Bioresource Technology 110 (2012) 264–272

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Bioresource Technology

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Ethanol production from Eucalyptus plantation thinnings

S. McIntosh a, T. Vancov a,b,⇑, J. Palmer a, M. Spain a

a NSW Department of Primary Industries, Wollongbar Primary Industries Institute, NSW, Australiab Primary Industries Innovation Centre, University of New England Armidale, NSW, Australia

a r t i c l e i n f o

Article history:Received 25 October 2011Received in revised form 18 January 2012Accepted 19 January 2012Available online 28 January 2012

Keywords:Cellic� Ctec 2Enzyme saccharificationEucalyptus dunniiEthanol fermentationPretreatment

0960-8524/$ - see front matter Crown Copyright � 2doi:10.1016/j.biortech.2012.01.114

⇑ Corresponding author. Address: Industry & InveHighway, Wollongbar, 2477 NSW, Australia. Tel.: +613264.

E-mail address: tony.vancov@industry.nsw.gov.au

a b s t r a c t

Conditions for optimal pretreatment of eucalypt (Eucalyptus dunnii) and spotted gum (Corymbia citriodora)forestry thinning residues for bioethanol production were empirically determined using a 33 factorialdesign. Up to 161 mg/g xylose (93% theoretical) was achieved at moderate combined severity factors(CSF) of 1.0–1.6. At CSF > 2.0, xylose levels declined, owing to degradation. Moreover at high CSF, depoly-merisation of cellulose was evident and corresponded to glucose (155 mg/g, �33% cellulose) recovery inprehydrolysate. Likewise, efficient saccharification with Cellic� CTec 2 cellulase correlated well withincreasing process severity. The best condition yielded 74% of the theoretical conversion and was attainedat the height of severity (CSF of 2.48). Saccharomyces cerevisiae efficiently fermented crude E. dunnii hydro-lysate within 30 h, yielding 18 g/L ethanol, representing a glucose to ethanol conversion rate of 0.475 g/g(92%). Based on our findings, eucalyptus forest thinnings represent a potential feedstock option for theemerging Australian biofuel industry.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Ethanol from renewable feedstocks is regarded as an ideal sup-plement and/or creditable replacement for fossil fuel. Typically,starch- and sugar-producing crops have been exploited as feed-stocks, but owing to its abundance and lower intrinsic value, ligno-cellulosic biomass is a far better resource for biofuel production. Ofthe diverse array of available lignocelluloses, trees are one of thebetter feedstock options, partly due to their higher cellulose den-sity and compositional uniformity. Moreover, trees possess a ligno-cellulosic energy conversion factor of 16 (compared to 1 and 8 forcorn and sugarcane, respectively), and can be grown on marginalland, thereby minimizing encroachment on food crop terrain(Fenning et al., 2008). Forest harvested residues represents apotentially large (4.2 Mt per annum) source of biomass in Australia(National Association of Forest Industries, 2005). Coupled with 2Mt of residues from timber-processing facilities means that around6.5 Mt of biofuel feedstock maybe available. Australia in particularhas a unique resource in hardwood species like eucalypts due to adiverse collection of approximately 700 native species. The geneticresources for this group remain largely Australian but eucalyptshave successfully proliferated in many parts of the world, particu-larly in many marginal environments that are not suited to foodcrop production.

012 Published by Elsevier Ltd. All r

stment NSW, 1243 Bruxner2 6626 1359; fax: +61 2 6628

(T. Vancov).

A potential drawback to using eucalypts or hardwoods as a feed-stock is the degree of difficultly associated with liberating cellulosefrom its lignin seal. This is by far the most costly step of the process,strongly influencing success and feasibility of prior and subsequentoperations (Wyman, 2007). Most pretreatment options are basedon physical or chemical approaches and some incorporate both toincrease efficiency. The current method of choice for a number oflignocellulose to ethanol operations is dilute acid hydrolysis whichsubjects biomass to low pH in combination with moderate to hightemperatures (Mosier et al., 2005). Dilute acid treatment predomi-nantly solubilises the hemicellulose fraction and disrupts thecrystalline structure of cellulose fibrils (Lee et al., 1999). Cellulasehydrolysis is thus enhanced by the resulting increase in porosityand overall surface area. Most biomass sources have been used asfeedstock and respond well to dilute acid pretreatment and enzymehydrolysis (Lloyd and Wyman, 2005). Few have reported usingdilute acid to pretreat wood from select Eucalyptus specieslet alone typical plantation thinnings. Thinnings encompass treesof low value that are selectively removed from plantations as a partof good crop management operations.

Therefore the aim of this work was to determine if dilute acidpretreatment could effectively be used to convert eucalypt forestthinnings into ethanol. The investigation is best summarised by:(1) determining the role of various pretreatment parameters andtheir effect on sugar solubilisation and generation of degradationor inhibitory compounds; (2) assessing enzymatic saccharificationof pretreated material and changes in sugar composition and therelationship to pretreatment parameters; and (3) evaluating thefermentation potential of sugar hydrolysates. Understanding these

ights reserved.

S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272 265

key elements will provide a set of tools for optimising the conver-sion of eucalypt residues to sugar streams. Measuring the effects ofage (6 vs 10 years) and species will be used by way of example asthis age range reflects current plantation thinning operations.Finally, besides the feedstock novelty factor, this is amongst thefirst papers to report the use of Novozyme’s cellulase blend Cellic�

CTec 2 on pretreated hardwoods and/or eucalypts.

2. Methods

2.1. Materials

Eucalypt thinnings used in this investigation were sourced fromcommercial plantations located in the Northern Rivers region,NSW, Australia. Dunn’s white gum (Eucalyptus dunnii) (ED_6) andspotted gum (Corymbia citriodora spp. Variagata) (CCV_6) wereboth aged 6 years and were sourced from the same plantation,whereas, the 10-year-old Dunn’s white gum (ED_10) was sourcedfrom an adjoining plantation grown in similar soil and climaticconditions. All samples were supplied by Southern Cross Univer-sity and Forests CRC.

A composite sample of 10 trees for each species and age combi-nation was used throughout the study. Selected trees representedvariation in the plantation at time of sampling. A 1 m long billetoriginating at breast height (1.3 m) was taken from each tree anddebarked. Individual billets were chipped and bagged separately.Wood chips were oven-dried at 50 �C for 96 h. Approximately5 kg of material from each of the 10 trees was combined to gener-ate representative samples and ground in a rotary mill (ThomasWiley Laboratory Mill). The grinds were sieved to select for a par-ticle size of61.4 mm (ASTM No. 14 sieve) and P1.0 mm (ASTM No.18 sieve). Milled material was stored at room temperature in air-tight containers and dried in a laboratory oven at 50 �C for 24 hprior to use.

All chemicals used were of reagent grade or higher and pur-chased from Sigma Chemical Co. (St. Louis, MO). Cellulase (Cellic�

CTec 2) was kindly supplied by Novozymes (Bagsværd, Denmark).

Table 1Experimental conditions of E. dunnii pretreatments and calculated combined severityfactors (CSF = log10[t � exp(T � 100/14.75)] � pH).

Experiment # H2SO4 (%) Process time (min) Temp. (�C) CSF pH

1 0 2 175 �2.39 4.902 0 2 185 �1.12 3.923 0 2 195 �0.83 3.934 0 5 175 �1.98 4.895 0 5 185 �0.46 3.666 0 5 195 �0.01 3.517 0 7 175 �0.89 3.948 0 7 185 �0.30 3.659 0 7 195 0.37 3.2710 0.25 2 175 1.03 1.4811 0.25 2 185 1.39 1.4112 0.25 2 195 1.60 1.5013 0.25 5 175 1.25 1.2614 0.25 5 185 1.72 1.4815 0.25 5 195 1.94 1.5616 0.25 7 175 1.55 1.5017 0.25 7 185 1.88 1.4718 0.25 7 195 2.31 1.3319 0.50 2 175 1.26 1.2520 0.50 2 185 1.64 1.1621 0.50 2 195 2.18 0.9222 0.50 5 175 1.77 1.1423 0.50 5 185 2.01 1.1924 0.50 5 195 2.39 1.1125 0.50 7 175 1.92 1.1326 0.50 7 185 2.17 1.1827 0.50 7 195 2.48 1.16

2.2. Pretreatment

A 3 � 3 � 3 factorial design was employed to evaluate the effectof variations in acid strength, temperature and time during pre-treatment. The experimental design is summarised in Table 1. Ini-tially, 6-year old E. dunnii samples were used to optimise the rapidmicrowave-assisted pretreatment variables such as acid-catalystconcentrations, processing temperatures and residence times.Previously reported studies on xylan solubility of eucalypt species(Canettieri et al., 2007; Emmel et al., 2003; Garrote et al., 2003;Romani et al., 2010), were used to design operational conditions.

Milled wood samples at a solid loading of 10% (w/v) weresoaked in sulphuric acid (H2SO4) at 0.25% and 0.5% (v/v), alongwith water controls for 60 min at room temperature. Pretreat-ments were performed in triplicate at either 175, 185, 195 �C usinga laboratory microwave (ETHOS PLUS, 1000 Watt, Milestone Inc.,Italy) with residence times of 2, 5 and 7 min. Microwave cookingcycles included an initial 12 min heating ramp to the target tem-perature and followed by a 15 min venting to cool. Following vent-ing all reaction vessels were rapidly cooled to room temperature ina cold water bath. The pH of the pretreated material was recordedand adjusted to pH 5.2 using a saturated Ca(OH)2 solution. Sampleswere stored at �20 �C.

2.3. Enzymes and Saccharifications

Cellulase (Cellic� CTec 2) enzyme loads are quoted as % proteinon cellulose (w/w). The protein concentration of Cellic� CTec 2 wasdetermined as 160 mg protein/mL using a commercial bicinchoni-nic acid (BCA) protein assay reagent kit (Pierce Products, USA). Thedensity of the enzyme solution was 1.16 g/mL. Total cellulaseactivity of 132 FPU/mL was confirmed for Cellic� CTec 2 usingthe filter paper assay as described by Adney and Baker (2008).

For enzyme iso-dosing experiments, pretreated slurries (unsep-arated soluble and insoluble fractions) were adjusted to pH 5.2with Ca(OH)2 and dosed with 1.25% Cellic� CTec2 (protein on cel-lulose). Hydrolysis was performed at 50 �C on a rotating wheel at40 rpm for 48 h. In enzyme dose–response experiments, pretreatedsolids (insoluble fractions) were recovered by filtration throughglass microfiber filters, washed with distilled water and resus-pended in 50 mM citrate buffer pH 5.2 plus appropriately dilutedenzymes (0.208–4.17%). Hydrolysis mixtures were incubated at50 �C on a rotating wheel at 40 rpm for up to 96 h. Enzyme saccha-rified hydrolysate samples were withdrawn at 24 h time intervalsand immediately chilled on ice, centrifuged at 8000g for 5 min,filtered and stored at �20 �C.

2.4. Analysis methods

Neutral detergent fibre (NDF), acid detergent fibre (ADF), aciddetergent lignin (ADL) and acid insoluble ash (AIA) were deter-mined for untreated wood samples using the ANKOM TechnologyMethods as reported by McIntosh and Vancov (2010). The differ-ence between NDF and ADF estimates detergent hemicellulose.Detergent cellulose was calculated by subtracting the values for(ADL + AIA) from ADF. Specific carbohydrate and lignin contentsof untreated materials were determined following concentratedacid hydrolysis as described by Sluiter et al. (2008). Likewise, waterand ethanol soluble material were extracted from untreated mate-rial and quantified according to the method described by Sluiteret al. (2008) (Table 2).

The composition of hydrolysates was determined using highperformance liquid chromatography (HPLC). The separation (HPLC)system consisted of a solvent delivery system (Controller 600Waters, Milford, MA) equipped with an auto sampler (717, Waters)

Table 2Chemical composition of untreated Eucalyptus spp.

Component E. dunnii (y6)a E. dunnii (y10)a CCV (y6)a E. globulusa,b E. grandisa,c

Glucan 47.5 47.2 48.5 46.3 44.6Xylan 17.31 16.7 17.1 16.6 15.33Galactan 1.2 1.2 1.3 na naMannan 1.5 1.3 1.4 na naArabinan 0.5 0.5 0.4 0.5 naLignin (acid soluble) 3.4 4.09 4.19 3.5 25.8 (total)Lignin (acid insoluble) 27 26.2 24.36 23 naExtractives 7 8 14.5 2.4 naAsh 0.95 0.94 0.8 0.2 naHemicellulose 23.8 24.9 25.5 na naCellulose 44.3 42.2 45.8 na na

na, not available.a Composition percentages are on dry-weight basis.b Taken from (Garrote et al., 2007).c Taken from (Emmel et al., 2003).

266 S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272

and a refractive index detector (410 differential refractometer,Waters) managed by the Waters Empower� software program.

Sugars were analysed using either a Sugar-Pak 1™ (6.5 � 300mm, Waters) or an RHM-Monosaccharide (7.8 � 300 mm, Rezex)column, fitted with pre-columns, IC-Pak Ion Exclusion guard-Pak(Waters) or Carbo-H guard cartridge (Rezex), respectively. TheSugar-Pak 1™ column was maintained at 70 �C, and sugars wereeluted with a mobile phase consisting of degassed Milli-Q filteredwater containing 50 mg/L Ca-EDTA at a constant (or isocratic) flowrate of 0.5 mL/min. The RHM-Monosaccharide column was main-tained at 60 �C, and sugars, acetic acid, levulinic acid, formic acid,furfural, 4-hydroxymethylfurfural and ethanol were eluted withan isocratic mobile phase consisting of degassed Milli-Q filteredwater containing 0.005 N H2SO4 at a flow rate of 0.6 mL/min. Therefractive index detector was maintained at 50 �C for allapplications.

Peaks detected by the refractive index detector were identifiedby retention time matching, and quantified by comparison withanalytical standards analysed in the batch.

2.5. Combined severity factor

A combined severity factor (CSF) was used to integrate theeffects of hydrolysis times, temperature and acid concentration/pH into a single variable (Chum et al., 1990). The CSF is defined as:

log CSF ¼ log R00 � pH ð1Þ

R00 represents the severity factor which and is determined byEq. (2) whilst pH relates to that of aqueous solution postpretreatment.

R00 ¼ t � exp½ðTH � TRÞ�=14:75 ð2Þ

where t is pretreatment time in minutes, TH is the pretreatmenttemperature in �C, TR is the reference temperature, most often100 �C. Table 1 summarises pretreatment conditions and associatedCSF values.

2.6. Fermentations

Saccharomyces cerevisiae ATCC 58447 was used in fermentationstudies and was routinely cultured at 30 �C on YPD agar plates(20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, 16 g/L agar).Acid pretreated and enzyme saccharified E. dunnii (ED_6) hydroly-sates were adjusted to pH 5.05 using NaOH. Any un-hydrolysedfibre and gypsum (CaSO4) was removed by filtration (0.22 lm) priorto use. Inoculums for yeast shake-flask studies were prepared byselecting a single colony from YPD culture plates and inoculating

into 10 mL pre-seed medium. The media consisted of 5 mL YPDbroth + 5 mL filter-sterilised hydrolysate containing 2 g/L KH2PO4,5 g/L yeast extract, 10 g/L peptone and 1 g/L MgSO4 at pH 5.05.Following incubation at 30 �C for 24 h, a 1% (v/v) aliquot of thepre-seed culture was used to inoculate a second seed medium (com-position as above). When the optical density (OD660) reading of thesecond seed culture reached between 0.8 and 1.0, a 10% (v/v) aliquotwas used to inoculate the main fermentation medium. The yeastfermentation media consisted of filter-sterilised hydrolysate con-taining 2 g/L KH2PO4, 5 g/L yeast extract, 10 g/L peptone and 1 g/LMgSO4. The pH was 5.05 and was not adjusted. Fermentations wereconducted in 250 mL Schott Duran� bottles with a working volumeof 200 mL and were incubated at 30 �C with slow agitation. Sampleswere taken at regular time intervals for measurement of opticaldensity, biomass, glucose, and ethanol concentration.

3. Results and discussion

3.1. Compositional analysis of eucalypt species

The chemical composition of eucalyptus is influenced by manyfactors including species, geographical location, silviculture prac-tices, seasonal conditions, stage of harvest and analytical proce-dures. The compositional analysis of ED_6, ED_10 and CCV_6,summarised in Table 2, reveals a high carbohydrate contentapproaching 70% on a dry weight basis. Estimates based on deter-gent fibre analysis of test eucalypt samples revealed between 42–46% cellulose and 23–26% hemicellulose on a dry weight basis.Compositional analysis following concentrated sulphuric acidhydrolysis revealed approximately 47–48% glucan, 16–17% xylan,5% minor sugars and 30% lignins. The amount of extractives ap-pears to be the only difference between samples, with CCV_6 closeto double ED levels. No storage carbohydrates were recorded inwater soluble extractives. Composition profiles of ED and CCV arewithin reported values for other eucalyptus species (Emmelet al., 2003; Garrote et al., 2007).

3.2. Sulphuric acid pretreatment and composition of prehydrolysates

In the absence of an acid catalyst (i.e. water only) at very lowCSF values (ranging from �2.39 to 0.37), low levels of monomericxylose were recovered in pretreatment hydrolysates (prehydroly-sates), corresponding to 2% of the available xylan (data not shown).This observation does not necessarily reflect poor hemicellulosesolubilisation; on the contrary, several studies have demonstratedthat in the absence of an acid catalyst xylo-oligosaccharides arepreferentially recovered instead of monomeric C5 sugars (Garrote

S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272 267

et al., 2007; Romani et al., 2010; Yu et al., 2010). Our results do,however, demonstrate the need for an acid catalyst for cost-effec-tive recovery of monomeric pentose sugars.

It is clearly evident that under test conditions (Fig. 1A) thehemicellulose fraction has been effectively solubilised to monosac-charides with high xylose recoveries. At lower CSF (1.0–1.6) xyloserelease increased with severity; however, at higher CSF (>2.0) xy-lose levels declined, presumably due to degradation. Glucose levelsprogressively rose with severity, which is indicative of cellulosedisruption. These trends are common in several reported pretreat-ment studies (Emmel et al., 2003; Kabel et al., 2007; Lloyd andWyman, 2005). Xylose levels peaked between 1.6 and 2.0 CSF,yielding a maximum of 162 mg/g original dry material. This repre-sents a recovery of 95% of the theoretical xylose yield in monosac-charide form. Although the maximum was attained by pretreatingeucalypt samples at 185 �C for 5 min in 0.25% H2SO4, it is notewor-thy that relatively high xylose levels were recovered from a rangeof pretreatment conditions.

Although each pretreatment variable contributed to sugarrecovery, temperature had the greatest impact followed by acidstrength and then reaction time. However, degrees of flexibilityamongst pretreatment variable settings were noted in the recovery

0

50

100

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200

250

0.95 1.15 1.35 1.55

0.95 1.15 1.35 1.55

CS

CSF

Suga

r rel

ease

(mg/

g)

xyloseglucosetotal

A

0

10

20

30

40

50

60

70

Inhi

bito

rs (m

g/g)

furfuralacetic acidformic acidHMFlevulinic acid

B

Fig. 1. Xylose, glucose and total combined sugar yield (A) and yield of furfural, acetic aci(6-year-old E. dunnii) prehydrolysates (10% w/v solid load) presented as a function of C

of maximum pentose levels. Generally, elevating acid concentra-tion facilitated lower pretreatment temperatures provided resi-dency times were high. For example, when samples werepretreated in the presence of 0.5% H2SO4 for 7 min, a lower operat-ing temperature (175 �C) was sufficient to release high xylose lev-els (91%). Time had a more pronounced effect on xylose recoveriesat either end of the study’s temperature range (i.e. 175 and 195 �C).At 175 �C, xylan conversion increased 2.4-fold by extending reac-tion time from 2 to 7 min. However, at 195 �C (and to a lesser ex-tent at 185 �C) a negative response to time was evident. Extendingresidence times to 5–7 min significantly (p < 0.05) decreased xy-lose yields. This sugar loss was more pronounced in combinationwith higher acid doses (0.5%) and increased severity (CSF P 2.15).Indeed, pretreating eucalypt samples under upper most extremeconditions at CSF = 2.48 (0.5% H2SO4/195 �C/7 min) resulted in anapproximately 3-fold decrease in xylose levels. Other side chainsugars like arabinose, mannose and galactose were quantified atlow levels as pretreatment severity increased and were completelyundetectable in prehydrolysates beyond a CSF of 1.8. Therefore CSFseems to be a good predictor for xylose yields in prehydrolysates.

Depolymerisation of cellulose was also evident with the re-lease of monomeric glucose into prehydrolysates (Fig. 1A). As with

1.75 1.95 2.15 2.35

1.75 1.95 2.15 2.35

F

d, formic acid, HMF and levulinic acid (B) (mg/g original material) obtained in ED_6SF (CSF = log10[t � exp(T � 100/14.75)] � pH).

268 S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272

hemicellulose, increasing pretreatment severity led to a greaterrelease and recovery of glucose. At around CSF = 1.6, a pointwhere maximum hemicellulose solubilisation take places, thebeginning of significant cellulose depolymerisation was wit-nessed. From CSF 1.6 to 2.0, glucose release steadily grew andthereafter climbed in an exponential fashion with increasing pre-treatment severity. As with hemicellulose solubilisation, tempera-ture had the greatest impact on cellulose depolymerisation.Synergy between the variables was evident, with temperatureand acid strength conferring higher glucose yields. For example,at 195 �C and 7 min the glucose release increased 1.6-fold from100 mg/g to a maximum of 162 mg/g when acid levels wereraised from 0.25% (CSF = 2.31) to 0.5% (CSF = 2.48). Based on thecompositional analysis of ED-6, this equates to approximately34% of the cellulose fraction. Incidentally, this is greater thanother reported instances in pretreatment studies conducted oneucalypts (Emmel et al., 2003; Ramos et al., 1992). The high de-gree of cellulose depolymerisation in our study may reflect theage (immature and undeveloped at 6 years) of the sample, theeucalypt species and/or the nature of pretreatment regime. Anotable difference between solubilisation of the hemicelluloseand cellulose fractions was the fact that the latter did not reachits potential maximum under the study conditions. This area ofcellulose depolymerisation during pretreatment of eucalypt thin-nings warrants further detailed investigation. Studies bySöderström and co-workers (2003) show that up to 40% cellulosehydrolysis was possible in softwoods at high CSF’s (3.1–3.2), al-beit, at the expense of pentose sugars and accumulation of degra-dation products (i.e. furfural, formic acid). They reported thatabove CSF = 3.2, glucose yields dramatically declined through deg-radation to HMF and levulinic acid.

Acid-based pretreatments are known to liberate compoundsthat negatively impact downstream processes such as enzymehydrolysis, fermentation and waste disposal (Kim et al., 2011;Palmqvist and Hahn-Hägerdal, 2000). These inhibitory compoundsoriginate from the release of and subsequent degradation of carbo-hydrates and lignins, producing carboxylic acids, furans and phe-nolic compounds. Formation of these compounds is directlyproportional to pretreatment severity (Tengborg et al., 1998). InFig. 1B, formation of furfural, HMF, acetic, levulinic and formicacids is presented as a function of CSF.

Acetic acid was identified as the most abundant compound withlevels reaching a maximum of 60 mg/g. Unlike the other com-pounds, acetic acid initially rose with severity, however, beyondCSF = 1.6 its concentration remained stationary and was indepen-dent of pretreatment severity. This is not unexpected as there isonly a limited number of acetyl groups associated with xylan andcorrelates well with hemicellulose solubilisation and xylose re-lease (Garrote et al., 2001).

The accumulation of furfural and 5-HMF, the two major degra-dation products of pentose and hexose sugars, respectively, in-creased with pretreatment severity particularly in the presenceof 0.5% H2SO4. Treatment conditions which triggered furfural accu-mulation strongly correlated to conditions associated with thecommencement of xylose loss (CSF P 2.0). In contrast, the appear-ance of low levels of HMF does not appear to correlate with anyglucose loss during pretreatment but presumably results fromthe degradation of minor hexose sugars associated with the hemi-cellulose fraction. These results support similar observations in thepretreatment of both hard and softwoods (Emmel et al., 2003; Gar-rote et al., 2001; Söderström et al., 2003).

Sugar degradation products such as levulinic and formic acidaccumulated at low concentrations and were influenced by pre-treatment severity. The presence of formic acid suggests that pre-vailing conditions favoured complete sugar degradation. Levulinicacid is an intermediary degradation compound. Incidentally, acetic

acid at trace levels was the only potential inhibitory compound de-tected in prehydrolysates treated with water only.

3.3. Enzymatic hydrolysis of pretreated material

Pretreatment processes typically lead to high sugar contents inprehydrolysates (mostly xylose) and solid residues (cellulose)which are acquiescent to enzymatic hydrolysis. The success ofenzymatic hydrolysis generally depends in part on the pretreat-ment’s capacity to remove cellulase-specific barriers (Jeoh et al.,2007). To evaluate the effectiveness of acid pretreatment on cellu-lose to glucose conversion, pretreated slurries were subsequentlyused in fixed enzyme iso-dosing trials. To avert maximum sugar re-lease and expose important pretreatment variable(s), enzyme as-says were conducted at low doses (�10.5 FPU/g cellulose) andrelatively short hydrolysis times (48 h). Prehydrolysate and solidresidue slurries served as a basis for this work to mimic a consol-idated process of lignocellulosic ethanol production with reducedsteps and improved water utility (cellulosic ethanol estimated at6 gallons water per gallon ethanol (Aden et al., 2002)). Moreover,residual oligosaccharides in the liquid fraction should undergo en-zyme hydrolysis and thereby increase the final yield of fermentablemonosaccharides.

Initially, enzyme hydrolysis of water pretreated material(CSF 6 0.37) yielded low cellulose conversion rates with maxi-mums reaching 18%. These were marginally better than those ofun-pretreated eucalypt samples which yielded a 6% conversion(data not shown). Pretreating in the presence of an acid catalystgreatly improved enzymatic hydrolysis of cellulose and release ofmonomeric glucose. In fact, cellulose hydrolysis and hence glucoseyields correlate well with increasing process severity (Fig. 2). Thebest results were attained at maximum severity (CSF = 2.48), lead-ing to a 74% conversion of theoretical cellulose. However, pretreat-ment conditions for optimal cellulose hydrolysis may have yet tobe established, judging by the evident trend in Fig. 2 (glucose levelsproportionally rising with severity).

With respect to severity, increasing reaction temperature signif-icantly effected cellulose hydrolysis more so than acid strength.Based on the extent of xylan removal and cellulose depolymerisa-tion (high glucose levels identified in prehydrolysates), enzymatichydrolysis efficiency was expected to significantly improve withincreasing acid strength and temperature (severity). Closer exami-nation of data from saccharification only (Fig. 2) reveals glucoseplateauing around 35–42% at CSF P 1.6. This plateau effect is unu-sual and contradicts typically reported trends between severityand cellulose hydrolysis (Emmel et al., 2003; Romani et al., 2010;Söderström et al., 2003). In these studies, glucose yields declinedas pretreatment severity increased, principally through the crea-tion of degradation products and lack of cellulose availability.The fact that significant levels of HMF or levulinic acid were not de-tected in our trials precludes these degradation products as a basisfor reduced enzymatic yields. The low enzyme dose employed inthis study was also excluded as a limiting factor in the hydrolysis(refer to Fig. 3).

To determine if extended reaction times and greater enzymeloads can improve glucan hydrolysis, enzyme dose response trialswere undertaken using eucalypt slurries pretreated at a CSF of 1.9.Enzyme dosage boundaries were set accordingly: low dosage(0.208% protein on cellulose) trials embodied levels prescribed bythe manufacturer for commercially viable cellulose hydrolysis,whilst higher dosages (4.17% protein on cellulose) served as anindication of the upper limits of enzymatically accessible cellulosein pretreated mixtures. Fig. 3 shows that cellulose hydrolysis re-sponds well to increasing enzyme dose with close to theoreticalglucose yields (92%) with enzyme doses corresponding to 4.17%protein on cellulose. Lower enzyme doses, particularly those

0

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0.95 1.15 1.35 1.55 1.75 1.95 2.15 2.35

CSF

Glu

cose

yie

ld (w

t % c

ellu

lose

)

pretreatmentsaccharificationtotal

Fig. 2. Contribution of glucose (% theoretical) in hydrolysates obtained after ED_6 (6-year-old E. dunnii) pretreatment (10% w/v solid load) and enzyme saccharification(50 �C; pH 5.2; 48 h) presented as a function of CSF (CSF = log10[t � exp(T � 100/14.75)] � pH). Cellic� CTec 2 was added at a dose of 1.25% protein on cellulose. Hydrolysiswas performed at 50 �C for 48 h.

0

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0 10 20 30 40 50 60 70 80 90 100Time (h)

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cose

yie

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t % c

ellu

lose

)

0.208% CT0.416% CT0.833% CT1.390% CT2.083% CT4.170% CT0.208% CT0.416% CT0.833% CT1.666% CT

Fig. 3. Glucose yields (% theoretical) from enzymatic saccharifications (10% w/v; 50 �C, pH 5.2) of acid pretreated (CSF = 1.9) ED_6 (6-year-old E. dunnii) slurries using variousenzyme combinations. Enzyme combinations and dosage expressed as % protein on cellulose. Cellic� CTec 2 has been abbreviated as CT. Dotted lines represent enzymesaccharifications using only the insoluble fractions post pretreatment as substrates. Data represents averages of two separate experiments.

S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272 269

recommended by the supplier (60.411% protein on cellulose),proved to be ineffective in cellulose hydrolysis. Given that the sub-strate is mostly devoid of hemicellulose, suggests that enzymeactivity at low dosage was probably hindered by persistent sub-strate specific barriers such as lignin and/or enzyme inactivatorsreleased or generated during pretreatment.

In an attempt to explain these observed low glucose release,post-pretreated insoluble cellulose fractions were washed and sub-jected to similar dose response enzyme saccharifications. The re-sults clearly show a significant improvement in the overall rateof glucose release and final yield over the course of all enzyme dosetrials. In effect enzyme loads could be reduced by as much as 2.5-fold whilst maintaining and/or enhancing rates of release and finalglucose yields. These observations demonstrate and confirm thatsoluble compounds generated during pretreatment are inhibitoryto cellulase activity. In fact, two recent investigations by Kim

et al. (2011) and Ximenes et al. (2011) identified a range of solublecompounds in prehydrolysates responsible for inhibiting and deac-tivating fungal cellulases by as much as 80%. Nevertheless, solubleinhibitors don’t account for the relatively poor glucose yields at-tained from washed substrates and high enzyme loads (60.416%protein/g cellulose). Possibly, enzyme activity was also impededby the presence of acid insoluble lignin (37% in the substrate).

3.4. Comparison between eucalypt species and age

The chemical composition of lignocellulose significantly influ-ences the success of its bioconversion into ethanol and differs con-siderably depending on its origin. Aside from genetic andenvironmental factors, the nature and age of eucalypt species gen-erally influences its elemental make-up (da Silva Perez et al., 2010).To investigate the effects of these latter traits, pretreatment and

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Fig. 4. Total glucose yield from enzymatic saccharification (10% w/v; 50 �C, pH 5.2) of ED_6 (6-year-old E. dunnii), ED_10 (10-year-old E. dunnii) and CCV_6 (6-year-old C.citriodora) pretreated at CSF = 1.60 (195�/0.25% H2SO4/2 min) or CSF = 2.48 (195�/0.5% H2SO4/7 min). Cellic� CTec 2 was added at a dose of 1.25% protein on cellulose.Contribution to total glucose -pretreatment (black) and enzyme saccharification (grey). Data represents the average response of at least three independent experiments.

270 S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272

enzymatic saccharification comparative studies of 6-year-old C.citriodora spp. Variagata (CCV_6) and 10-year-old E. dunnii(ED_10) samples against ED_6 were undertaken. These three sam-ples represent common Australian forestry plantation thinnings.Two different pretreatment regimes were employed; a low severity(CSF = 1.60; 0.25% H2SO4/195 �C/2 min) treatment designed formaximize pentose sugar recovery, and a high severity treatment(CSF = 2.48; 0.5% H2SO4/195 �C/7 min) for optimal glucose recov-ery. Total glucose yields following pretreatment and enzymatichydrolysis are shown in Fig. 4.

Pretreatment of the three samples revealed similar levels ofhemicellulose solubilisation with xylose yields ranging between160 mg/g ± 3 mg/g (low severity) and 52 mg/g ± 3 mg/g (highseverity). Likewise, glucose yields in prehydrolysates were compa-rable (Fig. 4) indicating no observable differences between sub-strates during pretreatment. For samples subjected to lowseverity conditions, total glucose yields were fairly consistentacross the board with ED_6 liberating slightly more glucose. Confi-dence testing (T-test) of the data revealed yields were not signifi-cantly different (p < 0.05). Under high severity settings, glucoserelease significantly increased with ED_6 yielding more than CCV,which inturn released more than ED_10. In general, both 6-year-old samples outperformed the 10-year-old sample. Confidencetesting confirmed that ED_6 and ED_10 were significantly differentfrom one another; but not from CCV. Although their chemical com-positional profiles appear to be similar (e.g. ED_6 and ED_10) theabove findings suggest that the eucalypt tree’s age at harvestingmay significantly effect cellulose hydrolysis (San Martín and Aguil-era, 1988). This leads to the question as to whether it is a structuraleffect. Changes in cellulose crystallinity, crystal size and degree ofpolymerisation during tree maturation have been cited as probablecauses (Hallac and Ragauskas, 2010; Jahan and Mun, 2005). Thecorrelation between a tree’s age and its recalcitrance may in factbe unique to specific plant species. A recent detailed study on Pop-ulus concluded that tree age had no effect on cellulose digestibility(DeMartini and Wyman, 2011).

3.5. Hydrolysate fermentations

Rapid and efficient fermentation of recovered sugars is oftenlimited because of the complex nature of the lignocellulosic

hydrolysates. A combination of mixed monosaccharides and arange of toxic compounds generated during pretreatment makefor a rather unfavourable environment for microbial growth result-ing in low ethanol titres and productivities. The fermentation char-acteristics of eucalypt (ED_6) hydrolysate sugars using S. cerevisiaeATCC 58447 are presented in Fig. 5.

In several previous fermentations with ED_6 hydrolysates test-ing both yeast and various Zymomonas mobilis stains, it was foundthat ATCC 58447 could be cultured in crude hydrolysate, albeit atslower rates than in a control media (data not shown). This was re-flected in its relatively low specific growth rate (0.06 h�1), glucoseuptake rate (1.39 g/g/h), and ethanol production rate which was0.67 g per gram of dry weight (DW) per hour. It took approximately30 h for S. cerevisiae to produce 18 g/L ethanol from 38 g of glucose(92% efficiency), a feat normally accomplished in less than half thetime in starch-based fermentations (Sánchez and Cardona, 2008).Xylose concentrations are not reported because S. cerevisiae ATCC58447 cannot utilise xylose.

The relatively poor performance of S. cerevisiae in the crudehydrolysate can be attributed to deficiency in essential nutrients,unfavourable pH conditions during the course of the fermentationand, in part, to the presence of growth inhibitory compounds. Cellgrowth and ensuing ethanol production in lignocellulosic hydroly-sate has been shown to be strongly dependent on the mass of theinitial cell inoculum and/or pH (Palmqvist and Hahn-Hägerdal,2000). Presumably, undissociated weak acid concentrations ac-crued during the fermentation, effectively lowering the pH.Improving fermentation performance through detoxification ofcrude hydrolysates, increasing nutrient supplements and main-taining pH are subject of current studies.

3.6. Conclusion

Although optimal pretreatment conditions for Eucalyptus hemi-cellulose and cellulose hydrolysis were found to be dissimilar, sig-nificant cellulose depolymerisation was detected under moderateseverity conditions (CSF P 2.0). We postulate that this event isassociated with and reflects the young age and species of test euca-lypts. With further development, a modified process could mini-mise and/or eliminate enzyme saccharification, thus deliveringappreciable cost savings. Although optimised for corn stover, CTec

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Fig. 5. Fermentation profile of S. cerevisiae in ED_6 hydrolysate prepared by pretreating 10% solids (w/v) with 0.25% H2SO4 for 2 min at 195 �C followed by enzymesaccharification (50 �C, pH 5.2, 72 h) with Cellic� CTec 2 at 2.083% protein on cellulose. Data represents averages of two separate experiments.

S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272 271

2 cellulase preparations performed well on pretreated eucalyptthinnings. Fermentation trials confirmed the technical feasibilityof converting eucalyptus forestry residues into ethanol, reinforcingits feedstock potential for an emerging Australian biofuel industry.

Acknowledgements

We gratefully acknowledge the financial support provided byDepartment of Agriculture, Fisheries and Forestry for this workand the support of NSW Department of Primary Industries, Austra-lia. We express our gratitude to Mr. Steve Morris for providing sta-tistical advice and assistance in the presentation of the data and toGraeme Palmer from SCU/CRC Forestry for harvesting and supplingeucalypt forestry thinning residues.

References

Aden, A., Ruth, M., Ibsen, K., Jechura, J., Neeves, K., Sheehan, J., Wallace, B.,Montague, L., Slayton, A., Lukas, J., 2002. Lignocellulosic biomass to ethanolprocess design and economics utilizing co-current dilute acid prehydrolysis andenzymatic hydrolysis for corn stover. National Renewable Energy LaboratoryTechnical Report NREL/TP-580-32438. Available from: <http://www1.eere.energy.gov/biomass/pdfs/32438.pdf> (accessed 11.11.2008).

Adney, B., Baker, J., 2008. Measurement of cellulase activity: National RenewableEnergy Laboratory Technical Report NREL/TP-510-42628. Available from:<http://www.nrel.gov/biomass/pdfs/42628.pdf> (accessed 11.11.2008).

Canettieri, E.V., Rocha, G.J., de Moraes, de Carvalho Jr., J.J.A., de Almeida e Silva, J.B.,2007. Optimization of acid hydrolysis from the hemicellulosic fraction ofEucalyptus grandis residue using response surface methodology. BioresourceTechnology 98, 422–428.

Chum, H., Johnson, D., Black, S., Overend, R., 1990. Pretreatment-catalyst effects andthe combined severity parameter. Applied Biochemistry and Biotechnology 24–25, 1–14.

da Silva Perez, D., Guillemain, A., Berthelot, A., N’Guyen-The, N., de Morogues, F.,Gomes, C., 2010. Evaluation of forestry biomass quality for the production ofsecond-generation biofuels. Cellulose Chemistry and Technology, 44, 1–14.

DeMartini, J.D., Wyman, C.E., 2011. Changes in composition and sugar release acrossthe annual rings of Populus wood and implications on recalcitrance. BioresourceTechnology 102, 1352–1358.

Emmel, A., Mathias, A.L., Wypych, F., Ramos, L.P., 2003. Fractionation of Eucalyptusgrandis chips by dilute acid-catalysed steam explosion. Bioresource Technology86, 105–115.

Fenning, T.M., Walter, C., Gartland, K.M.A., 2008. Forest biotech and climate change.Nature Biotechnology 26, 615–617.

Garrote, G., Dominguez, H., Parajó, J.C., 2001. Study on the deacetylation ofhemicelluloses during the hydrothermal processing of Eucalyptus wood.European Journal of Wood and Wood Products 59, 53–59.

Garrote, G., Eugenio, M.E., Díaz, M.J., Ariza, J., López, F., 2003. Hydrothermal andpulp processing of Eucalyptus. Bioresource Technology 88, 61–68.

Garrote, G., Kabel, M.A., Schols, H.A., Falque, E., Dominguez, H., Parajo, J.C., 2007.Effects of Eucalyptus globulus wood autohydrolysis conditions on the reactionproducts. Journal of Agricultural and Food Chemistry 55, 9006–9013.

Hallac, B.B., Ragauskas, A.J., 2010. Analyzing cellulose degree of polymerization andits relevancy to cellulosic ethanol. Biofuels, Bioproducts and Biorefining 5, 215–225.

Jahan, M., Mun, S., 2005. Effect of tree age on the cellulose structure of Nalita wood(Trema orientalis). Wood Science and Technology 39, 367–373.

Jeoh, T., Ishizawa, C., Davis, M., Himmel, M., Adney, W., Johnson, D., 2007. Cellulasedigestibility of pretreated biomass is limited by cellulose accessibility.Biotechnology and Bioengineering 98, 112–122.

Kabel, M.A., Bos, G., Zeevalking, J., Voragen, A.G.J., Schols, H.A., 2007. Effect ofpretreatment severity on xylan solubility and enzymatic breakdown of theremaining cellulose from wheat straw. Bioresource Technology 98, 2034–2042.

Kim, Y., Ximenes, E., Mosier, N.S., Ladisch, M.R., 2011. Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass. Enzyme andMicrobial Technology 48, 408–415.

Lee, Y.Y., Iyer, P., Torget, R.W., 1999. Dilute-acid hydrolysis of lignocellulosicbiomass. In: Recent Progress in Bioconversion of Lignocellulosics, (Tsao et al.,Eds). Advances in Biochemical Engineering/Biotechnology, vol. 65. Springer,New York, pp. 93–115.

Lloyd, T.A., Wyman, C.E., 2005. Combined sugar yields for dilute sulfuric acidpretreatment of corn stover followed by enzymatic hydrolysis of the remainingsolids. Bioresource Technology 96, 1967–1977.

McIntosh, S., Vancov, T., 2010. Enhanced enzyme saccharification of Sorghum bicolorstraw using dilute alkali pretreatment. Bioresource Technology 101, 6718–6727.

Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,2005. Features of promising technologies for pretreatment of lignocellulosicbiomass. Bioresource Technology 96, 673–686.

National Association of Forest Industries, 2005. Report 2 – The potential for wood ina sustainable and competitive Australian renewable energy industry. NationalAssociation of Forest Industries: Canberra. Available from: <http://www.nafi.com.au/Report%202.pdf> (accessed 09.03.2011).

Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosichydrolysates. I: inhibition and detoxification. Bioresource Technology 74, 17–24.

Ramos, L.P., Breuil, C., Kushner, D.J., Saddler, J.N., 1992. Steam pretreatmentconditions for effective enzymatic hydrolysis and recovery yields of Eucalyptusviminalis wood chips. Holzforschung 46, 149–154.

Romani, A., Garrote, G., Alonso, J.L., Parajo, J.C., 2010. Bioethanol production fromhydrothermally pretreated Eucalyptus globulus wood. Bioresource Technology101, 8706–8712.

San Martín, R., Aguilera, J.M., 1988. Steam pretreatment and enzymatic productionof D-glucose from Eucalyptus globulus. Biomass 15, 281–289.

Sánchez, Ó.J., Cardona, C.A., 2008. Trends in biotechnological production of fuelethanol from different feedstocks. Bioresource Technology 99, 5270–5295.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008.Determination of structural carbohydrates and lignin in biomass. NationalRenewable Energy Laboratory Technical Report NREL/TP-510-42618. Availablefrom: <http://www.nrel.gov/biomass/pdfs/42618.pdf> (accessed 11.11.2008).

Söderström, J., Pilcher, L., Galbe, M., Zacchi, G., 2003. Two-step steam pretreatmentof softwood by dilute H2SO4 impregnation for ethanol production. Biomass andBioenergy 24, 475–486.

Tengborg, C., Stenberg, K., Galbe, M., Zacchi, G., Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., 1998. Comparison of SO2 and H2SO4 impregnation of softwood

272 S. McIntosh et al. / Bioresource Technology 110 (2012) 264–272

prior to steam pretreatment on ethanol production. Applied Biochemistry andBiotechnology 70–72, 3–15.

Wyman, C.E., 2007. What is (and is not) vital to advancing cellulosic ethanol. Trendsin Biotechnology 25, 153–157.

Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2011. Inhibition of cellulases byphenols. Enzyme and Microbial Technology 46, 170–176.

Yu, Q., Zhuang, X., Yuan, Z., Wang, Q., Qi, W., Wang, W., Zhang, Y.H., Xu, J., Xu, H.,2010. Two-step liquid hot water pretreatment of Eucalyptus grandis to enhancesugar recovery and enzymatic digestibility of cellulose. Bioresource Technology101, 4895–4899.