• Extrusion

Continuous extrusion process, which involves a single or twin-screw extruder combining thermal, mechanical and chemical action, is regarded as a cost-effective pre-treatment method for enzymatic saccharification. What is more, the addition of enzymes during the extrusion process (bioextrusion) as new biomass pre-treatment technique for second generation bioethanol production has been proposed, resulting in a better sugar production [49]. With regards to its application to OP, extrusion in a twin-screw extruder was applied to OP to remove the extracts and the amorphous cellulose at 70 ◦C and sulphuric acid concentrations lower than 0.5 mol/dm3 [10]. The yield of sugars was low. However, combined with a subsequent dilute-acid hydrolysis with 1 mol/dm<sup>3</sup> H2SO4 of the resulting solid in a stirred-tank reactor, led to a sugar-rich hydrolysate [14]. Extrusion operation still needs to be optimised for the extrusion to be regarded as a single hydrolysis stage for bioethanol production.

• Dilute-acid hydrolysis

Dilute-acid pre-treatment, as one of the most important pre-treatments, has been widely applied to OP within the biorefinery concept. The aim of dilute acid is to solubilise the hemicellulose fraction without degrading cellulose as far as possible. With regards to the application of dilute-acid pre-treatment to OP, several authors have reported that hemicellulose depolymerised into a mixture of sugar oligomers and monomers under the acidic, thermal process while scarce alteration took place in lignin and cellulose structures [4,11,50,51]. In addition, cellulose porosity increased with the removal of hemicellulose and so enhanced enzymatic digestibility of the cellulose. The effects of temperature and acid concentration of dilute-acid pre-treatment on the subsequent simultaneous saccharification and fermentation of olive-pruning debris using response-surface methodology (RSM) have been studied. The pre-treatment led to a complete solubilisation of the hemicellulose. As a result, the cellulose percentage in the resulting solid was roughly 1.5 times as much as that for raw material [4]. According to the RSM, the highest overall ethanol yields would be obtained when the pre-treatment for olive-pruning debris were performed with 0.059 kmol/m<sup>3</sup> and 0.030 kmol/m<sup>3</sup> H2SO4 at 185 ◦C. Under these conditions, 15.3 kg and 14.5 kg ethanol would be generated from 100 kg olive-pruning debris, respectively [4]. Likewise, the effects of the reaction time (0–300 min), temperature (70–90 ◦C) and sulphuric acid concentration (0–0.05 kmol/m3) on the formation of d-glucose and d-xylose were evaluated by RSM [50]. Results showed that there were interactive effects between the three parameters on sugars production. The highest concentrations of d-glucose and d-xylose were achieved when

the highest temperature, acid concentration and residence time applied. The optimal conditions for generating d-xylose were 90 ◦C, 0.05 kmol/m3 H2SO4 and 300 min reaction time. Under these conditions, it was predicted that approximately 40% of the maximum attainable d-glucose and 60% of the potential d-xylose would be obtained [50]. The olive-pruning debris has been also hydrolyzed by 0.050–0.100 kmol/m<sup>3</sup> oxalic acid at 130–170 ◦C for 30 min using 1:10 dry raw material to organic acid ratio. However, the hydrolysate was not able to be fermented by the yeast *Pichia stipitis* CBS 6054 [14]. Similarly, the fermentation with *Pachysolen tannophilus* of the hydrolysates resulting from the hydrolysis with concentrated phosphoric acid of OP at 90 ◦C for 240 min led to very low bioethanol yields [52].

Dilute-acid pre-treatment has been assayed in combination with other pre-treatments, such as fungal pre-treatment [51] and autohydrolysis [15,48], to enhance the enzymatic hydrolysis of OP. For instance, the combination of fungal pre-treatment with a dilute-acid pre-treatment was studied [51]. It was found that the order of the pre-treatment combination has a relevant effect on the d-glucose yield of the subsequent enzymatic hydrolysis. The application of the best pre-treatments combination plus enzymatic hydrolysis to OP achieved 51% of the theoretical sugar yield. The afore mentioned best sequential pre-treatments were fungal pre-treatment with *Irpex lacteus* for 28 days followed by diluted-acid pre-treatment with 2% (*w*/*v*) H2SO4 at 130 ◦C for 90 min, which enhanced 34% the enzymatic hydrolysis yield compared with that of the application of solely the dilute-acid pre-treatment [51]. On the other hand, the application of an dilute-acid hydrolysis (90 ◦C, 0.05 kmol/m3 H2SO4) to the solid obtained after the autohydrolysis (200 ◦C, 0 min) of olive-pruning debris, neither increased the cellulose conversion nor led to lignin degradation [48]. By contrast, the application of the same dilute-acid pre-treatment to the resulting liquid from autohydrolysis rocketed the d-glucose and d-xylose extraction from OP. As a result, this sequential pre-treatments led to a phenolic compound-free pre-hydrolysate containing 114 g d-glucose and 78 g d-xylose per pre-treated kg of olive-tree pruning [48]. This fact has been verified by other authors, who stated that in all their experiments the percentage of lignin recovered was close to 100% [15]. These authors concluded that the dilute-acid hydrolysis of OP at 90 ◦C for 180 min leads to an almost complete hydrolysis of the hemicellulose when using a concentration of hydrochloric acid (HCl) exceeding 3.77%.
