Efficient organosolv pretreatment has been well developed for agricultural residues and hardwoods, rather than softwood biomass because they are considerably more recalcitrant to the pretreatment and enzymatic processes [57, 58]. In order to over­come the problems, organosolv combined with some other approaches have been attempted, such as the two-stage pretreatment approaches as follows:

1. Presoaking/pretreatment of biomass by dilute-acid before the organosolv process. Dilute acid-pretreated sugarcane bagasse was performed prior to an organosolv process using NaOH as a catalyst under the optimized conditions (60 min, 195 °C, using 30 % (v/v) ethanol), where 67.3 % (w/w) of the pretreated solid material was easily converted to glucose in 24 h [59]. The optimized dilute acid presoaking and aqueous ethanol organosolv treatment of Miscanthus led to a better recovery of xylans (~20 % (w/w) of dry mass against ~ 10 % (w/w) without presoaking), en­hanced dissolution of lignin in the aqueous ethanol, and increased enzymatic digestibility (98 % cellulose-to-glucose conversion against 80 % conversion without presoaking) [60].

2. Organosolv pretreatment followed by mechanical milling of pretreated substrates. The subsequent mechanical treatment of the aqueous ethanol-treated lodge — pole pine by mill refining decreased particle size and crystallinity but increased swelling and fiber delamination of the substrate. However, the hydrolysis process was not accelerated remarkably [61].

3. Organosolv pretreatment and substrate sulfonation of the product. After ethanol treatment, sulfonation of lodgepole pine could reduce none of the specific bind­ings of lignin to enzymes. Accordingly, it resulted in obvious enhancement of hydrolysis yields even at low enzyme loadings despite less removal of residual lignin. At the same time, enzyme recovery was increased as well [61].

Though some positive results were achieved with the combined method, it was impossible to check the efficiency of the developed methods such as acid presoaking and sulfonation on other kind of softwood. Additionally, alkali reagent, such as NaOH, NH3H2O, or triethanolamine, which has good ability for delignification rather than the improvement of hydrolysis, can be used in the presoaking prior to acid- catalyzed organosolv treatment [23, 62]. Moreover, it was reported that presoaking of straw by the reaction solvent under mild conditions could improve the subsequent organoslv delignification particularly in high solid-to-liquid ratios. This may account for the higher swelling of the material and increased accessibility of solvents to the reaction sites [63]. However, the effects of combined pretreatment on softwood need to be confirmed.