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"( ) COPYRIGHT STATEMENT

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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AUTHENTICITY STATEMENT

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

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E Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE Comparative evaluations of cellulosic raw materials for second generation bioethanol production Y.J. Jeon, Z. Xun and P.L. Rogers

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington, NSW, Australia

Keywords Abstract acid ⁄ enzyme pretreatment, bioethanol, fermentation, lignocellulosic materials, Aims: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic recombinant Zymomonas mobilis. raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. Correspondence Methods and Results: Dilute acid pretreatment (2% H2SO4) with 10% (w ⁄ v) Peter L. Rogers, School of Biotechnology and substrate loading was performed at 134C for 60 min followed by enzyme Biomolecular Sciences, The University of New hydrolysis at 60C. The results demonstrated that hydrolysis of herbaceous raw South Wales, Kensington, NSW 2052, Australia. E-mail: [email protected] materials resulted in higher sugar recoveries (up to 60–75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 2010 ⁄ 0710: received 29 April 2010, revised (pZB5) demonstrated that final ethanol concentrations and yields were also 29 July 2010 and accepted 5 August 2010 higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all doi:10.1111/j.1472-765X.2010.02923.x hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50C compared to 60C following acid pretreatment. Conclusions: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. Significance and Impact of the Study: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural ⁄ forestry biomass rather than food-based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.

Vandamme 2009). The aim of this study is to provide a Introduction comparative evaluation of eight different lignocellulosic Lignocellulosic raw materials can provide alternative feed- raw materials for their suitability for bioethanol produc- stocks for the sustainable production of bioethanol and tion. Such raw materials form the basis for second gener- other biochemicals with a wide range of economic and ation fermentation processes designed to use renewable environmental beneﬁts (Hahn-Ha¨gerdahl et al. 2007; agricultural ⁄ forestry biomass rather than food-based Thomsen and Haugaard-Nielsen 2008; Soetaert and resources (Himmel et al. 2007; Scharlemann and Laurance

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 1 Second generation bioethanol production Y.J. Jeon et al.

2008; Williams et al. 2008; Hayes 2009). A recombinant Table 1 The list of plant species used in this study and their sugar strain of Zymomonas mobilis capable of converting both compositions xylose and glucose to ethanol (Zhang et al. 1995) has Component (%)* been used, as previous studies have demonstrated that Raw materials Z. mobilis is capable of high specific rates of ethanol pro- (common name) Cellulose Hemicellulose References duction, as well as high ethanol yields and productivities Wheat straw 36 26 This study (Rogers et al. 2007). A recent report of an improved gen- 33Æ725Æ0 Ali et al. (1991) ome annotation for Z. mobilis (Yang et al. 2009), based Sugarcane bagasse 39Æ626Æ5 This study on an earlier study by Seo et al. (2005), provides an 35Æ035Æ8 Sasaki et al. (2003) excellent database for further genetic manipulation and Sorghum straw 32Æ4 27 This study Æ et al. strain enhancement of this micro-organism. Concentra- 35 1 24 Tellez-Lus (2002) Arundo donax 42Æ531Æ2 This study tions of potential inhibitors have been determined also 36 30 Pascoal Neto et al. (1997) for the various raw materials used in this study. Such Sugarcane tops 35 32 This study information provides possible reasons for reduced fer- Oil mallee 53 22 This study mentation productivities, as well as potential targets for Pine 49Æ813Æ1 This study further strain enhancement (Joachimsthal et al. 1998; Kim 45Æ322Æ2 Araque et al. (2008) et al. 2000a; Jeon et al. 2002; Pienkos and Zhang 2009; Eucalyptus 51Æ918Æ1 This study Æ Æ et al. Yang et al. 2010a). 46 8166 Garrote (2003) *Composition percentages are on dry-weight basis. Materials and methods acid pretreated hydrolysates was adjusted to 5Æ0 by addi- Raw materials and composition analysis tion of solid Ca(OH)2. The temperature for enzyme Sorghum straw (Sorghum bicolor), wheat straw (Triticum hydrolysis was initially selected at 60C to maximize the aestivum), sugarcane bagasse and sugarcane tops (SCT) b-glucosidase activity as optimal temperatures for the (Saccharum officinarum) were sourced from local farmers. cellulase (NS50013; Novozymes, Bagsværd, Denmark) The varieties are typical for Australia and were grown and b-glucosidase (NS50010; Novozymes) were in the using conventional farming practices. Hardwood (Euca- ranges of 45–60C and 45–70C, respectively, according lyptus dunnii) and softwood chips (Pinus elliotii) were to manufacturer’s instructions. The enzyme hydrolyses ) donated by Forestry New South Wales. Eucalyptus loxoph- were carried out at 60C with shaking at 180 rev min 1 leba ssp. lissophloia (oil mallee) was donated by Depart- for 22 h. For comparison, further studies on the effect of ment of Environment and Conservation (Western temperature on enzyme hydrolysis and sugar release were Australia). Arundo donax was donated by the South carried out at 50C. For both studies, 2% (v ⁄ v) cellulase Australian Research and Development Institute (SARDI, and 4% (v ⁄ v) b-glucosidase were used, the latter being South Australia). The biomass samples were analysed for designed to fully release the glucose. The Novozyme ) cellulose, hemicellulose and lignin using the detergent 50013 contained 700 EGU (endo-glucanase unit) g 1 of fibre method (Van Soest et al. 1991). Cellulose and hemi- total cellulase; the b-glucosidase activity of Novozyme ) celluloses contents for the various biomass samples are 50010 was 250 CbU (cellobiase unit) g 1. Recovery of listed in Table 1; these values being within the ranges for sugars (%) was calculated from the total sugars in the cellulose and hemicelluloses cited by Pienkos and Zhang final acid ⁄ enzyme hydrolysate samples and compared to (2009). the total available sugars based on dry-weight analyses (Table 1). Pretreatment Microbial strain for ethanol production All biomass material was dried at 55–60C for 48 h, milled and finely ground using a Micro Hammer-Cutter The recombinant strain of Zymomonas mobilis ZM4 Mill (Culatti AG, Zurich, Switzerland) and then passed (pZB5) (Zhang et al. 1995; Joachimsthal and Rogers through a 1Æ5- mm screen. Milled material was stored at 2000) was used in the fermentability tests. The recombi- room temperature in sealed containers. To evaluate the nant strain, which can utilize both xylose and glucose for sugar recovery yields from the hemicellulose and cellulose ethanol production, was kindly provided by Dr Min fractions, dilute acid pretreatments were performed in Zhang, National Renewable Energy Laboratory, Golden,

2% H2SO4 (v ⁄ v) with 10% (w ⁄ v) raw material at 134C Co. under a Materials Transfer Agreement. The strain was for 60 min. Prior to enzyme hydrolysis, the pH of the routinely cultured at 30C on agar plates containing

ª 2010 The Authors 2 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology Y.J. Jeon et al. Second generation bioethanol production

) ) ) 20 g l 1 xylose, 5 g l 1 yeast extract and 20 lgml 1 tetra- Standards containing analytical grade components were cycline. used periodically to conﬁrm calibration accuracy.

Fermentation studies Calculation of kinetic parameters The acid ⁄ enzyme-treated hydrolysates were adjusted to The maximum specific growth rates were calculated in ) pH 5 using 3 mol l 1 NaOH. To separate the liquid frac- the exponential phase of growth. The maximum specific tion, the solids fraction [unhydrolysed fibre and gypsum glucose uptake rates (qsmax,glucose) and maximum specific (CaSO4)] was removed by centrifugation at 7000 g for ethanol production rates (qpmax) were calculated over the 20 min. Inocula for fermentation were prepared by plat- exponential phase of growth based on the following for- ing Z. mobilis ZM4 (pZB5) from frozen cultures onto mulae: qsmax,glucose =(1⁄ xav)(Ds ⁄ Dt) and qpmax =(1⁄ xav) agar plates (composition described earlier). A single col- (Dp ⁄ Dt), where Ds and Dp are the changes in the glucose ony was selected and used to inoculate 10 ml of first seed and ethanol concentrations, respectively, over the time medium composed of per litre: 25 g xylose, 10 g yeast period Dt, and xav is the average biomass concentration extract and basal minerals (1 g MgSO4Æ7H2O; 1 g over Dt, where x, s and p are the concentrations of bio- (NH4)2SO4;2gKH2PO4). When an optical density of mass, glucose and ethanol, respectively. A660 =0Æ8–1Æ0 had been reached, a 1% (v ⁄ v) aliquot was In the major xylose utilization phase, following the used to inoculate the second seed medium composed of complete depletion of glucose, growth was very limited. the acid ⁄ enzyme hydrolysates supplemented with addi- During this phase, the formulae were modified to the fol- tional nutrients per litre: 2Æ5 g glucose, 12Æ5 g xylose, lowing: qsmax,xylose =(1⁄ xav)(Ds ⁄ Dt) where Ds is the 2Æ5 g yeast extract and basal minerals (concentrations as change in the xylose concentration during its maximum described earlier). When a further optical density of uptake rate over this time period Dt, and xav is the aver- A660 =0Æ8–1Æ0 had been reached, a 10% (v ⁄ v) aliquot of age biomass concentration over Dt. The overall yields for second seed culture was used to inoculate the main ethanol (Yp ⁄ s) production on sugar mixture media were fermentation medium. This was composed of the acid ⁄ based on the initial and final concentrations of biomass, enzyme hydrolysates supplemented with yeast extract sugars (glucose and xylose) and ethanol. ) (5 g l 1). For the plasmid maintenance, all media ) included 20 mg l 1 tetracycline. Fermentations were car- Results ried out in 100- ml Erlenmeyer flasks with working volumes of 50 ml. The batch cultures were performed at Results of acid ⁄ enzyme pretreatment 30C and initial pH 5Æ0 without agitation. Samples were taken at various time intervals to determine biomass, The results of the acid pretreatment followed by enzyme xylose, glucose, arabinose and ethanol concentrations. hydrolysis at 60C and pH 5Æ0 are summarized in Table 2. The results show that pretreatment and enzyme hydrolysis of the herbaceous raw materials resulted in Analytical procedures higher sugar recoveries when compared to those for Acid ⁄ enzyme hydrolysate samples were analysed for woody raw materials. Among the former, the hydrolysates glucose, xylose and arabinose as well as the degradation from wheat straw, sugarcane bagasse and sorghum straw products including acetate, furfural, hydroxymethylfural- (72–74%) showed the highest sugar recoveries. The dehyde (HMF), levulinic acid and formate by HPLC hydrolysate from A. donax (Adx) showed the lowest sugar using an Aminex column HPX-87H (300 · 7Æ8) (Bio- recovery (65%) among the herbaceous raw materials. The Rad, Richmond, CA, USA) equipped with a refractive woody raw materials showed poorer recoveries (<55%) index detector and a computer interfaced electronic inte- when compared to the herbaceous raw materials. grator. Separations were performed at 50C and eluted at ) ) 0Æ6 ml min 1 using 5 mmol l 1 H SO . Growth was mea- 2 4 Effect of reduced enzyme concentrations sured turbidometrically at 660 nm (1 cm light path). Dry cell mass was determined by conversion of optical density As enzyme costs are a major component in the cost of values at 660 nm to dry cell weights using a dry cell converting biomass to ethanol, a further evaluation was weight conversion constant value for ZM4 (pZB5) carried out with a 10-fold reduction in added enzymes, )1 (OD660nm 0Æ05 = 15 mg l ) (Kim et al. 2000b). Fermen- viz. 0Æ2% (v ⁄ v) cellulose and 0Æ4% (v ⁄ v) b-glucosidase at tation samples were analysed for glucose, xylose, arabi- 60C. However, it was found that decreased enzyme con- nose and ethanol concentrations by HPLC using a centrations resulted in a significant reduction in sugar refractive detector under the conditions described earlier. recovery yields. As a result, it was decided to continue

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 3 Second generation bioethanol production Y.J. Jeon et al.

Table 2 Sugar production for fermentation Total Sugar of raw materials (10% w ⁄ v) from acid ⁄ Glucose Xylose Arabinose sugar recovery enzyme pretreatment Raw material (g l)1) (g l)1) (g l)1) (g l)1) yield (%)*

Wheat straw 23Æ3193Æ045Æ374 Sugarcane bagasse 26Æ918Æ22Æ847Æ972 Sorghum straw 22Æ016Æ93Æ742Æ672 Arundo donax 17Æ723Æ32Æ243Æ259 Sugarcane tops 22Æ318Æ73Æ144Æ166 Oil mallee (Bark and 10Æ63Æ45Æ019Æ025 hard wood) Pine (soft wood) 10Æ619Æ71Æ531Æ851 Eucalyptus (hard wood) 8Æ914Æ60Æ323Æ834

Acid hydrolysis carried out using 2% H2SO4 (v ⁄ v) at 134C for 1 h. Enzyme hydrolysis carried out using 2% cellulase and 4% b-glucosidase at 60C for 22 h. *Sugar recovery yields were calculated based on composition (on dry-weight basis) of cellulose and hemicellulose (see Table 1). with the higher enzyme loadings although it was recog- found in this study were lower than the minimum inhibi- nized that further studies would be needed to determine tory levels reported by these authors, it is likely that optimal enzyme concentrations in view of the high cost fermentation rates and productivities in this study have of enzymes. been decreased not only by individual compounds but also by their combined inhibitory effects of such compounds (Palmqvist and Hahn-Hagerdal 2000a,b). Fermentation studies on acid ⁄ enzyme hydrolysates To evaluate the fermentability of these acid ⁄ enzyme Further studies on effect of temperature on enzyme hydrolysates, batch fermentation studies using a recombi- hydrolysis ⁄ sugar recoveries nant strain of Z. mobilis ZM4 (pZB5) were carried out. The results of the fermentation profiles with various raw As the optimal temperature for enzyme hydrolysis was materials are shown in Fig. 1(a,b), and an analysis of the undetermined, further studies on sugar recoveries with kinetic data is given in Table 3. From Table 3, it is evi- enzyme pretreatment at 50C rather than 60C were car- dent that all of the hydrolysates showed significantly ried out. The results from this evaluation showed that lower maximum specific rates of growth, sugar uptake increased sugar recovery yields of 5–25% could be and ethanol production compared to those for a glu- achieved at the lower temperature for enzyme hydrolysis cose ⁄ xylose medium of similar sugar concentrations. This (Table 5). Such conditions are likely to result in increased may be owing to low concentrations of essential minerals ethanol concentrations from the hydrolysates, and further ) in the hydrolysates (only 5 g l 1 yeast extract was added optimization of enzyme concentrations and hydrolysis to provide essential vitamins and growth factors) or to times as well as hydrolysis temperature are necessary to the presence of growth inhibitory compounds. The con- achieve high yields and reduce pretreatment costs. centrations of potential inhibitory compounds resulting from the hydrolysis (e.g. acetate, formate, furfural, HMF Discussion and conclusions and levulinic acid) have been determined (Table 4). Toxic effects caused by inhibitors derived from mild acid hydro- Of the various raw materials evaluated, the highest etha- lysis of lignocellulosic materials on strains of Z. mobilis nol yields and productivities were determined for wheat have been reported recently (Pienkos and Zhang 2009; straw and bagasse hydrolysates. Sorghum straw, SCT Yang et al. 2010a). Although the inhibitor concentrations and A. donax (Adx) hydrolysates were similar in their

Figure 1 (a) Fermentation proﬁles of ZM4 (pZB5) in various hydrolysates of grass-type raw materials using 2% H2SO4 at 134C for 1 h; enzyme hydrolysis using 2% cellulase and 4% b-glucosidase at 60C for 22 h. A: wheat straw; B: sugar cane bagasse; C: Sorghum straw; D: Arundo donax.( ) glucose; ( ) xylose; ( ) ethanol and ( ) biomass. (b) Fermentation proﬁles of ZM4 (pZB5) in various hydrolysates of sugar cane tops and various wood-based materials using 2% H2SO4 at 134C for 1 h; enzyme hydrolysis using 2% cellulase and 4% b-glucosidase at 60C for 22 h. A: sugar cane tops; B: oil mallee (Eucalyptus loxophleba); C: pine softwood (Pinus elliotii); D: eucalyptus (Eucalyptus dunnii). ( ) glucose; ( ) xylose; ( ) ethanol and ( ) biomass.

ª 2010 The Authors 4 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology Y.J. Jeon et al. Second generation bioethanol production

(a) 30 4 30 4 ) ) –1 –1 AB 3·5 3·5 25 25 3 3 ) ) 20 20 –1 –1

2·5 2·5 l g 15 2 15 2

1·5 1·5 10

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30 4 30 4 ) ) –1 CD 3·5 –1 3·5 25 25 3 3 ) ) 20 20 –1 –1 2·5 2·5 l g 15 2 15 2

1·5 1·5 10

10 Biomass ( Biomass (g l 1 1 5 5 0·5 0·5 Glucose, xylose, ethanol (g l

Glucose, xylose and ethanol (g l 0 0 0 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 Time (h) Time (h)

(b) 4

) 30 ) 30 4 –1 –1 AB 3·5 3·5 25 25 3 3 ) )

20 –1

20 –1 2·5 2·5 l g

15 2 15 2

1·5 1·5 10 10 Biomass ( Biomass (g l 1 1 5 5 0·5 0·5 Glucose, xylose and ethanol (g l Glucose, xylose and ethanol (g l 0 0 0 0 0204060 0204060 Time (h) Time (h)

30 4 )

) 30 4 –1 C 3·5 –1 D 25 3·5 25 3

) 3

20 ) –1

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2·5 l g 15 2 15 2 1·5 10 1·5 Biomass (g l 10 Biomass ( 1 1 5 5 0·5 0·5 Glucose, xylose and ethanol (g l 0 0 Glucose, xylose and ethanol (g l 0 0 0204060 0 204060

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 5 Second generation bioethanol production Y.J. Jeon et al.

Table 3 Kinetic analysis of ZM4 (pZB5) in various acid ⁄ enzyme hydrolysates of raw materials (10% (w ⁄ v) substrate; chemical pretreatment using

2% H2SO4 at 134C for 1 h; enzyme hydrolysis using 2% cellulase and 4% b-glucosidase at 60C for 22 h)

Wheat Bagasse Sorghum SCT Adx Oil mallee Pine Eucalyptus *Pure Biomass sources straw (10%) (10%) straw (10%) (6Æ7%) (10%) (6Æ7%) (10%) (10%) sugars

)1 Maximum specific growth rate lmax (h )0Æ19 0Æ15 0Æ11 0Æ14 0Æ11 0Æ16 0Æ06 0Æ08 0Æ43 Maximum specific glucose uptake 4Æ63Æ75Æ45Æ75Æ14Æ72Æ05Æ29Æ2 )1 )1 rate qsmax,glucose (g g h ) Maximum specific xylose uptake 0Æ49 0Æ30Æ60Æ76 1Æ30Æ35 0 0Æ32Æ6 )1 )1 rate qsmax,xylose (g g h ) Maximum specific EtOH rate 1Æ81Æ53Æ12Æ61Æ82Æ50Æ62Æ44Æ7 )1 )1 qpmax (g g h ) Total ethanol produced (g l)1)13Æ817Æ710Æ610Æ312Æ33Æ80Æ22Æ324 Maximum ethanol volumetric 1Æ01Æ20Æ52 0Æ49 0Æ40Æ22 0Æ10 0Æ12 3Æ18 productivity (g l)1 h)1) )1 Yp ⁄ s (g g )0Æ45 0Æ43 0Æ42 0Æ36 0Æ37 0Æ47 0Æ13 0Æ36 0Æ47 Ethanol yield (% theoretical) 88 84 82 71 72 92 25 71 92

SCT, sugarcane tops. *The data for rich medium containing 25 g l)1 glucose and 25 g l)1 xylose was reported by Joachimsthal and Rogers (2000).

Table 4 Inhibitory compounds derived from acid ⁄ enzyme hydroly- characteristics, while fermentation of the hydrolysates of ⁄ sates (10% w v) of various raw materials (Pretreatment conditions as the woody raw materials (oil mallee, pine and eucalyptus) in Table 3) resulted in relatively low ethanol concentrations and pro- Inhibitory compounds (g l)1) ductivities, the latter possibly because of higher concentrations of inhibitory compounds (e.g. higher furfural Acetate Formate Levulinic acid HMF Furfural concentrations in oil mallee). As previously reported Wheat straw 3Æ10Æ60Æ70Æ08 0Æ67 (Joachimsthal and Rogers 2000; Jeon et al. 2002; Yang Sugarcane Bagasse 4Æ10Æ70Æ80Æ10Æ74 et al. 2010b), improved strains of Z. mobilis can be devel- Æ Æ Æ Æ Æ Sorghum straw 2 3122606050 oped to partially overcome some of the inhibitory effects Sugarcane tops 2Æ10Æ20Æ20Æ14 0Æ14 that result from inhibitory components such as acetate. Adx 4Æ80Æ50Æ20Æ04 0Æ74 Eucalyptus 3Æ40Æ30Æ200Æ2 From this study, it is evident also that signiﬁcant R&D Pine 1Æ80Æ60Æ40Æ14 0Æ15 is needed in bioreactor design to achieve much higher Oil mallee 0Æ70Æ50Æ70Æ05 0Æ96 sugar concentrations in the hydrolysates. At 10% (w ⁄ v) raw material concentration, sugar recoveries of 80–90% HMF, hydroxymethylfuraldehyde. have been achieved with herbaceous materials (Table 5). )1 Table 5 Sugar production for fermentation of raw materials (10% However, higher sugar concentrations of 150–200 g l w ⁄ v) from acid ⁄ enzyme pretreatment at lower temperature for are needed for high productivity fermentations, and enzyme hydrolysis although biomass concentrations up to 15% (w ⁄ v) can be

Total Sugar hydrolysed in a simple stirred reactor, higher concentra- Glucose Xylose Arabinose sugar recovery tions above this are problematic because of the high levels Raw material (g l)1) (g l)1) (g l)1) (g l)1) yield (%)* of water adsorption by the ﬁne biomass particles. Coun- tercurrent bioreactor design, optimal conditions for the Wheat straw 26Æ6202Æ248Æ879 acid ⁄ enzyme hydrolysis and possibly higher operating Sugarcane bagasse 31Æ4242Æ457Æ887 Sorghum straw 27 22 5Æ154Æ191 temperatures for acid hydrolysis (e.g. for woody biomass) Arundo donax 25Æ021Æ41Æ547Æ965 as well as improved micro-organisms resistant to higher Sugarcane tops 25Æ816Æ94Æ146Æ870 inhibitor concentrations will all be needed for future pro- Oil mallee 11Æ23Æ65Æ820Æ627 cess enhancement and potential commercial viability Pine 11Æ118Æ61Æ230Æ949 (Galbe and Zacchi 2007). Eucalyptus 21Æ8160Æ338Æ154

Acid hydrolysis carried out using 2% H2SO4 (v ⁄ v) at 134C for 1 h. Enzyme hydrolysis carried out using 2% cellulase and 4% b-glucosi- Acknowledgements dase at 50C for 22 h. *Sugar recovery yields were calculated based on composition on dry- This research was carried out with support of the Australian weight basis of cellulose and hemicellulose (see Table 1). National Collaborative Research Infrastructure Strategy

ª 2010 The Authors 6 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology Y.J. Jeon et al. Second generation bioethanol production

(NCRIS) Biofuels Sub-Program and a Climate Action Pascoal Neto, C., Seca, A., Nunes, A.M., Coimbra, M.A., Grant (CAG) from the New South Wales Government. Domingues, F., Evtuguin, D., Silvestre, A. and Cavaleiro, J.A.S. (1997) Variations in chemical composition and structure of macromolecular components in different References morphological regions and maturity stages of Arundo 6 Ali, S.H., Asghar, S.M. and Shabbir, A.U. (1991) Neutral Sul- donax. Ind Crops Prod , 51–58. phite Pulping of Wheat Straw, Tappi Pulping Conference Pienkos, P.T. and Zhang, M. (2009) Role of pretreatment and Proceedings. Atlanta, GA: Tappi Press, p. 51. conditioning processes on toxicity of lignocellulosic 16 Araque, E., Parra, C., Freer, J., Contreras, D., Rodriguez, J., biomass hydrolysates. Cellulose , 743–762. Medonça, R. and Baeza, J. (2008) Evaluation of Organo- Rogers, P.L., Jeon, Y.J., Lee, K.J. and Lawford, H.G. (2007) solv pretreatment for the conversion of Pinus radiate D. Zymomonas mobilis for fuel ethanol and higher value 108 Don to ethanol. Enzyme Microb Technol 43, 214–219. products. Adv Biochem Eng Biotechnol , 263–288. Galbe, M. and Zacchi, G. (2007) Pretreatment of lignocellu- Sasaki, M., Adschiri, T. and Arai, K. (2003) Fractionation of losic materials for efficient bioethanol production. Adv sugarcane bagasse by hydrothermal treatment. Bioresour 86 Biochem Eng Biotechnol 108, 41–65. Technol , 301–304. Garrote, G., Eugenio, M.E., Dıáz, M.J., Ariza, J. and Lo´pez, F. Scharlemann, J.P.W. and Laurance, W.F. (2008) How Green 319 (2003) Hydrothermal and pulp processing of Eucalyptus. Are Biofuels? Science , 43–44. Bioresour Technol 88, 61–68. Seo, J.S., Chong, H.Y., Park, H.S., Yoon, K.O., Jung, C., Kim, Hahn-Ha¨gerdahl, B., Karhumaa, K., Fonseca, C., Spencer- J.J., Hong, J.H. and Kim, H. (2005) The genome sequence Martins, I. and Gorwa-Grauslund, M.F. 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