BR112016012592B1 - METHOD FOR THE FRACTIONATION OF LIGNOCELLULOSIC MATERIAL AND METHOD FOR THE PRODUCTION OF MICROBIAL LIPID - Google Patents

Abstract

METHOD FOR PROCESSING LIGNOCELLULOSIC MATERIAL USING AN ALKALINE DELIGNIFYING AGENT. The present invention relates to a method for obtaining sugar and lignin fractions from lignocellulosic materials and to a method for producing fermentation products using sugars obtained from lignocellulose. In particular, the present invention relates to a method for improving the sugar yield in the enzymatic hydrolyzate by introducing a hydrothermal hydrolysis pretreatment step and an alkaline delignification step.

Description

FIELD OF INVENTION

[001] The present invention relates to a method for obtaining sugar and lignin fractions from lignocellulosic materials and to a method for producing fermentation products using sugars obtained from lignocellulose.

BACKGROUND OF THE INVENTION

[002] Lignocellulose is the most abundant biopolymer on earth. Lignocellulose is the main structural component of woody plants and non-woody plants such as grass. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose and lignin. Large quantities of lignocellulosic waste are produced through forestry, wood and pulp and paper industries and agricultural practices (e.g. straw, crop residues, sugar cane bagasse, glume, shale) and many agro-industries. Furthermore, municipal waste contains fractions that can be considered as lignocellulose waste, such as waste paper or cardboard, garden waste or waste construction wood. Lignocellulosic waste, such as agricultural waste, offers highly sustainable non-food and non-ILUC (indirect land use change) alternatives for the production of biofuels. Furthermore, due to the great abundance and low prices, lignocellulosic waste is the preferred material for the production of biofuels. Furthermore, dedicated woody or herbaceous energy crops with biomass productivity have attracted interest as biofuel uses.

[003] The production of biofuels, especially ethanol, from lignocellulosic materials by microbial fermentations has been extensively studied. The biggest challenge for using lignocellulosic materials for the microbiological production of biofuels or biofuel inputs lies in the complexity of the lignocellulose material and its resistance to biodegradation. In lignocellulose, cellulose fibers (20 to 50% of plant dry weight) are embedded in a covalently found matrix of hemicellulose (20 to 40%), pectin (2 to 20%), and lignin (10 to 25%). %), forming a structure very resistant to biodegradation. Furthermore, hemicellulose sugar residues contain a varying mixture of hexoses (e.g., glucose, mannose, and galactose), and pentoses (e.g., arabinose and xylose) depending on the biomass.

[004] Certain microorganisms can produce lipids from organic molecules, such as sugars derived from lignocellulose. Certain microorganisms, typically yeast, fungi or bacteria, can efficiently convert both C6 and C5 sugars in lignocellulosic materials into oil. Oil produced by heterotrophic microorganisms is often called single-cell oil or microbial oil. The process of producing single-cell oil using heterotrophic microorganisms typically comprises cultivating microorganisms in aerated bioreactors, allowing the cells to accumulate lipids, cultivating lipid-rich cells, and recovering the oil from cells. Microorganism-based lipids (i.e., single-cell oils) can be used as feedstocks for the production of biofuels, such as biodiesel, renewable diesel, or biojet fuel.

[005] The economically viable production of biofuels from lignocellulosic materials by microbial fermentation requires an efficient conversion of the main carbohydrate constituents of lignocellulosic materials into biofuels. On the other hand, the economic viability of biofuel production requires that all major carbohydrate constituents of lignocellulosic material be converted into sugars, which are suitable for microbial production. Generally speaking, this means the breakdown (hydrolysis) of the polymeric structures of hemicellulose and cellulose to obtain monomeric sugars.

[006] The prior art describes several methods that can be used for producing sugars from lignocellulosic materials.

[007] Patent Publication US2008/032344 A1 describes a process for recovering cellulosic sugars and the near-native lignin coproduct from lignocellulosic biomass. The process comprises subjecting the raw material to autohydrolysis, the organosolv technique and enzymatic hydrolysis treatments to produce a cellulosic sugar solution comprising glucose, which is fermented with yeast and/or suitable recombinant organism to produce biofuel. and/or chemical product.

[008] Cunningham and Carr (1984) reported several technologies for removing hemicellulose and lignin from wheat straw to provide an improved cellulosic residue for enzymatic hydrolysis. They also disclosed a pretreatment method comprising the autohydrolysis of wheat straw followed by a subsequent alkali treatment with NaOH. The amount of NaOH used in the delignification of autohydrolyzed wheat straw is significantly higher than in the present invention. According to the teachings of Cunningham and Carr, delignification treatment of autohydrolyzed straw did not significantly enhance the conversion of cellulose to glucose in the enzymatic treatment step, while alkali treatment without the autohydrolysis step significantly enhanced the results of enzymatic hydrolysis (see Tables III and IV). Furthermore, the obtained hemicellulose and cellulose hydrolysates are not used in the production of single-cell oil as in the present invention.

[009] US Patent Publication 2013/143285 A1 describes a process for converting lignocellulosic input into fermentable sugars. The process comprises subjecting the lignocellulose input to treatment with alkali at a pH of 8 to 12, to a treatment with dilute acid and, finally, to enzymatic hydrolysis to produce a sugar solution comprising glucose, which is fermented. in a fermentation product.

[0010] US Patent Publication 2012/036768A1 describes a method for producing fermentable sugars from lignocellulosic materials, in which, in the method, a pretreated lignocellulosic material is subjected to a two-stage enzymatic hydrolysis treatment. The first enzymatic treatment comprises mixing the pretreated material with a first enzyme composition to produce a first hydrolysis mixture, which is thickened to increase the fiber concentration to provide a second hydrolysis mixture. The second enzymatic treatment comprises mixing the second hydrolysis mixture with a second enzyme composition to produce a liquid mixture containing fermentable sugars and a solid lignin phase.

[0011] One of the biggest challenges in producing lignocellulosic sugars from lignocellulosic material is providing a process that allows cost-efficient production of high-quality sugar hydrolysates that can be used without additional purification in production. of single cell oil. The high quality of sugar hydrolysates means that the amount of impurities, such as phenols and acids, must be below the concentration that is toxic to the microorganism used in fermentation. Cost efficiency requires that the consumption of hydrolysing agents, such as enzymes, be kept at a low level. This can be achieved, for example, by recycling cooking chemicals. Economic viability requires that the quality of side streams, which are not used as feedstock for microbial fermentation, be as high as possible to enable valorization of these streams.

[0012] State of the art lignocellulose pretreatment technologies were designed for anaerobic fermentations (cellulosic ethanol). Microbial oil production differs from anaerobic fermentations as it is an aerobic process (requires oxygen). This invention describes a lignocellulose fractionation process that has benefits especially for aerobic bioprocesses, such as microbial oil production.

SUMMARY OF THE INVENTION

[0013] It is an objective of the present invention to provide a lignocellulose fractionation process, which allows the production of high quality sugar hydrolysates usable as a carbon source in the production of microbial oil with heterotrophic microorganisms.

[0014] Another objective of the present invention is to provide a fractionation process with improved yield and productivity from the enzymatic hydrolysis of cellulose fractions.

[0015] A third objective of the present invention is to provide a fractionation process that produces a high quality lignin fraction.

[0016] A fourth object of the present invention is to provide a fractionation method that has an improved cost efficiency of the aerobic fermentation process due to the low amount of inert material (lignin) and inhibitory compounds in the sugar fraction used in fermentation. aerobics.

[0017] To achieve the objectives stated above, the invention is characterized by the features defined in the independent claims.

[0018] The first aspect of the present invention relates to a method of fractionating a lignocellulosic material, the method comprising

[0019] a) Subjecting the lignocellulosic material to a treatment in which the hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase,

[0020] b) Separate the first solid phase from the first liquid phase,

[0021] c) Subjecting the first solid phase to a delignification treatment in the presence of alkaline delignifying agent to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin,

[0022] d) Separate the second solid phase from the second liquid phase,

[0023] e) Subjecting the second solid phase to an enzymatic hydrolysis treatment in the presence of enzymes capable of hydrolyzing the hemicellulose and cellulose fractions of the second solid phase to produce a mixture comprising a third liquid phase and a third phase solid.

[0024] A second aspect of the present invention relates to a liquid phase in the form of an enzymatic hydrolyzate obtainable through the method of the present invention (the third liquid phase).

[0025] A third aspect of the present invention is a concentrated sugar hydrolyzate obtainable through the method of the present invention.

[0026] A fourth aspect refers to a fraction of precipitated lignin obtainable through the method of the invention.

[0027] An additional aspect of the present invention relates to a method for producing microbial lipid, the method comprising

[0028] (i) provide a cultivation medium that comprises the liquid phase of enzymatic hydrolyzate obtained through the method for the fractionation of a

[0029] lignocellulosic material (the third liquid phase) or the concentrated sugar hydrolyzate of the present invention,

[0030] (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe,

[0031] (iii) incubating said inoculated medium with said oleaginous microbe that allows the lipid to accumulate,

[0032] (vi) recovering the lipid from said oleaginous microbe.

[0033] Other preferred embodiments are presented in the dependent claims.

[0034] The invention is based on the findings that, by carrying out the treatment that dissolves the hemicellulose and separating the lignin before the enzymatic hydrolysis step by treatment with alkali, the total sugar yield increases. This is important since the objective of the invention is to extract as many sugars as possible from the lignocellulosic material. Another advantage of the invention is that the efficiency of enzymatic hydrolysis and the cost efficiency of aerobic fermentation are significantly improved.

[0035] The invention is also based on another finding that alkali treatment can be conducted with a very low amount of alkali agent compared to prior art descriptions.

[0036] The invention is also based on a finding that hemicellulosic sugars separated from lignocellulosic material before delignification can be used in microbial fermentation without any purification treatment.

[0037] The present invention provides the following advantages:

[0038] - high yield of total sugar, both cellulosic and hemicellulosic sugars are recovered efficiently

[0039] - a more efficient enzymatic hydrolysis of the cellulose fraction in terms of sugar yield and productivity,

[0040] - production of high quality lignin fraction, which allows the valorization of lignin

[0041] - increased cost efficiency of the aerobic fermentation process,

[0042] - production of high-quality microbial biomass residue from aerobic fermentation that comprises low amounts of lignin, which allows the use of the residue as animal feed,

[0043] - reduced consumption of alkaline agent in delignification and, subsequently, reduced consumption of acid in precipitation of dissolved lignin and in pH adjustment before enzymatic hydrolysis.

BRIEF DESCRIPTION OF FIGURES

[0044] Figures 1 to 4 show the process schemes, according to the embodiments of the invention.

[0045] Figure 5 shows the process scheme for enzymatic hydrolysis, according to the embodiments of the invention.

[0046] Figure 6 shows the total sugar yields from straw delignified by NaOH and self-hydrolyzed straw in batch hydrolysis after 24 h, 48 h and 72 h, after the 1st, 2nd and 3rd sequential hydrolysis and after solids recycling and an additional 24 h reaction.

[0047] Figure 7 shows the enzyme productivities in the hydrolysis of straw delignified by NaOH and self-hydrolyzed straw in batch hydrolysis, sequential hydrolysis and solids recycling.

[0048] Figure 8 shows the volumetric sugar productivities of batch hydrolysis, sequential hydrolysis and solids recycling.

[0049] Figure 9 shows the progression of solid recycling reactions with a 50% recycle rate in terms of hydrolysis (A), reaction volume (B) and reaction consistency (C).

[0050] Figure 10 shows the results of batch fermentation fed with Aspergillus oryzae in wheat straw hydrolysates.

[0051] Figure 11 shows the yield of solid residue from the autohydrolysis of wheat straw.

[0052] Figure 12 shows the concentration of total soluble sugar (g/l, left y axis) and potential microbial inhibitory substances; furfural, hydroxymethylfurfural (HMF) and soluble phenolics (g/l, right y axis) in the liquid fraction obtained from the autohydrolysis of wheat straw at 10% consistency (g dry matter of straw solids/g total ).

[0053] Table 1. Comparison of dry matter yield (DM yield) from self-hydrolyzed straw with different delignification treatments. The sugar yield from enzymatic hydrolysis of delignified straw was dependent on treatment conditions. The sugar yield below is determined both based on dry matter and autohydrolyzed straw and based on the carbohydrate content of autohydrolyzed straw.

[0054] Table 2. Composition of growth medium before feeding

[0055] Table 3. Composition of the inoculation medium, pH adjusted to 5.5.

[0056] Table 4. Composition of fermentation medium

DETAILED DESCRIPTION OF THE INVENTION

[0057] During the description of the embodiments of the invention, specific terminologies will be used for clarity. However, the invention is not intended to be limited to the specific terms selected, and it should be understood that each specific term includes all technical equivalents that operate in a similar way to accomplish the same purpose.

DEFINITIONS LIGNOCELLULOSIC MATERIAL

[0058] The terms "lignocellulosic biomass" or "lignocellulosic material" are intended to include, but are not limited to, woody or non-woody plants, herbaceous plants or other materials containing cellulose and/or hemicellulose: The materials may be agricultural residues (such as wheat straw, rice straw, glume, shales, corn crop residues, sugarcane bagasse, sugarcane tops and leaves), dedicated energy plantations (such as fast-growing grass, Miscanthus, Arundo donax, yellow grass, willow, water hyacinth, energy cane, energy sorghum), wood materials or waste (including sawmill waste or fractions and pulp and/or from paper mills, such as hemicellulose, used sulphide liquor, residual fiber and/or primary sludge), moss or peat or urban waste paper. The term lignocellulosic material also encompasses materials with a low lignin content, materials such as macroalgae biomass. Furthermore, the materials also comprise hemicellulose or cellulose fractions from industrial practices. The term lignocellulosic material covers any type of cellulose fraction. Raw materials or certain fractions, such as hemicellulose and/or cellulose, of raw materials from different origins, plant species or industrial processes can be mixed and used as raw materials for the cultivation of microorganism biomass in accordance with with this revelation. Typically, the lignin content in lignocellulose is greater than 5%. Lignocellulosic biomass may also contain starch, for example in the case of whole plants.

HYDROLYSIS

[0059] The term "hydrolysis" here refers to depolymerization by adding water to glycosidic bonds or ester bonds of non-monomeric carbohydrates to oligomers and monomers of sugar or carboxylic acids.

HYDROLYZED

[0060] The terms "hydrolyzed" or "hydrolyzed material" here refer to a material that has been subjected to hydrolysis.

LIGNOCELLULOSE HYDROLYZATE

[0061] The term "lignocellulose hydrolyzate" refers here to hydrolysis products of lignocellulose or lignocellulosic material comprising cellulose and/or hemicellulose, oligosaccharides, mono- and/or disaccharides, acetic acid, formic acid, other organic acids , furfural, hydroxymethylfurfural, levulinic acid, phenolic compounds, other hydrolysis and/or degradation products formed from lignin, cellulose, hemicellulose and/or other components of lignocellulose, nitrogen compounds originating from proteins , metals and/or non-hydrolyzed or partially hydrolyzed fragments of lignocellulose.

AROMATIC COMPOUNDS, PHENOLIC COMPOUNDS

[0062] Aromatic hydrocarbon refers here to a compound that has a ring structure, formed by covalent bonds between carbon atoms, which contains alternating conjugated double and single bonds in a ring structure. Aromatic hydrocarbon can also refer to a compound that has a ring structure, formed by covalent bonds between carbon atoms and non-carbon atoms, which contains alternating conjugated double and single bonds in a ring structure.

[0063] The term "phenolic compound" refers here to a compound that comprises at least one aromatic hydrocarbon group that contains at least one hydroxyl group (-OH) linked directly to the aromatic hydrocarbon group. In this application, the concentration of phenolic compound was measured with calorimetric analysis according to the Folin-Ciocalteu method (Waterhouse, 2002). Such compounds include, but are not limited to, phenolic compounds such as p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, 4-hydroxyacetophenone, acetovanilone, acetosyringone, 4-hydroxybenzaldehyde, vanillin, syringaldehyde, 4-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, phenol, guaiacol, syringol, hydroquinone, catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2,6-dimethylphenol, 2,4-dimethylphenol, 4 -ethylphenol, 3,4-dihydroxybenzaldehyde, 4-methylguaiacol, 4-vinylphenol, 4-ethyl-2-methylphenol, 4-allylphenol, 3-methoxycatechol, 2,6-dimethoxy-4-methylphenol, vanillic alcohol, homovanillin, homovanillic acid, 1-(4-hydroxy-3-methoxyphenyl)ethanol, 1-(4-hydroxy-3-methoxyphenyl)allene, vanillic acid methyl ester, 4-ethyl-2,6-dimethoxyphenol, 4-methylcatechol, 4 -ethylguaiacol, 4-propylphenol, 4-vinylguaiacol, 4-hydroxybenzyl alcohol, 3-hydroxy-2-methyl-(4H)-pyran-4-one, 3,5-dihydroxy-2-methyl-(4H)- pyran-4-one, 4-propenylphenol, 2,6-dimethoxy-4-propylphenol, dihydroconiferyl alcohol, homosyringaldehyde, 3,5-dimethoxy-4-hydroxybenzyl alcohol, 2,6-dimethoxy-4-propenylphenol, 1- (3,5-dimethoxy-4-hydroxyphenyl)ethanol, coniferyl aldehyde, syringylacetone, syringic acid methyl ester, propiosyringone, syringyl vinyl ketone, dihydrosynapyl alcohol, sinapaldehyde, 2,6-dimethoxyphenol, 1-(4-hydroxyphenyl) ethanol, eugenol, 5-ethylpyrogallol, 4-propylguaiacol, 1,4-dihydroxy-3-methoxybenzene, isoeugenol, 4-hydroxybenzoic acid methyl ester, guaiacylacetone, 2,6-dimethoxy-4-vinylphenol, propiovanilone, guaiacyl vinyl ketone, 4-allyl-2,6-dimethoxyphenol, and including all possible isomers, oligomeric and/or polymeric lignin, tannins, polyphenols, mixtures of phenolic compounds, covalently linked compounds comprising non-phenolic compounds and phenolic compounds.

[0064] The term "concentration of phenolic compounds" means the concentration of compounds (typically expressed as g/l) in aqueous solution as measured with the Folin-Ciocalteu method (Waterhouse, 2002)

HYDROTHERMAL TREATMENT

[0065] In the context of the present invention, the term "hydrothermal treatment" refers to the heat treatment of aqueous lignocellulose suspension at temperatures exceeding 50 °C. Hydrothermal treatment can be carried out under pressure in a pressurized reactor or at atmospheric pressure in a non-pressurized reactor. The pressure in the pressurized reactor can be generated by steam obtained from water when heated to the boiling point or by added pressurized gas phase. Hydrothermal treatment can be carried out in the presence of a catalyst or in the absence of a catalyst. Hydrothermal treatment in the absence of a catalyst (also called "autohydrolysis" or "AH") for the hydrolysis of lignocellulosic biomass without added catalyst when the aqueous suspension of lignocellulosic biomass is subjected to hydrothermal treatment at temperatures exceeding 120 °C under pressure.

[0066] "Self-hydrolyzed straw" refers here to the solid fraction that was obtained after the autohydrolysis of straw. The self-hydrolyzed straw may have been subjected to washing.

SEVERITY

[0067] The term "severity" here refers to a factor, which is calculated by equation 1 and which describes the hydrothermal conditions in terms of temperature and reaction time. S = Log(R0), where is the base temperature (100 °C).

DELIGNIFICATION TREATMENT

[0068] "Delignification treatment" refers here to a treatment that removes non-carbohydrate material, such as lignin, from lignocellulosic biomass. Delignification treatment also refers to a treatment that removes both carbohydrate and non-carbohydrate material as a mixture from lignocellulosic biomass.

STEAM EXPLOSION

[0069] In the context of the present invention, the term "steam explosion" refers to a treatment, in which the material is heated by high pressure steam (at temperatures between 110 °C and 250 °C, typically 140 to 230 °C) under pressure with or without the addition of chemicals (such as acids) and the material is maintained at the temperature for a certain time after which the pressure is released, causing an explosive decompression of the material. In this context, steam explosion is applied to lignocellulosic materials, and it typically results in a rupture of the rigid structure of the lignocellulose fibers, that is, the defibrillation of the cellulose fiber clusters.

ALKALINE DELIGNIFICATION AGENT

[0070] In the context of the present invention, the term "alkaline delignification agent" refers to a chemical compound or a mixture of chemical compounds that, when added to water, provides solutions with a hydrogen ion activity lower than that of than that of pure water, that is, a pH higher than 7.0. The alkaline delignifying agent may be selected from a group of compounds comprising, but not limited to, hydroxides, such as LiOH (lithium hydroxide), NaOH (sodium hydroxide), KOH (potassium hydroxide), Ca(OH )2 (calcium hydroxide), NH4OH (ammonium hydroxide), or compounds that can form hydroxide ions in water such as NH3 (ammonia) in liquid or gaseous state, carbonates such as HCO3- (bicarbonate ion), Li2CO3 (carbonate lithium), Na2CO3 (sodium carbonate), K2CO3 (potassium carbonate), sulfides such as Na2S (sodium sulfide), and the corresponding hydrates.

ALKALINE DELIGNIFICATION TREATMENT

[0071] In the context of the present invention, the term "alkaline delignification treatment" refers to lignocellulose treatment carried out in the presence of alkaline delignification agent, pH (initial pH) typically between 10 and 13. In alkaline delignification treatment , hydrogen peroxide (H2O2) can be used in combination with alkaline delignifying agent.

ENZYMATIC HYDROLYSIS

[0072] In the context of the present invention, the term "enzymatic hydrolysis" refers to the enzymatic treatment of lignocellulosic material comprising cellulose and/or hemicellulose, oligosaccharides, in which the enzymes facilitate the hydrolysis of cellulose and/or hemicellulose, oli - gosaccharides to obtain mono- and/or disaccharides. Typically, the enzymatic hydrolysis treatment of lignocellulosic material is conducted by subjecting the lignocellulosic material to a mixture of enzymes in the presence of water or a buffer. The enzyme mixture typically consists of, but is not limited to, 1,4-3-glucanases (endoglucanases and exoglucanases, or endocellulases and exocellulases), 1,4-3-glucosidases (cellobiases), and hemicellulose-degrading enzymes (hemicelulases). , xylanases, arabinases, etc.).

MICROBIAL LIPID OR LIPID

[0073] In the context of the present invention "microbial lipid", "lipid" or "intracellular lipid" refers to a fatty substance, the molecule of which generally contains, as a part, an aliphatic hydrocarbon chain, which dissolves in non-polar organic solvents but have insufficient solubility in water. Lipids are an essential group of large molecules in living cells. Lipids are, for example, fats, oils, waxes, wax esters, sterols, terpenoids, isoprenoids, carotenoids, polyhydroxyalkanoates, nucleic acids, fatty acids, fatty alcohols, fatty aldehydes, fatty acid esters, phospholipids, glycolipids, sphingolipids and acylglycerols, such as trialcylglycerols, diacylglycerols or monoacylglycerols. The preferred lipids in the present invention are fats, oils, waxes, acylglycerols and fatty acids and their derivatives, in particular triacylglycerols and wax esters. In the context of the present invention, lipids are synthesized by microbes and accumulated therein (intracellular lipids). In another embodiment of the invention, lipids are synthesized by microbes and excreted by them (extracellular lipids).

[0074] In connection with this invention, single cell oil is used as a synonym for lipids and fat.

[0075] The term "acylglycerol" refers to an ester of glycerol and fatty acids. Acylglycerols occur naturally as fats and fatty oils. Examples of acylglycerols include triacylglycerols (TAGs, triglycerides), diacylglycerols (diglycerides), and monoacylglycerols (monoglycerides).

SUGAR

[0076] In the context of the present invention, the term "sugar" refers here to oligomeric, dimeric and monomeric carbohydrates. Particularly, in this application, the term sugar refers to water-soluble oligomeric, dimeric or monomeric carbohydrates derived from lignocellulosic materials. The term “polymeric sugars” means carbohydrates that are in polymeric form and are not typically soluble in water.

SUGAR YIELD

[0077] In the context of the present invention, the term "sugar yield" refers here to the yield of oligomeric, dimeric or monomeric carbohydrates from specific materials. Particularly, in this application, the term sugar yield refers to the yield of water-soluble oligomeric, dimeric or monomeric carbohydrates derived from lignocellulosic materials.

SINGLE CELL OIL PRODUCTION PROCESS

[0078] "Single cell oil production process" refers here to a process, which comprises the steps of forming or allowing the growth of a lipid-synthesizing microorganism and allowing the then obtained organism mass produce and/or store (accumulate) lipid, recover cells from the liquid phase, and extract or recover lipids from cells. In certain cases, single cell oil may also be extracellular, such as excreted or released from cells into culture medium during or after cultivation. AEROBIC CULTIVATION

[0079] The term "aerobic cultivation" or "aerobic fermentation" refers to a cultivation in which the microorganism uses oxygen as a terminal electron acceptor for energy generation (that is, the microorganism uses aerobic respiration). Typically, in bioreactors, aerobic cultivation is carried out by adding oxygen or a gas mixture that contains oxygen (typically air), that is, the bioreactor is aerated. When microorganisms use aerobic respiration in cultivation, they can be referred to as "cultivation under aerobic conditions". Typically this occurs in aerated bioreactors.

OIL MICROBEE OR MICROORGANISM THAT PRODUCES OIL

[0080] The oleaginous microbes (also called oil-producing organisms) used in the present invention are selected from the group of bacteria, cyanobacteria, fungi, such as yeast and filamentous fungi, archaea or microalgae. Microorganisms can readily accumulate lipids or have been genetically modified to accumulate lipids or to enhance lipid accumulation.

[0081] Preferably, organisms have the ability to utilize C6 sugars and C5 sugars are used. Preferably, the organisms are yeast, filamentous fungi or bacteria.

[0082] In the context of the present invention, the ole aginous microorganism (oleaginous microbe) refers to a microorganism with the capacity to accumulate intercellular lipids so that the lipids add up to at least 15% (w/w) of the total biomass biomass (per cell dry weight) of the microbe when it is grown under suitable conditions. In a preferred embodiment, the oleaginous microbe has the capacity to accumulate at least 20% (w/w) of the total biomass of the microbe (by dry cell weight).

[0083] Preferred microorganism strains for the purposes of the present invention include, but are not limited to, the species and genera listed below:

[0084] According to one embodiment of the invention, the microbe is an oleaginous microbe with the ability to use sugars derived from lignocellulosic materials. Preferably, oleaginous organisms have the ability to utilize C6 sugars (six-carbon sugars, such as glucose, mannose and galactose) and C5 sugars (such as xylose and arabinose) in lignocellulosic hydrolysates. According to one embodiment of the invention, the oleaginous organism has the ability to utilize polymeric or oligomeric carbohydrates in lignocellulose or fractions thereof.

[0085] The preferred fungal (filamentous) strains are species of the genus Aspergillus, such as Aspergillus oryzae, Mortierella, such as Mortierella isabellina, Chaetomium, Claviceps, Cladosporidium, Cunninghamella, Emericella, Fusarium, Glomus, Mucor, Pseu - dozyma, Pythium, Rhizopus, such as Rhizopus oryzae, Tremella, Zygorhynchus, Humicola, Cladosporium, Malbranchea, Umbelopsis such as Umbelopsis isabellinae Ustilago. The most preferred fungal species are from the genera Aspergillus and/or Mortierella. Preferred fungi are fungi with the ability to produce lipids effectively.

[0086] The preferred yeast strains are those belonging to species of the genera Geotrichum, Deparyomyces, Pachysolen, Galactomyces, Hansenula, Leucosporidium, Sporobolomyces, Sporidiobolus, Waltomyces, Cryptococcus, such as Cryptococcus curvatus, Rhodosporidium, such as Rhodosporidium toruloi- des or Rhodosporidium fluviale, Rhodotorula such as Rhodotorula glutinis, Yarrowia such as Yarrowia lipolitica, Candida such as Candida curvata, Lipomyces such as Lipomyces starkeyi and Trichosporon such as Trichosporon cutaneum or Trichosporon pullulans. The most preferred yeasts are from the genera Lipomyces, Rhodosporidium and Cryptococcus. Preferred yeasts are those yeasts with the ability to produce lipids effectively.

[0087] The preferred bacteria are those that belong to species of the genera Rhodococcus, Acinetobacter and Streptomyces. Preferred bacteria are those bacteria with the ability to produce lipids effectively.

[0088] The most preferred algae are microalgae, such as species of microalgae from the genera comprising Brachiomonas, Crypthecodinium, Chlorella, Dunaliella, Hantzschia, Nannochloris, Nannochloropsis, Nitzschia, Prototheca, Scenedesmus, Schizochytrium, Traustrochytrium and Ulkenia . Preferred microalgae are those microalgae with the ability to grow heterotrophically and produce lipids effectively. Organisms belonging to the genera Schizochytrium, Thraustochytrium and Crypthecodinium and Ulkenia are sometimes called marine fungi.

[0089] According to another embodiment of the invention, lignocellulosic biomass carbohydrates are found mainly in monomeric form and organisms without the ability to use oligomeric or polymeric carbohydrates are used for the production of single cell oil. Such oil-producing organisms are selected from the group of bacteria, cyanobacteria, fungi such as yeast and filamentous fungi, archaea or microalgae. Microorganisms can readily accumulate lipids or have been genetically modified to accumulate lipids or to enhance lipid accumulation.

LIPID RECOVERY

[0090] "Oil recovery" or "lipid recovery" or "recovering lipid from an oleaginous microbe" refers to a process, in which lipid (intracellular lipid) is recovered by mechanical, chemical methods , thermomechanical or autocatalytic or by a combination of these methods from microorganism cells. Alternatively, "oil recovery" may mean the recovery of lipids produced extracellularly from the culture broth (fermentation).

SINGLE CELL MASS THAT CONTAINS LIPID

[0091] "Lipid-containing single cell mass" means a mass of single cell and cellular mycelium with a lipid content of preferably at least 10%, preferably at least 15% (w/w) or more of matter drying of microorganism biomass.

RESIDUAL CELL MASS

[0092] In the context of the present invention, "residual cell mass" refers to a solid, semi-solid or fluid fraction of material, which contains microorganisms treated for the recovery of intracellular lipids.

BIOFUEL

[0093] In the context of the present invention, "biofuel" refers to a solid, liquid or gaseous fuel derived primarily from biomass or biowaste and is different from fossil fuels, which are derived from the organic remains of plants and prehistoric animals.

[0094] According to EU directive 2003/30/EU, "biodiesel" refers to a methyl ester produced from vegetable oil or animal oil, of diesel quality to be used as biofuel. More broadly, biodiesel refers to long-chain esters, such as methyl, ethyl or propyl esters, from diesel-grade vegetable oil or animal oil. Biodiesel can also be produced from microorganism lipids, where the microorganism lipid can originate from a bacteria, a fungus (yeast or mold), an alga or another microorganism.

RENEWABLE DIESEL

[0095] "Renewable diesel" refers to a fuel that is produced from a treatment with hydrogen and lipids of animal, vegetable or microorganism origin, or mixtures thereof, in which the microorganism lipid An organism can originate from a bacterium, a fungus (a yeast or a filamentous fungus), an alga or another microorganism. Renewable diesel can also be produced from waxes derived from biomass by gasification and Fischer-Tropsch synthesis. Optionally, in addition to hydrogen treatment, isomerization or other processing steps can be carried out. The renewable diesel process can also be used to produce jet fuel and/or gasoline. The production of renewable diesel has been described in patent publications EP 1396531, EP1398364, EP 1741767 and EP1741768.

[0096] Biodiesel or renewable diesel can be mixed with fossil fuels. Suitable additives such as preservatives and antioxidants can be added to the fuel product.

LUBRICANT

[0097] "Lubricant" refers to a substance, such as grease, lipid or oil that reduces friction when applied as a surface coating to moving parts. Two other main functions of a lubricant are removing heat and dissolving impurities. Lubricant applications include, but are not limited to, uses in internal combustion engines as engine oils, fuel additives, driven devices such as pumps and hydraulic equipment, or in different types of bearings. Typically, lubricants contain 75 to 100% base oil and the rest are additives. Suitable additives are, for example, detergents, storage stabilizers, antioxidants, corrosion inhibitors, turbidity reducers, demulsifiers, antifoaming agents, cosolvents and lubricity additives (see, for example, US 7,691,792). The base oil for lubricant can originate from mineral oil, vegetable oil, animal oil or from a bacteria, fungus (yeast or a filamentous fungus), an alga or other microorganism. The base oil can also originate from waxes derived from biomass by gasification and Fischer-Tropsch synthesis. The viscosity index is used to characterize the base oil. Typically, a high viscosity index is preferred.

[0098] Lipids produced according to the method described in this invention can be used as an input for the production of biodiesel, renewable diesel, jet fuel or gasoline. Bio-diesel consists of fatty acid methyl esters, and is typically produced by transesterification. In transesterification, acylglycerols are converted into alkyl (methyl, ethyl or propyl) esters of long-chain fatty acids. Renewable diesel refers to fuel that is produced by hydrogen treatment (hydrogen deoxygenation, hydrogenation or hydroprocessing) of lipids. In hydrogen treatment, acylglycerols are converted to corresponding alkanes (paraffins). Alkanes (paraffins) can be further modified by isomerization or other process alternatives. The renewable diesel process can also be used to produce jet fuel and/or gasoline. Furthermore, lipid cracking can be carried out to produce biofuels. Furthermore, lipids can be used as biofuels directly in certain applications.

[0099] The lipids produced with the method can also be used as base oils for lubricants (lubricating oils) or as a starting material for the production of base oils for lubricants.

DRY MATTER

[00100] "DM" or "dry weight" refers here to dry matter and is a measure of the mass of a material when it has been subjected to a treatment that essentially removes water from the material (i.e., the material is completely dry).

CONSISTENCY

[00101] "Consistency" refers here to the ratio between the dry weight of solids and the total weight of the suspension.

METHOD FOR FRACTIONING A LIGNOCELLULOSIC MATERIAL

[00102] An object of the present invention is to provide a fractionation method, which has an improved cost efficiency of the aerobic fermentation process due to the low amount of inert material (lignin) and inhibitory compounds in the sugar fraction used in fermentation. aerobics.

[00103] The inventors have surprisingly discovered that alkaline delignification can be carried out effectively without excessive application of alkaline delignification agent if the lignocellulosic material is subjected to a hydrothermal, such as autohydrolysis, prior to delignification. alkaline. An advantage is reduced alkaline agent consumption in delignification and subsequently reduced acid consumption in precipitation of dissolved lignin and pH adjustment prior to enzymatic hydrolysis.

[00104] In a first aspect of the present invention it refers to a method of fractionating a lignocellulosic material, the method comprising

[00105] a) Subjecting the lignocellulosic material to a treatment in which the hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase,

[00106] b) Separate the first solid phase from the first liquid phase,

[00107] c) Subjecting the first solid phase to a delignification treatment in the presence of alkaline delignifying agent to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin,

[00108] d) Separate the second solid phase from the second liquid phase,

[00109] e) Subjecting the second solid phase to an enzymatic hydrolysis treatment in the presence of enzymes capable of hydrolyzing the hemicellulose and cellulose fractions of the second solid phase to produce a mixture comprising a third liquid phase in the form of an enzymatic hydrolyzate and a solid third phase.

[00110] In an additional step, the mixture of the third liquid phase comprising cellulosic carbohydrates can be separated from the third solid phase comprising lignin.

[00111] An embodiment of the method of the invention is illustrated schematically in Figure 1. Another embodiment of the method of the invention is illustrated schematically in Figure 2. The embodiment shown in Figure 2 includes a method of precipitation of soluble lignin (first liquid phase) obtained from alkaline treatment using acid.

STAGE OF PARTIAL REMOVAL OF HEMICELLULOSIC SUGARS FROM LIGNOCELLULOSIC MATERIAL

[00112] In step a) of the lignocellulosic material fractionation method, the lignocellulosic material is subjected to a treatment in which hemicellulose is at least partially removed from the lignocellulosic material to produce a first phase and a first solid phase. In step a) of the fractionation method, the hemicelluloses are at least partially dissolved and separated into a liquid phase (referred to as the first liquid phase)

[00113] The first liquid phase can be obtained directly from the lignocellulose treatment in which at least part of the hemicellulose becomes soluble in water (that is, the treatment results in a liquid phase). Alternatively, the treatment does not result in a liquid phase (i.e., it is carried out at such a high consistency that the liquid phase does not exist) and the first liquid phase is obtained by washing solid material that has undergone a treatment in which at least part of the hemicellulose becomes soluble in water. Alternatively, the first liquid phase can be produced directly from the treatment and, additionally, the solid phase is further washed and the resulting liquid is combined with the first liquid phase.

[00114] In another embodiment of the present invention, hemicellulose is at least partially removed from the lignocellulosic material by hydrothermal treatment. In a second embodiment, the hydrothermal treatment is conducted at a temperature between 100 and 250 °C, preferably between 140 and 240 °C, and more preferably between 140 and 200 °C. The intensification of hydrothermal treatment can be expressed in terms of severity, a term which is defined in this document. In a preferred embodiment, the hydrothermal treatment is conducted under conditions corresponding to severity between 2.0 and 4.5, preferably between 3.0 and 4.1, more preferably between 3.5 and 4.0.

[00115] In one embodiment of the present invention, hemicellulose is at least partially removed from the lignocellulosic material by autohydrolysis treatment. Autohydrolysis is typically carried out with a dry matter content of 5 to 60%, at temperatures between 140 and 240 °C for 1 to 120 minutes without the addition of acidic compounds that result in the dissolution of 5 to 40% of the matter content. dries into lignocellulosic material, including hemicellulosic carbohydrates. Typically, autohydrolysis dissolves 30 to 100% of the hemicellulosic carbohydrates of the lignocellulosic material, preferably >50%, more preferably >70%, more preferably >80%, even more preferably >90 %. The dissolved hemicellulose carbohydrates are at least partially in oligomeric form. More typically, autohydrolysis is carried out with dry matter content of 10 to 50% at 160 to 220 °C, depending on the lignocellulosic feedstock. After autohydrolysis, the solid and liquid phases are separated by any method, such as filtration, for example, pressure filtration, or by a pressure lathe. The solid fraction can be washed to remove dissolved hemicellulose from the solid phase.

[00116] According to another embodiment of the invention, the lignocellulosic material is subjected to a steam or steam explosion with or without the addition of acidic compounds, generally at temperatures between 110 and 250 °C, more typically at temperatures between 140 and 230°C. The treatment results in a dissolution of hemicellulosic carbohydrates. Optionally, the solid material from the vapor explosion is washed to recover dissolved hemicellulosic carbohydrates.

[00117] In one embodiment of the present invention, the lignocellulosic material is first subjected to hydrothermal treatment followed by a steam explosion step (exemplified in Figure 4). In another embodiment, the first solid phase is subjected to a steam explosion prior to delignification treatment in the presence of the alkaline delignification agent.

[00118] According to the invention, the treatment of lignocellulose, according to step a) (in which the hemicellulosic sugars become at least partially dissolved), allows the efficient recovery of hemicellulosic sugars for microbial cultivation, such as single cell oil production.

ALKALINE DELIGNIFICATION STAGE

[00119] In step c) of the lignocellulosic material fractionation method, the first solid phase (comprising the lignocellulosic material from which the hemicellulosic material has been partially removed) is subjected to an alkaline delignification step, in which at least less part of the lignin is solubilized. Alkaline delignification is carried out by subjecting lignocellulosic material from which hemicellulosic material has been partially removed to an alkaline delignification agent.

[00120] Alkali treatment is typically conducted by preparing a suspension comprising autohydrolyzed lignocellulose material, aqueous liquid, and one or more alkaline agents, or mixtures thereof, to provide a pH of the suspension above pH 7. Preferably, an alkaline chemical is added in an amount to provide the pH of the suspension between 10 and 13 at the beginning of the treatment. Typically, pH decreases during alkaline treatment.

[00121] The alkaline suspension is maintained at temperatures where the suspension contains at least one liquid phase. Incubation is not limited to any specific temperature but can be conducted over a wide temperature range isothermally or non-isothermally. Incubation is preferably carried out at temperatures from 25 °C, preferably between 30 and 160 °C.

[00122] Shaking is optionally conducted to increase heat transfer efficiency during incubation. The treatment time is selected according to the targeted degree of material dissolution. Preferably, the treatment time is half an hour to twenty hours.

[00123] After the reaction time, the second liquid phase and the second solid phase are separated using any method, such as, but not limited to, filtration, for example, pressure filtration or pressure lathe. The second solid phase is used for enzymatic hydrolysis to release sugars for additional use, such as microbial oil production. The second liquid phase can be treated to precipitate lignin, such as by acid treatment, and the precipitated lignin can be separated by any method.

[00124] In one embodiment of the present invention, the alkaline delignifying agent is selected from a group consisting of sodium hydroxides, sodium carbonates, potassium hydroxides, potassium carbonates, calcium hydroxides, calcium carbonates, hydroxide lithium, lithium carbonate, ammonium hydroxide, ammonia, sodium sulfide and the corresponding hydrates. In a preferred embodiment, the alkaline delignifying agent is sodium hydroxide or sodium carbonate.

[00125] As mentioned above, the inventors have surprisingly found that alkaline delignification can be carried out effectively without excessive application of alkaline delignification agent if the lignocellulosic material is subjected to a hydrothermal, such as autohydrolysis. , before alkaline delignification. Hydrothermal treatment results in a decreased acidity of the solid fraction (which comprises cellulose, lignin and residual hemicellulose). Therefore, in alkaline treatment less alkaline agent is required compared to the situation when hydrothermal treatment is not carried out. Therefore, alkaline treatment after hydrothermal treatment can be carried out at lower pH compared to the situation without autohydrolysis. Therefore, hydrothermal treatment is beneficial before alkaline treatment of lignocellulosic material. An advantage of applying lower amounts of the alkaline delignification agent is that less sugar is lost in the alkaline delignification step compared to a similar treatment conducted in the presence of high amounts of the alkaline delignification agent.

[00126] Consequently, in a preferred embodiment of the present invention, the concentration of the delignifying agent is 0.1 to 10% by weight, more preferably 0.1 to 4% by weight based on the amount of dry matter in the first phase solid. In a further embodiment, the alkaline delignifying agent is added to the first solid phase to obtain a suspension that has a pH above 7, preferably between 10 and 13.

[00127] In an additional embodiment, the delignification treatment is conducted at a temperature above 25, preferably between 30 and 160 °C

[00128] According to yet another embodiment of the invention, delignification of lignocellulosic material is carried out with ammonia with the delignification chemical. According to one embodiment of the invention, ammonia fiber expansion (AFEX) or ammonia recycle percolation is used with a temperature between 60 °C and 220 °C.

[00129] According to the invention, in the treatment of lignocellulose according to step c) alkaline delignification allows efficient enzymatic hydrolysis and efficient recovery of cellulosic sugars for microbial cultivation, such as the production of single cell oil.

ENZYMATIC HYDROLYSIS OF ALKALINE DELIGNIFICATION PRODUCT

[00130] In step e) of the lignocellulosic material fractionation method, the second solid phase obtained from the alkaline delignification step is subjected to enzymatic hydrolysis ENZYMATIC HYDROLYSIS

[00131] Enzymatic hydrolysis consists of incubating pre-treated straw or other substrate or raw material, with a mixture of enzymes, which typically consists of, but is not limited to, 1,4-3-glucanases (endoglucanases and exoglucanases , or endocellulases and exocellulases), 1,4-3-glucosidases (cellobiases) and enzymes that degrade hemicellulose (hemicelulases, xylanases, arabinases, etc.). Enzymes may or may not be commercial enzyme products. The pre-treated straw is mixed with water or buffer solution and the enzyme mixture in suitable proportions. Additives such as polyethylene glycol, detergents or other surface active agents, or proteins may or may not be added to the reaction. Any proportion of solids in the suspension (or "consistency") may be used, preferably 10 to 35%, or particularly 15 to 25%. The pH of the slurry is adjusted according to the optimum conditions for the enzyme mixture used. pH adjustment is performed before and/or during enzyme addition by adding acid or base at a suitable concentration, for example, H2SO4, HCl, HNO3, NaOH, NH3 or other acid or base. Additional pH adjustment may or may not be performed after enzyme addition and during hydrolysis.

[00132] A constant temperature is maintained during hydrolysis, according to the optimal conditions of the enzyme mixture, often 40 to 60 °C, or particularly 50 °C. The pretreated straw, water and/or buffer solution, and other constituents of the hydrolysis suspension may or may not be preheated to the reaction temperature before being added to the suspension. The suspension is agitated during the reaction by shaking, shaking, free-falling, or other means of disturbance.

[00133] In one embodiment of the present invention, the enzymatic hydrolysis treatment of lignocellulosic material is conducted by subjecting the lignocellulosic material to a mixture of enzymes in the presence of water or a buffer. The enzyme mixture typically consists of, but is not limited to, 1,4-3-glucanases (endoglucanases and exoglucanases, or endocellulases and exocellulases), 1,4-3-glucosidases (cellobiases) and hemicellulose-degrading enzymes (hemicellulose). - ses, xylanases, arabinases, etc.).

[00134] By subjecting the second solid phase obtained from the alkaline delignification step to an enzymatic hydrolysis step, the sugars are dissolved from the solid material. A solid-liquid separation by any method is carried out after enzymatic treatment that forms a third liquid phase that contains the sugars dissolved during hydrolysis and a third solid phase that comprises lignin and residual polymeric sugars (residual fiber). The third liquid phase is also called enzymatic hydrolyzate, which can be used for the production of microbial lipids.

[00135] Part of the hemicellulosic sugars separated from the material in step a) (for example, by hydrothermal treatment or autohydrolysis) can be introduced into the system and subjected to enzymatic hydrolysis together with the solid phase material obtained from step delignification (second solid phase).

[00136] Therefore, at least part of the first liquid phase can be added and mixed with the second solid phase before this mixture is subjected to the enzymatic hydrolysis treatment of step e). Therefore, in one embodiment of the invention, at least part of the first liquid phase is combined with the second solid phase and subjected to enzymatic hydrolysis treatment.

[00137] Alternatively, the enzymatic hydrolysis of the first liquid phase is carried out separately from the second solid phase.

[00138] In accordance with the invention, it has surprisingly been found that in the method of treating lignocellulose which includes the sequential steps of subjecting the lignocellulose to a treatment whereby hemicellulosic sugars become at least partially dissolved (such as autohydrolysis) and , then subjecting the solid phase obtained from this treatment to an alkaline delignification step (such as NaOH treatment) a higher overall (total) yield of sugar is obtained. High overall sugar yield is beneficial to the cost efficiency of microbial processes such as single cell oil production.

[00139] In one embodiment of the present invention, enzymatic hydrolysis is conducted as a batch hydrolysis.

BATCH HYDROLYSIS

[00140] Batch hydrolysis refers to a hydrolysis reaction, in which the reaction constituents are mixed to form a suspension or a slurry or paste, and incubated for a suitable period of time, after which the Solids are separated by filtration, centrifugation or other separation means and a liquid stream of soluble sugars, including glucose, xylose arabinose, galactose, mannose and others, and oligomers thereof, is acquired. Reaction constituents or other substances may or may not be added during the reaction. However, no liquid stream is separated from the slurry before the end of the reaction.

[00141] A batch reaction can also be carried out as a continuous process. In continuous batch hydrolysis, a constant stream of fractionated (pretreated) lignocellulosic material, liquid, enzymes, pH adjusting agents, and other reaction constituents is fed to the reactor while simultaneously a constant stream of slurry is removed. of the reactor, from which the liquid stream is separated. In a continuous batch hydrolysis, the reactor may or may not be subdivided into two or more reactors in series, through which the slurry constantly flows, particularly in order to improve the retention time distribution of the raw material. Conceptually, the solid material is separated from the liquid only after the reaction and no separation occurs during the reaction. Therefore, no separation occurs for the slurry flowing externally from the other reactors except for the slurry from the last reactor, and consequently no other separate liquid streams are removed from the slurry except for the liquid stream from the slurry separation. final external flow. However, additional feeding of reaction constituents can occur at any point in the process. If liquid separation occurs between two reactors, the reactors must be defined as separate reaction steps and the hydrolysis process must be defined as a sequential (stepwise) hydrolysis.

[00142] In a second embodiment, enzymatic hydrolysis is conducted as sequential hydrolysis, as illustrated in Figure 5.

SEQUENTIAL HYDROLYSIS

[00143] Sequential hydrolysis, also known as step hydrolysis, or two-stage hydrolysis, three-stage hydrolysis, or multi-stage hydrolysis, etc., consists of a sequence of batch reactions in series, in which between the reactions in batch, a liquid stream is separated from the slurry and the concentrated solid stream is fed to the next batch reaction and mixed with fresh water and/or buffer solution. The addition of fresh enzymes and other reaction constituents may or may not occur in the second, third, or later reaction. The reaction time may be the same or different in subsequent reaction steps. The process flow of a sequential reaction is shown in Figure 5.

[00144] Similar to that described for batch reactions, sequential hydrolyses can be carried out as a continuous process and the single feed steps of sequential hydrolyses can be subdivided into separate series reactors through which the slurry constantly flows . Liquid separation and addition of fresh liquid may or may not occur between reactors. Conceptually, whether the liquid is separated and the liquid is or is not replaced with fresh liquid between two reactors, the reactors must be defined as separate reaction steps.

[00145] In yet an additional embodiment, the enzymatic hydrolysis is conducted as a solid recycling hydrolysis, as illustrated in Figure 5.

RECYCLING OF RESIDUAL SOLIDS IN ENZYMATIC HYDROLYSIS (OR "SOLIDS RECYCLING")

[00146] A hydrolysis process with recycling of residual solids, or "solids recycling", includes a hydrolysis reaction, after which a liquid stream is separated from the slurry and a proportion of the concentrated solid stream is recycled back to the same reactor (Figure 5). The proportion of waste solids recycled is called the recycle rate and is denoted by r in Figure 5 and in Equations 2 and 3. At a constant raw material feed rate, solids recycling extends the reaction time of the solid material according to a geometric series, presented in Equation 2, where tn is the average reaction time of the solids after n subsequent recycle reactions, t0 is the reaction time of a single reaction, or the retention time of the slurry in the reactor, and r is the recycle rate. Eventually, a recycle process at constant feed and recycle rates and a constant retention time will reach a steady state, in which the average reaction time of the solid material can be calculated from Equation 3.

[00147] For example, at a 50% recycle rate in a 24 h reaction, the average reaction time of the solids will be 48 h in a steady state.

[00148] The recycling reaction can be carried out as a batch reaction, after which solids recycling occurs, or as a continuous process, in which the process constituents are constantly fed to the reactor, a constant external flow of Slurry occurs and the externally flowing slurry is separated into a liquid and a concentrated waste solid stream and a steady stream of waste solids is recycled back to the reactor. The recycle reaction may or may not be subdivided into separate reactors in series in order to improve residence time distribution and separation of liquid from or after the reactors and additional feeding of process constituents to these reactors may or may not occur. Additional sequential reaction steps may or may not be included after or before or during the solids recycling reaction.

[00149] The enzymatic hydrolyzed liquid obtained in step e) can be subjected to a hydrolyzate concentration step, such as by evaporation, to obtain a concentrated hydrolyzate. Therefore, in one embodiment, the method according to the invention includes further comprising a step of concentrating the third liquid phase. Preferably, the concentration of the third liquid phase is carried out by evaporation.

LIQUID ENZYMATIC HYDROLYZATE (LIQUID THIRD PHASE)

[00150] The liquid phase comprising cellulosic sugars can be used in the preparation of a culture medium, such as a culture medium for use in a method for producing microbial lipids, as described herein.

[00151] Consequently, a second aspect of the present invention relates to a liquid phase in the form of an enzymatic hydrolyzate obtainable through the method of the present invention (the third liquid phase).

[00152] Alternatively, the liquid phase of the enzymatic hydrolyzate can be concentrated to the obtained liquor that has a higher concentration of sugars. Thus, a further aspect of the present invention is a concentrated sugar hydrolyzate obtainable by the method of the present invention.

[00153] The enzymatic hydrolyzate from the first liquid phase (comprising hemicellulosic sugar monomers) and the enzymatic hydrolyzate, the third liquid phase (comprising cellulosic sugars) can be used in the culture broth alone or mixed as carbon sources for single cell oil production.

PRECIPITATED LIGNIN FRACTION (OBTAINED FROM THE SECOND LIQUID PHASE)

[00154] Precipitated lignin can be obtained by treating the second liquid phase comprising acid-soluble lignin and separated from the precipitated pure lignin, as illustrated in Figure 2.

[00155] Consequently, in one embodiment of the present invention the method includes an additional step of precipitating lignin from the second liquid phase by reducing the pH of the solution and the precipitated lignin is recovered by any suitable separation method, such as filtration.

[00156] The inventors of the present invention have found that the lignin fraction obtained by the method of the present invention is particularly pure (see Example 9).

[00157] Thus, a fourth aspect refers to a fraction of precipitated lignin obtainable through the method of the invention.

[00158] The separated lignin fraction can be used for value-added applications compared to the combustion value, such as in resin applications (e.g., phenolic resin applications) or polymer applications. Optionally, the separated lignin fraction is washed and/or further treated to purify it before use in value-added applications.

METHOD FOR PRODUCING MICROBIAL LIPID

[00159] An additional aspect of the present invention relates to a method for producing microbial lipid, the method comprising

[00160] (i) provide a cultivation medium comprising the Third liquid phase obtained by the method for fractionating a lignocellulosic material (which mainly comprises cellulosic carbohydrates) or the concentrated sugar hydrolyzate of the present invention.

[00161] (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe,

[00162] (iii) incubating said inoculated medium with said oleaginous microbe that allows the lipid to accumulate,

[00163] (vi) recovering the lipid from said oleaginous microbe.

[00164] The method of the invention is also referred to as a single-cell oil production process. The method of the present invention may be part of a process for biofuel production, as described herein, in which the oil or at least part of the oil is supplied in the form of microbial oil by the method described herein.

[00165] According to the preferred embodiment of the invention, the cultivation medium comprises lignocellulosic sugars derived from cellulose and/or hemicellulose. According to the invention, both hemicellulose and/or cellulose fractions of lignocellulosic biomass are used as raw materials for the production of microbial oil (single cell oil) in the same process (bioreactor system). The process preferably uses oleaginous microbes that have the ability to utilize both C6 (e.g. glucose, mannose, galactose) and C5 (e.g. xylose, arabinose) sugars.

[00166] According to another embodiment of the invention, the cultivation medium comprises hemicellulosic sugars derived from lignocellulose. According to yet another embodiment of the invention, the hemicellulosic sugars are at least partially in oligomeric form when fed into a single oil production process.

[00167] In a preferred embodiment of the present invention, the method for producing microbial lipid according to the preceding claim, wherein the method comprises the steps of:

[00168] a) Subjecting the lignocellulosic material to a treatment in which the hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase,

[00169] b) Separate the first solid phase from the first liquid phase,

[00170] c) Subjecting the first solid phase to a delignification treatment in the presence of alkaline delignifying agent to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin,

[00171] d) Separate the second solid phase from the second liquid phase,

[00172] e) Subjecting the second solid phase to an enzymatic hydrolysis treatment to hydrolyze the hemicellulose and cellulose of the second solid phase to produce a mixture comprising a third liquid phase of enzymatic hydrolyzate and a third solid phase.

[00173] f) separate the third liquid phase and the third solid phase,

[00174] (i) provide a cultivation medium comprising the Third liquid phase of enzymatic hydrolyzate of step f) and optionally the first liquid phase or the enzymatic hydrolyzate thereof,

[00175] (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe,

[00176] (iii) incubating said inoculated medium with said oleaginous microbe that allows the lipid to accumulate,

[00177] (iv) recovering the lipid from said oleaginous microbe.

[00178] The microbes used by the method to produce microbial lipids are oleaginous microbes. Oleaginous microbes (as described herein) have the capacity to accumulate intracellular lipids such that the lipids increase by at least 15% (w/w) of the total biomass (by cell dry weight) of the microbe when the It is even grown under suitable conditions. In a preferred embodiment, the oleaginous microbe has the capacity to accumulate at least 20% (w/w) of the total biomass of the microbe (by dry cell weight). In one embodiment of the present invention, the oleaginous microbe used for lipid production is selected from a group comprising yeast and filamentous fungi. Preferably, a method for producing microbial lipid is carried out under aerobic condition. Thus, in one embodiment of the present invention, the incubation in step (iii) is conducted as aerobic cultivation, as described herein.

[00179] When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. However, the mere fact that certain measures are mentioned in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention provides for all possible combinations and permutations of the described embodiments.

[00180] The terms "comprising", "comprising" and "comprising" in this document are intended by the applicant to be optionally replaced by the terms "consisting of", "consist of" or " consists of", respectively, in all cases. ITEMS

[00181] In the following, the invention is described by non-limiting items

[00182] Item 1. Method for fractionating a lignocellulosic material, wherein the method comprises:

[00183] a) Subjecting the lignocellulosic material to a treatment in which the hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase,

[00184] b) Separate the first solid phase from the first liquid phase,

[00185] c) Subjecting the first solid phase to a delignification treatment in the presence of alkaline delignifying agent to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin,

[00186] d) Separate the second solid phase from the second liquid phase,

[00187] e) Subject the second solid phase to an enzymatic hydrolysis treatment in the presence of enzymes capable of hydrolyzing the hemicellulose and cellulose fractions of the second solid phase to produce a mixture comprising a third liquid phase and a third solid phase.

[00188] Item 2. Method, according to item 1, in which at least part of the first liquid phase is combined with the second solid phase and subjected to enzymatic hydrolysis treatment.

[00189] Item 3. Method, according to item 1, in which hemicellulose is at least partially removed from the lignocellulosic material by hydrothermal treatment.

[00190] Item 4. Method, according to item 1, in which hemicellulose is at least partially removed from the lignocellulosic material by autohydrolysis treatment.

[00191] Item 5. Method, according to any of the preceding items, in which the alkaline delignifying agent is selected from a group consisting of sodium hydroxide, sodium carbonate, potassium hydroxide, sodium carbonate potassium, calcium hydroxide, calcium carbonate, lithium hydroxide, lithium carbonate, ammonium hydroxide, ammonia, sodium sulfide and the corresponding hydrates.

[00192] Item 6. Method, according to any of the preceding items, in which the concentration of delignifying agent is from 0.1 to 10%, preferably 0.1 to 4% based on the amount of dry matter in the first solid phase.

[00193] Item 7. Method, according to any of the preceding items, in which the alkaline delignifying agent is added to the first solid phase to obtain a suspension having a pH above 7, preferably between 10 and 13.

[00194] Item 8. Method, according to any of the preceding items, in which the delignification treatment is conducted at a temperature above 25, preferably between 30 and 160 °C.

[00195] Item 9. Method, according to any of the preceding items, in which the hydrothermal treatment is conducted at a temperature between 100 and 250 °C, preferably between 140 and 240 °C, and, most preferably between 140 and 200 °C

[00196] Item 10. Method, according to any of the preceding items, in which the hydrothermal treatment is conducted under conditions corresponding to severity between 2.0 and 4.5, more preferably, between 3, 0 and 4.1 and, most preferably, between 3.5 and 4.0.

[00197] Item 11. Method, according to any of the preceding items, in which the first solid phase is subjected to a steam explosion before said delignification treatment.

[00198] Item 12. Method, according to any of the preceding items, in which the enzymatic hydrolysis is conducted as a batch hydrolysis.

[00199] Item 13. Method, according to any of the preceding items, in which the enzymatic hydrolysis is conducted as a sequential hydrolysis.

[00200] Item 14. Method according to any of the preceding claims, wherein the enzymatic hydrolysis is conducted as a solid recycling hydrolysis.

[00201] Item 15. Method, according to any of the preceding items, in which lignin is precipitated from the second liquid phase by reducing the pH of the solution and the precipitated lignin is recovered by any suitable separation method , such as filtration.

[00202] Item 16. Method, according to any of the preceding items, which additionally comprises a step of concentrating the third liquid phase.

[00203] Item 17. Liquid phase in the form of an enzymatic hydrolyzate obtainable by the method as defined in any of the preceding items.

[00204] Item 18. Concentrated sugar hydrolyzate obtainable by the method as defined in item 16.

[00205] Item 19. Fraction of lignin obtainable by the method as defined in item 15.

[00206] Item 20. Method for producing microbial lipid, wherein the method comprises

[00207] (i) provide a cultivation medium comprising the third liquid phase of item 17 or the concentrated sugar hydrolyzate of item 18,

[00208] (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe,

[00209] (iii) incubating said inoculated medium with said oleaginous microbe that allows the lipid to accumulate,

[00210] (iv) recovering the lipid from said oleaginous microbe.

[00211] Item 21. Method for producing microbial lipid, according to the preceding item, wherein the method comprises:

[00212] a) subjecting the lignocellulosic material to a treatment in which hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase,

[00213] b) separate the first solid phase from the first liquid phase,

[00214] c) subjecting the first solid phase to a delignification treatment in the presence of alkaline delignifying agent to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin,

[00215] d) separate the second solid phase from the second liquid phase,

[00216] e) subjecting the second solid phase to an enzymatic hydrolysis treatment to hydrolyze the hemicellulose and cellulose of the second solid phase to produce a mixture comprising a third liquid phase of enzymatic hydrolyzate and a third solid phase.

[00217] f) separating the third liquid phase of an enzymatic hydrolyzate from the third solid phase.

[00218] (i) provide a cultivation medium comprising the Third liquid phase of enzymatic hydrolyzate from step f),

[00219] (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe,

[00220] (iii) incubating said inoculated medium with said oleaginous microbe that allows the lipid to accumulate,

[00221] (iv) recovering the lipid from said oleaginous microbe.

[00222] Item 22. Method, according to any of items 20 or 21, in which the microorganism used for the production of lipids is selected from a group comprising yeast and filamentous fungi.

EXAMPLES

[00223] The invention is illustrated by the following non-limiting examples. The invention can be applied to other lignocellulosic raw materials in addition to those illustrated in the examples. It should be understood that the embodiments given in the above description and examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention.

EXAMPLE 1 SELF-HYDROLYSIS (WITH PRE-ADJUSTED PH) OF WHEAT STRAW

[00224] A suspension was prepared by mixing 20 g of wheat straw previously ground to pass through a 1 mm screen and 180 g of water. The suspension was adjusted with acetic acid to 4.5. The suspension was transferred into an autoclave reactor which was then non-isothermally heated with a heating jacket to a temperature between 170 °C and 200 °C with continuous stirring. Temperature data during heating were recorded and used to calculate the severity of autohydrolysis (Equation 1). The reactor was cooled to approximately 50 °C, and the suspension was manually recovered for filtration. The liquid fraction was separated from the solid fraction and furfural and hydroxymethylfurfural (HMF) in the liquid fraction were measured using HPLC. The total sugar concentration (g/l) in the liquid fraction was determined after diluting acid hydrolysis that converts oligomeric and polymeric sugars into monosaccharides. The solid fraction was washed with water (0.5 dm3) and pressed. The solid residue obtained was weighed, sampled for dry matter determination, and the solid residue yield (%) was calculated as the weight ratio of solid residue to the wheat straw weighed for the autohydrolysis treatment ( 100%*g of dry wheat straw/g of dry solid residue). The soluble phenolic substances in the liquid were determined using the Folin-Ciocalteu method with guiaiacol as standard.

[00225] The results shown in Figure 11 and Figure 12 summarize the results. The solid residue yield decreased with the severity of autohydrolysis with 67% yield at the highest severity (Log(R0)=4.4) (Figure 11). The concentration of mosaccharide sugars in the liquid fraction is first increased and then decreased with increasing severity of autohydrolysis. The maximum sugar concentration (23.1 g/l) was obtained when the severity of autohydrolysis was Log(R0)=3.8. In addition to this severity of autohydrolysis, the sugar concentration in the liquid fraction decreased drastically and the concentration of furfural and HMF increased suddenly, reaching concentrations of 4.8 g/l and 0.3 g/l, respectively. In contrast, for the sudden generation of furfural and HMF, the concentration of soluble phenolic compounds is progressively increased from 0.5 g/l to 2.0 g/l with increasing severity of autohydrolysis.

[00226] This example shows that optimal autohydrolysis conditions in terms of severity of autohydrolysis (Log(R0)) can be selected to avoid excess formation of furfural, HMF and soluble phenolic compounds while maximizing the concentration of monosaccharides in the liquid fraction.

EXAMPLE 2

[00227] A suspension was prepared by mixing 33.8 kg of chopped wheat straw that contains 11% moisture with 350 kg of tap water that yields a consistency of 8.5%. The suspension was heated to approximately 60 °C, and the liquid fraction separated from the solids through filtration in a stirred Nutsche Filter. The solid fraction (31.2 kg of dry matter) was mixed with water to yield 192.4 kg of suspension at 5% consistency. The suspension was heated to 180 °C and cooled to room temperature which yields severity of S=4.15. The solid fraction was separated from the liquid fraction in a decanter centrifuge. The solid fraction was washed with water, and the washed insoluble fraction of "self-hydrolyzed straw" (15.3 kg of dry matter) was recovered using a decanter centrifuge and stored in a freezer. Based on HPLC analysis, the liquid fraction (6.3 kg of dry matter) contained hemicellulose-derived sugars approximately half of its dry matter.

[00228] The example shows that autohydrolysis allows the separation of hemicellulosic sugars from lignocellulosic material prior to enzymatic hydrolysis of the solid fraction containing cellulose.

[00229] The separation of the liquid and solid fractions and the optional washing of the solid fraction separate soluble phenolic substances and organic acids released in autohydrolysis from the autohydrolyzed solid fraction. As a result, the consumption of alkaline delignification chemical in the subsequent delignification step is synergistically decreased.

[00230] The solid fraction produced from this experiment was subsequently used in delignification tests, which resulted in a solid fraction with a high carbohydrate content and low lignin content.

EXAMPLE 3

[00231] A suspension was prepared by mixing 10.5 kg of ground straw at 7.3% moisture and 54.1 kg of tap water in a 100 dm3 container. After storage at room temperature for 18 h, 64.2 kg of the suspension was weighed into a 250 dm3 horizontal cylindrical stirred autoclave reactor. The reactor was closed and heated within 75 min to 140 °C, held at 140 °C for 5 h, and cooled to room temperature within 30 min. The hydrothermally treated suspension was removed from the reactor, and the liquid and solid fractions were separated by filtration. The solid fraction was washed with tap water and pressed using a hydropress. The pressed solid fraction (20.9 kg) had 42.7% dry matter content.

[00232] The solid fraction produced from this experiment was subsequently used in delignification tests, which resulted in a solid fraction with a high carbohydrate content and low lignin content.

SELF-HYDROLYZED STRAW DELIGNIFICATION

[00233] Autohydrolyzed straw contains considerable amounts of lignin that causes inhibitory effects on enzymatic hydrolysis. For this reason the solid material from autohydrolysis is extracted under alkaline conditions to remove lignin prior to enzymatic hydrolysis.

EXAMPLE 4

[00234] A suspension was prepared by mixing 20.5 kg (8 kg dry matter) of autohydrolyzed straw from Example 2 with 262 g of granular NaOH and tap water in a 180 dm3 stainless steel reactor equipped with a stirring unit. The suspension at 6.6% consistency was treated by heating the reactor to 95 °C in 30 min followed by isothermal treatment at 95 °C for 1 h with continuous stirring. The extracted solid fraction was separated from the liquid fraction through filtration in a centrifuge, washed with tap water, pressed and the AH straw extracted with NaOH (23.64 kg, 27% dry matter content) was stored at 6 °C. The liquid fraction ("NaOH delignification solution") containing 1.62 kg of dissolved material from autohydrolyzed straw was stored at 6 °C.

[00235] Enzymatic hydrolysis of AH straw extracted with NaOH with an enzyme dose of 35 μl/g Flashzyme Plus (Roal Oy, Finland), which was equal to 6 FPU (filter paper unit)/g DM of cellulase activity , yielded 65.0% sugar yield from autohydrolyzed straw carbohydrates. This is an improved result compared to 61.0% carbohydrate sugar yield from self-hydrolyzed straw without delignification.

[00236] The sugar yield is calculated as the ratio of the total amount of monosaccharides released in enzymatic hydrolysis of AH straw extracted with NaOH to the total amount of monosaccharides released from similar weight of AH straw in acid hydrolysis multiplied by the matter yield dry AH straw extracted with NaOH from delignification treatment.

[00237] This example shows that the same amount of enzyme produces a higher yield of sugar in enzymatic hydrolysis, when lignin is removed before enzymatic hydrolysis by alkaline treatment (NaOH). The example also shows that part of the lignin is dissolved in an alkaline liquid fraction and that the lignin can be recovered through precipitation.

EXAMPLE 5

[00238] A suspension at 3.6% consistency was prepared by mixing self-hydrolyzed straw from Example 2 (403 g, 156 g dry matter) with 4050 g tap water and 37.5 g Na2CO3 solid in a glass reactor equipped with a magnetic stirrer. The reactor was heated to 95 to 100 °C in 30 min and treated for 4 h at 95 to 100 °C with continuous stirring of the suspension. 812 g of Na2CO3-extracted solid fraction ("Na2CO3-extracted AH straw") having a dry matter content of 15% were separated from the liquid fraction (3,325 g) by filtration, washed with tap water, pressed and stored at 6°C. The yield of the solid fraction (DM of delignified straw/DM of self-hydrolyzed straw) was 78%. The liquid fraction (Na2CO3 delignification solution) had a pH of 9.8 and was stored at 6 °C. The amount of carbonate used is higher than NaOH since carbonate is a weaker base than hydroxide.

[00239] Enzymatic hydrolysis of AH straw extracted with Na2CO3 with an enzyme dose of 35 μl/g Flashzyme Plus (Roal Oy, Finland), which was equal to 6 FPU (filter paper unit)/g DM activity of cellulase, yielded 59.1% sugar yield from autohydrolyzed straw carbohydrates. This was less than 65.0% sugar yield with NaOH-extracted AH straw from autohydrolyzed straw carbohydrates or 61.0% sugar yield from autohydrolyzed straw carbohydrates without delignification. .

[00240] The sugar yield is calculated as the ratio of the total amount of monosaccharides released in enzymatic hydrolysis to the total amount of monosaccharides released from similar weight of AH straw in acid hydrolysis multiplied by the dry matter yield of AH straw extracted with Na2CO3 a from delignification treatment.

[00241] Compared to self-hydrolyzed straw that was subjected to enzymatic hydrolysis without delignification, the advantage of delignification with Na2CO3 was that the additional lignin fraction (designification solution with Na2CO3) was obtained.

[00242] This example shows that the same amount of enzyme produces the highest yield of sugar in enzymatic hydrolysis, when lignin is removed before enzymatic hydrolysis by alkaline treatment (Na2CO3). The example also shows that part of the lignin is dissolved in an alkaline liquid fraction and that the lignin can be recovered through precipitation.

EXAMPLE 6

[00243] A suspension was prepared by mixing 10.0 kg (4.41 kg dry matter) of self-hydrolyzed straw from Example 3 with tap water (29.5 kg) and 3.401 g of 50% ( w/w) of aqueous NaOH. The suspension was treated in a similar reactor and thermal conditions, as described above in Example 3. The solid fraction ("straw delignified with NaOH") was separated through filtration, and after washing it had 30.4% carbon content. dry matter. The liquid fraction ("NaOH delignification solution") had pH 13.0 and was stored at 6 °C.

EXAMPLE 7

[00244] The purpose of the example was to study the effect of alkaline treatment of autohydrolyzed straw on enzymatic hydrolysis and sugar yield from autohydrolyzed straw. To achieve the same, a suspension at 7.5% consistency was prepared by mixing 15 g of self-hydrolyzed straw dry matter from Example 1 with deionized water and chemicals listed in Table 1. The different alkaline chemicals were used to adjust the pH before heating to the indicated value. The suspension was quickly heated to 90 °C in a microwave oven. The hot suspension was filtered to separate the solid and liquid fractions. The solid fraction was washed with water and the filter cake was stirred until homogeneity, and its dry matter content was measured. The washed solid fraction (10 g of dry matter) was hydrolyzed with 350 μl (6 FPU/g) of Flashzyme Plus cellulolytic preparation (Roal, Finland) in 10% consistency in 0.0 Na acetate buffer. 05 M at pH 5 in the presence of potassium sorbate. Hydrolysis was performed in a 250 mL conical flask placed in a thermostatic incubator set at 50 °C, the reaction was continued with shaking at 200 rpm for 72 h. The released monomeric sugars were determined from the liquid fraction by HPLC. The dry matter and sugar yields obtained are presented in Table 1.

[00245] The example shows that delignification of autohydrolyzed straw increases the sugar yield from autohydrolyzed straw through enzymatic hydrolysis even when dry matter losses in delignification treatment are taken into account. Another advantage of delignification treatment is the generation of soluble lignin fraction suitable for further processing.

[00246] The surprising finding of the example was that only a low amount of chemical is needed in delignification of autohydrolyzed straw. Another surprising finding was that the pH of the liquid fraction isolated from the delignified solid fraction was relatively low, meaning that a lower amount of acid is needed to precipitate lignin from solution by lowering the pH.

EXAMPLES OF ENZYMATIC HYDROLYSIS OF SELF-HYDROLYZED AND/OR DELIGNIFIED STRAW EXAMPLE 8

[00247] A comparison between NaOH-delignified straw (material from Example 6) and self-hydrolyzed straw (material from Example 1) was performed in three different enzyme hydrolysis processes, including batch hydrolysis, sequential hydrolysis and solids recycling in a constant process feed. An enzyme mixture was used, which comprises 85% cellulase (Econase CE, Roal Oy), 10% cellobiase (Novozyme 188, Sigma/Novozymes) and 5% xylanase (GC140, Genencor). The reactions were performed at pH 5 and at a temperature of 50°C on a shaker at 200 rpm.

BATCH HYDROLYSIS

[00248] A batch hydrolysis was performed for the NaOH delignified straw and the self-hydrolyzed straw at 10% consistency and at 8% consistency (where "consistency" was the proportion of insoluble solids in the slurry of reaction, w/w). An enzyme dose was used that contained an enzyme activity of 9 FPU/g DM pretreated straw. The sugar yields (the anhydrous sugars released as a percentage of the total polymeric carbohydrates in the material), are shown in Figure 6. After 24h, 48h and 72h of hydrolysis, the sugar yields from straw delignified with NaOH at 8% and 10% % consistency on average 50%, 65% and 74%, whereas with self-hydrolyzed straw, yields at 8% and 10% consistency average 49%, 56% and 59%, respectively, which shows that a yield Higher sugar content can be obtained from NaOH-delignified straw with the same amount of enzyme as that from self-hydrolyzed straw. Yields were generally slightly higher at the lowest consistency.

[00249] The volumetric productivities shown in Figure 8 of the 48 h and 72 h batch hydrolysis of straw delignified with Na-OH was 1.32 g/l/h (grams of sugar per liter of reaction slurry per hour) and 0.82 g/l/h, respectively, which were considerably higher compared to the volumetric productivities of 0.74 g/l/h and 0.42 g/l/h, respectively, obtained from of self-hydrolyzed straw.

[00250] The enzyme productivity (kg of sugar obtained with one liter of enzyme) of the reactions is shown in Figure 7. The enzyme productivity in the 72-h batch hydrolysis of straw delignified with NaOH was 4.2 kg/I of enzyme which was almost double compared to the productivity of 2.2 kg/I of enzyme with auto-hydrolyzed straw.

[00251] This example indicates that a higher sugar yield, enzyme productivity and volumetric productivity were obtained from delignified straw compared to self-hydrolyzed straw in a batch enzymatic hydrolysis.

SEQUENTIAL HYDROLYSIS

[00252] The straw delignified with NaOH and the self-hydrolyzed straw were hydrolyzed by a three-step hydrolysis that consisted of three sequential 24-hour reactions. Between reactions, the liquid was separated from the solids and the solid residue was mixed with fresh liquid and enzymes. The proportion of solids in the total process ("the total consistency" or "the feed consistency") was 10% in the first two reactions and 8% in the three reactions. As the amount of total liquid was divided between the reactions, the actual consistencies of the first, second and third reactions were 14.3%, 12.5% and 10.6% with NaOH delignified straw and 14.4%, 12 .9% and 12.8% with self-hydrolyzed straw, respectively. A similar dose and enzyme cocktail were used, as described in "Batch hydrolysis of delignified straw with NaOH". Two-thirds (66.6%) of the total enzyme was applied in the first reaction and one-third (33.3%) in the second reaction.

[00253] The sugar yield (the anhydrous sugars released as a percentage of the total polymeric carbohydrates in the material), as shown in Figure 6, after the first, second and third reactions was 35%, 57% and 70%, respectively, of straw delignified with NaOH and 36%, 55% and 65%, respectively, of self-hydrolyzed straw. Although the yield was slightly higher from self-hydrolyzed straw after the first reaction, the yield from NaOH-delignified straw clearly prevailed after the second and third reactions. The total volumetric productivity (Figure 8) after two and three sequential 24-h reactions was 1.74 g/l/h and 1.61 g/l/h, respectively, with NaOH-delignified straw, which was considerably higher in compared to the productivity of 1.10 g/l/h and 0.94 g/l/h, respectively, of self-hydrolyzed straw. The enzyme productivity after three 24-h reactions with NaOH-delignified straw was 4.4 kg/I enzyme, which was drastically higher compared to the enzyme productivity of 2.4 kg/I with self-delignified straw. hydrolyzed (Figure 7).

[00254] This example indicates that a higher sugar yield, enzyme productivity and volumetric productivity were obtained from delignified straw compared to self-hydrolyzed straw in a sequential enzymatic hydrolysis.

HYDROLYSIS WITH SOLIDS RECYCLING

[00255] Straw delignified with NaOH and self-hydrolyzed straw were hydrolyzed in a solids recycling process. Multiple subsequent reactions were performed, in which fresh pretreated straw, enzyme and liquid were mixed to 10% consistency. In the mixture of fresh reaction constituents, 50% of the solid residue separated from the previous reaction was mixed. Recycling the solid waste led to an increase in the actual reaction consistency, which was increased to 12.3% with NaOH-delignified straw and to 13.2% with self-hydrolyzed straw. Six subsequent recycling reactions were performed in order to reach an equilibrium state, in which the reaction volume, consistency and hydrolysis yield remained constant between subsequent reactions. When the steady state was reached, the part of the solid residue that was not recycled was mixed with a constant amount of fresh liquid and an additional 24h reaction was performed, at a consistency of 10.5% with Na-OH delignified straw. and 13.1% with self-hydrolyzed straw. The total solids proportion (or "the total consistency" or "the feed consistency") was 10% in the recycling reaction and 8% in the process including the recycling reaction and the additional reaction.

[00256] The yield of sugar released as anhydrous sugars as a percentage of the total polymeric carbohydrates in the material is shown in Figure 6. In the initial reaction that did not contain any recycled material it was 51% and 50% for straw delignified with Na-OH and the self-hydrolyzed straw, respectively. After six subsequent reactions with 50% solid residue recycling, the hydrolysis yield was increased to 57% and 55% with NaOH-delignified straw and auto-hydrolyzed straw, respectively. After the additional reaction, the total sugar yield was 69% and 65% with NaOH-delignified straw and autohydrolyzed straw, respectively, thus showing generally higher yields for NaOH-delignified straw. The volumetric productivity of the recycling reaction alone with NaOH-delignified straw and self-hydrolyzed straw was 1.99 g/l/h and 1.17 g/l/h, respectively, and including the additional reaction, 1.77 g/h l/h and 0.99 g/l/h, respectively, showing considerably higher volumetric productivities with straw delignified with NaOH. The enzyme productivity of the recycling reaction alone with NaOH-delignified straw and autohydrolyzed straw was 3.2 kg/I and 2.0 kg/I, respectively, and when the additional reaction was included, 3.8 kg/I I and 2.4 kg/I, respectively, showing drastically higher enzyme productivity for NaOH-delignified straw compared to autohydrolyzed straw.

[00257] The progression of hydrolysis in subsequent reactions is shown in Figure 9a. The reaction volume progression is shown in Figure 9b. The progression of reaction consistency is shown in Figure 9c.

[00258] This example indicates that a higher sugar yield, enzyme productivity and volumetric productivity were obtained from delignified straw compared to self-hydrolyzed straw in an enzymatic hydrolysis process with solids recycling.

CONCLUSIONS FROM EXAMPLE 8

[00259] 1. The overall sugar yield was generally higher for NaOH-delignified straw compared to self-hydrolyzed straw in all different enzymatic hydrolysis processes.

[00260] 2. Since volumetric productivity was higher with NaOH-delignified straw compared to self-hydrolyzed straw in all different processes, smaller reactors and separators are required for a given sugar production rate in straw hydrolysis delignified with NaOH compared to self-hydrolyzed straw.

[00261] 3. Since enzyme productivity was higher with NaOH-delignified straw compared to self-hydrolyzed straw in all different processes, the enzyme cost per kg of sugar obtained is lower with NaOH-delignified straw than than with self-hydrolyzed straw.

[00262] 4. Equal amounts of water per pretreated straw were used in this example. It can be concluded that solids recycling and sequential hydrolysis lead to equal hydrolysis yields but increased volumetric productivities compared to batch hydrolysis.

EXAMPLE 9. LIGNIN QUALITY

[00263] Autohydrolyzed straw (material from Example 2) was hydrolyzed in a three-step sequential hydrolysis, as described in Example 8, at a feed consistency of 20% with an enzyme dose of 9 FPU , with the enzyme cocktail described in example 9. A sugar yield (the anhydrous sugars as a percentage of the total polymeric carbohydrates in the material) of 64.4% was obtained, which was equal to 34.8% straw DM self-hydrolyzed. Residual solids from enzymatic hydrolysis comprised 27.2% sugars, 55.5% lignin and 8.8% ash.

[00264] Autohydrolyzed straw (Material from Example 2) (40.7 g of dry matter) was extracted with 0.2 M aqueous NaOH. Extraction was performed at 7.7% consistency in a stirred glass reactor refluxed at 95 to 100 °C for 4 h. The liquid fraction was separated from the solid fraction extracted through filtration. The liquid fraction obtained (657.6 g) was acidified with 12 M sulfuric acid to pH 4.9 in order to precipitate the dissolved lignin. The precipitated lignin was separated from the liquid fraction by centrifugation, washed with acidified water and freeze-dried. The lignin fraction obtained represented 35.9% of the lignin originally present in the autohydrolyzed straw. The lignin fraction had a total lignin content (sum of acid-insoluble lignin and acid-soluble lignin) of 90.9% and 4.0% ash compared to 55.5% lignin content and 8. .8% ash content of residual solids after enzymatic hydrolysis.

[00265] This example shows that the advantage of the delignification treatment is the optional isolation of solid lignin fraction that has a notably higher lignin content compared to the lignin content of the enzymatic hydrolysis residue. The higher purity of lignin is advantageous for using lignin in higher value applications compared to combustion.

USE OF LIGNOCELLULOSIC SUGARS FOR THE PRODUCTION OF LIPIDS EXAMPLE 10 PREPARATION OF SELF-HYDROLYSIS LIQUID HYDROLYZATES C

[00266] The autohydrolysis reaction for wheat straw and the subsequent isolation of hemicellulose oligosaccharides was carried out to produce liquid fraction for fermentation, and the solid fraction susceptible to enzymatic hydrolysis. To achieve this, 35.7 kg of wheat straw (89.8% dry matter content) was mixed with 240 kg of water supply suspension at 11.6% consistency in a 500 dm3 stirred tank reactor. . The suspension was heated to 180 °C followed by cooling to below 100 °C. The hydrothermally treated suspension was discharged from the reactor and the first liquid fraction separated from the solid fraction using a decanter centrifuge. The solid fraction was washed with acidic water suspension adjusted to pH 4 with phosphoric acid. The solid fraction was separated from the second liquid fraction in the decanter centrifuge. The first and second liquid fractions were combined and concentrated in a falling film evaporator to yield 18.3 kg of concentrated autohydrolysis liquid which forms autohydrolysis liquid C which contains hemicellulose sugars partially in the form oligomeric and has 42% dry matter content and 38 °Bx refractometric dry substance. The washed solid fraction (96.7 kg which has 23.0% dry matter content) was used as feed material for enzymatic hydrolysis to produce cellulose hydrolyzate for cultivation.

[00267] Part of the phenolic compounds in the contained autohydrolysis liquid concentrate was removed by treating the liquid by adding 40 g/l of activated carbon, mixing gently for 20 hours at 4 °C and finally filtering the carbon using 400 um filtration cloth.

[00268] The enzymatic hydrolyzate from wheat straw cellulose fraction was prepared from the solid fraction containing cellulose (after washing) from the autohydrolysis experiment in which the autohydrolysis liquid C was prepared . The solid fraction washed from autohydrolysis treatment that forms autohydrolysis liquid C (17.3 kg having 23.1% dry matter content) was weighed in a 40 dm3 stirred tank reactor. and mixed with 14.7 kg of water and 10 mL of 50% NaOH (w/w) to yield a suspension at 12.5% consistency and at pH 5. The reactor was heated and maintained at 50 °C and 216 mL of enzyme mixture comprising 82% cellulose (Econase CE, Roal Oy), 10% cellobiase (Novozyme 188, Sigma/Novozymes) and 7% xylanase (GC140, Genencor). During the enzymatic treatment, the suspension was periodically stirred three times per hour for 5 min. After 48 h residence time, the suspension was supplemented with fresh enzyme mixture equivalent to 10% of the initial enzyme dosage and having similar proportions of individual enzymes. After a residence time of 72 h at 50 °C, the liquid fraction was separated from the solid fraction through filtration using a hydropress. The solid fraction was washed once with water and the liquid fraction was again separated from the solid fraction. The liquid fractions were combined and concentrated by evaporation under reduced pressure. The cellulosic hydrolyzate concentrate (1.57 kg) contained 220 g/l total sugar.

[00269] Cellulose hydrolyzate containing monomeric sugars was used as such in cultivation.

SINGLE CELL OIL PRODUCTION

[00270] The experiments were carried out using a lipid that produces the fungal strain Aspergillus oryzae. From the sporulated fungus grown on PDA plates, a spore suspension was made by adding 12 mL of sterile water and the spores were scraped with an inoculation loop into the liquid. 24 mL of the spore suspension was used directly for inoculating the fermenter. The average composition is presented in Table 5. Purified autohydrolysis liquid C (hemicellulose solution, hemicellulose sugars) and cellulose hydrolyzate from the same experiment were used in the cultivation. The cultivation was carried out in Biostat B plus a 5 I fermenter in a volume of 3 I, and during it the agitation was set at 500 rpm, the pH was maintained at 5.5 with 3 M NaOH, the aeration was 1 vvm and the temperature was 35 °C during growth, in lipid production it was reduced to 28 °C. TABLE 2 COMPOSITION OF GROWTH MEDIA

[00271] After inoculation it took about 30 hours before the fungus began to grow actively. During cultivation, the hemicellulose solution was added in small batches, and after 95 h of cultivation the feeds were changed to cellulosic hydrolyzate. During cultivation, a total of 236 g of hemicellulose and 484 g of cellulose hydrolyzate were added. Part of the sugars was left unused at the end of fermentation. In 167 h, when the cultivation ended, there was 16 g/l of biomass, of which 43% were lipids. It can be concluded that the production of microbial oil from wheat straw hemicellulose and cellulose sugars was successful.

EXAMPLE 11 - PRODUCTION OF MICROBIAL OIL IN HEMICELLULOSIC SUGARS PREPARATION OF HYDROLYZATE, SELF-HYDROLYSIS LIQUID D

[00272] A suspension was prepared by mixing 10.5 kg of ground wheat straw (92.7% dry matter content) and 54.1 kg of tap water in a 100 dm3 container. After storage at room temperature for 18 h, 64.2 kg of the suspension was weighed into a 250 dm3 horizontal cylindrical stirred autoclave reactor. The reactor was closed and heated within 75 min to 140 °C, held at 140 °C for 5 h, and cooled to room temperature within 30 min. The hydrothermally treated suspension was manually discharged from the reactor, and the first liquid fraction was separated from the first solid fraction through filtration. The first solid fraction was washed twice with tap water and pressed using a hydropress that yields the washed solid fraction. The washed solid fraction (20.9 kg) had 42.7% dry matter content. The first liquid fraction was combined with wash water and concentrated in a falling film evaporator to 11.5% (w/w) dry matter content. The concentrated liquid, autohydrolysis liquid D, contained 49.3% total sugar from the total dry matter of the concentrated liquid, as determined after dilute acid hydrolysis (4% w/w sulfuric acid, 121 °C, 1 h) by high-performance liquid chromatography (HPLC). The relative proportions of anhydrous xylose, anhydrous arabinose, anhydrous glucose and anhydrous galactose of the total sugar content were 57%, 19%, 13% and 11%, respectively.

[00273] After that, the autohydrolysis liquid D containing hemicellulosic sugars partially in oligomeric form was used in cultivation experiments as such without purification.

SINGLE CELL OIL PRODUCTION

[00274] The experiments were carried out using a lipid that produces the fungal strain A.oryzae. From the sporulated fungus grown on PDA plates, a spore suspension was made by adding 12 mL of sterile water and the spores were scraped with an inoculation loop into the liquid. 24 mL of the spore suspension was used to inoculate 6 flasks. The average composition is presented in Table 3. The inoculated flasks were incubated at 30 °C with shaking at 160 rpm for 1 day and then used for fermenter inoculation. TABLE 3 COMPOSITION OF THE INOCULATION MEDIUM, PH ADJUSTED TO 5.5.

[00275] Autohydrolysis liquid D (which contains hemicellulosic sugars partially in oligomeric form) was used and it contained 4.2 g/l of phenolic compounds based on analysis with the Folin-Ciocalteu method (Waterhouse , 2002). The cultivation was carried out in Biostat B plus a 5 I fermenter in a volume of 3 I, and during it the agitation was set at 400 rpm, the pH was maintained at 5.5 with 3 M Na-OH, the aeration was 1 vvm and a temperature of 30 °C. The average composition is presented in Table 4. TABLE 4 COMPOSITION OF FERMENTATION MEDIA

RESULTS:

[00276] During cultivation, the hemicellulosic solution was added in small batches. In total 150 g of hemicellulose were added. Part of the sugars was left unused at the end of fermentation. In 142 h, when the cultivation ended, there was 14 g/l of biomass, of which 21% were lipids. It can be concluded that the production of microbial oil from wheat hemicellulosic sugars (partially in oligomeric form) was successful. The production of microbial oil from hemicellulosic sugars has been successful without purification of the hydrolyzate (other than evaporation used in sugar concentration). During fermentation, the concentration of phenolic compounds was 2.8 g/l.

Claims (7)

1. Method for fractionating a lignocellulosic material, wherein the method is characterized by the fact that it comprises: a) Subjecting the lignocellulosic material to an autohydrolysis treatment in which the hemicellulose is at least partially removed from the lignocellulosic material to produce a mixture comprising a first liquid phase and a first solid phase, b) Separate the first solid phase from the first liquid phase, c) Subject the first solid phase to a delignification treatment in the presence of delignifying agent alkaline to produce a mixture comprising a second solid phase and a second liquid phase containing solubilized lignin, in which delignification occurs without steam explosion, and in which the concentration of delignifying agent is 0.1 to 10% by weight based on the amount of dry matter in the first solid phase, d) Separate the second solid phase from the second liquid phase, e) Subject the second solid phase to an enzymatic hydrolysis treatment in the presence of enzymes capable of hydrolyzing the hemicellulose fractions and cellulose from the second solid phase to produce a mixture comprising a third liquid enzymatic hydrolyzate phase and a third solid phase, and wherein the second solid phase is not treated with any acid prior to enzymatic hydrolysis, and wherein the lignin is precipitated from the second liquid phase by reducing the pH of the solution and the precipitated lignin is recovered by filtration. 2. Method according to claim 1, characterized in that the alkaline delignifying agent is selected from a group consisting of sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, lime hydroxide - heat, calcium carbonate, lithium hydroxide, lithium carbonate, ammonium hydroxide, ammonia, sodium sulfide and the corresponding hydrates. 3. Method, according to claim 1 or 2, characterized by the fact that the concentration of delignification agent is 0.1 to 4% by weight based on the amount of dry matter in the first solid phase. 4. Method according to any one of claims 1 to 3, characterized in that the alkaline delignifying agent is added to the first solid phase to obtain a suspension having a pH above 7, or between 10 and 13. 5. Method according to any one of claims 1 to 4, characterized by the fact that the first solid phase is subjected to a steam explosion before said delignification treatment. 6. Method according to any one of claims 1 to 5, characterized in that it additionally comprises a step of concentrating the third liquid phase. 7. Method for producing microbial lipid from a third liquid phase of enzymatic hydrolyzate, obtained by the method as defined in claim 1, characterized by the fact that it comprises: f) separating the third liquid phase of enzymatic hydrolyzate from of the third solid phase, (i) provide a culture medium comprising the third liquid phase of enzymatic hydrolyzate from step f), (ii) provide a fermentation broth by inoculating the culture medium with an oleaginous microbe, (iii) incubating said inoculated medium with said oleaginous microbe which allows the lipid to accumulate, and (iv) recovering the lipid from said oleaginous microbe.