MODIFIED LIGNIN AND SEPARATION METHODS

Abstract

A method of processing a lignocellulosic feedstock includes heating the lignocellulosic feedstock in a solvent mixture comprising an acid and at least 80% by volume of n-butanol to produce a reaction mixture including a butylated lignin. A butylated lignin product wherein at least 85% of the β-O-4 linkages include n-butyl ether groups can be prepared. Cellulose and butylated hemicellulose products can also be obtained.

Claims

1. A method of processing a lignocellulosic feedstock, the method comprising: heating the lignocellulosic feedstock in in a solvent mixture comprising an acid and at least 80% by volume of n-butanol to produce a reaction mixture including a butylated lignin.

2. The method according to claim 1 wherein the solvent mixture comprises at least 80% by volume of n-butanol and an aqueous acid.

3. The method according to claim 1, wherein heating is continued until the conversion to butyl ethers of the benzylic hydroxyl functional groups of the β-O-4 linkages on the lignin is at least 85%, as determined by .sup.1H—.sup.13C—HSQC NMR analysis.

4. The method according to claim 1, wherein the acid employed in the solvent mixture is selected from the group consisting of hydrochloric acid (HCl), hydroiodic acid (HI), hydrobromic acid (HBr), perchloric acid (HClO.sub.4), nitric acid (HNO.sub.3), sulfuric acid (H.sub.2SO.sub.4) and mixtures thereof.

5. The method according to claim 1, wherein the concentration of acid in the solvent mixture is from 0.05 M to 3 M.

6. The method according to claim 1, wherein the lignocellulosic feedstock is heated in a solvent mixture comprising only n-butanol, water and the acid.

7. The method according to claim 1, wherein the lignocellulosic feedstock is biomass from trees.

8. The method according to claim 1, wherein the lignocellulosic feedstock is dried to less than 20% moisture.

9. The method according to claim 1, wherein the heating is carried out at reflux and at ambient pressure.

10. The method according to claim 1, wherein after the heating step the reaction mixture is filtered to remove a solid cellulose product leaving a filtrate containing butylated lignin.

11. The method according to claim 10 wherein a butylated lignin product is obtained from the filtrate.

12. The method according to claim 11 wherein the butylated lignin product is obtained by concentrating the filtrate, mixing the residue with a water miscible solvent, and adding the resulting mixture to water, to precipitate the butylated lignin product.

13. The method according to claim 12 wherein acetone, or an acetone/water mixture is used as the water miscible solvent.

14. The method according to claim 13 wherein sodium sulfate is used to aid flocculation of the butylated lignin precipitate.

15. The method according to claim 12, wherein a hemicellulose product is obtained from the filtrate remaining after removal of the precipitated butylated lignin product.

16. A reaction mixture comprising a butylated lignin obtainable from or obtained from a method according to claim 1.

17. A butylated lignin product wherein at least 85% of the β-O-4 linkages include n-butyl ether groups as determined by .sup.1H—.sup.13C—HSQC NMR analysis.

18. The butylated lignin product of claim 17 wherein there are at least 20 β-O-4 linkages per 100 C9 units as determined by .sup.1H—.sup.13C—HSQC NMR.

19. A butylated lignin product wherein there are at least 20 β-O-4 linkages per 100 C9 units as determined by .sup.1H-.sup.13C—HSQC NMR.

20. A butylated lignin product obtainable from or obtained from a method according to claim 11.

21. A cellulose product obtainable from or obtained from a method according to claim 10.

22. A butylated hemicellulose product obtainable from or obtained from a method according to claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIGS. 1aand 1b illustrate results from NMR experiments showing beech wood and pulp obtained from beech wood;

[0059] FIG. 2 shows graphically, results from experiments in enzymatic hydrolysis of beech pulp;

[0060] FIG. 3 shows graphically, results from experiments to depolymerise lignin derivatives; and

[0061] FIGS. 4a and 4b show a .sup.1H—.sup.13C—HSQC NMR analysis of a butylated lignin obtained from beech wood.

DESCRIPTION OF SOME PREFERRED EMBODIMENTS AND EXPERIMENTAL RESULTS

Pre-Treatment of Lignocellulosic Biomass to Provide Pulp, Lignin and Hemicellulose Products

[0062] Lignocellulosic biomass may be treated with an acidic medium as a pre-treatment. The objective is to dissolve lignin and hemicelluloses, to allow isolation of a pulp comprising mostly cellulose, for further processing. Lignin and hemicellulose products may also be obtained.

[0063] Previous studies by Bauer et al. have demonstrated that 95% ethanol containing 0.2 M HCl at reflux could extract up to 50% of the initial lignin in Miscanthus gigantheus biomass, with little xylan or glucan removal. (Ref 1)

[0064] As delignification is known to improve the susceptibility of cellulosic pulp to enzymatic hydrolysis, the pre-treatment of a range of lignocellulosic materials under similar conditions was investigated.

[0065] Beech (hardwood), Douglas fir (softwood) and walnut shell (endocarp) substrates were examined as being complementary to the previously studied herbaceous Miscanthus giganteus. These woody biomass sources are potentially important biorefinery feedstocks which possess a high energy density, do not compete for agricultural land and can be harvested year round.

Materials

[0066] The lignocellulosic feedstocks used throughout were obtained from commercial suppliers: beech and Douglas fir sawdusts were obtained from Hot Smoked (Useful Stuff Ltd), UK and the ground walnut shell was a kind donation from A. & E. Connock, UK. Commercially available rice husks were obtained from a brewing supplies company. All materials were used as received.

Lignocellulosic Feedstock Processing—Beech Sawdust, Douglas Fir Sawdust, and Ground Walnut Shell—as Woody Substrate Examples

[0067] Biomass samples were weighed into a round bottom flask followed by the addition of alcoholic solvent and aqueous 4 M HCl. The ratio of alcohol to aqueous acid was fixed at 95:5 and the biomass loading was kept constant at 1 g per 10 mL of solvent. The samples were then heated at reflux with stirring for 6 hours under an ambient atmosphere for both ethanol and n-butanol treatments. Results for ethanol/HCl treatment in a closed vessel at 110° C. are also shown. The samples were then allowed to cool and filtered. The solid residue (pulp) was washed with ethanol (10 mL/g), air dried and then further dried under vacuum at 70° C. for 14 hours. The pulp was then analysed as further described below The separated liquor was then concentrated in vacuo, taken up in acetone (ca. 1.5 ml/g of biomass used, 1 vol) and precipitated by dropwise addition to rapidly stirring water (20 vols).

[0068] The ethanol lignins formed easily filterable precipitates whilst n-butanol lignins benefited from the addition of a small volume of sat. aqueous Na.sub.2SO.sub.4 to flocculate the lignins to an easily filterable form. The crude lignins were then collected by filtration, washed with water and then dried under vacuum over anhydrous CaCl.sub.2 for 16 hours. The lignin products were analysed as discussed below.

[0069] A hem icellulose fraction was isolated by concentration of the water fraction following precipitation of the lignin product. The resulting viscous oil was then dried in a vacuum oven at 70° C. for 24 hours. The hemicellulose products were analysed as discussed below.

Lignocellulosic Feedstock Processing—Rice Husk—as Grassy Substrate Example

[0070] The process generally follows that used for the woody feed stocks. To unextracted biomass (commercially available rice husks) was added n-butanol: 0.2 M (final concentration) HCl (95:5, 10 mL/g). The mixture was heated at a gentle reflux (ca. 100° C.) for 6 h and filtered when cooled. The residual pulp was washed with a solution of acetone:H.sub.2O (9:1 v/v), typically employing from 10 to 100 mL solvent per gram of pulp, and the combined filtrates were concentrated in vacuo to yield a gum-like residue. The residue was dissolved in the minimum amount of acetone: H.sub.2O (9:1, 5 mL per gram of residue) and added dropwise to H.sub.2O (50 mL per gram of residue) to precipitate lignin. This precipitation therefore uses a lower solvent to water ratio (1:10 of the acetone/water (dissolving solvent) to water (precipitating solvent) rather than 1:20 as employed in the experiments with woody substrates.

[0071] The crude precipitate was collected by filtration and dried in a vacuum oven at 50° C. for 16 h. The dried precipitate was dissolved in acetone: MeOH (9:1, 5 mL/g) and added dropwise to petroleum ether (50 mL/g). The precipitate was collected by filtration and dried in a vacuum oven at 50° C. for 16 h to give a brown powder.

[0072] When the process was scaled from 4 g rice husk to 400 g and 4 kg scales, the work up procedures were modified to facilitate recovery of hemicelluloses and provide easier recovery of the butylated lignin product. Precipitated lignin was removed by centrifugation. The aqueous filtrate was then added to a 0.5 v/v quantity of ethyl acetate and the solvents separated after mixing and allowing the layers to settle.

[0073] A high proportion of the modified hemicellulose sugars ˜50%) were removed in the ethyl acetate layer. If desired, the remaining hemicellulose sugars in the aqueous filtrate layer can be recovered by further extraction procedures and/or concentration. Concentration can be in vacuo or by blowing air or other gas through the mixture.

Results

[0074] Results for the four feedstocks examined are summarised in Table 1, below.

TABLE-US-00001 TABLE 1 Results of high alcohol biomass pre-treatments. Cellulose: Lignin Hemicellulose: Temp Pulp Yield Yield Entry Biomass Lignin (wt %) Solvent (° C.) (wt %) (wt %)  1  Beech 39:20:25 (Ref 2) ethanol reflux 85 4.1  2* butanol reflux 46 21  3  ethanol 110 49 10.6  4  Walnut 21:19:33 (Ref 2) ethanol reflux 86 6.1  5* butanol reflux 35 32  6  ethanol 110 46 19  7  Douglas 50:18:28 (Ref 2) ethanol reflux 89 5.5 Fir  8* butanol reflux 57 17  9  ethanol 110 79 5.5 10  Rice 59:27:14 butanol reflux 58 14.5 Husk Conditions: 10 mL/g loading, 0.2 M HCl, 95:5 ROH/H.sub.2O, 6 hours. *average of 3 repeat extractions

[0075] Solvent mixtures were made by mixing 95% of the alcohol (ethanol or n-butanol) with 5% 4M aqueous HCl to give a mixture with a 0,2 M HCl content.

[0076] 5

[0077] Treatment using 95% ethanol containing 0.2 M HCl at reflux for 6 hours with these substrates proved disappointing (Table 1, Entries 1, 4 and 7). Isolated yields of lignin were low in all cases accounting for only approximately 16-20% of the Klason lignin and pulp yields were correspondingly high. The Klason lignin content of a biomass is a pulp industry standard method for determining lignin involving a sulfuric acid treatment where the insoluble residue after the process is complete is defined as the Klason lignin content.

[0078] These results are consistent with the greater recalcitrance of woody biomass compared to herbaceous materials and highlights the more challenging nature of such feedstocks.

[0079] When the ethanol was replaced by n-butanol the isolated yields of lignin improved dramatically and unexpectedly whilst the corresponding pulp yields fell significantly (Table 1, Entries 2, 5, 8). Reaction was carried out with a mixture of 95% n-butanol and 5% 4 M aqueous HCl by volume. (i.e. a 0.2 M HCl strength in the mixture), reflux for 6 hours.

[0080] The isolated yield of lignin from beech and walnut shell was significantly higher than from Douglas fir based on the previously reported Klason lignin content of these feedstocks, accounting for 84, 97 and 61 wt % of the original lignin respectively. This likely reflects the known greater recalcitrance of softwood feedstocks.

[0081] In addition the lower pulp yields obtained in these pre-treatments indicate that efficient solubilisation of hemicelluloses occurs during the process. Although similar pulp yields and intermediate lignin yields could be obtained using ethanol at elevated temperatures in a sealed system (Table 1, Entries 3, 6 and 9), this introduces the added complexity of having to run the process under elevated pressures.

[0082] In proposed structural models of woody biomass the hemicelluloses and lignin form an intimate matrix (lignin-carbohydrate complex) around the cellulose fibres. Without wishing to be bound by theory, it seems likely that the efficient solubilisation of the hemicelluloses is an important step during the pre-treatment process to allow for high levels of delignification. Without it, solvent access to lignified tissues may be blocked by insoluble carbohydrates preventing lignin solubilisation.

[0083] Significant defibration of the biomass samples was only observed after either n-butanol or high temperature ethanol pre-treatments. This is consistent with the loss in structural integrity of the wood fibres after removal of both the lignin and hemicelluloses which leads to mechanical deconstruction of the wood particles under the stirring condition of the extractions.

Pulp Characterisation

[0084] In order to better understand the compositional changes occurring during the pulping process a 2D HSQC NMR study was undertaken. For this analysis beech wood was selected as it has previously been identified as a potential European biorefinery feedstock and has been used in a number of other pre-treatment studies.

[0085] An AcBr/AcOH reagent mixture was used to solubilise and derivitize all the cell wall components prior to NMR analysis without the need for extensive planetary ball milling. This mixture is well known to solubilise lignocellulosic materials and is usually used for the determination of lignin content or DFRC analysis (Derivatization Followed by Reductive Cleavage).

[0086] For this analysis beech wood was selected as a model substrate as it has previously been identified as a potential European biorefinery feedstock and has been used in a number of other pre-treatment studies. The results of this study are presented visually in FIGS. 1a, 1b. FIG. 1ashows .sup.1H—.sup.13C—HSQC NMR analysis of A) derivatised whole beech cell walls and B) derivatised pulp obtained from a butanol based treatment of beech wood. FIG. 1b shows the structural units identified by their .sup.1H—.sup.13C—HSQC NMR signals in FIG. 1a. Thus for example LigS.sub.2/6 refers to the C—H signals from LigS positions 2 and 6.

[0087] As can be seen in B) of FIG. 1a the lignin signals (Lig) are largely absent in the derivatised pulp product. The positions where essentially absent lignin signals would appear are marked by dashed ellipses. Note that the signals LigS.sub.2/6 shown in B) are magnified four times as indicated on the figure by the magnifying glass symbol and ‘×4’.

[0088] A comparison of the 2D HSQC NMR spectra for the whole beech wood against spectra for the pulp obtained from the n-butanol treatment procedure of Table 1 above was made. As indicated in FIG. 1a semi-quantitative analysis indicates reductions of 97% for lignin and 88% for xylan derived components relative to the cellulose derivatives, when comparing the pulp to the whole wood. This is consistent with the observed pulp yields.

[0089] The quality of the pulp obtained was further examined by enzymatic hydrolysis. Enzymatic hydrolysis was carried out at 50° C. as follows: To a glass vial was added substrate (250 mg), pH 5.5 acetate buffer (5 mL, 50 mM containing 0.02 wt % NaN.sub.3) and CTec 2 cellulase preparation (22 FPU). Shaken hydrolyses were conducted using a Buchi Syncore parallel synthesis reactor set at 300 rpm. Stirred hydrolyses were performed using a magnetic stirrer/hotplate. 100 μL samples were taken at 24, 48, 72 and 120 hours and the amount of reducing sugars released was determined using the DNS method relative to a glucose calibration curve.

[0090] The enzymatic hydrolysis of beech pulp obtained by the n-butanol treatment is shown graphically in FIG. 2.

[0091] At a 5 wt % consistency and an enzyme loading of 22 FPU (filter paper units) per gram of Cellic® CTec2 (Novozyme, Denmark) the crude pulp (labelled SH—CP) gave a reducing sugar yield of 77 g per 100 g after 120 hours. The rate of hydrolysis was significantly improved by pre-milling the pulp (labelled SH-G) to increase the enzyme accessible surface area giving a reducing sugar yield of 56 g per 100 g after 24 hours compared to 40 g per 100 g for the crude pulp, although final sugar yields only marginally improved to 82 g per 100 g. Combining pre-milling with additional mechanical stirring during the hydrolysis (labelled ST-G) further improved the rate (82 g per 100 g after 24 hours) and final sugar yields (94 g per 100 g) compared to standard shaking. The beneficial effect of stirring likely originates from further mechanical defibration of the pulp during the hydrolysis. This data clearly shows that n-butanol derived pulps are highly digestible and therefore potentially relevant for biorefineries where the released monomeric sugars could be subjected to fermentation to bioethanol/butanol or used as starting materials for chemical synthesis.

Lignin Characterisation

[0092] The lignins obtained were characterised by gel permeation chromatography (GPC) and .sup.1H—.sup.13C HSQC NMR which gives detailed structural and compositional information. The results of these analyses are summarised in Table 2 below. For comparison purposes a beech technical ethanosolv lignin prepared using current ethanol organosolv pre-treatment conditions (Ref 3) was also included (Table 2, Entry 1).

TABLE-US-00002 TABLE 2 2D HSQC NMR and GPC analysis of lignins. Aromatics Linkages (per 100 C.sub.9 units) (%) β-O-4- Σ β-aryl GPC (Da) Entry Lignin S G H β-O-4 OR β-β β-5 % OR ether Mn Mw PD Beech  1  Technical 76  24 0 7 10 3 4 57 17 976 2069 2.1  2  EtOH 61  39 0 22  41 7 4 65 63 1086  2767 2.5  3* n-BuOH 81  19 0 3 49 4 1 95 51 975 2483 2.5  4  EtOH 81  19 0 2 41 5 1 96 43 927 1846 2.0 (110° C.) Walnut shell  5  EtOH 57  33 10  17  41 5 8 71 58 880 2536 2.9  6* n-BuOH 74  25 1 3 49 3 2 97 52 1018  2717 2.7  7  EtOH 74  24 2 2 49 5 3 95 51 959 2060 2.1 (110° C.) Douglas Fir  8  EtOH  0 100 0 9 43 2 13  84 52 886 2200 2.5  9* n-BuOH  0 100 0 0 51 2 5 100  51 861 2628 2.5 10  EtOH  0 100 0 2 49 2 6 96 51 918 2341 2.6 (110° C.) Rice husk 11  n-BuOH 17  56 27 3 45 1 3 94 48 1865  3221 1.7 *average of 3 repeat extractions.

[0093] In the table the aromatic groups of the lignins are designated S, G and H as usual: denoting aromatics derived from sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol respectively. All the lignins isolated in this study were found to have undergone structural modification. Specifically, the α-OH group in the most abundant β-O-4 linkage became substituted by the alcohol solvent β-O-4-OR where R is Et or n-Bu).

[0094] The % OR figure gives a measure of the extent of etherification of the β-O-4 linkages in the lignin. It is calculated as the % of the total number of β-O-4 linkages per 100 C.sub.9 units that are etherified i.e. 100×β-O-4-OR/[β-O-4 +13-O-4-OR]).

[0095] The extent of this substitution depends both on the chemical composition of the lignin, with G units being more reactive than S (Table 2, Entries 2 and 6 vs 9), and on the severity of the pulping conditions, with higher temperatures resulting in more extensive substitution (Table 2, Entry 2 vs 3). This modification is clearly observable in the 2D HSQC NMR spectra obtained for these lignins. See FIG. 4 which shows a butylated beech wood derived lignin and is discussed further below under the heading “Analysis of Lignins”

[0096] Other than extensive α-OH substitution, the obtained lignins are remarkably free from further degradation, particularly compared to the technical lignin (Table 2, Entry 1). A semi-quantitative assessment of the amount of β-aryl ethers revealed that in all cases large amounts of this type of linkage are retained (43-63 per 100 C.sub.9 units compared to 17 for the technical lignin). (The terminology “per 100 C.sub.9 units” refers to the C.sub.9 units (nine carbon groups—6 aromatic and 3 side chain) that make up a lignin.)

[0097] As most selective lignin depolymerisation processes target cleavage of the β-aryl ether bonds this is a good measure of the potential of these lignins to undergo selective depolymerisation to aromatic chemicals. It was also observed that the S:G ratio increases under more severe pre-treatment conditions which also correlates with a decrease in β-5 linkages whilst β-β linkages appear to remain quite constant.

[0098] GPC analysis revealed that all the lignins had relatively similar apparent molecular weights and quite similar polydispersities. However, it should be noted that such analysis does not adjust for differences in the degree and nature of α-substitution. In the case of rice husks the lignin has a higher molecular weight, although the polydispersity is lower.

Lignin Valorisation

De-Etherification

[0099] Although the relatively high β-aryl ether content of the produced lignin products make them attractive targets for depolymerisation to aromatic chemicals, most current methods for this have been developed using ‘native like’ lignins, i.e. with α-OH groups in the β-O-4 linkages.

[0100] The α-etherification of lignins treated with alcohols is reversible, enabling the possibility of transforming the modified lignins into native-like lignins. Treating ether modified lignins under mild acidic aqueous organosolv type conditions (0.1 M HCl in 2:1 dioxane/water at 100° C.) can reverse α-etherification to give native-like lignins.

[0101] The ether modified lignin (200 mg) was dissolved in 1,4-dioxane/water (2:1, 5 mL) containing 0.1 M HCl and heated in a sealed vial at 100° C. (oil bath temperature) for 4 hours for ethanol and 6 hours for n-butanol lignins. After this time the lignin was recovered by precipitation in water (40 mL), collected by filtration and allowed to air dry overnight. Mass recovery for ethanol lignins was 60-70 wt %, for n-butanol lignins 55-60 wt %.

[0102] This process was effective on ethanol and n-butanol lignins obtained from both Douglas fir and walnut shell. In these experiments this process worked best for ethanol over butanol and Douglas fir over walnut shell lignins reflecting the lower initial degree of α-etherification in ethanol lignins (Table 2) and the greater reactivity of G units compared to the S units. In all cases a decrease in total β-aryl ether content was observed indicating some acid mediated degradation occurs during this process.

Direct Acidolysis to Monomers

[0103] The use of a triflic acid mediated lignin depolymerisation method has been reported by Deuss et al. (ref 4, 5). Initially, using Bi(OTf).sub.3 as a convenient alternative to triflic acid, it was found that modified lignins were efficiently depolymerised to give the expected acetals in 1,4-dioxane in moderate to good yields without any need to pre-process the lignin. The reaction is illustrated in Scheme 3, below. FIG. 3 shows the results graphically.

##STR00003##

[0104] For GC analysis: the lignin (100 mg) was dissolved in 1,4-dioxane (1.5 mL) containing ethylene glycol (100 mg). Bismuth triflate (5 mg) and n-octadecane (20 μL, 0.25 M in 1,4-dioxane, 0.005 mmol) was then added and the mixture heated at 150° C. (oil bath temperature) for 15 minutes in a sealed tube. The reaction was then cooled and concentrated in vacuo. The oily residue was then extracted with hot (ca. 70° C.) toluene (4×3 mL) and passed through a small plug of Na.sub.2SO.sub.4 and then concentrated in vacuo. GC—FID analysis was performed on a solution of the concentrated samples in dichloromethane.

[0105] At a preparative scale: walnut ethanol etherified lignin (1.0 g) was dissolved in 1,4-dioxane (15 mL) containing ethylene glycol (1.0 g) and bismuth triflate (50 mg) was then added and the mixture heated at 150° C. (oil bath temperature) for 15 minutes in a sealed tube. The reaction was then cooled and concentrated in vacuo. The oily residue was then extracted with hot (ca. 70° C.) toluene (7×15 mL), dried with Na.sub.2SO.sub.4 and then concentrated in vacuo. The oily residue was then dry loaded using a small quantity of silica on to a 10 g silica gel column and eluted using a gradient of 0-50% EtOAc/petroleum ether over 50 column volumes.

[0106] The highest yields (as determined by GC analysis) of acetals were obtained for ethanol extracted lignins i.e. where R=Et in Scheme 2. With beech and walnut shell ethanol lignins gave 17 and 18 wt % yields of acetals respectively, whilst Douglas fir gave 11 wt %.

[0107] This result is consistent with 2D HSQC analysis which indicated beech and walnut shell lignins had slightly higher β-aryl ether contents than the Douglas fir lignin (Table 2, Entries 2, 5 and 8) and the fact that softwood lignins are known to have higher amounts of cross linking between aromatic units (i.e. 5-5 and 4-O-5 linkages). The n-butanol extracted lignins followed the same pattern, although overall yields were lower which probably results from a combination of lower β-aryl ether content and higher amounts of α-butoxylation (Table 2, Entries 3, 6, 9) which increases the molecular weight of the lignin.

[0108] Comparison of the beech butanol lignin and a technical beech ethanol lignin show that n-butanol lignin is approximately 4 times higher yielding, highlighting their potential value for the production of mono-aromatic chemicals. Additionally, Bi(OTf).sub.3 could be replaced with cheaper and safer MsOH or TsOH with only a small drop in yield (FIG. 3).

[0109] The yields of both P1 and P2 were almost identical using these acids compared to Bi(OTO.sub.3 and the decrease in overall yield was the result of reduced P3 production.

[0110] The improved solubility of n-butanol lignins may allow the use of alternative solvents in such a process. The use of 2-MeTHF as a reaction solvent was demonstrated. This is a greener alternative to 1,4-dioxane, However the acetal yields were found to be significantly lower in these initial experiments.

[0111] A scale up of this reaction to a 1 gram scale using Bi(OTf).sub.3 as a catalyst in 1,4-dioxane allowed isolation of a combined 12.3 wt % yield of P1-3 acetals from walnut shell ethanol lignin after purification by column chromatography.

Hemicellulose Fraction

[0112] After isolation of the cellulose pulp and lignin a water soluble fraction is obtained containing most of the hemicelluloses. For the most promising n-butanol pre-treatments these water soluble fractions were analysed by .sup.13C NMR. This analysis revealed that they are, in the case of beechand walnut shell, composed almost exclusively of butyl-xylosides, whilst the Douglas fir extract contained butyl-mannosides as well as butyl-xylosides. These compounds were identified by comparison to authentic samples and correspond well to the known compositions of the native hemicelluloses in these substrates. Such butylated hemicellulose compounds are potentially valuable biorefinery products which may have applications in the synthesis of bio-renewable surfactants and wetting agents which can have properties similar or superior to petrochemical derived products.

Analysis of Lignins

NMR Spectroscopy

[0113] For lignin NMR analysis 100-110 mg of the lignin concerned was dissolved 0.6 mL of d.sub.6-acetone.

[0114] Wood and pulp analysis: The sample for analysis (125 mg-ground <0.5 mm) is added to AcOH/AcBr (4:1, 10 mL) and heated at 50° C. with sonication (Ultrawave Q-Series ultrasonic bath) until a clear solution is obtained (approx. 3 hour). This was best achieved using the sonicator but the same result can be achieved using conventional heating and stirring over a longer time. The sample is then concentrated in vacuo (caution HBr fumes) and further dried under a stream of air for 15 minutes. The sample is then dissolved in d.sub.6-acetone (0.7 mL), centrifuged and then used for NMR analysis immediately. The samples cannot be stored for extended periods.

[0115] NMR analysis was performed on a Bruker Ascend 700 MHz or 500 MHz spectrometers equipped with CPP TCl and CPP BBO probes respectively following previously reported protocols (Ref 6-the contents of which are incorporated by reference herein).

[0116] In summary, the .sup.1H—.sup.13C—HSQC experiment was acquired using the standard Bruker pulse sequence ‘hsqcetgpsp.3’ (phase-sensitive gradient-edited-2 D HSQC using adiabatic pulses for inversion and refocusing). Composite pulse sequence ‘garp4’ was used for broadband decoupling during acquisition. 2048 data points was acquired over 12 ppm spectral width (acquisition time 170 ms) in F2 dimension with 1 s interscan delay and the d4 delay was set to 1.8 ms (¼J, J =140 Hz). The number of scans is chosen to give a suitable signal-to-noise ratio—in this case 4 to 24 were used depending on the sample and instrumentation used for the analysis. The spectra were processed using squared cosinebell in both dimensions and LPfc linear prediction (32 coefficients) in F1.

[0117] Semi-quantitative analysis of the volume integrals for the lignin and carbohydrate linkages was performed using MestReNova 9.0 for lignins and TopSpin 3.1 (Windows) for cell walls.

[0118] Specifically for lignin the volume integrals of the benzylic (alpha, α) .sup.1H-.sup.13C correlations for both the unsubstituted β-O-4 linkages and the β-O-4 etherified linkages (β-O-4-OR where R is Et or n-Bu), were compared to the sum of the volume integrals of the S.sub.2/6, G.sub.2 and H.sub.2/6 positions on the lignin aromatic components (adjusted for the number of protons) to give the number of linkages of each type of β-O-4 linkage per 100 C.sub.9 units.

[0119] FIG. 4a shows a .sup.1H—.sup.13C—HSQC NMR analysis of a butylated lignin obtained from Beech wood. The signals used for calculation of the % butylated β-O-4 linkages per 100 C.sub.9 units are designated as i1 and i2 for the S and G aromatic signals and i3 for the alpha C—H signal of the butylated β-O-4-OR groups. FIG. 4b shows the nomenclature used for the signals of the various C.sub.9 structural units present in the sample The alpha C—H signal of the butylated β-O-4-OR groups are in structural unit A′.

[0120] The calculation of the number of unsubstituted β-O-4 linkages and substituted (butylated) linkages β-O-4-OR per 100 C.sub.9 units is based on the volume integrals I indicated by dashed boxes in the FIG. 4a.

[0121] For β-O-4-OR per 100 C.sub.9 units=100[i3/{(i½)+i2}];

[0122] For β-O-4 (unsubstituted) per 100 C.sub.9 units=100×[i4/{(i½)+i2}]

[0123] Graphical figures were prepared using Abobe Illustrator from spectra exported from MestReNova/TopSpin in the pdf format.

GPC Analysis

[0124] GPC analysis was carried out on a Shimadzu HPLC/GPC system equipped with a CBM-20A communications bus, DGU-20A degassing unit, LC-20AD pump, SIL-20A auto sampler, CTO-20A column oven and SPD-20A UV-Vis dectector. Samples were analysed using a Phenogel 5 μ 50A (300×7.8 mm) and Phenogel 5 μ 500A (300×7.8 mm) columns connected in series and eluted with inhibitor free THF (1 mL/min) with a column oven temperature of 30 ° C. Samples (10 mg) were dissolved in THF (1 mL) and filtered (0.45 μm PTFE syringe filter) before analysis. Analysis was performed using the GPC postrun data analysis module implemented in Shimadzu's LabSolutions software.

REFERENCES

[0125] 1. Bauer, S.; Sorek, H.; Mitchell, V. D.; Ibáñez, A. B.; Wemmer, D. E. J. Agric. Food Chem. 2012, 60, 8203.

[0126] 2. Shuai, L.; Questell-Santiago, Y. M.; Luterbacher, J. S. Green Chem. 2016, 18, 937; Antal, M. J.; Allen, S. G.; Dai, X.; Shimizu, B.; Tam, M. S.; Grønli, M. Industrial & Engineering Chemistry Research 2000, 39, 4024; and

[0127] Lee, J. Journal of Biotechnology 1997, 56, 1.

[0128] 3. Huijgen, W. J. J.; Telysheva, G.; Arshanitsa, A.; Gosselink, R. J. A.; de Wild, P. J. Industrial Crops and Products 2014, 59, 85: and Wildschut, J.; Smit, A. T.; Reith, J. H.; Huijgen, W. J. J. Bioresour. Technol. 2013, 135, 58.

[0129] 4. Deuss, P. J.; Scott, M.; Tran, F.; Westwood, N. J.; de Vries, J. G.; Barta, K. J. Am. Chem. Soc. 2015.

[0130] 5. Lahive, C. W.; Deuss, P. J.; Lancefield, C. S.; Sun, Z.; Cordes, D. B.; Young, C. M.; Tran, F.; Slawin, A. M. Z.; de Vries, J. G.; Kamer, P. C. J.; Westwood, N. J.; Barta, K. J. Am. Chem. Soc. 2016.

[0131] 6. Tran, F.; Lancefield, C. S.; Kamer, P. C. J.; Lebl, T.; Westwood, N. J. Green Chem. 2015, 17, 244.