Process for the treatment of lignocellulosic biomass

Abstract

Lignocellulosic biomass can be fractionated for the purpose of increasing cellulose purity in the pulp, increasing native lignin content of the isolated lignin, and improving cellulose hydrolysis, by performing the steps of: (a) extracting the biomass with an extracting liquid comprising at least 20 wt % of a first organic solvent at a temperature below 100° C.; (b) treating the extracted biomass with a treatment liquid comprising a second organic solvent selected from lower alcohols, ethers and ketones, optionally water and optionally an acid, at a temperature between 120° C. and 280° C., and, optionally: (c) subjecting a cellulose-enriched product stream resulting from step (b) to enzymatic hydrolysis. The first and second organic solvent may be different or the same; in particular they comprise ethanol or acetone.

Claims

1. A process for fractionating lignocellulosic biomass, comprising: (a) extracting the biomass, in fresh or dried form, with an extracting liquid comprising at least 50 wt % of a first organic solvent selected from lower alcohols and ketones at a temperature between 15° C. and 80° C., wherein the amount of extracting liquid is 0.5-20 L per kg biomass; (b) separating the extraction liquid from the extracted biomass, wherein the extraction liquid comprises lipophilic extractives selected from lipophilic proteins, fatty acids, triglycerides, waxes, terpenes, and resin acids; and (c) subjecting the extracted biomass from (b) to organosolv with a treatment liquid comprising a second organic solvent selected from lower alcohols, ethers and ketones, water and optionally an acid, at a temperature between 120° C. and 280° C., wherein a pulp comprising less lignin than the biomass and a liquor comprising lignin are obtained.

2. The process according to claim 1, wherein the first organic solvent comprises ethanol or acetone.

3. The process according to claim 2, wherein the first organic solvent comprises ethanol.

4. The process according to claim 1, wherein the extracting liquid comprises between 0 and 30 vol. % of water.

5. The process according to claim 1, wherein the extraction (a) is performed at a temperature between 30° C. and 75° C.

6. The process according to claim 1, wherein the treatment (c) is performed at a temperature between 125° C. and 250° C.

7. The process according to claim 1, wherein the treatment (c) is performed at a temperature between 120° C. and 170° C.

8. The process according to claim 1, wherein the first organic solvent is the same as the second organic solvent.

9. The process according to claim 8, wherein both the first and the second solvent comprise ethanol or acetone.

10. The process according to claim 1, wherein the extraction (a) is preceded by a pre-extraction in which the biomass is extracted with water at a temperature between 20° C. and 100° C., to obtain an extract comprising hydrophilic extractives selected from salts and water-soluble proteins.

11. The process according to claim 10, wherein the pre-extraction temperature is between 20° C. and 60° C.

12. The process according to claim 1, wherein the lignocellulosic biomass is selected from herbaceous biomass, softwood, hardwood and combinations thereof.

13. The process according to claim 12, wherein the second organic solvent is selected from lower alcohols and ethers.

14. The process according to claim 1, wherein the treatment liquid comprises: (i) 25-80 wt % of the second organic solvent, based on total weight of the treatment liquid; (ii) 20-75 wt % water, based on total weight of the treatment liquid; and (iii) at most 500 g of an acid, per kg dry weight of the biomass.

15. The process according to claim 1, further comprising: (d) subjecting the pulp to enzymatic hydrolysis.

16. A process for fractionation of herbaceous biomass, comprising: (a) extracting the biomass, in fresh or dried form, with an extracting liquid comprising at least 50 wt % of a first organic solvent selected from lower alcohols and ketones at a temperature between 15° C. and 80° C., wherein the amount of extracting liquid is 0.5-20 L per kg biomass; (b) separating the extraction liquid from the extracted biomass, wherein the extraction liquid comprises lipophilic extractives selected from lipophilic proteins, fatty acids, triglycerides, waxes, terpenes, and resin acids; and (c) subjecting the extracted biomass from (b) to organosolv with a treatment liquid comprising a second organic solvent selected from lower alcohols, ethers and ketones, and further comprises water and an acid, at a temperature between 120° C. and 170° C., and at a pH between 1 and 5, wherein a pulp comprising less lignin than the biomass and a liquor comprising lignin are obtained.

17. The process according to claim 16, further comprising: (d) subjecting the pulp to enzymatic hydrolysis.

18. The process according to claim 16, wherein the extraction (a) is preceded by a pre-extraction in which the biomass is extracted with water at a temperature between 20° C. and 100° C., and the hydrolysis (d) is performed in the presence of an aqueous extract obtained in pre-extraction.

19. The process according to claim 12, wherein the lignocellulosic biomass comprises herbaceous biomass.

20. The process according to claim 1, wherein the fresh or dried biomass is chopped or milled prior to (a).

21. The process according to claim 16, wherein the fresh or dried biomass is chopped or milled prior to (a).

22. The process according to claim 16, wherein the extraction (a) is performed at a temperature between 30° C. and 60° C.

23. The process according to claim 5, wherein the extraction (a) is performed at a temperature between 30° C. and 60° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 summarizes the results obtained from the enzymatic hydrolysis of example 1 (batch 1=aqueous extracted biomass, batch 2=aqueous and organic extracted biomass, control=non-extracted biomass).

(2) FIG. 2 summarizes the results obtained from the enzymatic hydrolysis of example 3.

EXAMPLES

(3) The following examples are intended to illustrate the invention, not to limit the scope.

Example 1

(Pre-)Extraction+Low Temperature Organosolv+Enzymatic Hydrolysis

(4) A first batch (batch 1) of wheat straw was pre-extracted using 10 L water per kg biomass. A second batch (batch 2) of wheat straw was first pre-extracted using the same conditions as for the first batch, and subsequently extracted with 10 L ethanol (containing about 4 wt % water) per kg biomass. Both pre-extraction and extraction were performed using a Soxhlet extractor. Both batches 1 and 2 of wheat straw were subjected to organosolv, as well as a batch of wheat straw, which has not undergone (pre-)extraction (control). Organosolv conditions employed: 140° C.; 120 min; solvent=ethanol/water (60/40 w/w); liquid/solid ratio=10 L/kg; 50 mM H.sub.2SO.sub.4 for the (pre-)extracted wheat straw; 60 mM H.sub.2SO.sub.4 for the control experiment. The increased H.sub.2SO.sub.4 concentration for the control experiment is to counteract the higher acid-neutralisation capacity of the mineral part of the original biomass, which is lowered during pre-extraction by (partial) removal of the mineral part.

(5) During organosolv of the control experiment, formation of balls of fatty acids and/or waxy material was observed, which hindered the fractionation of straw into the lignin-enriched liquor and the cellulose-enriched pulp, giving i.a. rise to a lower cellulose content of the pulp. These balls were not observed during organosolv of the second batch of extracted wheat straw.

(6) The negative effects of the extractives present in biomass on organosolv are reflected in the pulp yield, xylan hydrolysis and delignification degrees obtained in the organosolv process, as shown in table 2, and in the compositions of the cellulose pulp obtained during organosolv, as shown in table 3. Compositions are determined using the method described in W. J. J. Huijgen, A. T. Smit, P. J. de Wild, H. den Uil, BioResource Technology, 2012, 114, 389-398. The four components of the pulp given in Table 3 make up approximately 90 wt % of the pulp, and the remaining 10 wt % may include uronic acids, acetyl groups and extractives (non-structural components such as peptides, lipids, DNA, chlorophyll).

(7) TABLE-US-00002 TABLE 2 Pulp yield and fractionation degrees control batch 1 batch 2 Biomass recovery after extraction N.A. 91.7 wt % 87.6 wt % Pulp yield* 50.4 wt % 47.6 wt % 40.2 wt % Xylan hydrolysis** 78.5% 82.4% 92.8% Delignification*** 73.9% 70.8% 87.5% *Based on dry weight of the fresh biomass before (pre-)extraction. **Degree of xylan hydrolysis, based on amount of xylan present in the fresh biomass. ***Degree of lignin removal, based on lignin present in the fresh biomass.

(8) TABLE-US-00003 TABLE 3 Cellulose pulp composition (in wt % based on dry weight) component control batch 1 batch 2 glucan 63.8 68.7 78.9 xylan 8.5 7.4 3.5 lignin 9.2 10.8 5.5 ash 6.8 4.5 4.6

(9) Pulp yields are higher for the control experiment and the water pre-extracted batch 1, which are indicative of a less effective fractionation (i.e. more impurities are present in the cellulose pulp). This is confirmed in the degree of delignification and xylan hydrolysis, and in the compositions of the pulp, as shown in table 3. Ethanol extraction prior to organosolv results in a cellulose pulp comprising significantly increased amount of cellulose and significantly decreased amounts of lignin and xylan, when compared with cellulose pulp obtained by organosolv of biomass pre-extracted with water or not (pre-)extracted at all. Water pre-extraction leads to a significant reduction in ash content of the cellulose pulp. In addition, water pre-extraction and ethanol extraction give rise to more hydrolysis of the hemicellulose (xylan) during organosolv. Reduced amounts of lignin imply a more effective fractionation or delignification of the biomass (separation of lignin from cellulose).

(10) The cellulose-enriched pulp was subsequently subjected to enzymatic hydrolysis (conditions: 20 FPU per gram pulp of cellulase enzyme (ACCELLERASE® 1500, cellulase enzyme composition from DuPont Industrial Biosciences); 1.50 g pulp per 50.0 mL water buffered at pH 4.8; time=72 h). The progress of the enzymatic hydrolysis was monitored by determination of the glucose concentration at various intervals for 72 h. FIG. 1 summarizes the results obtained using the cellulose-enriched pulp, obtained by organosolv of batch 1, batch 2 and the control experiment. Water pre-extraction (batch 1) slightly increases the final glucose concentration, when compared to the control experiment, while ethanol extraction (batch 2) markedly increases the obtained final glucose concentration. Thus, employing organic extraction prior to organosolv increases the glucose concentration during enzymatic hydrolysis. Notably, in all experiments the final glucose concentration was already achieved after 24 h, which implies that the cellulose in the pulp obtained by the low temperature organosolv process of this example is readily accessible by the cellulase enzyme. Thus, the organosolv process is efficiently performed at reduced temperatures, such as at 140° C. or lower, preferably in combination with organic extraction prior to organosolv.

Example 2

(Pre-)Extraction+Organosolv

(11) Two batches (batches 1 and 3) of wheat straw (<1 cm) were pre-extracted using 10 L water per kg biomass and subsequently extracted with 10 L ethanol (containing about 4 wt % water) per kg biomass. Two other batches (batches 2 and 4) of wheat straw were not extracted (control). Both pre-extraction and extraction of batches 1 and 3 were performed in a counter-current stage-wise mode. All batches 1-4 of wheat straw were subjected to organosolv. Organosolv conditions employed for batches 1 and 2: low temperature organosolv at 140° C.; 90 min; solvent=ethanol/water (60/40 w/w); liquid/solid ratio=5 L/kg; 100 mM H.sub.2SO.sub.4 for the (pre-)extracted batch 1; 120 mM H.sub.2SO.sub.4 for the control batch 2. Organosolv conditions employed for batches 3 and 4: high temperature organosolv at 190° C.; 60 min; solvent=ethanol/water (60/40 w/w); liquid/solid ratio=5 L/kg; 30 mM H.sub.2SO.sub.4 for the (pre-)extracted batch 3; 60 mM H.sub.2SO.sub.4 for the control batch 2. The increased H.sub.2SO.sub.4 concentration for the control experiment is to counteract the higher acid-neutralisation capacity of the mineral part of the original biomass, which is lowered during pre-extraction by (partial) removal of the mineral part. In order to ensure proper wetting of the biomass when using a liquid-solid ratio of 5 L/kg, the wheat straw was placed in a porous metal basket and compressed with a mechanical press. The basket was placed in the autoclave below the level of the liquor. The liquid-solid ratio of 5 L/kg refers to the overall ratio in the autoclave.

(12) The negative effects of the extractives present in biomass on organosolv are reflected in the pulp yield and delignification degrees obtained in the organosolv process, as shown in table 4, and in the compositions of the cellulose pulp obtained during organosolv, as shown in table 5. Compositions are determined using the method described in W. J. J. Huijgen, A. T. Smit, P. J. de Wild, H. den Uil, BioResource Technology, 2012, 114, 389-398. The four components of the pulp given in Table 5 make up approximately 90 wt % of the pulp, and the remaining 10 wt % may include uronic acids, acetyl groups and extractives (non-structural components such as peptides, lipids, DNA, chlorophyll).

(13) TABLE-US-00004 TABLE 4 Pulp yield and fractionation degrees batch 1 batch 2 batch 3 batch 4 Biomass recovery after 90.6 wt % N.A. 90.6 wt % N.A. extraction Pulp yield* 41.7 wt % 48.7 wt % 39.0 wt % 46.4 wt % Delignification** 83.6% 76.0% 78.4% 72.6% Xylan hydrolysis*** 86.7% 72.3% 95.9% 79.5% lignin yields**** in pulp 16.4 wt % 24.0 wt % 21.6 wt % 27.4 wt % from liquor 71.2 wt % 84.0 wt % 72.4 wt % 90.2 wt % *Based on dry weight of the fresh biomass before (pre-)extraction. **Degree of lignin removal, based on lignin present in the fresh biomass. ***Degree of xylan hydrolysis based on amount of xylan present in the fresh biomass. ****Lignin mass balance, based on total weight of lignin present in the fresh biomass. Some residual lignin was found in the pulp (see also table 5). The remainder ended up in the liquor and was precipitated by addition of the liquor to 3 parts of water per part of liquor (w/w).

(14) TABLE-US-00005 TABLE 5 Cellulose pulp composition (in wt % based on dry weight) Component batch 1 batch 2 batch 3 batch 4 Glucan 71.2 65.0 76.5 64.8 Xylan 7.3 13.0 2.4 10.1 Lignin 6.3 7.9 8.9 9.4 Ash 4.6 6.3 4.3 7.5

(15) Both for low temperature as for high temperature organosolv, pulp yields are higher for the control experiments, which is indicative of a less effective fractionation (i.e. more impurities are present in the cellulose pulp). This is confirmed in the degree of delignification and xylan hydrolysis, which are higher for the (pre-)extracted batches, and in the compositions of the pulp, as shown in table 5. Extraction prior to organosolv results in a cellulose pulp comprising significantly increased amount of cellulose (based on measured glucose monomeric units) and significantly decreased amounts of lignin, xylan and ash, when compared with cellulose pulp obtained by organosolv of non-extracted wheat straw. Reduced amounts of lignin imply a more effective fractionation or delignification of the biomass (separation of lignin from cellulose). It is expected that the pulp obtained from batch 1 and 3 performs better in the enzymatic hydrolysis, compared to the pulp obtained form batch 2 and 4 respectively, in line with the results of example 1. The lignin mass balances in table 4 confirm that much less lignin ended up in the pulp when extraction was performed prior to organosolv. Notably, the total weight of the lignin obtained after organosolv exceeded 100 wt % for the control experiments, which indicates that the formation of “pseudo-lignin” (due to many reactions occurred during organosolv between lignin and for example non-structural components from the biomass and degradation products of e.g. xylan), and/or significant co-precipitation of lipophilic extractives such as waxes and fatty acids.

(16) Although the pulp obtained from extracted biomass using high temperature organosolv at 190° C. contained the highest amount of glucan, lower amounts of lignin were surprisingly found for the low temperature organosolv pulp, matching the highest degree of delignification for batch 1 in table 4. Thus, although it is generally established in the art that the effectiveness of the organosolv fractionation decreases with decreasing temperature, it is now surprisingly found that organosolv can also be performed at lower temperatures such as 140° C. with excellent fractionation, especially when an extraction step is performed prior to the organosolv step. The results of example 2 also demonstrate that the process according to the invention provides excellent fractionation even when the more challenging condition of using 5 L treatment liquid per kg biomass (liquid/solid ratio=5 L/kg) is applied. It is generally accepted that the extent of fractionation decreases but the industrial applicability increases when the amount of treatment liquid per kg biomass decreases. The process according to the invention thus provides effective organosolv fractionation even at the reduced temperature of 140° C. and at a liquid/solid ratio of 5 L treatment liquid per kg biomass.

Example 3

(Pre-)Extraction+Low Temperature Organosolv+Enzymatic Hydrolysis

(17) Wheat straw was chopped into pieces of about 1 cm length, and was divided into eight batches which received different treatments as summarised in Table 6. Pre-extraction was performed on batches 2, 5 and 7, which involved extraction with 10 L water per kg biomass, and subsequently with 10 L ethanol or acetone per kg of the original biomass prior to aqueous extraction. Both pre-extraction and extraction of batches 1 and 3 were performed in a counter-current stage-wise mode. Batches 1-8 were subsequently subjected to organosolv at the indicated temperature, using the solvent system and treatment time as given in Table 6. The liquid/solid ratio was 10 L per kg biomass. Sulfuric acid was added to the treatment liquid of batches 1-7. The increased H.sub.2SO.sub.4 concentration for the batches which did not undergo pre-extraction (1, 3, 4, 6) was applied to counteract the higher acid-neutralisation capacity of the mineral part of the original biomass, which is otherwise lowered during pre-extraction by (partial) removal of the mineral part. For batch 8, no acid was added, and organosolv was performed auto-catalytically at a temperature of 205° C. Batch 1 is identical to the control of example 1, and batch 2 is identical to batch 2 of example 1.

(18) TABLE-US-00006 TABLE 6 Pre-extraction and treatment conditions of wheat straw organosolv T H.sub.2SO.sub.4 batch pre-extraction t (min) (° C.) solvent system (w/w) (mM) 1 no 120 140 ethanol/water (60/40) 60 2 water; ethanol 120 140 ethanol/water (60/40) 50 3 no 120 140 acetone/water (50/50) 60 4 no 60 140 acetone/water (50/50) 60 5 water; acetone 60 140 acetone/water (50/50) 50 6 no 60 140 acetone/water (60/40) 60 7 water; acetone 60 140 acetone/water (60/40) 50 8 no 60 205 acetone/water (50/50) 0

(19) During organosolv with ethanol/water as treatment liquid (batch 1), the formation of balls of fatty acids and/or waxy material was observed, which hindered the fractionation of straw into the lignin-enriched liquor and the cellulose-enriched pulp, giving i.a. rise to a lower glucan concentration in the pulp. Only when pre-extracted biomass was subjected to ethanol/water organosolv (batch 2), no waxy balls were observed. In batches 3-7, wherein acetone/water was used as treatment liquid, no such balls were observed, even without pre-extraction.

(20) The results of the organosolv regarding the pulp are given in Table 7. Pulp yields, delignification percentages and pulp compositions are acceptable for all experiments. Surprisingly, the additional step of pre-extracting the biomass gave a greater extent of delignification and higher glucan purity of the pulp compared to non-pre-extracted biomass. This applies both to the ethanol organosolv (batch 2 vs. batch 1) and to the acetone organosolv (batch 5 vs. batch 4 and batch 7 vs. batch 6). Reducing the reaction time from 120 to 60 minutes of acetone organosolv (batches 3 vs. 4) gave surprisingly similar results in terms of delignification and pulp compositions, while pulp yield increases. High temperature organosolv (batch 8) gave similar results in terms of pulp composition and delignification, indicating that reducing the temperature does not negatively affect the performance of the organosolv fractionation.

(21) The form of the lignin (hydroxyl content) obtained as precipitate from the liquor obtained after organosolv is also given in Table 7. First of all, the lignin obtained by low temperature organosolv with acetone shows an increased content of hydroxyl groups, when compared to low temperature organosolv with ethanol as solvent, indicative of more native lignin and reduced formation of pseudo-lignins. In addition, the hydroxyl content of the lignin is markedly increased when compared to lignin obtained with high temperature organosolv at about 200° C., which is about 4 mmol/g lignin. The hydroxyl content of the lignin was determined via the wet chemical method as described by Zakis et al., “Functional analysis of lignins and their derivatives”, TAPPI Press, Atlanta, 1994, page 94.

(22) Compositions were determined using the method described in W. J. J. Huijgen, A. T. Smit, P. J. de Wild, H. den Uil, BioResource Technology, 2012, 114, 389-398. The four components of the pulp given in Table 7 make up approximately 90 wt % of the pulp, and the remaining 10 wt % may include uronic acids, acetyl groups and extractives (non-structural components such as peptides, lipids, DNA, chlorophyll).

(23) TABLE-US-00007 TABLE 7 Pulp yield and composition, hydroxyl content of lignin pulp pulp composition yield delignification (wt %)**** batch (wt %)* (%)** OH*** glucan xylan lignin ash 1 50.4 73.9 6.0 63.8 8.5 9.2 6.8 2 40.2 87.5 6.4 78.9 3.5 5.5 4.6 3 43.0 73.1 6.9 73.9 3.1 11.0 3.2 4 50.1 69.8 6.7 68.6 6.5 10.6 5.3 5 43.0 82.1 7.9 75.8 3.9 7.3 6.0 6 48.6 74.2 5.7 69.1 5.6 9.4 6.5 7 42.0 84.3 7.0 77.8 3.2 6.6 4.9 8 48.7 78.6 nd 65.4 7.8 7.0 11.1 *Based on dry weight of the fresh biomass, before pre-extraction. **Degree of lignin removal, based on lignin present in the fresh biomass. ***OH content in mmol per g lignin; nd = not determined. ****Based on dry weight of cellulose pulp.

(24) Some xylan and glucan degradation products were detected in the lignin-containing liquor. The products obtained from xylan and glucan are given in Table 8, in xylose and glucose equivalents respectively. In general, more residual xylan is found in the pulp when no pre-extraction is performed, when ethanol is used instead of acetone (batches 1 and 2), and when organosolv is performed autocatalytically at high temperature (batch 8). The hydrolysis and degradation products of xylan are found in the liquor. Importantly, the yield of monomeric xylose is greatly increased when ethanol is replaced by acetone, and no ethyl xylosides are formed using low temperature organosolv with acetone. Some furfural is detected in all liquors. Most, if not all of the remaining hemicellulose will have been converted to soluble xylooligosaccharides (XOS), which end up in the liquor. Regarding the glucan products, the major difference between ethanol and acetone as organic solvent is the formation of glucosides in the hydroxylic solvent, while no ethylglucosides are formed using the non-hydroxylic solvent.

(25) The most significant effect associated with reducing the temperature of the organosolv reaction from 205° C. to 140° C. is the increase in xylose yield, while the xylan hydrolysis degree remains more or less the same. This indicates that xylan degrades beyond its monomeric sugars at high temperature into undesirable by-products which have not been measured. Likewise, glucan is degraded to some extent at high temperature, and the degradation products are not retrieved as glucose monomers or hydroxymethylfuran (HMF). Thus, the glucose monomers that are formed during organosolv degrade into undesirable by-products at 205° C.

(26) TABLE-US-00008 TABLE 8 Distribution of xylan and glucan products xylan (%)* glucan (%)** batch xylan Xyl furfural EX glucan Glc HMF EG 1 21.5 32.9 7.7 38.1 90.7 2.5 bdl 4.6 2 7.2 36.9 11.1 46.6 89.5 3.0 bdl 5.4 3 6.8 81.3 15.4 — 89.8 7.3 0.8 — 4 16.4 73.2 7.3 — 97.2 5.6 0.5 — 5 8.4 76.7 9.7 — 92.1 5.6 0.3 — 6 13.6 71.9 9.4 — 94.8 5.7 0.5 — 7 6.7 70.2 12.6 — 92.4 6.0 0.5 — 8 17.7 0.6 3.8 — 92.0 bdl 0.2 — *In xylose equivalents; moles of product based on total moles of xylose monomeric units present in xylan in the fresh biomass, before pre-extraction. Total percentages above 100% result from measuring inaccuracies. Xylan is found in the pulp, the rest in the liquor. Xyl = xylose; EX = ethyl xylosides; **In glucose equivalents; moles of product based on total moles of glucose monomeric units present in glucan in the fresh biomass, before pre-extraction. Total percentages above 100% result from measuring inaccuracies. Glucan is found in the pulp, the rest in the liquor. Glc = glucose; HMF = 5-hydroxymethyl-furfural; EG = ethyl-glucosides; bdl = below detection limit.

(27) The cellulose-enriched pulp obtained by organosolv fractionation of each of the batches was subsequently subjected to enzymatic hydrolysis. Conditions: 10 FPU per gram pulp (for batches 1 and 2: 20 FPU/g, for batch 8: 38 FPU/g) of cellulase enzyme (ACCELLERASE® 1500 (batches 1-7) or ACCELLERASE® 1000 (batch 8), cellulase enzyme composition from DuPont Industrial Biosciences); 1.50 g pulp (dry weight) per 50.0 mL water buffered at pH 4.8; time=72 h. The progress of the enzymatic hydrolysis was monitored by determination of the glucose yield (as wt % based on total dry weight of the pulp at t=0 h) at various intervals up to t=72 h. FIG. 2 summarises the results obtained using the cellulose-enriched pulp obtained by organosolv of batches 1-8. Maximum glucose conversion values for batches 1, 2 and 8 were obtained after 24 h, in view of the increased enzyme load. The cellulose pulp obtained by organosolv at reduced temperatures is readily hydrolysed with a relatively low enzyme load of 10 FPU/g. Batches 4-7, subjected to low temperature organosolv with acetone for 60 minutes, were hydrolysed more or less similarly to batch 3 which was subjected to organosolv for 120 minutes. From a cost-effectiveness point of view, it is beneficial to perform the organosolv reaction for a shorter period of time, for which the process according to the invention is well suited.

(28) The pulp obtained with high temperature organosolv gave a lower yield in glucose, especially since the final conversion was already obtained after 24 h, while the other batches did not reach the final glucose conversion at the end of the measurement at t=72 h. The pulp obtained by ethanol organosolv of non-pre-extracted biomass (batch 1) gave slightly lower glucose yield than the pulp obtained by acetone organosolv of batches 3, 5 and 7, especially in view of the expected further conversion of these batches after t=72 h. The pulp obtained from ethanol organosolv of pre-extracted biomass (batch 2) gave the highest glucose yield, based on total weight of the pulp, which is due to its high glucan content (see Table 7) and a higher enzyme activity. The total glucose yield based on the glucan present in the fresh biomass is more or less similar for all experiments. Thus, it is concluded that the organosolv process is efficiently performed at reduced temperatures, such as at 140° C. or lower, using acetone as organic solvent, and that the cellulose pulps obtained therewith are efficiently hydrolysed to glucose.