Artículo 2 ASA H franco

7/29/2019 Artculo 2 ASA H franco

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Research Article

Received: 4 August 2011 Revised: 20 September 2011 Accepted: 28 September 2011 Published online in Wiley Online Library: 8 February 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.2761

Alkaline sulfite/anthraquinone pretreatment

followed by disk refining ofPinus radiataand Pinus caribaea wood chips for biochemicalethanol production

Heriberto Franco,a,b Andre Ferraz,c Adriane M. F. Milagres,c

Walter Carvalho,cJuanita Freer,a,dJaime Baezaa,d

and Regis Teixeira Mendoncaa,b

Abstract

BACKGROUND: Alkaline sulfite/anthraquinone (ASA) cooking of Pinus radiata and Pinus caribaea wood chips followed bydisk refining was used as a pretreatment for the production of low lignified and high fibrillated pulps. The pulps producedwith different delignification degrees and refined at different energy inputs (250, 750 and 1600 Wh) were saccharified withcellulases and fermented to ethanol with Saccharomyces cerevisiae using separated hydrolysis and fermentation (SHF) orsemi-simultaneous saccharification and fermentation (SSSF) processes.

RESULTS: Delignification of ASA pulps was between 25% and 50%, with low glucans losses. Pulp yield was from 70 to 78% forpulps of P. radiata and 60% for the pulp of P. caribaea. Pulps obtained after refining were evaluated in assays of enzymatichydrolysis. Glucans-to-glucose conversion varied from 20 to 70%, depending on the degree of delignification and fibrillationof the pulps. The best ASA pulp of P. radiata was used in SHF and SSSF experiments of ethanol production. Such experimentsproduced maximum ethanol concentration of 20 g L1, which represented roughly 90%of glucose conversion andan estimatedamount of 260 L ethanol ton1 wood. P. caribaea pulp also presented good performance in the enzymatic hydrolysis andfermentation but, due to the low amount of cellulose present, only 140 L ethanol would be obtained from each tonof wood.

CONCLUSION: ASAcooking followed by disk refining wasshown to be an efficient pretreatment process, which generateda lowlignified and high-fibrillated substrate that allowed the production of ethanol from the softwoods with high conversion yields.c 2012 Society of Chemical Industry

Keywords: Pinus radiata; Pinuscaribaea; ASA pretreatment; disk refining; enzymatic hydrolysis; ethanol fermentation

INTRODUCTIONIn recent years, several countries worldwide (Brazil, USA, Canada,

Japan, India, China and Spain, Sweden, among others) have

developed their internalethanol marketsand established plansfor

its use as a single-fuel or as an oxygenated additive to gasoline. 1

Biofuels offer many potential benefits, including energy security,

balance of trade, low greenhouse gas emissions, renewability, jobs

and community development, among others.2 World demand

for fuel ethanol in 2015 is estimated to range between 65

and 90 billion liters. Today, the world supply is mainly derived

from US corn or Brazilian sugarcane, with the production in

Brazil and USA estimated to be in the range of 28 to 35 billion

liters and 23 to 28 billion liters, respectively. 3 Other sources of

biomass can be used for ethanol production: woods, grasses,

wood wastes, agriculture wastes, and waste paper, among others.

The current annual world biomass potential is 6.49 billion tons,

of which 2.48 billion tons are already being used; the excess

biomass, 4.01 billion tons, can be used for modern biofuel

production.4

Bioethanol produced from lignocellulosic biomass is an inter-

esting alternative to those produced from sucrose and starch,

since lignocellulosic materials are not used as foods and are also

less expensive than conventional agricultural feedstocks.5 How-

ever, lignocellulosic biomass is more recalcitrant to microbial and

Correspondence to: Regis Teixeira Mendonca, Biotechnology Center, Universi-

dad de Concepci on, Casilla 160-C, Concepci on, Chile. E-mail: [email protected]

a Biotechnology Center, Universidad de Concepci on, Casilla 160-C, Concepci on,

Chile

b Faculty of Forest Sciences, Universidad de Concepci on, Casilla 160-C,

Concepci on, Chile

c DepartamentodeBiotecnologa, Escolade Engenhariade Lorena,Universidade

de Sao Paulo, Estrada Municipaldo Campinho, s/n CP 116,12602-810,Lorena,SP, Brasil

d Faculty of Chemical Sciences, Universidad de Concepci on, Casilla 160-C,

Concepci on, Chile

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www.soci.org H Franco etal.

enzymatic conversions than non-woody biomass. This is partic-

ularly true for softwoods due to the structural and chemical

complexity of the wood cell walls.6

For conversion of wood to ethanol, a complex pretreatment

process to transform the biopolymers (specifically cellullose) into

fermentable sugars is required. The main goalof any pretreatment

is to alter or remove structural and compositional impediments to

hydrolysisandsubsequentbioconversionprocessesimprovingthe

rates and yields of enzymatic hydrolysisand fermentation.7 Chem-

ical pretreatments have the primary goal of enhancing enzyme

accessibility to the cellulose by solubilizing the hemicelluloses and

lignin, and to a lesser degree, decreasing thedegreeof depolymer-

ization andcrystallinityof thecellulosic component.8,9 All chemical

pulping processes in commercial use today involve the removal

of lignin to produce pulp forvarious paper products, altough such

processes could be considered as potentialpretreatment methods

for generating fermentable sugars.10

The use of cooking liquors with NaOH and Na2SO3 as in

chemithermomechanical pulping (CTMP), sometimes with the

addition of small amounts of anthraquinone as in alkaline sul-

fite/anthraquinone pulping (ASA), has proven to be effective in

solubilizing and increasing lignin hydrophilicity while promotingoxidative stabilization of polysaccharides, leading to high delig-

nification rates and pulp yields.11,12 The conventional method of

CTMP manufacture is based on pre-impregnation of softwood

chips with alkali sodium sulfite solution before mild cooking and

pressurized disc refining. The sulfite treatment of wood chips

results in the sulfonation of lignin, which causes swelling and

weakening of the lignin matrix and, consequently, affects the

defibration of the wood chips, leading to a pulp characterized

by large and flexible fibers.13 Alkaline pretreatment conditions

also increase the fiber hydrophilicity by generating new carboxyl

groups and by sulfonation of the lignin, particularly in the regions

between the fibers.14,15,16

Recently, sulfite pretreatment to overcome recalcitrance of

lignocellulose (SPORL process) was applied to pretreat spruce,producing glucan to glucose yields of 91% and overall monosac-

charides (hexoses and pentoses) recovery of 88%.17 Enzymatic

hydrolysis of red pine SPORL pulps with enzyme loadings of 14.6

FPU Celluclast and 22.5 CBU Novozyme 188 per gram of substrate

resultedalso in highglucoseyields.18 SPORL-pretreated lodgepole

pine also retained 88% of the glucans in the solid fraction, whose

enzymatic hydrolysis yielded about 80% of glucose at 10% sub-

strate loading.19 Mild acidic conditions used in the SPORL process

led to dissolution of most of the hemicelluloses in wood, as well

as, some glucans from cellulose.18,19The alkaline medium and the

use of anthraquinone in ASA cooking is more selective towards

lignin removal, preserving a higher amount of carbohydrates from

degradation and providing higher pulp yields.12

With less organicmatter in the black liquor, less BOD is expected to be present in

the effluents from ASA process.

The present study was conducted to evaluate the pretreatment

conditions of P. radiata wood chips with ASA followed by disk

refining on the enzymatic hydrolysis of the solids and on the

fermentation of the sugars for bioethanol production. In addition,

the same process was also evaluated in a tropical pine species,

P. caribaea. The use of both softwood species is justified because

the P. radiata is widely used in pulping processes and distributed

in temperate regions, while P. caribaea is a species that grows

only in tropical climates. In this way, it is important to perform

a comparison of the ethanol production for these species with

potential to be used as bioenergy plantations.

MATERIALS AND METHODSRaw material

P. radiata and P. caribaea samples were chipped and screened to

approximately 2.0 cm2.5 cm 0.5 cm.The wood chips wereair-

dried until10% (w/w) moisture, and stored in plastic bagsuntil use.

P.radiata wood chips (from trees approximately11 years old)were

provided by a Chilean pulp mill located in the Bo B o Province. P.

caribaea with an estimated age of 25 years was harvested from a

plantation located in Chiriqu, Panama.

Alkaline sulfite/anthraquinone (ASA) pretreatment

Several ASA pretreatments (P) of pine wood chips were carried

out in different cooking conditions as detailed in Table 1. P-1 was

carried out in 2 L Erlenmeyer flasks with 200 g of wood chips

each and wood/liquor ratio of 1 : 6 (w/v). Liquor impregnation

of wood chips was performed applying vacuum to the flasks for

30 min. The Erlenmeyers were introduced in an autoclave where

the reaction carried out at 120C for 120 min. Wood chips for ASA

pretreatments P-2, P-3 and P4 were impregnated in the same way

but, attheend ofthe30 min vacuum, thewood chipstogether with

the liquor were transferred to 800 mL stainless steel reactors. The

reactors were tightly closed and an additional 15 min of vacuumwas applied. The reactors were placed in a silicone oil bath

equipped with an electrical heating source and thermocouple.

Cooking was performed at 170 C for different times as described

in Table 1. For each condition, quadruplicates were carried out

in order to obtain enough pretreated material for the next steps

of the study. After each reaction, the liquor was drained and the

cooked biomass waswashed with tap water. Theresidual material

was disintegrated in a 10 L laboratory blender (Metvisa, BMG-

Brasil) for 1 h with 8 L of water. After disintegrating, the biomass

was washed inside an 1000 150 mm diameter PVC column

with a 200 mesh screen at the bottom, to avoid losses of fines

(particles smaller than 0.2 mm). Fines initially passing through the

screen were pumped back to the column top. Filtrate recirculation

permitted the formation of a fiber mat at the column base that

retained fines. Water recirculation was stopped when wash water

was free of turbidity. After this point, additional biomass was

applied to the column and fresh water was passed through the

biomass until the wash water reached a neutral pH. The washed

material was centrifuged to a consistency of approximately 30%

(w/w). Water releasedduring the centrifugation stepwas collected

andusedas dilutingagentin thesubsequent disk refining process.

Pretreated material was suspended in water to a final volume of

25 L (approximately 2.0% consistency) and refined in a Bauer MD-

3000 disk refiner (REGMED, Brazil) with a disc clearanceof 0.1 mm.

Table 1. Cooking conditions of alkaline sulfite/anthraquinone pre-treatment of pine wood chips

Sample ExperimentNaOH

(%, w.b.)Na2SO3(%, w.b.)

Temperature(C)

Cookingtime(min)

P. radiata P-1 8.5 16.5 120 120

P-2 8.5 16.5 170 30

P-3 7.5 17.5 170 30

P-4 7.5 17.5 170 45

P. caribaea P-5 7.5 17.5 170 45

w.b.=on oven-dry wood basis.All experimentswere performed withaddition of 0.1% anthraquinone (w/od wood).

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ASA and disk refining preatment of pine wood chips for bioethanol production www.soci.org

Refining was performed up to 250 Wh, 750 Wh and 1600 Wh of

energy consumption by the disk refiner. Refined samples were

assayed for fibrillation degree by Canadian Standard Freeness

(CSF) procedure20 and centrifuged to 30% consistency for further

use. The CSF, freeness or fibrillation degree determination is a

procedure frequently used by the pulp and paper industry to give

a measure of the rate at which a dilute suspension of pulp may be

drained. The refining energy means the electric energy consumed

by the disc refiner used to refine (fibrillate) the wood materialobtained from the ASA pretreatment.

Chemical characterization of wood chips and ASA samples

Approximately 3 g of milled sample (40/60 mesh) was extracted

with 95% ethanol for 6 h in a Soxhlet apparatus. Extractive-free

wood and ASA pulp samples were hydrolyzed with 72% (w/w)

sulfuric acid at 30 C for 1 h (300 mg of sampleand 3 mLof sulfuric

acid). The acid was diluted by addition of 79 mL water, and the

mixture was heated at 121 C and 1 atm for 1 h. The resulting

material was cooled and filtered through a porous glass filter

number 3. The solids were dried to a constant weight at 105 C,

and quantified as insoluble lignin. The soluble lignin in the filtrate

was read in a standard UV cuvette (1 cm path length) at 205 nm.An absorptivity (extinction coefficient) value of 105 L g1. cm was

used to calculate the amount of acid soluble lignin present in the

hydrolysate. Concentrations of monomeric sugars in the soluble

fraction were determined by HPLC using a BIO-RAD HPX-87H

column at 45 C eluted at a rate of 0.6 mL min1 with 5 mmol L1

sulfuric acid. Sugars were detected with a temperature controlled

RI detector.21 The factors used to convert sugar monomers to

anhydromonomers were 0.90 for hexoses and 0.88 for pentoses.

All samples were analyzed in triplicate.

Enzymatic hydrolysis

Enzymatic hydrolysis of ASA pulps was performed using a

mixture of commercial enzyme preparations, namely Celluclast

and Novozym 188 (Novozymes, Denmark), at dosages of 8.8

FPU g1 pulp (d.w.) plus 40 IU of-glucosidase g1 pulp. Each

hydrolysis experiment was carried out in 125 mL Erlenmeyer flasks

containing 2 g of pulp (d.w.) and 20 mL of 50 mmol L1 sodium-

acetate buffer at pH 4.8 plus theenzyme solution(final consistency

of 10%). The Erlenmeyer flasks were incubated at 45 C under

reciprocal agitationof 150 cycles min1. Thereaction wasstopped

at defined periods from 24 to 72 h by heating the reaction flask

at 100 C for 5 min in a water bath, followed by centrifugation of

the suspension at 10 000 g for 15 min. For each hydrolysis time,

three replicate experiments were run. Hydrolysates were assayed

for glucose, cellobiose and hemicelluloses content (mannose plus

xylose) usingthe previously described HPLC procedure. Glucans to

glucoseandhemicelluloses(mainlymannans)tomonosaccharidesconversions were calculated considering 0.9 as the hydrolysis

factorduetowaterincorporationtothecarbohydratepolymer.The

P-4 pulp was also enzymatically hydrolysed with 20 FPU Celluclast

plus 40 UI of-glucosidase g1 of pulp in a 1 L Erlenmeyer flask

at 10% substrate consistency (50 g pulp suspended in 500 mL

of 0.05 mol L1 citrate buffer solution at pH 4.8). The flask was

incubated at 45 C under reciprocal agitation of 150 cycles per

min. The reaction was stopped at defined periods from 24 to

72 h by heating the reaction flask to 100 C for 5 min, followed

by centrifugation at 10 000 g for 15 min. The hydrolysate resulting

from this experiment was assayed for sugar content by HPLC and

used for bioethanol production in the separated hydrolysis and

fermentation experiment.

Separate hydrolysis and fermentation (SHF) and semi-simultaneous saccharification and fermentation (SSSF)

Separate hydrolysis and fermentation (SHF) was carried out using

the hydrolysate of the P-4 pulp from the scale-up assay. 49 mL

of the hydrolysate were added to a 250 mL Erlenmeyer flask and

sterilized at 111 C for 15 min. Afterwards, the pH was adjusted to

4.8 with 1 mL of 50 mmol L1 citrate buffer and the fermentation

medium was supplemented with malt extract (3 g L1), peptone

(5 g L1) and yeast extract (3 g L1). An initial concentration of

5 g L1 of commercial S. cerevisiae was used in the fermentation.

The fermentation medium was incubated in a water bath without

agitation at 30 C. Samples werewithdrawn at 24, 48 and 72 h and

analyzed by HPLC for ethanol and residual sugars. Experiments

were performed in triplicate.

Semi-simultaneous saccharification and fermentation (SSF) of

P-4 pulp (from the refining at 750 Wh) was performed with 5 g

pulp (d.w.) at 10% substrate consistency in a 250 mL Erlenmeyer

flask. The pulp was suspended in 50 mL of 50 mmol L1 citrate

buffersolution (pH4.8) with 8.8 or 20 FPU g1 Celluclast, plus 40 IU

g1 -glucosidase in both cases. Flasks were incubated in a water

bathat 45C under reciprocal agitation of 150 cycles min1 for 24

and 72 h as a pre-hydrolysis step. Further, the same medium wassupplemented with malt extract (3 g L1), peptone (5 g L1) and

yeast extract (3 g L1), and inoculated with 5 g L1 commercial S.

cerevisiae. Fermentation was performed at 30 C without agitation

for 24 h. Samples were collected, filtered and analyzed for ethanol

and residual sugars by HPLC. An additional SSSF experiment in

the same conditions as described above was performed in the

presence of 20 IU Megazyme mannanase (endo-1,4-mannanase

from Megazyme Int. Ltd, Ireland) g1 pulp.

RESULTS AND DISCUSSIONASA pulping followed by disk refining

Alkaline sulfite/anthraquinone (ASA) pulping followed by disk

refining of P. radiata and P. caribaea wood chips was used asa pretreatment aiming to obtain homogeneous and fibrillated

materialusefulfor cellulose enzymatic hydrolysis andfermentation

to bioethanol. Varied pretreatment conditions were evaluated

to obtain partial lignin removal without significant loss of

carbohydrates (especially glucans). The chemical composition

of the wood chips and pulps obtained after ASA pulping/disk

refining areshown in Table 2. For P. radiata, pulp yield varied from

71% to 78%, which was in the range expected for semi-chemical

pulping.22 Glucan losses were low and varied between 0% (P-1)

and 13% (P-2). Delignification during ASA cooking ranged from

25% to 50% for P-1 to P-4, while hemicelluloses solubilization was

between 50% and 58%.

The wood chips partially delignified by the ASA process weredefibrated in a laboratory blender and further refined in a disk

refiner aiming to fibrillate the wood fibers and increase the

superficial areaof pulps forfurthersaccharification.Fibrillationwas

carried out at three different energy consumptions in the refiner

(250, 750 and 1600 Wh) as shown in Fig. 1. All pulps presented

poor fibrilation at low energy input, giving approximately 800 mL

of freeness at 250 Wh. P-1 showed low degree of refining also at

750 Wh (770 mL of freeness), probably due to insufficient lignin

removalfromthewoodchipsduringthecookingstage.13 PulpsP-2,

P-3and P-4fromP.radiata presentedsimilar refining performance,

reaching freeness values of 5090 mL at 1600 Wh of energy

consumption. After refining at 1600 Wh, the pulps usually present

high amounts of milled fibers and fine particulated materials.23

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Table 2. Chemical components (g per100 g of original wood) in pine wood chips and ASA pulps

Sample Glucans Hemicelluloses Lignin Extractives Pulp yield

P. radiata Wood 44.1 0.2 21.4 0.3 29.1 0.7 2.4 0.2

P-1 44.6 0.1 12.4 0.1 21.9 0.4 n.d. 78.1

P-2 38 2 10.8 0.6 19.0 0.9 n.d. 72.8

P-3 41 2 11.3 0.6 18.2 0.2 n.d. 74.3

P-4 42 2 11.5 0.2 14.7 0.4 n.d. 71.3P. caribaea Wood 33.9 0.4 12.9 0.3 25.8 0.8 21.7 0.4

P-5 27.7 0.7 7.7 0.6 22.3 0.1 n.d. 59.9

n.d.= not determined.

Figure 1. Fibrillation degree of ASA pulps of Pinus radiata at differentenergy input during disk refining.

In fact, a more fibrilated and preserved fiber appearance was

observedfor pulps P-2to P-4refined at 750 Wh,whereas thepulps

obtained at 1600 Wh presented a typical sludge appearance.

Enzymatic hydrolysis

P-1 to P-4 ASA pulps produced after disk-refining at different

energy inputs were evaluated in enzymatic hydrolysis assays

(Fig. 2). For pulp P-1, only refining at 1600 Wh was able to

produce a fibrillated material suitable for enzymatic hydrolysis,

but the fibrous material was hydrolyzed to only a limited extent

of 30% after 72 h. This material contained the highest residual

lignin and hemicellulose contents (Table 2), which may have

hindered enzyme infiltration.24,25 For the pulps P-2 and P-3, which

underwent similar lignin removals of 3537%, the increase in

refining levels also provided increases in the glucan conversion

efficiencies, which were 50 60% for samples refined at 750 and1600 Wh (Fig. 2). The highest glucan conversion efficiency was

observed with the P-4 pulp refined at 750 Wh (70% after 72 h

of hydrolysis). This hydrolysis efficiency was slightly increased

to 75.5% when the Celluclast load was increased from 8.8 FPU

g1 pulp to 20 FPU g1 pulp (data not shown). It is expected

that the P-4 pulp had a higher sulfonation degree, making

lignin more hydrophilic and decreasing non-specific hydrophobic

binding of the enzymes to the lignin. The high hemicelluloses

removal observed for the pulp P-4 (up to 50%) should also have

increased the accessibility of cellulases to celluloseallowinghigher

conversion during saccharification.18,26,27 In the P-4 pulp, lignin

removal reached 50%. Prolonging the refining stage to 1600 Wh

of energy consumption with this pulp gave a sludge material

that was less susceptible to enzymatic hydrolysis than the fibrous

material obtained at moderate refining levels (Figure 2).

There was a direct correlation between lignin removal during

pretreatment and the efficiency of enzymatic hydrolysis of the

pulps (Fig. 3). The level of lignin removal was clearly more

important for efficient enzymatic hydrolysis than the fibrillation

levels obtained during disk refining. At high refining levels, the

fiber dimensions are reduced, the cell wall partially or fullycollapses and thelignin of middle lamella becomes more exposed

than in other fiber fractions.13 These effects could increase the

unproductive binding of enzymes to lignin and decrease the

glucan conversion.14 This data is in agreement with previous

reports showing that lignin is a major hindrance to cellulose

hydrolysis by cellulases.27,28,29

SHF and SSSF

Based on the efficient enzymatic conversion of glucans to glucose

in the P-4 pulps, this material was used as substrate in sepa-

rate hydrolysis and fermentation (SHF) and semi-simultaneous

saccharification and fermentation (SSSF). For SHF, the pulp was

hydrolyzedunderoptimalconditionsfortheenzymeactivity(45Cand pH 4.8) for 24 h or 72 h. The resulting sugar broth was sep-

arated from the fiber residues and fermented by S. cerevisiae at

30 C. Initial concentration of glucose in the sugar broths was 40

and 44 g L1 for hydrolysis periods of 24 and 72 h, respectively.

The time-course of the fermentations showed that the ethanol

concentration reached values of 1820 g L1 after 24 h of fer-

mentation. Glucose was completely consumed during this period.

The conversion of glucose to ethanol in these experiments was

approximately 90%, whereas the low ethanol concentration in

the fermented broth is a result of the low pulp consistency used

(10%). The maximum ethanol amount that could be produced

from P. radiata is approximately 320 L ton1 wood, considering to-

tal conversion of the glucose present in the wood and 0.51 g g1

as the conversion factor of glucose into ethanol. The ethanol

yield obtained from the sugar broth prepared from P-4 pulp was

90%. Based on this data, the calculated ethanol production from

P. radiata would be 260 L ton1 wood, corresponding to a final

yield of 80% of the theoretical. The difference is accounted for

by incomplete conversion of glucans in the enzymatic hydrolysis

and a small amount of glucose used for yeast maintenance and

growth.30

In the SSSF process, the pulp was first pre-hydrolyzed with the

enzymes for different periods of time at 45 C followed by yeast

and nutrients addition (in the same flask) and the temperature for

fermentationwas thenlowered to 30 C. Different combinations of

enzyme loads and pre-hydrolysis times were evaluated, including



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Figure 2. Enzymatic hydrolysisof ASA pulps (P-1, P-2, P-3 andP-4) obtained after diskrefining at different energyinputs (250, 750 and 1600 Wh). Enzymeload per gram of pulp of 8.8 FPU Celluclast plus 40 IU beta-glucosidade; 10% pulp consistency. Error bars represent the variation of three hydrolysisreplicates. When not visible, the error bars were smaller than the symbol size.

Figure 3. Effect of lignin removal after ASA cooking in the enzymatichydrolysis ofPinusradiatapulps produced at differentenergy inputsin thedisk refining. Enzyme load per gram of pulp of 8.8 FPU Celluclast plus 40IU beta-glucosidade; 10% pulp consistency.

one assay with the addition of a Megazyme mannanase in the

medium to determine if some increase in ethanol yield could

be obtained by the release of some glucose from the residual

glucomannans in the pulp (Table 3). As a consequence of the

different conditions used, the ethanol production varied from

15 to 22 g L1 (for a range of enzymatic hydrolysis times 24

to 72 h and 24 h fermentation). The addition of mannanases

did not seem to have a significant impact on the increase of

ethanol production since the results obtained in the assay P-4E

were similar to the ones obtained in the assay P-4D. Longer pre-

hydrolysis times were more important and favored the further

fermentation process. Moreover, the decrease in temperature

during fermentation probably reduced enzymes activity and only

the sugars released during the pre-hydrolysis were fermented.

Results obtained with the processes SHF and SSF were similar,

with over 90% of the glucans present in the pulp converted to

ethanol. Ethanol yields obtained in this work were very similar to

those reported with the SPORL process, 270 L ton1 wood in SSF

with 10% solids. The results were also higher than those obtained

in separate hydrolysis and fermentation for corn stover pretreated

with ammonia fiber expansion (AFEX) using S. cerevisiae 424A

(LNH-ST) in which an ethanol yield of 242 L ton1 biomass was

obtained.31

Fermentation ofPinus caribaea pulps from ASA/disk refiningprocess

P. caribaea is a pine species that grows in tropical areas and

can tolerate drought periods of up to 6 months, temperatures of20 to 27 C and rainfalls ranging from 1000 to 1800 mm yr1.32

This softwood could be a raw material for biofuels production

in tropical places, such as Central America. In the present study,

the composition of the 25 years old P. caribaea wood sample

presented a relatively low amount of cellulose and hemicelluloses

(34% and 13%, respectively), 26% lignin and a very high amount

of ethanol-soluble extractives (22%) compared with the wood

from the 11 years old P. radiata, as shown in Table 2. Similar

values for lignin and extractives (26.3% and 23.6%, respectively)

in P. caribaea have been previously reported.33 ASA cooking and

disk refining were performed under the same conditions as P-4

pulp (Table 1), which was the one that showed the most suitable

characteristics for fermentation. A delignification of only 14% was



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Table 3. Ethanol produced by SSF ofPinusradiata ASA pulps (P-4 refined at 750 Wh) after pre-hydrolysis for different times and enzyme loads

SampleCelluclast

(FPU g1) pulp)-glucosidase(IU g1 pulp)

Mannanase(IU g1 pulp)

Pre-hydrolysistime (h)

Ethanol after24 h fermentation

(g L1)

P-4A 8.8 40 72 21.5 0.6

P-4B 20 40 24 15.5 0.9

P-4C 20 40 48 18 1

P-4D 20 40 72 20.7 0.4

P-4E 20 40 20 72 22.0 0.1

Pulp consistency was 10% in all cases.

achieved after cooking, and 730 mL of freeness was obtained for

the pulp refined at 750 Wh. The pulp (P-5) also presented a low

amount of glucans (28%, on wood basis). After 72 h of enzymatic

hydrolysis (20 FPU Celluclast g1 pulp plus 40 IU -glucosidase/g

pulp) at 10% consistency the glucan-to-glucose conversion was

72%. SSF of the P-5 pulp performed after 72 h of pre-hydrolysis

resulted in an ethanol yield of 16 g L1 (approximately 140 L ton1

wood). Results of ethanol production were directly related withthelow amount of cellulose present in theP.caribaea wood, which

make this species less suitable as a raw material for bioethanol

production when compared with P. radiata.

CONCLUSIONSThe alkalinesulfite/anthraquinonepretreatmentofP.radiata wood

chips followed by disk refining was shown to be an effective

pretreatment, able to reduce the lignin content in the wood by

up to 50% with low loss of glucans and high pulp yield (over

70%). Depending of the fibrillation degree, the ASA pulps were

saccharified by cellulases withconversion yields up to 70%. During

the SHF or SSSF processes, the conversion of glucans to ethanol

was over 90%, indicating that the substrate was easily convertedto biofuel when a consistency of 10% was used. P. caribaea

pretreated at similar conditions was not as good as P. radiata due

to thehighamount of extractives andthe lowamount of cellulose,

which generated a lower ethanol yield when compared with thst

obtained from P. radiata pulps.

ACKNOWLEDGEMENTSH. Franco thanks the National Research Program 2005-2010 of

SENACYT-IFARHU, Panama for a PhD grant and a fellowship

from MECESUP, Chile (grant UCO-0702). Financial support from

FONDECYT in Chile (grant 1070492) and from FAPESP in Brazil

(grant 08/56256-5) are also acknowledged.

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