high consistency enzymatic hydrolysis of hardwood substrates

Bioresource Technology 100 (2009) 5890–5897

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Bioresource Technology

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High consistency enzymatic hydrolysis of hardwood substrates

Xiao Zhang a,*, Wenjuan Qin b, Michael G. Paice a, John N. Saddler b

a FPInnovations Paprican Division (PAPRICAN), 570 boul. St-Jean, Pointe-Claire, Quebec, Canada H9R 3J9b Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancovuer, BC, Canada V6T 1Z4

a r t i c l e i n f o

Article history:Received 16 September 2008Received in revised form 1 May 2009Accepted 20 June 2009Available online 29 July 2009

Keywords:High consistency hydrolysisLignocellulosic substratesFermentationEthanolRheological problem

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.06.082

* Corresponding author.E-mail address: [email protected] (X. Z

a b s t r a c t

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lig-nocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW)and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20%substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 henzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucoseconcentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation ofUBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipmentto carry out high consistency hydrolysis can overcome the rheological problems and greatly increase thesugar and ethanol concentrations after the hydrolysis and fermentation.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Bioconversion of renewable lignocellulosic feedstock, such aswood and agricultural residues, to liquid fuel provides a possiblemeans of reducing greenhouse gasses and alleviating the pressurefrom fossil fuel shortage (Wyman and Hinman, 1990; Galbe andZacchi, 2002). A typical lignocellulose-to-ethanol process basedon biological routes consists of at least four major steps: partialor complete removal of lignin and hemicellulose by pretreatment,enzymatic hydrolysis of polysaccharides to sugar monomers, fer-mentation of sugars to ethanol, and recovery of ethanol from theprocess stream. Despite significant advances in both scientificunderstanding and process engineering towards the commerciali-zation of such a bioconversion process, an economically feasibleindustrial biomass-to-biofuel process is yet to be developed (Sunand Cheng, 2002; Van Wyk, 2001). While most of the process stepsneed to be further optimized, the enzyme hydrolysis step has beenidentified as a major techno-economical bottleneck in the entirewood-to-ethanol bioconversion process. Conventional enzymatichydrolysis of lignocellulosic materials is typically carried out at asubstrate consistency below 5% solids content. This results in a su-gar concentration below 5% in the hydrolysate and, subsequently, afinal ethanol concentration less than 2% (w/w) after fermentation.In contrast, enzymatic hydrolysis and fermentation of starch-basedsubstrates (e.g. corn) is commonly performed at a substrate load-ing above 20% of dry matter and over 10% (w/w) final ethanol con-centration is typically obtained after fermentation.

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hang).

It is likely that increasing substrate loading during hydrolysis oflignocellulose will lead to increased sugar concentration and high-er final ethanol content after fermentation. This approach willbring economic savings to the bioconversion process, such asreducing capital and operational cost for hydrolysis and fermenta-tion, and minimizing energy consumption during distillation/evap-oration and other downstream processes. A previous techno-economic assessment has suggested that an increase in substrateloading from 5% to 8% (w/w) can reduce the total production costby nearly 20% (Stenberg et al., 2000; Wingren et al., 2003). A fur-ther increase in substrate loading will provide even more signifi-cant cost saving. However, the studies carried out at that timedid not achieve an effective hydrolysis at a substrate consistencyabove 10% using either separate hydrolysis and fermentation(SHF) or simultaneous saccharification and fermentation (SSF) ap-proaches. The study recognized a number of barriers to high con-sistency hydrolysis including (1) high concentration of fibrousmaterials reduces mass transfer rate; (2) high substrate consis-tency leads to high concentration of inhibitory substances; and(3) high sugar concentration causes severe end-products inhibitioneffects.

Unlike starch-based feedstock, the lignocellulosic substrate is afibrous material with a high degree of polymerization (DP). In awater suspension, fibrous substrates can interact with each otherand form fibre flocs or, on a larger scale, fibre networks. This leadsto a considerable increase in the viscosity of the substrate matrix,and creates a so-called ‘‘rheological problem” when the masstransfer rate in the substrate matrix is significantly hindered.Due to the limited amount of free water present in the matrix, ittakes a much longer time to liquefy the matrix and carry out

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effective hydrolysis. Rheological problems associated with mixingpulp fibre suspensions have long been recognized in pulp and pa-per manufacturing. Dealing with high substrate consistency is acommon practice in wood pulp bleaching. Industrial bleachingequipment is designed to handle pulps at various consistencies,typically up to a maximum of 35%. Medium or high consistencymixing devices can effectively break down fibre flocs and networksformed in pulp suspensions above 20% consistency.

In this study, we examined the feasibility of using a peg mixerto carry out enzyme hydrolysis of lignocelluloses at high substrateconsistency. Two substrates were used, unbleached hardwood pulp(UBHW) and organosolv pretreated poplar (OPP).

Diagram 1. The inner chamber of the laboratory peg mixer.

2. Methods

2.1. Substrates

Unbleached hardwood kraft pulp (UBHW) was obtained from aCanadian kraft pulp mill. The organosolv pretreated poplar (OPP)was prepared in Paprican’s pilot plant by cooking poplar woodchips in 50% (w/w) aqueous ethanol solution with 1.25% H2SO4

as catalyst at 170 �C for 60 min (Pan et al., 2006).The extractives content of UBHW and OPP was determined by

PAPTAC (Pulp and Paper Technical Association of Canada) standardprocedures (STANDARD G.13 and G.20) using acetone as a solvent.The total lignin content (acid soluble lignin and acid in-soluble lig-nin) of UBHW and OPP was measured following PAPTAC standardprocedures G.8 and G.9. The filtrate obtained from lignin analysiswas collected and used for sugar analysis. The sugar monomers inthe filtrate, including arabinose, galactose, glucose, xylose, andmannose, were separated by an anion exchange column (DionexCarboPacTM PA1) on a Dionex DX-600 Ion Chromatograph system(Dionex, Sunnyvale, CA) equipped with an AS50 autosampler anda GP50 gradient pump. De-ionized water was used as an eluent ata flow rate of 1 mL/min; 1 M NaOH was used to equilibrate the col-umn after elution of sugars. To optimize baseline stability anddetector sensitivity, 0.2 M NaOH was added post column. Afterbeing filtered through 0.45 lm nylon syringe filters (Chromato-graphic Specialties Inc.), a 20 ll sample was injected on the column.The sugar monomers were monitored by a ED50 electrochemicaldetector with parameters set for pulsed amperometric detection.Sugar standards were prepared and analyzed using the same proce-dure in order to calibrate the instrument before sample analysis.The Chromeleon 6.5 software was used to control the chromato-graph system and quantify sugar concentrations.

The viscosity of UBHW and OPP was measured following theScandinavian pulp, paper and board standard testing processSCAN-C15:62. The degree of polymerization of cellulose (DP) pres-ent in UBHW and OPP was estimated using the viscosity value ofthe respective pulp samples.

2.2. Enzymes

Celluclast 1.5L (cellulase) and Novozym 188 (b-glucosidase)were obtained from Novozymes North America (Franklinton, NC).The activity of Celluclast is 80 filter paper units per milliliter(FPU/mL). The activity of Novozym 188 is 450 cellobiase unitsper milliliter (CBU/mL). The enzyme dosage was 20 FPU cellulasesupplemented with 80 CBU of b-glucosidase per gram of cellulosein the substrate.

2.3. Enzymatic hydrolysis in shake flasks

The batch hydrolysis experiments were carried out in 500-mlflasks. The reaction solution contained 200 mM acetate buffer

(pH 4.8) with differing concentrations of the substrates and en-zyme dosages described earlier. All the flasks were fixed in a con-trolled environment incubator shaker (New Brunswick ScientificCo., Edison, NJ, USA). The enzymatic hydrolyses were carried outat 50 �C with a rotating speed of 200 rpm for up to 96 h at varioussubstrate consistencies. The cellulose-to-glucose conversion yieldis defined as the glucose amount in the liquid phase product di-vided by the cellulose content (as glucose) in the substrate.

2.4. Enzymatic hydrolysis in peg mixer

A laboratory peg mixer (Diagram 1) manufactured by the Pulpand Paper Research Institute of Canada (Pointe Claire, QC) wasused in this study for carrying out high consistency hydrolysis ofUBHW and OPP. The peg mixer has a working volume of 9000 land approximately 800 g (oven dried weight) substrate was usedfor batch hydrolysis under the same conditions as the shake flasks(temperature, pH and enzyme dosage) except that the mixingspeed was set at 20 rpm. Prior to the hydrolysis, the substrate, en-zyme and buffer were mixed thoroughly in a Hobart mixer (Hobart,North York ON) before they were transferred to the peg mixer.

2.5. Fermentation of the hydrolysate

An industrial adapted Saccharomyces cerevisiae strain (T2 yeastprovided by Tembec Inc., Témiscaming, Québec) was used for fer-mentation. The yeast cells were inoculated into 250 ml of YEPDmedium (Yeast extract 1%, Peptone 2% and glucose 2%), incubatedat 30 �C in a rotary shaker (200 rpm) for 24 h. The yeast cells werecollected by centrifugation at 5000g for 10 min at 4 �. The pelletwas washed three times with 10 ml phage storage buffer (PSB).Yeast cells from this preparation were then inoculated into 60 mlof pre-hydrolysate or pure glucose solution. The final cell concen-tration was 5.5 mg/mL.

The pH of the glucose controls and the hydrolysates was ad-justed to 6.5 using 50% NaOH prior to the fermentation after theaddition of 0.3% yeast extract, 0.5% peptone and (NH4)2HPO4 to afinal concentration of 20 mM. The fermentation experiment wascarried out in 125 ml serum bottles containing 60 ml of hydroly-sate. Fermentation was carried out in a rotary shaker (New Bruns-wick Scientific Co., Edison, NJ, USA) at 30 � for up to 96 h.

2.6. Sugar, ethanol and inhibitors analysis

During the hydrolysis, 0.5 ml aliquots were taken at differentreaction times, and immediately filtered through a 0.45 lm mem-

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5892 X. Zhang et al. / Bioresource Technology 100 (2009) 5890–5897

brane filter. The glucose concentration in the resulting filtrate wasthen determined by the above-mentioned ion chromatographmethod. All values are averages obtained from experiments per-formed in duplicate.

Aliquots of 0.5 ml were taken periodically from the fermenta-tion broth to determine the ethanol and glucose concentration.Samples were first centrifuged to remove the yeast cells and thenfiltered through a 0.45 lm membrane filter (Millipore, Bedford,MA) The ethanol concentration was determined by gas chromatog-raphy as previously described (Robinson et al., 2002) using a HP6890 series GC system. The glucose concentration was measuredby the same HPLC method as described in the previous section.

2.7. Data analysis and number of sample replicates

All the results reported for batch hydrolysis and fermentationare the mean values of at least six replicates based on two batchesof repeated experiments under the same conditions. Three sampleswere taken for each time point. The mean and standard deviationwere calculated by Origin� version 7SR2 (OriginLab Corp., North-ampton, MA).

3. Results

3.1. Chemical composition of unbleached hardwood pulp (UBHW) andorganosolv pretreated poplar (OPP)

As shown in Table 1, UBHW has a cellulose content of approxi-mately 80% with 19.6% of xylan. The pulp contains a small amountof lignin with low extractives content. The UBHW represents an‘‘ideal” pretreated wood substrate as it contains a minimum con-tent of lignin and other contaminants. To test whether the samehigh consistency hydrolysis approach can be applied to other sub-strates, an organosolv pretreated poplar (OPP) was prepared usingpretreatment conditions described previously (Pan et al., 2006).The chemical composition of the organosolv pretreated poplar(OPP) is also shown in Table 1. The OPP contains approximately87% cellulose with little xylan. The lignin content of OPP is slightlyhigher than that of UBHW. The most significant difference betweenthe UBHW and OPP is the significant amount of acetone extractivesdetected in OPP. Most of these extractives are likely derived fromlow molecular weight phenolic compounds which are soluble inacetone. Previous work (Pan et al., 2006) suggested a higher lignincontent in OPP substrate than that found in the current study.However, their lignin analysis was performed on samples without

Table 1The chemical composition of unbleached hardwood pulp (UBHW) and organosolvpretreated poplar (OPP).

Component (Weight percentage %)

Unbleached hardwood pulp (UBHW)Acetone extractives 0.15 ± 0.02Cellulose (as glucan) 79.1 ± 0.4Cellulose (as glucose) 84.3 ± 0.4Xylan 19.6 ± 0.4Lignin

Acid soluble 0.63 ± 0.04Acid in-soluble 1.06 ± 0.05

Organosolv pretreated poplar (OPP)Acetone extractives 8.17 ± 0.06Cellulose (as glucan) 86.5 ± 0.4Cellulose (as glucose) 92.3 ± 0.4Xylan 1.46 ± 0.03Lignin

Acid soluble 0.34 ± 0.01Acid in-soluble 2.08 ± 0.01

solvent extraction where these phenolic compounds can be precip-itated during acid hydrolysis and contribute to the total lignincontent.

3.2. Hydrolysis of UBHW at 2% and 5% consistency in shake flasks

The hydrolysability of UBHW was first determined in shakeflasks at 2% and 5% (w/w) consistencies. As shown in Fig. 1A,hydrolysis at 2% substrate consistency for 48 h resulted in a glu-cose concentration of about 17 g/L, while hydrolysis at 5% sub-strate consistency produced approximately 40 g/L glucose in thefinal hydrolysate. When the percentage of cellulose-to-glucoseconversion was determined (Fig. 1B), it was found that most ofthe cellulose present in 2% UBHW substrate was converted to glu-cose within 24 h of incubation with 20 FPU/g and 80 CBU/g of en-zyme loading. Increasing substrate consistency to 5% led to aslightly lower cellulose-to-glucose conversion rate, approximately95% after 48 h. This is probably due to end-product inhibition ef-fects from the glucose and cellobiose produced (Xiao et al., 2004).

3.3. High consistency hydrolysis of unbleached hardwood pulp(UBHW)

We next examined the possibility of hydrolyzing UBHW at dif-ferent substrate consistencies, from 2% to 20% at 3% intervals (Ta-

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Fig. 1. Enzymatic hydrolysis of UBHW (unbleached hardwood pulp) at 2% and 5%substrate consistencies in shake flasks, based on (A) glucose concentration formedand (B) percent cellulose conversion.

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Fig. 2. Enzymatic hydrolysis of UBHW (unbleached hardwood pulp) at 2% and 20%substrate consistencies in a peg mixer, based on (A) glucose concentration formedand (B) percent cellulose conversion.


ble 2). It was observed that increasing pulp consistency resulted ina decrease in the amount of free water in the substrate matrix. Thehigher the initial consistency, the longer it takes to liquefy the sub-strate matrix (Table 2). In shake flasks at 20% substrate consis-tency, the UBHW only liquefies after 40 h incubation in thepresence of cellulase enzymes. These experiments demonstratethat the shake-flask method is not suitable for evaluating high con-sistency hydrolysis of lignocellulosic feedstock.

A peg mixer (Diagram 1), which is commonly used for oxygendelignification, was found to be capable of providing effective mix-ing of UBHW at high consistency. As shown in Table 2, it only took1 h to liquefy UBHW at 20% consistency in the peg mixer comparedto 40 h in a shake flask at the same enzyme loading. The hydrolys-ability of UBHW at 20% substrate consistency in the peg mixer wasthen evaluated. It was anticipated that a high substrate loading willraise the cellobiose concentration in the hydrolysate which will inturn elevate the end-product inhibition effects on cellulase en-zymes (cellobiohydrolases and endoglucanases). Therefore, wechose a mixture of cellulase and b-glucosidase at an enzyme load-ing of 20 FPU of cellulase with 80 CBU of b-glucosidase per gram ofcellulose. As shown in Fig. 2A, a significant increase in glucose con-centration was obtained during hydrolysis of UBHW at 20% consis-tency. The glucose content reached 144 g/L after 96 h ofincubation. This is the highest glucose concentration ever reportedfrom batch hydrolysis of a lignocellulose substrate.

Hydrolysis of UBHW at 2% consistency was also carried out inthe peg mixer (Fig. 2) to compare with the results obtained fromshake flask experiments (Fig. 1). Similar hydrolysis profiles(Fig. 2) were obtained and both attained 100% cellulose-to-glucoseconversion after 24 h of incubation. However, at 20% substrate con-sistency, the cellulose-to-glucose conversion rate is only about 84%after 96 h of hydrolysis. Extending the hydrolysis to longer time re-sulted in little increase in glucose concentration (data not shown).Typically, cellulase hydrolysis of cellulose follows a two-phasecurve, with an initial logarithmic phase and a subsequent asymp-totic phase (Ramos et al., 1993). A number of factors contributeto the slow conversion rate in the later hydrolysis phase. Amongthese factors, end-products such as cellobiose and glucose wereshown to play a major role in hindering hydrolysis (Tengborg etal., 2001). It is anticipated that the end-products inhibition effectwill become severe at high substrate loading. A previous studyhas demonstrated that the presence of 100 g/L glucose in thehydrolysate can reduce the efficiency of cellulase hydrolysis by80% (Xiao et al., 2004). The lower conversion rate at 20% consis-tency compared to 2% is mainly due to the inhibition effects fromthe high glucose content in the hydrolysate (Xiao et al., 2004).

3.4. High consistency hydrolysis of organosolv pretreated poplar (OPP)

The OPP was hydrolyzed at both 2% and 20% substrate consis-tencies under the same conditions as applied to UBHW in thepeg mixer. As shown in Fig. 3, the OPP demonstrates a high hydro-lysability at 2 % substrate consistency. The substrate released16.8 g/L of glucose after 12 h enzyme hydrolysis (Fig. 3A) whichrepresents 91% of the available cellulose (as glucose) in the OPP(Fig. 3B). A complete conversion of all the cellulose-to-glucosewas obtained after 60 h of enzymatic hydrolysis. Hydrolysis ofOPP at 20% substrate consistency yielded a significantly higher glu-

Table 2The influence of substrate consistencies on liquefaction time during hydrolysis of UBHW

Hydrolysis in shake flasks

Substrate consistency (%) 2 5 8 11Liquefaction time (h) 0 0 2.5 6

cose concentration. The glucose content reached 158 g/L in thehydrolysate after 48 h of enzyme hydrolysis which is even higherthan that obtained from UBHW. The amount of released glucoseafter 48 h of hydrolysis corresponded to a cellulose-to-glucose con-version of about 85%. There is little increase in sugar concentrationbeyond 48 h hydrolysis of OPP at 20% substrate consistency. Highconsistency hydrolysis of OPP at lower enzyme loadings, 3 and10 FPU/g, was also tested (Fig. 4). It is apparent that decreasing en-zyme loading led to reduced sugar concentration and lower cellu-lose-to-glucose conversion rate. However, a 50% reduction inenzyme dosage only reduced final glucose concentration by about21% (from 158.2 to 124.5 g/L). Even at 3 FPU/g of enzyme loading, itis still possible to obtain a glucose concentration above 80 g/L(Fig. 4A) with 43% cellulose-to-glucose conversion yield (Fig. 4B)after 96 h of hydrolysis.

The hydrolysability of OPP at 30% substrate consistency wasalso determined (Fig. 5). As shown in Fig. 5, increasing OPP consis-tency from 20% to 30% resulted in a large increase in glucose con-

(unbleached hardwood pulp) in shake flasks and peg mixer.

Hydrolysis in peg mixer

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Fig. 3. Hydrolysis of organosolv pretreated poplar (OPP) at 2% and 20% substrateconsistency in a peg mixer, based on (A) glucose concentration formed and (B)percent cellulose conversion.

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Fig. 5. Comparison of glucose concentrations obtained from hydrolysis of OPP at20% and 30% substrate consistencies using 20 FPU/g of cellulose loading.


centration during the entire hydrolysis course with a final glucoseconcentration of 21% at 96 h of hydrolysis. However, the cellulose-to-glucose conversion yield is lower than that obtained at 20%. Itshould be mentioned that the initial OPP substrate has a solid con-tent slightly below 30% (�27%). Therefore, this substrate has to bedewatered by pressing prior to enzyme hydrolysis. Althoughopportunities exist to further increase high sugar concentrationat higher substrate consistencies, 20–30% is likely to be the practi-cal substrate consistency range to carry out high consistencyhydrolysis of lignocellulosic substrates, as most of the substratewould have solids content in this range after the pretreatmentstage.

3.5. Fermentability of the hydrolysate obtained from high consistencyhydrolysis of UBHW and OPP

The hydrolysates obtained from hydrolysis of UBHW and OPP at20% consistency represent the highest glucose concentrations thathave been obtained from batch enzymatic hydrolysis of lignocellu-loses. There has been little information on the fermentability of‘‘realistic” hydrolysate with such high sugar concentrations. Itcan also be expected that high substrate loading may lead to an in-creased amount of potential inhibitors in the hydrolysates. There-fore, it is critical to determine how well yeast will ferment thesehydrolysates. The hydrolysates obtained from 48 h hydrolysis of

UBHW and OPP substrates at 20% consistency were collected andused for the subsequent fermentation experiments. Two glucosesolutions were prepared as controls at the sugar concentrationspresent in UBHW and OPP hydrolysates. The initial glucose concen-


tration of UBHW hydrolysate prior to fermentation was about112 g/L and the control pure glucose solution was 110 g/L. The fer-mentation experiment was carried out for 96 h; the glucose reduc-tion and ethanol production were determined during thefermentation. The yeast showed a high fermentability on pure glu-cose solution. Nearly all the sugars were metabolized after 12 hfermentation (Fig. 6). The ethanol production reached approxi-mately 44 g/L at this time and started to level off (Fig. 7). The finalethanol concentration (after 96 h) in the fermentation broth was48.4 g/L which is about 86% of the theoretical glucose-to-ethanolconversion yield (based on a theoretical yield of 0.51 g of ethanolper gram of glucose). The yeast was also able to effectively utilizeglucose in UBHW hydrolysate to produce a significant amount ofethanol. Compared to glucose control, there was an initial lagphase observed in the glucose reduction and ethanol productionduring UBHW hydrolysate fermentation. The depletion of glucoseoccurred after 36 h of fermentation with an ethanol productionof 46 g/L at the same time. The final ethanol concentration (after96 h) was 50.4 g/L which is 88% of the theoretical yield.

The fermentation of OPP hydrolysate was then tested under thesame conditions and compared to a control medium containing150 g/L of pure glucose. The initial glucose concentration in OPPhydrolysate was about 149 g/L. The yeast again demonstrated a

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Fig. 6. The decrease in glucose concentration during fermentation of UBHWhydrolysate to ethanol by Saccharomyces cerevisiae.

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Fig. 7. The production of ethanol during Saccharomyces cerevisiae fermentation ofUBHW (unbleached hardwood pulp) hydrolysate.

good capability to ferment concentrated glucose solution. Almostall the glucose was used up within the initial 12 h of the fermenta-tion with an ethanol production of nearly 60 g/L (Figs. 8 and 9). Ahigher final ethanol concentration (after 96 h), 62.3 g/L, was ob-tained compared to the previous glucose control (Fig. 9). Howeverthe conversion yield was lower, 81% vs. 86% of the theoretical yield.Again, an initial lag phase was observed during OPP hydrolysatefermentation compared to the control medium with similar glu-cose content. The maximum ethanol production was achieved after24 h of fermentation. The final ethanol concentration (after 96 h)from fermenting OPP hydrolysate was 63.1 g/L which is equivalentto 83% of the theoretical yield.

4. Discussion

High consistency hydrolysis and fermentation (20% and above)is a common practice in current starch-based bioethanol produc-tion. However, the characteristics of lignocellulosic substrates aredifferent from starch-based feedstock. At low consistencies (<4%),the fibrous materials are suspended in abundant free water whichmakes the suspensions easy to be mixed and transferred. However,once the substrate consistency increases to 8%, a greater degree of

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Fig. 8. The decrease in glucose concentration during fermentation of OPP (organo-solv pretreated poplar) hydrolysate.

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Fig. 9. The production of ethanol during Saccharomyces cerevisiae fermentation ofOPP (organosolv pretreated poplar) hydrolysate.

Table 3The amount of potential inhibitory compounds present in UBHW (unbleachedhardwood pulp) and OPP (organosolv pretreated poplar) hydrolysates.

Concentration UBHW hydrolysate OPP hydrolysate

Acetic acid (mmol/L) 53.6 109.4Total phenolic (g/L) 2.1 5.2


fibre–fibre interactions occur, and this leads to a substantial in-crease in the strength of the fibre network. As a result, the charac-ter of the suspension changes from one mass of fibres in water towet fibre aggregates surrounded by gas (Duff and Titchener,1975). This creates the so-called ‘‘rheological problem” during mix-ing and significantly reduces the amount of free water available forhydrolysis. As shown in Table 2, a significant increase in the lique-faction time was observed at higher substrate consistency in shakeflasks. It is apparent that effective mixing to break down the fibrenetwork is a critical step to achieving high consistency hydrolysisof lignocellulosic substrates. The peg mixer is conventional equip-ment employed in the pulp and paper industry to provide effectivemixing of medium-consistency (8–15%) pulp. The results haveshown that this equipment can greatly improve the liquefactionrate of UBHW substrate in the presence of cellulase. Using a pegmixer for high consistency hydrolysis resolved the technical issuerelated to mixing.

During the preparation of this manuscript, two studies dealingwith the topic of high consistency hydrolysis were published inthe literature. Jorgensen and colleagues (Jorgensen et al., 2007)employed an in-house chamber to carry out liquefaction of wheatstraw at 40% consistency. After liquefaction, the straw slurry wassubjected to subsequent saccharification and fermentation ineither SHF or SSF configuration. Forty percent substrate consis-tency is apparently the highest solid loading that has been at-tempted so far. However, in a practical fibre processing industry(e.g. pulp and paper industry), a pulp consistency between 20%and 25% is typically encountered. Therefore, we chose to use 20%substrate consistency to examine hydrolysability of wood sub-strate in a peg mixer. As shown in Diagram 1, it appears that thepeg mixer can provide a similar mixing effect as the equipment de-signed by the Danish group; both apply a rotating shaft to breakdown fibre floc and disintegrate fibre networks. Although in ourstudy a lower substrate consistency was used, significantly higherglucose concentrations were obtained from hydrolysis of UBHWand OPP, at 144 g/L (or 144 g/kg), 158 g/L (or 158 g/kg) respectivelyafter 96 h at 20 FPU of cellulase loading. The density is estimated at1.06 kg/L based on measurement of a pure glucose solution of150 g/L. While low enzyme loadings, 3 and 10 FPU/g of cellulose,still yield 80.5 and 124.5 g/L of glucose in the final hydrolysates.In Jorgensen’s study, 86 and 76 g/kg glucose were produced byhydrolyzing wheat straw at 40% and 20% substrate consistency,respectively. The enzyme loading used in Jorgensen’s study(7 FPU per gram of dry matter) is equivalent to 13.5 FPU per gramof cellulose as the substrate has a cellulose content of 52%. A highb-glucosidase dosage was supplied (ratio of 5:1 between CBU andFPU) in Jorgensen’s study, while in our study, a CBU:FPU ratio of4:1 was applied. In another recent study, Cara et al. (2007) carriedout enzymatic hydrolysis of pretreated olive tree biomass at a sub-strate consistency of up to 30%. The study reported the productionof 73 g/L of glucose after 72 h hydrolysis of delignified LHW (liquidhot water)-pretreated olive tree biomass. Cara’s study employed15 FPU cellulase with 15 CBU of b-glucosidase per gram of sub-strate on delignified LHW-pretreated olive tree biomass, whichhas a cellulose content of 56.7%. It is acknowledged that the sub-strates used in these two studies are different from UBHW andOPP which contain higher cellulose content.

Comparing UBHW and OPP, it was surprising to find that OPPdemonstrated a better hydrolysability at high consistency thanUBHW. The initial high cellulose content likely contributed to thehigh glucose concentration observed during OPP hydrolysis.Although, a similar cellulose-to-glucose yield (84% vs. 85%) was ob-tained after hydrolyzing UBHW and OPP at high consistency for96 h, OPP demonstrated a higher initial reaction rate during hydro-lysis. The initial velocity (Vi) calculated based on the reaction rateobtained from the first hour of hydrolysis of OPP, is 0.204 g/g/h

(gram of glucose produced from 1 g of cellulose in an hour),whereas the Vi from UBHW is 0.146 g/g/h. In order to understandthis difference, we further measured the viscosity and determinedthe DP (degree of polymerization) of cellulose present in both sub-strates. It was found that OPP cellulose has extremely low viscosity(2.67 mPa s) and DP (207), while UBHW cellulose has a viscosity of40.3 mPa s and a DP of 1643. OPP was obtained from pulping woodchips at high temperature under acidic conditions. It is likely thatthis pretreatment significantly degraded the cellulose macromole-cules making them susceptible to cellulase hydrolysis. However,from the results obtained, it was not apparent how the xylan inUBHW and acetone extractives in OPP affected hydrolysability ofthe substrates at high consistency. The effect of these componentson high consistency hydrolysis is currently under investigation.

High consistency hydrolysis not only significantly reduces thecapital cost for installation of a hydrolysis vessel; more impor-tantly it also provides a concentrated glucose stream for the subse-quent fermentation which will lead to a significant savings in thedistillation cost. A previous study indicated that increasing glucoseconcentrations from 1.5% to 16% can lead to a sixfold reduction inthe distillation cost after fermentation Zacchi and Axelsson(1989)). S. cerevisiae can typically tolerate a high ethanol concen-tration up to 180 g/L (Lin and Tanaka, 2006). As seen from the re-sults, this industrial adapted S. cerevisiae effectively fermented thetwo pure glucose controls and assimilated most of the glucosewithin 12 h. The increase in initial glucose concentration from110 g/L (pure glucose control for UBHW) to 150 g/L (pure glucosecontrol for OPP) lowered the final ethanol yield from 0.44 g/g(gram of ethanol per gram of glucose) to 0.42 g/g which is probablydue to the inhibition effect from initial glucose concentration (Tha-tipamala et al., 1992). It was found that substrate (glucose) inhibi-tion starts to have a significant impact on fermentation yield atglucose concentrations over 150 g/L. The overall product yield isaround 0.45 when the substrate concentration is below 150 g/L.However the fermentation yield decreases linearly with in the in-crease in substrate concentration once the substrate concentrationexceeds 150 g/L (Thatipamala et al., 1992).

A slower initial rate compared to pure glucose controls was ob-served during fermentation of lignocellulosic hydrolysates. This isprobably due to the initial adjustment of yeast cells to the hydroly-sate conditions. The amount of potential inhibitors, including aceticacid, phenolic compounds, furfural and hydroxymethylfurfuralwere determined. As shown in Table 3, there is an appreciableamount of acetic acid and phenolic compounds present in bothhydrolysates with negligible amounts of furfural and hydroxymeth-ylfurfural. However, the final ethanol yield obtained from the twohydrolysates was not compromised by the presence of these inhib-itors. In fact the ethanol concentration obtained from this study isthe highest that has been ever reported in the literature from ligno-cellulose based feedstock. It is also interesting to note that,although the OPP hydrolysate has a higher acetic acid and total phe-nolic content than UBHW, its exhibits a superior fermentability. Forexample, the maximum ethanol yield was obtained after 24 h offermentation of OPP hydrolysate, while it took 36 h to reach to eth-anol production peak in UBHW hydrolysate. The organic acids andphenolic compounds are generally considered as inhibitors to yeast.However, the concentration effect of these compounds on yeast fer-mentation is still under debate. For example, the presence of


100 mmol/L of acetic acid in the media was shown to increaserather than decrease the ethanol yield from approximately 0.41–0.45 g/g by S. cerevisiae (Larsson et al., 1999). Also different typesof phenolic compounds could exhibit different effects on S. cerevisi-ae fermentation (Palmqvist and Hahn-Hagerdal, 2000). As men-tioned, the S. cerevisiae strain used in current study has beenadapted to spent sulphite liquor which has a high phenolic com-pounds and acetic acid content. Therefore, it is not surprising thatthe yeast can generate a high ethanol yield from sugars in thetwo hydrolysates which have a relatively low phenolic compoundsand acetic acid content compared to typical spent sulphite liquor.

5. Conclusions

We have demonstrated that a peg mixer, commonly employedin pulping processes, can be used for successful high consistencyhydrolysis of lignocellulosic substrates. Hydrolysis of unbleachedhardwood pulp (UBHW) and organosolv pretreated poplar (OPP)at 20% substrate consistency led to a high glucose concentrationin the hydrolysate. Enzymatic hydrolysis of OPP for 48 h resultedin a hydrolysate with 158 g/L of glucose content. This is the highestglucose concentration that has been obtained from enzymatichydrolysis of lignocellulosic substrate. Fermentation of UBHWand OPP hydrolysates with high glucose content led to high ethanolconcentrations in the final fermentation broth, much higher thanthose reported in previous literature. Applying pulping equipment,which is typically designed to handle high and medium-consis-tency fibrous materials, for high consistency hydrolysis provides apractical means to overcome the rheological problems encounteredin laboratory shake flask experiments. This brings the biomass con-version research a step closer to industrial implementation. Thehigh consistency hydrolysis and fermentation presents a new ap-proach to lignocellulose hydrolysis and fermentation and opensup new windows to examine substrate–enzyme interactions.

Acknowledgement

We would like to thank Novozymes North America Inc. for pro-viding enzymes. Funding was provided by Natural Resources Can-ada and the National Science and Engineering Research Council ofCanada.

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