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Title:
METHODS AND SYSTEMS FOR FRACTIONAL DISSOLUTION OF LIGNINS IN SEGMENTED CONTINUOUS FLOW MODE
Document Type and Number:
WIPO Patent Application WO/2020/128034
Kind Code:
A1
Abstract:
The invention relates to a system and a method for continuous segmented flow-based fractional dissolution of lignins, functionalized lignins, fractions of lignins, and/or fractions of functionalized lignins.

Inventors:
CRESTINI CLAUDIA (IT)
LANGE HEIKO (IT)
ARGYROPOULOS DIMITRIS (US)
Application Number:
PCT/EP2019/086784
Publication Date:
June 25, 2020
Filing Date:
December 20, 2019
Export Citation:
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Assignee:
UNIV CA FOSCARI (IT)
UNIV NORTH CAROLINA STATE (US)
International Classes:
C08H7/00; B01D11/02; C08L97/00
Domestic Patent References:
WO2018224598A12018-12-13
WO2015178771A12015-11-26
WO2015178771A12015-11-26
WO2011154293A12011-12-15
Foreign References:
GB2491169A2012-11-28
US20110262985A12011-10-27
US7465791B12008-12-16
US20100170504A12010-07-08
Other References:
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Attorney, Agent or Firm:
CATTANEO, Elisabetta et al. (IT)
Download PDF:
Claims:
Claims:

1. A system for continuous segmented flow-based fractional dissolution of lignins, functionalized lignins, fractions of lignins, and/or fractions of functionalized lignins, the system comprising at least the following components:

at least one solvent reservoir for containing at least one solvent effective to solubilize at least a portion of an isolated lignin material;

at least one pump downstream from the solvent reservoir, the pump configured to pump the at least one solvent or mixture of solvents at a selectable flow rate against constant or variable back-pressure;

at least one lignin sample holder downstream from the pump, the sample holder configured to contain the isolated lignin material and solvent or mixture of solvents and configured to allow outflow of the solvent and solubilized lignin fractions while retaining insoluble fractions; at least one detector downstream from the lignin sample holder configured to detect organic material in a flowing solvent;

at least one collection vessel configured to collect a volume fraction of solubilized lignin in a solvent; and

tubing configured to connect the above components and to flow the solvent, mixture of solvents, and a mixture of one or more solvents and a solubilized lignin fraction.

2. The system of claim 1 , further comprising a controller in data communication with the pump, the lignin sample holder, the detector, and the collection vessel, the controller programmed to allow real-time control and variability of release and mixture of solvents from the solvent reservoir, to allow real-time monitoring of lignin concentration in the solvent based on data received from the detector; and to control collection of different fractions of solubilized lignin based on data received from the detector.

3. The system of claim 1 or 2, wherein the system comprises two or more solvent reservoirs, each containing a different solvent and wherein the pump is configured to pump one solvent or a mixture of solvents from the two or more solvent reservoirs to the lignin sample holder.

4. The system of anyone of claims 1-3, wherein the system comprises 2 to 4 solvent reservoirs, each containing a different solvent, and wherein the pump is configured to pump one solvent or a mixture of 2-4 different solvents from the solvent reservoirs to the lignin sample holder.

5. The system of anyone of claims 1-4, wherein the at least one solvent is selected from the group consisting of an organic solvent, a mixture of organic solvents, a water-based solvent and a mixture of water based solvents.

6. The system of anyone of claims 1-5, wherein the lignin sample holder is a column having an inner volume.

7. The system of claim 6, wherein the column has an invariable end-piece and a variable endpiece, each endpiece comprising a connector configured to connect the column to the tubing, the variable endpiece configured to adjust to reduce the inner volume of the column to maintain a packing density of the lignin material, and wherein at least one endpiece includes a filter material to retain insoluble materials within the column.

8. The system of anyone of claims 1-7, wherein the system includes two or more lignin sample holders connected via a multiway port to tubing from the pump and tubing to the detector, wherein the two or more lignin sample holders are connected in parallel or in series.

9. The system of anyone of claims 1-8, wherein the isolated lignin material is a lignin, functionalized lignin or fraction of lignin or functionalized lignin from an industrial or laboratory process.

10. The system of anyone of claims 1-9, wherein the lignin, functionalized lignin or fraction of lignin or functionalized lignin is selected from the group consisting of kraft lignin, organosolv lignin, lignosulphonates, and soda lignins.

11. The system of anyone of claims 1-10, wherein the at least one detector is selected from the group consisting of a PDA detector, an Rl detector, and ATR-FTIR detector, and MALLS detector and a combination of two or more of these detectors.

12. The system of claim 11 , wherein the at least one detector is a PDA detector.

13. The system of anyone of claims 1-12, wherein the system further includes a back pressure regulator.

14. The system of anyone of claims 1-13, wherein the system further includes one or more manual and/or remotely controlled dialer points configured to allow switching the flow of a solvent stream, wherein the dialer point is located between the pump and the sample holder, between the sample holder and the detector, or both.

15. The system of anyone of claims 1-14, further comprising a heater configured to accommodate one or more of the lignin sample holders and in which the sample holders can be heated, cooled, or kept at ambient temperature.

16. The system of anyone of claims 1-15, further comprising a sonicator configured to provide sonication of the isolated lignin samples while in the sample holders.

17. The system of anyone of claims 1-16, wherein the lignins, functionalized lignins or fractions of lignin or functionalized lignin can be extracted by solvent streams comprising organic or inorganic solutes at a concentration in the range fromO.001 to 100 mol/L.

18. The system of anyone of claims 1-17, wherein the system is configured such that the at least one lignin sample holders can be flown through in alternating directions without process interruption and re-mounting, by using switch valves, loops, or a combination thereof.

19. The system of anyone of claims 1-18, wherein the lignin, functionalized lignin or fraction of lignin or functionalized lignin has a weight average molecular weight (Mw) in the range of from 180 Da to 1000000 Da.

20. The system of anyone of claims 1-19, wherein the lignin, functionalized lignin or fraction of lignin or functionalized lignin has a number average molecular weight (Mn) in the range of from 180 Da to 500000 Da.

21. The system of anyone of claims 1-20, wherein the lignin, functionalized lignin or fraction of lignin or functionalized lignin comprises from 0.01 mmol/g to 10 mmol/g aromatic hydroxyl content.

22. The system of anyone of claims 1-21 , wherein the lignin, functionalized lignin or fraction of lignin or functionalized lignin comprises from 0.1 mmol/g to 20 mmol/g aliphatic hydroxyl content. 23. A method of continuous segmented flow-based fractional dissolution of lignins, functionalized lignins or fractions of lignin or functionalized lignin through the system of anyone of claims 1-22 comprising:

loading an isolated lignin sample into at least one sample holder of the system;

providing one or more solvents capable of dissolving at least a portion of the lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin;

programming the system with parameters selected to optimize fractional dissolution of the lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin; and

using the system to obtain one or more fractions of lignin from the isolated lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin, wherein the one or more fractions of lignin have a difference in one or more of the following from the isolated lignin sample: weight average molecular weight, number average molecular weight, aromatic hydroxyl content, and heat stability.

Description:
METHODS AND SYSTEMS FOR FRACTIONAL DISSOLUTION OF LIGNINS IN

SEGMENTED CONTINUOUS FLOW MODE

FIELD OF THE INVENTION

The present invention relates to variously scalable continuous fractionation methods and systems applicable to isolated lignins and lignin-derivatives, including functionalized lignins and fractions of lignins as well as fractions of functionalized lignins.

BACKGROUND

Almost one third of the mass of lignocellulosic biomass is made of polyphenolic oligomers and polymers, which are, for the most part, lignins. 2 Since lignocellulosic biomass is already present in nature with a myriad of different specifics, 3 · 4 many industrial processes have been developed that are aimed at isolating the cellulose and hemi-cellulose parts of various types of lignocellulosic biomass. Many of these processes further modify the structure of the lignin component, which is often obtained as a rather low-quality by-product in biorefinery processes that were optimized with respect to obtaining/isolating the cellulose components. 5 · 6 Lignins produced from such processes are generally referred to in the industry as“technical lignins.” Most technical lignins are used as additives in various low value applications, such as binders, dispersants, adhesives, and other fillers in various industrial applications. Recently, bigger global players and promising newcomers in the biorefinery business have started to view the lignin-containing streams from these processes as an additional source for augmented revenues. 6

This interest in lignins as a revenue stream has led to the isolation and production of higher quality lignins for use in various applications in material science, functional cosmetics, biomedical devices, etc. However, most lignins isolated from biorefinery processes have unpredictable polymerization characteristics, variable degrees of delignification and other variable characteristics that prevent use in higher value applications. Additionally, higher value applications call for more detailed knowledge of the technical lignins to be employed in the process, including structural features, thermal characteristics, etc. The structural complexity, augmented reactivity, and thermal instability of most technical lignins have prevented widespread use of such lignins in higher end uses.

Fractionation of lignin can provide lignin fractions of variable molecular weight and functionality, and lignins isolated from these fractions exhibit or can be modified to exhibit an industrially acceptable polydispersity, variable functionality, and improved thermal stability amenable to use in some higher value applications. However, conventional fractionation processes are batch-type processes and are not readily adaptable to providing a continuous run. The technical problem is that of overcoming the drawbacks of the prior art batch-type processes, thus providing a continuous run process for producing lignins.

SUMMARY OF THE INVENTION

Therefore, the present invention concerns a system for continuous segmented flow-based fractional dissolution of lignins, functionalized lignins, fractions of lignins, and/or fractions of functionalized lignins, the system comprising at least the following components:

at least one solvent reservoir for containing at least one solvent effective to solubilize at least a portion of an isolated lignin material;

at least one pump downstream from the solvent reservoir, the pump configured to pump the at least one solvent or mixture of solvents at a selectable flow rate against constant or variable back-pressure;

at least one lignin sample holder downstream from the pump, the sample holder configured to contain the isolated lignin material and solvent or mixture of solvents and configured to allow outflow of the solvent and solubilized lignin fractions while retaining insoluble fractions; at least one detector downstream from the lignin sample holder configured to detect organic material in a flowing solvent;

at least one collection vessel configured to collect a volume fraction of solubilized lignin in a solvent; and

tubing configured to connect the above components and to flow the solvent, mixture of solvents, and a mixture of one or more solvents and a solubilized lignin fraction.

Advantageous aspects of the system of the invention are indicated in dependent claims 2-22, as it will be detailed in the embodiments reported below.

In a second aspect thereof, this invention moreover provides a method of continuous segmented flow-based fractional dissolution of lignins, functionalized lignins or fractions of lignin or functionalized lignin through the system of anyone of claims 1-22 comprising:

loading an isolated lignin sample into at least one sample holder of the system;

providing one or more solvents capable of dissolving at least a portion of the lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin;

programming the system with parameters selected to optimize fractional dissolution of the lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin; and

using the system to obtain one or more fractions of lignin from the isolated lignin sample representing a lignin, a functionalized lignin or fraction of lignin or functionalized lignin, wherein the one or more fractions of lignin have a difference in one or more of the following from the isolated lignin sample: weight average molecular weight, number average molecular weight, aromatic hydroxyl content, and heat stability.

Advantageous aspects of the method of the invention will be apparent from the embodiments reported below.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

FIGS. 1A-1C illustrate some lignin structures showing characteristic interunit bonding motifs and functional groups for different types of lignin: (A) branched polymeric lignin (outdated view); (B) linear chains of oligomeric milled wood lignin (MWL); 11 (C) complex structure of Lignoboost softwood kraft lignin (SWKL). 12 Not shown is the generic structures for lignosulfonates, since their structure is not yet fully understood.

FIGS. 2A-AD illustrate examples of different lignin fractionation processes: (A) typical work-flow for an ultrafiltration approach; 49 (B) typical work-flow for a fractional precipitation approach starting from a soluble fraction; 51 (C) typical work-flow of a chromatography-based fractionation, 52 (D) typical work flow of a fractionated solubilisation/filtration. 44

FIGS. 3A-B illustrate an embodiment of a continuous segmented flow system for fractional dissolution of lignins according to the present disclosure. FIG. 3A is a schematic diagram of the system, and FIG. 3B is a schematic diagram of an embodiment of column ends, showing an embodiment having fixed and adjustable endpieces.

FIG. 4 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), ASKL (small dash) and AIKL (large dash).

FIG. 5 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), MSKL (small dash) and MIKL (large dash).

FIG. 6 is a graph illustrating a comparison of GPC-elution profiles for PWL (solid), ASWL (small dash) and AIWL (large dash).

FIG. 7 is a graph illustrating a comparison of GPC-elution profiles for PWL (solid), MSWL (small dash) and MIWL (large dash).

FIG. 8 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), SKL fractions (various) and IKL (medium dash).

FIG. 9 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), SKL fractions (various) and IKL (medium dash). FIG. 10 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), SWL fractions (various) and IWL (medium dash).

FIG. 11 is a graph illustrating a comparison of GPC-elution profiles for PWL (solid), SWL fractions (various) and IWL (medium dash).

FIG. 12 is a graph illustrating a comparison of GPC-elution profiles for PKL (solid), SKL fractions (various) and IKL (medium dash).

FIG. 13 is a graph illustrating a comparison of GPC-elution profiles for PWL (solid), SWL fractions (various) and IWL (medium dash).

FIG. 14 is a graph illustrating a comparison of GPC-elution profiles for PWL (solid), SWL fractions (various) and IWL (medium dash).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of organic chemistry, chemical engineering, industrial engineering, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications and patents that are incorporated by reference, where noted, are incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. Any terms not specifically defined within the instant application, including terms of art, are interpreted as would be understood by one of ordinary skill in the relevant art; thus, is not intended for any such terms to be defined by a lexicographical definition in any cited art, whether or not incorporated by reference herein, including but not limited to, published patents and patent applications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. It must be noted that, as used in the specification and the appended claims, the singular forms “a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of cells. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "consisting essentially of" or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of" or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

As used herein,“isolated” means removed or separated from the native environment. Therefore, isolated lignin indicates the lignin is separated from its natural environment (e.g., plant source) but are not necessarily purified. For instance, an isolated lignin may be included in a byproduct from an industrial process or it may be subjected to additional purification and separation steps. As used herein, the term “technical lignin” indicates various lignins produced/isolated as a byproduct from various industrial processes such as, but not limited to physical and/or chemical processing of lignocellulose materials (e.g., for paper production, biofuel production, etc.). Technical lignins can include, but are not limited to, lignosulphonates (LS) (from sulfite pulping), kraft lignins (KL) (from kraft pulping), organosolv lignins (OSL) (from processing with organic solvents), soda lignins (SL) (from soda pulping), etc. As described herein, “technical lignins” typically exhibit more variable polydispersity, higher reactivity, lower molecular weight, thermal instability than unprocessed lignin.

As used herein, the term “functionalized lignins” includes all forms of chemically, biotechnologically, or physically altered or derivatized lignins on the basis of a fractional dissolution in various organic and/or aqueous solvents and/or mixtures thereof, homogeneous or heterogeneous under the employed flow conditions

The following is a list of abbreviations used in the present disclosure: AIKL, acetone insoluble kraft lignin; AIWL, acetone insoluble wheat straw lignin; ASKL, acetone soluble kraft lignin; ASWL, acetone soluble wheat straw lignin; ATR, attenuated total reflectance; BPR, back pressure regulator; CI-TMDP, 2-chloro-4,4,5,5-tetramethyl-1 ,3,2-dioxaphospholane; DMF, dimethyl formamide; DCM, dichloromethane; DMAc, dimethyl acetamide; DMSO, dimethyl sulfoxide; GPC, gel permeation chromatography; GVL, g-valerolactone; HPLC, high pressure liquid chromatography; HW, hardwood; IKL, insoluble kraft lignin; I R, infrared; IWL, insoluble wheat straw lignin; KL, kraft lignin; LS, lignosulphonate; MALLS, multiangle laser light scattering; MIKL, methanol insoluble kraft lignin; MIWL, methanol insoluble wheat straw lignin; Mn, number average molecular weight; MSKL, methanol soluble kraft lignin; Mw, weight average molecular weight; MW, molecular weight; MSWL, methanol soluble wheat straw lignin; MWL, milled wood lignin; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; OSL, organosolv lignin; PDA, polydiode array; PD, polydispersity; PEEK, polyether ether ketone; PKL, parent kraft lignin; PTFE, poly(tetrafluoroethylene); PVF, polyvinyl fluoride; PWL, parent wheat straw lignin; Rl, refractive index; RS, rice straw; SKL, soluble kraft lignin; SL, soda lignin; SW, softwood; SWL, soluble wheat straw lignin; SWKL, softwood kraft lignin; THF, tetrahydrofurane; UV, ultraviolet.

In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, embodiments of the present disclosure, in some aspects, relate to methods and systems for variously scalable continuous fractionation methods for isolated technical lignins and lignin- derivatives. The methods and systems of the present disclosure can be run in continuous flow mode for increased efficiency and output of fractionated technical lignins.

Many industrial processes developed for the isolation and cellulose and hemi-cellulose portions of lignocellulosic biomass also produce lignin as an unused byproduct. Such processes, focused on the isolation of the cellulose/hemi-cellulose components, produce a lignin byproduct that usually includes lignin components having a modified structure and considered a low-quality byproduct. However, as interest in higher quality lignins has increased due to potential applications other sectors, biorefinery players have started to focus on producing higher quality lignin streams from their processes as a secondary revenue stream.

For instance, different, higher quality lignins are now more readily available for potential applications in sectors of material science, functional cosmetics, and even biomedical devices 7 {e g·, the LignoBoost lignin, which is obtained by a novel process for the precipitation of lignin after standard kraft pulping, 8 and lignins obtained in industrialized organosolv pulping processes, e.g., Alcell lignin 9 and CIMV Biolignin™. 10 ) Some characteristic structures of important types of lignins are shown in FIGS. 1A-1C, while Table 1 gives an overview over some characteristic key figures for various types of lignin.

Table 1 : Important technical lignins and their main characterising features.

lignin lignin isolation process typical M w T g (°C) solubility source type (kDa)

softwood, kraft lignin kraft pulping SW: 5-20 13 - 15 HW: 83 17 SW: aq. pH >10, aq. hardwood HW: 4 15 2.6- 91 18 144 19 organic solvents 3

2.7 16 165 20

softwood, ligno- sulfite pulping 5-60 21 · 22 HW: 47 23 aq. pH > 3, aq. hardwood sulfonates organic solvents softwood, organosolv ethanol-water HW: 4 15 2-5 24 HW: 98 20 polar organic hardwood lignin organosolv SW: 2-11 24 solvents

pulping Grass: 3-7 25 · 26 wheat soda lignin soda pulping HW: 7-14 24 WS: 116 18 aq. pH>10, partial: straw, SW: 12-14 24 135 28 150 29 aq. organic solvents bagasse, 2-6 27 RS: 155 29

etc.

wheat steam- autohydrolysis or 12-20 25-90 18 high: Aq. pH >10, straw, explosion acid-catalyzed (grass) 15 partial: aq. organic bagasse, lignin pretreatment and solvents

etc. solvent extraction

various biorefinery lignin residues unknown unknown poorly soluble in all biomasses residual recovered after common solvents, lignin saccharification of soluble in some pretreated ionic liquids biomass

a: depending on lignin purity typically soluble in aq. acetone, tetrahydrofuran, dioxane,

DMSO. HW, hardwood. SW, softwood. WS, wheat straw. RS, rice straw.

Higher value applications, however, call for a detailed structural understanding of the technically produced lignin of choice, and the determination of basic thermal characteristics. 30 · Correlating structural features like, e.g., the abundance of free phenolic residues with observed thermal behaviours, has proven to be a powerful tool. For instance, thermal properties of an industrial lignin, and thereby its processibility in extruder-based applications, can be tuned by modifying the concentration of free phenolic hydroxyl groups. 31-35

While the functional groups play a significant role with respect to the type of application, the polymer characteristics are also important, especially the number average molecular weight (Mn) and the polydispersity (PD). Most isolated lignins are characterized by a polydispersity that a priori prevents any use in higher value applications, independent of the Mn, which additionally differs significantly depending on the isolation process. 5 One way to arrive at lignins that exhibit, at least, an industrially acceptable polydispersity is fractionation of lignin. This idea of fractionating lignins has been investigated on various samples since the 1980s, 36 after initial attempts in the early 1950s; 37 recently, however, it re-gained momentum in connection with more specified investigations for industrial applications of lignins. Reported versions include sequential precipitation out of alkaline solutions, 38 fractional precipitation of re-dissolved kraft lignin in a gradually changed binary solvent system, 39 (sequential) extractions using different solvents 38 · 40- 46 or just plain water 47 followed by adsorption as well as the fractionation by ultrafiltration of black liquor using ceramic membranes 48-50 as versatile options. FIGS. 2A-2D show flow diagrams of four common fractionation techniques: flow-based ultrafiltration, fractionated precipitation, chromatography-based fractionation, and fractionated solubilisation/filtration.

The aforementioned techniques succeed in dividing a given batch of lignin into fractions that differ in molecular weight characteristics and that eventually additionally differ in terms of the functional group distributions. These processes, however, suffer, to various extents, from the difficulty of providing a continuous run. In terms of fractional precipitation (method A), the continuous production of solids that need to be filtered off is hard to realise. At best, a discontinuous process is possible. In the case of ultrafiltration techniques (method B), membrane clogging is a known problem in continuous processing. In terms of chromatography-like/chromatography-based fractionation of lignins (method C), the presence of the remaining insoluble material, eventually in the form of a mixture with material forming the stationary phase in the chromatography set-ups renders it necessary to renew the set-up when loading a new batch of material to be fractionated. In WO2015178771A1 a column-based method for fractionation of lignins uses organic solvents or mixtures of organic solvents with increasing polarity. The lignin is mixed to various extents with inert materials inside the column. This method does not appear to be scalable, nor is it amendable to be remotely and/or fully controlled for automation. Insoluble lignin cannot be directly isolated, since it is mixed with an inert excipient material. 52 US20110262985A1 reports an extruder- containing process that is suitable for harvesting various streams of biomass from a feedstock, under eventual concomitant structural change of the biomass. The document does not describe fractionation of lignin, let alone fractionation of lignin in a continuous process. 53 US7465791 B1 details a ‘modular process’ for organosolv fractionation of lignocellulosic feedstocks into component parts and further processing of said component parts into at least fuel-grade ethanol and four classes of lignin derivatives. 54 US20100170504A1 describes a biorefinery process for the fractionation of lignocellulosic biomass into cellulose, hemicellulose sugars, lignin, and acetic acid. Fermentable hemicellulose sugars, low-molecular-weight lignin, and purified acetic acid are also major products of the process and system, but the lignin is not further fractionated in this process. 55 None of the above-described processes are feasible on a large scale in a continuously run process.

Thus, one purpose of the methods of the present disclosure is to provide a process for fractionation of technical lignins that is adaptable to a continuous flow model. As described in greater detail below, in the methods of the present disclosure, the process of fractionated precipitation can be inverted, inversed, and transferred into a segmented flow chemistry set-up to become a continuous process of fractionated dissolution that can be fully automated and run continuously. Furthermore, the methods of the present disclosure are scalable by optimization of the dimensions of the set-up as well as the overall run time.

The examples described in greater detail below also provide confirmation that the fractions of various technical lignins realized in the continuous process of the present disclosure are comparable in quality and quantity to fractions obtained in comparable non-continuous, batch- based fractional precipitation.

The methods and systems of the present disclosure relate to the fractionation of technical lignins in a scalable and continuous fashion. In embodiments, methods of the present disclosure are referred to herein as “continuous segmented flow-based fractional dissolution of lignins”. In embodiments, the system for continuous segmented flow-based fractional dissolution of lignins of the present disclosure includes at least the following elements: a pump, and lignin-sample holder, at least one detector capable of detecting organic material in a solvent stream, and one or more collection vessels. An embodiment of a system 10 of the present disclosure is illustrated in FIG. 3A, showing a pump module 20, a lignin sample holder module 30 (including a number of lignin sample holders 32, a detector module 40, and a collection module 50.

In embodiments, the pump module 20 includes at least one pump 22 suitable for pumping various solvents as well as solvents mixed with various lignin fractions. In embodiments, the pump is capable of pumping at various flow rates and against various back-pressures. In embodiments, the solvents to be pumped can include a single solvent (e.g., a pure solvent), and in embodiments, the solvents can include freely selectable miscible and/or immiscible mixtures of two or more solvents. As shown in FIG. 3A, the solvents can be contained in solvent reservoirs 24 and fed to or drawn by the pump 22 from the one or more solvent reservoirs 24 via tubing 26.

In embodiments, the lignin-sample holder module 30 is placed downstream to the pump module 20 (in embodiments, immediately downstream from the pump 22). The lignin sample holder module includes one or more sample holders 32 is configured to hold lignin sample materials, such as technical lignins. In embodiments, the sample holder is made of materials compatible with the solvents chosen for the dissolution protocol and suitable to withstand the backpressures generated in the system. In embodiments the lignin-sample holder can be one or more columns 32, such as the column illustrated in FIG. 3B and described in greater detail below. In embodiments, the sample holder(s) 30 can be housed in an oven 34. In the present disclosure, the term“columns” may be used interchangeably with“lignin sample holder;” however, it will be appreciated by the skilled artisan, that containers other than column-shaped containers can be used in embodiments of the present disclosure. In embodiments, the sample can flow from the pump 20 to the sample holders/columns 32 via tubing 26 and from the sample holders to the one or more detectors 42 via tubing 36.

In embodiments, the detector module 40 includes an in-line detector 42 or a series of one or more detectors. In embodiments, the detector is placed downstream to the lignin sample holder. In embodiments, the detector is suitable for the facile detection of organic material in the flowing solvent. The organic material detected can be UV-active or UV-inactive material, or a combination. In embodiments the detector is also in data communication with a controller 44, as described in greater detail below.

In embodiments, the collection module 50 includes one or more collection vessels 52 configured for collecting various volume fractions and placed downstream to the detector(s) 42. In embodiments, the system includes two or more collection vessels, and each is configured to collect a different fraction of lignin.

In the methods and systems of the present disclosure, the at least a portion of the technical lignins in the sample can display certain characteristics such as, but not limited to the following.

At least a portion of the technical lignins have solubility in at least one solvent, such as, but not limited to, an organic solvent, a water-based solvent, an ionic liquid solvent, or a combination thereof. In embodiments, the technical lignins can have an average molecular weight ranging from 180 to 1000000 Da. In embodiments, the technical lignins can have impurity contents of up to 80%, with impurities being selected from organic substances, inorganic substances, and mixtures thereof. Depending on the conditions selected, fractionation can be achieved and fractions released by one or more of the following:

(a) by adjusting/gradually changing the pH of aqueous buffer systems;

(b) by adjusting/gradually changing the salinity of aqueous salt solutions;

(c) by adjusting/gradually changing the polarity of the solvents used, and/or by mixing solvents and/or solvent systems;

(d) by working in pressurized systems in combination with one of the options from a) to c);

(e) by working at various temperatures in combination with one of the options from a) to c);

(f) by additionally using molecular weight-sensitive filtering solid or flexible membranes;

(g) by combing any one or more of options a) to g).

In embodiments, the system allows monitoring of lignin fractions in real time and inline by one or more of the following: a photo diode array (PDA) detector; an attenuated total reflectance (ATR) infrared (IR) detector; a refractive index (Rl) detector; and a multi angle laser light scattering (MALLS) detector. In embodiments, the system includes at least the PDA detector. In embodiments, the system includes the PDA detector and ATR IR detector. In embodiments, the system includes at least the PDA detector and one or more of the ATR I R detector, the Rl detector, and/or the MALLS detector.

In embodiments, the system is monitored and controlled using a computer (e.g., personal computing device or other computing device) equipped with suitable software. As described in greater detail in the examples below, the process was found to be easily adopted to accommodate fractionation and/or purification of chemically or biotechnologically altered lignins from industrial and laboratory productions.

In embodiments of systems and methods of the present disclosure for continuous segmented fractional dissolution of lignins, some of the following elements are described below (and with reference to FIGS. 3A and 3B) in greater detail in reference to some embodiments.

Module Pump (20): The system of the present disclosure includes a solvent delivery module for transporting solvents used for the fractionated dissolution protocol from solvent reservoirs into the system. This module is referred to herein as a“pump” and may include one or more pumps 22. The material of the pump(s) 22 is compatible with the solvents and/or solvent systems used and compatible with the chemical and physical conditions attributable to dissolved lignin solutions in the chosen set-up and system. In embodiments, the pump can be a conventional HPLC piston pump, a peristaltic pump, or even a simple syringe pump so long as the pump is strong enough to work against the back pressure generated by the system and the lignin-filled column. The materials of the pump are compatible with the solvents used. In embodiments a pump of the present disclosure can be made of various materials, depending on the construction, such as, but not limited to, stainless steel, chromium, rubber for seals, plexiglas components and other suitable materials. In embodiments, the pump is configured for pumping at various flow rates against changeable back-pressures. In embodiments, the pump is configured to furnish isocratic or gradient flows of solvent systems including at least one solvent suitable to dissolve lignin molecules. Bothe isocratic and gradient flows of solvent systems can vary in flow rate during the fractional dissolution protocol at various flow rates.

In embodiments, the pump is in communication with and can be controlled by a computer-assisted controller unit 44 that allows read-out and real-time remote operation and/or changes of crucial parameters such as, but not limited to: the pressure the pump has to work against, the flow rate, the solvent mixture, etc. In embodiments, the computer-assisted controller unit allows real-time changes of flow-rates and current solvent mixture. In embodiments, the pump is a mechanical pump, such as, but not limited to, a piston pump. In embodiments, the pump is equipped with a suitable degasser unit capable of providing solvents substantially free of gas bubbles. In embodiments the pump module can also include a mixing element that can mix the solvents coming from the pumps. In embodiments, the mixing element can also be in communication with the controller 44 to control mixing speed, time, etc.

Module column (lignin sample holder module 30): The system of the present disclosure also includes at least one lignin sample holder 32 in which a weighted quantity of isolated lignin can be loaded in dry form. As set forth above, in embodiments, the lignin sample holder is a column, but may take the form of other containers as well. In embodiments, the column does not include an inert filler material to be mixed with the lignin. In the present disclosure the lignin sample holder is also referred to as a column. In embodiments, as illustrated in FIG. 3B, the column 32 is closed at one end (e.g., a bottom end) with a dedicated invariable/fixed end-piece 33 that is configured to prevent lignin particles from clogging the tubing. In embodiments, the other end of the column is closed with an adjustable end-piece 35 that is configured to prevent lignin particles from clogging the tubing. The end-pieces provide mechanically robust connection ports to attach the tubing. In embodiments, the sample holders/columns are made of materials such as, but not limited to, glass or stainless steel, or other material suitable to withstand the chemical and physical demands posed by the chosen fractional dissolution protocol.

In embodiments, the invariable and variable end-pieces allow incorporation of a non-filtering solid porous material. For example, in some embodiments, such non-filtering solid porous material can be a frits made from PTFE, PVF, cellulose, regenerated cellulose, ceramics, silicates. In embodiments, the variable endpiece is adjusted in such a way that the lignin in the column is densely packed. Adjustment can be optimized during fractionated dissolution runs. In embodiments, invariable and variable end-pieces allow incorporation of a filtering solid porous material. In embodiments, the invariable and variable end-pieces allow additional incorporation of and/or a dialysis filter membrane as well. In embodiments, the adjustable endpiece gains its adjustability by a screw mechanism, which can be a remotely controllable screw mechanism (controlled by, e.g., the controller 44).

In embodiments the sample holders/columns 32 can be housed inside a heatable chamber, herein called a column oven 34. In embodiments, the temperature setting of the column oven can be realised remotely in real-time (by, e.g., the controller 44). In other embodiments, the columns 32 can instead be submerged in an ultrasonication bath instead of a column oven, such as to assist dissolution by sonication during segmented continuous flow fractional dissolution.

In embodiments (to facilitate scale-up of the lignin fractionation methods of the present disclosure), the system also includes one or more controllable switch points 38 (also referred to herein as“column dialers”). In embodiments, such as illustrated in FIG. 3A, a plurality of packed columns 32 are connected to a main flow line (e.g., tubing) using two remotely controllable switch points/column dialers 38, placed before/upstream of the column(s) 32 and one after/downstream of the columns. In embodiments, each column dialer can be remotely controlled (e.g., by controller 44), including control on the basis of real-time detector signalling. In embodiments, the column dialers also allow manual control.

Module detector (40): Downstream of the column-module 30, systems of the present disclosure include at least one or a series of detectors 42. In embodiments the detectors are located inline with the sample holder module 30. The detectors 42 are able to monitor contents in the liquid sample stream exiting the column. The detector can include one or more types of detectors configured for i) detecting lignin (purified or in the presence of other biomass compounds and/or in the presence of inorganic or organic impurities), including, for example monomeric, oligomeric, and polymeric species, and/or ii) allowing a monitoring of the concentration of a specific component in the stream of liquid exiting the column. In embodiments detector cells can be heated. In embodiments, a combination of PDA- and Rl-detectors can be included. In embodiments, a combination of PDA- and ATR-IR-detectors are used. In yet other embodiments, a combination of PDA- and ATR-IR- and MALLS-detector is used. In embodiments, one or more detectors 42 are linked to a controller 44 (e.g., a computer), which communicates detection results in real time in a way that allows real-time feedback for pump control.

Back Pressure Regulators: In embodiments, the system can also include one or more back pressure regulators 46. In embodiments, the sample/column module 30 is facultatively followed by a device for controlling overall system pressure, called a back pressure regulator (BPR). The BPR allows adjustment of overall system pressure, which can allow maintenance of any solvent system chosen for fractionated dissolution in the liquid state, even when the fractionation is run at temperatures higher than the boiling points of the solvents used or any positive azeotrope a given solvent system may form inside the system. In embodiments a facultative first BPR is installed between the sample columns 32 and the detector 42 in case column operating pressures override maximum pressure limits of downstream located detector modules.

In embodiments, independent of the inclusion of a facultative first BPR, the detector module 40 can be followed by a device for controlling overall system pressure, which is referred to herein as a closing back pressure regulator (closing BPR). In embodiments, a closing BPR can be included downstream of the one or more detectors 42. In embodiments, a closing BPR allows adjustment of overall system pressure to maintain any solvent system chosen for fractionated dissolution in the liquid state even when the fractionation is run at temperatures higher than the boiling points of the solvents used or any positive azeotrope a given solvent system may form inside the set up. In embodiments of the present disclosure, the system can include both a facultative BPR and a closing BPR. In such embodiments, the closing BPR is chosen in such a way that it does prevent any liquid from entering in a gaseous state in the detector module while only adding a minimum of additional pressuring to the entire system used for fractional dissolution.

Module collection (50): Downstream from the detector module 40 and optional closing BPR 46, the system includes a collection module 50 having one or more collection vessels 52 configured for collecting several individual batches of liquid of variable volume including solubilized lignin fractions. The collection module 50 may also be referred to herein as the“fraction collector.” In embodiments, the fraction collector is remotely controlled by a computer-assisted system (e.g., the controller 44) that allows real-time reaction of the fraction collector to respond to the real-time monitored detector signals. For instance, in response to a signal from the detector 42 that a different fraction is being pumped through the system, the controller could signal to direct the flow to a different collection vessel 52.

Tubing: Connection of various modules is provided by conduit (also referred to herein as tubing), to convey the lignin samples/fractions from one module to the next. For instance, as in the embodiment illustrated in FIG. 3A, tubing 26 carries lignins samples from the sample reservoirs to the pump and then carries lignin samples from the pump 22 to the columns 32. Then, the samples of solubilized lignin exit the columns 32 and proceed to the detector 42 via tubing 36, and from the detector to the fraction collection vessels 52 via tubing 36. In embodiments the tubing has appropriate chemical and physical characteristics sufficient to withstand the physical and chemical demands set by the fractionated dissolution set-up. In embodiments, the tubing includes various types of appropriate connectors, more-way valves, switches, stop cocks. In embodiments the tubing material is chosen from the group including, but not limited to, stainless steel tubing, PEEK-tubing, PTFE-tubing or combinations. In embodiments the inner diameters of the tubing are chosen in such a way that the tubing as such does not cause additional pressuring of the system. In embodiments, tubing junctions are constructed in such a way that undesired clogging of tubing is minimized (e.g., by providing a mesh or sieve at various inlets and outlets between the modules described above, see for example, FIG. 3B).

The system of the present disclosure provides many advantages, such as the ability to operate in continuous mode, to use two or more solvents or mixtures thereof at one time, to provide multiple columns in order to process more than one lignin sample at a time, to provide real time control of the concentration of solvent, real-time control of the direction and rate of flow, to allow real-time control of system pressure, to provide real-time monitoring of the fractions being obtained and collected, to allow remote control of automated fraction collection, and so forth. In embodiments, the system of the present disclosures is configured, with the pumps, tubing, optional switches, back flow regulators, and other components such that it can be operated where the direction of flow of the solvent stream is in an anti-gravitational direction. Also, in embodiments, the solvents used in the system can be used under critical conditions.

Lignins:

In embodiments of the present disclosure, isolated lignin that has not been chemically and/or physically and/or biotechnologically and/or biologically altered after initial isolation: (a) has a number average molecular weight (M n ) of from 250 Da to 100000 Da, as for example determined by GPC as described below; and (b) has a molar ratio of aromatic hydroxyl content to aliphatic hydroxyl content in the range of from 30/1 to 1/30 as for example determined by quantitative 31 P NMR as described below. In embodiments, the lignin includes from 0 mmol/g to 8 mmol/g aromatic hydroxyl content as determined by quantitative 31 P NMR as described below. In embodiments, the lignin displays solubility against at least one organic or aqueous phase-based solvent and/or solvent system at physical conditions realizable in the above-described system of the present disclosure (this includes explicitly super-critical solvents).

In embodiments of the present disclosure, lignins that have been chemically and/or physically and/or biotechnologically and/or biologically altered after initial isolation (e.g., technical lignins): have a number average molecular weight (M n ) of from 250 Da to 10000000 Da as for example determined by GPC as described below; and (b) has a molar ratio of aromatic hydroxyl content to aliphatic hydroxyl content in the range of from 30/1 to 1/30 as for example determined by quantitative 31 P NMR as described below. In embodiments, the lignin displays solubility against at least one organic or aqueous phase-based solvent and/or solvent system at physical conditions realizable in the above-described system of the present disclosure (this includes explicitly super critical solvents).

In embodiments the lignin material to be fractionated in the systems and methods of the present disclsoure is used in the provided form and is not mixed with additional inert mateirals, and the like.

Organic solvents: Organic solvents can be used as single solvent or in various combinations including one or more solvents in various volume ratios. Some typical solvents include but are not limited to: i) protic polar solvents like acetic acid, formic acid, methanol, ethanol, propanol; ii) aprotic polar solvents like acetone, tetrahydrofurane, g-valerolactone, diethyl ether, methyl tert- butyl ether, dioxane, dichloromethane, trichloromethane, tetrachloromethane, chlorinated ethanes and propanes; iii) apolar aprotic solvents like pentane, hexane, cyclohexane; iv) aromatic solvents like toluene, xylenes, mesitylene, halogenated benzenes; and v) solvents from the groups of aliphatic and aromatic /V-heterocycles and /V-containing alkyl derivatives. Various physical characteristics of a single solvent or mixtures of solvents determine a priori or real-time adjustments to the system appropriate for fractionated dissolution. It is noted that adequate choices of BPRs allow use of solvents and solvent mixtures, including those forming negative azeotropes, under critical conditions such as to avoid gas formation in the lines. High viscosity organic solvents, e.g., dimethylsulfoxide (DMSO), A/./V-dimethyl formamide (DMF), A/./V-dimethyl acetamide (DMAc), g-valerolactone (GVL) can be used utilizing heatable but otherwise analogously working equipment.

In some embodiments in which a mixture of solvents is used, solvents can be combined homogeneously in any ratio while in liquid state under the conditions applied for the fractionated dissolution. In embodiments, solvent mixtures can be recycled during isolation of dissolved fractionated lignin. Also, in embodiments, during recycling, solvents can be separated. In some embodiments, during recycling, solvent mixtures can be recycled as mixture with variations in volume ratios compared to starting mixes not exceeding 2%.

Aqueous solvents. In embodiments, fractional dissolution of lignins can be realised in methods and systems of the present disclosures using water-based solvent systems of various pH and/or various salinity and/or various Lewis acidity or basicity and/ orvarious ratios and concentrations of chaotropic and kosmotropic salts, wherein chaos- and kosmotropic refers to characteristics described by Hofmeister (incorporated by reference herein). 56 Method of segmented continuous fractional dissolution in flow: In embodiments, fractionation hardware set-ups can resemble the following illustrative example: fractional dissolutions are performed using a Shimadzu instrument including a controller unit (CBM-20A), a pumping unit (LC 20AT), a degasser unit (DGU-20A3), a column oven (CTO-20AC), a photo diode array detector (SPD-M20A), and a refractive index detector (RID-10A). In this illustrative embodiment, the instrumental set-up can be controlled using the Shimadzu LabSolution software package (Version 5.42 SP3).

In an embodiment of the methods of the present disclosures, a known quantity of a solid dry lignin is placed in a column that is closed at one end with a fixed endpiece. After filling, the column is closed at the other end using an adjustable endpiece. Both endpieces are equipped with a porous frit or membrane made of a material that is compatible with the desired fractionation protocol in order to prevent escape of solid lignin particles into the tubing. In embodiments, the column can placed in a column holder inside a column oven, in an oil bath or other environment for even heat distribution; however, in embodiments where heat is not needed, no oven, etc. is needed. In embodiment, the tubing is connected in a manner to line-up the various columns. As an illustrative example, a simple set-up of chosen modules as described above are arranged in the following order: degaser - pump- column dialer in column oven - single column in column oven - column dialer in column oven- PDA detector - Rl-detector - back pressure regulator - fraction collector. The desired solvent gradient system is programmed, and the segmented continuous fraction is started. Fractions are collected according to absorbance signal changes monitored by the inline PDA- and Rl-detectors. During the process, some losses of solid mass inside the column are compensated by adjusting the via a screw mechanism adjustable endpiece in a way that the remaining solids stay densly packed inside the column.

Once the fractions, according to the solvent mixture soluble parts, have been obtained, the fractions can be concentrated and solvent can be recycled. In embodiments, solid remains in the column are collected as insoluble fraction. Once dried, fraction yields are determined. Obtained fractions are analysed at least by means of GPC and quantitative 31 P NMR.

Method of measuring M n and M w : The number average molecular weight, M n , as well as the weight average molecular weight, M w , can be determined using gel permeation chromatography (GPC). Briefly described, lignin samples are dissolved in HPLC-grade dimethylsulfoxide (DMSO) (Chromasolv®, Sigma-Aldrich) containing 0.1 % (m/v) lithium chloride (LiCI) and filtered over a 0.45 pm syringe filter prior to injection into a 20 pL sample loop. T ypical analysis set-ups resemble the following specific example: GPC-analyses are performed using a Shimadzu instrument consisting of a controller unit (CBM-20A), a pumping unit (LC 20AT), a degasser unit (DGU-20A3), a column oven (CTO-20AC), a diode array detector (SPD-M20A), and a refractive index detector (RID-10A); the instrumental set-up is controlled using the Shimadzu LabSolution software package (Version 5.42 SP3). For analysis, an analytical GPC column PLgel 5 pm MiniMIX-C column (Agilent, 250 c 4.6 mm) is used. HPLC-grade DMSO (Chromasolv®, Sigma-Aldrich) containing 0.1 % (m/v) LiCI is used as eluent (isocratic at 0.25 mL min -1 , at 70°C). Standard calibration is performed with polystyrene sulfonate standards (Sigma Aldrich, MW range 0.43 - 2.60 x10 6 g mol -1 ) in acid form, and lower calibration limits are verified by the use of monomeric and dimeric lignin models. Final analyses of each sample is performed using the intensities of the UV signal at l = 280 nm employing a tailor-made MS Excel-based table calculation, in which the number average molecular weight (M n ) and the weight average molecular weight (M w )) is calculated based on the measured absorption (in a.u.) at a given time (min) after corrections for baseline drift. 57

M n is calculated according to the formula in which M n is the number average molecular weight

W j is obtained waw j = - with M being molecular weight

hi being the signal intensity of a given logM measurement point

V being the volume of the curve over a given logM interval d(logM).

M j is a given molecular weight.

The analysis is run in triplicate, and final values are obtained as the standard average.

M w is calculated according to the formula in which M w is the number average molecular weight

W j is obtained waw j = - h t with M being the molecular weight

hi being the signal intensity of a given logM measurement point

V being the volume of the curve over a given logM interval d(logM).

M j is a given molecular weight.

The analysis is run in triplicate, and final values are obtained as the standard average. Eventually necessary adjustment of M n and M w with respect to the desired applications is achieved by mechanical breaking of polymeric lignin using a ball mill, by chemically or enzymatically polymerising oligomeric lignin.

Method of measuring aromatic hydroxyl and aliphatic hydroxyl content: In embodiments, a procedure similar to the one published by Granata, et al. (J. Argric. Food chem 1995, incorporated herein by reference) can be used. 58 A solvent mixture of pyridine and (CDC ) (1.6: 1 v/v) is prepared under anhydrous conditions. The NMR solvent mixture is stored over molecular sieves (4 A) under an argon atmosphere. Cholesterol is used as internal standard at a concentration of 0.1 mol/L in the aforementioned NMR solvent mixture. 50 mg of Cr(lll) acetyl acetonate are added as relaxation agent to this standard solution.

Approx. 25 mg of the lignin sample are accurately weighed in a volumetric flask and suspended in 400 pl_ of the above prepared solvent solution. One hundred microliters of the internal standard solution are added, followed by 100 mI_ of 2-chloro-4,4,5,5-tetramethyl-1 ,3,2-dioxaphospholane (CI-TMDP). The flask is tightly closed, and the mixture is stirred for 120 min at ambient temperature. 31 P NMR spectra are recorded using suitable equipment, similar or identical to the following example: On a Bruker 300 MHz NMR spectrometer, the probe temperature is set to 20°C. To eliminate NOE effects, the inverse gated decoupling technique can be used. Typical spectral parameters for quantitative studies are as follows: 90° pulse width and sweep width of 6600 Hz. The spectra are accumulated with a delay of 15 s between successive pulses. Line broadening of 4 Hz is applied, and a drift correction is performed prior to Fourier transform. Chemical shifts are expressed in parts per million from 85 % H3PO4 as an external reference. All chemical shifts reported are relative to the reaction product of water with CI-TMDP, which has been observed to give a sharp signal in pyridine/CDCI 3 at 132.2 ppm. To obtain a good resolution of the spectra, a total of 256 scans are acquired. The maximum standard deviation of the reported data is 0.02 mmol/g, while the maximum standard error is 0.01 mmol/g. 58

Quantification on the basis of the signal areas at the characteristic shift regions (in ppm, as reported elsewhere 58 ) is done using a tailor-made table calculation in which the abundances, given in mmol/g, of the different delineable phosphitylated hydroxyl groups are determined on the basis of the integral obtained for the signal of the internal standard, that is present in the analysis sample at a concentration of 0.1 m, creating a signal at the interval ranging from 152.2 ppm to 151.6 ppm. The area underneath the peak related to the internal standard is set to a value of 1.0 during peak integration within the standard processing of the crude NMR data, allowing for determining abundances using simple rule-of-proportion mathematics under consideration of the accurate weight of the sample used for this analysis. The analysis is run in triplicate, and final values are obtained as the standard average.

Additional details regarding the methods, compositions, and organisms of the present disclosure are provided in the Examples below. The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any “preferred” embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and protected by the following claims.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1 % to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term“about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase“about‘x’ to‘y’” includes“about‘x’ to about‘y’”. EXAMPLES

Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLE 1— Segmented continuous flow fractionation of a softwood kraft lignin using isocratic flow of acetone at room temperature

1 g of a commercialised softwood kraft lignin (KL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this kraft lignin is fractionated in segmented continuous flow mode at room temperature using acetone as single solvent, using an isochratic flow at 2.0 ml/min for 45 min, corresponding to 90 mL of solvent in total. Once the inline-PDA- detector indicates that no lignin is washed out any more, acetone soluble kraft lignin (ASKL) is isolated from the acetone fraction by distilling of the acetone; recovered acetone could be used for consecutive fractionation runs. Acetone insoluble kraft lignin (AIKL) is recuperated from the column and air-dried. FIG. 4 shows the overlay of the GPC races, Table 2 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy. Table 2: Numerical data for GPC and 31 P NMR analysis of softwood kraft lignin fractions obtained in isocratic flow using acetone as single solvent.

a: sample not fully soluble.

EXAMPLE 2— Segmented continuous flow fractionation of a softwood kraft lignin using isocratic flow of methanol at room temperature

1 g of a commercialised softwood kraft lignin (KL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this kraft lignin is fractionated in segmented continuous flow mode at room temperature using acetone as single solvent, using an isochratic flow at 0.2 ml/min for 390 min, corresponding to 78 ml_ of solvent in total. Once the inline-PDA- detector indicates that no lignin is washed out any more, methanol soluble kraft lignin (MSKL) is isolated from the acetone fraction by distilling of the acetone; recovered acetone could be used for consecutive fractionation runs. Methanol insoluble kraft lignin (MIKL) is recuperated from the column and air-dried. FIG. 5 shows the overlay of the GPC races, and Table 3 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 3: Numerical data for GPC and 31 P NMR analysis of softwood kraft lignin fractions obtained in isocratic flow using methanol as single solvent.

a: sample not fully soluble.

EXAMPLE 3— Segmented continuous flow fractionation of a wheat straw organosolv lignin using isocratic flow of acetone at room temperature

1 g of a commercialised wheat straw organosolv lignin (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at room temperature using acetone as single solvent, using an isocratic flow at 0.2 ml/min for 420 min, corresponding to 84 mL of solvent in total. Once the inline-PDA-detector indicates that no lignin is washed out any more, acetone soluble wheat straw organosolv lignin (ASWL) is isolated from the acetone fraction by distilling of the acetone; recovered acetone could be used for consecutive fractionation runs. Acetone insoluble wheat straw organosolv lignin (AIWL) is recuperated from the column and air-dried. FIG. 6 shows the overlay of the GPC traces, and Table 4 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy after phosphitylation using 2-CI- TMDP. Table 4: Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in isocratic flow using acetone as single solvent.

a: sample not fully soluble.

EXAMPLE 4— Segmented continuous flow fractionation of a wheat straw organosolv lignin using isocratic flow of methanol at room temperature

1 g of a commercialised wheat straw organosolv lignin (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at room temperature using acetone as single solvent, using an isochratic flow at 0.2 ml/min for 300 min, corresponding to 60 mL of solvent in total. Once the inline-PDA-detector indicates that no lignin is washed out any more, methanol soluble wheat straw organosolv lignin (MSWL) is isolated from the acetone fraction by distilling of the acetone; recovered acetone could be used for consecutive fractionation runs. Methanol insoluble wheat straw organosolv lignin (MIWL) is recuperated from the column and air-dried. FIG. 7 shows the overlay of the GPC traces, and Table 5 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 5: Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in isocratic flow using acetone as single solvent.

a: sample not fully soluble. EXAMPLE 5— Segmented continuous flow fractionation of a softwood kraft lignin using a gradient elution of hexane-acetone at room temperature

1 g of a commercialised softwood kraft lignin (KL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, kraft lignin is fractionated in segmented continuous flow mode at room temperature using the following hexane-acetone mixtures at room temperature at a constant flow rate of 0.75 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run-time was 150 min, corresponding to a total of 112.5 mL of solvent used. Each time the inline-PDA-detector indicates that lignin is no longer being washed out with the current solvent mixture, solvent mixture is switched to next more polar one. Softwood kraft lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble kraft lignin (IKL) is recuperated from the column and air-dried. FIG. 8 shows the overlay of the GPC traces, Table 6 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy. Table 6: Numerical data for GPC and 31 P NMR analysis of softwood kraft lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

EXAMPLE 6— Segmented continuous flow fractionation of a softwood kraft lignin using a gradient elution of hexane-acetone at elevated temperature

1 g of a commercialised softwood kraft lignin (KL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this kraft lignin is fractionated in segmented continuous flow mode at room temperature using the following hexane-acetone mixtures at 70°C at a constant flow rate of 0.75 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run-time was 150 min, corresponding to a total of 112.5 mL of solvent used. Every time the inline- PDA-detector indicates that no more lignin is being washed with the current solvent mixture, the solvent mixture is switched to the next more polar one. Kraft lignin fractions are isolated from the various solvent mixtures by distilling off the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble softwood kraft lignin (IKL) is recuperated from the column and air-dried. FIG. 9 shows the overlay of the GPC traces, and table 7 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 7: Numerical data for GPC and 31 P NMR analysis of softwood kraft lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

EXAMPLE 7— Segmented continuous flow fractionation of a wheat straw organosolv lignin using a gradient elution of hexane-acetone at room temperature

1 g of a commercialised softwood kraft lignin (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at room temperature using the following hexane-acetone mixtures at room temperature at a constant flow rate of 0.75 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run-time was 230 min, corresponding to a total of 172.5 mL of solvent used. Every time the inline-PDA-detector indicates that no lignin is being washed with the current solvent mixture, the solvent mixture is switched to next more polar one. Organosolv lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble wheat straw organosolv lignin (IWL) is recuperated from the column and air-dried. FIG. 10 shows the overlay of the GPC traces, and Table 8 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 8: Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

EXAMPLE 8— Segmented continuous flow fractionation of a wheat straw organosolv lignin using a gradient elution of hexane-acetone at elevated temperature

1 g of a commercialised wheat straw organosolv lignin (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoro-ethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at 70° C using the following hexane-acetone mixtures at a constant flow rate of 0.75 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run-time was 230 min, corresponding to a total of 172.5 mL of solvent used. Every time the inline-PDA-detector indicates that no lignin is being washed with the current solvent mixture, solvent mixture is switched to next more polar one. Organosolv lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble wheat straw organosolv lignin (IWL) is recuperated from the column and air-dried. FIG. 11 shows the overlay of the GPC traces, and Table 9 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy Table 9: Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

EXAMPLE 9— Fast Segmented continuous flow fractionation of a softwood kraft lignin using a gradient elution of hexane-acetone at elevated temperature

1 g of a commercialised softwood kraft lignin (KL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this kraft lignin is fractionated in segmented continuous flow mode at 70° C using the following hexane-acetone at a constant flow rate of 1.5 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run-time was 75 min, corresponding to a total of 112.5 mL of total solvent used. Every time the inline-PDA-detector indicates that no lignin is being washed out any more with the current solvent mixture, the solvent mixture is switched to next more polar one. Finally, softwood kraft lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble softwood kraft lignin (IKL) is recuperated from the column and air-dried. FIG. 12 shows the overlay of the GPC traces, and Table 10 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy. Table 10: Numerical data for GPC and 31 P NMR analysis of softwood kraft lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

EXAMPLE 10— Fast Segmented continuous flow fractionation of a wheat straw organosolv lignin using a gradient elution of hexane-acetone at elevated temperature

1 g of a commercialised wheat straw organosolv lignin (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at 70° C using the following hexane-acetone mixtures at a constant flow rate of 1.5 ml/min: hexane/acetone = 100/0, 80/20, 50/50, 20/80, 0/100; total run time was 65 min, corresponding to a total of 97.5 mL of total solvent used. When the inline-PDA- detector indicates that lignin is no longer being washed out any more with the current solvent mixture, solvent mixture is switched to next more polar one. Wheat straw organosolv lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble wheat straw organosolv lignin (IWL) is recuperated from the column and air-dried. FIG. 13 shows the overlay of the GPC traces, and Table 11 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 11 : Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

EXAMPLE 11— Segmented continuous flow fractionation of a wheat straw organosolv lignin using various pure solvents at room temperature

1 g of a commercialised wheat straw organosolv (WL) are placed in a glass column equipped with one fixed and one adjustable endpiece carrying porous polytetrafluoroethylene frits. Following the detailed procedure above, using a basic set-up, this organosolv lignin is fractionated in segmented continuous flow mode at room temperature using the following pure solvents at room temperature at a constant flow rate of 0.75 ml/min in the given order: diethyl ether (Et 2 0), dichloromethane (DCM), methanol (MeOH); total run-time was 12 min Et20, 32 min DCM and 56 min MeOH, corresponding to a total of 75.5 mL of solvent used. When the inline-PDA-detector indicates that lignin is no longer being washed out with the current solvent, the solvent is switched to next one. Wheat straw organosolv lignin fractions are isolated from the various solvent mixtures by distilling of the solvent; recovered solvents could be used for consecutive fractionation runs. Insoluble wheat straw organosolv lignin (IWL) is recuperated from the column and air-dried. FIG. 14 shows the overlay of the GPC traces, and Table 12 lists data for M n and M w and abundancies of hydroxyl groups as determined by quantitative 31 P NMR spectroscopy.

Table 12: Numerical data for GPC and 31 P NMR analysis of wheat straw organosolv lignin fractions obtained in flow using a gradient elution system comprised of hexane-acetone mixtures.

a: sample not fully soluble.

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