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    • 专利摘要:
    The present invention relates to a process for the depolymerization of lignin, comprising a step of oxidation of non-phenolic lignin by bringing into contact, in at least one solvent, non-phenolic lignin, laccase, a redox mediator and an oxygen source, whereby a mixture comprising oxidized non-phenolic lignin and a depolymerization step of the oxidized non-phenolic lignin thus obtained is obtained by addition of an oxidizing agent, said non-phenolic lignin phenolic being obtained from phenolic lignin by functionalization of phenol functions of said phenolic lignin.
    公开号:FR3013713A1
    申请号:FR1361718
    申请日:2013-11-27
    公开日:2015-05-29
    发明作者:Stephane Grelier；Yoya Georges Koumba
    申请人:Centre National de la Recherche Scientifique CNRS；Universite des Sciences et Tech (Bordeaux 1)；Institut Polytechnique de Bordeaux；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a process for the depolymerization of lignin. Lignin is the second most abundant renewable biopolymer after cellulose, and together they make up more than 70% of the total biomass. The valuation of lignin is therefore an important issue. In the paper industry, large quantities of lignin are obtained as by-products or wastes, of which only a small part is recovered in chemical form. In the sugar industry, large quantities of lignin are also obtained in the form of waste during the extraction of sugar cane juice. Lignin is mainly used as a binder or dispersant, or for the preparation of biogas by a high temperature treatment (800 ° C - 1000 ° C). There are also lignin depolymerization methods involving chlorinated derivatives which are unsatisfactory from an environmental point of view. At present, there are few economically viable processes using lignin as a raw material for producing chemical compounds. There is therefore a need for a new lignin beneficiation process that is economically viable and environmentally acceptable. The object of the present invention is to propose a new process for the depolymerization of lignin which is efficient and adapted from an environmental point of view, allowing access to depolymerization products that can be used in various industrial fields, such as biorefineries, agrifood or cosmetics. Another object of the present invention is to provide a method for accessing, in a controlled manner, depolymerization products of various sizes such as 3,4-dimethoxybenzoic acid (methyl vanillic acid), veratraldehyde (methylated vanillin). ), veratrole (guaiacol methyl), or diformyl guaiacol methyl. The method of the present invention provides for sequentially combining the action of an enzyme with the action of an oxidizing agent.
    [0002] More particularly, the present invention relates to a process for the depolymerization of lignin comprising: a step of oxidation of non-phenolic lignin by bringing into contact, in at least one solvent, non-phenolic lignin, laccase, a mediator redox and an oxygen source, whereby a mixture comprising oxidized non-phenolic lignin is obtained, and a step of depolymerization of the oxidized non-phenolic lignin thus obtained, by addition of an oxidizing agent, said non-phenolic lignin being obtained from phenolic lignin by functionalization of phenol functions of said phenolic lignin. The method of the invention is non-microbiological, in the sense that it does not use microorganisms (such as fungi) endogenously expressing laccases.
    [0003] The inventors have observed, surprisingly, that the action on non-phenolic lignin of a laccase in the presence of a redox mediator and an oxygen source, followed by the addition of an oxidizing agent , has the effect of effectively depolymerizing the structure of lignin. In the first step of the process (oxidation step), the hydroxyl functions in the benzylic position of the non-phenolic lignin are oxidized to ketone functions. In this step, the polymeric structure of the lignin is not fragmented and substantially no C-C bond is cleaved. During the second step of the process (depolymerization step, also called fragmentation), C-C bonds of the polymeric structure of the lignin are broken.
    [0004] Without wishing to be bound to a particular theory, the C-C bonds thus broken are those which are in the vicinity of the ketone functions formed during the first step of the process. The process of the invention can be represented by the following illustrative scheme: 1st stage oxidation 2nd stage depolymerization R 'OR GOLD OR bonding oxidative cleavage in which the group -OR represents a functionalized phenol function of the non-phenolic lignin and the group -R 'represents a possible other substituent (different from a phenol function) of the phenyl group represented. The presence, number and type of -R 'group depend on the lignin used. It may for example be one or more methoxy groups. The diagram above is purely illustrative and only schematically reflects the structure of lignin. As will be explained below, the two process steps advantageously take place one after the other, in the same reactor. The reaction conditions of the steps of the process of the invention will be described later. In general, in the context of the present application, the term "reaction medium" means the medium in which the process steps take place and which comprises a lignin derivative, a laccase, a redox mediator, a source of oxygen, at least one solvent, and optionally an oxidizing agent (depending on whether it has already been added or not). By "bringing into contact" is thus meant the addition of a reagent to the reaction medium. Said reaction medium may also comprise other reagents as described in the present application. The raw material used in the process, namely non-phenolic lignin, will now be described. Non-Phenolic Lignin By "non-phenolic lignin" is meant a phenolic lignin derivative, which is the reaction product of the functionalization of phenol (Ph-OH) functions of a phenolic lignin (also simply called "lignin"). ). A non-phenolic lignin is a modified or functionalized lignin in which at least one phenol function is modified. Preferably, at least 50%, or even at least 60%, or even at least 70%, or even at least 80%, or even at least 90%, or even at least 95% or even at least 99% of the phenol functions of lignin. are modified. Preferably, the non-phenolic lignin does not have a free (non-functionalized) phenol function. A non-phenolic lignin is obtained by reaction of a phenolic lignin with a functionalizing agent capable of converting phenol functions of said phenolic lignin to nonreactive functions during the process oxidation step. The purpose of lignin functionalization is to protect the phenol functions during the oxidation step of the process and to prevent them from reacting. The functionalized phenol functions of the non-phenolic lignin are thus not reactive during the laccase oxidation step. In the context of the present invention, the term "functionalization of lignin" means the selective functionalization of phenol functions, that is to say that other hydroxyl functions of lignin (aliphatic alcohols in particular) are not functionalized. Preferably, the functionalization is complete, that is to say that all the phenol functions are functionalized. The inventors have found that by using phenolic lignin in accordance with the process of the invention, the depolymerization of lignin is improved. The alkylation is an example of functionalization suitable for carrying out the process. For this purpose, an alkylating agent is used as functionalization agent. According to one embodiment, alkylated lignin can be used as non-phenolic lignin. By "alkylated lignin" is meant non-phenolic lignin in which the phenol functions are functionalized with (typically C 1 -C 12) alkyls, to give alkoxy functions. Preferably, the alkylated lignin has no free phenol function, i.e. they are all (or almost all) as alkoxy functions. According to an advantageous embodiment, methylated lignin can be used as non-phenolic lignin. For this, a methylating agent is used as functionalization agent. By "methylated lignin" is meant alkylated lignin in which the phenol functions are functionalized with methyls to give methoxy functions. Preferably, the methylated lignin does not have a free phenol function, that is to say that they are all (or almost all) in the form of methoxy functions. After the functionalization of a phenolic lignin, in order to control the degree of functionalization and the selectivity of functionalization, it is possible to quantify by 31 P NMR the quantity of phenolic functions that may be remaining (which would therefore not have been functionalized). One method is described in particular in Granat et al. J. Agric. Food Chem., 1995, 43 (6), 1538-1544. In one embodiment, the non-phenolic lignin is an alkylated lignin which is obtained by bringing phenolic lignin and an alkylating agent into a basic aqueous solution.
    [0005] In the context of the present invention, an alkylating agent is a compound capable of functionalizing the phenol functions with alkyl groups, by substituting the hydrogen atoms of said phenol functions with alkyl groups (to give alkoxy functions).
    [0006] Preferably, an alkylating agent is used to alkylate selectively the phenol functions of lignin, that is to say that the other hydroxyl functions of lignin (secondary alcohols in particular) are not alkylated in alkoxy functions. Preferably, an alkylating agent is used to fully alkylate lignin phenol functions, i.e. all (or almost all) of the lignin phenol functions are alkylated to alkoxy functions. According to an advantageous embodiment, the non-phenolic lignin is a methylated lignin, which is preferably obtained by placing in a basic aqueous solution, lignin and a methylating agent. In the context of the present invention, a methylating agent is a compound capable of functionalizing the phenol functions with methyl groups, by substituting the hydrogen atoms of said phenol functions with methyl groups. Preferably, a methylating agent is used to selectively methylen the phenol functions of lignin, that is to say that the other hydroxyl functions of lignin (secondary alcohols in particular) are not methylated in methoxy functions. The bringing into contact with the methylating agent is typically carried out dropwise. The bringing into association is typically followed by a step of heating the reaction medium, at a temperature typically ranging from 50 ° C to 100 ° C. As the methylating agent, mention may be made of dimethyl sulphate (or dimethyl sulphate), dimethyl carbonate (DMC), methyl iodide and diazomethane. Preferably, dimethylsulphate is used, which allows to completely and selectively methylen the phenol functions of a phenolic lignin. The lignin methylation step may typically be performed according to a method described in Sadeghifar et al. Ind. Eng. Chem. Res. 2012, 51, 16713-16720. The lignin is dissolved in a basic aqueous solution, typically sodium hydroxide (for example 0.7 M). The resulting mixture is stirred, typically at room temperature, and a methylating agent is added, preferably dropwise (at about 50 equivalents of methylating agent based on the number of phenol function equivalents). The mixture is typically heated (e.g., 80 ° C) until reagents are consumed. The mixture is then acidified, filtered and the solid obtained is washed with distilled water and dried to provide the methylated lignin.
    [0007] Without wishing to be bound to a particular theory, the inventors have observed that the methylation of lignin, by selectively and completely blocking the phenol functions, makes it possible very effectively to avoid the polymerization reactions of lignin during the action of the lignin. laccase, and thus improve depolymerization.
    [0008] The effect of methylation is particularly illustrated in the comparative examples described below. A non-phenolic lignin (unmethylated) treated by the process of the invention is not depolymerized. Phenolic lignin In the context of the present invention, the term "phenolic lignin" or "lignin", a natural form of lignin, comprising free phenol functions (Ph-OH). The structure of the lignin is in the form of a complex three-dimensional network derived from the polymerization of base units which have a phenylpropane pattern as their backbone. Lignin therefore more generally refers to "lignins", depending on the basic units that constitute it. Among the basic units of lignin, also called monolignols, there may be mentioned mainly paracoumaryl alcohol, coniferyl alcohol and sinapyl alcohol. In the context of the present invention, the term "depolymerization" is understood to mean a reaction in which covalent bonds of the polymeric structure of the lignin are broken, leading to depolymerization products smaller than the starting lignin. By "smaller size" it is meant that the depolymerization products obtained according to the process of the invention have a lower molecular weight than the starting non-phenolic lignin. However, in the context of the present invention, the meaning of the term "depolymerization" should not be limited to the conversion of non-phenolic lignin to base units as described above. Typically, the depolymerization products of lignin obtained according to the process of the invention are formed of a base unit, a few base units, or even a few tens of base units. In general, mixtures of depolymerization products having a different number of base units are obtained. Generally, given the complex structure of lignin, the process of the invention provides a mixture of depolymerization products of various sizes and structures, which can carry different functional groups, such as ketone, aldehyde, acid, phenol groups. , alcohol or methoxy for example, preferably acid, methoxy, alcohol and ketone. Said depolymerization products may be defined as "monomers", "oligomers" or "fragments" of non-phenolic lignin.
    [0009] Thus, the term "depolymerization process" also refers to a "fragmentation process" of the lignin structure. The depolymerization products obtained according to the process of the invention have a size of between 100 g / mol and 5000 g / mol, preferably between 150 g / mol and 4000 g / mol, advantageously between 200 g / mol of 3 g / mol. 000 g / mol, preferably between 500 g / mol and 2500 g / mol. The mixture of lignin depolymerization products is typically characterized by its average molecular weight M ', which is the weighted average weighted average of each product, and has the formula: where n denotes the number of products i and M, denotes the molecular mass of product i. For a mixture of depolymerization products obtained according to the method of the invention, the average molecular weight, M ', can be measured by Size Exclusion Chromatography (SEC). The mixture of depolymerization products typically has an average molecular weight M of between 100 g / mol and 5000 g / mol, preferably between 150 g / mol and 4000 g / mol, advantageously between 200 g / mol of 3 g. 000 g / mol, preferably between 500 g / mol and 2500 g / mol. The mixture of depolymerization products obtained according to the process of the invention can also be characterized by the molar ratio between the mass average molecular weight M of said mixture and the weight average molecular weight of the starting lignin Mi, g. Typically, using the process of the invention, a molar ratio Mw / M ,, g of between 1/3 and 1/10 is obtained. The non-phenolic lignin used as raw material in the process of the invention can be obtained by any method of functionalization (alkylation, methylation, ...) known per se, from any available source of phenolic lignin, that it is commercial or extracted from industrial residues rich in lignin.
    [0010] As a source of phenolic lignin, it is possible to use the lignins described below, and preferably Kraft lignin (for example from black liquor), possibly purified, or sugarcane bagasse lignin, or else any other industrial lignin.
    [0011] The lignins that can be used in the context of the present invention are described in particular in Lignins and Lignans: Advances in Chemistry, Cyril Heitne and John Schmidt, CRC Press, Taylor & Francis Group, 2010. Advantageously, the lignin has been pretreated so as to make it soluble. in an aqueous medium.
    [0012] As phenolic lignin, Kraft lignin can be used, for example extracted from black liquor. Kraft lignins (also known as thiolignins) are water-soluble compounds derived from the industrial production of pulp using sulphate ions.
    [0013] The Kraft lignin that can be used in the process of the invention is typically obtained by extraction of black liquor, which is the cooking liquor resulting from the manufacture of Kraft paper. It is an aqueous solution consisting of dissolved lignin and hemicellulose residues from pulp, as well as other inorganic chemical compounds (dissolved salts).
    [0014] Purification of the black liquor is typically carried out as follows. The black liquor is acidified to precipitate the lignin. The solid obtained is centrifuged, isolated and extracted with ethanol. The liquid fractions are recovered and concentrated by evaporating the ethanol. The residue is washed with an acidic solution to remove the residual salts. This gives Kraft lignin.
    [0015] Kraft lignin extracted from black liquor may be further purified as follows to remove impurities (typically monophenol compounds and degradation residues). The Kraft lignin is brought into contact with tetrahydrofuran, the mixture obtained is filtered to remove the solid, the filtrate is concentrated by evaporating the solvent, and the solid obtained is then washed with diethyl ether to remove organic impurities, such as monophenol compounds. (vanillin, vanillic acid, ...) and degradation residues. The recovered solid is dried to give purified Kraft lignin. As phenolic lignin, it is also possible to use lignin from sugarcane bagasse.
    [0016] Cane bagasse is the fibrous residue of sugar cane obtained after extraction of the juice. It constitutes an important waste of the sugar industry, which is not sufficiently valued. The sugarcane bagasse lignin is commercially available (Solvay). It is also possible to use an industrial lignin source or mixtures of compounds comprising lignins, typically biomass mixtures comprising lignin and other constituents, such as cellulose and / or hemicellulose, or else a mixture of Kraft lignins. The lignin used in the process of the invention typically has a weight average molecular weight (MI, g) of from 1,000 to 10,000 g / mol, preferably from 1,000 to 5,000 g / mol. The mass-average molecular mass of the starting lignin (MI, g) represents the weighted mass average of the weight of each lignin size, and has the formula: ## EQU1 ## in which n denotes the number of molecules lignin of size J and M denotes the molecular weight of a lignin of size j. This value is usually indicated by the lignin supplier or can be determined by size exclusion chromatography as described above. The steps of the process of the invention will now be described. Laccase oxidation step The first step of the process of the invention consists of bringing into contact, in at least one solvent, non-phenolic lignin as described above, a laccase, a redox mediator and a source of oxygen. This step leads to the oxidation of the non-phenolic lignin by the action of the laccase / mediator system in the presence of an oxygen source, whereby a mixture comprising oxidized non-phenolic lignin is obtained. By "oxidized non-phenolic lignin" is meant a non-phenolic lignin as defined above in which the hydroxyl functions in the benzylic position have been selectively converted into ketone functions.
    [0017] During this step, the non-phenolic lignin is essentially not depolymerized, that is to say that this step does not affect (or very little) the polymeric structure of the lignin, in particular it does not break ( or very few) CC links.
    [0018] Laccase Laccases (EC 1.10.3.2) are a family of enzymes found in many plants, fungi and microorganisms. In vivo, laccases have oxidative activity and act as a catalyst in an enzymatic oxidation process. The laccases used in the process of the invention may be derived from plants, fungi or microorganisms. Laccases from fungi include laccases of the genera Aspergillus, Neurospora (eg Crassa Neurospora), Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes (eg Trametes villosa and Trametes versicolor), Rhizoctonia (eg Rhizoctonia). solani), Coprinus (for example Coprinus cinereus, Coprinus comatus, Coprinus friesii and Coprinus plicatilis), Psathyrella (for example Psathyrella condelleana), Panaeolus (for example Panaeolus papilionaceus), Myceliophthora (for example Myceliophthora thermophila), Schytalidium (for example Schytalidium thermophilum). ), Polyporus (for example Polyporus pinsitus), Phlebia (for example Radiata phlebia), Pycnoporus (for example Pycnoporus cinnabarinus) or Coriolus (for example Coriolus hirsutus). The laccases derived from bacteria are, for example, from Bacillus. Preferably, a laccase from Trametes versicolor, marketed by Sigma Aldrich, is used. The ratio of the amount of laccase contacted (in mg) on the initial amount of non-phenolic lignin present in the reaction medium (in grams) is generally between 0.1 / 1 and 5/1. In the context of their use in vitro, for the oxidation of a substrate consisting of bulky molecules (typically with a molecular weight greater than 5,000 g / mol), laccases are generally associated with a "redox mediator", which is a chemical compound of small size (typically of molecular weight less than 1000 g / mol) acting as a redox intermediate between the laccase molecules and the substrate molecules to be oxidized. Redox mediators are described in particular in Bourbonnais R. Appl. Enviro. Microbiol. 1995, 61, 1876-1880.
    [0019] Redox mediators are also referred to as electron transfer agents because they facilitate the electronic transfer between the laccases and the substrate to be oxidized. The principle of redox mediation is a technology known per se.
    [0020] In the context of lignin treatment with laccases, in the absence of a redox mediator, the chemical interactions between laccases, which are large proteins, and lignin molecules, which are large polymers. , would be disadvantaged. A redox mediator is added to accelerate the laccase catalysed enzymatic oxidation process. The ratio of the amount of redox mediator used (in mmol) relative to the amount of laccase placed in the presence (in mg) is generally between 1/1 and 1/50. Preferably, the redox mediator used according to US Pat. The process of the invention is 2,2-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). This mediator is particularly suitable for the oxidation of lignin by laccases. Unlike other redox mediators, such as hydroxybenzotriazole (HOBt) which degrades rapidly in the presence of laccase by losing its mediator activity (HOBt becomes HBt by loss of oxygen atom), ABTS has a better activity 20 as a redox mediator. The following redox mediators may also be suitable for carrying out the invention: violet acid, hydroxyanthranilic acid, TEMPO, N-hydroxyphthalimide (Chakar et al., Can J. Chem 82: 344-352 (2004)). Source of oxygen By "oxygen source" is meant a reagent capable of regenerating (reoxidizing) the active sites of the laccase involved in the enzymatic oxidation process mentioned above, which ultimately provides the non-phenolic lignin oxidized from non-phenolic lignin. By "oxygen" is meant here oxygen (O 2). Preferably, the oxygen source is a gas comprising oxygen, such as air or pure oxygen. As a source of oxygen, mention may be made of pure oxygen (O 2), which is brought into contact, by bubbling at atmospheric pressure or under a pressure of a few bars, in the reaction medium comprising, at the beginning of the oxidation step, non-phenolic lignin, a laccase, a redox mediator and at least one solvent. It is thus possible to saturate the reaction medium advantageously with dissolved oxygen.
    [0021] As an oxygen source, mention may also be made of air or any mixture of oxygen enriched gas. By "placing in the presence of an oxygen source" is meant the introduction of said source of oxygen into the reaction medium of the process of the invention. Said introduction can be carried out punctually or prolonged, preferably prolonged, the objective being to saturate the reaction medium with dissolved oxygen. Alternatively, it is possible to use, instead of the oxygen source, any oxidant able to regenerate (reoxidize) the active sites of the laccase involved in the enzymatic oxidative process described above.
    [0022] The first stage of the process is advantageously carried out under conditions of pH and temperature appropriate to the reactivity of the laccase used, that is to say under conditions of pH and temperature which do not distort the properties of the laccase.
    [0023] Preferably, the first step of the process of the invention is carried out in an acid medium, typically in a buffer at pH = 4. Preferably, the first step of the process of the invention is carried out at a temperature of between 20 ° C. and 60 ° C, preferably around 40 ° C. These conditions are particularly suitable for the implementation of laccase from Trametes versicolor. The optimal pH and temperature conditions associated with the type of laccase used can be adopted, these conditions being generally known for a given laccase.
    [0024] The first step of the process according to the invention is carried out in the presence of at least one solvent. According to one embodiment, the solvent is a mixture comprising water and a polar organic solvent. Preferably, the solvent is a mixture comprising a buffer solution of acidic pH (typically at pH = 4) and a polar organic solvent. The use of such a mixture as a solvent has the advantage of solubilizing the non-phenolic lignin and all the reagents brought together. This gives a homogeneous reaction mixture in which the chemical interactions are facilitated. According to this embodiment, the solvent preferably comprises between 30% and 70% by volume of water, advantageously between 40% and 60%, relative to the total volume of solvent.
    [0025] The additional solvent is typically composed of a polar organic solvent, preferably sufficiently volatile to be separated from the depolymerization products by evaporation, optionally under reduced pressure. The solvent is typically a mixture of water to solubilize laccase and an ether capable of solubilizing non-phenolic lignin, such as dioxane. A dioxane / water mixture (1/1) can typically be used. The first step is carried out according to a dilution such that for 1 g of non-phenolic lignin, a volume of solvent of 10 ml to 100 ml is used, preferably 30 ml to 70 ml, typically 50 ml. Those skilled in the art will be able to adapt the dilution of the reaction medium depending on the solubility of the starting non-phenolic lignin, the reaction temperature and / or the viscosity of the reaction mixture. Depolymerization Step The inventors have found that by bringing the oxidized non-phenolic lignin from the first step of the process of the invention into the presence of an oxidizing agent, the structure of the lignin is efficiently depolymerized to obtain products. depolymerization of lignin. By "oxidizing agent" is meant a substance capable of oxidizing the species present in the reaction medium. Preferably, the oxidizing agent is nucleophilic. Examples of nucleophilic oxidizing agents that may be mentioned include peroxides, such as hydrogen peroxide (H 2 O 2) and benzoyl peroxide, or compounds such as O 3, KMnO 4 and NaO 4, or any conventionally nucleophilic oxidant. used in organic chemistry. The oxidizing agent is preferably hydrogen peroxide (H2O2). Hydrogen peroxide is typically available as a 35 weight percent aqueous solution, which is equivalent to a molar concentration of about 10 M. Preferably, typically when the oxidizing agent is hydrogen peroxide, the ratio of the amount of oxidizing agent placed in the presence (in mol) on the amount of oxidized non-phenolic lignin present in the reaction medium (in grams) is between 0.01 / 1 and 0.02 / 1. The second stage of the process is preferably carried out in a basic medium, especially when the oxidizing agent is hydrogen peroxide. Thus, when the first process step has taken place in a neutral or acidic pH medium, it is then necessary to pass into a basic medium, preferably before the oxidizing agent is brought into contact. To pass in basic medium can be used any method known per se, such as the addition of a basic aqueous solution, typically sodium hydroxide, in the reaction medium. Implementation of the method A typical implementation of the method of the invention will now be described. The process steps are typically carried out one after the other in the same reactor. Alternatively, these two steps can be carried out separately, in two separate reactors.
    [0026] According to a particular embodiment, in a reactor equipped with a heating system, a stirring means, and optionally a refrigerant system, the non-phenolic lignin and a part of the solvent are introduced and the mixture is agitated. 'to complete dissolution. A solution of redox mediator is then added to a portion of the solvent, and then a laccase solution is added to the remainder of the solvent. The oxygen source is then brought into contact with the catalyst, preferably continuously, typically by bubbling in the reaction medium when the oxygen source is gaseous. The temperature of the mixture is typically maintained between 20 ° C and 60 ° C, preferably around 40 ° C, until complete oxidation of the non-phenolic lignin is achieved. At this point, the first step of the process is completed. The oxidized non-phenolic lignin can then be recovered. Alternatively, one can advantageously continue the process by performing the second step in the same reactor. According to this mode, one can place in basic pH by basifying if necessary the reaction medium. The oxidizing agent can then be added, preferably continuously, typically dropwise, when the oxidizing agent is a solution. The temperature of the mixture is typically maintained above 70 ° C, typically around 90 ° C.
    [0027] The progress of the reaction is followed by SEC (steric exclusion chromatography). Stirring and heating of the reaction medium is continued until the desired degree of depolymerization is obtained. Once the depolymerization is complete, the depolymerization products of the lignin can be recovered, using standard purification techniques in the field, namely filtration, extraction, distillation and / or chromatographic separation. In particular, the solvent may be removed by evaporation, possibly under reduced pressure. The method of the invention will now be illustrated by means of examples and comparative examples. EXAMPLES Reagents Black liquor (supplied by SMURFIT Kappa) Dimethylsulfate (marketed by Sigma Aldrich) Cane bagasse lignin (supplied by SOLVAY) 2,2-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS (marketed by Sigma Aldrich) Trametes versicolor Laccase (marketed by Sigma Aldrich) Acetate buffer (pH = 4, 50 mM) Hydrogen Peroxide (35%) (commercially available from Sigma Aldrich) Example 1 - Kraft Lignin Extraction From the black liquor Kraft lignin was extracted from the cooking liquor (marketed by SMURFIT KAPPA) by acid treatment, so as to precipitate it. Ethanol post-treatment removes the residual sodium salts. 800 g of black liquor are dissolved in 2 L of distilled water and the mixture is slowly acidified (pH = 13 to pH = 1.5) with a 6N HCl solution. The mixture is then centrifuged at 4000 rpm for 10 minutes, the pellet is recovered and treated again with a solution of HCl at pH = 1.5 and centrifuged again (repeat 3 times with HCl at pH = 1.3). Ethanol is then added to the solid obtained. After filtration, the ethanol phase is evaporated and the residue is washed with a solution of HCl at pH = 1.5 to remove the residual salts. After centrifugation, the pellet is recovered and lyophilized. 180 g of Kraft lignin is obtained. From the 180 g of Kraft lignin thus obtained, 10 g are taken and dissolved in 200 ml of tetrahydrofuran (THF). After stirring for 30 minutes under ultrasound, the mixture is filtered to remove 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 (Et2O). A fraction remains insoluble in Et 2 O (8.1 g) and the soluble fraction (0.9 g) is analyzed by GC-MS after silylation to make the compounds volatile. This analysis shows the presence of monophenols such as vanillin or vanillic acid and residues of the degradation of sugars. The insoluble fraction is composed of purified Kraft lignin, which can be used later for the methylation and depolymerization process of the present invention. Example 2A - Methylation of Kraft Lignin Kraft lignin (1 g) was dissolved in 20 ml of a 0.7 M NaOH solution (0.56 g) and the mixture was stirred at room temperature for 10 minutes, followed by 8 ml of dimethylsulphate ((Me0) 2SO2) are slowly added (drops). Stirring is continued for 30 minutes at room temperature. The reaction mixture is then heated at 80 ° C for 4 hours, adding 0.7 M NaOH solution to homogenize the reaction medium (about 10 mL of additional 0.7 M NaOH is added). After 4 hours of reaction, the mixture is brought back to room temperature and acidified with a 2M solution of HCl to pH = 2. The crude reaction product is filtered and the solid obtained is washed with water. distilled and dried by lyophilization. 0.874 g of methylated lignin is obtained. Example 2B - Methylation of sugarcane bagasse lignin The sugarcane bagasse lignin was supplied by SOLVAY was used without prior pretreatment. The lignin of bagasse is treated under the conditions described in Example 2A. 0.92 g of methylated lignin is obtained. Example 3A - Methyl lignin depolymerization (derived from Kraft lignin) 1 g of methylated lignin obtained in Example 2A was dissolved in 25 ml of dioxane and the mixture was stirred at room temperature until complete dissolution. Then, 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; mM), which is added to the methylated lignin solution. A solution of 5 mg of laccase in 200 ml of acetate buffer is then prepared, 24 ml of this solution is taken and slowly added to the medium containing the methylated lignin at 40 ° C. After addition of the laccase, oxygen gas is introduced into the mixture by bubbling for 1 hour at 40 ° C, using a flask filled with oxygen.
    [0028] After reacting for 22 hours at 40 ° C., 1 ml of a 3M solution of NaOH is added, then 1 ml of an aqueous solution of hydrogen peroxide (35 ° A, 10 mmol) is added slowly over mixture being stirred at 90 ° C. The progress of the reaction is followed by SEC (steric exclusion chromatography). The analysis by SEC is carried out using three columns of type TSK-gel (3000 PW, 4000 PW, 3000 PW) coupled in series, with 1 M sodium hydroxide up to pH = 12 and NaN 3 (3%) in osmosis water as eluent. The flow rate is 1 mL / min and the detection is by UV at a wavelength of 280 nm. The first step of the reaction (action of the laccase / ABTS system) leads to a low depolymerization. The second step (action of hydrogen peroxide) leads to a strong depolymerization, with the formation of low molecular weight molecules that have been isolated and identified. After 40 hours of reaction, the mixture is brought to pH = 6-7 using a 1N solution of HCl and the crude reaction product is extracted with dichloromethane (3 × 50 ml). After drying the organic phase over sodium sulphate (Na.sub.2SO.sub.4) and evaporation of the volatile solvents, the crude product is purified by flash chromatography (using 99/1 to 90/10 dichloromethane / methanol as eluent). 30 mg of 3,4-dimethoxybenzoic acid were thus isolated from 1 g of methylated lignin obtained in Example 2A. Comparative Example 1 For comparison, the purified Kraft lignin obtained in Example 1 was subjected to the action of the laccase / ABTS system, directly applying the reaction conditions described in Example 3A (i.e. ie, without methylation of the lignin). The progress of the reaction is followed by SEC (conditions described above): t (hours) Mw Mn M, 132 0 661 902 616 2 1 2 3 2 3 1 2 3 1 Mp: peak of molecular weight, in g / mol Mn: number average molecular weight, in g / mol MA, weight average molecular weight, in g / mol It is observed that the average molecular weight of the species in presence increases with the reaction time. 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 weight of the lignin. Example 3B - Depolymerization of methylated lignin (from bagasse lignin) The methylated lignin obtained in Example 2B was treated by applying the reaction conditions described in Example 3A (the action of laccase is prolonged for 65 hours, after which we add the hydrogen peroxide). The progress of the reaction is followed by SEC (conditions described above): t (hours) Mp Mn M, 0 5054 1414 10245 2 4806 759 8681 65 3555 269 6857 70 2443 1601 4907 80 1525 130 2112 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 low molecular weight phenols that have not been isolated and identified. Comparative Example 2 By way of comparison, bagasse lignin was subjected to the action of the laccase / ABTS system, directly applying the reaction conditions described in Example 3A (i.e. without performing methylation of lignin). The progress of the reaction is followed by SEC (conditions described above): t (hours) Mp Mn M, 0 2254 145 4957 1 4048 417 8245 24 9242 4282 1732 9 It is observed that the average molecular mass of the species in the presence increases with the reaction time.
    [0029] 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 weight of the lignin.
    [0030] Comparative Example 3 By way of comparison, the methylated lignin obtained in Example 2B was subjected to the action of hydrogen peroxide in a basic medium, without subjecting it to the action of the laccase / ABTS system before. 1 g of methylated lignin obtained in Example 2B is dissolved in 25 ml of dioxane and the mixture is stirred at ambient temperature until complete dissolution. Then 25 mL of osmosis water is added to the mixture. 1 ml of a 3M solution of NaOH is added, then 1 ml of an aqueous solution of hydrogen peroxide (35%, 10 mmol) is slowly added, the mixture being stirred at 90 ° C.
    [0031] The progress of the reaction is followed by SEC (conditions described above): t (hours) Mp Mn M, 0 5054 1414 10245 5 2541 404 4651 2394 153 4725 A less significant depolymerization of methylated lignin is observed it is pretreated by the action of the laccase / ABTS system. Without wishing to be bound to a particular theory, this partial depolymerization is explained by the fact that, in lignin (and thus in methylated lignin), a certain percentage of hydroxyl functions intended to be oxidized by the bringing into the presence of the laccase system / mediator are already in the oxidized state, that is to say in the form of ketones. The oxidative disruption of the C-C bonds in the vicinity of these ketone functions causes partial depolymerization.
    [0032] This example, however, shows that the first step of the process is necessary to optimize the depolymerizing action of hydrogen peroxide. Comparative analysis of bagasse lignins obtained after Examples 2B, 3B and at the end of Comparative Example 3 shows that the fragmentation of lignin is most effective when lignin is successively subjected to methylated by the action of the laccase / ABTS system and then by the action of hydrogen peroxide: Lignin of Example 2B, not depolymerized Mhg = 10245 Lignin of Example 3B, treated with laccase + H202 M, = 2112 Lignine of Comparative Example 3, treated with H 2 O 2 M, = 4725
    权利要求:
    Claims (1)
    [0001]
    REVENDICATIONS1. A process for the depolymerization of lignin, comprising: a step of oxidizing non-phenolic lignin by bringing non-phenolic lignin, laccase, a redox mediator and a dye source into at least one solvent oxygen, whereby a mixture comprising oxidized lignin is obtained, and a depolymerization step of the oxidized lignin thus obtained, by the addition of an oxidizing agent, said non-phenolic lignin being obtained from phenolic lignin by functionalization of phenol functions of said phenolic lignin. The process of claim 1 wherein the non-phenolic lignin is alkylated lignin. The process of any one of claims 1 or 2, wherein the non-phenolic lignin is methylated lignin. The process according to claim 3, wherein the methylated lignin is obtained by bringing phenolic lignin and a methylating agent into a basic aqueous solution. The process of Claim 4 wherein the methylating agent is dimethylsulfate. A process as claimed in any one of claims 1 to 5, wherein the source of oxygen is pure oxygen or air. A process according to any one of claims 1 to 6, wherein the redox mediator is 2,2-azino-bis- (3-ethylbenzothiazoline-6-sulphonic acid). The process of any one of claims 1 to 7, wherein the oxidizing agent is hydrogen peroxide. 152. 3. 20 4. 5. 25 6. 30 7. 8.9. Process according to any one of Claims 1 to 8, in which the oxidation step is carried out in a basic medium. The process of any one of claims 1 to 9, wherein the non-phenolic lignin is derived from the functionalization of Kraft lignin phenol functions or sugarcane bagasse lignin.
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    同族专利:
    公开号 | 公开日
    JP2017505754A|2017-02-23|
    US10059731B2|2018-08-28|
    PT3074569T|2019-08-01|
    WO2015078920A1|2015-06-04|
    JP6474138B2|2019-02-27|
    SG11201604262SA|2016-07-28|
    EP3074569A1|2016-10-05|
    US20160376300A1|2016-12-29|
    CN105899728A|2016-08-24|
    ES2742814T3|2020-02-17|
    EP3074569B1|2019-05-01|
    BR112016012027A2|2018-02-06|
    FR3013713B1|2016-01-15|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1361718A|FR3013713B1|2013-11-27|2013-11-27|PROCESS FOR DEPOLYMERIZATION OF LIGNIN WITH LACCASES|FR1361718A| FR3013713B1|2013-11-27|2013-11-27|PROCESS FOR DEPOLYMERIZATION OF LIGNIN WITH LACCASES|
    PT14806579T| PT3074569T|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
    EP14806579.0A| EP3074569B1|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
    PCT/EP2014/075680| WO2015078920A1|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
    JP2016534941A| JP6474138B2|2013-11-27|2014-11-26|Method for depolymerization of lignin with laccase|
    ES14806579T| ES2742814T3|2013-11-27|2014-11-26|Depolymerization procedure of lignin with lacases|
    US15/039,796| US10059731B2|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
    CN201480073131.0A| CN105899728A|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
    BR112016012027-2A| BR112016012027B1|2013-11-27|2014-11-26|LIGIN DEPOLYMERIZATION PROCESS|
    SG11201604262SA| SG11201604262SA|2013-11-27|2014-11-26|Process for depolymerization of lignin by laccases|
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