WO2016016234A1

WO2016016234A1 - Method of pre-treatment of lignocellulosic materials

Info

Publication number: WO2016016234A1
Application number: PCT/EP2015/067259
Authority: WO
Inventors: Peter Johannes Marie Baets; David Sanchez Garcia; Willem Jacob Groot; André Banier De Haan
Original assignee: Purac Biochem Bv
Priority date: 2014-07-28
Filing date: 2015-07-28
Publication date: 2016-02-04

Abstract

The present invention relates a process for treating a lignocellulosic material to render it amenable to biologically mediated transformation, said process comprising: i) providing a lignocellulosic material; ii) mixing said lignocellulosic material with an alkaline agent in the presence of water to form a reaction mixture having a solids content, said alkaline agent comprising a caustic magnesium salt; and, iii) heating said reaction mixture such that said solids are held at a temperature of from 130° C to 250°C for a time period of from 1 minute to 600 minutes. The caustic magnesium salt is preferentially selected from MgO, Mg (OH) 2, MgCO₃, Mg (HCO₃) 2 and mixtures thereof.

Description

Method of Pre-Treatment of Lignocellulosic Materials

Field of the Invention

The present invention is directed to a process for the chemical treatment of a lignocellulosic material using a caustic magnesium salt. More particularly, it is directed to a process of chemical treatment using a caustic magnesium salt which precedes either the saccharxfication or the simultaneous saccharxfication and fermentation of a lignocellulosic material .

Background of the Invention

Organic acids are widely used in the food, pharmaceutical, plastics and textile industries. Lactic acid, for instance, is used as a source of lactic acid polymers which find utility as biodegradable plastics and of which the physical properties can be controlled by adjusting the proportions of the L (+ ) - and D (-) -lactides ,

Organic acids can be produced by fermentation but the economics of such production is strongly dependent upon the cost of the raw materials . It is , for instance , very expensive when refined sugars and starch are used as the fermentation feedstock . Lignocellulosic biomass, which has no competing food value, is a lower-cost, alternative feedstock having wide availability and the potential to be sourced sustainably . However, there is still a need in the art to improve the commercial scale fermentation of lignocellulosic biomass for organic acid production and, in particular, lactic acid production .

The present invention is concerned with methods of treatment of lignocellulosic materials which render the materials more amenable to biologically mediated transformations . More particularly, it is concerned with methods of treatment which render the lignocellulosic materials more amenable to at least one of : enzymatic hydrolysis of carbohydrate components to sugars by saccharolytic enzymes present in the pretreated biomass ; microbial hydrolysis by microorganisms capable of the fermentation of hexose sugars such as glucose, mannose , and galactose; and, microbial hydrolysis by microorganisms capable of the fermentation of pentose sugars such as xylose and arabinose.

Approximately 90% of the dry weight of most plant materials is stored in the form of cellulose, hemicellulose, lignin and pectin, with the remainder being constituted by proteins, ash and extractives such as non-structural sugars, nitrogenous materials, chlorophyll and waxes.

Cellulose is the main structural constituent in plant cell walls. It exists mainly in a crystalline form and is typically found in an organized fibrous structure: the linear cellulose polymer consists of D-glucose subunits linked to each other by β- (1, 4) -glycosidic bonds; cellobiose is the repeat unit established through this linkage, and it constitutes cellulose chains; in turn, the long-chain cellulose polymers are linked together by hydrogen and van der Waals bonds, which cause the cellulose to be packed into microfibrils; and, hemicelluloses and lignin then cover said microfibrils. Fermentable D-glucose can be produced through the action of either acid or enzymes breaking the β~ (1, 4 ) - glycosidic linkages and the amorphous form of cellulose is more susceptible to such enzymatic degradation . However, high cellulose crystallinity, low accessible surface area, protection by lignin, and sheathing by hemicellulose all contribute to the resistance of cellulose in lignocellulosic biomass to hydrolysis .

Hemicellulose is mainly differentiated from cellulose in that hemicellulose has branches with short lateral chains consisting of different sugars . These monosaccharides include pentoses (xylose, rhamnose , and arabinose ) , hexoses (glucose, mannose, and galactose ) , and uronic acids (e.g. , 4-o-methylglucuronic, D-glucuronic, and D- galactouronic acids ) . The backbone of hemicellulose is either a homopolymer or a heteropolymer with short branches linked by β - ( 1 , 4 ) - glycosidic bonds and occasionally β - (1, 3) -glycosidic bonds . Also, hemicelluloses can have some degree of acetylation . Lignin is a complex, three-dimensional polymer constituted by phenylpropanoid subunits linked together by a variety of ether and carbon-carbon bonds. Lignin is intimately interlaced with hemicelluloses in the plant cell wall forming a matrix to cover the crystalline cellulose microfibrils. Whilst it imparts structural support and impermeability to the cell wall, its presence concomitantly provides a protective barrier that prevents plant cell destruction by fungi and those bacteria necessary for the conversion of biomass to organic acids. Lignin' s aromatic nature and complex structure make lignin degradation very difficult. Both lignin and lignin-derived compounds have a detrimental effect on the enzymatic hydrolysis of biomass because they physically hinder the accessibility of cellulases ; they also bind cellulases and lead to their inactivation .

Pre-treatment methods to break down lignin are thus essential for the effective enzymatic and microbial hydrolysis of lignocellulose and thus for the conversion of lignocellulose into organic acids such as lactic acid, succinic acid and acetic acid. Known pre-treatment methods can be roughly divided into different categories: physical (milling and grinding), physicochemical (steam pre-treatment / auto-hydrolysis, hydro-thermolysis, and wet oxidation) , chemical (alkali, dilute acid, oxidizing agents, and organic solvents) , biological, electrical, or a combination of these. The present invention is concerned with a chemical pre-treatment process utilizing an alkaline agent.

Compared with acid pre-treatment processes , alkaline processes are considered to cause less sugar degradation, and many of the caustic salts can be recovered and / or regenerated . Kong et al . Effects of cell-wall acetate , xylan backbone, and lignin on enzymatic hydrolysis of aspen wood, Appl. Biochem . Biotechnol . 1992 , 34/35, 23-35 reported that alkalis remove acetyl groups from hemicellulose (mainly xylan) , thereby reducing the steric hindrance of hydrolytic enzymes and greatly enhancing carbohydrate digestibility . Historically, sodium, potassium, calcium, and ammonium hydroxides have been preferred as alkaline pre-treatment agents and, of these, sodium hydroxide has been the most studied, as documented in, for instance: Fox, D. J et al . , Comparison of alkali and steam (acid) pretreatments of lignocellulosic materials to increase enzymic susceptibility: Evaluation under optimized pretreatment conditions J, Chem. Tech. Biotech. 1989, 44, 135-146; and, MacDonald, D. G. et al . Alkali treatment of corn stover to improve sugar production by enzymatic hydrolysis Biotechnol. Bioeng. 1983, 25, 2067-2076.

Calcium hydroxide (slake lime) has also found utility as a pre-treatment agent, mainly on account of the facts that it is relatively inexpensive (per kilogram) and that it is possible to recover calcium from an aqueous reaction system as insoluble calcium carbonate by neutralizing it with inexpensive carbon dioxide; the calcium hydroxide can subsequently be regenerated using established lime kiln technology. Lime pre-treatment does however tend to increase the crystallinity index of the pre-treated lignocellulosic biomass . Whilst this may not have an effect on ultimate sugar yields from enzymatic hydrolysis, the crystallinity significantly affects initial hydrolysis rates as reported in Chang et al . Fundamental factors affecting biomass enzymatic reactivity, Ap l . Biochem. Biotechnol. 2000, 84-86, 5-37.

Further as reported by Kim et al. Effect of structural features on enzyme digestibility of corn stover, Bioresour. Technol . 2006, 97, 583-591, the delignification of a given lignocellulosic material with calcium hydroxide can vary significantly with oxidative conditions and temperature. This brings into question the efficacy of calcium hydroxide in industrial processes where oxidative conditions cannot easily be moderated and where the lignocellulosic feedstock may be derived from more than one plant material or source, noting that the composition of lignin, hemicellulose and cellulose can vary from one plant species to another and, for a single plant type may vary with age and stage of growth . WO2013/062407 (Wageningen University et al . ) describes a process for the conversion of lignocellulosic material into an organic acid comprising an alkaline pre-treatment step and a fermentation step. Whilst the document purports magnesium oxide or magnesium hydroxide could be used in the alkaline pre-treatment step, this is not exemplified. Rather this document only demonstrates the use of calcium oxide or calcium hydroxide in a pre-treatment step which occurs at a temperature of from 20° to 115 °C . For the purposes of achieving water balance in the process, the liquid phase obtained in the fermentation step must be recycled to the alkaline pre-treatment and / or the fermentation step.

Statement of the Invention

In accordance with a first aspect of the invention there is provided a process for treating a lignocellulosic material to render it amenable to biologically mediated transformation, said process comprising: i) providing a lignocellulosic material; ii) mixing said lignocellulosic material in the presence of water with an alkaline agent to form a reaction mixture having a solids content, said alkaline agent comprising a caustic magnesium salt; and, iii) heating said reaction mixture such that said solids are held at a temperature of from 130°C to 250°C for a time period of from 1 minute to 600 minutes ,

The lignocellulosic material as provided in step i) may, optionally, have been subjected to one or more of pre-extraction, acid hydrolysis and mechanical comminution. Independently, the lignocellulosic material may further be characterized by having a cellulose content of from 20 to 70 wt.%, based on the dry weight of the material and / or a combined cellulose and hemi cellulose content of from 30 to 99 wt , % , preferably 35 to 95 wt.% based on the dry weight of the material.

To promote effective hydrolysis of the lignocellulosic material, the amount of alkaline agent added is determined such that the concentration of the caustic magnesium salt in the reaction mixture is at least 0.1 wt.% and more usually from 0.1 to 50 wt . %, based on the dry weight of the lignocellulosic material (w/w) . Moreover, it is preferred that the alkaline agent consists of caustic magnesium salt and more particularly, consists of caustic magnesium salt selected from MgO, Mg (OH) 2, MgCCb, Mg (HCO3) 2 and mixtures thereof.

The mixing of the lignocellulosic material with the alkaline agent in the presence of water should preferably yield a reaction mixture defined by at least one of: a total solids concentration of from 1 to 70% (w/w) ; and, a pH of from 8.0 to 14,0.

Without being bound by theory, it is contended that the above-defined thermal treatment of the lignocellulosic material in the presence of the caustic magnesium salt facilitates the subsequent saccharification and, optionally fermentation of that lignocellulosic material by performing at least one of: efficiently degrading lignin; increasing the porosity of the lignocellulosic materials; eliminating non-productive enzyme adsorption sites; increasing enzymatic and / or microbial access to cellulose and hemicellulose ; reducing the crystallinity of the cellulose; minimizing the degradation or loss of carbohydrate; and, minimizing the formation of by-products that are inhibitory to the subsequent saccharification and fermentation processes ,

In accordance with a second aspect of the present invention, the treated lignocellulosic material obtained by the above-defined process is used as a substrate for digestion by one or more of acid hydrolysis, enzymatic hydrolysis and microbial hydrolysis. More particularly, the treated lignocellulosic material is used as a substrate for at least microbial hydrolysis, wherein organic acids are obtained from the transformation of carbohydrates derived from the treated lignocellulosic material by a microorganism via fermentation.

As an embodiment of this use, there is provided a method for producing a fermentation product comprising organic acid from lignocellulosic material, said method comprising: a) treating a lignocellulosic material with an alkaline agent in the presenee of water in accordance with the above-defined process; b) saccharifying the treated aqueous lignocellulosic biomass in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic biomass comprising fermentable carbohydrates and an insoluble lignocellulosic fraction; c) fermenting the saccharified aqueous lignocellulosic biomass in the presence of organic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising the magnesium salt of said organic acid and a solid lignocellulosic fraction; and, d) recovering organic acid from the fermentation broth.

There is also provided a method for producing a fermentation product comprising lactic acid from lignocellulosic material, said method comprising: a) treating a lignocellulosic material with an alkaline agent in the presence of water in accordance with the above-defined process, said alkaline agent comprising a caustic magnesium salt; b) saccharif ing the treated aqueous lignocellulosic material in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic material comprising fermentable carbohydrates and a solid lignocellulosic fraction; c) fermenting the saccharified aqueous lignocellulosic material in the presence of lactic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; and, d) recovering lactic acid and/or a lactate salt from the fermentation broth.

In the recited methods of producing a fermentation product comprising organic acid or, in particular lactic acid, the saccharification [b) ] and the fermentation [ c) ] are carried out either simultaneously or sequentially. The solid lignocellulosic fraction obtained in both these steps comprises unhydrolyzed cellulose and hemicellulose and undissolved lignin fractions.

Definitions For the purposes of the instant description "an alkaline agent comprising a caustic magnesium salt" is also referred to as "magnesium-containing alkaline agent", or simply as "alkaline agent". The alkaline agent may be added to the lignocellulosic material in solid form, in the form of an aqueous solution or in the form of an aqueous slurry (e.g. having the caustic magnesium salt partially dissolved in water and partially in solid form) ,

The alkaline agent may comprise up to 50 wt . % , for example up to 20 wt . % , based on the dry weight of the alkaline agent, of one or more caustic salts other than a caustic magnesium salt, such as a caustic sodium salt, a caustic potassium salt, caustic calcium salt and/or a caustic ammonium salt. Specific examples of supplementary caustic salts include Ba (OH) 2, NaOH, Na₂C0₃, NaHC0₃, KOH, K₂C0₃₍ KHC0₃, CaO, Ca (OH) 2, CaC0₃, Ca (HC0₃) 2, NH4OH, (NH₄)₂C0₃, and (NH₄)HC0₃. It is however preferred that the alkaline agent comprises more than 90 wt . % , preferably more than 95 wt . % and more preferably more than 98 wt.%, based on the dry weight of the alkaline agent , of caustic magnesium salt , In particular, the alkaline agent may consist solely of caustic magnesium salt . For example, the alkaline agent may consist of MgO and / or Mg (OH) 2.

Particle size measurements as given herein for the caustic magnesium salt may be measured by laser light scattering using a well-known particle size analyzer such as CILAS 1064 instrument, available from Compagnie Industrielle des Lasers . Further standard techniques for analyzing particles sizes in the sub-millimeter range include the use of standard sieve shaker, microscopy and laser diffraction; these may equally be employed herein .

Where stated herein, the "solids content" or "dry weight" of the lignocellulosic material is determined by heating that material in air at 105°C to a constant weight, in accordance with ASTM E1756 - 08.

For the purpose of this specification, the length of time the aqueous phase remains in the alkaline hydrolysis reactor is determined simply by dividing the working volume of the reactor by the volumetric mixture or slurry flow feed rate to the reactor. The length of time the un- hydrolyzed solids are retained in the hydrolysis reactor is determined based on the solids concentration in the feed, at the exit and within the hydrolysis reactor, as is known in the art. The solids retention time may equally be calculated by using a tracer compound that binds to the solids.

As used herein, the "organic acid" means a substituted or unsubstituted alkyl group containing one or more carboxyl groups (-COOH) . The term "organic acid" is intended to include the salts and esters of the acids unless expressly stated otherwise.

A non-exclusive list of examples of organic acids which can be recovered in the inventive process include: lactic acid; 2, 5-furandicarboxylic acid (FDCA) ; keto-L-gluconic acid; tartaric acid; citric acid; acetic acid; adipic acid; maleic acid; malic acid; malonic acid; succinic acid; salicylic acid; glycolic acid; glutaric acid; gluconic acid; benzoic acid; formic acid; propionic acid; pivalic acid; oxalic acid; toluic acid; stearic acid; ascorbic acid; parmoic acid; glutamic acid; fumaric acid; and, mixtures thereof. The use of the pre-treated lignocellulosic material of the present invention is particularly suitable for the recovery of lactic acid, succinic acid, propionic acid, acetic acid, 2 , 5-furandicarboxylic acid (FDCA) and mixtures thereof.

The term "lactic acid" in this application refers to 2 -hydroxypropionic acid with the chemical formula C3H6O3. The salt form of lactic acid is referred to as "lactate" regardless of the neutralizing agent, e.g. calcium carbonate or ammonium hydroxide. As referred to herein, lactic acid can refer to either stereoisomeric form of lactic acid (L-lactic acid or D-lactic acid) . The term lactate can also refer to either stereoisomeric form of lactate (L-lactate or D-lactate ) . When referring to lactic acid production this includes the production of either a single stereoisomer of lactic acid or lactate or a mixture of both stereoisomers of lactic acid or lactate. As used herein, the term "fermentable carbohydrates" refers to carbohydrates which can be fermented by an organic acid producing microorganism. Generally, fermentable carbohydrates are C5 sugars, Ce sugars, oligomers thereof (e.g. dimeric C12 sugars) and/or polymers thereof. By C5 sugars and Ce sugars is meant saccharides with 5 and 6 carbon atoms, respectively, and by C12 sugars is meant saccharides with 12 carbon atoms (e.g. a disaccharide) .

The carbohydrates that a specific microorganism can ferment are either commonly known to the person of ordinary skill in the art or are easily accessible in the published, background literature. For completeness, common carbohydrates fermentable by lactic acid producing microorganisms include but are not limited to: C₅ sugars such as arabinose, xylose and ribose; Ce sugars such as glucose, fructose, galactose, rhamnose and mannose; and, C₁₂ sugars such as sucrose, maltose and isomaltose .

The content of fermentable carbohydrates in biomass may be determined by methods known in the art. Particularly instructive disclosures are: Milne et al . , Sourcebook of Methods of Analysis for Biomass Conversion and Biomass Conversion Processes, SERI/SP-220-3548. Golden, CO: Solar Energy Research Institute, February 1990; and National Renewable Energy Authority Determination of Structural Carbohydrates and Lignin in Biomass; Laboratory Analytical Procedure (LAP), Revised August 2012, http : //www. nrel■ go /docs /gen/fyl3/42618 ,pdf .

As used herein the term "simultaneous saccharification and fermentation" is intended to mean the simultaneous enzymatic hydrolysis of oligomeric and polymeric carbohydrates of the pre-treated lignocellulosic material into fermentable saccharides together with the further conversion of saccharides into the fermentation product by one or more microorganism ( s ) .

Caustic Magnesium Salt The caustic magnesium salt of the present invention comprises one or more compounds selected from the group consisting of magnesium oxide (MgO) , magnesium hydroxide (Mg{0H)₂), magnesium carbonate (MgC0₃) , magnesium hydrocarbonate (Mg (HCO₃) 2) , alkaline magnesium silicate, trimagnesium phosphate, and mono-magnesium phosphate. Preferably, the caustic magnesium salt is selected from the group consisting of MgO, Mg (OH) ₂, MgCOa, Mg (HCO₃) 2 and mixtures thereof. More preferably, the caustic magnesium salt comprises or consists of MgO and / or Mg (OH) 2.

The caustic magnesium salt may be provided, where applicable, in an aqueous solution. However, the caustic magnesium salts may equally provided in either their solid, particulate form or as an aqueous dispersion thereof.

Provision of the Lignocellulosic Material

The feedstock for the process of the present invention is a lignocellulosic material which broadly includes any material containing cellulose, hemicellulose and lignin, such as may be derived from plant biomass . It is preferred that the lignocellulosic feedstocks be characterized by a cellulose content of from 20 or 30 wt . % to 70 wt.%, based on the dry weight of the material and / or a combined cellulose and hemicellulose content of from 30 to 99 wt.%, preferably from 35 to 95 wt.% based on the dry weight of the material.

Exemplary but non-limiting lignocellulosic materials include: j atrophia; rapeseed; grasses, in particular C4 grasses such as switch grass , cord grass , rye grass, miscanthus , reed canary grass and combinations thereof; palm fronds ; sugar processing residues , including bagasse and beet pulp; agricultural residues, including in particular rice hulls , rice straw, corn, corn fiber, corn cobs, corn stover, wheat, wheat straw, maize, maize stover, sorghum, sorghum stover, sweet sorghum, sweet sorghum stover, soybean stover, barley straw, canola straw, oat straw and oat hulls ; forestry biomass such as recycled wood pulp fiber, sawdust, timber, hardwood, softwood, and combinations thereof; and, cellulosic waste materials such as waste paper, newsprint, cardboard, paper pulp, paper mill residue and the like.

Preferred lignocellulosic materials are selected from the group consisting of; wheat straw; sugarcane bagasse; corn stover; and, mixtures thereof .

The lignocellulosic feedstock may comprise particles, fibers or other residues of one material alone or, alternatively, may originate from a plurality of different materials. It is also envisaged that the lignocellulosic feedstock be fresh, partially dried or fully dried. In certain circumstances it may be advantageous to use fresh lignocellulosic material; the natural or bound water content of that material can reduce or obviate the need to add water in forming the pre-treatment reaction mixture of the present invention.

The physical pre-treatment of lignocellulosic materials - which may precede, be combined with or be integrated with the recited pre-treatment with the caustic magnesium salt - is not precluded by the present invention. For instance, the lignocellulosic materials may be subjected to steam injection, torrefaction, pyrolysis or γ-irradiation . More usually, the materials may be subjected to mechanical comminution by one or more of shredding, chipping, grinding, compression/expansion, extrusion and milling, including vibratory ball milling: these processes may serve to: reduce the particle size of the lignocellulosic materials ; increase the surface area of the material and its accessibility to hydrolytic enzymes; and, reduce cellulose crystallinity.

In an embodiment, the lignocellulosic material may either possess or be comminuted to a particle size of from 0.1 to 250 mm and preferably from 0.1 to 50 mm. Grinding may for instance be used to reduce the average particle size of the materials to from 10-30 mm; milling may achieve yet smaller particle sizes, having an average size of from 0.1 to 2 mm for instance. The desirable particle size distribution or comminution ratio of the lignocellulose materials depends in part on determining an acceptable energy consumption of the mechanical comminution step based on the predicted yield of lactic acid. The teaching of Cadoche, L. et al . Assessment of size reduction as a preliminary step in the production of ethanol from lignocellulosic wastes Biol. Wastes 1989, 30, 153-157 may be instructive in evaluating energy consumption for the size reduction of hardwoods and agricultural wastes as a function of final particle size and comminution ratio.

In an embodiment of the invention, the optionally physically pre-treated lignocellulosic material, is subjected to a pre-extraction step. As used herein, the term "pre-extraction" refers to any process or technique which is applied prior to the defined pre-treatment step with the intent of removing soluble components from the lignocellulosic bxomass , Whilst such pre-extraction will generally be applied to remove non-fermentable, soluble components contained in the bxomass, such as proteins, amino acids and soluble inorganic components, it is also envisaged that pre- extraction may be employed to remove soluble, fermentable components from the bxomass.

Whilst it is not preferred, a step of acid hydrolysis may precede the contacting of the lignocellulosic material with the caustic magnesium salt, whereby the caustic salt may act to neutralize at least part of the acid present. Such an acid hydrolysis step is generally effected by contacting the feedstock with an acidic aqueous solution stream which may include one or more of an inorganic acid, an organic acid, an amino acid, a mineral acid, a Bronsted acid and a Lewis acid. More usually, the acid may be sulfuric acid, sulfonic acid, phosphoric acid, nitric acid, acetic acid, lactic acid, formic acid, oxalic acid, succinic acid, levulinic acid, carbonic acid, glycolic acid, uronic acid, glucaric acid, hydrofluoric acid, hydrochloric acid, boric acid, boron trifluoride, or any combination of these acids. Solutions containing acidic salts, such as aluminum sulfate, ferric sulfate, aluminum nitrate or ferric nitrate may also be useful. Whilst concentrated acids may be powerful agents for cellulose hydrolysis, they are concomitantly toxic, corrosive and hazardous, and are therefore not preferred for use herein. Desirably therefore, when used, an aqueous acidic, pre-treatment solution should have a concentration of less than 8 wt , % , for instance less than 4 wt . % . A dilute acid pre-treatment step may typically occur at a temperature of from 150 to 200°C.

The person of ordinary skill in the art will of course be able to determine an appropriate contact or residence time, acid concentration and contact temperature for particular acids and ligncellulosic feedstocks. And the following teachings may inter alia be instructive in this regard: Esteghlalian, A. et al. Modelling and optimization of the dilute-sulfuric-acid pretreatment of corn stover, poplar and switchgrass, Bioresour. Technol . 1997, 59, 129-136. (77); Hinman, N. D, et al . Preliminary estimate of the cost of ethanol production for SSF technology, Appl, Biochem. Biotechnol. 1992, 34/35, 639-649; and, Brennan, A, H, et al , High temperature acid hydrolysis of biomass using an engineering-scale plug flow reactor: Result of low solids testing, Biotechnol. Bioeng. Symp. 1986, 17, 53-70.

Treatment with Caustic Magnesium Salt

In accordance with the process of the present invention, the lignocellulosic material, optionally treated as described hereinabove, is combined with the alkaline agent in the presence of water to form a reaction mixture .

The amount of alkaline agent so combined is determined such that the concentration of the caustic magnesium salt in the reaction mixture or slurry is at least 0,1 wt . % (w/w) , preferably at least 0.5 wt . % (w/w) , based on the dry weight of the lignocellulosic material. Typically, the amount of alkaline agent is determined such that the concentration of the caustic magnesium salt is at most 50 wt . % (w/w), based on the dry weight of the lignocellulosic material. Good results have, for instance, been obtained when the concentration of the caustic magnesium salt in the pre-treatment reaction mixture is from 0.5 to 40% (w/w) and particularly from 5 to 30% (w/w) .

The reaction mixture or slurry formed should typically have a solids content of from 1 to 70% (w/w) , for example from 10 to 60% (w/w) and preferably from 20 to 50% (w/w) . The reaction mixture or slurry may be further characterized by a pH of from 8.0 to 14.0, preferably from 8,5 to 13.0 and more preferably from 9,0 to 12.0. As would be recognized by a person of ordinary skill in the art, a pH greater than 9 may be achieved by adding one or more caustic salts, other than the caustic magnesium salt(s) to the reaction mixture or slurry.

These characteristics of magnesium salt concentration, solids content and pH of the reaction mixture or slurry are not mutually exclusive; the mixture may possess any combination of the defined properties. A person of skill in the art will also recognize that the amounts of water and caustic magnesium salt are result effective variables and thus the most preferred amounts thereof might differ from those values stated depending on the type of lignocellulosic material.

As previously noted, the alkaline agent may be added in a solid, particulate form before or after any water which is required to adjust the solids content to an appropriate level. Alternatively, the alkaline agent may be added as an aqueous solution or dispersion but this variant does not preclude the further addition of either water or particulate, solid alkaline agent to the mixture.

In an important embodiment of the present invention, the alkaline agent in the form a solid, aqueous solution or aqueous suspension is provided at a temperature above 25 °C for mixing with the lignocellulosic biomass; temperatures of from 25°C to 300 °C, or from 100⁰ C to 250 ° C are feasible. This embodiment enables the caustic magnesium salt(s) to be sourced from thermal processes performed in situ without intermediate loss of thermal energy. By way of example, magnesium oxide sourced from in situ calcination may be utilized in this manner.

Suitable reactors or reaction vessels within which the reaction mixture may be disposed should be closed and pressurizable but preferably allow for any carbon dioxide formed during the reaction to be vented, either continuously or periodically. A person of ordinary skill in the art will be able to make an appropriate determination of a suitable reactor - or a suitable series of reactors - based on whether the reaction is to be performed as a batch process or an essentially continuous process with continuous feeding of reaction mixture and withdrawal of the hydrolysis product. Without intention to limit the present invention, examples of suitable reactors include: horizontal reactors with screw transport of the biomass; vertical tower reactors such as those disclosed, for example, in UK Patent Nos . GB 706,686 and GB 812,832.

In both batch and continuous processes, it is of course necessary to adequately mix the water, caustic salt and lignocellulosic material. It may also be desirable to cause mixing of the liquid phase, containing the lignocellulosic material, and any gas phase per se which may be present in the reactor. Adequate mixing may be achieved by, for instance, mechanical stirring, liquid phase recirculation or by selecting an appropriate flow rate through a tubular reactor.

Heat may be supplied to the reaction mixture by any suitable method including but not limited to: steam heating; induction heating; microwave heating; immersion of the reactor or reaction vessel in an appropriate heating bath; by means of a thermally conductive material which either contacts the reactor or reaction vessel or is immersed within the mixture and through which heated fluid is passed; or, similarly, by means of one or more electrical resistance heating elements contacting the outside of the reactor or reaction vessel and/or being immersed in the reaction medium. The reactor (s) or reaction vessels may optionally be p re-warmed prior to the introduction of the lignocellulosic material . In the present invention, the aqueous salt / biomass reaction mixture is maintained in the reaction vessel (s) at a temperature of from 130 °C to 250°C, such as from 140°C or from 170°C to 250°C, and preferably from 170°C to 230°C. The reaction mixture should be at a pressure within the vessel (s) such that boiling of the liquid, aqueous medium does not occur under the temperature conditions in question. The total residence time in the reactor (s) at the above recited temperature and, as defined for the solids present within the reaction mixture, should be from 1 to 600 minutes, and more usually be from 1 to 480 minutes. As will be recognized, the residence time of the water within the reactor (s) may differ from that of the solids, depending on the type of reactor employed.

The preferred residence time will depend significantly upon the selected temperature, pH and type of lignocellulosic material. Selecting a higher temperature within the defined range can reduce the required residence time to achieve an effective hydrolysis. Analogously, operating at a higher pH within the recited range will also permit a reduced residence time ,

By way of illustration good results have been obtained where the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% (w/w) and a pH of from 9.0 to 12, and said solids are held at a temperature of from 130°C to 250 °C for a time period of from 1 minute to 240 minutes, preferably 1 to 30 minutes. Similarly, independent of the pH, good results have been obtained the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% (w/w) and where said solids of the reaction mixture are held a temperature of: either i) from 170° to 230 °C for a period of from 1 to 240 minutes, preferably from 1 to 120 minutes; or, ii) from 140° to 170°C for a period of from 180 to 600 minutes, preferably from 240 to 480 minutes.

After the requisite residence time the product of the alkaline hydrolysis is collected for further processing. Whilst it is not obligatory, the lignocellulosic feedstock may be processed, after the pre-treatment with caustic magnesium salt(s), to obtain a solids stream comprising the pretreated feedstock and an aqueous stream comprising soluble components. This may be carried out by washing the pretreated feedstock with an aqueous solution to produce a wash stream, and a solids stream comprising the pretreated feedstock. Alternatively, the pretreated feedstock may be subjected to a solids- liquid separation, using known methods such as centrifugation, microfiltration, plate and frame filtration, crossflow filtration, pressure filtration, vacuum filtration and the like. The aqueous stream thus obtained can itself be separately subjected to the fermentation to ferment the available sugars: for example, xylose present in this stream may be fermented to ethanol, xylitol , lactic acid, butanol , or a mixture thereof .

In one aspect, the present invention is directed to the use of the lignocellulosic material, pre-treated in the above described manner, as a substrate for digestion by one or more of acid hydrolysis, enzymatic hydrolysis and microbial hydrolysis. More particularly, the treated lignocellulosic material is used as a substrate for at least microbial hydrolysis, wherein organic acids are obtained from the transformation of carbohydrates derived from the treated lignocellulosic material by a microorganism via fermentation. An intermediate step of wet oxidation, where the pre-treated material is contacted with oxygen at an elevated temperature of from 150 to 185 °C, for instance, before enzymatic and / or microbial hydrolysis is not precluded by the present invention. Further, as is well-known in the art, the H, temperature, nutrient level and carbohydrate content of the pre-treated lignocellulosic material or the solids stream comprising the lignocellulosic material may be moderated to facilitate enzymatic hydrolysis and / or microbial fermentation .

In preparation for biologically mediated transformation, the pretreated lignocellulosic feedstock or the solids stream comprising the pretreated feedstock is typically slurried in an aqueous solution such as process water, fresh water, steam condensate or process recycle streams. The aqueous slurry should ideally have a solids concentration that enables it to be pumped and the tolerated concentration of pretreated lignocellulosic feedstock in the slurry will then depend upon inter alia the particle size and water retention of the feedstock and the pump capacity .

In practice, the solids concentration is usually from 3 to 30 wt . % , based on the total weight of the slurry, with a solids concentration from 10 to 25 wt . % being preferred. Where required, the concentration of suspended or undissolved solids can be determined by filtering a sample of the slurry using glass microfiber filter paper, washing the filter cake with water, and drying the cake overnight. It is further preferred that the fibrous or particulate solids comprise from 20 or from 30 wt . % to 70 wt. % cellulose.

In an exemplary biologically mediated transformation, the pre-treated lignocellulosic material is processed by: saccharif ing the treated aqueous lignocellulosic biomass in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic biomass comprising fermentable carbohydrates and an insoluble lignocellulosic fraction; fermenting the saccharified aqueous lignocellulosic biomass in the presence of organic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising the magnesium salt of said organic acid and a solid lignocellulosic fraction; and, recovering organic acid from the fermentation broth. The saccharification and fermentation steps may be carried out simultaneously or sequentially. The solid lignocellulosic fraction derived from the saccharification and fermentation steps comprises unhydrolyzed cellulose and hemicellulose and undissolved lignin fractions .

In an important embodiment of the present invention, the pre-treated lignocellulosic material is processed by: saccharifying the treated aqueous lignocellulosic biomass in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic biomass comprising fermentable sugars and an insoluble lignocellulosic fraction; fermenting the saccharified aqueous lignocellulosic biomass in the presence of lactic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; and, recovering lactic acid and/or a lactate salt from the fermentation broth. The saccharification and fermentation steps may be carried out simultaneously or sequentially. The solid lignocellulosic fraction derived from the saccharification and fermentation steps comprises unhydrolyzed cellulose and hemicellulose and undissolved lignin f actions .

Saccharifcation generally uses one or more enzymes selected from the group consisting of: cellulases such as CBHl , CBH2 , EG, and BGL; GH61 polypeptides having cellulolytic enhancing activity as described in, for example WO2005/074647 , WO 2008/148131, and WO 2011/035027; hemieellulases ; expansins ; esterases, such as acetylxylan esterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate; laccases ; ligninoiytic enzymes; pectinases ; peroxidases; proteases; amylolytic accessory enzymes; inulinases , levanases ; and swollenins . Where enzymatic hydrolysis or saccharification occurs separately from fermentation, the teaching of the following documents may, by way of example, be instructive: US 20130122554 (Honda Motor Co, Ltd.) and Japanese Patent Laid-Open Nos . 2006-101829 and 2008-271962.

The fermentation of the optionally saccharified lignocellulosic material - hereinafter the fermentation medium - will now be described in more detail .

Fermentation or Simultaneous Saccharification and Fermentation As is known in the art, the fermentation medium may be provided with additional fermentable carbohydrates. This may be necessary if the content of fermentable carbohydrates, as measured by high-pH anion exchange chromatography based on a calibration against appropriate C5, C6 and/or C12 sugar standards, is considered to be too low. It is also possible to combine a primary slurry having a relatively low fermentable carbohydrate content with a secondary slurry having a relatively high fermentable carbohydrate content.

It is also known to supplement the fermentation medium with additional nutrients besides the lignocellulosic material. Such additional nutrients, which may be added in solid form or as solutions or dispersions in water, typically comprise one or more of: mineral salts, in particular sources of mineral nitrogen, phosphate, sulfur and trace elements such as zinc, magnesium, calcium, manganese, potassium, sodium, boric, iron, cobalt, copper, molybdenum, nickel and aluminum; organic nitrogen, for example yeast autolysates and hydrolysates , plant protein hydrolysates, and, animal protein hydrolysates. Such organic nitrogen sources generally provide nitrogen in the form of free amino acids, oligopeptides, peptides, vitamins and traces of enzyme cofactors; the addition of such species to the medium in pure form is also not precluded.

During fermentation, carbohydrate degrading enzymes, as previously mentioned, may be added to the fermentation broth to assist the degradation of fermentable carbohydrates, especially those in polymeric form. This concept of simultaneous saccharification and fermentation is described in, for example, WO 03/095659 and US2013236933 (Huang et al. ) .

Prior to inoculation, the pH of the fermentation medium should be adjusted to a pH suitable for fermentation with the microorganism of choice. By adding an appropriate compound - usually an acid such as sulfuric acid, nitric acid, hydrochloric acid or, preferably lactic acid - to the fermentation medium, the pH thereof is adjusted to a value of, usually, from 2 to 10. For illustration, pH values of the medium are typically from 5 or from 6 to 8 for simultaneous saccharification and fermentation, and are typically from 2 to 5 for so-called low pH fermentations. Where the lignocellulosic material was subjected to acid hydrolysis prior to being mixed and treated with the caustic magnesium salt, the neutralization of acid with that salt may, at this stage, mitigate the amount of the pH adjustant required to bring the pH to a desirable level.

In an interesting embodiment, the pH of the fermentation medium may be adjusted by the addition of an amount of lignocellulosic material which itself has an acidic pH on account of being treated by acid hydrolysis, such as described hereinabove.

The fermentation medium is fermented by means of an organic acid producing microorganism - bacteria, yeasts and / or fungi, for instance - in the presence of a caustic magnesium salt to provide a fermentation broth containing the magnesium salt of the organic acid. The fermentation is generally performed by incubating the fermentation medium with the microorganism at a suitable temperature for a suitable period of time.

The processes will hereinafter be described with specific reference to the preferred production of lactic acid and the processing of magnesium lactate. Whilst various modifications of the described processes may be required to derive alternative or additional organic acids, such as acetic or succinic acids, and to process the magnesium salts thereof, such modifications are easily effected by a person of ordinary skill in the art .

Suitable lactic acid producing microorganisms may include bacteria, fungi and yeasts, and may be selected from microorganisms that are either homolactic lactic acid producers or heterofermentative microorganisms which produce lactic acid. The microorganisms may be genetically engineered to produce or overproduce lactic acid.

Examples of such microorganisms include, but are not limited to: bacterial species of the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus,

Vagococcus, Weissella , Bacillus (including Bacillus coagulans, Bacillus licheniformis, Bacillus smithii, Bacillus thermolactis and Bacillus thermoamylovorans) , Geobacillus (including Geobacillus stearothermophilus and Geobacillus thermoglucosidans) ,

Caldicellulosiruptor (including Caldicellulosiruptor saccharolyticus) , Clostridium (including Clostridium thermocellum) , Thermoanaerobacterium (including Thermoanaerobacterium saccharolyticum) , Thermoanaerobacter and Escherichia (including Escherichia coli) ; and, fungal and yeast species from the genera Saccharomyces (including Saccharomyes cerevisiae) ,

Kluyveromyces (including Kluyveromyces lactis and Kluyveromyces marxianus) , Issatchenkia (including Issatchenkia orientalis) , Pichia (including Bichia stipitis) , Candida (including Candida boidinii, Candida magnolia , Candida methanosorbosa , Candida sonorensis and Candida utilis) and Rhizopus (including Rhizopus arrhizus, Rhizopus microspores and Rhizopus oryzae) .

Bacterial genera that are of particular interest are Lactobacillus, Bacillus (including Bacillus coagulans, Bacillus licheniformis, Bacillus smithii, Bacillus thermolactis and Bacillus thermoamylovorans) , Geobacillus (including Geobacillus stearothermophilus and Geobacillus thermoglucosidans) and Escherichia (including Escherichia coli) . Additionally or alternatively, preferred bacterial species are those that display optimal growth at a pH in the range of about 6 to about 8.

The incubation temperature may depend on the microorganism used. For example, the optimum temperature to be used may be established by analyzing the activity of the fermentation microorganism under different temperature conditions. In general, the temperature may be within the range from 30 to 80 °C; preferably a temperature with the range from 40 to 75°C is used and more preferably a temperature of from 45 to 70°C. As is known in the art, the pH of the fermentation medium may be adjusted and controlled during the fermentation: a drop in pH below a critical value, depending upon the microorganism used in the process, could damage the metabolic process of the microorganism and bring the fermentation process to a stop. Generally, the pH is adjusted during fermentation so as to maintained with the aforementioned ranges of from 4 to 9 or, preferentially, from 5 to 8.

Herein the adjustment of pH is effected by a caustic magnesium salt preferably selected from MgO, Mg (OH) 2, MgCCb, Mg (HCO3) 2 and mixtures thereof; the caustic magnesium salt neutralizes the lactic acid excreted by the microorganisms during fermentation, thereby generating a magnesium lactate salt. Optionally, a portion of the caustic magnesium salt present during fermentation may have been provided by or be residual from the pre-treatment step. Additionally or alternatively, the caustic magnesium salt may be purposively added during the course of fermentation. The addition of minor, supplementary amounts of other caustic salts, in particular caustic salts of alkali and alkaline earth metals is not precluded.

The person of ordinary skill in the art will be aware that magnesium lactate crystals can form during fermentation where the concentration of the salt is sufficiently high. Whether or not precipitation of magnesium lactate occurs will thereby depend on the concentration of fermentable carbohydrates in the fermentation medium, the fermentation temperature, the concentration of other constituents of the fermentation medium and the dilution factor of the added caustic magnesium salt. Typically, magnesium lactate remains soluble in a fermentation broth at a concentration of at the most 9.5 wt . % when measured at a temperature of 80°C.

The fermentation is allowed to proceed for a period of from 4 hours to 1 week; an exemplary period of from 8 hours to 3 days might be mentioned. The fermentation might equally be stopped when the content of fermentable carbohydrates in the liquid phase of the fermentation broth is below 5 g/1, for example below 2g/l or lg/1. The amount of fermentable carbohydrates may be monitored by subjecting extracted samples of the fermentation broth to a solid/liquid separation step, thereby removing any solids from the liquid phase; a chromatogram of that liquid phase is then obtained by high-pH anion exchange chromatography using a suitable device such as a pulsed amperometric detector (HPAEC-PAD) . The carbohydrate composition of the liquid phase is then determined based on a calibration performed by using appropriate standards (e.g. C₅, C₆ and/or C12 sugar standards) . Generally, the molar yield of lactic acid produced relative to the fermentable carbohydrates consumed (e.g. C5, Ce and/or C12 sugars) is from 70 to 100 % , in particular from 80 to 100 % .

The fermentation of a fermentation medium comprising the pre-treated lignocellulosic material in combination with caustic magnesium salt can result in a fermentation broth comprising magnesium lactate in a concentration at which the magnesium lactate is only present in solution. However, it is preferred for crystallization of magnesium lactate to occur within the fermentation broth.

It is possible for the fermentation broth - comprising dissolved and crystalline magnesium lactate, the lignocellulosic fraction and biomass

- to be recovered in toto and subj ected to further processing either without prior treatment of the broth or after a concentration step whereby water is evaporated from the broth under ambient or reduced pressure to further crystallize out magnesium lactate .

The present invention also does not preclude methods of recovery of the magnesium lactate by which that salt is partially purified . For instance, in an exemplary method, the fermentation broth comprising dissolved and crystalline magnesium lactate is subj ected to a solid / liquid separation; the solids thus obtained - which include magnesium lactate, lignin and biomass - are then further processed; and, the mother liquor ma be recycled to the biologically mediated transformation process - for instance to the enzymatic hydrolysis and / or the fermentation steps

- to provide water balance thereto , In an alternative partial purification method, dissolved magnesium lactate may be isolated from a fermentation broth by sequentially performing: i) a solid/liquid separation step, optionally conducted at a temperature of from 20 to 75°C or from 30 to 60°C, by which the magnesium lactate-containing fermentation broth is treated by one or more of flotation, sedimentation, flocculation, centrifugation, filtration and decantation, to provide a magnesium lactate-containing medium which is separated from the biomass and other solid impurities which remain in the solid residue; and, ii) a concentration step to provide magnesium lactate crystals from the magnesium lactate medium. This concentration step ii) may be performed by removal of water under ambient or reduced pressure or by means of evaporative crystallization. Alternatively, this concentration step may be performed by evaporation followed by cooling crystallization. Salts originating from the lignoeellulosic materials remain in the liquid phase and do not thereby co-precipitate or co-crystallize with the magnesium lactate crystals. The magnesium lactate crystals formed may be separated by solid/liquid separation and washed.

There is no however intention in the present invention to limit the methods by which magnesium lactate may be concentrated and recovered from the fermentation broth. The term recovered is intended to encompass both isolation of magnesium lactate from the broth and the extraction of magnesium lactate as a solid, solution or suspension in combination with the biomass residue from the fermentation step. And one or more known or conventional methods of recovery like liquid/liquid extraction, nanofiltration, active carbon treatment, distillation and re- crystallization, adsorption, electro-dialysis, membrane separation, reactive extraction and esterification can be adopted herein. Further suitable methods are disclosed in inter alia WO2005/123647 and WO 2011/095631 (both Purac Biochem B.V.), the disclosures of which are incorporated herein by reference. And such methods may equally find utility in further purification steps applied to the magnesium lactate. The recovered yield of lactic acid in the form of magnesium lactate crystals is usually from 50 wt . % or even from 70 wt . % to 99 wt . % , based on the amount of lactic acid produced during fermentation.

Whilst the industrial utility of organic acids and, in particular, of lactic acid are well known, those impurities originating from the pre- extraction, pre-treatment and / or fermentation steps of the present invention may also find utility after appropriate physical and / or chemical processing. It is envisaged that residual cellulose and lignin may serve as boiler fuel for electricity or steam production. Further, black liquor gasification of lignin is a recent commercial development. Certain impurities may be used as fertilizer, particularly magnesium rich fertilizer. And carbon dioxide released in the fermentation process may be captured for sale, for example, to the beverage industry.

The present invention is further illustrated by the following Example, without being limited thereto or thereby.

Examples

Raw Materials and Their Analysis:

Bagasse was provided by Purac Thailand Ltd. (Rayong, Thailand).

Ground wheat straw was obtained from a local supplier. For Examples 1 to 9 below, the wheat straw was milled using a Retch Cutting Mill (SM100) and then screened to attain a median particle size in the range from 500 microns to 1 mm. For Examples 10 to 12 below, the wheat straw was milled using hammer mill (Apex Commuting Mill) with a screen size of 1.5mm.

Using a Mettler Toledo Advanced Moisture Analyzer, the dry weight contents of selected 0.5 - 2. Og milled wheat straw samples shed - were measured .

The wheat straw and bagasse were analyzed for carbohydrate, acid soluble lignin, acid insoluble lignin and ash contents in accordance with the procedure given in: Determination of Structural Carbohydrates and Lignin in Biomass : Laboratory Analytical Procedure (LAP), National Renewable Energy Authority (August 2012) http: //www , n el . gov/docs/gen/fy!3/42618. pdf; and, Determination of Ash in Biomass: Laboratory Analytical Procedure (LAP), National Renewable Energy Authority (January 2008) http: //www, nrel . gov/docs/gen/fy08/42622.pdf. Where applicable, current practice relating to said procedures may be found at

Where applicable, glucose was determined using the Megazyme D-Glucose assay kit (glucose oxidase/peroxidase; GOPODj employing a pulsed amperometric detector (Roche/Hitachi GOD-PAD) and spectrophotometry (Hitachi U-2800, 540 nm) . Xylose may be determined using the Megazyme D-xylose kit and spectrophotometry (Hitachi U-2800, 34 Onm) .

The theoretical maximum yield of glucose from wheat straw was determined to be 37.1 wt . % , based on the total dry weight ,

Examples 1 - 9

Pre-Treatment : Weighed 12.45g portions of the milled wheat straw or bagasse were separately slurried in 150ml demineralised water, following which the solid magnesium oxide or, where applicable, solid calcium oxide or sodium hydroxide were added thereto; no f rther basic compounds were added . For completeness , it is noted that in Example 9 below, the NaOH was added to the milled wheat straw after the solid magnesium oxide, thereby raising the pH of that sample ,

The pre-treatment of said samples was carried out in a double wall stainless steel , stirred reactor (Buchi Autoclave ) . The reactor was rated for 60 Bar and was equipped with a pressure safety spring . Heating was carried out using hot oil up to 190 °C .

The properties of each separate portion and the different conditions of temperature and residence time to which they were subj ected are shown in Table 1 herein-below . All samples were continuously stirred for the requisite residence time in the reactor . Further all samples that were treated with caustic magnesium or calcium oxide had an initial pH of from 8.5-9.4 prior to any heating step.

Preparation for Enzymatic Hydrolysis : The thus pre-treated solids were subjected to a first solid/liquid separation step using a Buchner filter under reduced pressure (approx. 200mBar) . The liquid fraction was collected for analysis. The solids were collected, dispersed in water and neutralized to a pH of from 6-7 with lactic acid.

Next the solids were subjected to a two-stage second solid / liquid separation step using a filtering centrifuge (Hermle Sieva 2) equipped with a 5 micron filter cloth. In a first stage, the centrifuge is initiated at 5000 rpm before being increased to 10000 rpm; the filtrate is collected and added to the centrifuge again; samples of the then derived filtrate and filter cake are collected for analysis. In a second stage, the centrifuge is re-initiated with the addition of 1 litre of demineralized water thereto. The separated solid fraction was then collected; its dry matter content was measured before being subjected to enzymatic hydrolysis.

Enzymatic Hydrolysis: At a dry matter content of 10% (w/w) , the pre- treated solids were hydrolyzed in 50 rtiL polypropylene tubes with a cellulase enzyme mix CMAX available from Dyadic. A potassium phosphate buffer (pH, 6.4) was employed and, furthermore, sodium azide {0.02 %, w/w) was present to prevent microbial infection of the hydrolyzate. The added amount of enzyme was varied in the experiments - as indicated in Table 1 - with enzyme loading of 20mg/g dry weight being more usual, noting that this loading should ensure satisfactory release of carbohydrates (NREL, 2011) .

The hydrolyzation reactions were incubated at 52 °C at 300 rpm. After 24 hours, 48 hours and 72 hours, duplicate 0.2 ml samples were taken and filtered using a micro plate; the duplicate supernatants were then analyzed.

The concentrations of glucose are given in Table 1 below. Table 1

It is clear from Table 1 that the elevated temperature pre- treatment of the wheat straw with caustic magnesium salt facilitates the subsequent hydrolysis of this biomass , as evidenced by the attained concentrations of glucose.

Example 10

This Example is intended to demonstrate the scaling up of the pre-treatment process to accommodate larger amounts of lignocellulosic material.

Pre-Treatment : Weighed 1.6 kg portions of the milled wheat straw were separately slurried in 13.4 litres of demineralised water, following which the solid magnesium oxide was added thereto; no further basic compounds were added.

The pre-treatment of said samples was carried out in a double wall stainless steel, jacketed reactor equipped with an anchor propeller (50 litre Buchi Autoclave) . The reactor was rated at greater than 20 Bar and was equipped with a pressure safety spring. Heating to 190 °C was carried out using both direct injection of pressurized steam and hot oil circulated through the jacket. The reaction mixture was stirred continuously during its residence time in the reactor .

After the reaction was performed, the reactor was cooled using oil circulated in the jacket followed by a rapid cooling effected by releasing the pressure of the reactor. The contents of the reactor were then collected.

The properties of the collected material and the conditions of temperature and residence time to which that material had been subjected are shown in Table 2 herein-below . Further the samples that were treated with caustic magnesium oxide in this manner had an initial pH of from 8.5-9.4 prior to any heating step.

Preparation for Enzymatic Hydrolysis: The thus pre-treated, solids were subjected to a first solid/liquid separation step under gravity using a 1mm screen, the liquid fraction being collected in a 120 liter vessel. The solids were collected, dispersed in water in a further 120 liter vessel and neutralized to a pH of from 6-7 with lactic acid (50 wt . % aqueous solution} .

The slurry formed was again filtered under gravity as described above (1 nun screen) except that 26 liters of demineralized water was sprinkled evenly over the filter cake. The filter cake was divided into six portions (SPs) each of which was pressed at an applied pressure of 250 Bar using a bench press filter (Fischer MachineFabriek) . Each separated solid fraction was then collected; the cakes were subsequently broken up, homogenized and distributed over two containers (SPl , SP2) . The dry matter content of each container was measured and found to be from 38-42% (w/w) .

Enzymatic Hydrolysis: The pre-treated solids were hydrolyzed in polypropylene tubes with a cellulase enzyme mix CMAX4 available from Dyadic; the pre-treated solids were added so as to be in an amount of 10 wt.%, by dry weight (c. 1 g dry weight ) . A potassium phosphate buffer (pH, 6.4) was employed and, furthermore, sodium azide (0.02 % , w/w) was present to prevent microbial infection of the hydrolyzate . The enzyme loading was 20mg/g dry weight .

The hydrolyzation reactions were incubated at 52 °C at 300 rpm. After 24 hours and 48 hours , duplicate 0.2 ml samples were taken and filtered using a micro plate ; the duplicate supernatants were then analyzed .

The concentration of glucose after 48 hours is given in Table 2 below . Table 2

It will be apparent to those skilled in the art, upon consideration of the specification that various modifications can be made in the disclosed embodiments without departing from the scope of the invention. It is therefore intended that the embodiments and examples be considered illustrative only, with the true scope of the invention being indicated by the following claims.

Claims

1. A process for treating a lignocellulosic material to render it amenable to biologically mediated transformation, said process comprising :

i) providing a lignocellulosic material;

ii) mixing said lignocellulosic material with an alkaline agent in the presence of water to form a reaction mixture having a solids content, said alkaline agent comprising a caustic magnesium salt; and,

iii) heating said reaction mixture such that said solids are held at a temperature of from 130°C to 250°C for a time period of from 1 minute to 600 minutes.

2. The process according to claim 1, wherein said lignocellulosic material as provided in step i) has been subjected to one or more of pre-extraction, steam pre-treatment, acid hydrolysis and mechanical comminution,

3. The process according to claim 1 or claim 2, wherein said lignocellulosic material as provided in step i) is particulate and has an average particle size of from 0.1 to 250 mm, preferably 0,1 to 50 mm,

4. The process according to any one of claims 1 to 3, wherein said lignocellulosic material as provided in step i) has a cellulose content of from 20 to 70 t.%, based on the dry weight of the material .

5. The process according to any one of claims 1 to 4, wherein the sum of the cellulose and hemicellulose content of said lignocellulosic material provided in step i) is from 30 to 99 wt.%.

6. The method according to any one of claims 1 to 5, wherein the alkaline agent comprises or consists of a caustic magnesium salt selected from MgO, Mg (OH) 2, MgC0₃, Mg (HC0₃) 2 and mixtures thereof.

7. The method according to any one of claims 1 to 6, wherein the concentration of the caustic magnesium salt in the reaction mixture is from 0.1 to 50 wt . %, based on the dry weight of the lignocellulosic material (w/w) .

8. The method according to claim 7, wherein the concentration of the caustic magnesium salt is from 0.5 to 40% (w/w), preferably from 5 to 25% (w/w) .

9. The method according to any one of claims 1 to 8, wherein the total solids concentration of the reaction mixture is from 1 to 70% (w/w) , preferably from 10 to 60% (w/w) and more preferably from 20 to 50% (w/w) .

10. The method according to any one of claims 1 to 9, wherein said reaction mixture has a pH of from 8.0 to 14.0, preferably from 8.5 to 13.0 and more preferably from 9.0 to 12.0.

11. The method according to claim 10, wherein:

the reaction mixture has a pH of from 9.0 to 12.0 and, in step iii) , said solids are held at a temperature of from

130 °C to 250⁰ C for a time period of from 1 minute to 240 minutes, preferably from 1 to 30 minutes.

12. The method according to any one of claims 1 to 10, wherein: the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% (w/w) and,

said solids of the reaction mixture are held at a temperature of from 140° to 170⁰ C for a period of from 180 to 600 minutes, preferably from 240 to 480 minutes.

13. The method according to any one of claims 1 to 10, wherein: the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% ( w/w ) ; and,

said solids of the reaction mixture are held at a temperature of from 170 °C to 230°C for a period of from 1 to 240 minutes, preferably from 1 to 120 minutes.

14. A treated lignocellulosic material obtained by the process of any one of claims 1 to 13.

15. Use of the lignocellulosic material as defined in claim 14 as a substrate for digestion by one or more of acid hydrolysis, enzymatic hydrolysis and microbial hydrolysis.

16. A method for producing a fermentation product comprising organic acid from lignocellulosic material, said method comprising; a) treating a lignocellulosic material with an alkaline agent in the presence of water in accordance with the method defined in any one of claims 1 to 13;

b) saccharif ing the treated aqueous lignocellulosic biomass in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic biomass comprising fermentable carbohydrates and an insoluble lignocellulosic fraction;

c) fermenting the saccharified aqueous lignocellulosic biomass in the presence of organic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising the magnesium salt of said organic acid and a solid lignocellulosic fraction; and,

d) recovering organic acid from the fermentation broth, wherein the saccharxficatxon and fermentation steps may be carried out simultaneously or sequentially.

17. A method for producing a fermentation product comprising lactic acid from lignocellulosic material, said method comprising: a) treating a lignocellulosxc material with an alkaline agent in the presence of water in accordance with the method defined in any one of claims 1 to 13;

b) saccharifying the treated aqueous lignocellulosxc material in the presence of a hydrolytic enzyme to provide an saccharified aqueous lignocellulosxc material comprising fermentable carbohydrates and a solid lignocellulosxc fraction;

c) fermenting the saccharified aqueous lignocellulosxc material in the presence of lactic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; and,

d) recovering lactic acid and/or a lactate salt from the fermentation broth,

wherein said saccharification and said fermentation are carried out simultaneously or sequentially.

18. The method according to claim 16 or claim 17, wherein the caustic magnesium salt of step c) is independently selected from MgO, Mg (OH) 2 , MgC0₃, Mg (HC0₃) ₂ and mixtures thereof.

19. The process according to any one of claims 16 to 18, comprising the steps of:

a) i) treating a first portion of lignocellulosic material with an alkaline agent in the presence of water in accordance with the method defined in any one of claims 1 to 13;

a) ii) treating a second portion of lignocellulosic material with an acid; and,

a) iii) mixing the treated first and second portions of lignocellulosic material prior to the saccharification and / or fermentation steps .

20. The method according to any one of claims 16 to 19 comprising simultaneous saccharification and fermentation.