Process for depolymerization of lignin by laccases

Abstract

A method for depolymerization of lignin includes oxidizing non-phenolic lignin by putting into presence, in at least one solvent, non-phenolic lignin, laccase, redox mediator and a source of oxygen, whereby a mixture including oxidized non-phenolic lignin is obtained, and further includes depolymerization of the oxidized non-phenolic lignin thereby obtained, by adding an oxidizer. The non-phenolic lignin is obtained from phenolic lignin by functionalization of phenol functions of the phenolic lignin.

Claims

1. A method for depolymerization of lignin, comprising: oxidizing a non-phenolic lignin by adding together the non-phenolic lignin, laccase, a redox mediator and an oxygen source in at least one solvent, thereby obtaining a mixture comprising oxidized lignin; and depolymerizing the oxidized lignin in the obtained mixture by adding an oxidizer, wherein said non-phenolic lignin is prepared by alkylation of phenol functional groups of a phenolic lignin.

2. The method according to claim 1 wherein the non-phenolic lignin is methylated lignin.

3. The method according to claim 2, wherein the methylated lignin is obtained by adding together the phenolic lignin and a methylation agent in a basic aqueous solution.

4. The method according to claim 3, wherein the methylation agent is dimethyl sulfate.

5. The method according to claim 1, wherein the oxygen source is pure oxygen or air.

6. The method according to claim 1, wherein the redox mediator is 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid).

7. The method according to claim 1, wherein the oxidizer is hydrogen peroxide.

8. The method according to claim 1, wherein the oxidizing is carried out in a basic medium.

9. The method according to claim 1, wherein the non-phenolic lignin stems from the alkylation of phenol functional groups of a Kraft lignin or sugarcane bagasse lignin.

Description

EXAMPLES

(1) Reagents Black liquor (provided by SMURFIT Kappa) Dimethyl sulfate (marketed by Sigma Aldrich) Sugarcane bagasse lignin (provided by SOLVAY) 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (marketed by Sigma Aldrich) A laccase from Trametes versicolor (marketed by Sigma Aldrich) Acetate buffer (pH=4; 50 mM) Hydrogen peroxide (35%) (marketed by Sigma Aldrich)

Example 1

Extraction of Kraft Lignin from the Black Liquor

(2) The Kraft lignin was extracted from the baking liquor (marketed by SMURFIT KAPPA) with an acid treatment, so as to cause its precipitation. A post-treatment with ethanol gives the possibility of removing the residual sodium salts.

(3) 800 g of black liquor are dissolved in 2 of distilled water and the mixture is slowly acidified (from pH=13 to pH=1.5) by means of a 6N HCl solution. The mixture is then centrifuged at 4,000 rpm for 10 minutes, the pellet is recovered and again treated with a HCl solution at pH=1.5 and again centrifuged (3 times treated with HCl at pH=1.3). Ethanol is then added to the obtained solid. After filtration, the ethanol phase is evaporated and the residue is washed with an HCl solution at pH=1.5 for removing the residual salts. After centrifugation, the pellet is recovered and freeze-dried. A 180 g of Kraft lignin are obtained.

(4) From the 180 g of Kraft lignin which are thereby obtained, 10 g are sampled which are dissolved in 200 ml of tetrahydrofurane (THF). After stirring for 30 minutes with ultrasound, the mixture is filtered for removing the white solid deposit, insoluble in THF. After evaporation of the THF, 9.2 g of a brown solid are obtained. This brown solid is dissolved in 100 ml of diethyl ether (Et.sub.2O). A fraction remains insoluble in Et.sub.2O (8.1 g) and the soluble fraction (0.9 g) is analyzed by GC-MS after silylation in order to make these compounds volatile.

(5) This analysis demonstrates the presence of monophenols like vanillin or vanillic acid and residues from the degradation of the sugars. The insoluble fraction consists of purified Kraft lignin, which may be used subsequently for the methylation and the depolymerization method of the present invention.

Example 2A

Methylation of Kraft Lignin

(6) 1 g of Kraft lignin is dissolved in 20 ml of a 0.7 M NaOH solution (0.56 g) and the mixture is stirred at room temperature for 10 minutes, and then 0.8 mL of dimethylsulfate ((MeO).sub.2SO.sub.2) are slowly added (drop wise). Stirring is continued for 30 minutes at room temperature. The reaction mixture is then heated to 80? C. for 4 hours, while adding a 0.7 M NaOH solution for homogenizing the reaction medium (about 10 mL of additional 0.7 M NaOH are added). After 4 hours of reaction, the mixture is brought back to room temperature and acidified with a 2 M HCl solution down to a pH=2. The reaction crude is filtered and the obtained solid is washed with distilled water and then dried by freeze-drying. 0.874 g of methylated lignin are obtained.

Example 2B

Methylation of the Sugarcane Bagasse Lignin

(7) The sugarcane bagasse lignin was provided by SOLVAY, was used without any preliminary pre-treatment.

(8) The bagasse lignin is treated under the conditions described in Example 2A. 0.92 g of methylated lignin are obtained.

Example 3A

Depolymerization of Methylated Lignin (Derived from Kraft Lignin)

(9) 1 g of methylated lignin obtained in Example 2A is dissolved in 25 mL of dioxane and the mixture is stirred at room temperature until full dissolution.

(10) Next, a solution of 51 mg of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (0.1 mmol) in 1 ml of acetate buffer (pH=4; 50 mM) is prepared which is added to the methylated lignin solution.

(11) Next, a solution of 5 mg of laccase in 200 mL of acetate buffer is then prepared, 24 ml of this solution are sampled which are slowly added to the medium containing the methylated lignin at 40? C. After adding the laccase, oxygen gas is introduced into the mixture by bubbling for 1 hour at 40? C., by using a flask filled with oxygen.

(12) After 22 hours of reaction at 40? C., 1 mL of a 3 M NaOH solution is added, and then 1 mL of an aqueous solution of hydrogen peroxide (35%, 10 mmol) is slowly added, the mixture being stirred at 90? C.

(13) The progression of the reaction is tracked by SEC (size exclusion chromatography). The analysis by SEC is conducted by using three columns of the TSK-gel type (3000 PW, 4000 PW, 3000 PW) coupled in series, with 1 M of sodium hydroxide until pH=12 and NaN.sub.3 (3%) in osmosed water as an eluant. The flow rate is 1 mL/min and the detection is ensured by UV at a wavelength of 280 nm.

(14) The first step of the reaction (action of the laccase/ABTS system) leads to low depolymerization. The second step (action of hydrogen peroxide) leads to strong depolymerization, with the formation of molecules of low molecular mass which were isolated and identified.

(15) After 40 hours of reaction, the mixture is brought back to pH=6-7 by means of a 1 N HCl solution and the reaction crude is extracted with dichloromethane (3?50 ml). After drying the organic phase on sodium sulfate (Na.sub.2SO.sub.4) and evaporation of the volatile solvents, the crude is purified by flash chromatography (with as an eluant: dichloromethane/methanol 99/1 to 90/10).

(16) 30 mg of 3,4-dimethoxybenzoic acid were thus isolated from 1 g of methylated lignin obtained in Example 2A.

Comparative Example 1

(17) As a comparison, the purified Kraft lignin obtained in Example 1 was subject to the action of the laccase/ABTS system, by directly applying the reaction conditions described in Example 3A (i.e. without carrying out methylation of the lignin).

(18) The progression of the reaction is tracked by SEC (conditions described above):

(19) TABLE-US-00001 t (hours) M.sub.p M.sub.n M.sub.w 0 661 902 1326 2 842 1254 1662 73 905 1036 1821 M.sub.p: molecular weight peak, in g/mol M.sub.n: number average molecular mass, in g/mol M.sub.w: mass average molecular mass, in g/mol

(20) It is observed that the average molecular mass of the species in presence increases with the duration of reaction.

(21) Thus, when the action of the laccase/ABTS system is carried out on non-methylated Kraft lignin, no depolymerization is observed, but on the contrary an increase in the molecular mass of the lignin.

Example 3B

Depolymerization of Methylated Lignin (Derived from Bagasse Lignin)

(22) The methylated lignin obtained in Example 2B is treated by applying the reaction conditions described in Example 3A (the action of the laccase is prolonged for 65 hours, after which hydrogen peroxide is added).

(23) The progression of the reaction is tracked by SEC (conditions described above):

(24) TABLE-US-00002 t (hours) M.sub.p M.sub.n M.sub.w 0 5054 1414 10245 2 4806 759 8681 65 3555 269 6857 70 2443 1601 4907 80 1525 130 2112

(25) The first step of the reaction (laccase/ABTS) leads to low depolymerization. The second step (addition of hydrogen peroxide at t=65 h) leads to depolymerization with the production of phenols of low molecular mass which were not isolated and identified.

Comparative Example 2

(26) As a comparison, the bagasse lignin is subject to the action of the laccase/ABTS system, by directly applying the reaction conditions described in Example 3A (i.e. without carrying out methylation of lignin).

(27) The progression of the reaction is tracked by SEC (conditions described above):

(28) TABLE-US-00003 t (hours) M.sub.p M.sub.n M.sub.w 0 2254 145 4957 1 4048 417 8245 24 9242 4282 17329

(29) It is observed that the average molecular mass of the species in presence increases with the duration of reaction.

(30) Thus, when the action of the laccase/ABTS system is carried out on non-methylated bagasse lignin, no depolymerization is observed, but on the contrary an increase in the molecular mass of the lignin.

Comparative Example 3

(31) As a comparison, the methylated lignin obtained in Example 2B was subject to the action of hydrogen peroxide in a basic medium, without subjecting it to the action of the laccase/ABTS system beforehand.

(32) 1 g of methylated lignin obtained in Example 2B is dissolved in 25 mL of dioxane and the mixture is stirred at room temperature until full dissolution.

(33) Next, 25 mL of osmosed water are added to the mixture.

(34) 1 mL of a 3 M NaOH solution is added, and then 1 ml of an aqueous solution of hydrogen peroxide (35%, 10 mmol) is slowly added, the mixture being stirred at 90? C.

(35) The progression of the reaction is tracked by SEC (conditions described above):

(36) TABLE-US-00004 t (hours) M.sub.p M.sub.n M.sub.w 0 5054 1414 10245 5 2541 404 4651 15 2394 153 4725

(37) A less significant depolymerization of the methylated lignin is observed than when it is treated beforehand with the action of the laccase/ABTS system.

(38) Without intending to be bound to a particular theory, this partial depolymerization is explained by the fact that, in the lignin (and therefore in the methylated lignin), a certain percentage of hydroxyl functions intended to be oxidized by being put in the presence of the laccase/mediator system are already in an oxidized states, i.e. as ketones. The oxidizing rupture of the CC bonds in the vicinity of these ketone functions causes partial depolymerization.

(39) This example nevertheless shows that the first step of the method is required for optimizing the depolymerizing action of the hydrogen peroxide.

(40) The comparative analysis of the bagasse lignins obtained at the end of Examples 2B, 3B and at the end of comparative Example 3 shows that the fragmentation of lignin is most efficient when the methylated lignin is successively subject to the action of the laccase/ABTS system and then to the action of hydrogen peroxide:

(41) TABLE-US-00005 Lignin of Example 2B, non-depolymerized M.sub.lig = 10245 Lignin of Example 3B, treated with laccase + H.sub.2O.sub.2 M.sub.w = 2112 Lignin of the comparative Example 3, treated with H.sub.2O.sub.2 M.sub.w = 4725