From the investigations presented above it has been concluded that the max­imum yields of mannose, the main hemicellulose sugar in softwood, and of glucose are not obtained at the same degree of severity. The optimal yield of mannose is obtained at a lower severity than that required for maximum di­gestibility of the cellulose in the subsequent enzymatic hydrolysis step. This suggests two-stage steam pretreatment, in which the first stage is performed at low severity to hydrolyse the hemicellulose, and a second stage, at a higher degree of severity, in which the solid material from the first step is pretreated again [82].

Although there are several studies on two-stage acid hydrolysis of soft­wood, the number of studies on two-stage steam pretreatment is scarcer. Soderstrom et al. [85-87] performed a thorough investigation on the two — stage pretreatment of spruce using either SO2 impregnation or H2SO4 im­pregnation in both steps, as well as H2SO4 in the first stage and SO2 in the second. The highest sugar yields were achieved for two-step pretreat­ment with either SO2 impregnation or H2SO4 impregnation in both steps (see Fig. 2). A wide range of pretreatment conditions resulted in similar sugar yields of about 50 g per 100 g raw material.

The highest sugar yield was 51.7 g per 100 g, corresponding to 80% of the theoretical, obtained for pretreatment conditions of 190 °C for 2 min and 210 °C for 5 min. This yield (in %) is slightly lower than that reported by Nguyen et al. [ 14]. However, the amount of sugar obtained expressed as grams per 100 g dry raw material is higher. Ngyuen et al. stated that they obtained a sugar yield of 82%, which, in their case, corresponds to 46 g/100 g dry raw

material. They used a cellulase activity of 60 FPU/g cellulose, which is more than twice the amount that was used in the study shown in Fig. 2. The max­imum overall sugar yield obtained with two-step pretreatment using H2SO4 in both stages was only slightly lower, 77% of theoretical.

Besides overall sugar yield it is also of importance to investigate the fer — mentability of the pretreated materials. Impregnation with dilute H2SO4 fol­lowed by pretreatment at a high combined severity (i. e. high temperature and/or long residence time) resulted in materials that were not fermentable. Impregnation with SO2, however, was successful in creating fermentable ma­terials for all investigated pretreatment severities.

The two-step pretreatment results in a higher ethanol yield than does the one-step pretreatment, and it has also the advantage of lower requirement of enzymes and water in the SSF step. Major drawbacks are, however, the higher capital cost and the higher energy consumption. In a study by Wingren et al. [90] the overall ethanol production cost was shown to be very much de­pendent on the way the two pretreatment steps are performed, especially if the pressure is released or not between the steps, and also on the dry matter (WIS) content in the second step. The lowest cost estimated for the two-stage process, 3.90 SEK/L, which was about 6% lower than that for the one-stage process, requires a high ethanol yield, high concentration of WIS in the fil­ter cake between the steps, and that the sugars being fed to the second step will not become degraded. The higher yield has been demonstrated experi­mentally, but the two other assumptions still need to be verified on the pilot scale.

5

Conclusions

In conclusion, a large number of pretreatment methods have been investi­gated and developed during the last 10 years, resulting in high recovery of sugars and rather high overall ethanol yields. However, most of the results were obtained in studies using batch-operating equipment on a rather small scale. Enzymatic hydrolysis has also, in most cases, been assessed at low sub­strate concentration.

One problem with the data produced so far is the difficulty in comparing methods, as the assessment is performed in different ways. In most cases the pretreatment is not assessed under realistic process conditions. The whole process must be considered as the various pretreatment methods give dif­ferent types of materials: hemicellulose sugars can be obtained either in the liquid as monomer or oligomer sugars, or in the solid material to various ex­tents; lignin can be either in the liquid or remain in the solid; the composition and amount/concentration of possible inhibitory compounds also vary. This will affect how the enzymatic hydrolysis should be performed (e. g. with or without hemicellulases), how the lignin is recovered and also the use of the lignin co-product.

For agricultural residues a large number of pretreatment methods result in high sugar yields while for wood, and especially softwood, the number of feasible methods is smaller. Acid hydrolysis and steam pretreatment with acid catalyst seem to be the methods that can be used for all types of raw ma­terials, but the drawback is the high equipment cost and the formation of inhibitors. This requires further improvement and also a better integration with the enzymatic hydrolysis development, as improved enzyme mixtures may lead to less severe pretreatment conditions and thereby lower cost and reduce formation of inhibitory compounds.

To verify the technology the next step is to implement all of these improve­ments in a pilot-scale process with all steps integrated into a continuous pilot plant. This will provide better data for assessment and for scale-up to a demo­or full-scale process. It will also give better information on how various pre­treatment conditions will affect all the other process steps, i. e. enzymatic hydrolysis, fermentation, downstream processing and wastewater treatment, as well as product and co-product quality.