A biorefinery process

Abstract

A process to extract lignin and / or cellulose fibres from a lignocellulosic biomass substrate comprising the steps of: pre-treating the lignocellulosic biomass substrate with a binary acidic deep eutec

Description

Field of the Invention The present invention relates to a biorefinery process, in particular a process to extract lignin and / or cellulose fibres from a lignocellulosic biomass substrate and a process for conversion of cellulose fibres to nanocellulose. Background to the Invention Lignin is the second most abundant bioorganic polymer after cellulose. Lignin is not naturally available in its isolated form, but it is physically combined with cellulose and hemicellulose. It is a widely available, sustainable, and inexpensive material. Lignin polymer is a by-product which is potentially available in comparatively large amounts, meaning that the valorisation of lignin in composite materials for any high-value application could have huge economic benefits (Mariana et al 2021) however there are challenges associated with its recovery. Cellulose nanomaterials are another highly valuable product. Cellulose nanomaterials are cellulosic materials with nano-scale dimensions usually extracted from lignocellulosic biomass. Usually, this material possesses various attractive characteristics such as non-toxic nature, excellent biocompatibility, remarkable optical properties, high aspect ratio, superior mechanical capabilities, tailorable surface chemistry and many others. Besides, it has potential use in various fields, such as application in packaging materials to construction composites, electronics devices to biomedical scaffolds, and many others. Thus, due to its growing applications and demand, there is a need to develop sustainable processes for the production of nanocellulose. AU2020100319A4 discloses a method for preparing cellulose nanofibrils by combining the treatments of deep eutectic solvent pre-treatment (choline chloride and urea [in a molten state]) with high-pressure homogenization micro fluidization to prepare cellulose nanofibrils in place of traditional mechanical processing alone (high energy consumption) or chemical or biological pre-treatments (reactive products are harmful to the environment). This method does not entirely remove the use of expensive machinery. CN113880958A discloses a method for preparing kelp residue nano-crystal cellulose by utilizing eutectic solvent and ultrasound. This method discloses the preparation and use of imidazole and glycerol (deep eutectic solvent 1) and choline chloride and oxalic acid dihydrate (deep eutectic solvent 2). KR20210117531A discloses a manufacturing method of cellulose nanofibril using Betaine-Lactic Acid Deep Eutectic Solvent from wood flour. CN112482069A discloses a method for preparing high-yield dissolving pulp by using eutectic solvent (lactic acid and choline chloride). This method does not appear to disclose the utilization of the separated lignin, nor does it disclose the processing of nanocellulose. CN115197342A discloses a deep eutectic solvent preparation (choline chloride, p-chlorobenzenesulfonic acid and ethylene glycol) and the method thereof and application of deep eutectic solvent in depolymerizing and separating wood fiber raw materials. This approach uses a hydrothermal coupling treatment method of deep eutectic solvent and acetic acid, which can separate the extraction and depolymerization of hemicellulose and the extraction and separation of lignin to obtain additional Xylo-oligosaccharides with higher value, and at the same time in order to improve the separation efficiency of lignin and cellulose in lignocellulosic raw materials. CN112029115A discloses a method for separating and extracting lignin by in-situ synthesis of deep eutectic solvent. It is an object of the invention to overcome at least one of the above-referenced problems and provide a process for extracting lignin or cellulose fibres from a lignocellulosic biomass substrate that is more efficient and environmentally friendly. Summary of the Invention The invention provides an efficient biorefinery process for the extraction of cellulose fibres and lignin from lignocellulosic biomass, especially from agricultural wastes. The invention comprises the application of a binary acidic deep eutectic solvent for the extraction of cellulose and lignin. Further, the invention also describes the application of a hydrated ternary deep eutectic solvent in the hydrolysis of cellulose fibres for conversion to nanocellulose. In a first aspect, there is provided a process to extract lignin and / or cellulose fibres from a lignocellulosic biomass substrate comprising the steps of: A) pre-treating the lignocellulosic biomass substrate with a binary acidic deep eutectic solvent to produce an intermediate product, wherein the binary acidic deep eutectic solvent typically comprises betaine hydrochloride and acid; B) separating of the intermediate product into the liquid phase and the solid phase; and C) treating the liquid phase to extract lignin by precipitation, or D) treating the solid product to extract cellulose fibres. The invention addresses environmental concerns associated with solvent use by employing deep eutectic solvents that are considered to be green in nature and have minimal environmental impact. By doing this, it makes the process of extracting biomaterials more sustainable and less harmful to the environment. A further advantage of the invention is the simplicity and efficiency in preparing the deep eutectic solvents. The process is notably straightforward, requiring minimal time (less than 10 minutes) compared to the synthesis of other reported deep eutectic solvents, contributing to the overall efficiency and practicality of the invention. A further advantage of the invention is the extended stability of the synthesized deep eutectic solvent (> 3 weeks) at room temperature. This remarkable stability period enhances the reliability and usability of the solvents, contributing to the overall effectiveness of the invention. In any embodiment, the process comprises steps C and D. In any embodiment, step A comprises heating the lignocellulosic biomass substrate with a binary acidic deep eutectic solvent. In any embodiment, the binary acidic deep eutectic solvent comprises betaine hydrochloride and formic acid. In any embodiment, a molar ratio of betaine hydrochloride to acid is 1:10 to 1:20. In any embodiment, the molar ratio of betaine hydrochloride to acid is 1:15. In any embodiment, the lignin extract is obtained from the liquid phase by the addition of water to the liquid phase to produce a diluted liquid phase, resting of the diluted liquid phase to allow precipitation of the lignin, and separation of the precipitated lignin. In any embodiment, the lignin extract has a purity of at least 60%, 70% or 80%. In any embodiment, the process comprises recovery of at least 40%, 45% or 50% of the lignin content in the lignocellulosic biomass substrate (w / w). In a y embodiment, the lignocellulosic biomass substrate comprises at least 15%, 17% or 19% lignin (w / w). In any embodiment, the cellulose fibre extract is obtained from the solid product by treating the solid product with an oxidising agent (and optionally heat) to produce a slurry, separating the slurry into a slurry liquid phase and a slurry solid phase, and washing of the slurry solid phase with water to produce a solid product, wherein the solid product comprises the cellulose fibres. In any embodiment, the oxidising agent is selected from a hypochlorite salt and hydrogen peroxide. In any embodiment, the oxidising agent is a hypochlorite salt (e.g. (sodium hypochlorite) comprising at least 1%, 1.5% or 2% active chlorine. In any embodiment, the cellulose fibre extract has a purity of at least 70%, 80% or 90%. In any embodiment, the process comprises recovery of at least 50%, 60%, 70% or 77% of the cellulose fibre content in the lignocellulosic biomass substrate (w / w). In any embodiment, the lignocellulosic biomass substrate comprises at least 35%, 40% or 44% cellulose (w / w). In any embodiment, the cellulose fibres are converted to a nanocellulose product by treating the cellulose fibres with a hydrated ternary deep eutectic solvent optionally followed by an ultrasound treatment. In any embodiment, the hydrated ternary deep eutectic solvent comprises betaine hydrochloride. In any embodiment, the hydrated ternary deep eutectic solvent comprises betaine hydrochloride, acid and water. The acid may be selected from oxalic acid and tartaric acid, or a (di)hydrate thereof. In any embodiment, the acid in the hydrated ternary deep eutectic solvent comprises tartaric acid, preferably L-(+)-tartaric acid. In any embodiment, the hydrated ternary deep eutectic solvent comprises betaine hydrochloride, acid and water mixed in a 5-15:5-15:100-300 molar ratio. In any embodiment, the hydrated ternary deep eutectic solvent comprises betaine hydrochloride, acid and water mixed in an approximate 10:10:200 molar ratio. In any embodiment, a ratio of the lignocellulosic biomass substrate to the binary acidic deep eutectic solvent is in the range 1:10 to 1:30 w / w. In any embodiment, the ratio of the lignocellulosic biomass substrate to the binary acidic deep eutectic solvent ratio in the range 1:15 to 1:25 w / w. In any embodiment, the process comprises an initial step of preparing the lignocellulosic biomass substrate (e.g. straw) comprising size reducing the substrate, and optionally sieving the size reduced substrate. In another aspect, the invention provides a purified lignin extract obtained by the process of the invention. In any embodiment, the purified lignin extract has a purity of at least 60%, 70% or 80 % (w / w). In another aspect, the invention provides a purified cellulose fibre extract obtained by the process of the invention. In any embodiment, the purified cellulose fibre extract has a purity of at least 60%, 70% or 80 % (w / w). In another aspect, the invention provides a binary acidic deep eutectic solvent comprising (or consisting essentially of) betaine hydrochloride and formic acid, at 1:15 molar ratio. In another aspect, the invention provides a ternary deep eutectic solvent comprising betaine hydrochloride, acid and water mixed in a 5-15:5-15:100-300 molar ratio. In any embodiment, the acid is selected from tartaric acid, oxalic acid, and (di)hydrates thereof. In any embodiment, the acid is L-(+)-tartaric acid. In any embodiment, the hydrated ternary deep eutectic solvent comprises betaine hydrochloride, acid and water mixed in an approximate 10:10:200 molar ratio. Brief Description of the Figures Figure 1: A flow diagram representing the process of one embodiment of the invention. Figure 2: A flow diagram representing the process of obtaining a nanocellulose product according to another embodiment of the invention. Detailed Description of the Invention All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. Definitions and general preferences Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art: Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps. As used herein the term “Deep eutectic solvent” refers to the combining of two components: one acting as the hydrogen bond donor and the other as the hydrogen bond acceptor. These two components are therefore held together mostly through an intermolecular hydrogen bond interaction. As used herein the term “Biomass” refers to a renewable organic material which is derived from plants and animals. As used herein the term “Lignocellulosic biomass” refers to a plant or plant-based material which is generally not used for human food or animal feed purposes primarily including agricultural residues, energy crops, forestry residues, hay, stray, grass, and yard trimmings. As used herein the term “Lignin” refers to an organic polymer that forms important structural materials in the tissues of the majority of plants and is physically combined with cellulose and hemicellulose in its natural form and requires physical and / or chemical extraction. As used herein the term “Cellulose fibres” refers to fibres obtained from cellulose. As used herein the term “Acid” refers to a substance containing hydrogen ions (and is capable of donating a proton) and has a pH less than 7. Examples of acids include but are not limited to formic acid, L-(+)-Tartaric acid, oxalic acid, and (di)hydrates thereof.. As used herein the term “Purified lignin extract” refers to lignin that has been isolated from its natural source and contains a concentrated form of lignin, typically having a % purity of at least 60%. 70% or 80% (w / w). As used herein the term “Purified cellulose fibres” refers to cellulose fibres that have been isolated from its natural source and contains a concentrated form of cellulose fibres, typically having a % purity of at least 70%, 75% or 80% (w / w).. As used herein the term “Nanocellulose” refers to cellulose that has been isolated from its natural source and further broken down generally by hydrolysis to the nanoscale into a nanomaterial, usually within the range 1 to 100 nm. As used herein the term “Oxidising agent” refers to a substance in a redox reaction that “gains” an electron from a reducing agent. Oxidising agents will “oxidise” other substances by making that substance lose electrons. Oxidising agents employed in the present invention include, for example, hypochlorite and hydrogen peroxide As used herein the term “Separating” as applied to a composition refers to gaining both the liquid phase and the solid phase which were previously combined in one mixture. This can be achieved by various techniques including but not limited to, centrifugation, and various filtration techniques including coarse filtration and membrane filtration such as cation and anion exchange. As used herein the term “% purity” as applied to lignin extract refers to the amount by weight of lignin in the extract. The same definition applies to the % purity of the cellulose fibre extract. As used herein, the term “% recovery” as applied to the lignin extract refers to the weight % of lignin in the lignocellulosic biomass substrate that is recovered in the lignin extract as a result of the process of the invention. Thus, if 100g of lignocellulosic biomass substrate contains 20g lignin, and the lignin extract contains 15g lignin, the % recovery would be 75%. The same definition applies to the % recovery of cellulose fibres. Exemplification The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention. Preparation of Biomass samples: - Barley straw was used as the biomass in this work. - The straw samples were cut into small pieces and ground using a high-speed grinder (Manufacturer: CGOLDENWALL, China; 2400 W). - The ground samples were passed through a 1 mm sieve. Subsequently, the samples were stored in zip-lock bags for future use. Synthesis of Binary Acidic Deep Eutectic Solvent: - The binary acidic deep eutectic solvent was prepared using Betaine hydrochloride (Merck, CAS: 590-46-5; Molecular Weight: 153.61 g / mol; Physical state: powder or crystals) and Formic acid (Merck, CAS: 64-18-6; Molecular Weight: 46.03 g / mol; Physical state: liquid). - Betaine hydrochloride and formic acid were mixed at a 1:15 molar ratio. - Exactly 100 g of betaine hydrochloride was weighed and transferred to a beaker. Then, 449 g of formic acid was slowly added to the beaker. - The betaine hydrochloride-formic acid mixture was heated in a hot plate at 80°C with continuous stirring at 300 rpm until a transparent liquid was formed. The process required less than 10 minutes to form the transparent solvent. The process was carried out in a fume hood. - Once the solvent was formed, it was allowed to cool down and then transferred to a Duran glass bottle for storage. The prepared solvent is stable at room temperature for at least one month. Synthesis of Hydrated Ternary Deep Eutectic Solvent - The hydrated ternary deep eutectic solvent was prepared using Betaine hydrochloride (Merck Ireland; CAS: 590-46-5; Molecular Weight: 153.61 g / mol; Physical state: powder or crystals), L-(+)-Tartaricacid (Fisher Scientific Ireland; CAS: 87-69-4; Molecular Weight: 150.09 g / mol; Physical state: solid) and deionised water (Molecular Weight: 18 g / mol). -Betaine hydrochloride, L-(+)-Tartaric acid and water were mixed at a 1:1:20 molar ratio. - Exactly 50 g of betaine hydrochloride and 48.85 g of L-(+)-Tartaric acid were weighed and transferred to a beaker. Then, 117.18 g of deionised water was added to the beaker. The mixture was heated in a hot plate at 80TD with continuous stirring at 300 rpm until a transparent liquid was formed. The process required less than 10 minutes to form the transparent solvent. The process was carried out in a fume hood. - Once the solvent was formed, it was allowed to cool down and then transferred to a Duran glass bottle for storage. The prepared solvent is stable at room temperature for at least three weeks. Pretreatment of biomass samples using binary acidic deep eutectic solvent -The binary deep eutectic solvent (DES) consisting of Betaine hydrochloride and formic acid (1:15 molar ratio) was utilised for the pretreatment of biomass. - The biomass to DES ratio of 1:20 w / w was used in the pretreatment process. Exactly 10 g of Barley straw was mixed with 200 g of DES in a Duran glass bottle. -A water bath with shaking mechanism was used as the heat source for the pretreatment process. -The bottle containing the sample was kept in the water bath. The treatment was carried out at 90 TD for 3 hours with continuous shaking at a speed of 120 rpm. - After the treatment was completed, the samples container was kept in the ice bath for 15 minutes to allow the sample to cool down. - After cooling, the solid and liquid portion of the treated samples were separated by centrifugation at 4500 x g for 20 minutes. The liquid portion was kept in a conical flask. - The solid residue was washed 5 times with water. Each washing step involved saturating the residue with a sufficient amount of water, ensuring thorough mixing, and subsequently subjecting the mixture to centrifugation at 4500 x g for a duration of 10 minutes. The supernatant obtained from this process was then combined with the liquid portion obtained during the initial centrifugation step conducted after the pretreatment was finished. - The washed solid residue was freeze dried for further downstream processing and composition analysis. Precipitation and recovery of lignin - The liquid portion obtained from pretreatment process was used in precipitation and recovery of lignin. - Water was added to the liquid portion at a volumetric ratio of 6:1, and the resulting mixture was then left undisturbed for a period of 1 day, allowing for the precipitation of solid components. -On the following day, the solid material was recovered through centrifugation at 4500 x g for 20 minutes. The supernatant was used in a rotary evaporator to recycle the deep eutectic solvent. -The solid material was washed two times with water. Each washing step involved saturating the residue with a sufficient amount of water, ensuring thorough mixing, and subsequently subjecting the mixture to centrifugation at 4500 x g for a duration of 10 minutes. - After washing, the solid material was freeze dried and the resulting solid material is the lignin. Purification of the cellulose-rich solid component - Sodium hypochlorite solution containing 2% active chlorine was used for the purification of the cellulose-rich solid component obtained from the pretreatment process. -The solid component was mixed with the solvent at a ratio of 1:20 w / v. The treatment was carried out in a water bath at 70 'C for 30 minutes with continuous shaking at a speed of 80 rpm. The process was carried out in a fume hood. - After the treatment, the solid and liquid portion were separated by centrifugation at 4500 x g for 10 minutes. The liquid portion was stored in chlorinated solvent waste container and disposed of as per the waste disposal guidelines. -The solid portion was washed two times with water. Each washing step involved saturating the residue with a sufficient amount of water, ensuring thorough mixing, and subsequently subjecting the mixture to centrifugation at 4500 x g for a duration of 10 minutes. -The solid component obtained from this process was high-purity cellulose fibres. The sample was freeze dried and was subsequently ground into fine powder using a high-speed grinder. Production of nanocellulose - The hydrated ternary deep eutectic solvent consisting of Betaine hydrochloride, L-(+)-Tartaric acid and water (1:1:20 molar ratio) was used for the hydrolysis of cellulose fibres obtained from Section D. - Exactly 0.2 g of dried cellulose fibres was mixed with 10 ml of deep eutectic solvent (DES) at a solid-to-DES ratio of 1:50 w / v. -The cellulose-DES mixture was mixed properly and then treated at 90 °C for 2 h in a shaking water bath using a shaking speed of 150 rpm. Post DES treatment, cold water was added to the cellulose-DES mixture (10 times the volume), and then centrifugation was carried out for 20 minutes at 4500 x g. The solid residue was further washed with deionised water for 3 times. Each washing step involved saturating the residue with a sufficient amount of water, ensuring thorough mixing, and subsequently subjecting the mixture to centrifugation at 4500 x g for a duration of 10 minutes. After washing, 200 ml of deionised water was mixed with the treated cellulose and the mixture was ultrasonicated for 2 hours using probe-type ultrasound (3 mm diameter). 5 Table 1: Cellulose yield and purity Parameters Content (%) $ Cellulose content in raw biomass 44.09 ±1.11 # Cellulose yield with respect to the initial processed weight of biomass 37.72 ± 0.68 * Cellulose purity 90.75 ± 2.88 * Cellulose recovery with respect to the initial cellulose content in raw biomass 77.64 ± 2.76 Data are represented as mean ± SD ($ 3 replications; # 4 replications; * 8 replications) Table 2: Lignin yield and purity Parameters Content (%) $ Lignin content in raw biomass 19.84 ±1.28 * Lignin solubilization during binary DES treatment 67.01 ± 4.61 * Pure lignin precipitation efficiency with respect to the amount of lignin solubilized during binary DES treatment 76.67 ± 2.92 # Lignin yield with respect to the initial processed weight of biomass 12.66 ±0.71 * Lignin purity 80.43 ±1.98 * Pure Lignin recovery with respect to the initial lignin content in raw biomass 51.33 ±3.48 10 Data are represented as mean ± SD ($ 3 replications; # 4 replications; * 8 replications) Equivalents The foregoing description details presently preferred embodiments of the present 5 invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto. 10

Claims

1. A process to extract lignin and / or cellulose fibres from a lignocellulosic biomass substrate comprising the steps of:(a) pre-treating the lignocellulosic biomass substrate with a binary acidic deep eutectic solvent to produce an intermediate product, wherein the binary acidic deep eutectic solvent comprises betaine hydrochloride and acid;(b) separating of the intermediate product into the liquid phase and the solid phase; and(c) treating the liquid phase to extract lignin by precipitation, or(d) treating the solid product to extract cellulose fibres.

2. A process according to Claim 1, comprising steps (c) and (d).

3. A process according to Claim 1 or 2, wherein the lignin is extracted from the liquid phase by addition of water to the liquid phase to produce a diluted liquid phase, resting of the diluted phase to allow precipitation of the lignin, and separation of the precipitated lignin.

4. A process according to any preceding Claim, wherein the cellulose fibres are extracted from the solid product by treating the solid product with an oxidising agent to produce a slurry, separating the slurry into a slurry liquid phase and a slurry solid phase, washing of the slurry solid phase with water to produce a solid product, wherein the solid product comprises the cellulose fibres.

5. A process according to any preceding Claim, wherein the cellulose fibres are converted to a nanocellulose product by treating the cellulose fibres with a hydrated ternary deep eutectic solvent followed by an ultrasound trreatment.

6. A process according to Claim 5, wherein the hydrated ternary deep eutectic solvent comprises Betaine hydrochloride, L-(+)-Tartaric acid and water.

7. A process according to any preceding Claim, wherein the binary acidic deep eutectic solvent comprises betaine hydrochloride and formic acid.

8. A process according to any preceding Claim, wherein a molar ratio of betaine hydrochloride to acid is 1:10 to 1:20.

9. A process according to Claim 8; wherein the molar ratio of betaine hydrochloride to acid is 1:15.

10. A process according to any preceding Claim, wherein a ratio of the lignocellulosic biomass substrate to the binary acidic deep eutectic solvent is in the range 1:10 to 1:30 w / w.11 .A process according to Claim 10, where in the ratio of the lignocellulosic biomass substrate to the binary acidic deep eutectic solvent ratio in the range 1:15 to 1:25 w / w.

12. A purified lignin extract obtained by a process of any of Claims 1 to 11.

13. A purified lignin extract according to Claim 12, wherein the purified lignin extract has a purity of at least 80 % w / w.

14. A purified cellulose fibre extract obtained by a process of any of Claims 1 to 11.

15. A binary acidic deep eutectic solvent comprising betaine hydrochloride and formic acid, at molar ratio of about 1:15.19

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