bioethanol from scbh - status & pespective

Bioresource Technology 101 (2010) 4754–4766

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Bioresource Technology

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Production of bioethanol from sugarcane bagasse: Status and perspectives

C.A. Cardona *, J.A. Quintero, I.C. PazDepartamento de Ingeniería Química, Universidad Nacional de Colombia Sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia

a r t i c l e i n f o

Article history:Received 15 August 2009Received in revised form 22 October 2009Accepted 23 October 2009Available online 28 November 2009

Keywords:Sugarcane bagasseEthanolPretreatmentLignocellulosicStability

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.10.097

* Corresponding author. Tel.: +57 6 8879300x50417E-mail address: [email protected] (C.A. Card

a b s t r a c t

Lignocellulosic biomass is considered as the future feedstock for ethanol production because of its lowcost and its huge availability. One of the major lignocellulosic materials found in great quantities to beconsidered, especially in tropical countries, is sugarcane bagasse (SCB). This work deals with its currentand potential transformation to sugars and ethanol, considering pretreatment technologies, detoxifica-tion methods and biological transformation. Some modeling aspects are exposed briefly. Finally stabilityis discussed for considering the high nonlinear phenomena such as multiplicity and oscillations, whichmake more complex the control as a result of the inhibition problems during fermentation when furfuraland formic acid from SCB hydrolysis are not absent.

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1. Introduction

For large-scale biological production of fuel ethanol, it is desir-able to use cheaper and more abundant substrates. When produc-ing ethanol from maize (made up from starch chains) or sugarcane(in the form of either cane juice or molasses) the raw material con-stitutes about 40–70% of the production cost (Sendelius, 2005;Quintero et al., 2008). By using waste products from forestry, agri-culture and industry, the costs of the feedstocks may be reduced.Lignocellulose (complex polymer made up from three carbohy-drates: cellulose hemicelluloses and lignin) is considered as anattractive feedstock for the production of fuel ethanol, because ofits availability in large quantities at low cost (Cardona and Sánchez,2007; Cheng et al., 2008) and for reducing competition with foodbut not necessarily with feed. Today the production cost of ethanolfrom lignocellulose is still too high, which is the major reason whyethanol from this feedstock has not made its breakthrough yet.

Many lignocellulosic materials have been tested for bioethanolproduction as was reviewed by Sánchez and Cardona (2008). Ingeneral, prospective lignocellulosic materials for fuel ethanol pro-duction can be divided into six main groups: crop residues (canebagasse, corn stover, wheat straw, rice straw, rice hulls, barleystraw, sweet sorghum bagasse, olive stones and pulp), hardwood(aspen and poplar), softwood (pine and spruce), cellulose wastes(newsprint, waste office paper and recycled paper sludge), herba-ceous biomass (alfalfa hay, switchgrass, reed canary grass, coastalBermudagrass and thimothy grass), and municipal solid wastes

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(MSW). Numerous studies for developing large-scale productionof ethanol from lignocellulosics have been carried out in the world.However, the main limiting factor is the higher degree of complex-ity inherent to the processing of this feedstock. This is related tothe nature and composition of lignocellulosic biomass (which con-tain up to 75% of cellulose and hemicelluloses). Cellulose andhemicelluloses should be broken down into fermentable sugarsin order to be converted into ethanol or other valuable products(xylans, xylitol, hydrogen and enzymes). But this degradation pro-cess is complicated, energy-consuming and non-completely devel-oped (Sánchez and Cardona, 2008). With the advent of moderngenetics and other tools the cost of producing sugars from these re-calcitrant fractions and converting them into products like ethanolcan be significantly reduced in the future.

Several reviews have been published on the theme of fuel eth-anol production especially from lignocellulosic biomass (Lin andTanaka, 2006; Cardona and Sánchez, 2007; Sánchez and Cardona,2008). Lignocellulosic materials from different crop residues havebeen used for conversion to ethanol. One of the major lignocellu-losic materials found in great quantities to be considered, espe-cially in tropical countries, is sugarcane bagasse (SCB), thefibrous residue obtained after extracting the juice from sugar cane(Saccharum officinarum) in the sugar production process (Martínet al., 2007a).

SCB is produced in large quantities by the sugar and alcoholindustries in Brazil (Martínez et al., 2003; Hernández-Salas et al.,2009), India (Martínez et al., 2003; Chandel et al., 2007), Cuba(Martínez et al., 2003), China (Martínez et al., 2003; Cheng et al.,2008), México (Hernández-Salas et al., 2009), Indonesia (Restutiand Michaelowa, 2007) and Colombia (Quintero et al., 2008). In

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general, 1 ton of sugarcane generates 280 kg of bagasse, and5.4 � 108 dry tons of sugarcane are processed annually throughoutthe world (Cerqueira et al., 2007). About 50% of this residue is usedin distillery plants as a source of energy (Pandey et al., 2000); theremainder is stockpiled. Therefore, because of the importance ofSCB as an industrial waste, there is great interest in developingmethods for the biological production of fuel and chemicals thatoffer economic, environmental, and strategic advantages (Adsulet al., 2004).

In the approximately 80 sugarcane producing countries there isa potential to make better use of the SCB. Subjected to improvedenergy efficiency, sugar producers could supply energy either asco-generated electricity, or as fuel ethanol through cellulosehydrolysis followed by fermentation (Botha and Blottnitz, 2006).The most common use for SCB is the energy production by com-bustion (Ramjeawon, 2008). In addition, SCB can be used also toproduce chemical compounds such as furfural or hydroxymethyl-furfural (Almazán et al., 2001), paper paste (Pattra et al., 2008) orethanol (Laser et al., 2002). The use of SCB in chemistry and bio-technology has been reviewed elsewhere (e.g. Pandey et al., 2000).

As raw material, SCB should be analyzed from composition,structure and surface properties. SCB is primarily composed of lig-nin (20–30%), cellulose (40–45%) and hemicelluloses (30–35%)(Peng et al., 2009). Because of its lower ash content, 1.9% (Li etal., 2002), bagasse offers numerous advantages compared withother agro-based residues such as paddy straw, 16% (Goh et al.,2009), rice straw, 14.5% (Guo et al., 2009) and wheat straw, 9.2%(Zhao and Bai, 2009). Work on structure and surface characteriza-tion of SCB has not been done extensively, but some works can befound (Zhao et al., 2007; Quintero and Cardona, 2009). In a previ-ous work (Quintero and Cardona, 2009) SCB was obtained from asmall sugarcane juice factory and milled for its structural analysis.Obtained fibers had smooth surface layers and characteristic elon-gations with lengths over 200 lm (this was obtained from SEMmicrographs with in a JEOL JSM-5910LV microscope). XRD analysis(Rigaku MiniFlex II unit with CuKa used at 30 kV and 15 mA, dif-fraction angle ranged from 35� to 2� with a scan speed of 5�/min)showed that crust and marrow bagasse exhibit different structuresand crystallinity. Crust bagasse presents two diffraction peaks at 2hvalues of 18.04� and 21.9�, while marrow bagasse presents only apeak at 21.86�, characteristic of the cellulose structures. It isimportant to note, that most of the developments in SCB transfor-mation to sugars and ethanol have the common scientific basiswith other lignocellulosic materials, due to the fact that there arenot considerable qualitative differences in composition andstructure.

Overall fuel ethanol production from SCB includes five mainsteps: biomass pretreatment, cellulose hydrolysis, fermentationof hexoses, separation and effluent treatment (see Fig. 1). Further-more, detoxification and fermentation of pentoses released duringthe pretreatment step can be carried out. Solid fraction from pre-treatment contains the cellulose which is later hydrolisated, andliquid fraction contains the hemicellulose hydrolysate. Once cellu-lose hydrolysis is completed, the resulting hydrolysate is fer-mented and converted into ethanol. This process is calledseparate hydrolysis and fermentation (SHF). SHF is one of theconfigurations that have been tested more extensively. Pentosefermentation, when it is carried out, is accomplished in an inde-pendent unit. The need of separate fermentations is due to thefact that pentose utilizing microorganisms ferment pentoses andhexoses slower than microorganisms that only assimilate hex-oses. Moreover, these microorganisms are more sensitive to theinhibitors and to the produced ethanol. For this reason, the hemi-cellulose hydrolysate resulting from pretreatment should bedetoxified. If the fermentation of the hemicelluloses and cellulosehydrolysates is carried out in a separate way, less liquid volumes

of hydrolysate have to be detoxified. The ideal organism for theproduction of ethanol would be the one which can utilize pentoseand hexose sugars generated by lignocellulose hydrolysis (Chan-del et al., 2007).

Present paper deals with the uses, pretreatment and biologi-cal transformation of SCB into added value products, emphasiz-ing on fuel ethanol production. Potential uses of lignocellulosicbiomass depend on its composition and in some extend of itsavailability. Moreover, the required pretreatment is a functionof the structure complexity. Main pretreatment methods forSCB are presented. Potential applications of bagasse hydrolysateand the detoxification methods are discussed. Finally, somemodeling and stability aspects are considered. Separation andpurification, and effluent treatment technologies are not dis-cussed in this paper, because, these technologies are well estab-lished for other types of raw material. Additionally effluentproduct and wastes are similar, despite the highly variations inraw material composition.

2. Pretreatment methods

Lignocellulosic materials do not contain monosaccharidesreadily available for bioconversion. Instead they contain polysac-charides, such as cellulose and hemicelluloses, which have to behydrolyzed, by means of acids or enzymes, to fermentable sug-ars. Enzymatic hydrolysis is a promising way for obtaining sug-ars from lignocellulosic materials, but the low enzymaticaccessibility of the native cellulose is a key problem for bio-mass-to-ethanol processes. Cellulose in plants is closely associ-ated with hemicelluloses and lignin. The lignin is partlycovalently associated with hemicelluloses, thus preventing theaccess of hydrolytic agents to cellulose. In addition, the crystal-line structure of cellulose itself represents an extra obstacle tohydrolysis. A pretreatment is required for removing lignin andhemicelluloses, reducing cellulose crystallinity and increasingthe porosity of the material (Keller et al., 2003). This enhancesthe enzymatic susceptibility of cellulose. An effective pretreat-ment must preserve the utility of the hemicelluloses and avoidthe formation of inhibitors (Laser et al., 2002). An economicalpretreatment should use inexpensive chemicals and require sim-ple equipment and procedures (Martín et al., 2007a).

Several pretreatment methods have been investigated for dif-ferent lignocellulosic materials (reviewed by Sun and Cheng(2002); Cardona and Sánchez (2007); Sánchez and Cardona(2008)), steam explosion, solvent extraction, and thermal pretreat-ment using acids or bases (Mosier et al., 2005); along with biolog-ical pretreatments with white rot fungi (Itoh et al., 2003). Amongall these methods, acid pretreatment is still the method of choicein several model processes. Pretreatment methods already investi-gated for bagasse include acid pretreatment with different acids(Gámez et al., 2004, 2006; Rodriguez-Chong et al., 2004; Chandelet al., 2007; Cheng et al., 2008; Pattra et al., 2008; Hernández-Salaset al., 2009), steam explosion (Martín et al., 2002a; Sendelius,2005; Hernández-Salas et al., 2009), alkaline treatment (Hernán-dez-Salas et al., 2009, alkaline dewaxed (Peng et al., 2009), biolog-ical treatment (Li et al., 2002; Camassola and Dillon, 2009), wetoxidation (Martín et al., 2007a), organosolv pretreatment (Pasquiniet al., 2005a, b; Pereira et al., 2007; Tu et al., 2008), liquid hot waterpretreatment (Laser et al., 2002) and pretreatments with peraceticacid (Teixeira et al., 1999) or with ammonia water (Kurakake et al.,2001). Table 1 shows some of the most pretreatment methods usedfor bagasse exploitation with their respective operation conditionsand in some cases sugar yields. Higher yields are presented withacid hydrolysis. Little information is presented for alkaline pre-treatment because delignification is its main objective.

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Fig. 1. Processes scheme of fuel ethanol production from sugarcane bagasse. Possibilities for reaction–reaction integration are shown inside the shaded boxes: CF, co-fermentation; SSF, simultaneous saccharification and fermentation; SSCF, simultaneous saccharification and co-fermentation.

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2.1. Acid pretreatment

The hydrolysis with dilute acids (sulphuric, hydrochloric or ace-tic acid are habitual, typically 1–10% weight) is usually called acidhydrolysis or prehydrolysis and consists in the hydrolysis of thehemicellulosic fraction at moderate temperature (in the range100–150 �C). The hemicellulose fraction of SCB represents up to35% of the total carbohydrates that can be readily hydrolyzed tomonomeric sugars by dilute acid. However, the concentration ofreducing sugar in the hydrolysate is relatively low due to high li-quid/solid ratio during the acid hydrolysis. So the hydrolysateshould be concentrated before fermentation (Cheng et al., 2008).

The acid medium attacks the polysaccharides, especially hemi-celluloses that are easier to be hydrolyzed than cellulose. There-fore, the cellulose and lignin fractions remain almost unaltered inthe solid phase and can be further processed, being consideredsuitable for SCB pretreatment as shown by Gámez et al. (2004,2006). Depending on the operational conditions, the liquid phaseof the hydrolysates will be constituted by sugar (xylose, glucoseand arabinose), products of decomposition of the hemicelluloses(such as oligomers from the polymers and acetic acid generatedfrom the hydrolysis of acetyl groups linked to sugars) and/or thedecomposition products from monosaccharides (such as furfural,product of dehydration of pentoses, and 5-hydroxymethylfurfural(HMF), product of dehydration of hexoses) (Gámez et al., 2006).These products are growth inhibitors of microorganisms. There-fore, the hydrolysates can be used as fermentation media if theconcentration of inhibitors remains low (Gámez et al., 2004). Themost used acid is H2SO4, among other acids that can be used suchas HCl or HNO3.

2.1.1. Acid pretreatment using sulphuric acid (H2SO4)Pattra et al. (2008) has evaluated the hydrolysis of SCB using

H2SO4 at various concentrations (0.25–7.0% volume) and reactiontimes (15–240 min) at 121 �C, 1.5 kg/cm2 in autoclave. Optimalconditions obtained were 0.5% H2SO4 and 60 min, which yielded24.5 g/L of total sugar. At these conditions the highest glucose con-centration was obtained: 11 g glucose/L; 11.29 g xylose/L; 2.22 garabinose/L; 2.48 g acetic acid/L and 0.12 g furfural/L were ob-

tained. An increase from 0.5% to 1.0% H2SO4 did not affect the glu-cose concentration in SCB hemicellulose hydrolysate, but whenH2SO4 concentration was between 1.0 and 5% H2SO4 glucose con-centration decreased. Xylose was found as the main sugar in SCBhemicellulose hydrolysate. In order to increase the reducing sugarproduction from in the SCB and acid recovering, Cheng et al. (2008)has proposed an acid recycle process and detoxification of hydroly-sate performed by electrodialysis. The main problem encounteredwhen treating the lignocellulose with acids is the formation of fur-an derivatives and other non identified toxic products. This is par-ticularly true in the case of xylans, very easily leading to furfuralproduction.

2.1.2. Acid pretreatment using hydrochloric acid (HCl)Hydrochloric acid has been used for pretreatment of different

lignocellulosics (e.g. sorghum straw, SCB, ryegrass and palm oilwastes), however, environmental impact and corrosive propertiesstrongly limits its application. SCB hydrolysis with HCl shows high-er yields (see Table 1) compared to other lignocellulosics (Hernán-dez-Salas et al., 2009) and converting more than 30% by weight toreducing sugars.

2.1.3. Acid pretreatment using phosphoric acid (H3PO4)The interest in the use of H3PO4 is that after neutralization of

hydrolysates with NaOH, the salt formed is sodium phosphate(Gámez et al., 2006). This salt can remain in the hydrolysates be-cause it is used as nutrient by microorganisms. Therefore, a filtra-tion operation of is not needed with the consequent advantages:improvement of process profitability (avoiding salts removal anddecreasing the amount of nutrients needed for fermentation) andpositive impact to the environment (the salt formed is not awaste). Gámez et al. (2006) have evaluated the hydrolysis of SCBwith phosphoric acid under mild conditions (see Table 1). Usingthese conditions, 17.6 g of xylose/L; 2.6 g of arabinose/L; 3.0 g ofglucose/L, 1.2 g furfural/L and 4.0 g acetic acid/L were obtained.The efficiency in these conditions was 4.46 g sugars/g inhibitorsand the mass fraction of sugars for dissolved solids in liquid phasewas up to 55%. The rate of xylose release increased with the phos-phoric acid concentration. Xylose concentrations in hydrolysates at

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Table 1Implemented pretreatments for sugarcane bagasse exploitation.

Pretreatment Agent Conditions Yield Remarks References

%w/wof SCBa

g/L

Dilute acid HCl Acid concentration (1.2% v/v) mL of acid solution/g ofbagasse by weight: 15:1. Operation at 121 �C and 1.1 kg/cm2 for 4 h

37.21 ND For depithed bagasse morethan 30% by weight wasconverted to reducing sugars

Hernández-Salas et al.(2009)

Acid concentration (1.2% v/v) mL of acid solution/g ofbagasse by weight: 15:1. Operation at 121 �C and 1.1 kg/cm2 for 4 h

35.37 ND For pith bagasse Hernández-Salas et al.(2009)

Acid concentration (2.5% v/v) fibers size between 2.2and 10 mm. Operation at 140 �C for 30 min. Solid to liquid ratio of 1:10

ND 30.29 Chandel et al. (2007)

H2SO4 Acid concentration (1.25%, w/w). Operation at 121 �C during 2 h.The biomass at a solid loading of 10% (w/w)

ND 59.1 Cheng et al. (2008)

Acid concentration (0.5%). Operation at 121 �C, 1.5 kg/cm2

during 60 minND 24.5 Pattra et al. (2008)

H3PO4 Acid concentration (4%). Operation at 122 �C during 300 min.Water/solid ratio of 8 (g water/g sugarcane bagasse on dry basis)

ND 23.2 Gámez et al. (2006)

HNO3 Acid concentration (6%). Operation at 122 �C for 9.3 min ND 23.51 Rodriguez-Chong et al.(2004)

Alkaline–enzymepretreatment

NaOH Base concentration (2% w/v) mL of solution/g of bagasse: 5:1NaOH: 50 mg/g of bagasse. Operation at 121 �C, 1.1 kg/cm2

during 4 h. 0.19 mL of enzyme per gram of bagasse

13–18 ND Hernández-Salas et al.(2009)

Alkaline pretreatment NaOH Base concentration 3%, solid to liquid ratio of 1:25 (g/mL) Operation at50 �C for 3 h

27.65 ND For dewaxed sugarcane bagasse.74.9% of the original hemicelluloses werehydrolyzed. Xylose was the predominantsugar (79.2–96.7% of total sugars)

Peng et al. (2009)

Steam explosion Water Operation at 121 �C and 1.1 kg/cm2 for 4 h ND ND Hernández-Salas et al.(2009)

Water, SO2 and H2SO4 SO2 concentration 2% by weight of water in the bagasse. Acidconcentration 0.25 g H2SO4 per 100 g dry matter. 180 �C during 5 min

ND ND Glucose and xylose yields in average86.3% and 72.0%, respectively

Sendelius (2005)

Wet oxidation Water and oxygen Operation at 195 �C during 15 min, alkaline pH. Oxygen pressure: 12 bar 11.6 ND Yielding a solid material with nearly 70%cellulose content, hemicellulosessolubilization: 93%of and 50% of lignin. Enzymaticconvertibility ofcellulose of around 75%

Martín et al., 2007a

Water and oxygen Operation at 185 �C, 5 min, acidic pH. Oxygen pressure: 12 bar 16.1 ND Xylose was the main sugar obtained Martín et al. (2007a)Organosolv pretreatment Supercritical CO2 and 1-

butanol-water mixtureOperation at 7 MPa and 190 �C. 60% of butanol in the solvent mixture.Reaction time 105 min

ND ND Delignification extent: 94.5% Pasquini et al. (2005a)

Supercritical CO2 andethanol–water mixture

Operation at 16.0 MPa and 190 �C. Ethanol–water (1:1/v:v). Reactiontimes in the range: 30–150 min

ND ND Pulping yield: 32.7%; Residual Klasonlignin: 8.7%. Delignification extent: 88.4%

Pasquini et al. (2005b)

Dimethyl formamide(DMF)

Operation at 200–210 �C for 150 min and 40–60% DMF ND ND Delignification extent: 82.7%. Solidmaterial with nearly 83.53% of a-cellulose

Rezayati-Charani et al.(2006)

a SCB: Sugarcane bagasse; ND: non-data available.

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60 min of reaction were 6.1, 7.3 and 8.6 g/L using H3PO4 concentra-tions of 2%, 4% and 6%, respectively.

2.1.4. Acid pretreatment using nitric acid (HNO3)Comparison of results obtained using sulphuric and hydrochlo-

ric acids (see Table 1) demonstrated that the nitric acid presentssimilar results for hydrolysis under the evaluated conditions byRodriguez-Chong et al. (2004) (acid concentration, 2–6%; reactiontime, 0–300 min; and temperature, 100–128 �C). Optimal condi-tions obtained from kinetic models by Rodriguez-Chong et al.(2004) were: 122 �C, 6% HNO3 and 9.3 min. Using these conditions,18.6 g xylose/L; 2.04 g arabinose/L; 2.87 g glucose/L; 0.9 g aceticacid/L and 1.32 g furfural/L were obtained. The highest xylose con-centration (21.0 g/l) was reached after 180 min.

2.2. Alkaline pretreatment

Alkaline pretreatment of SCB digests the lignin matrix andmakes cellulose and hemicellulose available to enzyme degrada-tion (Pandey et al., 2000). Alkali treatment of lignocellulosic sub-stances such as cereal straw and bagasse disrupts the cell wall bydissolving hemicelluloses, lignin, and silica, by hydrolyzing uronicand acetic esters, and by swelling cellulose. Last decreases the crys-tallinity of cellulose. By this process, straw and bagasse can be sim-ply fractionated into alkali-soluble lignin, hemicelluloses andresidue, which makes easy to utilize them for more valuable prod-ucts. The end residue (mainly cellulose) can be used to produceeither paper or cellulose derivatives. Recently, some importantapplications for hemicelluloses, such as the production of xylans,have been proposed (Peng et al., 2009). They have evaluated thesequential treatments of dewaxed bagasse with water and 1%and 3% NaOH aqueous solutions yielded 25.1% hemicelluloses frombagasse and accounted for 74.9% of the original hemicelluloses.These results indicated that 1% and 3% NaOH aqueous solutions un-der these conditions promoted a substantial dissolution of thehemicellulosic polysaccharides and lignin macromolecules.

2.3. Thermal pretreatment

Fractionation and solubilization studies of lignocellulosic mate-rials by thermal treatments have shown the efficiency of this tech-nology to improve the yields of extraction of hemicelluloses.Boussarsar et al. (2009) have evaluated the SCB conversion byhydrothermal treatment. Optimal conditions were 170 �C for 2 h,reaching higher solubilization of hemicellulose than that at150 �C and lower degradation of sugar monomers than 190 �C.However, analysis of thermal hydrolysates shows the presence ofxylan oligomers and polymers with large chains. On the otherhand, Sendelius (2005) has evaluated the steam pretreatment con-ditions with respect to final ethanol yield, using SCB as feedstock.The variables considered were temperature (180, 190 and205 �C), time (5 and 10 min) and impregnating agents (water, 2%SO2 by weight of water in the bagasse and 0.25 g H2SO4 per 100 gdry matter). The most prominent tested pretreatment conditionwas: SO2-impregnation with a temperature of 180 �C during5 min, which gave a glucose yields in average 86.3% and xyloseyields in average 72.0%. The fermentation of these hydrolyzedmaterials gave an overall ethanol yield of 80%, based on theoreticalvalue.

2.4. Biological pretreatment

It is generally known that microorganisms degrade untreatedbagasse slowly; therefore, isolation of efficient strains is regardedas an important research area for lignin degradation in SCB. Themost promising microorganisms for biological pretreatment are

the white rot fungi, microorganism that belong to the class Basid-iomycetes and that are capable of degrading a lignocellulose sub-strate (Pan et al., 2005). Camassola and Dillon (2009) pretreatedSCB with the white rot fungus Pleurotus sajor-caju PS2001. Subse-quently, they evaluated the use of this biologically pretreated ba-gasse for the production of cellulases and xylanases by thefungus Penicillium echinulatum. Despite the environmental advan-tages offered by this type of pretreatment, biological pretreatmentusing the fungus P. sajor-caju PS2001 was not effective since theenzymatic activities with biologically pretreated SCB were lowerthan the control treatments carried out with untreated SCB andcellulose. Also, although the enzymatic activities of the culturewith biologically pretreated bagasse were lower than the culturescarried out with untreated SCB, it should be noted that the produc-tion of enzymes of the cellulose and hemicellulase complex afterthe production of the mushrooms is another way to add value tothis agricultural residue.

A marine fungus, Phlebia sp. MG-60, which has been screenedfrom mangrove stands, proved to have excellent lignin degradationability and selectivity. Li et al. (2002) have incubated this marinefungus, with SCB. With this pretreatment more than 50% of ligninin the SCB was degraded by Phlebia sp. MG-60, and less than 10% ofthe holocellulose was lost. Without Kirk medium addition, Phlebiasp. MG-60 did not show higher delignification ability or better del-ignification selectivity than the other white rot fungi. However,when Kirk medium was added to the culture instead of sterilizedwater, outstanding delignification capability and excellent selec-tive property to delignify SCB were observed. Thus, with properaddition of a nutrient such as Kirk medium, Phlebia sp. MG-60could efficiently degrade lignin in SCB while holocellulose wasscarcely damaged. Kirk medium composition was: 1% (w/v) glu-cose, 1 g/L KH2PO4, 1 g/L Ca(H2PO4), 221 mg/L ammonium tartrate,500 mg/L MgSO4�7H2O, 1 mg/L thiamine–HCl and 10 ml Kirk min-eral solution (Kirk et al., 1978). Other microorganisms evaluatedin the degradation of bagasse are several white rot fungi: Phanero-chaete chrysosporium ME-466, Phanerochaete sordida YK-624, andCeriporia sp. MZ-340.

2.5. Wet oxidation

Wet oxidation (WO) is the process of treating material withwater and either air or oxygen at temperatures above 120 �C.Two types of reactions occur during WO: a low-temperaturehydrolytic reaction and a high-temperature oxidative reaction. Ithas been demonstrated that combination of alkali and WO reducesthe formation of toxic furaldehydes and phenol aldehydes (Klinkeet al., 2002). In a recent work, the enzymatic convertibility andthe fermentability of bagasse pretreated by WO at different pH val-ues were investigated (Martín et al., 2006). Martín et al. (2007a)have investigated different conditions of wet oxidation (WO) pre-treatment on fractionation and enzymatic convertibility of SCB.Variable factors studied were pH, temperature and reaction time,while pressure (12 bar) was kept constant. The pH was adjustedby adding Na2CO3 or H2SO4. The highest cellulose content, nearly70%, was obtained in the pretreatment at 195 �C, 15 min and alka-line pH. The highest sugar yield in the liquid fraction, 16.1 g/100 g,was obtained at 185 �C; 5 min and acidic pH (see Table 1). Cellu-lose enrichment was reached due to removal of hemicellulosesand lignin, as can be deduced from the high degrees of solubiliza-tion of hemicelluloses and lignin achieved in the pretreatmentsleading to fibers with higher cellulose content. Although the anal-ysis of the solid fraction in most of the pretreatments showed highdegrees of hemicelluloses solubilization, the content of free sugarsin the liquid fraction was very low. It is known that wet oxidationmainly catalyses the transfer of hemicelluloses from the solidphase to the liquid phase, but it does not catalyse the hydrolysis


of the liberated hemicelluloses molecules. The products ofhemicelluloses hydrolysis during WO are not monosaccharides,but sugar oligomers. Reactive oxygen species such as N-methyl-morpholine-N-oxide (Kuo and Lee, 2009), sodium hypochloriteand hydrogen peroxide (Lee et al., 2009) in solution have beeninvestigated for its ability to oxidize sugarcane bagasse.

2.6. Organosolv pretreatment

Organic solvent or organosolv pulping processes are alterna-tives to soda or kraft pulping to delignify lignocellulosic materialsfor the production of paper pulp. For the industrial processes (Kraftand Soda), the burning step is of fundamental importance to re-cover the inorganic chemicals employed in the pulping. In theorganosolv process, the exclusive utilization of organic solvent/water mixtures eliminates the need to burn the liquor and allowsthe isolation of the lignins (by distillation of the organic solvent)(Pereira et al., 2007). Formic acid, a typical organosolv system,has been examined under atmospheric pressure to pulp bagasse fi-bers. Tu et al. (2008) showed that efficient bagasse pulping wasachieved when the formic acid concentration was limited to 90%(v/v). The delignification of bagasse by 90% formic acid was almostcompleted after approximately 80 min. Dimethyl formamide hasbeen also used for organosolv pulping of bagasse (Rezayati-charaniand Mohammadi-Rovshandeh, 2005; Rezayati-Charani et al.,2006). Other organosolv alternative is its combination with super-critical carbon dioxide. Organosolv-CO2 pulping consists in the uti-lization of pressurized carbon dioxide as an important part of thepulping liquor (50% alcohol/water mixture and 50% carbon diox-ide). This process combines the utilization of a lower amount of or-ganic solvent and facilitates the lignin recovery, by the release ofpressure after pulping. This process produces pulp with lowerstrength properties but in similar yields and in shorter times whencompared with the industrial processes (Pereira et al., 2007).

SCB delignification was studied combining the utilization of car-bon dioxide at high pressures and solvent mixtures, methanol/water, ethanol/water and n-propanol/water (Pasquini et al.,2005b). The utilization of these different alcohols produced pulpswith similar delignification extent but with a continuous decreasein pulp yield with the increase of the alcohol’s chain length (Pasqu-ini et al., 2005a). To extent the study of the effect of the co-solvent(alcohol/water) in the delignification process Pasquini et al.(2005a, b) have described the utilization of CO2 at sub- and super-critical conditions with 1-butanol/water and ethanol/water as co-solvents in the delignification of SCB. For 1-butanol/water casethe higher delignification extent (94.5%) was obtained at 7 MPa,190 �C, 105 min and 60% 1-butanol in the co-solvent mixture.The results also indicate a low selectivity of the process once thelignin removal was accomplished by an extensive hydrolysis ofthe polysaccharide fraction. The best compromise between ligninremoval and polysaccharide preservation was obtained at highpressures and low content of 1-butanol in the co-solvent mixture.For ethanol/water mixture the best results were obtained at16.0 MPa and 190 �C. Under these conditions the delignificationextent was in the order 88.4% for SCB.

2.7. Final remarks

Dilute sulphuric acid pretreatment has been successfully devel-oped given that high reaction rates can be achieved improving sig-nificantly the subsequent process of cellulose hydrolysis. However,the costs of this type of pretreatment are still higher. The mainadvantage of dilute acid pretreatment is the higher recovery ofsugars derived from hemicelluloses, but concentration of reducingsugars is relatively low due to high liquid to solid ratio. Other

drawbacks include the formation of furan derivatives and othertoxic products and the need of an additional concentration step.

Alkaline pretreatment decreases the polymerization degree andcrystallinity of cellulose by the destruction of links between ligninand other polymers, and breakdown of lignin. Its costs are so highthat these methods are not competitive for large-scale plants. Bio-logical pretreatment has low energy requirements and mild envi-ronmental conditions. However, these processes are too slowlimiting its application at industrial level.

Wet oxidation and organosolv pretreatment are the most per-spective technologies for SCB hydrolysis at the near future, becauseboth lead to high degree of solubilization of hemicelluloses and lig-nin, and formation of degradation products is avoided. Last impliesthe elimination of the detoxification stage. However in the case oforganosolv pretreatment large reaction time and high pressure areneeded. Moreover, for wet oxidation, products of hydrolysis areoligomers.

3. Cellulose hydrolysis

Cellulose obtained from pretreatment should be degraded intoglucose (saccharification) using acids or enzymes. In the formercase, concentrated or dilute acids can be used. If dilute acids(H2SO4 and HCl) are employed, temperatures of 200–240 �C at1.5% acid concentrations are required to hydrolyze the crystallinecellulose, but the degradation of glucose into HMF and othernon-desired products is unavoidable under these conditions. Onevariant of the acid hydrolysis is the use of extremely low acidand high-temperature conditions during batch processes (Ojumuand Ogunkunle, 2005). However, cellulose hydrolysis is currentlycarried out using microbial cellulolytic enzymes. Enzymatic hydro-lysis has demonstrated better results for the subsequent fermenta-tion because no degradation components of glucose are formedalthough the process is slower.

Commercial enzymes have been used to convert SCB to fer-mentable sugars. Enzymatic hydrolysis of cellulosic materials bycellulase enzymes is the most promising approach to get highproduct yields critical to economic success (Lynd et al., 1996). Tohelp the enzymes to perform well and degrade the lignocelluloseefficiently, the fibers in the raw material need to be accessible tothe enzymes. A pretreatment in some way is needed to exposethe fibers. If the pretreatment is too harsh, liberated sugars canbe degraded to enzyme- and yeast-inhibiting compounds loweringthe overall yields. On the other hand, if too weak pretreatmentconditions are used this will result in low enzyme accessibilityand the same drawbacks. Several pretreatment methods have beenevaluated jointly with enzymatic hydrolysis (saccharification).Among them are alkaline pretreatment (Hernández-Salas et al.,2009), steam explosion (Sendelius, 2005; Hernández-Salas et al.,2009) and wet oxidation (Martín et al., 2007a).

Hernández-Salas et al. (2009) had optimized an enzyme formu-lation to process SCB and agave bagasse, which contained Cellu-clast, Novozyme and Viscozyme L. From alkaline–enzymatichydrolysis of SCB samples, a reduced level of reducing sugar yieldwas obtained (11–20%) compared to agave bagasse (12–58%). Glu-cose concentration was higher in hydrolysates derived from thealkaline–enzymatic treatment. Martín et al. (2002a) used a mixtureof endo-glucanases and cellobiases to saccharify steam pretreatedSCB. The obtained hydrolysate had a sugar composition similar tothat reported from chemically treated bagasse.

Martín et al. (2007a) has evaluated the effect of wet oxidationpretreatment on fractionation and enzymatic convertibility ofSCB. Pretreatment conditions improved the enzymatic convertibil-ity of cellulose. The highest convertibility, 74.9% was achieved inthe hydrolysis of the material obtained by pretreatment at

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195 �C, 15 min and alkaline pH. Some xylan convertibility was alsoobserved. Doubling the hydrolysis time from 24 to 48 h led only tosome additional conversion, since most of cellulose was alreadyhydrolyzed during the first 24 h. The low increase of the convert-ibility at 48 h might be an indication of some degree of denatur-ation or inactivation of the cellulases. This pretreatment gavealso the highest overall glucose yield, 68.9%, which takes into ac-count not only the glucose formed during the enzymatic hydroly-sis, but also the losses occurred during the pretreatment. Theincrease of the enzymatic convertibility is probably related to thelow content of lignin and hemicelluloses and the high cellulosecontent of the remaining solid material. The solubilization of hemi-celluloses and lignin and the destruction of their association withcellulose have certainly led to an increase of the accessibility of cel-lulose to enzymes. Some destruction of the crystalline structure ofcellulose and the decrease of its degree of polymerization are otherevents leading to improvements of the enzymatic convertibilitythat occurred during pretreatment. Lignin acts as a competitiveadsorbent for cellulases and reduces the activity of the adsorbedenzymes (Martín et al., 2007a).

4. Detoxification

During pretreatment of lignocellulosics, in addition to the sug-ars, aliphatic acids (acetic, formic and levulinic acid), furan deriva-tives furfural and HMF, and phenolic compounds are formed. Theexistence of these substances is more probably when acid and/orhigh-temperatures are used. These compounds are known to affectethanol fermentation performance. Furfural could be generated asa degradation product from pentoses. It was found that furfuralcontents increase with the concentration of the acid catalysts suchas H2SO4 (Pattra et al., 2008).

Table 2Methods for sugarcane bagasse hydrolysate detoxification.

Method Agents PreviousPretreatment

Conditions

Alkaline detoxification Overlimingwith Ca(OH)2

Steam-explosiondilute acid

pH 9–10.5 then pHadjustment to 5.5–6.5with H2SO4 or HCl

Overliming Acidhydrolysis

ND

Combined alkalinedetoxification

KOH andsodium sulfite

Acidhydrolysis

pH 10, then pHadjustment to 6.5 withHCl and addition of 1%sodium sulfite at 90 �C

Microbialdetoxification

Trichodermareesei

Steam-explosion

ND

Electrodialysis Chargedmembranesand anelectricalpotentialdifference

Acidhydrolysis

Pre-evaporation at100 �C during 15 min.Electrodialysisoperation at 20 V. Flowrate 50 L/h

Ion exchange resin Commercialanionexchange resin

Acidhydrolysis

Resin to hydrolisateratio (w/w): 1:10.Regular stirring for 1 hat room temperature

Activated charcoal Activatedcharcoal

Acidhydrolysis

ND

Enzymes treatment Laccase from C.stercoreus

Acidhydrolysis

Incubated in orbitalshaker at 100 rpm for4 h at 30 �C

ND: non-data available.

Another inhibitory substances founded in SCB hemicellulosehydrolysate is acetic acid. Acetic acid can be generated when thehydrolysis reaction takes place at the acetyl group of hemicellulose(Rodriguez-Chong et al., 2004). Generally, acetic acid is inhibitoryto yeast when its concentration is between 4 and 10 g/L. Maximumconcentration of acetic acid obtained for SCB hydrolysates fromacid hydrolysis pretreatment with 6% H2SO4 during 60 min was2.72 g/L, value lower than that for a toxic effect (Pattra et al.,2008). While using 4% H3PO4 during 300 min, the highest valuewas 4.0 g acetic acid/L (Gámez et al., 2006). On the other hand, arelative low furfural concentration (1.5 g/L) was obtained using6% H3PO4 at 300 �C, although it is over the limit (1.0 g/L) for yeastinhibition. This shows that the decomposition of pentoses to furfu-ral is low and confirms the selectivity of this treatment using phos-phoric acid.

Several detoxification methods like neutralization, overlimingwith calcium hydroxide, activated charcoal, ion exchange resins(Carvalheiro et al., 2005) and enzymatic detoxification usinglaccase (Chandel et al., 2007) are known for removing variousinhibitory compounds from lignocellulosic hydrolysates. Table 2presents main detoxification methods implemented in SCB hydrol-ysates with their corresponding operation conditions. Percentagesof toxic compounds removal are shown. Few methods can removeenough quantities of all toxic substances.

4.1. Neutralization

In the operation of neutralization, it is usual to add chemicalsthat neutralize the acids of the hydrolysates, forming salts. Thesesalts have low solubility and are normally removed by filtration.The concentration of hydrolysates by evaporation is usual to in-crease the sugar concentration. In this operation, besides water,

Removal (%) Remarks References

Furfural (51%),HMF (51%),phenoliccompounds (41%),Acetic acid (0%)

Sánchez and Cardona(2008)

Furans (45.8%),phenolics(35.87%)

Chandel et al. (2007)

ND Reduction of ketones andaldehydes, removal of volatilecompounds

Palmqvist and Hahn-Hägerdal (2000)

Phenoliccompounds (80%)

Palmqvist and Hahn-Hägerdal (2000)

Furfural (45%),acetic acid (90%)

Losses of sugar are less of 5% Cheng et al. (2008)

Furans (63.4%),phenolics (75.8%),acetic acid (85.2%)


Furans (38.7%),phenolic (57%),acetic acid (46.8%)


Phenolics (77.5%) Does not affect furans andacetic acid content. Negligibleloss in total sugars andmaximum removal phenoliccompounds


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small amounts of growth inhibitors such as acetic acid, furfural andHMF are removed (Carvalho et al., 2002). A detoxification opera-tion by adsorption on charcoal can remove the inhibitors. In thisoperation, phenolic compounds proceeding from lignin can alsobe removed.

4.2. Overliming

Overliming the hydrolysate has been effective as a detoxifica-tion process due to partial removal of toxic inhibitors, such as fur-fural and 5-hydroxymethylfurfural, although the wholemechanism is not well understood. During overliming, sulphuricacid is removed from the initial hydrolysate by adding lime to ad-just the pH and precipitation as gypsum. However, it has been ob-served that the concentrations of acetic acid before and after thedetoxifying treatment were not altered significantly (Keikhhosroet al., 2006). Another potential drawback of overliming is sugar lossdue to hydroxide-catalysed degradation reactions and conversionof sugars into unfermentable compounds (Carvalho et al., 2005).Moreover, the acid cannot be reused any more because it has be-come salt (Ali et al., 2006). Chandel et al. (2007) have also demon-strated that acetic acid concentration is not altered usingoverliming but this method led to the removal of furans (45.8%)and phenolics (35.87%).

4.3. Adsorption with activated charcoal

Charcoal adsorption decreases the concentrations of both aceticacid and phenolics derived from the SCB hydrolysate. Treatmentwith activated charcoal caused 38.7%, 57% and 46.8% reduction infurans, phenolics and acetic acid, respectively (Chandel et al.,2007).

4.4. Ion exchange resins

Ion exchange treatment has demonstrated to be an efficientmethod for removing furans (63.4%), total phenolics (75.8%) andacetic acid (85.2%) from a SCB hydrolysate.

4.5. Enzymatic detoxification

Treatment with the enzymes like laccase, obtained from the lig-ninolytic fungus Trametes versicolor, has been shown to increasethe ethanol productivity in a hemicellulose hydrolysate of SCB(Chandel et al., 2007). The laccase treatment led to selective re-moval of total phenolics by 77.5% without affecting furans and ace-tic acid content of the hydrolysate.

4.6. Electrodialysis

Another detoxification method more currently used is electro-dialysis (ED), which is an electrochemical separation process inwhich electrically charged membranes and an electrical potentialdifference are applied to separate ionic species from an aqueoussolution and other uncharged components. Cheng et al. (2008)have evaluated the detoxification of SCB acid hydrolysate by boil-ing and electrodialysis resulted in a better fermentability of thehydrolysate (see Table 3). Volatile compounds, such as furfural,were stripped by boiling, while acetic acid and sulphuric acid wereremoved by electrodialysis. After treatment by electrodialysis, 90%of acetic acid in hydrolysate was removed. The losses of glucose,xylose, arabinose, galactose, mannose and cellobiose were lowerthan 5%.

The sulphuric acid and acetic acid in concentrated compartmentof ED device are collected and separated by distillation and the sul-phuric acid can be reused, which will save the operation cost and

have no environmental impact. ED process reduces the loss of su-gar and makes the production of ethanol easier, however, due tothe instrument cost, the economical evaluation of ED detoxifica-tion is required to be studied further in the actual production ofethanol (Cheng et al., 2008).

Chandel et al. (2007) have evaluated the efficiency of variousdetoxification methods (ion exchange treatment, activated char-coal, laccase, overliming and neutralization) (see Table 2) for theremoval of inhibitors from SCB hydrolysate and eventually forimproving the fermentation of hydrolysate to ethanol using Can-dida shehatae. Overliming and laccase did not cause any affect onacetic acid levels. Laccase treatment brought about negligible lossin total sugars and maximum removal of phenolic compoundspresent in acid hydrolysate. Ion exchange treated hydrolysate gavemaximum ethanol concentration (8.67 g/L), followed by activatedcharcoal (7.43 g/L), laccase treatment (6.50 g/L), overliming(5.19 g/L), and neutralized hydrolysate (3.46 g/L). The neutraliza-tion of acid hydrolysate alone did not remove toxic compoundsto the desired levels, resulting in poor ethanol yield of 0.22 g/g(see Table 3).

5. Ethanol production by fermentation

5.1. Production technologies

The configuration employed for fermenting biomass hydroly-sates involves a sequential process where the hydrolysis of cellu-lose and the fermentation are carried out in different units(Sánchez and Cardona, 2008). This configuration is known as sep-arate hydrolysis and fermentation (SHF). When this sequential pro-cess is employed, solid fraction of pretreated lignocellulosicmaterial undergoes hydrolysis (saccharification). This fraction con-tains the cellulose in a form accessible to acids or enzymes. Oncehydrolysis is completed, the resulting cellulose hydrolysate is fer-mented and converted into ethanol. Saccharomyces cerevisiae isthe most employed microorganism for fermenting the hydroly-sates of lignocellulosic biomass. This yeast ferments the hexosescontained in the hydrolysate but not the pentoses. One of the mainfeatures of SHF process is that each step can be performed at itsoptimal operating conditions (especially temperature and pH).

When a technological flowsheet involving a SHF process is em-ployed, the detoxified hemicellulose hydrolysate can be unifiedwith the cellulose hydrolysate coming from the enzymatic reactor.To increase the amount of sugars converted into ethanol, yeastassimilating the xylose besides glucose can be employed, but inthis case the biomass utilization rates are lower than that of micro-organisms that only assimilate hexoses. This is explained by thediauxic growth of this type of yeast. To offset this effect, sequentialfermentations are employed and both fermentations are per-formed independently (co-fermentation) (see Fig. 1). One of themain challenges in pentose fermentation lies in the fact that theproductivities of pentose utilizing microorganisms are less thanthose of hexose-fermenting ones.

The co-fermentation of lignocellulosic hydrolysates representsother technological option for utilizing all the sugars released dur-ing biomass pretreatment and cellulose hydrolysis. This kind ofcultivation process aims at the complete assimilation of all the sug-ars resulting from lignocellulosic degradation by the microbialcells and consists in the use of a mixture of two or more compatiblemicroorganisms that assimilate both the hexoses and pentosespresent in the medium. This means that the fermentation is carriedout by a mixed culture.

Conversion of cellulose into ethanol can be carried out throughSSF (see Fig. 1) as in the case of starch. For this conversion, severalenzymes with cellulolytic activity (basically endo-glucanases, cel-

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Table 3Sugarcane bagasse hydrolisate fermentation.

PreviousPretreatment

Detoxificationmethod

Microorganism Conditions Ethanol yield Remarks References

% (w/w of RSa) g/L

Acid HydrolysisWith H2SO4

Electrodialysis PachysolentannophilusDW06

Batch fermentation at30 �C for 14 h with apH of 5. Air flow0.1 vvm. Agitation:150 rpm.

34 19 Table 4. Sugarcane bagasse hydrolisatefermentation. Productivity of 0.53 g/L h. Xylose consumption was total andonly 60% of arabinose was assimilated

Cheng et al. (2008)

Withoutdetoxification

PachysolentannophilusDW06

Batch fermentation at30 �C for 14 h with apH of 5. Air flow0.1 vvm. Agitation:150 rpm.

0.03 1.9 Sugar fermented was only 9% Cheng et al. (2008)

Acid hydrolysiswith HCl


Non-recombinantSaccharomycescerevisiae

Batch fermentation at30 �C during 48 h.

14.11 5 For depithed bagasse Hernández-Salaset al. (2009)



Batch fermentation at30 �C during 48 h.

15.72 4.7 For pith bagasse Hernández-Salaset al. (2009)

Ion exchangeresin

Candidashehatae NCIM3501

Batch fermentation at30 �C during 24 h and150 rpm.

48 8.67 Chandel et al. (2007)

Activatedcharcoal



42 7.43 Chandel et al. (2007)

Enzymes(laccase fromC. stercoreus)



37 6.50 Chandel et al. (2007)

Acid hydrolysiswith HCl

Overliming Candidashehatae NCIM3501

Batch fermentation at30 �C during 24 h and150 rpm

30 5.19 Chandel et al. (2007)

Neutralization Candidashehatae NCIM3501

Batch fermentation at30 �C during 24 h and150 rpm

22 3.46 Chandel et al. (2007)

Alkaline treatmentand enzymaticsaccharification



Operation at 30 �Cduring 48 h

32.57 12.5 For depithed bagasse Hernández-Salaset al. (2009)




25.76 12.9 For pith bagasse Hernández-Salaset al. (2009)

H2SO4-catalysedsteampretreatment


Adapted xylose-utilizingrecombinantSaccharomycescerevisiae


38 ND Pretreatment was followed byenzymatic hydrolysis. Strain wasisolated from an adaptation culturewith increasing concentrations ofinhibitors. Ethanol productivity:2.55 g/(g h)

Martín et al. (2007b)


Non-adaptedxylose-utilizingrecombinantSaccharomycescerevisiae


18 ND Pretreatment was followed byenzymatic hydrolysis. Ethanolproductivity: 1.15 g/(g h)

Martín et al. (2007b)

a RS: Reducing sugars; ND: non-data available.


lobiohydrolases and b-glucosidase) are added to the suspensionobtained by mixing water with the solid fraction resulting fromthe pretreatment step and that contains cellulose and lignin. Inthe same way, process microorganisms (yeasts) are added to thismixture in the bioreactor where SSF is accomplished for immedi-ately converting the formed glucose into ethanol. The increasedethanol concentration in the culture broth allows the reductionof energy costs during distillation. In addition, SSF offers an easieroperation and a lower equipment requirement than the sequentialprocess since no hydrolysis reactors are needed. Nevertheless, SSFhas the inconvenient that the optimal conditions for hydrolysis andfermentation are different, which implies a difficult control andoptimization of process parameters (Claassen et al., 1999). In addi-tion, larger amounts of exogenous enzymes are required (Cardonaand Sánchez, 2007).

In the case of lignocellulosic biomass, a very promising inte-grated configuration for bioethanol production is the inclusion ofpentose fermentation in the SSF. This process is known as simulta-neous saccharification and co-fermentation (SSCF) (see Fig. 1). Thisconfiguration implies a higher degree of intensification through itsreaction–reaction integration. In this case, the hydrolysis of cellu-lose, the fermentation of glucose released, and the fermentationof pentoses present in the feed stream is simultaneously accom-plished in a same single unit. Besides the effectiveness of employedcellulases, the key factor in SSCF is the utilization of an efficientethanol-producing microorganism with the ability of assimilatingnot only hexoses (mainly glucose), but also pentoses (mainlyxylose) released during the pretreatment step as a result of thehemicellulose hydrolysis. Therefore, genetically modified microor-ganisms has been developed and successfully proven in SSCF


processes for ethanol production from lignocellulosic materials(Cardona and Sánchez, 2007). Recombinant strains of Escherichiacoli, Zymomonas mobilis and S. cerevisiae capable of hexose andpentose catabolism and high ethanol production have also beenconstructed. Their use has made the conversion of lignocelluloseto ethanol economically feasible (Mohagheghi et al., 2002; Martínet al., 2002b).

5.2. Production results

SCB has proven to be a feasible raw material for fuel ethanolproduction due its relative low lignin content and high productionof sugars by appropriate pretreatments. Some of the more cur-rently advances in fuel ethanol production using bagasse reportedalcohol yields up to 48% (% w/w of reducing sugars) (see Table 3).Hernández-Salas et al. (2009) have pretreated the whole SCB anddifferent fractions of it by dilute acid (HCl) and alkaline pretreat-ment (NaOH). Selected hydrolysates were fermented with a non-recombinant strain of S. cerevisiae and maximum alcohol yield byfermentation (32.6%) was obtained from the hydrolysate of sugar-cane depithed bagasse. Yields for other fractions are shown in Ta-ble 3.

By the other side, using Pachysolen tannophilus DW06 for fer-menting SCB hydrolysate obtained from acid pretreatment(H2SO4) and detoxified with electrodialysis, it was possible to ob-tain an ethanol yield of 34% (Cheng et al., 2008). Higher yields wereobtained by Chandel et al. (2007) with C. shehatae NCIM 3501 fer-menting hydrolysates of SCB obtained with dilute acid (HCl) pre-treatment and different detoxification methods: 48% with anindustrial ion exchange resin (DIAION HPA 25, Mitsubishi ChemicalCorporation, Japan), 42% with activated charcoal and 37% with lac-case (from C. stercoreus). Lower ethanol yields were obtained withoverliming (30%) and neutralization (22%).

Table 4Revenue obtained from using 1 ton of sugarcane bagasse for fuel ethanol productionor electricity cogeneration in Colombia.

Ethanol Units Electricity Units

Yield per ton of SCB 150–236a L 200–600a kWhProduction Cost 0.26–0.33b US$/L 0.028–0.034c US$/kWhSelling price* 0.930–0.980d US$/L 0.033–0.07c,e US$/kWhRevenue 0.6–0.720 US$/L �0.001–0.042 US$/kWhRevenue per ton of SCB 90–170 US$ �0.2–25.2 US$

a Botha and Blottnitz (2006).b Luo et al. (2009).c Ministry of the Environment (2005).d Proexport Colombia (2008).e Federación de Biocombustibles (2009).

6. Energy cogeneration

Energy cogeneration is well established process in sugar indus-try, due to the high quantity of SCB available, which is composed of50% fibre, 48% moisture and 2% sugars. It is normally burnt to gen-erate steam and electricity to meet the energy requirements of thecane sugar factory. The bagasse has a gross calorific value of19.25 MJ/kg at zero moisture and 9.95 MJ/kg at 48% moisture.The net calorific value of bagasse at 48% moisture is around 8 MJ/kg. The fact that the sugar cane plant provides its own source of en-ergy from sugar production in the form of bagasse has long been aspecial feature of the sugar industry. In the traditional approach,sugar factories co-generate just enough steam and electricity tomeet their needs. With the availability of advanced co-generationtechnologies, sugar factories today can produce surplus electricityfor sale to the national grid or directly to other electricity users(Quintero et al., 2008; Ramjeawon, 2008). However, in some coun-tries bagasse is usually burned in low-efficiency boilers to avoidthe need to handle surplus bagasse, and the cogeneration systemswork on back pressure steam turbines (BPST) with low pressure atlow-temperature (typically 1.9–21 MPa, 573 K), the process doesnot produce energy in an efficient, cost-effective manner (Ramjea-won, 2008).

Other potential lignocellulosic by-product from sugar factoryare the sugar cane agricultural residues (SCAR), which as a rule,are burned just before the harvest in order to facilitate the easierharvesting of cane stalks. Production of each million ton of raw su-gar could mean 50,000 ton of SCAR (that is, SCAR with a moisturecontent of 30% of sugarcane weight) with a lower heat value (LHV)of 10.5 MJ/kg and a bulk density of 180 kg/m3. This amount of SCARcould be substituted for the same quantity of bagasse, leaving the

surplus bagasse to be converted into another kind of energy carrier,such as ethanol, bio-oil, etc. (Ramjeawon, 2008).

Main reason for considering fuel ethanol production from SCB isthe social and industrial pressure about finding alternative rawmaterials and agro-industrial residues like SCB that are offered inhigh quantities in tropical countries. Table 4 shows an exampleof revenues (based on range values) that a sugar industry placedin a tropical country like Colombia, could obtain by using SCB forproducing either ethanol of electricity. As it can be see revenuesobtained from ethanol production are higher than that obtainedfrom electricity cogeneration even at low ethanol prices in themarket. However, this alternative is not profitable at industrial le-vel for the existing mills, because of the high capital investmentneeded and the low maturity of this technology. In addition, elec-tricity generated at cogeneration systems is very cheap for the milland heat energy is used in the whole process. Revenue obtainedfrom electricity cogeneration can be higher in other countries likeBrazil due to the lowest production cost (0.00109–0.00885 US$/kWh) (Moreira, 2000).

7. Xylanases and cellulases production

High cost commercial xylanases and cellulases used in the sac-charification step for SCB transformation to ethanol can beproduced from the same bagasse. Many microorganisms, includingfilamentous fungi, yeasts and bacteria, have been cultivated inmedia containing SCB or its hydrolysate. The use of SCB as low costraw material for xylanase production by Bacillus circulans D1 insubmerged fermentation has been investigated (Bocchini et al.,2005). The microorganism was cultivated in a mineral mediumcontaining hydrolysate of bagasse or grass as carbon source. Highproduction of enzymes was obtained during growth in media withbagasse hydrolysates (8.4 U/mL) and in media with grass hydroly-sates (7.5 U/mL). Xylanase production in media with hydrolysateswas very close to that obtained in xylan containing media(7.0 U/mL); and this fact confirms the feasibility of fermenting thisagro-industrial byproducts by B. circulans D1 as an alternative tosave costs on the enzyme production process.

The media containing hydrolysates of SCB, with initial sugarconcentration from 2.5 to 10.0 g/L, can be employed in place ofthe control medium, since they afforded xylanase productionsequal or higher than that obtained in the medium with xylan. Thisreplacement implies economical advantages for xylanase produc-tion process, mainly regarding to the commercial xylan high costand the availability and low cost of sugarcane (Bocchini et al.,2005). SCB hydrolysates is an efficient alternative to reduce thecosts of xylanase production in submerged fermentation, sincethese materials are often available in tropical countries, as an inex-pensive source of components that propitiate the bacterial growthand the enzyme production.


Other strains like Penicillium janthinellum NCIM 1171 and Trich-oderma viride NCIM 1051 have been evaluated in production of cel-lulase and xylanase enzymes from chemically treated SCB (Adsulet al., 2004). Higher xylanase and b-glucosidase activities were de-tected in the medium with bagasse as compared to the values ob-tained with pure cellulose powder. Bagasse treated with NaClO2

during 4 h at 70 �C gave high yields of xylanase (130 IU/ml) andb-glucosidase activities (2.3 IU/ml) for both P. janthinellum and T.viride (Adsul et al., 2004).

8. Mathematical modeling

The modeling of the hydrolysis of a polysaccharide is very com-plicate. Multiple factors related to the lignocellulosic material(size, particle shape, structure, accessibility of proton to heterocy-clic ether bond, etc.) and to the reaction medium (type of acid, con-centration, temperature, time, etc.) affect the hydrolysis. Thesolution of compromise between the complexity of a rigorousmodel and the search of equations modeling the empirical datain a simple and satisfactory way have conducted to the generaluse of pseudohomogeneous, irreversible and first order kineticsthat make easy the calculations without to sacrifice the theory ba-sis. The simplified models for the study of the kinetics in hydrolysisprocess using acids began with the work of Saeman for the hydro-lysis of douglas fir wood using sulphuric acid (Saeman, 1945). Thehydrolysis of cellulose was studied establishing the followingmodel:

Cellulose!k1 Glucose!k2 Decomposition products ðHMFÞ

where, k1 (min�1) is the rate for release of glucose from celluloseand k2 (min�1) is rate for glucose decomposition. This model con-siders the hydrolysis of cellulose to release glucose that in severeconditions is decomposed into HMF. Both reactions (release anddecomposition) were considered irreversible and first order. Themodel of Saeman was also applied to the hydrolysis of the hemicel-lulosic fraction (Téllez-Luis et al., 2002). The model of Saeman canbe applied to other polysaccharides; therefore the model can begeneralized for the decomposition of any polymer. The generalizedpolymer could be cellulose, xylan, araban, etc. Kinetic parameters ofthe above mentioned models for hydrolysis pretreatment withphosphoric acid (Gámez et al., 2006) and nitric acid (Rodriguez-Chong et al., 2004) has been reported. Other efforts have been madein modeling delignification process of bagasse with formic acid (Tuet al., 2008).

9. Stability of fermentation systems based on SCB

In general, more efficient pretreatment technologies, detoxifica-tion methods and the construction of microorganism strains capa-ble to ferment lignocellulosic materials have different advancesduring last years. However, other restrictions of the fermentationprocesses related to original microorganism have not been passed.One of them is the existence of nonlinear phenomena such as mul-tiplicity and oscillation. The complexity of stability can be in-creased as a result of the inhibition problems duringfermentation when furfural and formic acid from SCB hydrolysisare present in the bioreactor. It is generally considered that thenonlinear phenomena are unfavorable for stable operations inindustrial fermentation. The nonlinear analysis of oscillatory fer-mentations with Z. mobilis indicates that with a change in theparameters, these simple oscillations bifurcate in more complexphenomena such as totally developed chaos (Garhyan and Elnas-haie, 2004). Recombinant strains of Z. mobilis are common microor-ganisms used in SCB transformation to ethanol (Mohagheghi et al.,

2002) and oscillations can be expected as an important issue in thebioreactor design and control.

From different studies about behavior of continuous cultures ofS. cerevisiae, it has been established that various operation vari-ables influence the stability of processes with this microorganism.In anaerobic cultures, it was seen that the inhibitory action of eth-anol leads to unstable states. Efforts for using yeast strains with theability to ferment all lignocellulose-derived sugars include the useand modification of S. cerevisiae with high inhibitor tolerance toacetic acid, furfural and formic acid (Hahn-Hägerdal et al., 2007).In the case of SCB hydrolysis, acetic acid is the key inhibitor formedat higher proportion. There is no study in literature about the influ-ence of this type of inhibitors on stability, but massive productionof bioethanol from SCB needs this type of information for processdesign and performance. If this inhibition is increased by aceticacid during fermentation of sugars from SCB hydrolysis not onlyethanol production can be reduced, but also stability characteris-tics of the process get more complex. For example, addition of ace-tate (10 g/L) or furfural (2 g/L), in concentrations similar to thosefound in SCB hydrolysates, decreased cell mass formation andgrowth rate in almost all strains of industrial S. cerevisiae (Garay-Arroyo et al., 2004). The wide variability of responses to thedifferent environmental stress conditions tested show that nogeneral rules can be assumed for different S. cerevisiae strains,and that these responses are highly dependent on their geneticand environmental backgrounds. From here stability of the processcan be predicted to be complex.

Trends in bioethanol production from SCB and other lignocellu-losic materials show the high potential of process integration. Thedevelopment of processes to produce ethanol by coupled sacchar-ification and fermentation of SCB can be analyzed. Here, the aceticacid and furfural removal should be coupled to the entire process(simultaneous sacharification and cofermentation process – SSCF)and stability problems derived from the presence of these inhibi-tors must be accounted. The study of steady states would allowdetermining the optimal operation conditions of both processes.

Until now, there are not reports about stability studies forSCB hydrolysis and fermentation to ethanol. It is an importantchallenge in the development of technologies for ethanol pro-duction from SCB. Moreover, nowadays, due to wide variety offeedstocks, industrial yeast strains are exposed to constant envi-ronmental changes including ethanol accumulated along the pro-cess, solute concentration, medium ionic strength, and toxins orinhibitory substances that can affect the stability of fermentation(Garay-Arroyo et al., 2004; Zhao and Bai, 2009). Therefore, thedesign of new processes that involve fermentation processesmust be accompanied of a stability study to formulate suitableoperation strategies.

10. Perspectives, challenges and conclusions

An increased use of biofuels would contribute to sustainabledevelopment by reducing greenhouse-gas emissions and the useof non-renewable resources. In recent years it has been suggestedthat, instead of traditional feedstocks, cellulosic biomass (celluloseand hemicellulose), including SCB could be used as an ideally inex-pensive and abundantly available source of sugar for fermentationinto transportation fuel ethanol. The efficiency of biomass conver-sion to ethanol depends upon the ability of the microorganismused in the process to utilize these diverse carbon sources andamount of fraction present in biomass. The cost of ethanol produc-tion from SCB is relatively high based on current technologies.

Pretreatment continues to be the most important step in etha-nol production from SCB. It is expected, in the next future, thattechnologies can impact more easily this step rather than genetic


modification to sugarcane strains (with the purpose of reducinglignin in the stalks). This is explained by the fact that the develop-ment and transfer of this type of sugarcane strains to the agricul-ture should take long time. Additionally, sugar factories linked tothe sugarcane growing sector (usually owning most of the crops)can choice the status quo instead of investing in new projects.Promising pretreatment methods as organosolv and wet oxidationwill be the most studied in the coming years. Cellulose hydrolysisdevelopments are high dependent on the pretreatment methodfrom the point of view of the obtained enzyme accessibility andinhibitors production. New developments are more related to theefficient in situ cellulases production from the SCB to be used inthe hydrolysis step.

Detoxification developments in the next years will be weak(appointing to integration in one step of different detoxificationmethods) in contrast to the genetic modifications of fermentingmicroorganisms that can tolerate desired concentrations of inhibi-tors. Integrated configurations as SSF and SSCF are the top efficienttechnologies to be analyzed and confirmed at pilot and industriallevels before its wide use in industry. Energy cogeneration as wellas xylanases and cellulases production from SCB will stay as a realalternative for adding high value to this residue. At the same way,energy cogeneration for the sugar factory needings and electricitysupply to the grid will be always the main barrier to the use of SCBfor ethanol production. Other perspective of research and develop-ment in ethanol production from SCB is the analysis of the stabilityin bioreactors in the form of single units as well as integrated con-figurations. Most of the sugarcane juice and molasses based etha-nol production process suffer a lot of problems regardingstability. The existence of new inhibitors in the ethanol processfrom SCB supposes new stability problems for this technology. Sothe design and introduction of high scale SCB use for ethanol pro-duction in the industry requires serious analysis of this problem.

According to the above mentioned perspectives first main chal-lenge for successful use of SCB as raw material in fuel ethanol pro-duction is to reduce hydrolysis costs to make SCB a cheapersubstrate like molasses and other directly fermentable materials.Second challenge is process optimization, including detoxificationtechnologies and in situ cellulase enzyme production. Third chal-lenge includes maintaining a stable performance of the geneticallyengineered microorganisms in commercial scale fermentationoperations. The future trends for improving the pretreatment oflignocellulosic feedstocks also include the production of geneti-cally modified plant materials with higher carbohydrate contentor modified plant structure to facilitate pretreatment in milderconditions or using hemicellulases. Other challenges are not de-scribed in this paper, but the most important concern is how topropose bioethanol production from SCB as a real, economicaland environmental alternative to burning or cogeneration in sugarmills. Although bioethanol production has been greatly improvedby new technologies, there are still challenges that need furtherinvestigations and developing more efficient pretreatment tech-nologies for the lignocellulosic biomass and integrating the opti-mal components into economic ethanol production systems.

Acknowledgements

The authors express their acknowledgments to the National Uni-versity of Colombia at Manizales for funding different research pro-jects in fuel ethanol production and lignocellulosics exploitation.

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