KR20100091217A - Method for producing lipid - Google Patents

Abstract

The present invention relates to a method for forming a lipid or lipid mixture from an organic source material comprising cellulose, hemicellulose, starch, non-starch polysaccharides, any mixtures thereof or degradation products thereof. According to the method, the source material is treated one or more times with an aqueous solution of water, acid or alkali and separated into precipitates and filtrates. The precipitate obtained from the treatment described above may be subjected to mechanical or thermo-mechanical grinding, or the precipitate may be treated with a strong acid, or the precipitate may be acidified and mechanically or thermo-mechanically ground. After this treatment, it is separated into a precipitate and a filtrate. Lipid-producing microorganisms are contacted in the culture medium with the source material or the filtrate obtained, and the microbial cells begin to produce lipids, which lipids are recovered.

Description

Lipid Production Method {METHOD FOR PRODUCING LIPID}

The present invention relates to a process for producing a lipid or lipid mixture from organic raw materials according to the preamble of claim 1. The invention also relates to a biofuel according to claim 18, including the use of a lipid or lipid mixture produced by the process as a biofuel according to claim 17. The invention also relates to a municipal sewage purification method according to claim 19.

It is generally known that the use of traffic fuels made from fossil raw materials is extremely large and consumption continues to increase. Thus, aspects of sustainable development, environmental impacts and justifications for fossil energy resources have emerged as a legitimate global task. In this reference frame, renewable alternative raw materials for traffic fuels are emerging as a growing concern.

The first step in fuel production based on renewable natural resources is to attempt to replace, at least in part, fossil raw materials with organic raw materials. Like this approach, one skilled in the art can imagine a problem that is very difficult to solve. Although only part of the fossil raw material is replaced, the amount of organic raw material required is quite large, in proportion to the current consumption of the fossil raw material. In some respects, it has already been observed that unilateral and large-scale consumption of organic natural resources or areas cleared under cultivation for this purpose will cause intractable consequences in the balance of natural biodiversity and primary production of food. . The conversion of organic materials into forms that can be used for the production of traffic fuels in an energy-efficient manner also presents technical challenges.

Particularly advantageous source sources for traffic fuels will be organic fats, in particular triacyl glycerol, since their energy content is considerably higher than, for example, the corresponding carbohydrate or alcohol content. In addition, they can be converted into components of traffic fuels such as diesel fuel, biodiesel or renewable diesel through the most well known and relatively effective chemical processes. However, the lack of reserves of natural fat raw materials presents a suggested factor. At best, on the basis of current local resources, only minor industrial production of biofuels is feasible. Thus, the increase in fat reserve requires a very significant increase in the cultivation of fatty plants. Such a big change in the field of cultivation and production of local plants, in turn, has a strong impact on the balance of the food economy of the world market. This necessity, still at the speculative level, is already manifesting itself as a large increase in the price of raw materials for food and forage.

The total amount of naturally renewable organic mass is very large; Calculated as the amount of carbon, this is significantly greater than the annual usage of fossil carbon as a transportation fuel. However, the main part of this renewable mass, about 60%, consists of a material that still contains a significant amount of oxygen, so the value of this fuel is quite low.

Based on the known art, the use of relatively low energy carbohydrates as high-energy compounds comprising further reduced hydrocarbon chains is already theoretically possible, in particular in known chemical applications such as gasification techniques, extreme It is known to exist as an energy-intensive method of (R. Agrawal, NR Singh, FH Ribeiro and WN Delgass 2007. "Sustainable fuel for the transportation sector", PNAS 104: 4828-4833, and WO 2006/117317) ), Where the total efficiency between supply and production is still low. The corresponding underlying problem also relates to biological methods according to the known art, which are used to convert hexose sugars in carbohydrates into higher-energy compounds. One example thereof is the production of alcohols, in particular ethanol, as described in the specifications of US 2002/0185447, US 5637502, and WO 03/038067, among others.

Patent specification US 2004/0231661 also describes the treatment of materials comprising lignocellulosic by water and acidic extracts and by hydrolysis, whereby xylose and glucose are formed, which are used for the production of ethanol. Can be. Patent specification US 5,221,357 describes the treatment of materials comprising cellulose and hemicellulose by acidic hydrolysis, and the treatment by acidic hydrolysis to produce monosaccharides such as pentose and hexose, as well as mechanical treatment of solid phases, It can be used to prepare ethanol. Patent specification US 4,752,579 describes the treatment of corn seed husks for separating monosaccharides by acid and / or alkali and enzymatic hydrolysis.

Some specifications describe the production of lipids by microbial fermentation from different organic materials. The patent specification US 4,368,056 proposes the use of microbial glycerides, which are produced during fermentation during the production of biodiesel and the use of carbohydrates present in low content in industrial wastes, butanol fermentation. Dai et al., Dai, C. Tao, J., Xie, F., Dai, Y. and Zhao, M. Biodiesel generation from oleagenous yeast Rhodotorula glutinis with xylose assimiating capacity. Afr. J. Biotechnol. VoI 6 (18), pp. 2130-2134, 19 September 2007] Rhodotorula As raw materials for the production of lipids by yeast, straw of plant material such as corn stalks, leaves and rice, obtained by the above treatment, by grinding and acid treatment, and by use of filtrate and washing with water. The extraction of carbohydrates present in the present invention is described. Lipids obtained from microorganisms are used for the production of biodiesel. Angelbauer et al., Angerbauser, C, Sierbenhofer, M. Mittelbach, M. and Guebtz, GM Conversion of sludge into lipids by Lipomyces starkeyi for biodiesel production. Bioresource Technology 99 (2008) 3051-3056, in turn, describes its use in lipid production by Lipomyces yeast in biodiesel raw materials, including treatment of wastewater sludge with alkalis and acids.

Problems encountered with the above-mentioned methods include the fact that the yield of sugars that can be used by microorganisms is still low or that the method is applied to the use of biomaterials as raw materials of low importance to the need for large-scale production. It is evident that the quantitative goals set for the production of biofuels cannot be achieved by the methods described above and the source materials used.

Thus, there is still a great need for new technical solutions, in particular solutions that can be used more comprehensively than before, to convert the world's renewable organic carbohydrate reserves into compounds with high energy content, regardless of their chemical or structural composition. exist. It will be particularly important to address how to effectively convert carbohydrates present in various organic materials into high energy content compounds such as fats, which are more transformable into the use of transportation fuels or raw materials for their use.

It is an object of the present invention to provide a new solution to the problem of the method of converting organic biomaterials into compounds with high energy content.

In particular, it is an object of the present invention to provide a solution to the problem of the method of converting carbohydrate components obtainable from organic biomaterials into lipids suitable for the production of biodiesel.

More precisely, the method according to the invention is characterized in that which is specified in the characterized part of claim 1.

In turn, the use according to the invention is characterized by the one described in claim 17 and the biofuel described in claim 18.

The municipal sewage purification method according to the present invention is characterized by what is stated in claim 19.

The present invention is based on the observation that when dealing with biomass in different ways for the recovery of cellulose and hemicellulose fractions, microbial populations appeared more quickly in the fraction, and in addition, the cellulose and hemicellulose fractions were degraded. Surprisingly, in the carbohydrate fraction described above, it has been found that the microorganism is also growing, with ATP citrate lyase enzyme (EC 2.3.3.8, previously EC 4.1.3.8), which microorganism is Lipids, in particular triacyl glycerol, were collected into their cells. Based on this observation, the present invention treats a biomaterial to separate cellulose and hemicellulose contained in the biomaterial from the rest of the biomaterial, and hydrolyzes the cellulose so that the hydrolysis product is suitable for the growth of lipid-collecting microorganisms. And a method for producing a lipid, and a method for using the lipid formed by the method as a raw material in the production of biodiesel.

In accordance with the description of the present invention, carbohydrates suitable for use by lipid-synthesizing microorganisms may be produced from organic sources derived from various sources and / or carbohydrate fractions may be produced from lipids that may be produced by microorganisms.

According to the invention, such carbohydrates can in particular be produced from biomaterials comprising hemicellulose, cellulose, starch or non-starch polysaccharides. Carbohydrates suitable for use by microorganisms are in particular monosaccharides and oligosaccharides comprising both hexose and pentose sugars. If the lipid-producing microorganism has been chosen to enable the use of carbohydrate polymers, the carbohydrates may also be in polymer form.

Among the various forms, wood contains the largest amount of renewable biomass that can be recovered at present. The use of wood, in particular its mechanical or thermo-mechanical treatment or other manufacture or production of mechanical mass from wood, is large-scale and, as a process, produces many carbohydrate-containing minor flows. These side cuts in the wood-refining industry have been found to have very low economic added value; In many cases, the costs are incurred, such as the environmental burden caused by their removal. As a technical problem, such side flow is particularly problematic. Side flows are bulky, but their carbohydrate content is generally low. Like diluted water-based carbohydrate solutions, this is not suitable for processes aimed at utilizing carbohydrates in solution by chemical methods.

Accordingly, the object of the present invention is also a method using a large scale industrial side flow comprising a biomaterial, which currently requires an expensive purification procedure or remains unutilized by the current process but still has the potential as an energy source. To provide a solution to the problem.

In the process according to the invention, cellulose, hemicellulose, starch, both, mixtures of some or their products, or, alternatively, starch or non-starch linked to the starch or non-starch carbohydrates themselves or to cellulose or hemicellulose materials. Organic source materials, including carbohydrates, are processed. The source material may be pre-treated mechanically, thermo-mechanically, physically, chemically, biologically, or by a combination of these treatments, or may be suitable for use by itself. When carbohydrates in polymer form are included, they are preferably treated with water or an acid or an alkali, or a combination thereof. After such preliminary treatment in accordance with the present disclosure, the mixture is divided into filtrate and solids, ie precipitates (FIG. 1), and the filtrate or two fractions are recovered. It is preferable to resume treatment of the source material, which is carried out with water or acid or alkali, or a combination thereof, and to bind the filtrates together after the separation of the precipitates. The filtrate obtained by the treatment comprising alkali is passed on its own or after the aforementioned recycling, preferably into a mixture, and acid treatment of the source material is carried out to increase the content of soluble monosaccharides. Any precipitate or filtrate or source material itself produced in this treatment, or a combination of the source material and the filtrate or precipitate or filtrate or precipitate is single-cell after possible pre-treatment, such as neutralization, bleaching and filtration. It is used to produce lipids. The filtrate can also be combined, diluted or concentrated to achieve a composition for microorganisms that produces a suitable monosaccharide content and single-cell lipids.

From each alternative treatment of the source material, precipitates of various compositions are produced, depending on whether the treatment is treated in water or in the presence of acids or alkalis. In order to increase the production of sugar, the precipitate is preferably transferred by mechanical grinding, either by itself or in the presence of water, acid or alkali, from which the filtrate and precipitate are obtained again. Filtrates or precipitates or combinations thereof are used to produce single-cell lipids, or the precipitates are preferably treated with strong acid if desired. As a result of the strong acid treatment, the filtrate and precipitate are produced again from which the filtrate or precipitate or combination thereof can be transferred to the production of single-cell lipids. The precipitate can be treated with even stronger acid concentrations by combining the treatment and grinding. Filtrates or precipitates or combinations thereof, produced as a result of this treatment, can be used for the production of single-cell lipids. Precipitates can be removed, burned and used in the production of biofuels or their precursors by other methods. Each filtrate fraction or precipitate or source material can be used on its own or in various combinations to produce single-cell lipids. Precipitates obtained from the process steps described above may be retreated by the earliest or later process steps set forth herein. In order to increase the production of sugars, it is desirable to treat the precipitates with stronger acids or alkalis than in previous treatments.

In addition, enzyme treatment or fermentation of microorganisms can be carried out between the different process steps.

The invention also relates to a process for recycling the used alkali and acid again during the process.

The invention also relates to a process for recycling the single-cell biomass used in the process as the biomaterial intended by the invention when the produced lipid is recovered.

Economically available components can be recovered from the precipitate obtained from the treatment carried out according to the method. In turn, the precipitate can also be used in other microbial processes than in the production of lipids.

The method according to the present disclosure provides a solution to the problem of how the hexose and pentose monosaccharides contained in the carbohydrates of the biomass can be gradually delivered into smaller fractions containing higher amounts of monomer sugar units. The microorganisms that synthesize can utilize any fraction more effectively for lipid production. In connection with any process, insoluble matter, precipitates are separated from solubles, filtrates are produced accordingly, and the hexose and pentose sugars contained in the filtrate are either single-celled on their own or after the necessary pre-treatment. It can be used to produce single-cell lipids as a mixture of filtrates for the production of single-cell lipids. In addition to the filtrate, a precipitate or a combination of filtrate and precipitate can also be used for the production of single-cell lipids.

The overall advantages of the present invention can be used to apply to simple processes and unit operations, which are low energy, such as mixtures of hexose and pentose monosaccharides or oligomers formed thereby in industrial applications, energy efficient and environmental methods. It is ready to produce energy-rich chemical compounds, lipids, from compounds of biological origin containing.

In the process according to the invention, it is particularly preferred that the new surface is always exposed in the organic material by mechanical or thermo-mechanical grinding, which in turn can be treated with water or with acids or alkalis of different strengths, and thus solutions And sediment decomposition can be provided, from which more sugars can be obtained which can be used in the production of microbial lipids, especially with the solution.

When several separations of solution and sediment are combined, compared to single-phase hydrolysis methods, significantly improved output of hexose and pentose sugars is obtained for mechanical or thermo-mechanical grinding.

In the process according to the invention, the flows obtained from the various treatments neutralize each other, by combining with the filtrate obtained from the acid and alkali treatments, or by combining with the filtrate and the precipitate. Thus, the filtrate or precipitate or combinations thereof are themselves available without adjustment of acidity, or at least less need for adjustment of acidity. The possibility of binding each other of biomass from several different biomass and from different origins is also advantageous.

The necessity of the present invention is particularly emphasized in that the formed sugars available for lipid production, by themselves or in part, can be used naturally and microbiologically to produce other compounds such as alcohols.

A particular advantage of the present invention is that in addition to the use of side flows or main flows of mechanical and thermo-mechanical treatment of wood, it is also suitable for the use of other biomaterials that release carbohydrates to produce lipids.

The invention further relates to a process for forming a lipid mixture or lipid from a mixture according to the invention, produced by recycling the fibers once again, produced by thermo-mechanical treatment, alkali or acid treatment.

According to the present disclosure, other organic raw materials that are difficult to use on a large scale, which can be processed into lipids by microorganisms, either with or from oligosaccharides and pentose sugars, are for example recycled newspapers. Recycled fibers obtained from, beet pulp obtained from sugar beet, and straw (eg oats) of rice hulls and grains; Other similar devaluation parts of field-cultivated plants, sawdust, refined mechanical pulp, straw and peat, especially slightly cracked peat. Other organic materials that have never been used so far include biomass in swamps or underwater, biomass in the catchment area of cellulose plants, and biomass to municipally operated sludge plants, or other to waste or incineration. It is an organic municipal waste. This organic material can also be treated by replacing various embodiments of the invention such that the carbohydrates contained in the material become useful for the microorganism to produce single-cell biomass and lipids. For example, municipal waste can be treated by the process of the present invention, and among other advantages, the killing of harmful microorganisms in the treatment will be beneficial.

The essential point of the present invention is that carbohydrate-containing biomaterials, regardless of which organic material they are derived from, are treated such that monomer hexose and / or pentose sugars or oligomers suitable for single-cell lipid production are obtained from the carbohydrates. Is the point. It is preferable that the treatment by a combination of two or more treatments is performed.

The use of the method according to the invention is suitable in view of the need for large-scale production of biofuels, where the source material can be produced simultaneously from several different biomass, which source material is a cellmass of lipid-producing microorganisms. And production of lipids. Components suitable for the lipid production of microorganisms can be produced regardless of the composition, utility and structure of the biomass. A significant advantage of the present invention is also that, by the process according to the invention, the available sugars can be produced in high yields and the consumption of the chemical need for control of acidity can be reduced.

Compared with the prior art, the invention described herein provides a breakthrough technology that combines the conversion of biomaterials comprising both cellulose and hemicellulose into available hexose and pentose sugars. In the same embodiment, the present invention also makes it possible to use non-starch polysaccharides and monosaccharide units of starch comprised in this biomaterial as sugars available for the production of single-cell lipids. The invention is particularly feasible so that it can be applied on an industrial scale to materials produced by industry or the community and derived from renewable natural resources or their devalued side flows. The method according to the invention can be used to treat materials containing cellulose and hemicellulose in a controlled manner, from which precursors of single-cell lipids can be formed, which are caused by microorganisms that are stable to the production of single-cell lipids. Can be used.

In the following, the invention is explained in more detail by the accompanying drawings and detailed description.

1 shows the main steps of carrying out a method according to the invention.
2 shows the use by itself or in combination of hexose and pentose sugars for the production of cell masses and lipids.
FIG. 3 shows the production of lipids and the growth of yeast in the culture medium, where a mixture is added as a carbon source and for the production of lipids, which mixture is 10% of the filtrate obtained by and from alkaline treatment. It was produced from rice husk by acidic hydrolysis.
Figure 4 shows the growth of yeast in the culture medium, where commercially available pentose sugar was added as a carbon source.

"Carbohydrate" refers to an organic molecule comprising an aldehyde, an acid or a keto group, and several hydroxyl groups. Thus, the range of carbohydrates includes compounds described by the terms monosaccharides, oligosaccharides, polysaccharides, sugars, celluloses, hemicelluloses, starches and non-starch carbohydrates.

"Cellulose" is a long-chain polysaccharide whose primary structure consists of a polymer formed by β-1-4 bonds of glucose.

"Starch" is a long-chain polysaccharide and consists mainly of α-1-4 and α-1-6 glucose units.

As used herein, “usable sugars” refer to sugars to which microorganisms may increase, wherein lipid- and alcohol-producing microorganisms may produce lipids or alcohols.

"Hemicellulose" refers to a group of compounds consisting of several different hexose and pentose sugars such as galactose, mannose, glucose, xylose and arabinose.

A "monosaccharide" is a monomeric unit of carbohydrates, consisting of (CH ₂ O) _n , generally 3-9 carbon atoms and having stereochemical differences at one or more carbon atoms. This includes hexose sugars with six carbon atoms, such as glucose, galactose, mannose, fructose, and pentose sugars, such as xylose, ribose and arabinose, with five carbon atoms.

"Oligosaccharide" refers to a carbohydrate formed from two or more monosaccharides by O-glycoside linkages.

"Otanose" refers to a monosaccharide comprising five carbon atoms.

"Heat charcoal" refers to a monosaccharide comprising six carbon atoms.

"Hydrolysis" refers to the destruction of carbon-carbon, carbon-oxygen, carbon-nitrogen, or carbon-sulfur bonds under the influence of water, acid or alkali, whether or not water participates in the reaction. In enzymatic hydrolysis, the corresponding reaction is catalyzed by the enzyme. For example, hydrolysis is a reaction in which O-glycosidic bonds between monosaccharides of carbohydrates or peptide bonds between amino acids of proteins are disrupted.

With regard to "treatment with water, acid or alkali", this means that the organic material itself, or a product derived therefrom, is mechanically or thermomechanically extracted and treated, or a combination of these treatments in the presence of water, acid or alkali It means to be performed. According to Brønsted-Lowry acid alkali theory, an acid refers to a chemical substance, molecule, or ion that can give hydrogen ions (quantum), and an alkali can receive hydrogen ions (quantum) , Refers to a substance, molecule or ion. Acids are also referred to as Lewis acids that can accept electron pairs, and bases are referred to as Lewis alkalis that can give base pairs. By definition, the activity of substances such as acids or alkalis is not limited to aqueous solutions. In the present description, according to the above definitions, the terms “acid” and “alkali” also refer to acidic and alkaline catalysts. In the present description, an acid also refers to any acid phase, which may function as an acid, for example in gaseous, solid or liquid form (eg, aqueous solution). Correspondingly, alkali also refers to any alkaline state that can function as an alkali, for example gas, solid or liquid form (eg an aqueous solution).

As used herein, "organic source material" refers to any organic material produced in living organisms. Organic source materials in the present description are also called biomaterials.

In particular, organic source material refers to an organic material comprising polysaccharides. "Polysaccharide" refers to a carbohydrate polymer formed from monosaccharides, which may also include compounds other than monosaccharides. For example, polysaccharides are cellulose, hemicellulose and starch. Polysaccharides also include alginate, glucan, inulin and arabic gums, among others. Among other polysaccharides, mannane may also be mentioned. The source material may include polysaccharides by themselves or as a mixture, or may include degradation products thereof.

"Non-starch polysaccharide" refers to a carbohydrate whose molecular structure lacks or lacks the typical α-1-4 binding of starch. Non-starch polysaccharides are, for example, glucans, alginates, inulin and arabic gums. Non-starch polysaccharides also include hemicelluloses and celluloses. Other non-starch polysaccharides are, for example, carbohydrate polymers found in algae.

"Cultivated plant" refers to a plant planted and grown on land prepared for this for commercial purposes.

The term "lipid" refers to a fatty substance, which molecules, in part, comprise an aliphatic hydrocarbon chain and are soluble in organic solvents but insoluble in water.

In the present invention, lipids formed in microorganisms are mainly tri-, di-, or mono-acylglycerols, or sterol esters, but other lipids such as phospholipids, free fatty acids, sterols, polyprenol, sphingolipids, glycolipids And diphosphatidyl glycerol may also be formed intracellularly.

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

According to the EU Directive 2003/30 / EY, "biodiesel" refers to methyl-esters produced from vegetable or animal oils of diesel quality which can be used as biofuels.

Renewable diesel refers to hydrogen-treated lipids of animal, plant or microbial origin, which can be derived from bacteria, yeast, fungi, algae or another microorganism.

The source material of the process according to the invention can be cellulose, hemicellulose and biomass, preferably wood pulp (possibly comprising a binder), which is mechanical or thermo-mechanical or other physical method, or chemical, It can be produced enzymatically, or microbiologically or by a combination of these methods. Without modifications of the present invention, sugar beet pulp comprising non-starch polysaccharides, including starch, including, for example, potatoes, parts thereof, seeds of arable crops, corn and rice, each including beets It may also be the source material of the method according to the invention. Some of the plants comprising non-starch polysaccharides, for example β-glucan, are also suitable as source material. The method is also suitable for use of carbohydrates, such as alginates, derived from single-cell organisms as source material. The compositions of source materials described herein may also include various amounts of proteins and lipids, which may serve as source materials for the growth and lipid production of lipid-synthesizing microorganisms.

The source material of the process according to the invention can also be chaff of recycled fibers, sugar beet pulp and grains, for example oats, sawdust, purified mechanical pulp, peat, straw, obtained from newspaper recycling, for example. Other source materials useful in the process according to the invention include, for example, microbial masses such as single-cell biomass, biomass in swamps or water, including algae and microalgae, biomass in the catchment zone of cellulose plants, And biomass, or biological components, that go from municipal sewage to sludge plants operated, and are currently municipal incineration, composting, or other municipal wastes used in some other methods, which result in the extensive release of carbon contained in the waste as carbon dioxide. Cause.

According to a preferred embodiment of the present invention, the filtrate or a combination of filtrates, a precipitate or a combination of precipitates, obtained from the organic material (organic material itself or any combination thereof) according to the invention is transferred to the mixture and At least one step, wherein lipid production takes place. An alternative embodiment of the present invention is based on what kind of monosaccharide composition is desired for the growth of microorganisms and the production of lipids in a filtrate or a combination of filtrates, or a precipitate, a combination of precipitates, or a combination of filtrates or precipitates. Can be selected. Thus, the filtrate or precipitate is preferably treated with a biomaterial with a material selected from the group consisting of iii) water, ii) acid, and iii) alkali, followed by fiber-containing precipitates and fiber-free filtrates. Is separated from the group created by the separation.

Optionally, the precipitate is subjected to any one or more of the items (i), (ii) and (iii) again one or more times, and the obtained precipitate is preferably subjected to mechanical or thermo-mechanical grinding, and to the precipitate and filtrate. Separate.

Depending on which biomaterial has been used and which monosaccharides are desired, the biomaterial is treated with water, acid or alkali, preferably acid or alkali (generally by an aqueous solution of acid or alkali). If desired, this process can also be performed multiple times. The same biomaterial may also be treated sequentially with several different solutions, and compounds that enhance the separation and hydrolysis of carbohydrates may be added to the water.

The source materials listed in list group I below can be treated with water in the first step and with a mixture of water and acid if desired to improve the treatment results.

The biomaterials of group II are treated in the first step, preferably with acids.

The biomaterials of group III are treated in a first step, preferably with alkali. If the precipitate and the filtrate are recovered, the filtrate can be retreated with acid.

Group Ⅰ

Biomaterials derived from mechanical, thermomechanical, enzymatic or microbiological treatments of wood, or combinations of these treatments, or from cultivated plants.

Group II

Source material comprising recycled fibers, beet pulp, rice husk, straw, bran, grain granules, pre-cultivated plants, cultivated plants, TMP pulp, MDF pulp or starch or non-starch polysaccharides.

Group III

Sawdust, refined mechanical pulp, rice hulls, straw and woody plant parts, TMP pulp, MDF pulp, beet pulp, cultivated plants, which may include varying amounts of starch.

The treatment can be enhanced, for example, by adding one or more enzymes to the treatment solution, preferably to a treatment solution made with water. Enzyme treatment or microbial fermentation may also be added between the various process steps.

In the next step, the lipid-producing microorganism is contacted with any filtrate or precipitate or combination thereof in the culture medium, the microbial cells are allowed to produce lipids, and the lipids are recovered.

The fiber-containing precipitate obtained from the biomaterial treatment step described above is also preferably treated using a process in which the fiber-containing precipitate is mechanically ground and separated into fiber-containing precipitates and fiber-free filtrates.

In addition, the fiber-containing precipitate obtained from the mechanical grinding can be treated with a strong acid and separated into a fiber-containing precipitate and a fiber-free filtrate. After acid treatment, the precipitate can also be recycled back 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 can be milled mechanically or thermo-mechanically and separated into fiber-containing precipitates and fiber-free filtrates.

The filtrate or precipitate or combinations thereof, obtained from any of the treatments described above, can be added to the culture medium of lipid-producing microorganisms.

Generally, the total amount of sugar in the filtrate is 0.5-10% by weight. Of these, the sugars available for the production of biomass and lipids generally comprise 0.5% by weight, preferably at least 3% by weight, more preferably 4-5% by weight. For example, by grinding and re-extracting biomaterials, more sugars can be separated from the biomaterials, and in this way, the content of sugars can be increased to more favorable levels. However, the content 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 so that they first produce biomass and then produce lipids, or simultaneously produce biomass and lipids.

Depending on the origin of the biomaterial and the method of treatment (to be treated with water, acid, alkali), as shown in FIG. 2, hexose monosaccharides, pentose monosaccharides or both can be obtained in various ratios. From some biomaterials, mainly hexose sugar can be obtained, while from others, mainly octane sugar can be obtained. With the appropriate choice of microorganisms capable of producing lipids, filtrates or precipitates containing mainly hexose sugar or combinations thereof can be used for the production of cellular mass, and then filtrates containing mainly pentose sugars. Or precipitates or combinations thereof may be used for the production of lipids into the cell mass. Alternatively, lipids may be produced in cell masses from hexasaccharides. Cell masses and lipids can be produced from hexose. Correspondingly, by appropriate selection of microorganisms capable of producing lipids, filtrates or precipitates comprising mainly pentose sugars or combinations thereof can be used for the production of cell masses, and then filtrates comprising mainly hexasaccharides Or precipitates or combinations thereof may be used for the production of lipids into the cell mass. Alternatively, the lipid may be produced from the pentose into the cell mass, or the cell mass and the lipid may be produced from the pentose sugar. Both cell masses and lipids can also be produced from mixtures of pentose and hexose.

Treatment of Biomaterials

In the following, the treatment of biomaterials according to preferred embodiments of the invention is described. Typically this process is performed as a combination of two or more processes:

The source material is preferably extracted at a temperature of 90-100 ° C. Advantageous embodiments of acid extraction use 5-10% mineral acid, for example sulfuric acid, or organic acid, for example citric acid or acetic acid, and in alkaline extraction, preferably 0.5-2.0 M NaOH is used. . Treatment time can be extensive; Preferably it is 1-10 hours, generally 2-8 hours, most suitably 2-4 hours. Treatment with other treatments, such as enzymes, microorganisms, chemical oxidation or reduction, or a combination of these treatments, may also preferably be combined with water extraction.

According to the method, the precipitate produced in the extract is preferably mechanically at a temperature of 100-210 ° C., generally at 150-200 ° C., preferably for 2-20 minutes, usually 5-11 minutes. May be ground. The pressure is preferably 6-8 bar. The resulting mass is filtered and the filtrate is treated using the method described above to be suitable for the production of single-cell lipids. The precipitate can be delivered by acid treatment with strong acid, preferably treated with 40-72% sulfuric acid, suitably 65-70% sulfuric acid. In general, the treatment time is 2-8 hours, preferably 2-4 hours. The method can be carried out with any acid, depending on which proton-catalyzed hydrolysis is provided. Suitable acids are, for example, strong mineral acids, phosphoric acid, sulfuric acid, or oxygen acids of sulfur, nitrogen, chlorine, bromine and iodine.

The result of the hydrolysis is divided into filtrates treated to be suitable for the production of single-cell lipids, the precipitates can be transferred to a dilute acidic solution, preferably 5-10% sulfuric acid solution, and the milling temperature It may be performed at 170-200 ° C. and pressure 6-10 bar for 10-20 minutes. The mixture is divided into filtrate and precipitate, the filtrate is treated to be suitable for the production of single-cell lipids and the precipitate can be removed.

The described embodiments of the methods aim at the full utilization of carbohydrates present in the source material for the production of single-cell lipids. However, the method, which starts from the method of extraction of the source material, can also be implemented only for the selected part, for example when the fibrous material of the precipitate is also used for other purposes. The method according to the invention is preferably characterized in that it comprises the steps described throughout them, but the monosaccharide fractions produced by this run to the extent that they deviate from the basic method, or in the unit of operation, and in the production of single-cell lipids. With the use of, it is not limited from performing the part (s) of the method. The process step can also be attached to the method according to the above description, wherein the monosaccharide-containing fraction obtained from the source material can be used for the production of single-cell biomass or ethanol in addition to the production of lipids.

In the following description, several methods according to preferred embodiments of the invention are described. The same method can also be applied to other raw materials than those set forth above.

Ⅰ.

According to a preferred embodiment of the invention, wood fibers comprising pulverized wood, TMP pulp, sawdust or mechanical pulp are treated according to the following construction steps:

A. 100 g of wood 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. Carbohydrate yield in the solution ranges from 2% to 5%, depending on the method of making the wood fiber, and typically 4-5% for TMP fibers at high processing temperatures (170 ° C. or higher).

B. To increase the carbohydrate yield, the fiber fraction is taken at 1 liter of 5-10% acid (preferably 5%), at about pH 1 (eg mineral acid), at a temperature of 90-100 ° C., 2 Hydrolysis for -4 hours, preferably for 2 hours. Separating the remaining precipitate from the solution; Recover the solution.

C. Preferably, excess acid is decanted from the precipitate, and the precipitate is re-milled in a fiber deflector (eg wing or disc refiner). It is desirable to increase the temperature of the precipitate by pre-steaming for 1 minute and to maintain the concentrate of the mixture. The temperature is increased to 150-200 ° C., preferably 170 ° C., and the pressure is 6-8 bar (wing refiner). The grinding time is selected from the range of 2 to 15 minutes, depending on the wood. A 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 allowed to absorb at room temperature for 2-4 hours, preferably 2 hours. Excess acid is decanted and the precipitate is re-milled in a fiber separator (eg wing or disc refiner). It is then desirable to raise the temperature of the precipitate by pre-steaming for 1 minute, without spilling of the concentrate. The temperature is increased to 150-200 ° C., preferably 170 ° C., and the pressure is 6-8 bar (wing refiner). The grinding time is selected from the range of 2 to 15 minutes, depending on the wood. The mixture is filtered and the precipitate is separated from the solution. The solution is recovered and the precipitate is used as fuel.

The solution fraction obtained from any constituent step AD can be further processed by bleaching methods, pH adjustment, and other measurements that promote the growth of microorganisms, for example, by the removal of water, so that the solution obtained is cultured medium of microorganisms. It can be used within, and the microorganism is able to produce lipids. By combining with construction steps A-D, 40-65% of the original wood fiber material can be converted from the fiber material used as the source material into soluble form. Dissolved carbohydrates include glucose, galactose, mannose, xylose and arabinose units, a common part thereof, typical for the type of wood used.

II.

According to another preferred embodiment of the invention, 100 g of wood fibers, pulverized wood, recycled fibers, TMP pulp, sawdust or mechanical pulp are used, at a temperature of 90-100 ° C. for 2-4 hours, preferably For 3 hours, it is extracted in 1 liter of 4-8% alkaline solution, preferably in 1 M NaOH. The precipitate is separated from the solution at room temperature by filtration and two fractions are recovered. Depending on the fiber treatment of the source material, the carbohydrate yield in the solution is in the range of 5-8%. This solution is preferably treated by any of the following construction steps:

A. The vat liquor is itself re-used for the following fiber batch extraction in the above-described method and the solution is recovered.

B. The whole liquor is hydrolyzed with acid according to any of the steps B-D of the previous embodiment to treat the polysaccharides and oligo- and lignin dissolved therein.

C. The barrel liquor recycled according to step A is used according to step B of embodiment I.

In order to increase the monosaccharide output, the precipitate recovered from the alkaline treatment is preferably treated using any of the following methods or combinations thereof:

D. According to any of the steps B, C, D of previous embodiment I or according to both, the precipitate is treated. The mixture is filtered, the solution is neutralized, and both the precipitate and the solution are recovered.

E. The precipitate left in step A of embodiment II is transferred to an alkali, preferably the mixture for 2-8 minutes, preferably 6 minutes, at 4-10 bar pressure, preferably 8 bar, temperature 170 ° C. Re-treat under conditions, which are milled in. Filtration is performed and the solution is neutralized and recovered. By separate milling, the amount of dissolved material can increase to 27% of the amount of existing fibers in the source material.

The carbohydrate-containing solution, produced in each component step AE, comprising glucose, galactose, mannose, xylose, and arabinose units, is prepared by By removing, it is processed into a form suitable for lipid production, which is used as, or as part of, a culture medium for the microorganisms, which causes the microorganisms to produce lipids.

III.

According to a third preferred embodiment of the invention, wood fibers, pulverized wood, TMP pulp, sawdust, mechanical pulp, regenerated fibers (or neutralized extracts prepared according to step B above) are to be hydrolyzed and By adding 1 liter of the acid (e.g. mineral acid) per 100 g of material, at a pH of about 1, using a temperature of 90-100 ° C., for 2-4 hours, preferably for 2 hours, intensity 5 Hydrolysis with an acid having 10% (preferably 5%). The precipitate is separated from the solution by filtration. The solution contained 4-14% carbohydrate, calculated from the amount of fibers in the source material. The precipitate can be used as a fiber or can be further processed to increase the yield of monosaccharides according to step C or D or both steps of the first embodiment described above. The solution can be separated from the precipitate, neutralized, filtered, used on its own, or in concentrated form in the culture medium of the microorganism or part thereof, which will produce lipids.

Ⅳ.

According to a fourth preferred embodiment of the invention, using 100 g of wood fibers, pulverized wood, TMP pulp, sawdust, mechanical pulp or the precipitate remaining in step A of embodiment II or step AC of embodiment I, 5-10% of acid at pH 1 (eg mineral acid) is added and allowed to absorb for 2-4 hours, preferably for 2 hours. Excess acid is decanted and the precipitate is re-milled in a fiber separator (eg wing or disc refiner). The temperature of the precipitate is preferably increased by pre-steaming for 1 minute and prevents the concentrate from flowing out. The temperature is increased to 150-200 ° C., preferably 170 ° C., and the pressure is 6-8 bar (wing refiner). The grinding time is selected in the range of 2 to 15 minutes depending on the wood. The precipitate is separated from the solution by filtration. The remaining precipitate can be used as fuel or re-milled in the manner described above to increase the monosaccharide yield in the solution. The solution can be neutralized, filtered and used on its own or in concentrated form and used by the method described in embodiments I-III.

Ⅴ.

According to a fifth preferred embodiment of the invention, 40-72% strong acid, using 100 g of wood fibers, pulverized wood, TMP pulp, sawdust, mechanical pulp or residual precipitates according to any of the embodiments I-IV (Eg sulfuric acid) is added and allowed to absorb at room temperature for 2-4 hours, preferably 2 hours. Excess acid is decanted and the precipitate is re-milled in a fiber separator (eg wing or disc refiner). The temperature of the precipitate can be increased by pre-steaming for 1 minute, preventing the concentrate from flowing out. The temperature is increased to 150-200 ° C., preferably 170 ° C., and the pressure is 6-8 bar (wing refiner). The grinding time is selected in the range of 2 to 15 minutes depending on the wood. The carbohydrate mixture is filtered and the precipitate is separated from this solution. The portion of material dissolved from the precipitate used as source material is in the range of 40-65%. The remaining precipitate may be used as fuel or may be re-milled to increase monosaccharide yield. The solution is neutralized, filtered and treated according to any of the embodiments I-IV.

Alternatively, 40-72% of strong 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 heated at 100 ° C. for 4 hours at normal pressure. Further processing as above.

Lipid Production Using Microorganisms

The present invention, through the combination of filtrates produced at various constituent steps, allows for the widespread use of hexose and pentose monosaccharides, including carbohydrates contained in the source material, in lipids and single-cell biomass by microbiological methods. . Using pre-treatment, eg, washing, neutralizing, decolorizing or other post-treatment procedures, each recovered filtrate is used for the production of single-cell lipids on their own or alternatively in combination with various aqueous fractions. Used. Since the method of treating the source material is also part of the present invention, the present invention is also applicable to ethanol production.

The filtrate, ie the aqueous fraction, or any combination of filtrates is added to the microbial culture medium in which microorganisms are present or inoculated and allow the microorganisms to produce lipids. The lipids are recovered in the form of microbial masses, or the lipids are separated from the masses and both lipids and microbial masses separated therefrom are recovered. Lipids can be recovered using known methods by removing them from the cells or by destroying the cells. The lipid can be extracted from the disrupted cells using an organic solvent. A lipid recovery method applicable to the present invention is described (see, eg, Y. Lipids: Extraction, Quality Analysis, and Acceptability, Critical Reviews in Biotechnology, 12 (5/6); 463-491 by Z. Jacob). (1992)]. Preferred lipid recovery methods are phase separation. In addition, treatment of lipids formed with fatty acid esters in a microorganism can occur without prior homogenization of the microbial cells and subsequent separation of fat. Preferred embodiments of the present invention relate to a method of forming a lipid or lipid mixture from a carbohydrate mixture resulting from processing of an organic source material, which comprises the monomeric hexose sugar and the pentose sugar or oligomeric forms, and according to this method Adding the containing mixture to an aqueous culture medium in which the lipid-producing microorganisms are cultured, supplementing the medium with nutrients necessary for growth, inoculating the medium with the organism, culturing the organism and producing lipids The cell mass is recovered and the lipid or lipid mixture is separated from the cells or the adipose-containing cells or components thereof are used by themselves.

The method according to the invention provides particular flexibility for the production of microbial lipids. Fractions containing both hexose and pentose are natural carbon sources for many lipid-producing microorganisms. Thus, the method also has the advantage of allowing the microorganism to be broadly selected, such as based on lipid production capacity, yield of biomass, type of culture or culture conditions. Other components of microorganisms except lipids can be used energy efficiently in many different ways, thus improving the overall economic performance of the method according to the invention. A preferred method of using lipid-free microorganisms is to recycle them into culture media of hydrolysis and lipid-producing microorganisms or to use them as food or nutritional substances. It is also possible to separate various components, for example (special) sugars, colorants, β-glucans, sterols, sterol esters or proteins from lipid-free microbial masses.

The microorganism is selected from naturally or genetically modified fat-accumulating microorganisms, preferably from yeast, mold, bacteria and algae, more preferably from yeast and mold, and most preferably from yeast. It is essential that the microorganisms to be used must be able to produce lipids from hexose or pentose or both. The present invention thus encompasses all microorganisms whose lipid accumulation is based on the ATP: citrate lyase activity (EC 2.3.3.8) they contain.

Lipid that can be applied to the present invention synthesized in yeast (genera) comprises a speed of: Candida (Candida), Yarrow vias (Yarrowia), lipoic My process (Lipomyces), also torulra (Rhodotorula) And Cryptococcus , It is a strain that synthesizes the pentose xylose into lipids, such as Candida Culvata. curvata ) (D) (Evans, CT. and Ratledge, 1983. A comparison of the oleaginous yeast, Candida curvata , grown on different carbon sources in continuous and batch culture, Lipids 18 623-629), Rhodotorula gracilis ) (Yoon, S., Rhim, J., Choi, S., Ryu, D. and Rhee, J. 1982. Effect of Carbon and Nitrogen Sources on Lipid Production of Rhodotorula gracilis , J. Ferment . Technol . 60, 243-246) and Rhodosporidium toruloides ), Rhodotorula glutinis ) , Rhodotorula graminis ), Lipomyces starkeyi ), Lipomyces lipopearl lipofer ), Candida lipolytica ), Cryptococcus , Cryptococcus alvidus albidus ) , Trichosporon cutaneum and Trichosporon pullulans) (Fall, R., Phelps , P. and Spindler, D. 1984, the pentose arabinose lipids, including Bioconversion of Xylan to Triglycerides by Oil- Rich Yeasts. Appl. Environ. Mircobiol. 47, 1130-1134) Synthesizing System, Lipomyces Stakka starkeyi ) (Naganuma, T., Uzuka, Y. and Tanaka K. 1985. Physiological Factors Affecting Total Cell Number and Lipid Content of the Yeast, Lipomyces starkeyi, J. Gen. Appl . Microbiol. 31, 29-37).

Correspondingly, the genus of fat- accumulating fungi that can be applied to the present invention includes among others:

- aspergillosis (Aspergillus)

Saturday hatred (Chaetomium) in the car.

- Chloe Dos Forest Stadium (Clodosporidium)

- Kuhn corridors Melaka (Cunninghamella)

- AIME Lee Sela (Emericella)

- Fusarium (Fusarium)

- Mall tee Relais (Mortierella)

- non-cor (Mucor)

- Penny room Solarium (Penicillium)

- Petey Stadium (Pythium)

- Rizzo crispus (Rhizopus)

- Tree seconds deolma (Trichoderma)

Correspondingly, the genus of fat- accumulating bacteria that can be applied to the present invention includes, among others:

- ahsine tobak Termini (Acinetobacter)

- Tino evil Park Hotel (Actinobacter)

- I know times (Anabaena)

- Park Hotel (Arthrobacter) as alsseu

- Bacillus (Bacillus)

- Cross-tree Stadium (Clostridium)

- Flexi tumefaciens (Flexibacterium)

- Micro Caucus (Micrococcus)

- Mycobacterium (Mycobacterium)

- nokal Dia (Nocardia)

- No Stock (Nostoc)

- Oscar Silas Astoria (Oscillatoria)

- Capital Monastir (Pseudomonas)

- Rhodococcus (Rhodococcus)

- also emptiness Micro (Rhodomicrobium)

- also may Monastir (Rhodopseudomonas)

Shh and Nella (Shewanella)

- Streptomyces (Streptomyces)

- Vibrio (Vibrio)

Correspondingly, the fat-accumulating micro-algae genus that can be applied to the present invention includes among others:

- Boats Rio Caucus (Botryococcus)

- Bra Chino Monastir (Brachiomonas)

- Chlamydomonas (Chlamydomonas)

- Chlorella (Chlorella)

The creep to Cody nium (Crypthecodinium)

- Two Nari Ella (Dunaliella)

- euglena (Euglena)

Nano-keulroriseu (Nannochloris)

Nano-claw drop system (Nannochloropsis)

- Butterfly Kula (Navicula)

- Nichia (Nitzschia)

- Solarium eponymous Kit (Schizochytrium)

- skeletal retrograde nematic (Sceletonema)

- Sene des Sommers (Scenedesmus)

-Tetrahydro cell Miss (Tetraselmis)

- teurawooseu Torquay Yttrium (Thraustochytrium)

- Oh cry Kenny (Ulkenia)

According to a preferred embodiment of the invention, the microorganisms which synthesize fatty acids-containing lipids in their cells, preferably in an amount of 12-65% by weight of the dry weight of the cells, are used for lipid synthesis.

According to a particularly preferred embodiment of the invention, the lipid-free biomass formed in the present invention, treated in a method suitable for the microorganism, is used as a nutrient in the culture medium. In addition to this component, the culture medium may be supplied with a desired component for the microorganism used. In order to produce lipids, microorganisms generally require a carbon source, inter alia, wherein the carbon source in the present invention is a source material, a nitrogen source, for example an inorganic ammonium salt (for example ammonium sulfide) or an organic nitrogen source ( For example, amino nitrogen, yeast extract, or hydrolyzed cell masses, and micronutrient sources such as phosphates, sulfates, chlorides, vitamins or cationic sources (eg, Mg, K, Na, Ca, Fe). Or Cu ion source), which component may be added to the medium if necessary. When the method of the invention is applied, the lipid concentration of the cells is preferably 40% by weight, most preferably 65% by weight.

Biofuel Manufacturing

Fatty acid esters comprised in microbial-derived lipids may be treated to be suitable as biodiesel fuels by any known method. One preferred approach is to perform transesterification with short-chained alcohols, preferably with methanol, to obtain fatty alcohol esters.

Impurity side flows resulting from the transesterification and production of biodiesel or renewable diesel—side flows include alcoholic compounds, such as glycerol or non-esterified fatty acid salts. It is difficult to develop in an energy efficient way-silver can be re-used in the production of single-cell lipids, where the lipids are used on their own or as other glycerolipid-containing materials of biological origin. Can be recycled.

Advantage of the present invention

The advantages of the present invention include the fact that the equipment required for the present invention is simple, and the related art is known as far as manufacturing and operation is concerned. The process according to the invention is not limited to any production scale, but can be easily scaled up or down depending on the carbohydrate content and the content of the source material to be treated. The performance of the process for producing lipids does not require energy-consuming heating, pressurized unit operation or acid, alkaline or enzymatic catalysts as well as other chemical catalysts. The present invention only requires the internal cycle of the process according to the invention, or the chemical use which can be incorporated into the processing of such biomaterials. And since the diluted carbohydrate solution is suitable for use as such, as or in a microbial culture medium, the present invention does not require costly water-removal from the available sugar solution. The overall economic performance of the present invention is that lipid-free biomasses produced herein can be produced in a variety of different ways, such as the production of individual organic components, in addition to internal circulation, as feed, as feedstock, or as supplementary culture medium for microbial production. Improved by the fact that it has a purpose. The method is also suitable for the production of single-cell biomass and ethanol.

An advantage of the proper treatment of the biomass according to the invention is a more comprehensive hydrolysis of the organic material and thus better availability of the organic material for the production of single-cell lipids compared to the current technology. In addition, precipitates or filtrates obtained from acid and alkali treatments neutralize each other and reduce the chemical utilization required for neutralization.

The prior art solutions describe a method comprising a microbiological step for the production of ethanol, where the carbohydrate-containing material is used as such or converted to monosaccharides as raw material. Patent application publications US 2002/0185447 and US Pat. No. 5,637,502 also describe methods of treating carbohydrates, such as treatment with acids or alkalis followed by alcoholic fermentation. With regard to microbiological treatments, both methods are limited to the production of ethanol in which hexose sugar formed from polysaccharides is used. In US patent application publication US 2003/0096385, prenyl alcohols (geranyl and farnesyl derivatives of alcohols) are produced using microorganisms. Patent application publication WO 03/038067 describes a method by which genome modification of fungal microorganisms can yield organisms that can utilize pentose sugars. The publication aims only at ethanol production.

The present invention offers a novel solution to the development of the more significant aqueous side flows of thermo-mechanical wood processes, in particular in the pulp and paper industry, as raw materials for traffic fuels. The present invention contemplates the biological loading of carbohydrate-containing side flows produced in connection with the production of mechanical wood pulp, thus reducing the energy consumption of waste treatment. In a specific form, the present invention is an environmentally friendly solution for producing raw materials for traffic fuels, biodiesel or renewable diesel, such as from diluted carbohydrate-containing aqueous fractions produced in the TMP process, or from mechanical treatment of the corresponding wood. It is a lipid production method to provide.

The method also provides an alternative solution for developing other source materials. According to the method, recycled fibers such as printed paper, packaging materials and similar cellulose-based materials can be used as source materials.

Accordingly, the present invention is compared with the prior art by increasing the effectiveness of lipid sources and reducing the total demand for organic lipids from other sources, in accordance with the principles of sustainable development. As a result, the present invention increases the effectiveness of renewable natural resource-based biofuel feedstocks and performs their production costs to end-user levels acceptable to consumers.

Where total use of the source material is contemplated, the present invention is not limited to only the total use of carbohydrates included. The invention also includes the step of allowing the lipid component of the fraction extracted from the wood material to be recovered. If the source material 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 acid treatment of the source material, the lipids in the extracted fractions yield free fatty acids separated from the filtrate in the form of insoluble alkaline earth metal salts, such as Ca ⁺⁺ salts, and after separation of the precipitates, these lipids are converted into alcohol esters. Esterified. Correspondingly, the alkaline extract of the source material produces a fatty acid salt from the extracted fraction, which salt is water soluble and therefore mixed with the filtrate. In the present invention, the salt is converted into the form of an insoluble salt, such as a Ca ⁺⁺ salt, and the precipitate is separated from the aqueous phase and used for the preparation of fatty alcohol esterides.

The following examples are intended to illustrate the invention and are not to be construed as limiting the scope of the invention in any way. The present invention is also not limited to microbial strains used in any way. The invention can be practiced not only by the strains used but also by other strains of the same species or genus, or by strains of other microorganisms or species or genetically modified microbial strains. Lipid-producing microorganisms are generally available and can be found in several families of strains—eg, ATCC, DSM, and the like. Lipid-producing microorganisms and lipid production methods using microorganisms (including algae) are described in, for example, Single Cell Oils, eds. Z. Cohen and C. Ratledge, AOCS Press, 2005 and Microbial Lipids, eds. C. Ratledge and SG Wilkinson, vol. 1 and 2, Academic Press, 1988].

Example

Example  One

Each 100 g of wood fibers, ground wood, TMP pulp, sawdust and mechanical pulp were placed at 1 liter of boiling water, 90-100 ° C. for 2 hours. The solution was separated away from the fibrous material (hereafter precipitate). The precipitate was recovered and treated with various alternatives according to one of the alternatives set forth in Examples 4 or 5 to increase monosaccharide yield. The carbohydrate yield of the solution ranges from 2% to 6% depending on the source material and is typically 4% when spruce fibers crushed at high temperatures (above 170 ° C.) are used as the source material. Some of the solution was reused for the following fiber batch extraction without prior evaporation and some of the solution was concentrated by evaporation until the dry matter content reached about 20% by weight. The concentrated filtrate was recovered for the production of single-cell lipids. Dissolved carbohydrates are representative of the extracted species, which are divided into hexose and pentose sugars.

Example  2

Wood fibers, crushed wood, recycled fibers, TMP pulp, sawdust and mechanical pulp were each added to 1 liter of 5% alkaline solution (NaOH) and stirred at 90-100 ° C. for 3 hours. Filtration was carried out at normal temperature and the precipitate was further treated for production of monosaccharides according to Example 4. The extraction solutions were treated so that a portion of each solution was reused to process the next fiber batch, neutralized a portion, and some oligo- and polysaccharides, including some lignin, dissolved in the alkaline extraction solution, so some examples Hydrolysis in accordance with 4. Solutions obtained from different source materials by the alkaline treatment described above retained an average of 5% of the weight of the source material.

The precipitate obtained from the existing alkaline treatment was also retreated with alkali by repeating the previous alkaline treatment and ground for 6 minutes at 170 ° C. temperature at 8 bar pressure. With this additional milling, the amount of material dissolved in the alkaline solution could increase to an average of 27% of the amount of existing source material. After neutralization, hydrolysis and concentration, the solution darkened and treated with ion exchanger and activated charcoal for decolorization. The mixture was recovered and used for single-cell lipid production.

Example  3

Each 100 g of wood fibers, crushed wood, TMP or MDF pulp, sawdust, mechanical pulp, recycled fibers and neutralized extracts prepared according to Example 2 were prepared at 90-100 ° C. in 1 liter of 5% mineral acid, pH 1 Hydrolysis for 3 hours at. The precipitate was separated from the solution by filtration. The solution contained monosaccharides with an average of 10% of the source material content. Each solution was treated to neutralize some, filtered and concentrated by evaporation until the content of monosaccharides reached about 20% by weight and this concentrated solution was recovered for the production of single-cell lipids. As described in this example, a portion of the solution was reused per se for hydrolysis of the following source material batches. The precipitate was further treated to increase the yield of monosaccharides according to Example 5 below.

Example  4

Each 125 g of wood fibers, crushed wood, TMP pulp, MDF pulp, sawdust, mechanical pulp, straw, grain husks and precipitates according to Examples 1-3 were added to a sulfuric acid solution with a concentration of 10% and for 2 hours. Allow the acid to be absorbed. Decant the excess acid, transfer the mixture into the wing finder, pre-steam for 11 minutes to raise the temperature without flowing out of the concentrate, then increase the temperature to 160 ° C. and the pressure to 8 bar and wing refiner Grinding was carried out using. For spruce fibers a mixture grinding time of 11 minutes was chosen. After the grinding, a precipitate was separated from the solution. Each precipitate was partitioned so that some were used for incineration and some were regrind to increase the monosaccharide yield according to Example 5 below. Correspondingly, the solution was neutralized, filtered and concentrated by evaporation until the monosaccharide concentration reached about 20%. This solution was recovered for the production of single-cell lipids.

Example  5

The remaining precipitates according to Examples 1-4, including 125 g each of wood fibers, crushed wood, TMP pulp, sawdust, mechanical pulp, straw and grain fibers, were added to a 40% sulfuric acid solution and allowed to absorb for 2 hours at room temperature. The temperature of the precipitate was increased by decanting excess acid and grinding the precipitate in a wing fiber separator and presteaming for 11 minutes without flowing out of the concentrate. The temperature was then increased to 170 ° C. and the pressure reached 8 bar. The most suitable grinding time for straw and grain fibers was 11 minutes. The precipitate was separated from the solution. The proportion of dissolved material exceeded an average of 50% of the source material. The solution was neutralized, filtered, concentrated by evaporation and recovered for production of single-cell lipids. The precipitate, ie the remaining fibers, was partially incinerated and partially regrind according to this example to increase the yield of monosaccharides.

Concentrated sulfuric acid (72%) was also absorbed individually into the above-mentioned source material at room temperature for 2 hours of treatment time. The acid was then diluted to 5% and heated at 100 ° C. for 4 hours at normal pressure. Further treatment and use of the resulting fractions were carried out as above.

Example  6

Monosaccharides suitable for the production of single-cell lipids can be produced from chaff, straw and grain husks by direct hydrolysis with 5% acid, impregnated with stronger acids, hydrolyzed in 5% solution, or first It can be treated with alkali and hemicellulose and cellulose can be hydrolyzed separately as shown in the examples above. This treatment can be combined with an impregnation treatment with acid or alkali and subsequently repeated grinding in a wing or disc refiner to produce thermo-mechanical pulp. However, in this example, the rice husk, straw and grain husks (125 g each) were pre-steamed at 8 bar pressure for 11 minutes and treated to a thermo-mechanical pulp at a temperature of 170 ° C. in a wing refiner. The resulting thermo-mechanical pulp was hydrolyzed for 4 hours at 90-100 ° C., at normal pressure, in a volume of 5% acid (sulfuric acid), 1 liter. The solution fractions were separated by filtration and neutralization. Subsequently, the solution was concentrated and recovered for single-cell lipid production. Monosaccharide yield was an average of 50% of the source material. To increase the carbohydrate yield, the remaining precipitate after filtration was thermo-mechanically re-treated according to Example 5 and incineration was carried out in part.

Example  7

Rice husk, straw and grain husks (16 kg each) were treated in 100 liters of alkaline solution (1.2 M NaOH) at 90-100 ° C. for 4 hours. The mixture was divided into precipitate and solution. An average of 49-57% of the dry matter of the food source material remained in the solution. The alkaline solution was brought to 5% with sulfuric acid with respect to acid, which was hydrolyzed as in Example 6. A mixture of xylose and arabinose was mainly formed, which also contained small amounts of glucose and galactose. The monosaccharide content of the solution was 23-33% of the food source material. Prior to recovery for production of single-cell lipids, the solution was lightened by treating activated carbon.

The precipitate obtained by filtration was infiltrated with 5% acid (sulfuric acid) and ground in a wing refiner at pressure 6 bar and temperature 150 ° C. for 11 minutes. The precipitate was digested with soluble carbohydrates (primarily glucose and galactose) and recovered for the production of single-cell lipids after neutralization and filtration. A portion of the precipitate remaining after filtration, which was performed after acid hydrolysis, was still re-milled and then hydrolyzed at 10% acid according to Example 4. After this treatment, a solution comprising a total of 55-65% monosaccharides of the source material was obtained. This solution was recovered for the production of single-cell lipids.

Example  8

Steam-dried beet pulp was poured into the keg and enough boiling water was poured over it to allow the water to cover the beet pulp. The solution is cooled and water is separated from the solid by decanting. 3.6 mg / ml of dry matter was dissolved in the solution. The new batch was treated with some of the aqueous fractions, and after concentration, was partially recovered for production of single-cell lipids. The solid fraction obtained, comprising 16.7% dry matter, weighed 748 g of precipitate, correspondingly as dry matter, 125 g of dried beet pulp was treated with 0.4 M phosphoric acid (H ₃ PO ₄ , 500 ml) was infiltrated. Excess acidic solution was decanted and pre-steamed for 11 minutes before thermo-mechanical grinding was started. The mixture was ground for 11 minutes using a wing refiner. During grinding, the pressure was 8 bar and the temperature was initially 172 ° C., after 10 minutes of grinding time, 162 ° C. The mixture was removed from the refiner and filtered. The 2.26 liter solution fraction contained 2.75% dry matter. After filtration, 50% of the dry matter provided for the grinding was present in the solution. The solution mainly contained monosaccharides, glucose, galactose, arabinose and xylose. In addition, small amounts of oligosaccharides and larger molecular weight compounds with molecular weights ranging from 500 to 3000 were observed. The solution fractions were neutralized, concentrated and recovered for single-cell lipids.

The solid fiber pulp, precipitate (125 g dry matter), obtained from the treatment of the beet pulp, was allowed to soak in 15% hydrochloric acid at room temperature for 4 hours. Excess acidic solution was decanted and the pulp was regrind under the same conditions. After the filtration step, an additional 28% monosaccharides were observed to enter the solution phase. The precipitate was disposed of.

Example  9

The source material is a thermo-mechanically treated spruce fiber, weighing 46.8 g (about 1 liter of volume) and adding 0.2N NaOH to the top of it, additionally 5.6 g of Na ₂ CO ₃ and 1.3 g MgSO ₄ 6H ₂ O (500 ml) was added. This mixture was heated to 50 ° C. and yielded a small amount of glucose, xylose, galactose, arabinose, mannose and additional oligomers in the solution. The pH of this solution was about 11, which was filtered and washed with water. The wash and filtrate were combined and the solution was evaporated to 320 mL. The amount of dissolved material was 4.5% of the source material (dry material). To increase the content of monosaccharides, acid was added to the solution and a precipitate formed. This precipitate was separated from the solution, the latter was recovered and used for single-cell lipid production. The precipitate was hydrolyzed to monosaccharides in 5% acid as described in Example 3. Using hydrolysis, 1% monosaccharides were produced from the precipitate mass and the monosaccharides were placed in the solution and recovered for single-cell lipid production after bleaching.

Example  10

Measure 45 g batch (approximately 1 liter) of thermo-mechanically treated spruce fibers, add 0.2 N NaOH on top of it, and additionally 5.6 g Na ₂ CO ₃ and 1.3 g MgSO ₄ 6H ₂ O (500 ml) was added. The mixture was heated to 50 ° C., filtered and the precipitate washed with water. Wash and filtrates were mixed. To increase the content of monosaccharides, acid was added to the mixed filtrate and hydrolysis was carried out according to Example 3. After neutralization, the resulting hydrolysis product was recovered for single-cell lipid production. The washed precipitate was compressed to 300 ml, 1 liter citrate buffer, pH 4.8, and cellulase enzyme were added. Oligomers and glucose were produced in the mixture. The enzyme-treated mixture was recovered on its own or as a separate filtrate, solution and used for single-cell lipid production.

Example  11

125 g of oat rice husk batch was provided and it was thermo-mechanically ground at 170 ° C. and pressure 8 bar for 2 minutes. After this treatment, the precipitate was separated from the solution by filtration. This solution was recovered for use in single-cell lipid production. A 7 g batch of precipitate (fiber) was used and brought to 19% in strength against sulfuric acid and refluxed for 4 hours. Analysis showed that this treatment resulted in the release of up to 56% of the monosaccharides of the source material into the solution as mostly xylose, mannose, glucose, galactose and arabinose. The solution was recovered for the production of single-cell lipids.

Example  12

400 g of oat chaff were weighed and 3 liters of water and 200 g of NaOH were added. The mixture was kept stirring at 90-96 ° C. for 2 hours. This mixture was filtered through a cloth and the fibers separated. Neutralize the filtrate fractions (50 ml) and adjust its pH to 4.8 using citrate buffer (40 ml), add multifect xylanase (10 ml) and add the mixture to 50 The thermostat was thermostated at < RTI ID = 0.0 > Sugar was released into the solution, of which 25.2% was xylose and 11.8% was arabinose. In addition, after 50 hours of reaction time, oligomers were present in this solution. The total mixture was recovered for the production of single-cell lipids.

Example  13

125 g of beet pulp was infiltrated with 0.4 M sulfuric acid and after 12 hours, the excess acid was decanted. The pulp was transferred to a wing refiner, pre-steamed for 4 minutes and ground at 150 ° C. for 6 minutes at 6 bars. The mixture was neutralized and citric acid was added until the pH dropped to 4. 10 ml of Pectinase 4450U, ie 178 mg / ml of protein (Sigma) was added and the reaction was allowed to proceed at 25 ° C. for 24 hours. After this reaction, part of the mixture was recovered for production of single-cell lipids and part was filtered. The precipitate obtained from the filtration was returned to the grinding step and the solution fraction obtained from this treatment was treated with activated charcoal, 2 g / liter and then transferred to an anion exchange column. Monosaccharide solutions obtainable from this column were evaporated to 20% concentration on dry matter and recovered for production of single-cell lipids.

Example  14

Oat chaff was treated according to Example 7, where a mixture comprising glucose 23.8 g / L, xylose 93.3 g / L, arabinose 37.1 g / L and galactose 9.0 g / was produced. Mixture of lipid-synthetic yeast Yarrowia Ripolitica ( Yarrowia) lipolytica ) ATCC 20373 and Rhodotorula glutinis ) The culture medium for TKK 3031 per se was added as a 1: 1 dilution and as a corresponding dilution supplied with 11 g / L of glucose. The incubation period was 68 hours, the temperature was 28 ° C., the shake was 250 rpm and the culture volume was 50 ml. As shown in FIG. 3, the yeast did not require other nutrient additions, could grow, and synthesize lipids.

Example  15

Three Yeasts, Rhodotorula glutinis ), Yarrowia lipolitica lipolytica ) and Kluyveromyces marxianus (Anam. Candida kefyr) ATCC 42265 were incubated alone with xylose as the carbon source. The culture medium contained 20 g / L xylose, 10 g / L yeast extract and 20 g / L peptone. Cultivation was performed under shaking at 200 rpm at a volume of 50 ml and 25 ° C. 4 shows that all three strains can utilize pentose sugar as a carbon source.

Claims (19)

Producing lipids or lipid mixtures from organic source materials comprising polysaccharides, selected from the group comprising cellulose, hemicelluloses, starches, mixtures thereof, any mixtures or degradation products thereof or non-starch polysaccharides As a method,
treating said source material with a material selected from the group comprising a) iii) water, ii) acid, and iii) alkali, and then separating the precipitate and the filtrate and depositing the sediment obtained by the treatment as such Or mechanical or thermo-mechanical grinding in the presence of water, acid or alkali, separating the precipitate and filtrate, and alternatively, treating the precipitate with the treatment and / or iii), ii) or iii) section ( repeat grinding of any of the sections one or more times,
b) the filtrate obtained or the plurality of filtrates or precipitates obtained, or any combination obtained therefrom, and optionally the source material, in contact with a lipid-producing microorganism in a culture medium, as a result Where the microbial cells begin to produce lipids,
c) recovering said lipids,
Method of producing lipids or lipid mixtures. The method of claim 1,
A process for producing a lipid or mixture of lipids, characterized in that said precipitate is optionally further treated using a method comprising one or more of the following steps:
d) treating the precipitate obtained from section a) with strong acid and separating the precipitate from the filtrate, or alternatively,
e) acidify the precipitate obtained from section a) or d) and grind it mechanically or thermo-mechanically and separate the precipitate from the filtrate or, optionally, any of steps a), d) or e) Treating the precipitate obtained from the step one or more times in any order using the method according to any of steps a), d) or e),
f) the filtrate or precipitate obtained from section a), d) or e), or any combination obtained therefrom, and optionally, the source material, in contact with the lipid-producing microorganisms in the culture medium, Result in the microbial cells starting to produce lipids, and
g) recovering the lipids. 3. The method according to claim 1 or 2,
Wherein said source material is from a mechanical or thermo-mechanical treatment of wood or from a cultivated plant. 4. The method according to any one of claims 1 to 3,
Process for producing a lipid or lipid mixture, characterized in that the source material is treated with water or acid. The method according to claim 1 or 2,
Said source material is from a source selected from the group comprising reclaimed fiber, sugar beet pulp, rice hulls, straw, bran, grain granules, whole cultivable crops, cultivated plants, TMP pulp and MDF pulp Method of producing a lipid or lipid mixture. The method according to any one of claims 1, 2 or 5,
Wherein said source material is treated with an acid. 3. The method according to claim 1 or 2,
Wherein said source material is derived from a source selected from the group comprising sawdust, purified mechanical pulp, rice hulls, straw, TMP pulp, MDF pulp, sugar beet pulp, and cultivated plants that do not contain the required amount of starch, Method of producing lipids or lipid mixtures. The method according to any one of claims 1, 2 or 7,
Characterized in that the source material is treated with alkali. 3. The method according to claim 1 or 2,
The source material is from a source selected from the group comprising microbial mass, swampy or submerged biomass, biomass from catchments of cellulose plants, biomass from municipal waste and biomass from municipal sewage. Characterized in that the lipid or lipid mixture production method. The method according to any one of claims 1 to 9,
A process for producing a lipid or lipid mixture, characterized in that the filtrate comprises at least 0.5-1% by weight, at most 20-30% by weight, preferably 4-5% by weight, of sugars available during the production of lipids. 3. The method according to claim 1 or 2,
A process for producing a lipid or lipid mixture, characterized in that the treatment of any one of steps a), d) or e) is carried out one or more times. The method of claim 1,
When the source material is treated with alkali in iv) of section a), the obtained precipitate is re-treated with acid and the precipitate and the filtrate are separated. 3. The method according to claim 1 or 2,
A process for producing a lipid or lipid mixture, characterized in that when the fiber-containing precipitate is treated with a strong acid, the obtained precipitate is milled again mechanically or thermo-mechanically. The method according to any one of claims 1 to 13,
A method of producing a lipid or lipid mixture, characterized in that at least one enzyme is added to the biomaterial treatment solution. The method according to any one of claims 1 to 14,
Lipids or lipids, characterized in that the filtrate obtained from any of the above steps is further processed using methods that are more suitable for the growth of microorganisms, such as decolorization methods, pH adjustment and / or removal or addition of water. Method of producing a mixture. The method according to any one of claims 1 to 15,
Wherein said source material is pre-treated mechanically, thermo-mechanically, physically, chemically, biologically, or a combination of these treatments. Use of a lipid or lipid mixture produced using the method according to any one of claims 1 to 16 as a raw material for biofuel manufacture. A biofuel characterized in that the lipid produced using the method according to any one of claims 1 to 16 is used for its production. A municipal sewage treatment method comprising treating municipal sewage using the method according to any one of claims 1 to 16.

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