Producing high sugar concentrations from loblolly pine using wet explosion pretreatment

Bioresource Technology 121 (2012) 61–67

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Bioresource Technology

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Producing high sugar concentrations from loblolly pine using wetexplosion pretreatment

Diwakar Rana, Vandana Rana, Birgitte K. Ahring ⇑Bioproducts, Sciences and Engineering Laboratory (BSEL), Washington State University, Richland, WA 99354-1671, USA

h i g h l i g h t s

" Wet explosion pretreatment for thesugars release from loblolly pine.

" Conducted pretreatment at high drymatter (25%) of whole pretreatedslurry.

" Achieved highest sugar yields (96%)ever reported from loblolly pine.

0960-8524/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.biortech.2012.06.062

⇑ Corresponding author. Address: Center for Bioproington State University, 2710 University Drive, Richlan+1 509 372 7682; fax: +1 509 372 7690.

E-mail addresses: [email protected], [email protected]: http://www.tricity.wsu.edu/bsel (B.K. Ahring

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 March 2012Received in revised form 21 June 2012Accepted 23 June 2012Available online 1 July 2012

Keywords:Loblolly pineLignocellulosic biomassWet explosionPretreatmentSugar release

a b s t r a c t

We present quantitative analysis of pretreatment for obtaining high conversion and release of sugarsfrom loblolly pine. We use wet explosion (WEx): wet oxidation followed by steam explosion and enzy-matic hydrolysis (EH) at high dry matter to solubilize sugars. WEx was conducted at 25% (w/w) solidsin presence of oxygen at pressures 6.5–7.2 bar, temperatures 170–175 �C and residence time from 20to 22.5 min. EH of pretreated samples was performed by Cellic� Ctec2 (60 mg protein/g cellulose) andCellic� Htec2 enzymes (10% of Ctec2) at 50 �C for 72 h. At the optimal WEx condition 96% celluloseand nearly 100% hemicellulose yield were obtained. The final concentrations of monomeric sugars were152 g/L of glucose, 67 g/L of xylose, and 67 g/L of minor sugars (galactose, arabinose and mannose). Com-pared to previous work WEx seems to be superior for releasing high concentrations of monomeric sugars.

� 2012 Published by Elsevier Ltd.

1. Introduction

Pretreatment of softwood with the intent to release high con-centrations of sugars has been identified as a bottleneck for biore-fineries based on woody materials. The difficulty for accessing thebiomass after pretreatment has been attributed to a high lignincontent (Söderström et al., 2003) and crystallinity of cellulose(Bansal et al., 2010). Softwood is abundantly available in major

Elsevier Ltd.

ducts and Bioenergy, Wash-d, WA 99354-1671, USA. Tel.:

du (B.K. Ahring).).

parts of the world including the United States (Smith et al., 2002)and can be an excellent alternative feedstock for biofuels produc-tion in the future.

An optimized pretreatment of softwood would produce concen-trated sugars stream with a high yield which further is suitable fordownstream processing into liquid transportation fuels (LTF). Thetwo pathways that can lead to LTF from the released sugars in-cludes biochemical route includes fermentation (Regalbuto,2009), and catalytic route that includes aqueous phase reformingfollowed by catalytic conversion (Huber et al., 2005). Both technol-ogies uses concentrated sugars streams as a feedstock in order tobe economically viable. The concentrated sugar stream puts fewerburdens on equipment and catalysts for catalytic route, and thesimilar justification is good for biochemical pathway. Concentrated

62 D. Rana et al. / Bioresource Technology 121 (2012) 61–67

sugars lower the heating requirements (Kristensen et al., 2009b)which further lower the operating cost. Furthermore, it increasesthe volumetric efficiency of the equipment (Kristensen et al.,2009b) which again lowers the capital cost. Producing concen-trated sugars stream requires that enzymatic hydrolysis is per-formed at high dry matter (Georgieva et al., 2008). Since, higherdry matter has been reported to result in inhibition of enzymes(Georgieva et al., 2008; Kristensen et al., 2009a), it is importantto use a pretreatment process which only produces low concentra-tions of inhibitors as we otherwise will be wasting biomass and en-zymes without harvesting the maximum outcome. Last butcertainly not the least, pretreatment not only accounts for one ofthe most expensive unit operation in lignocellulosic biorefinerybut also affect the cost of downstream processes (Yang and Wy-man, 2008). In a biorefinery setting, various other products aregenerated besides biofuels such as ethanol that include hydrogenfrom xylose fermentation, methane from the anaerobic digestionof ethanol free effluent, liquid fertilizer after anaerobic digestion(Ahring and Westermann, 2007). It is, therefore, important not tosettle for any process which will not lead to a high production yieldof high value products – in the present study it is high sugar yield.

Several researchers tried to overcome the recalcitrance of soft-wood to release the monomeric sugars. Martínez et al. (1997) con-ducted the autohydrolysis, one-step pretreatment followed byenzymatic hydrolysis and obtained 44% glucose yield. Stenberg etal. (1998) attempted the use of SO2 addition together with steampretreatment and obtained 75% sugars yield at 2% DM. Two yearslater 75% maximum sugars yield were reported by Nguyen et al.(2000). However, it was conducted at 1% DM, which basically hasno merit for economic conversion into biofuels. In 2003,Söderström et al. (2003) made some improvement and obtained77% sugars yield. Zhu et al. (2009) reported above 90% glucoseyield from cellulose via SPORL pretreatment, involving impregna-tion with sodium bisulfite before pretreatment followed bymechanical milling. Enzymatic hydrolysis was, however, done at2% DM. One possible reason for the enzymatic hydrolysis at lowerDM is to see the maximal extent of hydrolysis in laboratory scale.Another possible reason for the enzymatic hydrolysis at lower drymatter could be attributed to the incompetent performance ofenzymes with higher DM’s. The enzyme efficiency has been im-proved significantly over the past few years. This allows for theuse of much higher dry matter concentration in biorefineries of vi-tal importance for the economics (Peterson et al., 2008).

We selected WEx pretreatment for mainly three reasons (1) thispretreatment attack the lignin structure which has been found toallow for low enzyme usage; (2) no chemical is added to run theprocess except water and oxygen/air and (3) no need to recoverand recycle the added chemicals after the pretreatment. At tem-peratures around 170 �C, oxygen generates heat that lowers theheating requirements (Ahring et al., 1996). WEx can also operateat higher dry matter concentration (Ahring and Munck, 2009)which further improves the process economy by giving high con-version at lower water consumption. As far as greener environ-ment is concerned, WEx does not require the use of hazardouschemicals and their subsequent recycling and recovery which addsan extra expense and complication to the process. For instance,AFEX requires a sophisticated system for recovery and recyclingof ammonia (Sendich et al., 2008) and SPROL requires the removalof sulfite or bisulfite (Zhu et al., 2010). Dilute acid pretreatment re-quires the presoaking or impregnation with SO2. As no SO2 recy-cling is encountered during any of the descriptions of dilute acidpretreatment found in the literature the result will be a pretreatedmaterial containing sulfur which potentially can be released to theatmosphere and result in acid rain (Lynch et al., 2000). Anotherproblematic characteristic of these chemicals is the fact that theycatalyze the degradation of the sugar monomers (Xiang et al.,

2004). As large portions of pretreated biomass will be hot (50–100 �C) for 1–2 h after pretreatment, the degradation at a slowerrate will continue during storage of pretreated biomass in thetanks before the enzymatic hydrolysis. WEx provides flexibilityto produce sugars streams to suit various routes – catalytic routeand biochemical route. The catalytic route will be sensitive to forinstance sulfur compounds but will have no problems accommo-dating reasonable quantity of HMF and furfural. The biochemicalroute will, however, be sensitive towards the presence of HMFand furfural, as these compounds are microbial inhibitors. The syn-ergistic effect of acetic acid (1.5 g/L), HMF (0.05 g/L) and furfural(0.15 g/L) has been found to inhibit the ethanol fermentation byPichia stipitis (Bellido et al., 2011). WEx can be tailored to producea hydrolysate which fits exactly the targeted conversion processi.e. for the biochemical route, lower pretreatment severity andfor the catalytic route higher pretreatment severity would producethe desired results. This of course had to be determined by rigorousoptimization studies for the targeted yields of sugars and microbialinhibitors.

In the present paper we will examine the process conditions forreleasing the highest amount of sugars from loblolly pine withminimum sugar degradation products. These findings will be ofinterest to researchers and engineers developing infrastructureready drop-in fuels via catalytic route or fermentation route, andinform the field of green and sustainable chemistry.

2. Methods

2.1. Raw material preparation

Loblolly pine chips were kindly provided by Iowa State Univer-sity, Ames, IA in early October, 2010. The chips were milled to2 mm particle size with a Retsch cutting mill SM 200 (RetschInc., PA, USA) and kept in buckets at room temperature prior tothe pretreatment. A portion of the raw material was milled to1 mm particle size and used for the compositional analysis. Thecompositional analysis was performed in replicates.

2.2. WEx pretreatment at WSU pretreatment pilot plant

Loblolly pine was pretreated in a pretreatment system as shownin Fig. 1. The system consisted of 10 L pretreatment reactor(equipped with hot oil heater and stirrer), and 100 L flash tank(equipped with a vapor outlet and chiller). The reactor was loadedwith loblolly pine and water to a total dry matter concentrationw/w of 25%. The reactor was then closed and oxygen was introducedinto the reactor at a pressure between 6.5 and 7.2 bar. After intro-ducing the oxygen, the pretreatment reactor was heated until thetemperature inside the reactor was between 170 and 175 �C. Thereactor heat-up time varied between 5 and 6 min. At the end ofwet oxidation reaction (after a reaction time of 20–22.5 min), theloblolly pine was flashed into 100 L flash tank connected with thereactor (see Fig. 1). Even though, temperature and time variationdoes not seem large, however, their combined effect in terms ofseverity is different. The sudden drop in pressure disrupted thestructure of loblolly pine making it more accessible for the enzymes.By applying the chiller system the temperature was lowered to 4 �Cwithin minutes to stop any further pretreatment reaction. Loblollypine samples resulting from the three different pretreatment runsfollowed by enzymatic hydrolysis are designated as LP1, LP2, andLP3. The conditions used for WEx pretreatment are as shown in Ta-ble 1. The conditions were chosen based on a long range of labora-tory runs made to define the target parameters for the presentstudy. The experiments were performed at neutral pH as no chemi-cals were added. For LP1 preparation, pretreatment reactor was

Sampling Point 1: Compositional Analysis of Raw Biomass

Oxygen Water

pH adjustmentEnzymes

Sampling Point 2: Sugars Analysis inthe pretreated solids and liquid

Released Sugars for further processing

Sampling Point 3: Sugars Analysis inthe enzymatically hydrolyzed solids and liquid

Pretreated Slurry

Chilled Water Outlet

Chilled Water Inlet

Hot Oil Outlet

Hot Oil Inlet Milled BiomassRaw Biomass Milling Pretreatment Reactor 10 L

Flash Tank 100 L

Bioreactor 15 / 100 L

Fig. 1. Process flow diagram of the pretreatment system.

Table 1Process conditions for WEx in this study with TS 25%.

SampleNo.

Oxygen pressure,PO2 (bar)

Reaction time, tR

(min)Reactiontemperature, T (�C)

LP1 6.5 22.5 175LP2 7.2 22 170LP3 7.2 20 170

D. Rana et al. / Bioresource Technology 121 (2012) 61–67 63

charged with 1100 g loblolly pine and 2990 ml water. Three suchbatches were mixed together thoroughly to produce Sample LP1.For the Sample LP2 preparation, pretreatment reactor was chargedwith 1700 g loblolly pine and 4560 ml water for the first batch andfor the three batches the reactor was charged with 1900 g of loblollypine and 5092 ml of water. The variation between the four batches isneglected because of a tight control over the process parametersduring WEx. The above four batches were mixed together thor-oughly to produce Sample LP2. For the Sample LP3 preparation, pre-treatment reactor was charged with 1700 g loblolly pine and4617 ml water. Four such batches were mixed together thoroughlyto produce Sample LP3.

2.3. Enzymatic hydrolysis

Enzymatic hydrolysis of the LP1 pretreated slurry was per-formed in a 15 L Applikon bioreactor at 50 �C, 950–1000 rpm for72 h. Enzymatic hydrolysis of the LP2 and LP3 pretreated slurrieswere performed in a 100 L bioreactor at 50 �C, 950–1000 rpm for72 h. The entire batches of LP1, LP2 and LP3 were enzymaticallyhydrolyzed. A mixture of two enzymes Cellic� Ctec2 and Htec2(kindly provided by Novozymes, USA) was used to determinethe convertibility of cellulose and hemicellulose into monomers.Cellic� Ctec2 at an enzyme loading of 60 mg protein/g of celluloseand Cellic� Htec2 at an enzyme loading of 10% of Cellic� Ctec2were added to the pretreated slurry. The total solids (TS) in the

pretreated slurry were 25% before the pH was adjusted to 5.0using 4 M KOH. The minor changes in the dry matter content ofpretreated slurry during the pretreatment flashing were neglectedbecause, the system was closed and it was cooled by chiller. Athigh dry matter content, the stirring was found to be difficult dur-ing the first hours after which the material got fully liquid andeasy to mix independent on the hydrolysis reactor used. Sampleswere withdrawn every 24 h until the hydrolysis time of 72 h, cen-trifuged, filtered (0.45 lm) and analyzed in duplicate for sugarsanalysis.

2.4. Analytical methods

A portion of slurry obtained after pretreatment of loblolly pinewas separated into solid and liquid fraction for sugars and ligninanalysis. The separation was performed with a bench top centri-fuge (Thermo Scientific, CR4i Jouan Centrifuge, 4750 rpm,10 min). After separation the liquid fraction was filtered through0.45 lm syringe filter whereas the solids were washed and driedin an incubator at 40 �C for 24 h before characterization. Total sug-ars, sugars degradation products, ash and lignin of pretreated andenzymatically hydrolyzed solids and liquids were determinedaccording to Laboratory Analytical Procedures (LAP) establishedby National Renewable Energy Laboratory (Sluiter et al., 2011).Sugars analysis were performed by high performance liquid chro-matography (HPLC) refractive index (IR) equipped with an AminexHPX-87P column (Bio-Rad Laboratories, CA, USA) at 83 �C withdeionized water (Thermo Scientific, Barnstead Nanopure, IA, USA)as an eluent with a flow rate of 1.0 ml/min. Sugar degradationproducts (HMF and Furfural) and short-chain organic acid (aceticacid) were measured by high performance liquid chromatography(HPLC) refractive index (IR) equipped with an Aminex HPX-87Hcolumn (Bio-Rad Laboratories, CA, USA) at 60 �C with 4 mMH2SO4 as an eluent with a flow rate of 0.6 ml/min.

Table 2Compositional analysis of raw loblolly pine.

Biomass % DM Standard deviation

Glucan 35.97 0.46Xylan 7.54 0.61Galactan 2.47 0.40Arabinan 1.57 0.16Mannan 8.15 0.23Lignin 30.65 0.78Ash 0.77 0.05Extractives 6.45 0.08

64 D. Rana et al. / Bioresource Technology 121 (2012) 61–67

2.5. Sugar yield determination for high solids hydrolysis

The glucose yield is determined by Eq. (1) which is a modifiedversion of equation suggested by Kristensen et al. (2009b) for highsolids enzymatic hydrolysis.

%Yield ¼ ðGluEH;LÞ þ 1:0526� ðCelEH;LÞ1:111� FcelluloseRB � ðini:solÞ � 100� fCorrection ð1Þ

where (GluEH,L) is the glucose concentration (g/L) in enzymatichydrolysate liquid, (CelEH,L) is the cellobiose concentration (g/L) inenzymatic hydrolysate liquid, FcelluloseRB is the fraction of cellulosein the raw biomass as determined by compositional analysis and(ini.sol) is the initial solids concentration (g/L) at which enzymatichydrolysis is performed and fCorrection is the yield correction factorat high dry matter. The yield correction factor eliminates the over-estimation of sugars yield as it considers (1) change in volume dur-ing enzymatic hydrolysis, and (2) non-uniformity of specific gravity

A

B

Fig. 2. Liquid fraction analysis in grams per liter, (A) sugars released duri

of liquid and biomass in enzymatic hydrolysate (Kristensen et al.,2009b). Ideally, correction factors must be determined for each sub-strate (Kristensen et al., 2009b). However, in the absence of suchdata for loblolly pine, we have assumed the similar curve for lob-lolly pine, which is a conservative approximation as the trend ofthe correction factor curve gets steeper as the cellulose concentra-tion increases.

3. Results and discussion

3.1. Raw material

The chemical composition of the loblolly pine is shown in Table2 (showed glucan 35.97%, xylan 7.54%, galactan 2.47%, arabinan1.57%, mannan 8.15%, and lignin 30.65%). The amount of acetatepresent in the raw biomass was found to be 2.86% DM. The compo-sition of raw material was used to quantify the effectiveness of ourpretreatment by calculating the percentage cellulose conversionafter pretreatment and enzymatic hydrolysis.

3.2. WEx pretreatment

The amount of sugars released in the liquid phase after WExpretreatment at the three different conditions is shown inFig. 2A. Amount of hemicellulose removed from the substratewas comparable for LP1 and LP3. However, approximately 40%more hemicellulose was removed for LP2, as can be revealed byFig. 2A. Even though, the pretreatment conditions of LP2 and LP3are in close range, it is their combined effect (severity) that

ng pretreatment, and (B) sugars released after enzymatic hydrolysis.

D. Rana et al. / Bioresource Technology 121 (2012) 61–67 65

produced different sugar yields. It was found through a range ofexperiments done on loblolly pine (data not shown) that 20 minor less of pretreatment time at 170 �C is not sufficient to open itsstructure. However, as soon as the pretreatment time is increasedby two more minutes, significant changes in the material wasfound leading to improvement in the release of sugars. This sug-gests that WEx pretreatment is chemical as well as mechanical innature in which the cell walls within the biomass resists the heatand oxygen for a certain time and then ultimately break more openand thereby easing the penetration of the enzymes. The movementof hemicellulose from the biomass into liquid phase led to the in-crease in glucan content in the pretreated solids as shown inFig. 3A. Glucan content was comparable for LP1 and LP2. However,LP3 showed approximately 35% lower glucan content in compari-son with LP1 and LP2. This lower glucan content suggests that low-er concentrations of the hemicellulose fractions was moved intoliquid phase during the WEx pretreatment, which can be furtherinterpreted from Fig. 2A. The content of xylan, galactan, arabinan,and mannan in the solid fraction decreased with the decrease inreaction time as shown by Fig. 3A. This manifests the need for long-er pretreatment time (22 min) in order to hydrolyze the hemicellu-lose. Lignin content resulting from the oxidative reduction wascomparable for the pretreatment conditions investigated due tothe close range of oxygen pressures used (6.5–7.2 bar) as shownin Table 1.

The concentration of furfural, HMF and acetic acid, whichare potential microbial inhibitors, was comparable for the

0

10

20

30

40

50

60

Com

posi

tion

% D

M

A

0

10

20

30

40

50

60

LP1 L

Com

posi

tion

% D

M

B

Fig. 3. Solid fraction composition as a percentage of dry matter, (A) sugars and lignin comhydrolysis.

pretreatment conditions investigated and was found to be withinacceptable range (Palmqvist et al., 1999). The production of aceticacid was attributed to the hydrolysis of acetyl groups attached toxylan chain (Mittal et al., 2009). The formation of acetic acid low-ered the pH of the pretreated slurry to approximately 2.0 (data notshown). The amount of furfural and HMF decreased as the reactiontime was decreased as shown in Fig. 4A. The amount of HMF waslower than furfural for the pretreatment conditions investigated,which agrees with the previous studies on pretreatment (Palmq-vist et al., 1999).

The amount of hydrolyzed hemicellulose decreased as theoxygen pressure (Table 1, LP1) or the reaction time (Table 1, LP3)is decreased. Lower reaction time and lower oxygen pressure bothcontributed to a lower production of microbial inhibitors as shownin Fig. 4A. This is in agreement with previously published work(Söderström et al., 2003) which shows that increasing the severityof the process produces more sugar degradation products. Theamount of hemicellulose solubilized dropped drastically at a com-parable reaction time with 0.7 bar dip in oxygen pressure from LP2to LP1 as shown in Fig. 2A.

3.3. Enzymatic hydrolysis of pretreated slurry

We investigated the enzymatic digestibility of the pretreatedslurry of LP1, LP2 and LP3 using conditions described in Section2.3. The results showed almost complete hydrolysis within 72 hwhich is attributed to opening of the structure of biomass during

P2 LP3

Glucan

Xylan

Galactan

Arabinan

Mannan

Lignin

position after pretreatment, and (B) sugars and lignin composition after enzymatic

0

0.5

1

1.5

2

2.5

3

3.5

Con

cent

rati

on g

/L

A

0

3

6

9

12

15

18

LP1 LP2 LP3

Con

cent

rati

on g

/L

BHMF

Furfural

Acetic Acid

Fig. 4. Sugar degradation products formed in liquid phase in grams per liter, (A)sugar degradation products formed during pretreatment, (B) sugar degradationproducts formed after enzymatic hydrolysis.

0

50

100

LP1 LP2 LP3

Yie

ld %

Cellulose

Hemicellulose

Fig. 5. Cellulose and hemicellulose conversion after WEx pretreatment followed byenzymatic hydrolysis for LP1, LP2 and LP3.

66 D. Rana et al. / Bioresource Technology 121 (2012) 61–67

WEx pretreatment along with the rather high dose of enzymesused during enzymatic hydrolysis. Recent experiments in our lab-oratory have shown that the same positive results can be obtainedwith lower enzyme loads using longer hydrolysis times.

The amount of sugars released in the liquid fraction is shown inFig. 2B. The overall sugars concentration takes into account dilu-tion due to 4 M KOH solution, and amount of enzymes added (cel-lulase and hemicellulase). The sugars present in the enzymesolutions has been subtracted from the sugar yields calculations.The highest sugars yield was achieved from the pretreated LP2

with 152 g/L of glucose, 67 g/L of xylose and 68 g/L of minor sugars,which corresponds to 96% cellulose and 105% hemicelluloseconversion (Fig. 5). We also achieved good sugar yields with LP1as depicted in Fig. 2B. However, LP3 released less sugars attributingto insufficient pretreatment, suggesting that 20 min of pretreat-ment time is not enough for wet explosion of loblolly pine.

The composition of the separated solids as shown in Fig. 3Bachieved significant hydrolysis and subsequent movement of cel-lulose through liquid phase which agrees with the amount of sug-ars measured in the liquid fraction. However, the amount of klasonlignin in the solid fraction increased slightly during enzymatichydrolysis of all three samples as shown in Fig. 3B suggesting thatenzymatic hydrolysis with cellulolytic enzymes has no effect onklason lignin solubilization. This slight increase in the klason lignincontent agrees with the previously published studies (Sannigrahiet al., 2011) in which the formation of pseudo-lignin (carbohydrateand lignin degradation products during the pretreatment) has beenfound to be responsible for the slight increase in the klason lignincontent.

The concentration of sugar degradation products formed afterenzyme hydrolysis is shown in the Fig. 4B. The amount of aceticacid, HMF and furfural was found to be within acceptable rangefor the fermentation route as defined by (Palmqvist et al., 1999).As previously stated, a catalytic route does not care about HMFand furfural as far as ‘‘aqueous phase reforming’’ for LTF productionis concerned (Huber et al., 2005).

The amount of HMF and furfural increased slightly during enzy-matic hydrolysis as shown in Fig. 4. This is attributed to dehydra-tion of monomeric sugars that were released from cellulose andhemicellulose respectively (Martín et al., 2007). The amount of ace-tic acid increased dramatically during enzymatic hydrolysis. This isdue to the hydrolysis of acetyl groups attached at the backbone ofhemicellulose (Martín et al., 2007).

The advantage of eliminating the needs of hazardous chemicalssuch as ammonia and sulfur dioxide will offer greener environ-ment which is an equally important question that science is tryingto answer to the mankind. Future research will explore the opti-mized process parameters to further improve the economics byreducing the amount of heat and oxygen required during WExpretreatment. The present study used high enzyme dosage of60 mg protein/g cellulose of Cellic� Ctec2 to investigate the maxi-mal extent of sugars released. The amount of enzymes dosage willbe optimized in our future research.

4. Conclusions

Our investigation revealed that WEx is a promising pretreat-ment method for producing high sugar yields from loblolly pine.152 g/L of glucose, 67 g/L of xylose, and 68 g/L of minor sugars, cor-responding to 96% glucose and nearly 100% hemicellulose yield,were obtained and this is the highest sugar yield ever reportedfor loblolly pine. EH was conducted at 25% TS which is a significantimprovement over the present EH studies which are often con-ducted at 2–20% TS in order to prevent enzyme inhibition.

Acknowledgements

This work was financially supported by the National AdvancedBiofuels Consortium and Department of Energy, Grant Award No.ZFT04064401.

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