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Bioorganosolv pretreatments of P. radiata by a brown rot fungus (Gloephyllum trabeum) and ethanolysis

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Bioorganosolv pretreatments of P. radiata by a brown rot fungus (Gloephyllum trabeum) and ethanolysis
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  Enzyme and Microbial Technology 47 (2010) 11–16 Contents lists available at ScienceDirect EnzymeandMicrobialTechnology  journal homepage: www.elsevier.com/locate/emt Bioorganosolv pretreatments of   P. radiata  by a brown rot fungus( Gloephyllum trabeum ) and ethanolysis Mariel Monrroy a , Jeniffer Iba˜nez a , b , Victoria Melin a , b , Jaime Baeza a , b ,Regis Teixeira Mendonc¸a a , c , David Contreras a , b , Juanita Freer a , b , ∗ a Renewable Resources Laboratory, Biotechnology Center, Universidad de Concepción, Casilla 160-C, Concepción, Chile b Faculty of Chemical Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile c Faculty of Forest Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile a r t i c l e i n f o  Article history: Received 29 November 2009Received in revised form 28 January 2010Accepted 29 January 2010 Keywords: Brown rot fungiOrganosolvBioethanol a b s t r a c t Pinus radiata  wood chips were pretreated with the brown rot fungus  Gloephyllum trabeum  for 3 weeksfollowed by an organosolv delignification with ethanol:water mixture at pH 2. The organosolv processwas assessed using biotreated material that showed a high viscosity loss and a mass loss ranging from6% to 8%. The experiment was designed to optimize the organosolv conditions, ethanol:water ratio andH factor (factor that combines temperature and time in one variable) to obtain the highest ethanol yieldbysimultaneousenzymaticsaccharificationandfermentation(SSF)for96hat10%consistency.Theopti-mized conditions for organosolv process for biotreated material were: ethanol:water mixture (60/40,v/v)and1156Hfactor(185 ◦ C,18min);theoptimizedconditionsforthecontrol(chipswithoutbiotreat-ment) were: ethanol:water mixture (60/40, v/v) and 6000 H factor (200 ◦ C, 32min). The experimentalethanol yields obtained at these conditions were 63.8% and 64.3% (wood basis) for biotreated mate-rial and control, respectively. The maximum amount of ethanol that could be produced from  P. radiata is 252gethanol/kgwood, assuming total glucose conversion into ethanol. The results indicate that theobtained ethanol was 161gethanol/kgwood from both materials. The biotreatment of the wood beforethe organosolv process improved solvent accessibility. To obtain the same ethanol yield, lower processseverity was required in the biotreated samples in comparison with the control. © 2010 Published by Elsevier Inc. 1. Introduction Oil depletion and the greenhouse effect have increased theneed for alternative non-fossil transportation fuels [1,2]. Research in bioethanol production from lignocellulosic biomass (LCB) hasgrown significantly over the last few decades. Lignocelluloseis the most abundant biomass that can be converted into liq-uid fuels by enzymatic hydrolysis and microbial fermentation.According to U.S. Department of Energy, it can be estimated thatterrestrial plants produce 13 × 10 10 metrictons of biomass peryear or about 6 times of the world’s energy requirement [3].LCB includes materials such as agricultural and forestry residues,municipal solid waste, and industrial wastes. LCB can be used asan inexpensive feedstock for production of renewable fuels andchemicals [4]. ∗ Corresponding author at: Centro de Biotecnología, Universidad de Concepción,Barrio Universitario s/n, Concepción, Chile. Tel.: +56 41 2204601;fax: +56 41 2204074. E-mail address:  jfreer@udec.cl (J. Freer). However,duetotheirstructuralfeatures,LCBhaslimitedacces-sibilitytoenzymesormicroorganisms.LCBismadeupofcellulose,hemicellulose, and lignin. Since the carbohydrate polymers aretightly bound to the lignin, mainly by hydrogen bonds but also bysomecovalentbonds[5],pretreatmentisrequiredtomakebiomassmoreaccessibletotheenzymesthatconvertthecarbohydratepoly-mers into fermentable sugars. In short, pretreatment alters thephysicalfeaturesandchemicalcomposition,specificallyimprovingenzymeaccessandeffectivenessbybreakingtheligninseal,remov-ing hemicellulose, disrupting the crystalline structure of cellulose,and expanding the structure to increase pore volume [1,6–8]. A large number of pretreatments have been tested by manyresearchers, which can be broadly classified into physical, chem-ical, physicochemical, biological and combined pretreatments[5,6,9–11]. Pretreatments using organic solvents have been reported as efficient processes. These pretreatments break theinternalligninandhemicellulosebondsandseparatetheligninandhemicellulose fractions that can be potentially converted to usefulproducts.Methanol,ethanol,butanol,n-butylamine,acetone,ethy-leneglycol,andothercompoundshavebeenusedintheorganosolvprocess [5,12]. These treatments have sometimes been combined with biological treatments [11,13]. 0141-0229/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2010.01.009  12  M. Monrroy et al. / Enzyme and Microbial Technology 47 (2010) 11–16 In biological pretreatments, natural wood attacking microor-ganisms that can degrade lignin and holocellulose are allowedto grow on the biomass, producing lignin–holocellulose complexdegradation. The main biological pretreatments include fungi andtheir enzymes. The principal organism used for biological pre-treatment of lignocellulose is white rot fungi [5,14]. These fungi have been shown to effectively disrupt the lignin–cellulose com-plex. Other types of wood rot fungi, such as the brown rot fungi,are responsible for naturally breaking down the highly orderedcellulose crystalline structure. These fungi preferentially degradethe wood polysaccharides, but also partially oxidize lignin [15]. They degrade holocellulose in an unusual manner, causing a rapiddecrease in degree of polymerization with a low mass loss. It hasbeenproposedthatinitialdepolymerizationiscausedbyproducingsmall, diffusible, extracellular oxidants (free radicals), operating ata distance from the hyphae [15,16]. As a result of the initial attack by brown rot fungi and thedepolymerization of the cellulose, wood strength rapidly decays.The aim of this work was to use a fungal pretreatment prior to theorganosolv process (ethanolysis) in order to improve the solventaccessibility, decreasing the H factor. The ethanolysis process wasoptimized for conversion of   Pinus radiata  into ethanol by SSF. 2. Experimental  2.1. Raw material and preparationP. radiata  D. Don samples were chipped and screened to approximately2.0cm × 2.5cm × 0.5cm. The wood chips were air-dried until reaching 10% (w/w)moisture, and then stored in plastic bags until its use. Prior to the biodegradationexperiments, wood chips were immersed in water for 36h and the excess of waterwasdrained.Moistwoodchipsweresterilized(121 ◦ C/30min)andbroughttoroomtemperature.  2.2. Fungus, inoculum preparation and wood biodegradationGloephyllumtrabeum (ATCC11539)wasusedforthewoodchipstreatment.Thestrain was maintained on 2% (w/v) malt extract, 0.5% (w/v) soybean peptone, 1.5%(w/v) agar culture plates at 24 ◦ C for 1 week. Then, fungal mycelium was grownin 2L Erlenmeyer flasks with 200mL liquid culture medium (sterilized at 121 ◦ C,30min) containing 2% (w/v) malt extract and 0.5% (w/v) soybean peptone. Eachflask was inoculated with 20 discs (8mm in diameter) of the strain pre-cultured insolid medium and maintained unshaken for 2 weeks at 25 ◦ C. The grown myceliumwas filtered and washed with 300mL sterilized water. Washed mycelium obtainedfrom several cultures was blended with 50mL of sterilized water in two cycles of 10s. The mycelium suspension was used to inoculate the sterilized wood chips inbioreactors.Eachbioreactorwasloadedwith300gwoodchipsandinoculatedwithasuspensionvolumecorrespondingto500mgoffungalmyceliumperkgofdrywood.Theinoculatedwoodchipswereincubatedinanacclimatizedroomat25 ◦ Cand55%relativehumidity(RH)for3weeks.Afterthebiodegradingtreatment,thesuperficialmycelium was removed from the wood chips by brushing and the decayed woodchipsweredriedat40 ◦ Cfor48h.Themoistureofeachsamplewasdeterminedandthecalculatedinitialandfinaldryweightswereusedtodeterminemasslossduetofungal biotreatment. The samples obtained in this process were named “biotreatedmaterial”.Controlwoodchipsampleswerepreparedunderthesameconditionsbutwithout fungus addition. All the experiments were carried out in sextuplicate.  2.3. Organosolv pretreatment (ethanolysis) Organosolv pretreatment was carried out in a 1-L Parr reactor (Moline, IL, USA)loaded with 40g of wood chips (dry weight base) of the biopretreated materialor control and 240mL of an ethanol (95%):water mixture (60/40, v/v) contain-ing sulfuric acid as catalyst (0.13%, w/v pH approximately 2). The solvent:woodratio inside the reactor was 6:1 (w/w). The H factor was 1156 (185 ◦ C) and 6000(200 ◦ C) for biotreated and control materials, respectively. The cooking severity inthe organosolv process was described by the H factor. This factor relates the timeandtemperatureinasinglevariable.Theseexperimentalconditionswerepreviouslydetermined by a factorial analysis, where these values were optimized for a maxi-malethanolyield.Theeffectoftheethanol:watermixture(50/60–70/30,v/v)andHfactor (800–6000) were evaluated (Table 1). The pressures obtained were 1.8MPa(at 185 ◦ C) and 2.5MPa (at 200 ◦ C) for biotreated material and control, respectively.Oncetheprocessended,thereactorwascooledinawater–icebath.Thesupernatantwasremovedbydecantationandthesolidpressedouton130-nylonmesh.Thesolidwas washed with water, disintegrated in a blender for 5min, and then passed by aFiber Classifier (Regmed, Brazil) to segregate fibers from the rejects. The pulp wascentrifuged. The samples were then stored in plastic bags at 4 ◦ C.  2.4. Chemical characterization of wood and organolsolv pulp samples Milledwoodsampleswereextractedwithacetonefor16haccordingtostandardTAPPI procedure T280 pm99. Wood and pulp samples were characterized for theircarbohydrate composition using the methodology described by Ferraz et al. [17]. Ina test tube, 300mg of extractive-free milled wood was weighed and 3mL of 72%(w/w) H 2 SO 4  was added. The hydrolysis was carried out in a water bath at 30 ◦ C for1h with stirring every 10min. Later, the acid was diluted to 4% (w/w) with 79mL of distilled water and the mixture transferred to a 250-mL Erlenmeyer flask andautoclaved for 1h at 121 ◦ C. The residual material was cooled and filtered througha sintered glass filter number 4. The solid fraction, insoluble lignin, was dried andweighed. The concentration of monomeric sugars in the soluble fraction was deter-mined by high-performance liquid chromatography (HPLC) in a Hewlett Packard1050usingaAminexHPX-87Hcolumn(Bio-Rad,Hercules,CA,USA),at45 ◦ C,elutedat0.6mL/minwith5mMH 2 SO 4  andwitharefractiveindexdetector.Glucose,man-noseandarabinosewereusedasexternalcalibrationstandards(sincetheretentiontimeandresponseformannose,galactoseandxylosearesimilar,thesethreesugarswereexpressedasmannose).Thefactorsusedtoconvertsugarmonomerstoanhy-dromonomerswere0.90forglucosetoglucanandformannosetomannan,and0.88for arabinose to arabinan. All samples were analyzed in triplicate.TheholocelluloseandviscosityweredeterminedaccordingtostandardTAPPIT9m54 and T230 om04.  2.5. Inoculum preparation The yeast strain used in this work was a thermal acclimatized (40 ◦ C)  Saccha-romyces cerevisiae  IR2T9 [9]. The inoculum was grown in 100mL of liquid cultureconsisting in glucose, 50g/L; yeast extract, 5g/L; peptone, 5g/L; KH 2 PO 4 , 1.0g/L;MgSO 4 · 7H 2 O, 0.50g/L; NH 4 Cl, 2g/L in a 500-mL Erlenmeyer flask. The culture wasincubated for 48h at 40 ◦ C in an orbital shaker at 150rpm.  2.6. Simultaneous saccharification and fermentation (SSF) The simultaneous saccharification and fermentation were performed at 10%substrate consistency. In a 125-mL Erlenmeyer flask, 3g dry weight of pretreatedmaterial (65% humidity) was suspended in a total reaction volume of 30mL 0.05M citrate buffer solution (pH 4.8). Nutrients, consisting in KH 2 PO 4 , 1.0g/L;MgSO 4 · 7H 2 O,0.50g/L;peptone,5.0g/L;yeastextract,5.0g/L,wereadded.Theenzy-matic hydrolysis of the samples was performed using a commercial preparationof   Trichoderma reesei  cellulases Celluclast (70FPU/mL; Novozymes, NC, USA), sup-plemented with a   -glucosidase Novozym 188 (230IU/mL; Novozymes, NC, USA),  Table 1 Experimental design for organosolv pretreatment of   P. radiata .Experimental number Control BiotreatedH factor Organic solvent (%) H factor Organic solvent (%)1 5000( − 1) 50( − 1) 900( − 1) 50( − 1)2 7000(1) 50( − 1) 1300(1) 50( − 1)3 5000( − 1) 70(1) 900( − 1) 70(1)4 7000(1) 70(1) 1300(1) 70(1)5 4586( − 1.41) 60(0) 817( − 1.41) 60(0)6 7423(1.41) 60(0) 1383(1.41) 60(0)7 6000(0) 46( − 1.41) 1100(0) 46( − 1.41)8 6000(0) 74(1.41) 1100(0) 74(1.41)9 6000(0) 60(0) 1100(0) 60(0)10 6000(0) 60(0) 1100(0) 60(0)11 6000(0) 60(0) 1100(0) 60(0)  M. Monrroy et al. / Enzyme and Microbial Technology 47 (2010) 11–16 13 wheretheamountsofenzymewere20FPUand40IU/gofpretreatedmaterial.Afterenzymeaddition,theyeastinoculumwasaddedwithacelldensity6.0g/L(3.5 × 10 8 to3.3 × 10 9 yeastcell/mL).SSFwasperformedat40 ◦ Cand150rpmfor96h.Samplesweretakenat24,48,72and96handanalyzedforethanolcontentbygaschromatog-raphy (GC) on a Perkin-Elmer autosystem XL Headspace using a FID detector and acolumnHPSMS30m.TheGCprogramwas:50 ◦ C × 3min;10 ◦ C/min,100 ◦ C × 1min;25 ◦ C/min, 125 ◦ C × 1min. The temperatures of the injector and detector were 200and 300 ◦ C, respectively. Ethanol yield were calculated as a percentage of the theo-reticalyield.Thetheoreticalyieldwascalculatedbydividingtheamountofethanolobtained (g) by the amount of glucose in the pretreated material (g), assumingthat all the potential glucose in the pretreated material is available for fermen-tation, with a fermentation yield of 0.51gethanol/gglucose multiplied by 100. Allthe determinations were realized in duplicate.  2.7. Response surface methodology In order to determine the conditions to obtain maximal ethanol yield from theorganosolv pulps, the variables organic solvent concentration and H factor werestudied. The influence of each variable was determined using response surfacemethodology(RSM)[18].Thismodelisbasedonacentralcompositecircumscribeddesignmadeofafactorialdesignandstarpoints.Thevariablevalueswerecodedandnormalized in unitary values: − 1 was defined as the lowest value of a variable and+1 was defined as the highest value. From the extreme variable values, the centralpoint (coded 0) was set and assayed in triplicate. Four star points distributed at adistance of 1.41 from the central point were included. The complete experimentaldesign for the reaction conditions of the two methods optimized for ethanol yieldis presented in Table 1.A second-order function that best describes the system’s behavior was deter-mined by a multiple lineal regression method (MLR). The statistical validation wasperformed by a one-way ANOVA test with 95% confidence level. The optimal con-dition values were determined based on the response surface calculated using theSIMPLEXmethod[18].AllthecalculationswereperformedwiththesoftwareModde7.0.0.1 (Umetrics, USA). 3. Results and discussion  3.1. Biodegradation of P. radiata Woodchipsof  P.radiata weredecayedby G.trabeum fordifferentperiods (data not shown). During the first month of biodegrada-tion, the mass loss ranged between 6% and 8%. Weight loss wascalculatedbythedifferencebetweentheweightsofthewoodsam-ple before and after the fungus treatment and the value obtainedwas 6%. We consider that this value is underestimated since dur-ing the fungus treatment, the cellulose chains were fragmented,and a water molecule is incorporated for each broken bond. Theweight loss calculated based on the main component content was8.2%. However, this value is overestimated; for example, the glu-can content is calculated by multiplying the glucose content inthe hydrolysate by 0.9 (162/180) to reflect the weight gain of glucose by the addition of water to the anhydroglucan duringhydrolysis. It is not considered that there are many chains, forthat reason more water is subtracted. The hemicellulose (man-nan+galactan+xylan) and glucans decreased approximately 5%and 3%, respectively. Holocellulose viscosity decreased linearlyover time until the third week (Fig. 1), but without a large car-bohydratemineralization.Thematerialbiotreatedfor3weekswasselected to assay the organosolv process. Table 2 summarizes thechemicalcompositionofthebiotreatedandcontrol P.radiata woodchips. Fig.1.  Holocelluloseviscosityof  P.radiata woodchipsunderbrowndegradationby G. trabeum .  3.2. Organosolv pulps characterization Table 3 summarizes the chemical composition and pulp yieldfor the organosolv pulps produced from biotreated material andcontrol.Thepulpyieldsforthebiotreatedmaterialwere30%lowerfor H factors between 4000 and 7000 (data not shown), probablydue to the process’s high severity. Therefore, H factors employedin the factorial design for the biotreated material were lower thanfor the control. The organosolv pulps yields obtained were 27–43%and 23–41% for the biotreated material and control, respectively.In general, there were few rejects (<5) in the pulping process. Theglucancontentwasslightlyhigherforcontrolpulps(81–85%)thanbiotreated pulps (75–80%). Almost all hemicelluloses were solubi-lized or degraded. The hemicelluloses remaining in the pulps werebelow 3%. The lignin content varied between 10% and 20% in thepulps, and was slightly higher for biotreated pulp.  3.3. Simultaneous saccharification and fermentation (SSF) The hydrolysis and fermentation of the cellulose were per-formed by SSF process. Previous studies have shown that SSFprocesses have several advantages over a separate hydrolysis andfermentation(SHF)process:lowerenzymeinhibitionbyhydrolysisproducts, less overall processing times, and higher ethanol yields.SSF is also considered a less capital intensive process [11,19]. In order to assure greater conversion in all experiments, the SSF wasevaluatedfor96h.Theethanolyieldsobtainedfromthebiotreatedpulps varied between 73% and 91% (pulp basis) and 51% and64% (wood basis). For the control sample, ethanol yields variedbetween 82% and 87% (pulp basis) and 43% and 65% (wood basis).The results indicated that the amounts of ethanol obtained were129–161gethanol/kgwoodand108–164gethanol/kgwoodforthebiotreated and control pulps, respectively. The ethanol yields frombiotreated and control pulps were very similar, but the organosolvprocess conditions were less severe for the biotreated samples. Aprobable explanation for this can be physical structural features of biotreated material, such as distribution of lignin in the biomassmatrix,crystallinity,porevolumeandbiomassparticlesize,wherethe material is more accessible in the organosolv process. Thesefeatures have been considered relevant for conversion of lignocel-lulosic biomass to liquid fuels [1,7,20].  Table 2 Chemical composition of wood chips from control and biotreated  P. radiata .Wood samples Glucan (%) Mannan+galactan+xylan (%) Arabinan (%) Lignin (%) Extractives (%) Viscosity    (dL/g)Control 44.5  ±  0.2 19.0  ±  0.4 1.2  ±  0.03 27.5  ±  0.6 1.5  ±  0.04 5.8  ±  0.1Biotreated 44.1  ±  0.1 15.4  ±  0.1 0.83  ±  0.02 29.4  ±  0.3 1.2  ±  0.02 2.5  ±  0.2Biotreated (cwl) a 41.5  ±  0.1 14.4  ±  0.4 0.8  ±  0.02 27.6  ±  0.3 1.1  ±  0.02 2.5  ±  0.2 a Values corrected for weight loss due to fungal biodegradation.  14  M. Monrroy et al. / Enzyme and Microbial Technology 47 (2010) 11–16  Table 3 Chemical composition of organosolv pulps from control and biotreated  P. radiata .Experimental number Wood samples H factor Organic solvent (%) Pulp yield (%) Rejects (%) Glucan (%) Mannan+galactan+xylan (%) Lignin (%)1 Control 5060 50 38 2 84  ±  1 1.6  ±  0.4 14.1  ±  0.42 Control 7000 50 27 0 81  ±  1 1.3  ±  0.3 16.5  ±  0.13 Control 5077 70 34 2 84.4  ±  1 1.6  ±  0.02 14.7  ±  0.24 Control 7008 70 23 1 82.6  ±  0.5 0.5  ±  0.01 15.4  ±  0.55 Control 4825 60 38 5 87  ±  1 1.3  ±  0.6 10.3  ±  0.16 Control 7423 60 33 1 84.8  ±  0.4 1.2  ±  0.3 11.4  ±  0.17 Control 6103 46 37 5 81  ±  2 0.6  ±  0.02 11.4  ±  0.18 Control 6049 74 38 5 83  ±  1 0.8  ±  0.02 14.4  ±  0.79 Control 6048 60 40 4 85  ±  1 0.6  ±  0.04 15.1  ±  0.110 Control 6051 60 41 2 84  ±  1 0.9  ±  0.1 13.5  ±  0.311 Control 6032 60 40 2 83  ±  1 0.6  ±  0.1 13.0  ±  0.31 Biotreated 900 50 34 7 78.4  ±  0.5 1.2  ±  0.01 16.5  ±  0.62 Biotreated 1312 50 37 6 78.2  ±  0.6 1.2  ±  0.05 17.3  ±  0.23 Biotreated 901 70 38 2 78  ±  0.2 2.7  ±  0.1 17.7  ±  0.24 Biotreated 1312 70 39 4 74.3  ±  0.2 2.2  ±  0.1 16.3  ±  0.25 Biotreated 811 60 40 4 76.2  ±  0.2 2.5  ±  0.1 16.7  ±  0.26 Biotreated 1368 60 41 4 78  ±  1 1.9  ±  0.1 16.5  ±  0.47 Biotreated 805 46 27 18 75.4  ±  0.4 1.2  ±  0.02 20.2  ±  0.38 Biotreated 1088 74 39 3 78.9  ±  0.5 1.2  ±  0.2 16.5  ±  0.59 Biotreated 1092 60 41 3 78.4  ±  0.6 1.6  ±  0.1 16.3  ±  0.110 Biotreated 1085 60 43 4 77.7  ±  0.1 2.3  ±  0.2 16.8  ±  0.111 Biotreated 1073 60 39 4 80  ±  1 2.2  ±  0.2 15.4  ±  0.4  3.4. Response surface methodology for the organosolv pretreatment  The main purpose of this study was to compare biotreated andcontrol material in terms of their response to organosolv pre-treatments in order to optimize these pretreatments to achievemaximum ethanol yield. From the experimental design data(Table 1) and its corresponding ethanol yield (Table 4), a quadratic polynomial was determined (Eqs. (1) and (2)) and validated by theANOVA test for the biotreated and control pulps. The variables inquadratic polynomial were scaled and centered.Biotreated material:  Y   =  64 . 27 ± 1 . 34 + 1 . 86 ± 0 . 84 Z 1 + 0 . 98 ± 0 . 98 Z 2 − 2 . 67 ± 1 . 02 Z 12 − 8 . 30 ± 1 . 04 Z 22 (1)Control:  Y   =  (122e 18 ± 410e 17 − 214e 17 ± 267e 17 Z 1 − 5 . 41e 16 ± 253e 17 Z 2 − 3 . 97e 17 ± 335e 17 Z 12 − 424 e 17 ± 2.97e 17 Z 22 ) 1/10 (2) Z = X  i − X  0 X  max − X  0 (3)where  Z  denotes the scaled and centered value from srcinal vari-ables ( X  ) (Eq. (3)),  Z 1  correspond to H factor,  Z 2  to the organicsolvent concentration (ethanol, %, v/v),  X  0  is midrange and  Y   is theethanol yield (%, wood basis). The error values corresponded to a95% confidence level. The response surfaces for the polynomials(plot inside the dominium limited for the experimental planning)are shown in Fig. 2.For the biotreated pulp, the linear terms for H factor and theorganic solvent concentration have positive coefficients, meaningthat the ethanol yield incremented with the increase in these vari-ables until a maximum value, as shown by the negative quadraticterms for all variables. According to the polynomial, the H factor isthe main determining factor for maximum ethanol yield. For thecontrol pulp, the linear terms for the H factor and the organic sol-vent concentration have negative coefficients, meaning that theethanol yield increases with the decrease in these variables untila maximum value. Considering the confidence intervals, the inter- Fig.2.  Surfaceresponsefortheethanolyield.(A)Controland(B)biotreatedsamples.  M. Monrroy et al. / Enzyme and Microbial Technology 47 (2010) 11–16 15  Table 4 Experimental ethanol yield and response polynomial calculated values.Experimental number Control (ethanol yield (%)) Biotreated (ethanol yield (%))Experimental Calculated Experimental Calculated1 62.5 60.6 51.6 50.42 43.0 54.7 54.9 54.23 53.1 59.5 52.8 52.44 36.8 51.5 55.3 56.15 63.0 62.6 56.6 56.36 56.1 50.7 61.8 61.67 57.3 58.2 36.5 37.78 60.0 55.9 49.5 49.09 63.5 64.4 63.8 64.310 63.7 64.4 63.1 64.311 65.6 64.4 64.6 64.3 actionbetweenvariablesdidnothaveasignificanteffectonethanolyield.Basedontheexperimentalconditionsusedintheexperimentaldesign, the polynomial was used to predict the ethanol yield. Theresponses were close to the experimental values (Table 4) with acorrelation coefficient ( r  2 )=0.99 and ( r  2 )=0.79 for the biotreatedand control pulps, respectively. These values together with theANOVA test statistically validated the model.The optimum values of the variables for maximal ethanol yieldweredeterminedbytheSIMPLEXmethodusingthemaximumval-uesoftheresponsesurface[18].Thepredictedvaluesforbiotreatedpulps were: 1156 H factor and 60% (v/v) organic solvent (at 185 ◦ Cfor 17min). According to the polynomial, the maximum ethanolyield was 64.6 ± 1.2% (at 95% confidence level), while the exper-imental ethanol yield for this pulp obtained at these conditionswas 63.8%, agreeing with the predicted value (Fig. 2). The pre-dicted values for control pulps were: 6000 H factor and 60% (v/v)organicsolvent(at200 ◦ Cfor32min).Accordingtothepolynomial,the ethanol yield was 64.4 ± 2.2%, while the experimental ethanolyield for this pulp obtained at these conditions was 64.3%, agree-ingwiththepredictedvalue(Fig.3).Ethanolproductionwas40g/L forbothmaterials(Fig.2).Themaximalethanolamountthatcouldbe produced from  P. radiata  is 252gethanol/kgwood consideringtotal conversion of glucose into ethanol. The results indicate thatthe amount of ethanol obtained is 161gethanol/kgwood for bothmaterials.Theethanolyieldobtainedinthisstudyissimilartotheamountobtained by Itoh et al. [13] and Mu˜noz et al. [11] from beech and  P.radiata , respectively, with white rot fungi although the organosolvconditions used in these studies were significantly higher. Itoh etal. [13] employed 200 ◦ C at 120min and 180 ◦ C at 60min for thecontrol and biotreated materials, respectively. Mu˜noz et al. [11] Fig.3.  EthanolproductionfromSSFof  P.radiata organosolvpulps.Ethanolyield(  )biotreated material and (  ) control. Ethanol amount (  ) biotreated material and(  ) control. employed 200 ◦ C at 60min for both materials. The present studyused organosolv at lower severity for the control (200 ◦ C, 32min)andforthebiotreatedmaterial(185 ◦ C,18min),obtainingthesameethanol yield. 4. Conclusions The biotreatment of the wood prior to the organosolv pro-cess improved solvent accessibility. A lower process severity wasrequired in the biotreated samples with brown rot fungus in com-parison with the control to obtain the same ethanol yield.  Acknowledgement FinancialsupportfromFONDECYT(Grant1080303)isgratefullyacknowledged. References [1] Kristensen J, Thygesen L, Felby C, Jorgensen H, Elder T. Cell-wall structuralchanges in wheat straw pretreated for bioethanol production. Biotechnol Bio-fuels 2008;1:5.[2] WymanCE,DaleBE,ElanderRT,HoltzappleM,LadischMR,LeeYY.Coordinateddevelopmentofleadingbiomasspretreatmenttechnologies.BioresourTechnol2005;96:1959–66.[3] U.S. Department of Energy, Oak Ridge National Laboratory, BioenergyInformation Network, Bioenergy Feedstock Development Program.Bioenergy Frequently Asked Questions. 2001. Available on-line athttp://bioenergy.ornl.gov/faqs/index.html.[4] Ramakrishnan A, Mala R. Bioethanol from lignocellulosic biomass: part IIIhydrolysis and fermentation. In: Pandey A, editor. Handbook of plant-basedbiofuels. Boca Raton: Taylor & Francis Group; 2009. p. 159–76.[5] Laxman RS, Lachke AH. Bioethanol from lignocellulosic biomass. In: Pandey A,editor. Handbook of plant-based biofuels. Boca Raton: Taylor & Francis Group;2009. p. 121–38.[6] Um BH, Karim MN, Henk LL. Effect of sulfuric and phosphoric acid pre-treatments on enzymatic hydrolysis of corn stover. Appl Biochem Biotechnol2003;105:115–25.[7] Zhu L, O’Dwyer JP, Chang VS, Granda CB, Holtzapple MT. Structuralfeatures affecting biomass enzymatic digestibility. Bioresour Technol2008;99:3817–28.[8] MosierNS,WymanC,DaleB,ElanderR,LeeYY,HoltzappleM,etal.Featuresof promising technologies for pretreatment of lignocellulosic biomass. BioresourTechnol 2005;96:673–86.[9] Araque E, Parra C, Freer J, Contreras D, Rodríguez J, Mendonc¸a R, et al. Evalua-tion of organosolv pretreatment for the conversion of   Pinus radiata  D. Don toethanol. Enzyme Microb Technol 2008;43:214–9.[10] Ballesteros I, Negro MJ, Oliva JM, Cabanas A, Manzanares P, BallesterosM. Ethanol production from steam-explosion pretreated wheat straw. ApplBiochem Biotechnol 2006, 129–132:496–508.[11] Mu˜nozC,Mendonc¸aR,BaezaJ,BerlinA,SaddlerJ,FreerJ.Bioethanolproductionfrombio-organosolvpulpsof  Pinusradiata and  Acaciadealbata .JChemTechnolBiotechnol 2007;82:767–74.[12] ZhaoX,ChengK,LiuD.Organosolvpretreatmentoflignocellulosicbiomassforenzymatic hydrolysis. Appl Microbiol Biotechnol 2009;82:815–27.[13] Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T. Bioorganosolve pretreat-ments for simultaneous saccharification and fermentation of beech wood byethanolysis and white rot fungi. J Biotechnol 2003;103:273–80.[14] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production:a review. Bioresour Technol 2002;83:1–11.
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