ENZYMATIC DEGRADATION OF CELLULOSIC SUBSTRATES IN THE PRESENCE OF LIGNOCELLULOSE MILLING PARTICLES

Abstract

A process and apparatus for the enzymatic degradation of a cellulosic substrate is disclosed. The process comprises agitating a composition with milling particles, wherein the milling particles are or comprise a lignocellulosic material and wherein the composition comprises: a. the cellulosic substrate; b. a cellulase enzyme; and c. a liquid medium.

Claims

1. A process for the enzymatic degradation of a cellulosic substrate, the process comprising agitating a composition with milling particles, wherein the milling particles are or comprise a lignocellulosic material and wherein the composition comprises: a. the cellulosic substrate; b. a cellulase enzyme; and c. a liquid medium.

2. A process according to claim 1, wherein the milling particles are or comprise any one of: wood, nutshells, husks, corn cobs, bamboos, fruit stones or any combination thereof.

3. A process according to claim 1, wherein the milling particles have a density of from 300 Kg/m.sup.3 to 1400 Kg/m.sup.3.

4. A process according to claim 1, wherein the lignocellulosic material has a lignin content from 15 wt. % to 40 wt. %.

5. A process according to claim 2, wherein the milling particles are or comprise wood and the wood has a Janka hardness greater than 1500 N.

6. A process according to claim 2, wherein the milling particles are or comprise wood, and the wood is optionally unrefined.

7. A process according to claim 1, wherein the milling particles have a size of from 1 mm to 400 mm.

8. A process according to claim 1, wherein the milling particles have an angular shape.

9. A process according to claim 1, wherein the milling particles are or comprise wood and the wood comprises pine, eucalyptus, poplar, acacia, rubberwood, willow, elm, birch, maple, walnut, cherry, apple, chestnut, beech, oak, hickory, alder, mesquite, bamboo or any combination thereof.

10. A process according to claim 1, wherein the cellulosic substrate is or comprises a waste feedstock.

11. A process according to claim 1, wherein the cellulosic substrate is or comprises cotton, flax, rayon, bamboo, sugarcane, sisal, abaca, jute, kenaf, banana, capok, coir, pina raffia, ramie, hemp or a mixture thereof.

12. A process according to claim 1, wherein the cellulosic substrate is or comprises paper, cardboard, pulp of a mixture thereof.

13. A process according to claim 1, wherein the cellulosic substrate is or comprises leaves or grasses.

14. A process according claim 1, wherein the cellulosic substrate has been refined prior to use.

15. A process according to claim 1, wherein the liquid medium is aqueous.

16. A process according to claim 15, wherein the liquid medium comprises at least 95 wt. % of water.

17. A process according to claim 1, wherein the agitation is performed for a period of from 6 hours to 72 hours.

18. A process according to claim 1, wherein the composition has a temperature of from 30 to 50° C. during agitation.

19. A process according to claim 1, wherein the composition comprises from 0.001 to 8 wt. % of cellulase enzymes.

20. A process according to claim 1, wherein the composition comprises from 1 to 40 wt. % of cellulosic substrate.

21. A process according to claim 1, wherein the ratio of milling particles to composition is from 1:20 to 1:1 by weight.

22. A process according to claim 1, wherein the cellulase enzyme is or comprises an endo-cellulase.

23. A process according to claim 1, wherein the cellulase enzyme is or comprises an exo-cellulase.

24. A process according to claim 1, comprising separating the milling particles from the composition after agitation.

25. A process according to claim 24, comprising re-using some or all of the milling particles in a subsequent process for the enzymatic degradation of a cellulosic substrate.

26. A process according to claim 25, wherein the re-using of milling particles in a subsequent process is performed with the addition of virgin milling particles in an amount of from 0.1 to 75% by weight of the total milling particle weight.

27. A process according to claim 1, wherein agitation is performed using a rotary drum or a stirrer.

28. A process according to claim 1, wherein the milling particles decrease in dry mass from 0.5% to 4% after agitation with the composition.

29. A process according to claim 1, wherein enzymatic degradation of the cellulosic substrate produces monosaccharides and/or oligosaccharides.

30. A process according to claim 29, comprising the step of separating the monosaccharides and/or oligosaccharides from the composition after agitation.

31. A process for the production of glucose from a cellulosic substrate, comprising the process according to claim 29, and wherein oligosaccharides are produced and wherein the oligosaccharides are converted to glucose by further enzymatic digestion.

32. A process for the production of one or more alcohols from a cellulosic substrate, comprising a process according to claim 31, and wherein the glucose is fermented to produce said one or more alcohols.

33. A process for the production of one or more biogases from a cellulosic substrate, comprising a process according to claim 29, and wherein the monosaccharides and/or oligosaccharides are converted to the one or more biogases by anaerobic digestion.

34. A process for the production of energy from a cellulosic substrate, comprising a process according to claim 32, and wherein the one or more alcohols are combusted to release energy.

35. An apparatus for the enzymatic degradation of a cellulosic substrate, the apparatus comprising a milling vessel charged with a composition and milling particles, wherein the milling particles are or comprise a lignocellulosic material; and wherein the composition comprises: a. the cellulosic substrate; b. a cellulase enzyme; and c. a liquid medium.

36. A process for the production of energy from a cellulosic substrate comprising a process according to claim 33 wherein the one or more biogases are combusted to release energy.

Description

[0108] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus, it will be appreciated that the features and preferences of the first aspect of the invention are also applicable to the second, third, fourth, fifth and sixth aspects of the invention. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0109] FIG. 1 is a block diagram showing a process for the degradation of a cellulosic substrate according to the first aspect.

[0110] FIG. 2 is a block diagram showing a process for the degradation of a cellulosic substrate according to an embodiment of the first aspect.

[0111] FIG. 3 is a block diagram illustrating processes according to the second, third and fifth aspects.

[0112] FIG. 1 illustrates a process 100 according to the first aspect of the invention. In FIG. 1, a cellulosic substrate 105 is comprised as part of a composition 103. The composition additionally comprises one or more cellulase enzymes 106 and a liquid medium 107. The cellulosic substrate is degraded by agitating the composition in the presence of lignocellulosic milling particles 104, which is or comprises a lignocellulosic material.

[0113] In an embodiment, agitation may be performed by charging a milling vessel with the composition 103 and lignocellulosic milling particles 104. The milling vessel may be a 40 m.sup.2 cylindrical rotary milling vessel configured to rotate around an axis of rotation aligned in the horizontal direction. The composition 103 may comprise 10,000 L of water 107, 10 Kg of enzymes 106 and 1,000 Kg of cellulosic substrate 105. The milling particles 104 may be 5,000 Kg of untreated and unrefined wood. The milling vessel may be rotated for a duration of 24 h and its contents may be maintained at a temperature of 45° C.

[0114] Agitation of the composition 103 in the presence of the milling particles 104 mechanically breaks up the cellulosic substrate 105 and causes the mechanical breakdown of, amongst others, crystalline cellulose, hemi cellulose and lignin components that may be present in the cellulosic substrate 105. This increases the surface area of the cellulosic substrate 105 and increases degradation of the cellulose in the substrate. Agitation of the composition 103 in the presence of the milling particles 104 also increases mixing of the enzymes 106 with the cellulosic substrate 105, the mechanical action of the agitation can drive the enzymes into the pores of the cellulosic substrate 105, further enhancing the rate of degradation. The degradation of the cellulosic substrate 105, by process 100 results in a degraded composition 102 which includes sugars produced by the enzymatic degradation of the cellulose in the cellulosic substrate 105. The degraded composition may also comprise lignin, hemi celluloses and undigested crystalline cellulose.

[0115] The milling particles 104 comprise a lignocellulosic material. During process 100, the agitation may cause small fragments to be broken off the milling particles. These fragments may additionally be mechanically degraded by the milling particles and the fragments enzymatically degraded by the cellulase enzymes. This degradation may produce sugars, lignin, hemi celluloses and other products similar to the digestion of the cellulosic substrate 105. Beneficially, the use of a lignocellulosic milling particles 104 removes the need for the fragments of the milling particles to be separated from the degraded composition 102 after degradation by process 100. Additionally, lignocellulosic milling particles 104 are cheaper than conventional milling particles such as alumina or steel balls, advantageously cause less wear of milling vessels, and have been demonstrated to be surprisingly more effective. Lignocellulosic milling particles are also lower in density and, advantageously, agitation with lignocellulosic milling particles uses less energy.

[0116] An embodiment of process 100 is shown in FIG. 2, wherein process 100 is followed by separation 120 of the lignocellulosic milling particles 104 from the degraded composition 102. Separation may be performed by filtering with a porous material (i.e. a mesh) for example. The separated lignocellulosic milling particles 104 may be reused 121 as the milling particle 104 in a subsequent iteration of process 100 with new cellulosic feedstock. Lignocellulosic material may be inherently porous and may have a considerably higher surface area compared to conventional milling particles. A significant quantity of enzymes 106 can become adsorbed onto the surface of the lignocellulosic milling particles 104; thus, re-use of lignocellulosic milling particles 104 can cause the re-use of a portion of the enzymes 106, thereby advantageously reducing the quantity of enzymes 106 that need to be added in further iterations of process 100.

[0117] After a plurality of re-uses 121 as a milling particle, the milling particles may be discarded, or may undergo a pre-treatment/refinement step 122 and may be used as a cellulosic substrate 105 for agitation with new milling particles 104 as part of another iteration of process 100. Thus, at end-of-life, no waste milling particles are generated.

[0118] FIG. 3 illustrates processes according to the second, third and fifth aspects of the invention. As explained above and illustrated in FIGS. 1 and 2, a process 100 for the degradation of a cellulosic substrate involves the agitation of a composition in the presence of lignocellulosic milling particles. The enzymatic degradation of the cellulose in the cellulosic substrate by the enzyme(s) in the composition results in the production of saccharides. In process 200 the saccharides are converted to glucose by further enzymatic degradation. As part of process 200, degraded composition 102 may be exposed to enzymes such as β-glucosidase, hemi cellulases or further cellulases for a period of time of from 1 to 6 hours. This may yield a composition 202 with a typical glucose content of from 1 to 9%. Thus, a second aspect comprises processes 100 and 200.

[0119] The glucose-containing composition 202 may undergo a subsequent process of fermentation 300 by a microorganism such as yeast. Fermentation 300 may convert some of the glucose and optionally some of the saccharides in the glucose-containing composition 202 to an alcohol-containing composition 302. Thus, a third aspect comprises processes 100, 200 and 300.

[0120] The alcohol may be distilled from the alcohol-containing composition 302 and may be combusted 400 to produce energy which may be in the form of heat and/or which may be converted to motion or electrical energy. Thus, a fifth aspect comprises processes 100, 200, 300 and 400.

Experimental Data

Milling Particles

[0121] Bar-be-quick® (Rectella International Ltd, Burnley, UK) hickory smoking wood chips of size between approximately 2 mm (shortest dimension) and approximately 4 cm (longest dimension) were used as the lignocellulosic (wood) milling particles. For each test, a dry weight of 145.6 g wood milling particles were first soaked in cold tap water in a static beaker overnight. On the morning of the test, wood milling particles were strained in a coarse sieve and remaining non-absorbed water was removed using a paper towel. Between consecutive interations, the wood chips were thoroughly rinsed with tap water to remove enzyme and non-degraded cotton particles.

[0122] Spherical stainless steel (SS) ball bearings (milling particles) of size 3.5 mm and 1.3 cm size were used for the comparative tests in an amount of 145.6 g when dry.

Cellulosic Substrate

[0123] White woven mercerised cotton sheet fabric (Whaleys Bradford Ltd, Bradford, UK) was used as the cellulosic substrate in the tests. Cotton sheet fabric was cut into small pieces of 1 cm in size.

Enzyme

[0124] The cellulase enzyme used in the tests was Cellusoft® LT 19500 L (Novozymes A/S, Bagsværd, Denmark). This enzyme product was determined to be a cellulase blend containing at least endoglucanase and β-glucosidase activities, as it demonstrated activity in the conversion of cellobiose to glucose.

Apparatus

[0125] The stirring device used was a Hei-Torque 400 overhead stirrer (Heidolph, Schwabach, Germany), which was held in a 90° horizontal axial position using a fixed frame and weight balancing. The milling vessel used was a 1.125 litre stainless steel ball mill pot (Capco, a division of Castle Broom Engineering Ltd, Ipswich, UK). The ball mill pot was modified by welding one stainless steel lifter of 2 cm height, and a nut in the centre of the base for connection to the stirring rod of the stirring device, so that rotation of the stirring device rotated the steel ball mill pot. The heating was provided by a Mini Kitchen fan assisted convection oven (Russell Hobbs, Failsworth, UK). The heating temperature setting was calibrated to maintain 340 ml of water at a temperature of 40±2° C. during 24 hours of rotation.

Combined Milling and Enzymatic Degradation

[0126] The stainless steel ball mill pot was first pre-heated in the oven for at least 2 hours, and at least 6 hours with the stainless steel milling particles. The process water as the liquid medium was buffered at pH 6 using 50 mM sodium citrate and citric acid, and pre-heated to around 40° C. using a hotplate. The pre-heated stainless steel ball mill pot was loaded with the milling particles, 340 ml of pre-heated buffer, 6.8 g (20 g/L) of cotton pieces, and 0.408 ml (6% by weight of substrate) of Cellusoft® LT 19500 L. The ball mill pot was then rotated at 83 rpm (equating to a centripetal force of 0.45 G on the inner surface of the milling vessel) for 24 hours and samples taken at the specified time points. Samples were heated at 80° C. for 15 minutes to denature the enzymes and stop the reaction.

[0127] In order to account for any non-specific background absorbance from either slight degradation of the wood chips, or the release of reducing substances from the wood chips, consecutive wood chips (no substrate) control tests were also performed.

[0128] A Brennenstuhl® PM 231 E wattmeter was connected to a multi-socket adaptor feeding both the stirrer and the oven, and the energy consumption was measured after 24 hours in kilowatt hours (kW.h) to one decimal place.

Oligosaccharide and Monosaccharide Concentration Determination

Dinitrosalicylic Acid Assay Method

[0129] Oligosaccharide and monosaccharide concentration was determined using the dinitrosalicylic acid (DNSA) assay. Solution part ‘a’ was prepared by dissolving 75 g sodium potassium tartrate in 125 ml deionized water. Solution part ‘b’ was prepared by dissolving 2.5 g of 3,5-dinitrosalicylic acid in 50 ml of 2 N NaOH solution. DNSA reagent was prepared by mixing solution parts a & b and raising the final volume to 250 ml with deionized water. The reagent was stored in a brown glass jar in a refrigerator. A calibration curve was prepared using a concentration range of pure cellobiose (0, 0.25, 0.5, 1, 2, 5, 10 and 20 g/L). To carry out the assay, 2 ml of DNSA reagent was added to 1 ml of each sample, shaken, and vials were placed in a bath of boiling water for 5 minutes to develop colour. Vials were then transferred into a bath of ice-cold water for 10 minutes to quench the colour change reaction and topped up with 9 ml of deionized water. UV-visible light transmission through the samples was measured in a quartz cuvette at 540 nm using a Konica-Minolta CM-3600A spectrophotometer.

Data Analysis

[0130] Percentage transmission (%T) values were converted into absorbance units using the equation Absorbance=2-LOG(%T). Absorbance values for buffer only (blank) and the appropriate consecutive wood chips (no substrate) control test samples were subtracted to account for non-specific background absorbance. Oligosaccharide and monosaccharide concentration was then determined using the gradient equation of the linear cellobiose calibration curve (0-20 g/L). To calculate the % conversion of cellulose to saccharides, the saccharide concentration was divided by the maximum theoretical saccharide release (19 g/L, assuming a 95% cellulose content of the cotton substrate) and multiplied by 100.

EXAMPLE 1

[0131] Table 1 shows the saccharide concentration in g/L at various time points during combined milling and enzyme degradation of cotton using an equivalent dry weight (145.6 g) of wood or comparative (stainless steel) milling particles.

TABLE-US-00001 TABLE 1 Oligosaccharide and monosaccharide release using an equivalent weight of milling particles Time, No milling 3.5 mm SS Wood h particles balls chips 2 0.04 0.61 0.65 4 0.20 1.46 1.66 6 0.32 2.07 2.22 24 1.32 5.30 6.46

[0132] This shows superior yeild of saccharides from the use of the wood chips as a milling particles compared to an equivalent weight of stainless steel balls.

[0133] Table 2 shows the % conversion of cellulose to oligosaccharides and monosaccharides at various time points during combined milling and enzyme degradation of cotton using an equivalent dry weight (145.6 g) of wood or comparative (stainless steel) milling particles.

TABLE-US-00002 TABLE 2 percent conversion of cellulose using an equivalent weight of milling particles Time, No milling 3.5 mm SS Wood h particles balls chips 2 0.2 3.2 3.4 4 1.1 7.7 8.7 6 1.7 10.9 11.7 24 7.0 27.9 34.0

[0134] This shows superior conversion of cellulose to oligosaccharides and monosaccharides from the use of the wood chips as a milling particles compared to an equivalent weight of stainless steel balls.

EXAMPLE 2

[0135] Table 3 shows both the saccharide concentration and the % conversion of cellulose to oligosaccharides and monosaccharides after 24 hours of combined milling and enzyme degradation of cotton using an equivalent volume (35%) of wood or comparative (stainless steel) milling particles. After five consecutive uses and air-drying for 48 hours, the dry weight of the wood chips was 141 g, indicating a weight loss of 3%.

TABLE-US-00003 TABLE 3 Oligosaccharide and monosaccharide release and % cellulose conversion using an equivalent volume of milling particles No 1.3 cm Wood Wood Wood Wood Wood Wood milling SS chips chips chips chips chips chips, particles balls 1 2 3 4 5 mean Saccharide 1.32 4.85 6.46 5.83 6.15 5.86 6.55 6.17 release, g/L Conversion, 7.0 25.5 34.0 30.7 32.3 30.9 34.5 32.5 %

[0136] This shows superior yeild of saccharides and superior conversion of cellulose to saccharides from the use of the wood chips as a milling particles compared to an equivalent volume of stainless steel balls. The minimal weight loss of the milling particles also demonstrates that the increase of yield and conversion is due to improved milling rather than the lignocellulose in the milling particle being itself converted to saccharides.

[0137] Table 4 shows the combined energy consumption in kilowatt hours (kW.h) for the heating and the horizontal axis rotation of the steel milling vessel after 24 hours of combined milling and enzyme degradation of cotton using an equivalent volume (35%) of wood or comparative (stainless steel) milling particles.

TABLE-US-00004 TABLE 4 Energy consumption using an equivalent volume of milling particles Energy consumption, Energy consumption/% kW .Math. h cellulose conversion 1.3 cm SS balls 1.5 0.056 Wood chips 1 1.3 0.038 Wood chips 2 1.3 0.042 Wood chips 3 1.4 0.043 Wood chips 4 1.4 0.045 Wood chips 5 1.3 0.038 Wood chips, mean 1.3 0.040

[0138] This shows a reduction in energy consumption from milling with a lignocellulosic milling particle compared to stainless steel balls.

EXAMPLE 3

Milling Particles

[0139] Natural bamboo circles were obtained from Bakerross.co.uk and sawn into pieces between approximately 2 cm (shortest dimension) and approximately 3 cm (longest dimension). These were used as the lignocellulosic milling particles. In order to prevent any background interference in the DNSA assay from the release of reducing substances from the bamboo pieces, they were first subjected to multiple days of washing by tumbling without enzyme or substrate. Samples were subjected to the DNSA assay to confirm that no more reducing substances were being released. For each test, a dry weight of 400 g bamboo milling particles were first soaked in cold tap water in a static beaker overnight. When stainless steel milling particles were used in comparative tests the amount used was the same volume as the bamboo milling particles. On the morning of the test, bamboo milling particles were strained in a coarse sieve and remaining non-absorbed water was removed using paper towel. In-between consecutive cycles, the bamboo milling particles were thoroughly rinsed with tap water to remove enzyme and non-degraded cotton particles. The bamboo milling particles were tested using the same methodology as for Examples 1 and 2.

[0140] Table 5 shows the reducing sugar concentration in g/L at various time points during combined milling and enzyme hydrolysis of cotton using an equivalent volume (35%) of bamboo pieces, wood chips or comparative (stainless steel) milling particles.

TABLE-US-00005 TABLE 5 Reducing sugar release using an equivalent volume of milling particles Time, No milling 1.3 cm SS Wood Bamboo h particles balls chips pieces 2 0.04 1.42 0.65 0.86 4 0.20 2.03 1.66 1.54 6 0.32 2.91 2.22 2.30 24 1.32 4.85 6.46 6.21

[0141] This shows a superior yield of sugar after 24 hours from the use of the bamboo pieces as milling particles compared to an equivalent volume of stainless steel balls, and that the performance is similar to wood chips.

[0142] Table 6 shows the % conversion of cellulose to reducing sugars at various time points during combined milling and enzyme hydrolysis of cotton using an equivalent volume (35%) of bamboo pieces, wood chips or comparative (stainless steel) milling particles.

TABLE-US-00006 TABLE 6 Percent conversion of cellulose using an equivalent volume of milling particles Time, No milling 1.3 cm SS Wood Bamboo h particles balls chips pieces 2 0.2 7.5 3.4 4.5 4 1.1 10.7 8.7 8.1 6 1.7 15.3 11.7 12.1 24 7.0 25.5 34.0 32.7

[0143] This shows superior conversion of cellulose to reducing sugars after 24 hours from the use of the bamboo pieces as milling particles compared to an equivalent volume of stainless steel balls, and that the performance is similar to wood chips.