The pretreatments described above such as steam explosion, liquid hot water, dilute acid, lime, and ammonia pretreatments are the most studied methods because they have potential as cost-effective pretreat­ments (Kazi et al., 2010; Mosier et al., 2005; Piccolo and Bezzo, 2009; Tao et al., 2011; Wyman et al., 2005). Other alternatives such as biological, ultrasonication, micro­wave, organosolvs, ILs, and combinatorial methods are also essayed; however, they are either low effective, long-time treatment or too expensive, and further inves­tigation and improvements have to be reached before they can be competitive. In this section multiple or combinatorial pretreatments and other alternative pretreatments will be discussed.

Biological pretreatments must decrease the time of the process in order to be competitive in an industrial concept of biorefinery; to reach this objective its combi­nation with chemicals and/or physical methods has been proposed by several studies. For example, the combination of a biological pretreatment by I. lacteus or P. subvermispora with a mild alkali pretreatment improved significantly ethanol production without the production of inhibitor compounds for downstream pro­cesses (Salvachua et al., 2011; Zhong et al., 2011). Other two-step pretreatment proposed consisted in a mild physical or chemical step (ultrasonic and H2O2) and a subsequent biological treatment by P. ostreatus, increasing significantly the lignin degradation compared to those of one-step pretreatments (Yu et al., 2009); also, pretreat­ment by white-rot fungi has been combined with organo — solv pretreatment in an ethanol production process from beech wood chips (Salvachua et al., 2011); the combina­tion of biological and mild acid pretreatment was reported as a promising method to improve enzymatic hydrolysis and ethanol production from water hyacinth with low lignin content (Ma et al., 2010). Another combi­nation of biological pretreatment with thermal processing for wheat straw consisted in a first phase of biodegrada­tion by P. chrysosporium (10 days) and a thermal decom­position using pyrolysis (Zeng et al., 2011). Also, a combination of fungal treatment with liquid hot water treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa (Wang et al., 2012).

Sugarcane bagasse is one of the most promising biomass considered in biorefineries; thus, several studies have proposed combined pretreatments. The ultrahigh-pressure explosion combined with alkaline treatment (0.5% NaOH) at 125 °C for 120 min signifi­cantly decreased the particle size and disrupted the microstructure, with a significant delignification and increased enzymatic digestibility of sugarcane bagasse (Chen et al., 2010). A combined treatment with dilute sulfuric acid and microwave heating up to 190 °C for 5 min has also been studied. This treatment resulted in an increment of the specific surface area of bagasse, almost complete removal of hemicellulose and signifi­cant reduction of the crystalline structure of cellulose (Chen et al., 2011), while microwave—alkali treatment at 450 W for 5 min resulted in almost 90% of lignin removal from the bagasse (Binod et al., 2012). Also, bagasse has been subjected to sono-assisted alkaline pre­treatment (Velmurugan and Muthukumar, 2012a). Acid, alkaline or sequential acid/alkaline solutions have been tested to conversion into bio-oil in a pyrolysis process at low-temperature conversion under He or O2/He atmo­spheres at 350—450 °C (Cunha et al., 2011). A two — stage process for delignification of sugarcane bagasse uses alkali and peracetic acid combination (Teixeira et al., 2000; Zhao et al., 2011b).

Same strategies (acidic/alkaline) have been proposed for corn stover. For example, a two-stage process consists of use of 0.07 wt% sulfuric acid at 170 °C,

2.5 ml/min for 30 min and ARP (15 wt% ammonia) at the same temperature, 5.0 ml/min for 60 min. In the first stage hemicellulose was recovered while in the following stage lignin was recovered. This treatment brought about enzymatic digestibility of 90% using 60 filter paper units/g glucan cellulase enzyme loadings (Kim, 2011). Another combined treatment proposed for corn stover is SAA (15 wt% ammonia) with solution con­taining also 20 wt% ethanol at 60 °C for 24 h preserving the hemicellulose in solid form (Kim et al., 2009). Also, the use of NaOH (0.3 N) and a step of particle size homogenization has reported a significant enhancement of enzymatic hydrolysis (Li et al., 2004). The synergistic effect of preimpregnation by sulfuric acid (3 wt%) and steam explosion (190 °C) has been investigated; after 48 h of digestion the yield of glucose was 93% of the theoretical (Zimbardi et al., 2007).

Sequential stages of autohydrolysis and ethanol— water mixtures were used to pretreat olive tree trimmings recovering up to 42% of the polysaccharides contained in the raw material (Requejo et al., 2011). Also, this process has been tested with uncatalyzed ethanol—water solu­tions of Eucalyptus globulus wood (Romani et al., 2011). Mixtures of ethanol/water/acetic acid in an autoclave have been also used (Teramoto et al., 2008). This combined process causes the solubilization of hemicelluloses and lignin, leaving solids enriched in cellulose. A treatment of ethanosolv catalyzed with FeCl3 (0.1 M) at 170 °C for 72 h has been proposed for barley straw allowing enzy­matic digestibility of 89%. This treatment had a particu­larly strong effect on enzymatic digestibility and cellulose recovery (Kim et al., 2010).

Another pretreatment at pH 1 (hydrochloric acid) and subsequently at pH 13 (sodium hydroxide) released 69% and 95% of the theoretical maximal amounts of glucose and xylose, respectively, from the straw and removal of 68% of the lignin (Pedersen et al., 2010). The opposite sequence alkaline stage (ammonia) followed by acidic stage (dilute sulfuric acid by percolation) has also been used to treated rice straw (Kim et al., 2011a).

Microwave-based heating (190 °C) was used to pre­treat switchgrass presoaked in alkali solutions (0.1 g/g) resulting in release of 90% of maximum potential sugars. This value was significantly higher than the one obtained with conventional heat and it was attributed to the disruption of recalcitrant structures under microwave heating (Hu and Wen, 2008).

Significant disintegration of lignocellulosic struc­ture of wheat, barley straw grinds, switchgrass and coastal bermuda grass has been reported with the microwave—chemical (NaOH or Ca(OH)2) pretreat­ments (Kashaninejad and Tabil, 2011; Keshwani and Cheng, 2010). Also, microwave-assisted pretreatment of woody biomass with ammonium molybdate acti­vated by H2O2 has also been proposed resulting in a selective delignifying system (Verma et al., 2011).

For hydrogen production from Miscanthus by Ther­motoga elfii, high delignification values were obtained by the combination of mechanical (one-step extrusion) and chemical pretreatments (NaOH at 70 °C) resulting in a 33% conversion into monosaccharides of the initial biomass after enzymatic hydrolysis (de Vrije et al.,

2002) .

A two-stage pretreatment method was proposed and tested for deconstruction of Miscanthus; first, biomass is pretreated at 50 °C, 1.0—4.0% alkaline peroxide solutions to remove up to 64% of hemicellulose and 64% of lignin. The remaining solids were subjected to a second pre­treatment at 121 °C with electrolyzed water (Wang et al., 2010).

On the other hand, application of a dehydration pro­cess to the mechanochemical pretreatment process of the bioethanol production system has been proposed for energy saving and cost reduction. However, the dehy­dration process has problems with the loss of sugars eluted in the liquid phase during the hydrothermal pro­cess (Yanagida et al., 2011).

Combination of hot compressed water (hydrothermal treatment) and mechanochemical milling, including a dewatering step for Eucalyptus and rice straw, has been proposed for ethanol production (Fujimoto et al., 2008; Hideno et al., 2012). Torrefaction is a mild thermal pre­treatment (T < 300° C) that improves biomass milling and storage properties (Chen et al., 2012; Fisher et al., 2012). This treatment has gained attention in recent years and some biomasses that have been treated include oil palm fiber and eucalyptus, Norwegian birch, spruce, Miscanthus and white oak sawdust; residues from coffee grain, sugarcane, sawdust and rice husk bagasse (Chen et al., 2012; Lu et al., 2012; Medic et al., 2012; Protasio et al., 2012; Srinivasan et al., 2012; Tapasvi et al., 2012; Tumuluru et al., 2012). Wet torrefaction (hot compressed water 200—260 °C) and dry (nitrogen, 250—300 °C) has been tested with Loblolly pine with mass yield of solid product ranging between 57% and 89%, and energy densification to 108—136% of the orig­inal feedstock (Yan et al., 2009).

Extrusion has also been used in combination with alkali (1.70%, w/v NaOH) soaking for pretreatment of prairie cord grass at a barrel temperature of 114 °C, 122 rpm screw speed resulted in an 82% of sugar recov­ery after enzymatic hydrolysis (Karunanithy and Muthu — kumarappan, 2011). An alkali-combined extrusion pretreatment of corn stover obtained glucose and xylose sugar yields of 86.8% and 50.5%, respectively. The condi­tions used were alkali loading of 0.04 g/g dry biomass, a screw speed of 80 rpm, residence time for extrusion is 27 min, temperature of 140 °C and washed with water (Zhang et al., 2012b). Also, glucose conversion of 95% was reported from soybean hulls using a thermomechan­ical extrusion pretreatment (screw speed 350 rpm, 80 °C and in-barrel moisture content 40% wt) (Yoo et al., 2011). A study of high-temperature (110—130 °C), concentrated — acid (5—30 wt.%) hydrolysis kinetics was undertaken for pretreated pine in a corotating twin-screw extruder reactor, obtaining more than 50% of the theoretical glucose in roughly 25 min (Miller and Hester, 2007). A successive pretreatment of ball-milled bamboo con­sisted in ultrasound treatment in ethanol solution at 20 ° C from 0 up to 50 min. After that the samples were dissolved with 7% NaOH/12% urea solutions at 12 °C, followed by successive extractions with dioxane, ethanol, and dimethyl sulfoxide (Li et al., 2010). Other treatments, such as SAA and proton beam irradiation, have been tested with rice straw and approximately 90% of the theoretical glucose conversion was obtained at 12 h (Kim et al., 2011b). Microwave pretreatment also has been combined with alkali to pretreat cashew apple bagasse founding that alkali exerted influence on glucose formation (Rodrigues et al., 2011).

A pretreatment method using ammonia and ILs reported a synergy effect for rice straw, achieving 82% of the cellulose recovery and 97% of the enzymatic glucose conversion with recycling of the ILs (Nguyen et al., 2010). Pretreatment of wheat straw with combined sulfuric acid (0—3%, w/v) and Tween-20 (concentration,

0— 1%) was evaluated with modification of lignin surface (Qi et al., 2010). Other surfactants, such as, Tween-80, dodecylbenzene sulfonic acid, and polyethylene glycol 4000, have also been used combined with diluted acid to treat corn stover and bagasse (Qing et al., 2010; Sindhu et al., 2012).

Other pretreatments include technology used in kraft pulp mills for the efficient conversion of lignocellulosic biomass into ethanol (Gonzalez et al., 2011). Sulfite pre­treatment to overcome recalcitrance of lignocellulose consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining (Li et al., 2012; Zhang et al., 2012a). Pretreat­ment of corn stalk with sulfite (7%) at a temperature of 180 °C for 30 min was successfully performed (Liu et al., 2011; Zhu et al., 2009). Silage preparation is a well-known procedure for preserving plant material; the effects of Fe(NO3)3 pretreatment conditions on sugar yields were investigated for corn stover silage. Ensiling techniques, with and without supplemental enzymes, also have been reported as a cost-effective pretreatment (Chen et al., 2007; Sun et al., 2011; Thomsen et al.,

2008) . Also, FeSO4 (0.1 mol/L at 180 °C for 20 min) was investigated as a catalyst for the pretreatment of corn sto­ver, observing significantly increased hemicellulose degradation in aqueous solutions with high xylose recovery and low cellulose removal (Zhao et al., 2011a). Lignocellulose pretreatment featuring modest reaction conditions (50 °C and atmospheric pressure) was demon­strated to fractionate lignocellulose to amorphous

cellulose, hemicellulose, lignin, and acetic acid by using a nonvolatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone) and water (Zhang et al., 2007).