Invasive Biomass Valorization Environmentally Retamo Espinoso

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Research ArticleReceived: 8 July 2012 Revised: 3 September 2012 Accepted: 16 September 2012 Published online in Wiley Online Library: 14 November 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.3963

Invasive biomass valorization: environmentallyfriendly processes for obtaining secondgeneration bioethanol and saccharides fromUlex europusIria Ana Ares-Peon,a Aloia Roman,a Gil Garrotea and Juan Carlos Parajob

Abstract

BACKGROUND: Ulex europus (UE) is a widespread invasive shrub species causing economic problems and environmentalhazards. This work deals with the valorization of UE by hydrothermal processing (to obtain hemicelluloses-derived saccharides)followed by simultaneous saccharification and fermentation of the resulting solids for manufacturing second generationbioethanol.

RESULTS: Hydrothermal processing of UE resulted in the solubilization of up to 21.5 wt% of the oven-dry raw material, leadingto the formation of hemicelluloses-derived saccharides as major reaction products. Treatments at various severities resultedin processed solids with enhanced susceptibility to enzymatic hydrolysis, allowing cellulose to glucose conversions up to 87%.Simultaneous saccharification and fermentation of solids pretreated under selected conditions, performed at various chargesand solid loadings, resulted in bioethanol conversions up to 82% of the stoichiometric amount, with volumetric concentrationshigher than 30 g ethanol L-1.

CONCLUSION: Hydrothermal processing of UE followed by simultaneous saccharification and fermentation of pretreated solidswas suitable for the selective separation of hemicelluloses as soluble saccharides and for the manufacture of second generationbioethanol at high yield from the pretreated solids.c 2012 Society of Chemical Industry

Keywords: autohydrolysis; bioethanol; simultaneous saccharification and fermentation; Ulex europus; valorization

INTRODUCTIONGorse (Ulex europus, UE) is a densely packed, prickly evergreenshrub less than 4 m tall. The International Union for Conservationof Nature recognizes gorse as one of the top 100 worst invasivespecies in the world. Gorse is abundant in Galicia (North West ofSpain), where it is spread over about 30% of the territory.1 Theproduction of gorse has been estimated at 0.71.9 metric tonsha-1 year-1,2 which corresponds to 6.2105 to 1.7106 metrictons gorse year-1 in our region. UE may grow altering the soilproperties, increasing erosion and becoming a significant firehazard,andoutcompeteotherplantswithsubsequent reduction inproductivity. Finding a profitable way for gorse valorizationwouldresult in environmental benefit and in improved development ofsustainable agricultural and silvicultural exploitation.

Owing to its woody nature, gorse can be included among thelignocellulosic materials (LCMs), 1,3 whose structural componentsinclude cellulose (a linear polymer made from glucose structuralunits), hemicelluloses (branched polymers made up of sugars andsubstituents) and lignin (a tridimensional polymer made up fromoxygenated phenylpropane structural units). Additionally, LCMscontain non-structural components (including extractives, ashesand protein), which are of minor interest for the objectives ofthis study. LCMs can be utilized according to the biorefinery

concept46 to obtain various fractions suitable for specificpurposes such as the manufacture of fuels and/or chemicals.

LCMs fractionation may be carried out starting with anautohydrolysis stage, in which the feedstock is treatedwith hot, compressed water to cause a number of effects,including hemicelluloses solubilization and structural alterationof the cellulose remaining in processed solids. Under selectedconditions, hemicelluloses can be selectively converted intosoluble saccharides (mainly of oligomeric nature, but alsomonosaccharides). When hardwoords or agricultural materialsare employed as autohydrolysis substrates, xylooligomers (XO)

are usually the target products.79 XO can be used for anumber of applications in the food, pharmaceutical or chemicalindustries,10,11 or subjected to hydrolysis to yield solutions

Correspondence to: G. Garrote, Department of Chemical Engineering, Facultyof Science, University of Vigo (Campus Ourense), As Lagoas, 32004 Ourense,Spain. E-mail: [email protected]

a Department of Chemical Engineering, Faculty of Science, University of Vigo(Campus Ourense), As Lagoas, 32004, Ourense, Spain

b CITI (Centro de Investigacion, Transferencia e Innovacion), University of Vigo,Tecnopole, San Cibrao das Vinas, Ourense, Spain

J Chem Technol Biotechnol 2013; 88: 9991006 www.soci.org c 2012 Society of Chemical Industry

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www.soci.org IA Ares-Peon et al.

of hemicellulosic sugars12,13 suitable for the manufacture offermentation media (for example, for lactic acid or bioethanol

production).1416

The solids from hydrothermal processing of LCMs are mainlymade up of cellulose and lignin, and are expected to presentincreased susceptibility to further processing, for example by

delignification17,18 or enzymatic methods.1922

Alternatively, theaqueousprocessingofLCMsmaybeconceivedas a pretreatment for enzymatic hydrolysis, as it meets a numberof favourable requirements,23 including savings in chemicals,limited corrosion, ability to cause structural alteration of LCMs,high selectivity with respect to cellulose solubilization and thepossibility of obtaining valuable products from hemicelluloses.

The production of bioethanol from pretreated solids canbe carried out by consecutive stages of enzymatic hydrolysisand fermentation (method known as separate hydrolysis andfermentation, SHF) or by a single stage with enzymes andmicroorganisms (referred to as simultaneous saccharification andfermentation, SSF). SSF has comparative advantages derivedfrom the lower operational costs, limited contamination risk anddecreased enzyme inhibition caused by glucose and cellobiose.Because of this, SSF has provided improved results over those of

SHF.2426

This work deals with the valorization of UE by aqueousfractionation (to recover hemicelluloses as soluble saccharides),andwiththefurtherutilizationof thepretreatedsolidsassubstratesfor bioethanol production by SSF.

MATERIAL AND METHODSRaw materialUE samples were collected locally, milled to pass an 8 mm screen,air-dried, homogenized in a single lot to avoid compositionaldifferences between samples, and stored in a dark and dry placeuntil use.

Analysis of raw materialUE samples from thehomogenized lotweremilled to aparticle sizeless than 0.5mmand subjected to the following assays: extractives(TAPPI T-264-cm-97 method), moisture (T-264-cm-97 method),ashes (T 211 om-93 method), and quantitative acid hydrolysis(T-249-cm-85 method). The liquid phase from the latter assaywas analyzed by high performance liquid chromatography (HPLC)for sugars and acetic acid (conditions: detector, refractive index;column, Aminex HPX-87H; mobile phase, 0.01 mol L-1 H2SO4; flowrate,0.6mLmin-1). Theconcentrationsofglucose,xylose,arabinoseand acetic acid were employed to calculate the sample contentsof glucan, xylan, arabinosyl substituents and acetyl groups. Thesolid phase from the quantitative acid hydrolysis was consideredas Klason lignin after correction for ashes. Analyses were carriedout in quadruplicate.

AutohydrolysisWater andUE samplesweremixedat aproportionof 8 kgwater perkg wood and heated in a stainless steel reactor of 1 gallon volume(Parr InstrumentsCompany,Moline, Illinois) following thestandardtemperature profile19 to reach the target temperature (denotedTMAX, in the range 150240

C). Then, the medium was cooled byflowing water through an internal stainless steel loop. Solids wereseparated by filtration, washed with water and employed for solid

yield determination (SY, g solid recovered per 100 g raw material,oven dry basis) and analyzed.

The operational range was selected to cover the conditionsleading from little to extensive hemicelluloses solubilization. Theharshness of treatments can be expressed in terms of severity(S0),27 calculated as:

So = log Ro = log [RoHEATING + RoCOOLING

= log

tMAX0

exp

(T (t) TREF

) dt

+tF

tMAX

exp

(T (t) TREF

) dt

(1)

where Ro is the severity factor, tMAX (min) is the time neededto achieve the target temperature TMAX (

C), tF (min) is the timeneeded for the whole heatingcooling period, and T(t) and T (t)represent the temperature profiles in the heating and coolingstages, respectively. Calculations were made using the valuesreported usually for and TREF (14.75

C and 100C, respectively).

Analysis of solid phase from autohydrolysisSamples of solids from autohydrolysis were air dried, milled to aparticle size

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Bioethanol and chemicals from Ulex Europus www.soci.org

experiments were performed in duplicate, and average results arereported.

The results achieved in the enzymatic hydrolysis can beconveniently compared in terms of the cellulose-to-glucoseconversion achieved at time t (CGCt), calculated as:

CGCt = 100 Gt G0Gn100 180162 LSR+1KL/100

(2)

where Gt is the glucose concentration (g L-1) at time t, G0 is theinitial glucose concentration, Gn is the glucan content of the solidphase (g glucan per 100 g solid phase, oven dry basis), 180/162 isthe glucose hydration factor, is density (g L-1), LSR is the liquor tosolid ratio employed in enzymatic hydrolysis (g liquid g-1 oven-drysolid), and KL is the Klason lignin content of solid phase (g ligninper 100 g solid phase, oven dry basis).

Yeast cultivation and inoculum preparationThestrainemployed forbioethanolproductionwasSaccharomycescerevisiaeCECT-1170,obtained fromtheSpanishCollectionofTypeCultures (Valencia, Spain). Cells were grown at 32C for 24 h in amedium containing 10 g glucose L-1, 5 g peptone L-1, 3 g maltextract L-1 and 3 g yeast extract L-1.

Simultaneous saccharification and fermentation (SSF) of solidphases from autohydrolysisSimultaneous saccharification and fermentation (SSF) assays werecarried out in duplicate in 250 mL Erlenmeyer flasks (orbitalshaking at 120 rpm, 35C, pH 5). SSF media were prepared bymixing the appropriate amounts of solid phases, water, buffer,nutrients and cells. Suspensions containingwater, buffer and solidsubstrateswereautoclavedat 121C for 15min separately fromthenutrients, and thermostated at 35C. At time 0, enzymes and cellswere added. For 100 mL of media, 10 mL of inocula (initial yeastconcentration, 1.0 g L-1) and 10 mL of nutrients (concentrations: 5g peptone L-1, 3 g yeast extract L-1 and 3 g malt extract L-1) wereadded. At selected fermentation times, samples were withdrawnfrom themedia, centrifuged at 5000 rpm for 5min, and aliquots ofsupernatants were assayed for sugars, acetic acid and ethanol byHPLC using the method described above.

RESULTS AND DISCUSSIONComposition of Ulex europusTable 1 shows compositional data for UE. Glucan was the mostabundant fraction, followed by lignin. Hemicelluloses includedxylan, arabinosyl substituents and acetyl groups.

The glucan content was similar to that of fast growinghardwoods such as Eucalyptus globulus8 or other invasive speciessuch as Arundo donax.30 The molar ratio xylose: acetyl groups: arabinose (10: 6.7 : 0.7) was also similar to that reported forEucalyptus wood.8

Hydrothermal processingTable 2 shows the results achieved for SY, NVC content, andsolid phase composition in treatments performed at selected So.The treatments at TMAX of 205, 215, 230 or 240

C were made intriplicate, and the average values are reported. Figures 1 and 2show the composition of the liquid phases, measured by theircontents of oligomers, monosaccharides, acetic acid, HMF and F.

Table 1. Composition of Ulex europus (data in wt% standarddeviation; data based on four replicates)

Content

Glucan 41.7 0.2Xylan 17.3 0.4Arabinan 1.2 0.3Acetyl groups 3.8 0.0Klason lignin 29.4 0.1Extracts 5.1 0.1Ashes 0.7 0.1

UE fractionationThe SY determined for experiments performed at So 3.2 wereclose to 91 g per100 g oven-dry UE. Harsher conditions leading toSo in the range 3.24.0 resulted in decreased yields (up to 70 g per100 g oven-dry UE), whereas severe conditions (So > 4) led to SYin the range 6569 g per 100 g oven-dry UE. As this latter value isbelow the joint contribution of lignin and glucan in the untreatedfeedstock, it can be inferred that at least one of these fractionswasdegraded in part under the corresponding conditions. NVC wasbelow 10 g per 100 g liquor operating at So < 3.2, in the range2025 g per 100 g liquor for treatments performed at So in therange 3.64.4, and decreased at higher severities owing to sugardecomposition.

Composition of spent solidsTable 2 also includes data concerning the composition of solidsfrom autohydrolysis. The most abundant component was glucan,whose percentage increased with severity up to 57.7 g per 100g solid in the sample obtained at So = 4.38. Higher severitiesresulted in decreased Gn contents, reaching 55 g per 100 g in theseverest experiment. The glucan recovery (measured as g glucanprocessed solids per 100 g glucan in UE) presented an averagevalue of 91.0%, confirming the occurrence of some solubilization.

The Klason lignin content of processed samples (KL, measuredas g Klason lignin per 100 g treated solids) increased with severity,reaching values above 40% in experiments performed at So 4. The average lignin recovery was near 100%, owing to theparticipation of condensation reactions under harsh conditions.

The removal of hemicelluloses from solid phase increasedsteadily with severity. High solubilisation was observed at So> 3.2, and complete removal was observed under the harshestconditions assayed. Acetyl groups and xylan showed closelyrelated variation patterns; whereas arabinosyl groups were splitoff under mild conditions, with complete removal at So = 3.2(conditions under which about 61% of the initial xylan and acetylgroups still remained in solid phase). The selective solubilisationof hemicelluloses occurring under mild and severe operationalconditions, resulted in the observed increase in glucan and lignincontents.

The above data confirm that selected autohydrolysis conditionsmay result in both extensive hemicelluloses removal and highretentions of cellulose and lignin in solid phase.

Liquid phase compositionThe major solutes in autohydrolysis liquors corresponded tohemicelluloses-derived compounds, including oligosaccharides,monosaccharides and sugar degradation products.

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Table 2. Effects achieved by hydrothermal processing: solid yield (SY , g treated solids per 100 g rawmaterial, oven dry basis), liquor content of nonvolatile compounds (NVC, g non-volatile compounds per 100 g raw material, oven dry basis), and composition of treated solids (expressed as g ofcomponent per 100 g treated solid, oven dry basis). Standard deviations are based on three replicates

TMAX (C) So (-) SY NVC Glucan Xylan Arabinan Acetyl groups Klason lignin

150 2.32 92.3 6.3 43.4 0.6 17.9 0.4 1.8 0.4 3.7 0.1 32.9 2.7160 2.61 91.7 6.2 46.2 0.4 19.0 0.3 1.9 0.1 3.7 0.0 33.2 2.7170 2.91 91.6 7.2 44.7 0.6 18.2 1.4 1.5 0.2 3.5 0.1 35.6 3.5180 3.20 89.3 9.5 44.8 0.6 17.6 0.1 1.4 0.1 3.6 0.0 35.6 1.6190 3.49 81.4 16.5 45.8 0.9 13.0 0.4 < 0.1 2.8 0.0 39.8 1.6195 3.64 75.1 23.1 46.8 0.0 9.6 0.6 < 0.1 1.9 0.0 38.4 0.6200 3.79 71.9 25.0 48.9 0.2 8.3 0.2 < 0.1 1.6 0.1 39.9 0.4205a 3.94 70.8 1.1 24.8 0.1 52.5 0.6 6.4 0.6 < 0.1 1.0 0.1 42.2 1.0210 4.08 66.6 22.5 53.7 0.6 5.3 0.0 < 0.1 0.8 0.0 41.8 0.5215a 4.23 69.2 1.9 22.2 0.4 56.0 0.2 5.1 0.1 < 0.1 0.7 0.0 39.2 2.0220 4.38 67.0 21.1 57.7 1.0 4.7 0.5 < 0.1 0.4 0.2 41.6 3.0225 4.52 65.9 14.7 55.9 0.8 4.1 0.3 < 0.1 0.3 0.0 45.7 1.1230a 4.67 65.6 0.5 13.1 0.2 57.1 1.2 2.5 0.4 < 0.1 0.2 0.0 42.1 3.5235 4.81 67.9 10.9 55.4 0.9 < 0.1 < 0.1 < 0.1 46.1 0.6240a 4.97 66.0 0.9 10.1 0.2 54.5 1.9 < 0.1 < 0.1 < 0.1 39.0 0.8a Average values of three replicates.

0

2

4

6

8

10

12

14

16

18

20

2 2.5 3 3.5 4 4.5 5So (dimensionless)

Conc

entra

tion

(g/L)

GO

XO

ArO

AcO

Figure 1. Dependence of the concentrations of GO (glucooligomers, g eq. glucose L-1), XO (xylooligomers, g eq. xylose L-1), ArO (arabinosyl moieties, geq. arabinose L-1) and AcO (acetyl groups linked to oligomers, g eq. acetic acid L-1) on So (severity).

Owing to the analytical method employed for oligosaccharidequantitation, which is based on total hydrolysis and furtherdetermination of the structural units, oligomers are referred to asglucooligomers (GO), xylo-oligomers (XO), arabinosyl units boundto oligomers (ArO) and acetyl groups esterified to oligomers(AcO). All of them were measured as monomer equivalents.Figure 1 shows the dependence of the concentrations of GO,XO, ArO and AcO on So, and Fig. 2 displays the interrelationshipbetween the concentrations of monosaccharides (glucose, xyloseand arabinose) and other compounds present in themedia (aceticacid, HMF and F) on So.

In general terms, a slight decrease in GO concentration wasobserved when So increased. Under mild operational conditions(So < 4.08), the GO concentration averaged 4.0 g L-1, whereasharsher treatments decreased the concentration to 1 g L-1. XOwere themajor liquor components,with concentrations increasingwith So in the range 3.644.08 (to reach a maximum of 19.6 g

L-1 at So = 3.79), and decreasing further up to almost completeconsumption. The variation pattern observed for AcO was closelyrelated to the one described for XO, with concentrations withinthe range 44.6 g L-1 in experiments performed at So 3.644.08(maximum value, 4.56 g AcO/L at So = 3.79), and further decreaseunder higher severity conditions. Comparatively, ArO reachedlimited concentrations (0.6 g L-1 as an average), and showed amaximum at So about 3.5. The behaviour of the overall oligomerconcentration was governed by the variation pattern observedfor XO, reaching concentrations in the range 2629 g L-1 at So of3.644.08 (maximum value, 29.0 g L-1 at So = 3.79, correspondingto 71% of the initial xylan, which is in agreement with resultsreported on the autohydrolysis of UE).31

Figure 2 shows that the monosaccharide concentrationsattained in the reaction media were comparatively limited: theglucose concentration (average value, 1.8 g L-1) remained fairlyconstant with So, even if a slight increase in concentration

wileyonlinelibrary.com/jctb c 2012 Society of Chemical Industry J Chem Technol Biotechnol 2013; 88: 9991006

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0

1

2

3

4

5

6

7


Conc

entra

tion

(g/L)

GlucoseXyloseArabinoseAcetic acidHMFF

Figure 2. Dependence of the concentrations of sugars (glucose, xylose and arabinose, g L-1), acetic acid (g L-1), F (furfural, g L-1) and HMF(hydroxymethylfurfural, g L-1) on So (severity).

(compatible with GO decomposition) was observed under harshconditions. Xylose, the most abundant sugar, increased itsconcentration with So (especially at So > 3.79) up to reach amaximum (5.0 g L-1 at So = 4.38), and then decreased with theseverity of treatments up to 1 g L-1. The concentration of arabinosewas below 1 g L-1, and reached a maximum under conditionsdefined by So in the range 3.644.23.

The concentrations of acetic acid, F and HMF increasedsignificantly at So > 4, to reach the following maxima at So4.55: acetic acid, 6.6 g L-1; HMF, 2.2 g-1; and F, .8 g L-1.

Enzymatic hydrolysis of Ulex europusThe solids from the various autohydrolysis treatments (seeTable 2) were assayed for susceptibility to enzymatichydrolysis. In agreement with literature reported on the scarceenzymatic digestibility of highly lignified substrates subjected toautohydrolysis,19,20 and in order to explore the effects obtainedby drastic hydrothermal processing, solids autohydrolyzed at So= 5.06 (obtained by processing at 230C for 10 min) were alsoassayed as substrates for enzymatic hydrolysis, but not achievedimprovements in the results (data not show).

The enzymatic assays were performed at liquid to solid ratios(LSR) of 6, 12 or 20 g liquid g-1 oven-dry autohydrolyzed UE,using enzyme to substrate ratios (ESR) of 6.2, 10.3 or 11.5 FPU g-1

oven-dry autohydrolyzed UE.Autohydrolyzed solids treated at So < 3.8 presented little

enzymatic digestibility, reaching CGC48 < 20%. This latterparameter increased to 70% when UE was treated at So = 4.67,and decreased under harsher conditions

The cellulose to glucose conversion was favoured by increasedvalues of LSR and ESR, the major effects being associated toLSR within the considered experimental domain. The kinetic ofenzymatic hydrolysis was interpreted on the basis of the followingequation:32

CGCt = CGCMAX tt + t1/2 (3)

where CGCMAX and t1/2 are fitting parameters measuring themaximum glucose conversion achievable at infinite reaction time,and t1/2 (h) measures the reaction time needed to reach a glucose

conversion corresponding to 50%of CGCMAX. Figures 3 and 4 showthe dependences of CGCMAX and t1/2 on So.

Solids pretreated undermild conditions (So < 3.8) led to CGCMAX< 30%, but as expected, 33,34 the susceptibility of substratesincreased with severity, to reach CGCMAX about 90% in the case ofthe solid autohydrolyzedat So =4.67.Harsher conditionsprovidedworse susceptibility of treated solids to enzymatic hydrolysis.The highest cellulose conversions were achieved in experimentsperformed at the lowest solid loadings, as reported in relatedstudies,35 with minor effects associated to variable ESR within thetested range. The experimental results of this work compare wellwith data reported for other LCMs of residual nature. For example,glucose conversions in the range 1576% have been achievedusing steam-pretreated olive tree trimmings (operating at ESR =15 FPU g-1 and LSR=20 g g-1); 36 whereas guayule residues treatedaccording to the AFEX process provided less than 30% celluloseconversion (operatingat ESR=12mgproteing-1 glucanandLSR=100 g g-1 glucan).37 Sugar cane bagasse subjected to consecutivestages of steam explosion and NaOH delignification allowed 72%cellulose conversion (operating at ESR = 20 FPU g-1 cellulose andLSR=11.3 g g-1).38

The kinetic parameter t1/2 decreased with severity from valueslonger than 40 h to reach a minimum (3.55 h) in the experimentcarried out at So = 4.97. The limited susceptibility observed forsolids from too severe treatments were well interpreted by thevariation pattern of t1/2 (which presented increased values forthese substrates). In a similar way, the favourable kinetic effectscaused by increased ESR and decreased LSR were reflected indecreased values of t1/2.

Bioethanol production by SSFBased on the results obtained in the above experiments, solidtreated at selected severities were employed for bioethanolproduction by SSF operating at the best ESR (in the range 4.1to 11.5 FPU g-1) and intermediate solid loading (in the range 6 to12 g g-1).

As representative examples, Fig. 5 shows the ethanolconcentration profiles for selected experiments, which presenteda typical shape with declining slope.

The ethanol yield (YE, g ethanol per 100 g potential ethanol),calculated for conversion of glucan into ethanol without


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10

20

30

40

50

60

70

80

90


CGCM

AX

(%)

(a)(b)(c)(d)

Figure 3. Dependence of the maximum glucose conversion (CGCMAX) on So (severity) for: (a) experiments carried out at LSR = 12 g g-1 and ESR = 6.2FPU g-1; (b) experiments carried out at LSR = 20 g g-1 and ESR = 10.3 FPU g-1; (c) experiments carried out at LSR = 6 g g-1 and ESR = 11.5 FPU g-1; (d)experiments carried out at LSR = 12 g g-1 and ESR = 11.5 FPU g-1.

0

10

20

30

40

50


t 1/2

(h)

(a)(b)(c)(d)

Figure 4. Dependence of the kinetic parameter t1/2 (h) on So (severity) for: (a) experiments carried out at LSR = 12 g g-1 and ESR = 6.2 FPU g-1; (b)experiments carried out at LSR = 20 g g-1 and ESR = 10.3 FPU g-1; (c) experiments carried out at LSR = 6 g g-1 and ESR = 11.5 FPU g-1; (d) experimentscarried out at LSR = 12 g g-1 and ESR = 11.5 FPU g-1.

degradation as:

YE = 100[

ethanol]

[ethanol

]POT

(4)

where [ethanol] is the highest ethanol concentration (g L-1)achieved in the experiment and [ethanol]POT is the potentialethanol concentration, calculated as:

[ethanol

]POT =

Gn

100 92162

LSR + 1 KL100

(5)

where Gn is the glucan content of solids (g glucan per 100 gsolid, oven dry basis), (92/162) is the stoichiometric factor, isthe density of the reaction medium (average value of 1005 2g L-1), LSR is the liquid-to-solid ratio in SSF experiments and KL isthe Klason lignin content of solids (g Klason lignin per 100 g solid,oven dry basis).

The results determined for the ethanol conversion (EC, definedas the percentage of ethanol obtained in experiments respect tothe ethanol that would result from total cellulose saccharificationand further stoichiometric conversion of glucose into ethanol),

are listed in Table 3. The dependence of EC on the operationalvariables presented trends closely related to the ones describedabove for CGC.

The maximum YE (83.3%) was achieved under the followingconditions: solid pretreated at So = 4.67; LSR and ESR fixed inthe highest values assayed. The maximum ethanol concentration(30.2 g L-1) was achieved for the same solid and the same ESR,but at LSR = 6 g g-1. This result was in the range reported as athreshold foreconomicprofitability,39 andcompareswellwithdatareported in related studies. For example, wastes from olive treespretreatedbyacidhydrolysis led to fermentationmediacontaining20.3 g ethanol L-1,40 whereas olive tree trimmings pretreated bysteamexplosionandalkalineperoxidedelignificationwereusedassubstrates to obtain 29.4 g ethanol L-1 with an ethanol conversionof 65.2%.41 An ethanol concentration of 25 g L-1 was reported fordelignified sugar cane bagasse.38

CONCLUSIONSThisworkprovidesanexperimental assessmenton thevalorizationof Ulex europus (an invasive species causing environmental


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15

20

25

30

0 20 40 60 80 100 120 140 160Time (h)

Conc

entra

tion

(g/L)

So = 4.03So = 4.38So = 4.67

Figure 5. Ethanol concentration profiles determined in SSF assays performed at LSR = 6 g g-1 and ESR = 11.5 FPU g-1.

Table 3. Data for SSFexperiments: pretreatment conditions (definedby severity,So); operational conditionsdefinedbyenzyme to substrateratio (ESR) and liquor to solid ratio (LSR); and ethanol yield (YE)

So(dimensionless) ESR(FPU g-1) LSR(g g-1) YE(%)

3.79 11.5 6 6.3 1.24.08 11.5 6 38.8 1.44.38 11.5 6 49.7 0.84.67 11.5 6 60.9 1.14.97 11.5 6 47.5 1.54.23 4.1 10 11.6 0.24.67 4.1 10 23.7 0.54.97 4.1 10 22.0 0.53.94 8.2 10 68.0 3.74.67 8.2 10 82.3 4.23.64 6.2 12 29.7 0.83.79 6.2 12 49.6 2.03.94 6.2 12 50.1 1.0

hazards) by means of an environmentally friendly process,based on consecutive stages of hydrothermal processing andSSF. The effects of the pretreatment conditions on the solubleproducts obtained from hemicelluloses and on the susceptibilityof pretreated solids to enzymatic hydrolysis were assessedunder conditions covering the experimental domain of practicalinterest. Under selected processing conditions, up to 21.5 wt%of the raw material was recovered as valuable oligomeric ormonomeric saccharides, whereas the kinetics and yields ofenzymatic hydrolysis presented a clearly defined optimal range.Based on this latter information, SSF studies were carried outto establish conditions leading to ethanol concentrations andconversions that compare well with those in the literature.

ACKNOWLEDGEMENTSThe authors are grateful to Xunta de Galicia for financial supportof this work, in the framework of the Research Project Use of forestresidues for biofuels production (reference 08REM002383PR).

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