JP2011502526A - Lipid production method - Google Patents

Abstract

The present invention relates to a method for producing lipids or lipid mixtures from organic raw materials containing cellulose, hemicellulose, starch, non-starch polysaccharides, any mixtures thereof or degradation products thereof. According to the method of the present invention, the organic raw material is treated one or more times with water, an aqueous solution of an acid, or an aqueous solution of an alkali, and a precipitate and a filtrate are separated. The precipitate obtained by the above treatment may be mechanically or thermomechanically ground, or the precipitate may be treated with a strong acid, or the precipitate is acidified and then mechanically or thermomechanically. You may grind into. After the treatment, the precipitate and the filtrate are separated. In the medium, the microorganism producing lipid is brought into contact with the organic raw material or the obtained filtrate to cause the microorganism cell to start producing lipid, and then the lipid is recovered.

Description

  The present invention relates to a method for producing a lipid or lipid mixture from an organic raw material according to the preamble of claim 1. The invention also relates to the use of a lipid or lipid mixture produced by the method as a biofuel according to claim 17 together with a biofuel according to claim 18. The invention further relates to a method for purifying urban sewage as claimed in claim 19.

  It is generally known that the use of transportation fuels produced from fossil raw materials is extremely diverse and that the consumption of these fuels is continuously increasing. Therefore, the adequacy of fossil energy resources, environmental effects, and the aspect of sustainable development that are friendly to the environment are quite obvious as an indispensable global challenge. From this point of view, it is attracting attention as an object of increasing interest in renewable raw materials for transportation fuels.

  The first step in producing fuels that rely on renewable natural resources is to attempt to at least partially replace fossil feeds with organic feeds. However, you can imagine that even this approach creates problems that are quite difficult to solve. Even if only a small part of the fossil raw material is replaced, the amount of the organic raw material required becomes extremely large in proportion to the current consumption of the fossil raw material. In connection with some of this, large-scale consumption or exploitation of cultivated land from only one aspect of organic natural resources for this purpose is a balance between natural biodiversity and primary food production. Has already been observed to have an impact on this, and it will be difficult to solve such problems. It is also technically difficult to convert the organic material into a form that can be used to produce transportation fuels in an energy efficient manner.

  A particularly preferred raw material source for transportation fuels is organic fats, especially triacylglycerol. The reason for this is that the energy content of organic fats is, for example, significantly higher than the energy content of the corresponding carbohydrate or alcohol. Furthermore, it is well known that organic fats can be converted into components such as transportation fuels, such as diesel fuel, biodiesel fuel or renewable diesel fuel, by relatively efficient chemical methods. However, the shortage of natural fat raw material is a limiting factor in this method. Based on current fatty resources, it is possible to some extent to produce biofuels industrially, albeit insufficiently. Therefore, in order to increase the amount of stored fat, it is necessary to greatly increase the amount of cultivation of a plant rich in fat. This major change in the production sector for the cultivation of fatty plants has a strong impact on the balance of the food economy in the global market. This need is only speculative, but has already emerged as the prices of food and feed ingredients have soared.

  The total amount of renewable organic mass in nature is very large, and when calculated as the amount of carbon, the total amount is significantly greater than the annual use of fossil carbon as transportation fuel. However, the majority of these renewable masses (about 60%) are composed of compounds containing a significant amount of oxygen, so their value as fuel is very low.

Based on known prior art, high energy compounds with shorter hydrocarbon chains can be obtained using chemicals with relatively low energy content, especially in chemical methods (eg, methods that consume very large amounts of energy). It is theoretically known to be obtained by certain vaporization techniques, etc., but in this case the overall efficiency between feed and yield is low (R. Agrawal, N. R. Sing, F. H. Ribeiro and W. N. Delagas, 2007, “Sustainably Available Fuels for the Transportation Sector”, PNAS , 104: 4828-4833, and International Patent Application Publication No. 2006/117317). .
Corresponding underlying problems also relate to biotechnological processes by known techniques used in converting hexose sugars contained in carbohydrates to high energy compounds. Examples of this include the production of alcohols, in particular ethanol, such examples being disclosed in US 2002/0185447, US 5537502 and WO 03/038067. It is described in the description.

  US Patent Application Publication No. 2004/0231661 discloses a process for producing xylose and glucose that can be used for ethanol preparation by subjecting a substance containing lignocellulose to water extraction and acid extraction, followed by hydrolysis. Is disclosed. US Pat. No. 5,221,357 describes the hydrolysis of hemicellulose and materials containing cellulose, mechanical treatment of the solid layer to effect acid hydrolysis, and monosaccharides that can be used to prepare ethanol, such as pentose and Disclosed is a process for producing hexose sugars. US Pat. No. 4,752,579 discloses a process for separating corn seed husks from monosaccharides by acid and / or alkali and enzymatic hydrolysis.

  Some specifications disclose methods of producing lipids from various organic materials using microbial fermentation methods. U.S. Pat. No. 4,368,056 proposes the use of carbohydrates present in low levels in industrial waste in butanol fermentation and the use of glycerides by microorganisms produced by fermentation during the production of biodiesel fuel. Publication by Dai et al. (Dai, C, Tao, J., Chef, F., Dai, Y and Tsao, M, “Generation of biodiesel fuel from oily yeast Rhodotorula glutinis with xylose assimilation ability” Afr. J. Biotechnology, Vol. 6 (No. 18), pages 2130 to 2134, September 19, 2007), is a plant material as a raw material for producing lipids by Rhodotorula yeast. A method of extracting carbohydrates contained in straw (for example, corn stalks, leaves of leaves and rice) by grinding and acid treatment, and using the filtrate and washing water obtained by the treatment to produce lipids using Rhodotorula yeast It is disclosed that it is used as a raw material. Lipids obtained from microorganisms are used in the production of biodiesel fuel. Angerbauer et al. Disclose utilizing wastewater sludge for the production of lipids by Lipomyces yeast, treating wastewater sludge with alkali and acid, and converting the sludge into a raw material for biodiesel fuel (Angerbauer, C., Sabenhofer, M, Mittelbach, M, Goubitz, GM, "Conversion of sludge to lipids by Lipomyces starkeyi to produce biodiesel fuel," BioResource Technology, Volume 99 (2008) See pages 3051 to 3056).

International Patent Application Publication No. 2006/117317 US Patent Application Publication No. 2002/0185447 US Pat. No. 5,637,502 International Patent Application Publication No. 03/038067 US Patent Application Publication No. 2004/0231661 US Pat. No. 5,221,357 US Pat. No. 4,752,579 US Pat. No. 4,368,056

R. Agrawal, N.A. R. Sing, F.M. H. Ribeiro and W.C. N. Delagas, 2007, “Sustainably Available Fuels for the Transportation Sector”, PNAS, 104: 4828-4833. Dai, C, Tao, J., Chef, F., Dai, Y and Tsao, M, “Production of Biodiesel Fuel from Oily Yeast Rhodotorula glutinis with Xylose Assimilation Capability” Afr. J. Bio Technology, Vol. 6 (No. 18), pp. 2130-2134, September 19, 2007 Angerbauer, C.I. Sabenhofer, M, Mittelbach, M, Goubitz, G. M, “Conversion of sludge to lipids by Lipomyces starkeyi to produce biodiesel fuel”, BioResource Technology, Vol. 99 (2008), pages 3051-3056

  The problems that arise in the above methods include the fact that the yield of sugars that can be utilized by microorganisms is low, and these methods apply to the use of biomaterials as raw materials that are less important for the demands of large-scale production. Is included. It is clear that the quantitative goals for the production of biofuels cannot be achieved by using the method described above and the raw materials used in the method.

  Thus, new technical solutions that can be used to convert renewable organic carbohydrates buried in the earth into compounds with higher energy content, regardless of their chemical or structural composition, There is still a strong need for solutions that can be applied more broadly than before. How can carbohydrates present in various organic substances be converted to compounds with higher energy content, such as fats that are more compatible with use as transportation fuels or raw materials for such use It is particularly important to solve the problem of whether or not it can be converted automatically.

SUMMARY An object of the present invention is to provide a new solution to the problem of how organic biomaterials can be converted to compounds with higher energy content.

  In particular, the object of the present invention is to provide a solution to the problem of how carbohydrate components obtainable from organic biomaterials can be converted into lipids suitable for the production of biodiesel fuel.

  More precisely, the method according to the invention is characterized by what is stated in the characterizing part of claim 1.

  Similarly, the use according to the invention is characterized by what is stated in claim 17, and the biofuel according to the invention is characterized by what is stated in claim 18.

  The method for purifying urban sewage according to the invention is characterized by what is stated in claim 19.

  The present invention is based on the observation that when biomass is treated in various ways to recover cellulose and hemicellulose fractions, the cellulose and hemicellulose fractions are further degraded when microbial populations appear more rapidly in the fractions. . Surprisingly, a microorganism with ATP citrate lyase enzyme (EC 2.3.3.8; formerly EC 4.1.3.8) also grows in the carbohydrate fraction described above, and the microorganism utilizes the enzyme. Thus, it was discovered that lipids, particularly triacylglycerol, are collected in cells. Based on these observations, according to the present invention, after the biomaterial is processed and the cellulose and hemicellulose contained therein are separated from the remaining components of the biomaterial, it is subjected to hydrolysis treatment, thereby producing a hydrolysis product. Of biomaterials to obtain hydrolysis products suitable for the growth of microorganisms that capture lipids and the production of lipids, and the lipids formed as described above as raw materials in the production of biodiesel fuel Developed use.

  According to the description of the present invention, carbohydrates suitable for use in lipid-synthesizing microorganisms can be produced from organic raw materials originating from various sources and / or lipids are produced by microorganisms. The resulting carbohydrate fraction can be generated. According to the present invention, such carbohydrates can be produced in particular from biomaterials containing hemicellulose, cellulose, starch or non-starch polysaccharides. Carbohydrates suitable for use by microorganisms are in particular monosaccharides and oligosaccharides containing both hexose sugars and pentose sugars. If a lipid-producing microorganism is selected that can utilize a carbohydrate polymer, the carbohydrate can be polymerized.

  Various forms of wood hold the most renewable biomass that can be recovered at present. The use of wood, especially the mechanical or thermomechanical treatment of wood or the production or production of mechanical mass from wood, takes place on a large scale, in the process producing large amounts of carbohydrate-containing sidestreams. Very little economic added value has been found for the side cuts that occur in the wood refining industry. In many cases, this type of slicing introduces additional costs. This is because the environmental load caused by the fragment must be reduced. As a technical challenge, there is a problem especially with sidestreams like this kind of snippet. Although the side stream is generated in large quantities, the carbohydrate content is generally low. Dilute aqueous carbohydrate solutions are less suitable for processes aimed at using carbohydrates in solution by chemical means.

  Accordingly, the object of the present invention is to provide a biomaterial containing biomaterials that currently requires expensive purification means or is not fully utilized in current methods but has potential as an energy source. It also provides a solution to the problem of how to use industrial substreams generated on a scale.

  In the process according to the invention, cellulose, hemicellulose, starch, a mixture of all of these, a mixture of parts thereof, or their degradation products, or starch or non-starch carbohydrate itself, or the carbohydrate combined with cellulose or hemicellulose. The organic raw material to contain is processed. The raw material can be subjected to mechanical, thermomechanical, physical, chemical or biological pretreatment, or a combination thereof, or it may be appropriate to use the raw material as it is. In the case of a raw material containing a polymerized carbohydrate, it is preferable to treat with water, acid or alkali, or a mixture thereof. After these pretreatments by the methods described herein, the mixture is separated into filtrate and solids. That is, the precipitate (FIG. 1) and the filtrate or both fractions are recovered. It is preferable to repeat the treatment of the raw material using water, acid or alkali, or a mixture thereof and separate the precipitate, and then mix the filtrate. The filtrate obtained in the treatment containing an alkali is subjected to acid treatment of the raw material in order to increase the amount of soluble monosaccharides in the state of the filtrate as it is or after being subjected to the above-mentioned repeated treatment to form a mixture. It is preferable. Any filtrate or precipitate produced in these processes, the raw material itself, a mixture of the raw material and the filtrate or precipitate, or a plurality of filtrates or precipitates may be subjected to suitable pretreatments such as neutralization, decolorization and filtration. It is used for the production of single cell lipids. The filtrate can be subjected to a mixing treatment, a dilution treatment or a concentration treatment in order to obtain a monosaccharide content and composition suitable for a microorganism producing a single cell lipid.

  Depending on whether the treatment is carried out with water or in the presence of an acid or alkali, a selectable treatment of the raw material produces precipitates having various compositions. In order to increase the yield of sugar, preferably the precipitate is subjected to a mechanical grinding process as it is or in the presence of water, acid or alkali, and the filtrate and the precipitate are again removed from the mechanical grinding process. can get. The filtrate, precipitate or mixture thereof is used to produce single cell lipids, and if desired, preferably the precipitate is treated with a strong acid. As a result of the acid treatment, a filtrate and a precipitate are produced again, and the filtrate or precipitate or a mixture thereof can be used for the production of single cell lipids. The precipitate can be subjected to treatment involving grinding under relatively high acid concentration conditions. The filtrate, precipitate or mixture thereof produced as a result of this treatment is used for the production of single cell lipids. The precipitate can be separated and incinerated or can be used to produce biofuels or their precursors by other methods. Each of the filtrate fractions, precipitates or raw materials can be used alone to produce single cell lipids, but can also be used as various mixtures. The precipitate obtained from the foregoing process steps can be processed again at an earlier or later process step as described herein. Therefore, it is preferred to treat the precipitate with an acid or alkali stronger than the acid or alkali used in the previous treatment in order to increase the sugar yield.

  Furthermore, enzyme treatment or microbial fermentation can take place between different process steps.

  The invention also relates to a process for recycling spent alkali and acid in the above process.

  The present invention also relates to a method of recirculating the single cell biomass used in the above method as the biomaterial according to the present invention when the produced lipid is recovered.

  The economically available components can be recovered from the precipitate obtained by the treatment performed by the method of the present invention. The filtrate can also be used in other microbiological methods other than lipid production.

  The method described herein can be used to fractionate hexose and pentose monosaccharides contained in biomass carbohydrates in a fraction that can be more efficiently utilized by lipid-synthesizing microorganisms to produce lipids. It provides a solution to the problem of whether it can be gradually changed into smaller fractions containing relatively large amounts of monomeric sugar units. In connection with any process step in which insoluble material (precipitate) is separated from soluble material and a filtrate is obtained, the hexose and pentose sugars contained in the filtrate are either intact or necessary. It can be used to produce single cell lipids in a filtrate mixture for producing single cell lipids obtained after various pretreatments. In addition to the filtrate, a precipitate, or a mixture of filtrate and precipitate, can be used to produce single cell lipids.

  The overall advantage of the present invention is that high energy compounds (ie, hexose sugars and pentose sugars or oligomers produced thereby, and mixtures thereof, such as low energy content biological origin compounds (ie, In order to produce lipids, unit operations and simple methods already used industrially can be applied in an energy efficient and environmentally friendly manner.

  In the process according to the invention, it is particularly preferred that the new surface of the organic material is always exposed by mechanical or thermomechanical grinding of the organic material that can be treated again with water or various concentrations of acid or alkali. As a result, a solution and a precipitate in the solution are prepared, and more saccharides that can be used for lipid production of microorganisms can be obtained from the solution and the precipitate, particularly the solution.

  When the solution and the precipitate are separated several times, significantly higher yields of hexose and pentose sugars are obtained by mechanical or thermomechanical grinding compared to single-phase hydrolysis methods.

  In the process according to the invention, the treatment streams obtained from the various treatments are neutralized with each other by mixing the filtrates obtained from the acid treatment and the alkali treatment or by mixing the filtrate and the precipitate. Therefore, the filtrate or the precipitate or a mixture thereof can be used as it is without adjusting the acidity, and at least the necessity for adjusting the acidity is low. It is also effective to mix several different biomass and biomass from different sources with each other.

  It is particularly desirable to develop a process that produces sugars that can be used for the production of lipids and that can be used fully or partially microbiologically to produce other compounds such as alcohols. It is emphasized.

  The unique advantages of the present invention are suitable for the use of alternative biomaterials that release carbohydrates to produce lipids, in addition to utilizing sidestreams or mainstreams of mechanical and thermomechanical treatment of wood. It is that.

  Furthermore, the present invention relates to a method for forming a lipid or lipid mixture from a mixture produced by recirculating fibers produced in thermomechanical treatment and alkali or acid treatment according to the method of the invention.

  According to the present invention, it is another organic raw material that can be converted into hexose sugar and pentose sugar, or an oligomer formed by them, and can be converted from these sugars to lipids by microorganisms, and is difficult to use on a large scale. Other organic raw materials are, for example, newspaper, beet pulp obtained from sugar beet, recycled fiber obtained from straw recycling such as rice husk and oats, sawdust, pulverized mechanical pulp, straw, and peat, similar to cultivated plants (Especially slightly decomposed peat). Other organic materials that are rarely used at present are biomass in swamps or subsurface biomass, biomass from cellulosic catchments, and biomass present in activated sludge plants from municipal waste, or sedimentary areas or incineration Other organic waste related to These organic materials are treated by modifying various aspects of the method of the present invention so that the carbohydrates contained in these materials are converted to a state useful for microorganisms that produce single cell biomass and lipids. May be. For example, municipal wastewater may be treated using the method of the present invention, which provides the advantage of killing particularly harmful microorganisms during treatment.

  An important aspect of the present invention is that regardless of the origin of the organic raw material, such that monomeric hexose sugars and / or pentose sugars, or oligomers thereof, suitable for producing single cell lipids are derived from carbohydrates, Biomaterials containing carbohydrates are to be processed. It is preferable to perform the treatment by combining two or more treatment methods.

  Using the method according to the present invention suitable for the scale required for mass production of biofuels, the production of cell mass of lipid producing microorganisms and the raw materials that can be used in the production of lipids are produced simultaneously from a variety of different biomass be able to. Ingredients suitable for microbial lipid production can be produced regardless of biomass composition, availability and structure. Also, the great advantage of the present invention brought about by using the method according to the present invention is that an effective sugar can be produced in a high yield and the consumption of chemicals necessary for adjusting the acidity can be reduced.

  Compared to conventional prior art, the invention described herein combines the conversion of biomaterials containing both cellulose and hemicellulose into hexose and pentose sugars suitable for use. Provide innovative technology. According to the same embodiment, the present invention also allows starch and non-starch monosaccharide units contained in biomaterials to be used to convert sugars useful for the production of single cell lipids. In particular, the present invention can be applied on an industrial scale to materials originating from renewable natural resources, or materials originating from substreams of natural resources that are less valuable from industrial facilities or municipalities. To be executed. The method according to the invention can also be used in the production of single cell lipids to treat materials containing cellulose and hemicellulose in a controlled manner so that precursors of single cell lipids that can be used by safe microorganisms are generated. .

  In the following, the invention will be described in more detail with the aid of the accompanying drawings and detailed description.

FIG. 1 shows the main working steps of the method according to the invention. FIG. 2 shows that hexose sugars and pentose sugars are used as such or in a mixed state to produce cell mass and lipids. FIG. 3 shows that the mixture of the rice husks subjected to alkali treatment and the filtrate obtained from the 10% acid hydrolysis was added as a carbon source for the production of lipids in yeast. 2 is a graph showing growth and lipid production. FIG. 4 is a graph showing yeast growth in a medium supplemented with a commercially available pentose sugar as a carbon source.

  “Carbohydrate” means an organic molecule having an aldehyde group, an acid radical or keto group and a plurality of hydroxyl groups. Accordingly, the range of carbohydrates includes compounds described in terms such as monosaccharides, oligosaccharides, polysaccharides, sugars, cellulose, hemicellulose, starch and non-starch carbohydrates.

  “Cellulose” is a long-chain polysaccharide having a primary structure composed of a polymer formed by β-1-4 bonds of glucose.

  “Starch” is a long-chain variety consisting mainly of α-1-4 and α-1-6 glucose units.

  In the present invention, “usable sugar” means a sugar that is used when a microorganism grows, and that can be used by lipid- and alcohol-producing microorganisms to produce lipid or alcohol.

  “Hemicellulose” means a group of compounds consisting of a plurality of different hexose sugars and pentose sugars such as galactose, mannose, glucose, xylose and arabinose.

A “monosaccharide” is a carbohydrate represented by (C—H ₂ O) n, generally consisting of 3 to 9 carbon atoms and having a stereochemical difference at one or more carbon atoms. Monomer unit. These include hexoses with 6 carbon atoms such as glucose, galactose, mannose, fructose and the like, and pentoses with 5 carbon atoms such as xylose, ribose and arabinose.

  “Oligosaccharide” means a carbohydrate produced from two or more monosaccharides by O-glycoside linkages.

  “Pentose sugar” means a monosaccharide containing 5 carbon atoms.

  “Hexose sugar” means a monosaccharide containing 6 carbon atoms.

  “Hydrolysis” means that a carbon-carbon bond, carbon-oxygen bond, carbon-nitrogen bond, or carbon-sulfur bond is affected by water, acid or alkali, regardless of the water participation in the reaction. Means to be disconnected. In the case of enzymatic hydrolysis, the corresponding cleavage reaction is catalyzed by the enzyme. Hydrolysis is, for example, a reaction in which an O-glycoside bond between monosaccharides of carbohydrates or a peptide bond between amino acids of proteins is cleaved.

  “Treatment with water, acid or alkali” means in this specification that the organic substance itself or a product derived from the organic substance is extracted in the presence of water, acid or alkali, and is mechanical or thermomechanical. Means that these processes are performed in combination, or a combination of these processes. According to Bronstead Raleigh's acid-alkali theory, acid means a chemical substance, molecule or ion that can donate a hydrogen ion (proton), and alkali means a substance, molecule or ion that can accept a hydrogen ion (proton). I mean ion. The acid is also called a so-called Lewis acid capable of accepting an electron pair, and the alkali is called a so-called Lewis alkali capable of donating a base pair. According to these definitions, the activity of the substance as acid or alkali is not limited to an aqueous solution. As used herein, the terms “acid” and “alkali” according to these definitions also mean acid and alkaline catalysts. As used herein, acid also refers to any acidic phase, including a gas phase, solid phase, or liquid phase (eg, an aqueous solution) that includes a component that functions as an acid. Correspondingly, alkali means any alkaline phase containing a component that functions as an alkali, such as the gas phase, the solid phase, or the liquid phase (eg, an aqueous solution).

  “Organic raw material” as used herein means any organic matter produced by a living organism. In the present specification, the organic raw material is also referred to as biomaterial.

  In particular, the organic raw material means an organic substance containing a polysaccharide. "Polysaccharide" means a carbohydrate polymer formed from monosaccharides that can also contain compounds other than monosaccharides. For example, polysaccharides are cellulose, hemicellulose and starch. Polysaccharides also contain, among other things, alginate, glucan, inulin and gum arabic. Among other polysaccharides, mannan is exemplified. The organic raw material may contain the polysaccharide as it is or as a mixture, or may contain these degradation products.

  “Non-starch polysaccharide” means a carbohydrate having a molecular structure that lacks α-1-4 bonds that are characteristic of starch, or a molecular structure with few such bonds. Non-starch polysaccharides are, for example, glucan, alginate, inulin and gum arabic. Non-starch polysaccharides also contain hemicellulose and cellulose. Examples of other non-starch polysaccharides include carbohydrate polymers contained in algae.

  “Cultivated plant” means a plant that is planted or sown for beneficial purposes in soil prepared for cultivation.

  The term “lipid” generally refers to an aliphatic substance that contains an aliphatic hydrocarbon chain as part of a molecule that dissolves in an organic solvent but hardly dissolves in water.

  In the present invention, lipids produced in microorganisms are mainly tri-, di- or mono-acylglycerols or sterol esters, but other lipids such as phospholipids, free fatty acids, sterols, polyprenol, sphingos. Lipids, glycolipids and diphosphatidylglycerol are also produced in the cells of microorganisms.

  The invention can be used for the production of biodiesel or renewable diesel fuel.

  According to EU-Direction 2003/30 / EY, “biodiesel fuel” means “methyl-ester produced from vegetable or animal oils with diesel fuel quality for use as biofuel”.

  Renewable diesel fuel refers to hydrogenated lipids of animal, plant or microbial origin, which may be of bacterial, yeast, mold, algae or other microbial origin.

  The raw materials used in the method according to the invention may be cellulose, hemicellulose and biomass, preferably mechanical or thermomechanical methods or other physical methods, or chemical, enzymatic or microbiological methods. Or a wood pulp that may contain a binder produced by a combination of these methods. Plant raw materials containing starch, for example, potato, various parts of potato, cultivatable crop seeds, corn, rice, sugar beet, and sugar beet pulp containing non-starch polysaccharides contained in similar plants Can be used as a raw material for the process according to the invention without modification of the process according to the invention. Various parts of plants containing non-starch polysaccharides such as β-glucan are also suitable as raw materials. In addition, the method of the present invention is suitable for using as a raw material a carbohydrate such as alginate that originates from a unicellular organism. Moreover, the raw material composition mentioned above contains the protein and lipid which can be used also as a raw material for the proliferation of a lipid synthesis microorganism and lipid production in various ratios.

  The raw materials used in the method according to the present invention are, for example, recycled fiber (waste paper pulp) obtained from recycling of newspaper, husks of cereals such as sugar beet pulp and oats, sawdust, refined mechanical pulp, peat and straw. May be. Other raw materials useful in the method according to the invention include, for example, microbial masses such as single-cell biomass, biomass in marshes or water containing algae and meat microalgae, biomass from the catchment area of a cellulose factory, activated sludge from municipal sewage Currently used in incineration, composting, or other methods that move to plants or municipal wastes that contain biological composites that release carbon contained in the waste as carbon dioxide over a wide range It is the municipal waste that has been.

According to a preferred embodiment of the present invention, the filtrate, filtrate mixture, precipitate or precipitate mixture obtained from one or more organic substances according to the method of the present invention is introduced into the mixture in which the production of lipids takes place. Including at least one step. Another embodiment of the method of the present invention is that what monosaccharide composition is preferred in the filtrate, filtrate mixture, precipitate, precipitate mixture, or filtrate and precipitate mixture in terms of microbial growth and lipid production. Can be selected by criteria. Accordingly, the biomaterial is treated with a filtrate or precipitate, preferably a material selected from the group comprising (i) to (iii) below:
(I) water,
(Ii) an acid, and (iii) an alkali,
It is then selected from the group produced by separating the precipitate containing the fiber and the filtrate containing no fiber.

  If desired, the precipitate is further subjected to treatment with any substance (i), (ii) or (iii) one or more times, preferably the resulting precipitate is mechanical or thermomechanical. Subjected to a mechanical grinding process, the precipitate and filtrate are separated.

  Depending on the type of biomaterial used and the desired monosaccharide, the biomaterial is treated with water, acid or alkali, preferably with acid or alkali, generally treated with an aqueous solution of acid or alkali. The If necessary, the processing may be performed a plurality of times. The same biomaterial may be treated sequentially with multiple different solutions, and compounds that promote carbohydrate separation and hydrolysis may be added to water.

  The raw materials described in the following list of Group I can be treated with water in the first step, and are treated with a mixture of water and acid in order to improve the treatment effect.

  Group II biomaterials are preferably treated with acid in the first step.

  Group III biomaterials are preferably treated with alkali in the first step. If the precipitate and filtrate are already recovered, the filtrate can be treated again with acid.

Group I
Biomaterials obtained by mechanical, thermomechanical, enzymatic or microbiological treatment of wood, biomaterials obtained by a combination of these treatment methods, or biomaterials obtained from cultivated plants.

Group II
Recycled fiber, beet pulp, rice husk, straw, bran, grain granules, all cultivated plants, cultivated plants, TMP pulp, MDF pulp, or raw materials containing starch or non-starch polysaccharides.

Group III
Sawdust, refined mechanical pulp, rice husk, straw, plant wood parts, TMP pulp, MDF pulp, beet pulp, cultivated plants that may contain various amounts of starch.

  For example, the treatment can be facilitated by adding one or more enzymes into the treatment solution, preferably an aqueous treatment solution. Enzymatic treatment or microbial fermentation treatment can be interposed between the various process steps.

  In a subsequent step, the lipid-producing microorganism is contacted with any filtrate, precipitate, or mixture thereof in the medium, causing the cells of the microorganism to produce lipid, which is then recovered.

  Preferably, the precipitate containing the fiber obtained from the above-described biomaterial treatment step is separated from the precipitate containing the fiber and the fiber-free filtrate by mechanically grinding the precipitate containing the fiber. Will be processed using the method.

  Furthermore, the fiber-containing precipitate obtained from mechanical grinding can be treated with a strong acid, and then the fiber-containing precipitate and the fiber-free filtrate can be separated. After acid treatment, the precipitate can be subjected to mechanical grinding again, or as described above, the precipitate can be used for lipid production using microorganisms.

  In addition, the biomaterial can be acidified and then mechanically or thermomechanically pulverized, and then the fiber-containing precipitate and fiber-free filtrate can be separated.

  The filtrate or precipitate obtained from any of the treatments described above, or a mixture thereof can be added to the medium for the lipid-producing microorganism.

  Generally, the total amount of sugar in the filtrate is 0.5 to 10% by weight. Of these, sugars that can be used for biomass and lipid production are generally at least 0.5 wt%, preferably at least 3 wt%, more preferably 4-5 wt%. For example, after the biomaterial is pulverized and then re-extracted, more sugar can be separated from the biomaterial, thus increasing the amount of sugar to a more effective amount. However, the amount of sugar in the filtrate is preferably less than 30% by weight, more preferably less than 20% by weight.

  Microorganisms capable of producing lipids can be grown such that they first produce biomass and then produce lipids or produce both biomass and lipids simultaneously.

  Depending on the origin of the biomaterial and the treatment method of the biomaterial (treatment method using water, acid or alkali), hexose monosaccharides, pentose monosaccharides or mixtures of various proportions as shown in FIG. ,Obtainable. From some biomaterials, hexose sugars can be obtained mainly, and from other biomaterials, pentose sugars can be obtained mainly. By appropriately selecting microorganisms capable of producing lipids, filtrates, precipitates or mixtures thereof mainly containing hexose sugars are used for the production of cell mass, and then filtrates, precipitates mainly containing pentose sugars Alternatively, a mixture of these can be used to produce lipids in the cell mass. Alternatively, lipids can be produced in cellular mass from hexoses. Cell mass and lipid can be produced from hexose. Correspondingly, by appropriate selection of microorganisms capable of producing lipids, filtrates, precipitates or mixtures thereof containing mainly pentose sugars can be used for the production of cell mass and then mainly containing hexose sugars. The filtrate, precipitate or mixture thereof can be used to produce lipids in the cell mass. Alternatively, lipids can be produced in the cell mass from pentose, or cell masses and lipids can be produced from pentose. Both cellular mass and lipids can be produced from a mixture of pentose and hexose.

In the following, the processing of biomaterials according to a preferred embodiment of the invention will be described. In general, the treatment is carried out by a combination of two or more treatment methods.

  Preferably, the raw material is extracted at a temperature of 90-100 ° C. In a preferred embodiment of the acid extraction, a mineral acid such as 5-10% sulfuric acid or an organic acid such as citric acid or acetic acid is used, preferably 0.5-2.0 M NaOH for alkaline extraction. Is used. The treatment time can be set widely, preferably 1 to 10 hours, generally 2 to 8 hours, most suitably 2 to 4 hours. Another treatment, such as treatment with enzymes, microorganisms, oxidizing agents or reducing agents, or a combination of these treatments, is preferably combined with water extraction.

  According to the method, the precipitate formed in the extraction is preferably mechanical at a temperature of 100-210 ° C., generally 150-200 ° C., preferably 2-20 minutes, generally 5-11 minutes. Can be crushed. Preferably, the pressure is 6-8 bar. The mass produced is filtered and the filtrate is processed using the methods described above to make it suitable for the production of single cell lipids. The precipitate can be acid treated with a strong acid, preferably 40-72% sulfuric acid, suitably 65-70% sulfuric acid. Usually, the treatment time is 2 to 8 hours, preferably 2 to 4 hours. The process can be performed with any acid that results in proton-catalyzed hydrolysis. Suitable acids are for example strong mineral acids, phosphoric acid, sulfuric acid or oxygen acids of sulfur, nitrogen, chlorine, bromine and iodine.

  The product of the hydrolysis is separated into a filtrate and a precipitate that are processed to make it suitable for the production of single cell lipids, which precipitate is preferably introduced into a dilute acidic solution such as 5-10% sulfuric acid solution, 170 It can be ground for 10-20 minutes at a temperature of ~ 200 ° C and a pressure of 6-10 bar. The mixture is separated into a filtrate and a precipitate, the filtrate is processed to suit the production of single cell lipids, and the precipitate can be removed.

  Embodiments of the foregoing method are directed to the overall use of carbohydrates present in the feedstock for the production of single cell lipids. However, when starting from a raw material extraction method, the method may be performed by selecting only a portion of the treated product, for example, when the fiber material in the precipitate is also used for another purpose. it can. The method according to the invention is preferably characterized in that it contains all the above-mentioned procedures, but is limited to the extent that it deviates from the basic method by carrying out one or more steps of the method according to the invention. Or the order of unit operations is not limited. Further, the method of the present invention is not limited to the use of the monosaccharide fraction produced by these operations in the production of single cell lipids. In addition to lipids, the process steps in which the fractions containing monosaccharides obtained from the raw material are used to produce single cell biomass or ethanol can also be associated with the method according to the invention.

  In the following description, several methods according to preferred embodiments of the present invention are disclosed. The method can be applied to raw materials other than the raw materials described in the explanation.

I.
According to a preferred embodiment of the present invention, xylem fibers, TMP pulp, sawdust or mechanical pulp containing ground xylem are treated by the following constituent steps:
A. 100 g of xylem fibers are extracted in 1 liter of water at 90-100 ° C. for 2-4 hours (preferably 2 hours). The precipitate is separated from the solution by filtration and the solution is recovered. The yield of carbohydrate in the solution is in the range of 2% to 5% depending on the method for producing xylem fibers, and is generally 4-5% for TMP fibers at higher processing temperatures (above 170 ° C.). It is.
B. In order to increase the yield of carbohydrates, the fiber fraction is obtained at a temperature of 90-100 ° C. in 1 liter of a solution of an acid (for example mineral acid) with a pH of about 1 and 5-10% (preferably 5%). For 2 to 4 hours (preferably 2 hours). The remaining precipitate is separated from the solution and the solution is recovered.
C. Excess acid is decanted from the precipitate and the precipitate is ground again in a steaming defibrator (eg, a wing or disc type pulverizer). Preferably, it is preferable to increase the temperature of the precipitate by pre-cooking for 1 minute to keep the condensate in the mixture. When using a blade type pulverizer, the temperature is increased to 150 to 200 ° C. (preferably 170 ° C.), and the pressure is set to 6 to 8 bar. The grinding time is selected from the range of 2 to 15 minutes depending on the type of wood. The precipitate is separated from the solution by filtration and the solution is recovered.
D. 40-72% strong acid (sulfuric acid) is added to the precipitate fraction and the strong acid is adsorbed to the fraction at room temperature for 2-4 hours (preferably 2 hours). Excess acid is decanted off and the precipitate is ground again in a steaming and defibrating machine (for example a wing or disc type pulverizer). It is preferred to increase the temperature of the precipitate by pre-cooking for 1 minute so that no condensate is discharged. When using a blade type pulverizer, the temperature is increased to 150 to 200 ° C. (preferably 170 ° C.), and the pressure is set to 6 to 8 bar. The grinding time is selected from a range of 2 to 15 minutes depending on the type of wood. The mixture is filtered and the precipitate is separated from the solution. The solution is collected and the precipitate is used as fuel.

  The solution fraction obtained from any of the constituent steps AD can be subjected to further processing using other means of promoting microbial growth, such as decolorization, pH adjustment, and water removal. By using the resulting solution in a microbial medium, the microorganism can produce lipids. By combining the constituent steps A to D, 40 to 65% of the xylem fiber raw material can be converted from the fiber used as the raw material into a solubilized product. Dissolved carbohydrates contain glucose units, galactose units, mannose units, xylose units and arabinose units, the mutual proportion of these units being typical values depending on the type of wood used.

II.
According to another preferred embodiment of the invention, 100 g of xylem fibers, ground wood, recycled fibers, TMP pulp, sawdust or mechanical pulp are used, these raw materials being 4-8% alkaline solution (preferably 1M NaOH solution) is extracted in 1 liter at a temperature of 90-100 ° C. for 2-4 hours (preferably 3 hours). The precipitate is filtered from the solution at room temperature and both fractions are collected. Depending on the treatment of the raw fiber, the yield of carbohydrate in the solution is in the range of 5-8%. Preferably, this solution is treated by any of the constituent steps described below:
A. By the above-described method, the vat solution is reused as it is in the subsequent fiber batch extraction step, and then the solution is recovered.
B. The lignin, oligosaccharide and polysaccharide dissolved in the solution are treated by hydrolyzing the vat solution with an acid according to any of the steps BD of the previous embodiments.
C. The vat solution recycled in step A is used according to step B of embodiment I.

In order to increase the yield of monosaccharides, preferably the precipitate recovered in the alkaline treatment is treated using any of the following methods or combinations thereof:
D. The precipitate is treated using any of Steps B, C, D of Embodiment I above, or all of these steps. The mixture is filtered, the solution is neutralized, and then both the precipitate and the solution are collected.
E. The remaining precipitate obtained from step A of embodiment II is retreated with alkali. In this case, preferably the mixture is reprocessed under conditions of grinding at a temperature of 170 ° C. and a pressure of 4-10 bar (preferably 8 bar) for 2-8 minutes (preferably 6 minutes). Filtration is performed and the solution is recovered after neutralization. With this additional grinding means, the amount of dissolved material can be increased to 27% of the initial amount of fiber in the raw material.

  The solution produced in each of the constituent steps A to E, which contains a carbohydrate containing a glucose unit, a galactose unit, a mannose unit, a xylose unit and an arabinose unit, is, for example, a decolorization method, a pH adjustment method, or By the method of removing water from the solution, it is prepared in a suitable state for lipid production by microorganisms. The solution is used as a culture medium or part of the culture medium for the microorganism to cause the microorganism to produce lipids.

III.
According to a third preferred embodiment of the present invention, xylem fibers, ground wood, TMP pulp, sawdust, mechanical pulp, recycled fibers (or neutralized extract prepared in step B above) are used, These raw materials are hydrolyzed with an acid having a concentration of 5-10% (preferably 5%). The hydrolysis is performed by adding 1 liter of the above-mentioned acid (for example, mineral acid) solution to 100 g of the material to be hydrolyzed, at a pH of about 1, and at a temperature of 90 to 100 ° C. for 2 to 4 hours (preferably 2 hours). The precipitate is separated from the solution by filtration. The solution contains 4-14% of the carbohydrate calculated from the fiber content of the raw material. The precipitate may be used as a fiber or further processed using steps C or D of the first embodiment described above, or both, to increase the yield of monosaccharides. Also good. The solution is separated from the precipitate, neutralized, filtered, and then allowed to produce lipids by using the microorganism as is or concentrated as a microorganism medium or part thereof.

IV.
According to a fourth preferred embodiment of the present invention, it is obtained from 100 g xylem fibers, ground wood, TMP pulp, sawdust, mechanical pulp, or steps A to C of embodiment I or step A of example II. Remaining precipitate is used. To these raw materials is added a solution of 5 to 10% acid (eg, mineral acid) having a pH of about 1, and the acid is absorbed for 2 to 4 hours (preferably 2 hours). Excess acid is removed by decantation, and the precipitate is ground again in a steaming defibrator (eg, a wing or disc type pulverizer). Preferably, the temperature of the precipitate is increased by pre-cooking for 1 minute so that the concentrate does not flow out. When using a blade type pulverizer, the temperature is increased to 150 to 200 ° C. (preferably 170 ° C.), and the pressure is set to 6 to 8 bar. The grinding time is selected from a range of 2 to 15 minutes depending on the type of wood. The precipitate is separated from the solution by filtration. The remaining precipitate can be used as a fuel or can be ground again by the procedure described above to increase the yield of monosaccharides in solution. The solution is neutralized, filtered, and then used as is or concentrated and is used by the methods described in embodiments I-III.

V.
According to a fifth preferred embodiment of the present invention, 100 g xylem fibers, ground wood, TMP pulp, sawdust, mechanical pulp, or a precipitate obtained by any of embodiments I-IV is used. A 40-72% acid (for example, sulfuric acid) solution is added to these ingredients and the acid is absorbed at room temperature for 2-4 hours (preferably 2 hours). Excess acid is removed by decantation, and the precipitate is ground again in a steaming defibrator (eg, a wing or disc type pulverizer). The temperature of the precipitate is raised by pre-cooking for 1 minute so that the concentrate does not flow out. When using a blade type pulverizer, the temperature is increased to 150 to 200 ° C. (preferably 170 ° C.), and the pressure is set to 6 to 8 bar. The grinding time is selected from the range of 2 to 15 minutes depending on the type of wood. The carbohydrate mixture is filtered and the precipitate is separated from the solution. The amount of lysate from the precipitate used as raw material ranges from 40 to 65%. The remaining precipitate can be used as a fuel or can be ground again to increase the yield of monosaccharides. The solution is neutralized, filtered, and then treated according to any embodiment I-IV.

  Alternatively, 40-72% concentrated sulfuric acid is absorbed into the precipitate at room temperature for 1-3 hours (preferably 1.5 hours). The acid is then diluted to 5% and the resulting mixture is boiled at 100 ° C. for 4 hours under atmospheric pressure. Further processing is as described above.

Production of lipids using microorganisms The present invention provides a microbiological method for carbohydrates, hexose monosaccharides and pentose monosaccharides contained in raw materials, obtained through a combination of filtrates produced in the various constituent steps described above. Can be used extensively for the production of lipids and single-cell biomass. Each recovered filtrate may be used directly to produce single-cell lipids after being subjected to pretreatment such as washing, neutralization, decolorization, or other post-treatment, or various aqueous fractions. May be used in combination. Due to the raw material processing method which is a part of the present invention, the present invention can also be applied to ethanol production.

  The microorganisms are made to produce lipids by adding the filtrate, ie the aqueous fraction, or any mixture of filtrates to the microorganism medium inoculated or inoculated with the microorganisms. The lipid is recovered in the form of a microbial mass, or the lipid is separated from the mass, and the lipid and the microbial mass separated from the lipid are recovered together. Lipids can be recovered using well-known methods of removing lipids from cells or disrupting cells. Lipids can be extracted from disrupted cells using organic solvents. Examples of lipid recovery methods applicable to the present invention include Z. Jacob, “Yeast Lipids: Extraction, Quality Analysis and Acceptability”, Critical Reviews in Biotechnology, Vol. 12 (5/6); pages 463-491 (1992). A preferred method for recovering lipids is the phase separation method. The treatment of converting lipids produced in microorganisms into fatty acid esters can be carried out without prior homogenization of microbial cells and subsequent fat separation.

  A preferred embodiment of the present invention relates to a method for producing lipids or lipid mixtures from a carbohydrate mixture produced in the processing of organic raw materials containing monomeric or oligomeric hexose sugars and pentose sugars. According to this method, the carbohydrate-containing mixture is added to an aqueous medium in which the lipid-producing microorganism is cultured, the medium is supplemented with nutrients necessary for the growth of the microorganism, and the medium is inoculated with the microorganism. The The microorganisms are cultured, the microorganisms produce lipids, and the cell mass is recovered. Lipids or lipid mixtures are separated from the cells or fat-containing cells, or these components are used as is.

  The method according to the present invention provides outstanding flexibility for microbiological lipid production. The fraction containing both hexose and pentose sugars is a natural carbon source for various lipid producing microorganisms. Therefore, this method also has an advantage that microorganisms selected from a wide range can be used based on, for example, lipid production capacity, biomass yield, culture method or culture conditions. Other components other than lipids produced by microorganisms can be used with high energy efficiency in a variety of different processes, thus improving the overall economic effect of the process according to the invention. A preferred method of using a lipid-free microbial mass is a method of hydrolyzing the mass and recycling it in the medium of a lipid-producing microorganism, or a method of use as a feed or nutrient. It is also possible to separate various components such as certain sugars, colorants, β-glucans, sterols, sterol esters or proteins from lipid-free microbial masses.

  The microorganism is selected from natural or genetically engineered fat-accumulating microorganisms, preferably yeasts, molds, bacteria and algae, more preferably yeasts, molds, most preferably yeasts. It is a prerequisite that the microorganisms used can produce lipids from hexose sugars or pentose sugars or both. Accordingly, the present invention encompasses all microorganisms that accumulate lipids based on ATP: citrate lyase activity (EC 2.3.3.8).

  Lipid synthesizing yeast genera that can be applied to the present invention include the following genera: Candida, Yarrowia, Lipomyces, Rhodotorula, or Cryptococcus. For this type of yeast, strains that synthesize xylose, a pentose sugar, and convert it into lipids, such as Candida curuvata (D) (by Evans, CT and Ratledge, 1983, “continuous culture”). And comparative study of oleaginous yeast "Candida cruvata" grown on different carbon sources in batch culture ", Lipids, Vol. 18, pp. 623-629), Rhodotorula gracilis (Yan , S., Rim, J., Choi, S., Ryu, D. and Lee, J., 1982, "Effects of carbon and nitrogen sources on lipid production in Rhodotorula gracilis", J. Ferment. Technol. 60, pp. 243-246), Rhodosporidium toruloides, Rhodotorula glutinis Rhodotorula graminis, Lipomyces starkeyi, Lipomyces lipofer, Candida lipolytica, Cryptococcus, Cryptococcus albidus Trichosporon pullulans (Fall, R., Phelps, P. and Spindler, D., 1984, “Biotransformation of xylan to triglycerides by oil-rich yeast”, Appl. Environ. Mircobiol. 47, pages 1130 to 1134) and strains that synthesize pentose sugar arabinose and convert it into lipids, such as Lipomyces starkeyi (Naganuma, T , Uzuka, Y. and Tanaka K., 1985. “Physiological factors affecting the total cell number and lipid content of the yeast“ Lipomyces starkei ”,” J. Gen. Appl. Microbiol., 31st. Volume, pages 29-37).

Similarly, the fat-accumulating molds applicable to the present invention include, among others, the following genera:
-Aspergillus
-Chaetomium
-Clodosporidium
-Cunninghamella
-Emericella
-Fusarium
-Mortierella
-Mucor
-Penicillium
-Pythium
-Rhizopus
-Trichoderma.

Similarly, the fat accumulating bacterial genera that can be applied to the present invention include, among others, the following genera:
-Acinetobacter
-Actinobacter
-Anabaena
-Arthrobacter
-Bacillus
-Clostridium
-Flexibacterium
-Micrococcus
-Mycobacterium
-Nocardia
-Nostoc
-Oscillatoria
-Pseudomonas
-Rhodococcus
-Rhodomicrobium
-Rhodopseudomonas
-Swanella
-Streptomyces
-Vibrio.

Similarly, fat accumulating meat microalgae applicable to the present invention include, among others:
-Botryococcus
-Brachiomonas
-Chlamydomonas
-Chlorella
-Crypthecodinium
-Dunaliella
-Euglena
-Nannochloris
-Nannochloropsis
-Navicula
-Nitzschia
-Schizochytrium
-Sceletonema
-Scenedesmus
-Tetraselmis
-Thraustochytrium
-Ulkenia.

  According to a preferred embodiment of the present invention, microorganisms that synthesize lipids containing fatty acids intracellularly, preferably in a proportion of 12 to 65% by weight of the dry weight of the cells, are used for lipid synthesis.

  According to a particularly preferred embodiment of the present invention, lipid-free biomass produced in the present invention by being processed in a manner suitable for microorganisms is used as a nutrient in the medium. In addition to these components, the medium can be supplemented with components preferred for the microorganisms used. In order to produce lipids, microorganisms generally include carbon sources (obtained from raw materials in the present invention), nitrogen sources, such as inorganic ammonium salts (eg, ammonium sulfate) or organic nitrogen sources (eg, amino nitrogen, yeast, among others). Extract or hydrolyzed cell mass) and micronutrients such as phosphate, sulfate, chloride, vitamin or cation source (eg Mg, K, Na, Ca, Fe or Cu ion source) etc. And Therefore, these components can be added to the medium as needed. When the method of the present invention is applied, the lipid concentration in the cells is preferably 40% by weight, more preferably 65% by weight.

Production of Biofuel The fatty acid ester contained in the microorganism-derived lipid can be processed to be suitable as a biodiesel fuel using any known method. One preferred method is a method in which a transesterification reaction is performed with a short-chain alcohol (preferably methanol) to obtain an alcohol ester of a fatty acid.

  An impure sidestream containing an alcohol compound, such as glycerol or a non-esterified fatty acid salt, which is difficult to use effectively in an energy efficient manner, and is a biodiesel fuel or renewable diesel fuel by transesterification The side stream produced in production can be used as is or reused to produce single cell lipids that are recycled into another glycerolipid-containing material of biological origin.

Advantages of the present invention The advantages of the present invention include that the equipment required for the method of the present invention is simple and that the techniques related to manufacturing and operation are known. The method according to the present invention is not limited to a specific production scale, and the scale can be easily increased or decreased according to the carbohydrate content and the amount of raw material to be processed. In order to carry out the method of the invention for producing lipids, no energy consuming heating, pressure unit operation, and no other chemical catalyst other than acid contact, alkaline catalyst or enzyme catalyst is required. The method only requires the use of chemicals incorporated into the internal cycle of the method according to the invention or the processing of biomaterials as described above. In the method of the present invention, since a diluted carbohydrate solution is suitable for use as it is in a microbial medium or as a microbial medium, it removes water from the usable sugar solution, resulting in an increase in cost. No processing is required. The overall economic effect of the method according to the invention is improved by the following facts. That is, the lipid-free biomass produced in the process can be used in many different applications in addition to its use in internal cycles, such as for producing individual organic components, for use as feed or feed ingredients, or It has use as a supplemental medium for producing microorganisms. The method is also suitable for the production of single cell biomass and ethanol.

  The advantages of the stepwise treatment of biomass according to the present invention are that the organic material can be more comprehensively hydrolyzed and the organic material is better used to produce single cell lipids compared to the prior art. It is. Furthermore, the filtrates or precipitates obtained from the acid treatment and alkali treatment are neutralized with each other, thereby reducing the amount of chemicals required for neutralization.

  Prior art solutions disclose a method comprising a microbial process in which a carbohydrate-containing substance is used as a raw material, either directly or converted to a monosaccharide, to produce ethanol. Patent application publication US 2002/0185447 and US Pat. No. 5,637,502 also disclose carbohydrate processing methods such as, for example, processing methods using acids or alkalis. After these treatments, alcohol fermentation is performed. With respect to microbiological processing, all of the above methods are limited to the production of ethanol using hexose sugars produced from polysaccharides. Patent application publication US 2003/0096385 discloses a method in which prenyl alcohol (granyl and farnesyl derivatives of alcohol) is produced using microorganisms. Patent application publication WO 03/038067 discloses a method for producing microorganisms that can utilize pentose sugars by modifying the genome of fungal microorganisms. The publication is only for the production of ethanol.

  The present invention provides a new solution for utilizing the relatively important aqueous side stream generated by thermomechanical wood processing, particularly in the pulp and paper industry, particularly as a feedstock for transportation fuels. The present invention reduces the side-stream biological burden including carbohydrates produced in connection with the production of groundwood pulp and the energy costs of wastewater treatment. In a specific embodiment, the present invention is for transport fuels, biodiesel fuels or renewable diesel fuels from, for example, a carbohydrate containing diluted aqueous fraction produced in a TMP process or corresponding mechanical processing of wood. The present invention relates to a lipid production method that provides an environmentally friendly solution for producing raw materials.

  The method also provides another solution for utilizing another raw material. According to the method, recycled fibers such as printing paper, packaging materials and similar materials based on cellulose can be used as raw materials.

  Therefore, compared to the prior art, the present invention is based on the principles of sustainable development by increasing the availability of lipid raw materials and reducing the total demand for organic lipids produced from other raw materials. Become. Thus, the present invention contributes to increasing the availability of raw materials for biofuels based on renewable natural resources and reducing their production costs to an extent acceptable to the end consumer.

For all uses of raw materials, the present invention is not limited to using all the carbohydrates contained in the raw materials. The method of the present invention also includes a step of recovering the lipid component of the extracted fraction in the recovered wood raw material. When the feed is treated with water, the filtrate separates the lipid components that can be recovered from the aqueous solution by methods well known to those skilled in the art. As a result of the acid treatment of the raw material, the lipids in the extraction fraction produce free fatty acids that are separated from the filtrate in the form of insoluble alkaline earth metal salts such as Ca ⁺⁺ salts. After separating the precipitate, the free fatty acid is transesterified to an alcohol ester. Correspondingly, fatty acid salts that are mixed in the filtrate because of water solubility are produced from the extracted fraction by alkaline extraction of the raw material. In the present invention, the salt is converted into the form of a water-insoluble salt (such as Ca ⁺⁺ salt), and the precipitate is separated from the aqueous phase and used to produce a fatty acid alcohol ester.

  The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, the present invention is not limited to the microorganism strain used. The present invention can be carried out using another strain of the same species or genus, a strain of another microbial genus or species, or a genetically engineered microbial strain, rather than using only the used strain. . Lipid-producing microorganisms are generally available, and this type of microorganism is available from multiple strain deposit agencies, such as ATCC, DSM, and the like. Lipid-producing microorganisms and methods for producing lipids using microorganisms (including algae) are described, for example, in the following publications: “Single Cell Oil”, edited by Z. Cohen and C. Latledge, AOCS Press, 2005, and “microbial lipids”, edited by C. Latledge and S. G Wilkinson, Volumes 1 and 2, Academic Press, 1988.

Example 1
Each 100 g of wood fiber, ground wood, TMP pulp, sawdust and mechanical pulp were stored in 1 liter of boiling water (90-100 ° C.) for 2 hours. The solution was separated from the fibrous material (hereinafter referred to as precipitate) by filtration. The precipitate was collected and treated according to one of the other methods described in Example 4 or 5 to increase the yield of monosaccharides. The carbohydrate yield of the solution ranged from 2% to 6% depending on the raw material. In addition, when the spruce fiber ground at high temperature (above 170 ° C.) is used as a raw material, the yield is generally 4%. A portion of the solution was reused without pre-evaporation to extract the next fiber batch, and a portion of the solution was evaporated and concentrated until a dry matter content of about 20% by weight was obtained. . The concentrated filtrate was collected to produce single cell lipids. Dissolved carbohydrate is characteristic of the tree species subjected to the extraction process, and the carbohydrate separated into hexose and pentose.

Example 2
Wood fiber, ground wood, regenerated fiber, TMP pulp, sawdust and mechanical pulp were each put into 1 liter of 5% alkaline solution (NaOH) and stirred at 90 ° C. to 100 ° C. for 3 hours. Filtration at room temperature and the precipitate was subjected to another treatment to produce monosaccharides according to Example 4. A portion of the extraction solution was reused when processing the next fiber batch to neutralize a portion of the solution and the lignin was dissolved with the oligosaccharide and polysaccharide in the alkaline extraction solution. Part of the solution was subjected to hydrolysis according to Example 4. The solutions obtained from different raw materials by the alkali treatment described above retained an average of 5% by weight of raw materials.

  Further, the precipitate obtained in the first alkali treatment was treated again with alkali by repeating the alkali treatment described above, and then pulverized for 6 minutes at a pressure of 8 bar and a temperature of 170 ° C. By this further grinding treatment, the amount of substance dissolved in the alkaline solution could be increased to an average of 27% of the initial amount of raw material. The solution turned black, and the solution was neutralized, hydrolyzed, and concentrated, and then treated with activated carbon and an ion exchanger for decolorization. The mixture was collected and used for the production of single cell lipids.

Example 3
Each 100 g xylem fiber, ground wood, TMP pulp or MDF pulp, sawdust, mechanical pulp, regenerated fiber, and neutralized extract prepared according to Example 2, 5% mineral at pH 1 Hydrolysis was carried out at 90-100 ° C. for 3 hours in 1 liter of acid solution. The precipitate was separated from the solution by filtration. The solution contained an average of 10% monosaccharides relative to the raw material. Some solutions were neutralized and concentrated by evaporation until about 20% by weight monosaccharide was obtained after filtration, and the concentrated solution was collected to produce single cell lipids. A portion of the solution was reused as is to hydrolyze subsequent batches of raw materials described in this example. Further processing of the precipitate according to Example 5 below increased the monosaccharide yield.

Example 4
125 g of xylem fibers, ground wood, TMP pulp, MDF pulp, sawdust, mechanical pulp, straw, cereal shells and precipitates according to Examples 1 to 3 in 10% strength sulfuric acid solution The acid was charged and absorbed for 2 hours. Excess acid was removed by decantation, the temperature was raised by introducing the mixture into a wing-type pulverizer and pre-cooking for 11 minutes so that the concentrate did not escape. Thereafter, the temperature was set to 160 ° C., the pressure was set to 8 bar, and a pulverization process using a blade type pulverizer was performed. For the spruce fiber, the mixture was subjected to a grinding process for 11 minutes. After grinding, the precipitate was separated from the solution. The mixture was divided, some of the precipitate was used for ashing, and some of the precipitate was subjected to grinding again to increase the yield of monosaccharides according to Example 5 below. Correspondingly, the solution was neutralized and, after filtration, concentrated by evaporation until a monosaccharide concentration of about 20% was achieved. These solutions were collected to produce single cell lipids.

Example 5
125 g of wood fiber, ground wood, TMP pulp, sawdust, mechanical pulp, straw, cereal fiber and residual precipitates from Examples 1 to 4 were charged into a 40% sulfuric acid solution and the acid Was absorbed at room temperature for 2 hours. Excess acid was removed by decantation, and the precipitate was pulverized with a wing-type steaming defibrator so that the temperature of the precipitate increased by pre-cooking for 11 minutes so that the concentrate did not flow out. The temperature was then raised to 170 ° C. and the pressure was 8 bar. The most suitable grinding time for straw and grain fibers was 11 minutes. The precipitate was separated from the solution. The percentage of dissolved material on average was greater than 50% of the raw material. The solution was neutralized, filtered and then concentrated by evaporation and collected to produce single cell lipids. The yield of monosaccharides was increased by ashing a portion of the precipitate (ie, residual fiber) and pulverizing a portion again according to this example.

  Moreover, high concentration sulfuric acid (72%) was absorbed in the above-mentioned raw material alone (treatment time at room temperature: 2 hours). Subsequently, the acid was diluted to 5% and boiled at normal pressure and 100 ° C. for 4 hours. Other treatments and use of the generated fractions were performed as described above.

Example 6
Monosaccharides suitable for the production of single cell lipids may be produced by directly hydrolyzing rice husk, straw and cereal husks using 5% acid, or impregnated with stronger acid to give 5% It may be produced by hydrolysis in a solution of the above, or may be produced by treatment with an alkali in advance and hydrolysis in a solution of 5%. It may be hydrolyzed. These treatments may be performed in combination with an acid or alkali impregnation treatment, and then repeatedly pulverized by a blade type or disc type pulverizer to produce a thermomechanical pulp. However, in this example, rice husk, straw and cereal husk (125 g each) were pre-cooked for 11 minutes at a pressure of 8 bar and processed at a temperature of 170 ° C. using a wing-type pulverizer to produce thermomechanical pulp. It was. The resulting thermomechanical pulp was hydrolyzed in 1 liter of 5% acid (sulfuric acid) solution (90-100 ° C., 4 hours at normal pressure). The solution fraction was separated by filtration and neutralized. Subsequently, the solution was concentrated and collected to produce single cell lipids. The yield of monosaccharides was 50% of the raw material on average. In order to increase the yield of carbohydrates, the precipitate remaining after filtration was thermomechanically reprocessed according to Example 5 and partly subjected to ashing.

Example 7
Rice husk, straw and cereal husk (16 kg each) were each treated in 90 liters of alkaline solution (1.2 M NaOH) at 90-100 ° C. for 4 hours. The mixture was separated into a precipitate and a solution. On average 49-57% of the feed dry matter remained in solution. The alkaline solution was made into a 5% acidic solution using sulfuric acid, and then hydrolyzed in the same manner as in Example 6. A mixture of xylose and arabinose, which also contains a small amount of glucose and galactose, mainly formed. The monosaccharide content of the solution was 23-33% of the feedstock. The color of the solution was thinned by the activated carbon treatment that was applied before collecting the solution to produce single cell lipids.

  The precipitate obtained by filtration was impregnated in a 5% acid (sulfuric acid) solution and pulverized in a blade type pulverizer at a pressure of 6 bar and a temperature of 150 ° C. for 11 minutes. After neutralization and filtration, the precipitate recovered to produce single cell lipids was broken down into water soluble carbohydrates (mainly glucose and galactose). A portion of the precipitate remaining after filtration performed after acid hydrolysis was ground again and then hydrolyzed with a 10% acid solution according to Example 4. After these treatments, a solution containing a total of 55 to 65% monosaccharides with respect to the raw material was obtained. The solution was collected to produce single cell lipids.

Example 8
Steam-dried beet pulp was added to the tub, and a sufficient amount of boiling water was poured over the tub to cover the beet pulp. The solution was allowed to cool before separating the water from the solid by decantation. 3.6 mg / mol dry substance was dissolved in water. A portion of the aqueous fraction was used for new batch processing, and after concentrating some of the aqueous fraction, it was collected to produce single cell lipids. The resulting solid fraction (precipitate) weighed 748 grams and contained 16.7% dry matter. The corresponding 125 g of dried beet pulp as the dry substance was impregnated with 0.4 M phosphoric acid solution (H ₃ PO ₄ , 500 ml) at room temperature for 18 hours. Excess acid solution was removed by decantation and pre-cooked for 11 minutes before starting thermomechanical grinding. The mixture was ground for 11 minutes using a wing-type refiner. During the grinding, the pressure was maintained at 8 bar, the initial temperature was 172 ° C., and the temperature was 162 ° C. when the grinding time was 10 minutes. The mixture was removed from the beater and then filtered. The solution fraction was 2.26 liters and contained 2.75% dry matter. 50% of the dry substance that was subjected to the grinding treatment was present in the solution after filtration. The solution mainly contained monosaccharides, glucose, galactose, arabinose and xylose. Further, a small amount of oligosaccharide having a molecular weight in the range of 500 to 3000 and a compound having a higher molecular weight were observed. The solution fraction was neutralized and concentrated and collected to produce single cell lipids.

  Solid fiber pulp [precipitate (125 g dry matter)] obtained from the above treatment of beet pulp was impregnated in 15% hydrochloric acid at room temperature for 4 hours. Excess acid solution was removed by decantation and the pulp was ground again under the same conditions. After the filtration step, it was observed that an additional 28% monosaccharide was incorporated into the solution layer. The precipitate was discarded.

Example 9
The raw material was spruce fiber that was thermomechanically processed and weighed 46.8 g (volume of about 1 liter), with 0.2N NaOH added to the top of the raw material, to which 5.6 g Na ₂ was added. CO ₃ and 1.3 g MgSO ₄ 6H ₂ O (500 ml) were added. The mixture was heated to 50 ° C. to produce a small amount of glucose, xylose, galactose, arabinose, mannose, and oligomers in the solution. The pH of the solution was about 11, and the solution was filtered and washed with water. The washing liquid and the filtrate were mixed and the solution was evaporated to 320 ml. The amount of dissolved substance was 4.5% (dry substance) of the raw material. In order to increase the amount of monosaccharide, acid was added to the solution to form a precipitate. The precipitate was separated from the solution and the latter was collected and used to produce single cell lipids. The precipitate was hydrolyzed to monosaccharides with the 5% acid described in Example 3. By using hydrolysis, 1% monosaccharides were produced from the precipitate mass, the monosaccharides were introduced into the solution and recovered after the decolorization treatment to produce single cell lipids.

Example 10
A 45 g batch (about 1 liter) of thermomechanically treated spruce fiber is weighed and a 0.2 N NaOH solution is added to the top of the fiber, to which another 5.6 g Na ₂ CO ₃ and 1 0.3 g MgSO ₄ 6H ₂ O (500 ml) was added. After the mixture was heated to 50 ° C. and filtered, the precipitate was washed with water. The washing solution and the filtration solution were mixed. In order to increase the amount of monosaccharide, acid was added to the mixed filtrate and hydrolysis was carried out according to Example 3. After neutralization, the hydrolysis product was recovered to produce single cell lipids. The washed precipitate was concentrated to 300 ml, 1 liter of citrate buffer (pH 4.8) was added, and cellulase enzyme was added. Oligomer and glucose were produced in the mixture. The enzyme treated mixture was collected as is and as a separated filtrate (solution) and used for the production of single cell lipids.

Example 11
A batch of 125 g of oat husk was prepared and the batch was thermomechanically ground at 170 ° C. and a pressure of 8 bar for 2 hours. After the treatment, the precipitate was separated from the solution by filtration. The solution was collected for single cell lipid use for production. A 7 g precipitate (fibrous) batch was used and the batch was added to 19% strength sulfuric acid and refluxed for 4 hours. As a result of analyzing the treated product, monosaccharides were present in the solution at a ratio of 56% of the raw material, and most were xylose, mannose, glucose, galactose and arabinose. The solution was recovered for use in single cell lipid production.

Example 12
400 g of oat chaff was weighed and 3 liters of water and 200 g NaOH were added. The mixture was maintained with stirring at a temperature of 90-96 ° C. for 2 hours. The mixture was filtered through a cloth to separate the fibers. The filtrate fraction (50 ml) was neutralized and the pH was adjusted to 4.8 with citrate buffer (40 ml). Multifect xylanase (10 ml) was then added and the mixture was maintained under isothermal conditions at 50 ° C. Sugar was released into the solution, of which 25.2% was xylose and 11.8% was arabinose. In addition, oligomers were also present in the solution after 50 hours of reaction time. All mixtures were recovered for use in the production of single cell lipids.

Example 13
125 g of beet pulp was impregnated with 0.4 M sulfuric acid, and after 12 hours, excess acid was removed by decantation. The pulp was put into a wing-type pulverizer, pre-cooked for 4 minutes in the wing-type pulverizer, and pulverized for 11 minutes at 150 ° C. and a pressure of 6 bar. The mixture was neutralized and citric acid was added until the pH value was 4. 10 ml of pectinase 4450U (ie 178 mg / ml protein (Sigma)) was added and the reaction was continued at 25 ° C. for 24 hours. After the reaction, a part of the mixture was recovered for production of single cell lipids, and a part of the mixture was filtered. The precipitate obtained by filtration was returned to the grinding stage and the solution fraction resulting from this treatment was treated with activated carbon (2 g / l) and then introduced into an anion exchange column. The monosaccharide solution obtained from the column was evaporated to a dry substance ratio of 20% and collected to produce single cell lipids.

Example 14
The oat chaff is treated according to Example 7 and contains 23.8 g / L glucose, 93.3 g / L xylose, 37.1 g / L arabinose, and 9.0 g / L galactose. To produce a mixture. This mixture was added as a medium for lipid synthesis yeast. The lipid synthesis yeasts Yarrowia lipolytica ATCC 20373 and Rhodotorula glutinis TKK3031 were diluted 1: 1 as they were, and 11 g / L glucose was supplemented to the corresponding dilution. The culture time was 68 hours, the mixture was shaken at a temperature of 28 ° C. and 250 rpm, and the volume of the culture solution was 50 ml. As shown in FIG. 3, it was found that the yeast can grow and synthesize lipids without the need for other nutrients.

Example 15
Three yeasts, Rhodotorula glutinis, Yarrowia lipolytica and Kluyveromyces marxianus (Anam. Candida kefyl) ATCC 42265, exclusively with xylose as carbon source Cultured. The medium contains 20 g / L xylose, 10 g / L yeast extract, and 20 g / L peptone. The culture was carried out by shaking at a volume of 50 ml of culture solution, a temperature of 25 ° C. and 200 rpm. FIG. 4 shows that all three strains can use pentose as a carbon source.

Claims (19)

Method for producing lipids or lipid mixtures from cellulose, hemicellulose, starch, a mixture of all of these or any mixture thereof or organic raw materials containing polysaccharides selected from the group comprising degradation products and non-starch polysaccharides Because
The method comprising the following steps a) to c):
a) The organic raw material
i) water,
ii) acids and
iii) treating with a substance selected from alkalis;
After the treatment, the precipitate and the filtrate are separated,
The precipitate obtained from the treatment is then either neat or mechanically or thermomechanically ground in the presence of water, acid or alkali and then the precipitate and filtrate are separated, or Subjecting the precipitate to one or more treatments with any of i) water, ii) acid or iii) alkali and / or grinding in the presence of the substance;
b) contacting the lipid-producing microorganism in the medium with one or more filtrates obtained in the above step, the precipitate, or the precipitate, or any mixture thereof and optionally the organic raw material, Let the cells of the microorganism start producing lipids, then c) recover the lipids. The process according to claim 1, characterized in that the treatment of the precipitate is also carried out using a process comprising one or more of the following steps d) to g) as required:
d) after treating the precipitate obtained from step a) with a strong acid and then separating the precipitate from the filtrate, or
e) after acidifying the precipitate obtained from step a) or d) and mechanically or thermomechanically grinding the acidified product, separating the precipitate from the filtrate or, if necessary,
Treatment of the precipitate obtained from any of steps a), d) or e) once or more times using the method according to any of steps a), d) or e) in any order. Do it,
f) In the medium, the lipid-producing microorganisms are filtered or precipitated from steps a), d) or e), or any mixture of filtrates and precipitates obtained from these steps, and if necessary By contacting the corresponding organic raw material, the cells of the microorganism are started to produce lipid, and then g) the lipid is recovered.   3. The method according to claim 1 or 2, wherein the raw material originates from a material obtained by mechanically or thermomechanically treating wood or a cultivated plant.   The method according to claim 1, wherein the raw material is treated with water or an acid.   The raw material originates from a raw material selected from the group comprising recycled fiber, sugar beet pulp, rice husk, straw, bran, grain, all culturable crops, cultivated plants, TMP pulp and MDF pulp The method according to claim 1 or 2.   6. Process according to claim 1, 2 or 5, characterized in that the raw material is treated with an acid.   The raw material originates from a raw material selected from the group comprising sawdust, refined mechanical pulp, rice husk, straw, TMP pulp, MDF pulp, sugar beet pulp and cultivated plants substantially free of starch Item 3. The method according to Item 1 or 2.   The method according to claim 1, 2 or 7, wherein the raw material is treated with an alkali.   The feedstock originates from a feedstock selected from the group comprising microbial mass, swamp or underwater biomass, biomass from a cellulose factory catchment, biomass from municipal waste and biomass from municipal sewage The method according to claim 1 or 2.   10. The filtrate according to claim 1, wherein the filtrate contains at least 0.5 to 1% by weight, 20 to 30% by weight, preferably 4 to 5% by weight, of sugars available in the production of lipids. The method described in 1.   The method according to claim 1 or 2, wherein the process in any one of steps a), d) or e) is performed once or a plurality of times.   The process according to claim 1, characterized in that when the raw material is treated with the alkali iii) of step a), the resulting precipitate is retreated with acid and then the precipitate and filtrate are separated. .   3. A process according to claim 1 or 2, characterized in that when the fiber-containing precipitate is treated with a strong acid, the resulting precipitate is ground again mechanically or thermomechanically.   The method according to claim 1, wherein one or more enzymes are added to the biomaterial treatment solution.   Using a method wherein the filtrate obtained from any step leads the filtrate to a more suitable state for microbial growth, such as decolorization, pH adjustment and / or water removal or addition, The method according to claim 1, wherein the method is processed.   16. The raw material is pretreated by mechanical treatment, thermomechanical treatment, physical treatment, chemical treatment, biological treatment, or any combination thereof. The method described in 1.   Use of a lipid or lipid mixture produced using a method according to any of claims 1 to 16 as a raw material for the production of biofuels.   A biofuel produced using a lipid produced using the method of any of claims 1-16.   A method for purifying urban sewage, wherein the city sewage is treated using the method according to any one of claims 1 to 16.

Images