.We hypothesize that lignosulfonate serves as a
surfactant to enhanceenzymatichydrolysis athigher concentrations and that this enhancement offsets itsinhibitive
effect from nonspecific binding of cellulase, when lignosulfonateis applied to lignocellulosic solid substrates.
Lignosulfonate can blocknonspecific bindingof cellulase by bound lignin on thesolid substrates, in thesame
manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly
proportional to the amount of lignosulfonate applied which is very important to practical applications. For a
SPORL-pretreatedlodgepole pinesolid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g
glucan with theapplication of itscorresponding pretreatment hydrolysate coupled with increasing hydrolysis pHto
above 5.5comparedwith only51% for thecontrol run without lignosulfonate atpH 5.0. The pH-inducedlignin
surface modification atpH 5.5furtherreduced nonspecific binding of cellulase by lignosulfonate.
Conclusions: The results reported inthis study suggest significant advantages for SPORL-pretreatmentin terms of
reducing water usage and enzyme dosage, and simplifying process integration, i.e., it should eliminate washing of
SPORL solid fraction for direct simultaneous enzymatic saccharification and combinedfermentationof enzymatic
and pretreatment hydrolysates (SSCombF). Elevated pH 5.5or higher, rather thanthe commonly believedoptimal
and widely practiced pH 4.8-5.0, should be used inconductingenzymatic saccharification of lignocelluloses.
Keywords: Enzymatic hydrolysis/saccharification, Lignin/lignosulfonate, Nonspecific/nonproductivebinding/
adsorption, Cellulase enzymes, Pretreatment
*Correspondence:[email protected]
2USForestService,ForestProductsLaboratory,Madison,WI,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Wangetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background reduce nonspecific binding of cellulase to the bound
The interactions between lignin and cellulase play a lignin fraction in lignocelluloses in the same manner as
major role in enzymatic hydrolysis of lignocelluloses for other surfactants to result in a gain in enzymatic hy-
sugar and biofuel production from biomass [1]. These drolysis efficiency. Furthermore, it is possible that the
interactions can be described as (1) lignin physical application of certain lignosulfonate, e.g., spent liquors
blockage to limit cellulose accessibility to cellulase [2], from SPORL pretreatments, with low nonspecific bind-
and (2) nonspecific adsorption or binding of cellulase ings of cellulase – hypothesized in the previous para-
enzymes to lignin [3-7]. Reported studies indicate that graph – may result in a net enhancement of enzymatic
these two mechanisms produced negative effects on en- saccharificationoflignocelluloses. The validationorcon-
zymatic saccharification of lignocelluloses. Pretreatment firmation of this argument has significant scientific and
of lignocelluloses such as Organosolv [8,9] and SPORL practical implications for sulfite pretreatment technolo-
[10] is able to partially remove lignin physical blockage gies, such as SPORL [10] that has been demonstrated
bysolubilizingafractionoflignininto thehemicellulosic robust performance for sugar and biofuel production
sugar stream (pretreatment spent liquor). However, fur- with very high yields from woody biomass including very
therprocessinglignocellulosestoremove ligninblockage recalcitrantsoftwoodspecies[24-26].Specifically,thesep-
such as by delignification is not only expensive but also aration of the SPORL pretreatment hydrolysate (spent li-
maynotbenecessary intermsofimprovingcellulose ac- quor) from the SPORL pretreated solid ligno cellulosic
cessibility. It is probably more effective to address the fraction and washing of the SPORL solid fraction would
issue of nonspecific binding of cellulase to lignin to fur- notbe required in order to enhance enzymatic saccharifi-
ther enhance enzymatic saccharification. One passive cation. This can significantly simplify biorefinery process
approach was to wash the solid fraction of pretreated integrationandsaveasignificantlyamountofwater.
lignocelluloses to eliminate nonspecific binding of cellu- Previously, we demonstrated that the application of
lase to free lignin (referring to lignin separated from divalent metal compound, such as Ca(II) applied for
solid lignocellulosic substrate), as commonly described neutralizing pretreatment hydrolysate, can eliminate
in the literature [11]. Another passive approach is to use nonspecific cellulase binding to lignosulfonate in the un-
surfactant, protein and metal compound to block bound washed SPORL pretreated aspen (hardwood) solids
lignin (referring to lignin retained in solid lignocellulosic [18,19]. The aspen lignosuflonate in the SPORL spent li-
substrate) and free lignin, reducing their nonspecific quor had a low degree of lignin sulfonation because of a
binding to cellulase [3,12-19]. However, washing is a sig- low sulfite loading of 3% on oven dry (od) wood used in
nificant environmental concern because the amount of pretreatment. This study is a step further in demonstrat-
water required is on the order of 10 m3 water/ton ligno- ing the role of lignin sulfonation in enhancing enzymatic
cellulose, based on pulp mill experience [19]. The appli- saccharification. SPORL spent liquor that contain ligno-
cations of surfactant and protein are both expensive at sulfonatewithhighdegreeofsulfonation,producedfrom
therequiredlevels. lodgepole pine (softwood) by SPORL at 8% sulfite load-
Hydrophobic interaction hasbeenidentifiedasthepri- ing on od wood, was directly mixed with SPORL pre-
mary driving force for protein adsorption [20]. Increas- treated lodgepole pine and dilute acid pretreated aspen.
ingthehydrophobicity ofa substrate resultsinenhanced The objectives of the present study are: (1) to verify the
adsorption of protein or cellulase [1,21]. This suggests two hypotheses proposed above by directly comparing
that nonspecific binding (adsorption) of cellulase (made enzymatic cellulose saccharification efficiencies of sev-
of protein) to lignin will be different for lignins of differ- eral SPORL-pretreated lignocellulosic substrates with
ent hydrophobicities. Sulfonated lignin, has good hydro- and without the applications of a SPORL-pretreatment
philic properties. We can hypothesize that sulfonated hydrolysate from lodgepole pine at different loadings;
lignin such as lignosulfonate in the sulfite pretreatment (2) to verify our previous finding [27,28]: significant im-
hydrolysates (spent liquor) [9,10] may produce less non- provement in cellulose saccharification when enzymatic
specific binding (adsorption) to cellulase enzymes. This hydrolysis of lignocelluloses is conducted at an elevated
hypothesis is indirectly corroborated by the excellent en- pH 5.5 – 6.2 as oppose to pH 4.8 – 5.0 as exclusively
zymatic digestibility of lignocellulosic substrates after used in the literature. The elevated pH study was con-
sulfite pretreatment such as SPORL[10], sulfite pulping ducted with the application of SPORL pretreatment hy-
[22],andligninsulfonation[23]. drolysate for further enhancement of saccharification
Lignosulfonate functions as a surfactant due to its efficiency.Thisworklaysthefoundationfordirectsimul-
stronghydrophilicity.Thispropertyhasbeenusedtode- taneous enzymatic saccharification and combined fer-
velop commercial surface modification products such as mentation (SSFCombF) of the whole lignocellulosic
dispersants and plasticizers. Therefore, we can further slurry at high solids loadings without a separation and
hypothesize that the application of lignosulfonate can washingstepafterpretreatment[28].
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Results and discussions recovery achieved at lignosulfonate concentration ap-
EnzymatichydrolysisofWhatmanpaperwiththe proximately 0.3 g/L as Klason lignin (Figure 2a). Al-
additionoflignosulfonates though a few data points have large standard deviations
Enzymatic hydrolysis of a pure cellulose substrate of for the duplicate data sets of 24 and 48 h, the results
Whatman paper can be inhibited by a commercial ligno- showedthesimilartrend.Thesephenomena suggestthat
sulfonate as revealed in our previous study [18]. This in- lignosulfonate can be inhibitive to enzymatic hydrolysis
hibitive phenomenon was verified in this study at high of cellulose due to nonspecific binding of cellulase as
concentrations upto 10 g/L using the same commercial previously known. The recovery of SED at high lignosul-
lignosulfonate (Figure 1). The pH of the buffer solution fonate concentrations can be explained first by the ionic
was4.8andthemeasuredpHinthehydrolysissuspension strength effect [29]. The neutralization of the pretreat-
was 4.53. The reduction in substrate enzymatic digestibil- ment hydrolysate using NaOH solution resulted in a Na+
ity(SED),definedasthepercentageofglucaninthecellu- concentration in the mixed enzymatic and pretreatment
losic solid substrate converted to glucose enzymatically, hydrolysate of 23.5 mmol/L. Our previous study found
wasrapid at lowlignosulfonate concentrationofless than that enzymatic hydrolysis of a pretreated lignocellulosic
0.5 g/L (observable after 6 h hydrolysis) and then slowed substrate can be improved by 4% at K+ concentration of
significantly, in agreement with our previous study [18]. 2 mmol/L [19]. Lignosulfonate as a surfactant may also
This reduction in enzymatic hydrolysis is due to the non- playaroletoenhance enzymatichydrolysisof pure cellu-
specificlignosulfonatebindingofcellulase. lose [3]. The extent of this positive effect of surfactant is
A very interesting phenomena was observed when a small forpurecellulosesamples,however, and varieswith
SPORL-pretreatment lodgepople hydrolysate (L-BD4- the amount of surfactant applied. This explains the grad-
T85-3) was added into the enzymatic hydrolysis suspen- ual recovery trend of SED with the amount of lignosulfo-
sion (buffered at pH=4.8) of Whatman paper. The nate added (Figure 2a and b). The fact that recovery of
SPORL hydrolysate, L-BD4-T85-3, was first neutralized SED did not take place in Figure 1 using a commercial
from pH approximately 1.5 to 4.8 using NaOH before lignosulfonate alone without the application of ions
adding to the Whatman paper suspension. The reduc- suggests that ionic strength mechanism may play an im-
tion in SED was also apparent (observable after 24 h portant role for the observed recovery of glucose concen-
hydrolysis) and substantial at low lignosulfonate concen- trationinFigure2a.
tration of less than 0.2 g/L as Klason lignin (Figure 2a). Our previous study indicated that a divalent metal can
However, SED started to recover when lignosulfoante form complex with lignosulfoante to reduce its affinity
concentration was further increased (the glucose in the to cellulase [18]. We conducted a separate study by neu-
pretreated hydrolysate was subtracted from the mea- tralizingthesameSPORL-pretreatmenthydrolysateusedin
sured glucose concentration in the mixed hydrolysate in theprevioussetofexperiments(Figure2a)usingCa(OH) .
2
calculating SED). The recovery was also rapid with 100% Thesametrendof SEDreductionandrecoverywiththe
addition of lignosulfonate as discussed in the previous
paragraph was observed. However, slightly higher SEDs
than that for the control run without lignosulfonate
were obtained after 48 hours hydrolysis when lignosul-
fonate concentration was greater than 0.3 g/L as Klason
lignin (Figure 2b). The difference in SED between runs
using NaOH (Figure 2a) and Ca(OH) neutralization
2
(Figure2b)werewithintheerrormargin,suggestingthe
lodgepole pine lignosulfonate produced using a high
sulfite dosage of 8% on wood may already has very low
affinity to cellulase due to its high degree of sulfonation
or hydrolphlicity. The formation of lignosulfonate-Ca(II)
complex on lignin nonspecific binding to cellulase is
not significant.
EnzymatichydrolysisofaSPORL-pretreatedlodgepolepine
withtheapplicationofSPORL-pretreatmenthydrolysates
The application of non-ionic surfactant can significantly
Figure1Effectoftheapplicationofacommerciallignosulfonate reduce nonspecific binding of cellulase by lignin to en-
onsubstrateenzymaticdigestibility(SED)ofWhatmanpaper
hance enzymatic hydrolysis of lignocellulsosic substrates
(purecellulose)atcellulaseloadingof15FPU/gglucan.
[3,5,17]. Similarly, the application of lignosulfonate, as a
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Figure2EffectoftheapplicationofaSPORL-pretreatedlodgepolepinehydrolysate(L-BD4-T85-3)onsubstrateenzymaticdigestibility
(SED)ofWhatmanpaper(purecellulose)atcellulaseloadingof15FPU/gglucan.(a)L-BD4-T85-3neutralizedusingNaOHtopH4.8;(b)L-
BD4-T85-3neutralizedusingCa(OH) topH4.8.
2
surfactant,can enhance enzymatic hydrolysisoflignocel- non-ionic surfactant at equivalent cellulase loadings [3].
lulosic substrates. A SPORL – pretreatment hydrolysate The amount of glucose in L-BD4-T85-3 (Table 1) was
from lodgepole pine (L-BD4-T85-3) was first neutralized subtracted from the measured glucose concentration in
using NaOH to pH 4.8 and then applied to enzymatic the mixed hydrolysate for calculating SED. Lignosulfo-
hydrolysis of a SPORL pretreated lodgepole pine solid nate concentration in the mixed hydrolysate at 1.0 g/L
substrate (SP-BD4) at 2% solids (i.e., dilution with as Klason lignin is equivalent to lignin concentration in
water). The results indicate cellulose conversion of SP- the whole slurry of the pretreated material when hydro-
BD4 represented by SED increased linearly with the in- lyzed at2%waterinsolublesolids.
crease in lignosulfonate concentration (> 0.2 g/L) in the Thegains inSED per unit mass oflignosulfonate load-
mixed hydrolysate (Figure 3a), in agreement with that ing, ΔSED/m , for the results presented in Figure 3a
lig
reported in the literature [3,19]. Furthermore, the in- were calculated under different lignosulfonate concen-
crease in SED, i.e., from approximately 49% to 72% at trations and various enzymatic hydrolysis durations. The
24 h, or from 52% to 78% (by 50%) at 72 h, by applying results indicate negative SED gains at low lignosulfonate
lignosulfonate at 1.0 g/L (as Klason lignin) was also loadings(<0.2g/L asKlasonlignin)andshorthydrolysis
qualitatively in agreement with the gain in SED of a SO duration of 6 h (Figure 3b). This again confirmed that
2
catalyzed steam exploded spruce substrate by applying lignosulfonate does have inhibitive effect on enzymatic
Figure3EffectoftheapplicationofaSPORL-pretreatedlodgepolepinehydrolysate(L-BD4-T85-3)onenzymaticcellulose
saccharificationofaSPORL-pretreatedlodgepolepine(SP-BD4)atcellulaseloadingof13FPU/gglucan.(a)Substrateenzymatic
digestibility(SED);(b)GainsinSEDpermgoflignosulfonateapplied(%L/g).
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Table1Listofsubstratesproducedalongwiththe with the increase in lignin concentration, has significant
productionconditions importance for simultaneous enzymatic saccharification
Sample Method Chemicalcharges T(°C) Duration@ and combined fermentation of the enzymatic and pretreat-
Label1 onwood(%) T(min) ment hydrolysates (SScombF) using the whole slurry at a
LodgepolePine–Solidcellulosicsubstrates highsolidsloadings.
SP-BD4 SPORL HSO:2.2 180 20 Three SPORL-pretreatment hydrolysates, i.e., L-BD4-
2 4
T85-1, L-BD4-T85-2, L-BD4-T85-3, obtained from three
NaHSO:8.0
3
pretreatmentswithdifferentpretreatmenttimes(Table1)
LodgepolePine–Liquidsubstrates:Pretreatmenthydrolysates
(spentliquor) were used to further verify the positive effects of the
application of lignosulfonate on enzymatic hydrolysis of
L-BD4-T85-1 SPORL HSO:2.2 185 5
2 4
lignocelluloses. Both NaOH and Ca(OH) were used to
L-BD4-T85-3 NaHSO:8.0 185 25 2
3 neutralize the hydrolysates to pH 4.8 before application.
L-BD4-T85-5 185 45
The results indicated that the enzymatic cellulose con-
Aspen–Solidcellulosicsubstrates version efficiency, or SED, of a SPORL-pretreated lodge-
SP-AS SPORL HSO:1.1 170 25 pole pine, SP-BD4, was increased by over 50% when
2 4
NaHSO:3.0 either one of the pretreatment hydrolysate was applied
3
(Figure 4a and b). The hydrolysate with shortest pre-
SPH-AS HSO:0 170 25
2 4
treatment duration of5 min(Table 1), i.e.,L-BD4-T85-1,
NaHSO:3.0
3 with the highest lignosulfonate concentration of 12.6 g/L
DA-AS Diluteacid(DA) HSO:1.1 170 25
2 4 as Klason lignin (Table 1) produced highest gain in en-
1SPstandsforSPORL;SPHstandsforSPORLwithsodiumbisulfiteonly;DA zymatic saccharification, i.e., SED at 72 h was increased
standsfordiluteacid.ASstandsforaspen.
from approximately 52% to 87%, or by 67% (Figure 4a).
hydrolysis. However, there are two competing processes, While the hydrolysate with the longest pretreatment
lignosulfonate adsorbed by cellulase to reduce cellulase duration of 45 min (Table 1), L-BD4-T85-5, with lowest
activityand lignosulfonateservesasa surfactant toblock lignosulfonate concentration of 9.7 g/L as Klason lignin
lignin on the solid substrate to prevent cellulase adsorp- (Table 1), produced smallest gain in SED at 72 h
tion on the solid substrate lignin. The second process increased by 48% (Figure 4a). This difference is mainly
prevails at high lignosulfonate concentrations and longer attributed to the differences in applied lignosulfonate
hydrolysis duration. The results also indicate that the SED dosage and in the composition of cellulase inhibitive
gain at 48 h per unit mass of lignosulfonate, ΔSED/m , compounds other than lignin in the pretreatment hydro-
lig
initiallydecreasedrapidlyandthendecreasedveryslowlyat lysates [30]. It is expected that a longer pretreatment
lignosulfonate concentration exceeding 0.2 g/L as Klason duration resulted in more inhibitive compounds (i.e.,
lignin.Atalignosulfonatedosageof1.0g/LasKlasonligin, furans) in the pretreatment hydrolysate, L-BD4-T85-5,
ΔSED/m increased from approximately 2% L/g at hy- and therefore a less enhancement of SED, than a shorter
lig
drolysis time of 1 h to 25% L/g at 48 h. The slow- pretreatment. The differences in the properties of the
diminishing effect, i.e., ΔSED/m decreased very slowing lignosulfonates due to the varied degree of sulfonation
lig
Figure4EffectoftheapplicationofdifferentSPORL-pretreatmentlodgepolepinehydrolysates(spentliquors)producedunder
differentpretreatmentdurationsonSEDofSP-BD4.(a)LiquorsneutralizedusingNaOHto4.8;(b)LiquorsneutralizedusingCa(OH) to4.8.
2
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under different pretreatment durations may also affect 13 FPU/g glucan after 72 h of hydrolysis for SP-BD4. The
SEDenhancement. SED of the corresponding control run(pH 4.8without lig-
Hydrolysate neutralization using Ca(OH) showed nosulfonate) was only 51%. When a similar substrate pro-
2
consistently but only slightly increased enhancement of duced from the same batch of lodgpole pine (BD4) wood
enzymatic saccharification at 72 h than those observed chips under the identical SPORL-pertreatment conditions,
using NaOH by comparing the results in Figure 4a to a cellulase loading of 24 FPU/g glucan (15 FPU/g sub-
those in Figure 4b. The fact that the differences in SED strate) was required to achieve 90% cellulose conversion
enhancements are pretty much within the measurement [31]. This suggests a savings in cellulase application of
errors suggests the lignosulfonate produced at a high 46% can be achieved by using lignosulfonate at elevated
sulfite dosage of 8% on wood may already has avery low pH. Furthermore, the increased pH is also favorable for
affinity to cellulase. The role of divalent metal to form yeastfermentationsthroughSSFCombF.
complex with lignosulfonate to reduce lignosulfonate A similar study was also conducted using a dilute
binding of cellulase [18,19] is not very important. This is acid- (DA-AS) and SPORL- (SP-AS) pretreated aspen
inagreementwith thatobservedfrom Figure2aandb. substrates with the addition of SPORL-pretreatment hy-
drolysate at lignosulfonate concentration of 1.0 g/L as
Improvedenzymatichydrolysisofalodgepoplepineby Klason lignin, L-BD4-T85-3, at elevated pH of 5.5. The
combiningwithitspretreatmenthydrolysateandusing results indicate that using a high pH alone can enhance
anelevatedpHof5.5 enzymatic saccharification as represented by SED gains
Previouslywedemonstratedthatenzymaticsaccharification (Figure 5b). With the application of L-BD4-T85-3, SED
efficiency of lignocelluloses can be increased significantly decreased slightly during the first 10 hours for both sub-
whenhydrolysiswasconductedatanelevatedpHapproxi- strates (by comparing the solid symbols on solid line
mately 5.0 or higher for SPORL-pretreated substrates with the corresponding open symbols on dash line), sug-
[27,28].WemixedthesameSPORL-pretreatedhydrolysate gesting lignoslfonate can be inhibitive to enzymes. But
(L-BD4-T85-3) with the SPORL-pretreated solid substrate the lignosulfonate can enhance enzymatic hydrolysis
(SP-BD4) to conduct enzymatic hydrolysis at an elevated after24hours.TheSEDof bothsubstratesafter24hours
buffer solution pH of 5.5. The results show that SED at were further enhanced at elevated pH 5.5. When com-
72hincreaseslinearlywithlignosulfonate concentrationin pared with the control run using buffer solution pH of
the mixed hydrolysate (Figure 5a). Furthermore, an add- 4.8 without the application of pretreatment hydrolysate
itional gain in SED at 72 h of 10 percentage points was (L-BD4-T85-3), the SED at 72 h were increased by 25
achieved when the pH of the buffer solution was elevated and 62% for the DA-AS and SP-AS, respectively. The
to5.5.Thisdemonstratesthattheapplicationoflignosulfo- pH effect on improving enzymatic saccharification is
nateatelevatedpHcanfurtherenhanceenzymatichydroly- more pronounced for the SPORL-pretreated sample
sisoflignocelluloses.Cellulosesaccharificationefficiencyor compared with the dilute acid pretreated sample, and
SED of 90% can be achieved at a cellulase loading of only agreeswith ourprevious study [28].
Figure5ComparisonsofSEDofdifferentpretreatedsolidsubstratesattwoenzymaticsaccharificationpHwiththeapplicationsofa
SPORL-lodgepolepinehydrolysate(L-BD4-T85-3).(a)SEDofSPORLpretreatedlodgepolepine(SP-BD4)at72hundervariousL-BD4-T85-3
loadingsexpressedasconcentrationoflignosulfonateinthemixedhydrolysate,Celluclaseloading:13FPU/gglucan;(b)Time-dependentSEDsof
SPORL-anddiluteacid-pretreatedaspensubstrates(DA-AS,SP-AS)atafixedL-BD4-T85-3loadingof1.0g/LoflignosulfonateasKlasonlignin,
Celluclaseloading:7.5FPU/gglucan.
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Commerciallignosulfonatetoenhanceenzymatic increasedbyapproximately75%comparedwithsaccharifi-
saccharificationoflignocellulose cationofthewashedsolidsubstratealoneatpH4.8.These
The commercial lignosulfonate (D748, LignoTech USA, resultsdemonstratethefeasibilityofeliminatingsolidsub-
Rothschild, WI) was applied to enzymatic hydrolysis of stratewashingfordirectsimultaneousenzymaticsacchari-
dilute acid-pretreated (DA-AS) and SPORL-pretreated fication and combined fermentation of enzymatic and
aspen (SPH-AS), and lodgepole pine (SP-BD4) solid sub- pretreatment hydrolysates(SSCombF)usingSPORLtech-
strates at lignosulfonate concentration of 10 g/L at an nologywithreducedwaterandenzymeuse.
elevated buffer solution pH of 5.5. The SED at 72 h was
increased by approximately 20 and 10% (Figure 6), re- Materials and methods
spectively, for SPH-AS and SP-BD4, indicating the com- All experiments were carried out according to the ex-
mercial lignosulfonate is also effective to increase perimentalprocessflowsimilartopreviousdescribedex-
enzymatic hydrolysis of lignocelluloses. However, no sig- cept the pretreatment spent liquor was pH adjusted
nificant effect was observed to the dilute acid pretreated before added to the solid fraction to conduct enzymatic
aspen (DA-AS) though slight inhibitive effect to reduce saccharification[32].
SED before 24 h and the graduate recovery of SED after
24 was observed, suggesting the interaction of lignosul- Materials
fonate with the bound lignin on the lignocellulose solids A lodgepole (Pinus contorta) tree killed by mountain
alsodependsonthestructure ofthesolidlignin. pine beetle (Dendroctonus ponderosae) (estimated infest-
ation age of 4 years, abbreviated BD4) was harvested
Conclusions from the Canyon Lakes Ranger District of the Arapaho–
Thisstudydemonstrateslignosulfonatehasa low affinity Roosevelt National Forest, Colorado. The details of the
to cellulase. As a surfactant, certain lignosulfonate, such tree were described in our previous studies [31,33].
as lignosulfonate from SPORLpretreatment of lodgepole Fresh aspen (Populus tremuloides) wood logs were
pine, can be applied to significantly enhance enzymatic obtained from northern Wisconsin, USA. All wood logs
hydrolysis of lignocelluloses. Elevated pH can also en- were shipped to the U.S. Forest Products Laboratory,
hance enzymatic saccharification of lignocelluloses. By Madison, Wisconsin, and chipped using a laboratory
directly mixing SPORL pretreatment hydrolysate of a chipper. The wood chips were then screened to remove
lodgepole pine with the corresponding SPORL pretreated all particles greater than 38 mm and less than 6 mm in
lodgepolepinesolidsubstrate,90%ofenzymaticsacchari- length. The thickness of the accepted chips ranged from
fication of pretreated lodgepole pine can be achieved at 1 to 5 mm. The chips were kept frozen at a temperature
pH 5.5 and cellulase loading of 13 FPU/g glucan, or ofabout−16°Cuntilused.
Celluclast 1.5 L and Novozyme 188 (β-glucosidase)
were generously provided by Novozymes North America
(Franklinton, NC). Sodium acetate buffer, sulfuric acid,
and sodium bisulfite were ACS reagent grade and used
asreceived from Sigma-Aldrich(St.Louis,MO).
Substrateproduction
Several lignocellulosic solid substrates were produced
fromboththelodgepolepineandaspenwoodchipsusing
pretreatment methods with different process chemistries.
Table 1 lists the various substrates produced along with
production process conditions. Because dilute acid is a
widely studied pretreatment and effective on many feed-
stocks except softwoods such as lodgepole pine used in
this study. We also produced solid substrates from aspen
(hardwood) using dilute acid and SPORL (Table 1) to
evaluate the effectiveness of the application of lignosulfo-
nate to enhance saccharfication of these aspen substrates.
Figure6Effectoftheapplicationofacommercial A laboratory wood pulping digester of capacity of 23 L
lignosulfonateontimedependentSEDsofpretreatedaspen wasusedtoconductpretreatmentasdescribedinourpre-
(SPH-AS,DA-AS)andapretreatedlodgepolepine(SP-BD4). vious study [10]. Thedigesterwasheatedbya steamjacket
Celluclaseloading7.5FPU/gglucanandhydrolysisbuffered
androtatedat2rpmformixing.Theovendry(od)weightof
atpH5.5.
woodchipsineachpretreatmentwas2kg.Thepretreatment
Wangetal.BiotechnologyforBiofuels2013,6:9 Page8of10
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Pretreatment
hydrolysate
(spent liquor)
e
at
r
st
b
u
Pretreatment Size d s
reduction oli
s S
al
c
mi Press
e
h
C Water
Steam
Figure7Aschematicexperimentalflowdiagram.
liquidtowoodratio(L/W)waskeptat3(v/w).The chemical hydrolysates were analyzed according to the methods
charges, reaction temperature, and duration of different describedlaterinthetext.TheresultsarelistedinTables2
pretreatments are listed in Table 1. and3.Itshouldbepointedoutthattheexperimentalflow
The pretreated wood chips remained intact and were showninFigure7istodemonstratetheconceptproposed
separated from the pretreatment hydrolysate (hemicellu- inthisstudy.Thefreshwaterusedfordiskmillingshould
losic sugar stream) by a screen. The pretreated wood be replaced by the pretreatment hydrolysate, i.e., the sep-
chips were disk milled using disk plates of pattern D2B- aration of pretreatment hydrolysate from the solids is not
505 with a plate gap of 0.25 mm and adjusted to a re- necessary, as practiced in our laboratory currently after
finer discharge consistency of 10% with dilution water theproposedconceptbeingproventhroughthisstudy.
(Figure 7). The energy consumption for disk milling was The pH of the pretreatment hydrolysates were adjusted
recordedasdescribedelsewhere[34,35].Thesize-reduced to 4.8 using NaOH before applied to the suspension of
solids were directly dewatered to a solids content of ap- solidsubstratetoconduct enzymatichydrolysis. Forcom-
proximately 30% by vacuum pressing in a canvas bag (as parisonpurposes,thepHofthepretreatmenthydrolysates
prewashing). The yields of solid substrate after washing werealsoadjustedto4.8usingCa(OH) insomehydroly-
2
werethendeterminedfromtheweightandmoisture con- sis experiments as indicated. The glucose was measured
tentofthecollectedsubstrate.Thechemicalcompositions using the glucose analyzer described in the “Analytical
of both the solid substrates along with the pretreatment methods”section.
A pure cellulosic substrate, a commercial Whatman
filter paper (Grade 3, Cat No 1003 150, Whatman Inter-
Table2Chemicalcompositionsandyieldsoftheuntreated
national,England)wasalsoused.The manufacturer spe-
andpretreatedlignocellulosicsubstrateslistedinTable1
cified ash content is 0.06%. High purity lignosulfonate
Samplelabel Klignin Glucan Xylan Mannan Solidsyield
(%) (%) (%) (%) (wt%) D748 from softwood sulfite pulping was donated by
LignoTech USA (Rothschild, WI).
Untreatedwood
BD4 28.6 41.9 5.5 11.7 100
FL 29.2 39.1 6.0 10.0 100 Table3Glucoseandlignosulfonateconcentrationsinthe
pretreatmenthydrolysates(hemicellulosicsugarstream)
Aspen 20.2 45.6 16.4 1.4 100
listedinTable1
Pretreatedwood
Liquidsubstrate Glucose(g/L) Lignosulfonateas
SP-BD4 34.7 57.4 1.5 0.6 58.6 Klasonlignin(g/L)
SP-AS 28.1 66.2 1.9 0.3 58.2 L-BD4-T85-1 2.95 12.6
SPH-AS 22.2 66.6 5.3 0.8 60.9 L-BD4-T85-3 5.34 11.1
DA-AS 30.0 61.6 3.3 0.4 63.5 L-BD4-T85-5 7.18 9.7
Wangetal.BiotechnologyforBiofuels2013,6:9 Page9of10
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Enzymatichydrolysis Competinginterests
Enzymatic hydrolysis was conducted using commercial Theauthorsdeclarethattheyhavenocompetinginterests.
enzymes at 2% substrate solids (w/v) in 50-mL of buffer
Authors’contributions
solutionsonashaker/incubator(ThermoFisherScientific,
ZJWconductedmostoftheexperiments.TQLinitiatedandassisted
Model4450,Waltham,MA)at50°Cand200rpm.Unless conductingthepHeffectexperiments.JYZdirectedtheresearchand
indicated,the pH of the buffersolutionof sodium acetate analyzedthedataandwrotetheentirepaper.Allauthorsreadandapproved
thefinalmanuscript.
was4.8.Celluclast1.5Lloadingsvariedbetween7.5to13
FPU/gglucan.TheratioofNovozyme188(β-glucosidase)
Authors’information
loading (in CBU) to Celluclast 1.5 L loading (FPU) was WangandLanwerevisitingPh.DstudentsattheUSDAForestService
maintained at 1.5 for all experiments. Selected hydrolysis (USDA-FS),ForestProductsLab(FPL),fromSouthChinaUniversityof
experiments were carried out in duplicates to ensure ex- Technology,GuangZhou,China.WangiscurrentlywithKeyLabofPaper
Science&Technology,ShandongPolytechnicUniversity,Jinan,China.Zhuis
perimentalrepeatability.Hydrolysatewassampledperiod- aScientificTeamLeaderattheUSDA-FS-FPL.Heisaco-inventortheSPORL
ically for glucose concentration analysis. Each data point pretreatmentprocessandpublishesextensivelyinwoodybiomass
istheaverageoftworeplicates.Theaveragerelativestand- bioconversionforbiofuel,fiber,andnanocellulosicmaterials.Zhuisan
electedFellowoftheInternationalAcademyofWoodScience(IAWS)andan
arddeviationwasapproximately2%. officeroftheForestProductsDivisionoftheAmericanInstituteofChemical
Engineering(AIChE)andtheTechnicalAssociationofthePulpandPaper
Industry(TAPPI).Heservesontheeditorialboardsofseveraltechnical
journals.ThisworkwasconductedonofficialgovernmenttimeofZhu.
Analyticalmethods
The chemical compositions of the original and pre-
Acknowledgements
treated biomass were analyzed by the Analytical and Mi- ThisworkwaspartiallysupportedbyaUSDASmallBusinessInnovative
croscopy Laboratory of the Forest Products Laboratory Research(SBIR)PhaseIIproject(ContractNumber:2010-33610-21589)to
BiopulpingInternational,Inc.Thisprojectprovidedpartialfinancialsupport
as described previously [31]. All lignocelulosic samples
toWangandfullsupporttoLanfortheirvisitingappointmentsattheUS
were Wiley milled (model #2, Arthur Thomas Co, Phile- ForestService(USFS),ForestProductsLaboratory(FPL).TheChinese
delphia, PA). The milled sample of 20 mesh (~1 mm) in ScholarshipCouncilprovidedtheotherpartialsupportforWang.We
acknowledgeFredMatt(FPL)forcarryingoutcarbohydrateanalyses.
size was hydrolyzed in two stages using sulfuric acid of
72% (v/v) at 30°C for 1 h and 3.6% (v/v) at 120°C for Authordetails
1 h. The hydrolysate supernatant and remaining solids 1KeyLabofPaperScience&Technology,ShandongPolytechnicUniversity,
Jinan,China.2USForestService,ForestProductsLaboratory,Madison,WI,
are then filtered through a Gooch Crucible lined with a
USA.3CollegeofLightIndustryandFoodSciences,SouthChinaUniversityof
21mmWhatmanfilterintoavolumetricflask.Thesuper- Technology,Guangzhou,China.
natant was used for carbohydrate analysis using high-
Received:31July2012Accepted:13September2012
performanceanionexchangechromatographywithpulsed
Published:28January2013
amperometricdetection(HPAEC-PAD)[36].Klasonlignin
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