CN101910407A - Method for producing lipid - Google Patents

Abstract

The invention relates to a method for forming a lipid or a lipid mixture from an organic source material comprising cellulose, hemicellulose, starch, non-starch polysaccharide, any mixture thereof or degradation products thereof. According to the method, the source material is treated one or more times with water, an aqueous solution of acid or alkali, and the precipitate and the filtrate are separated. The precipitate obtained from the treatments mentioned above can be subjected to mechanical or thermo-mechanical grinding, or the precipitate can be treated with a strong acid, or the precipitate can be acidified and ground mechanically or thermo-mechanically. After the treatments, the precipitate and the filtrate are separated. A lipid-producing microorganism is contacted with the source material or the obtained filtrate(s) in a culture medium, whereby the microorganism cells begin to produce lipid, and the lipids are recovered.

Description

Produce the method for lipid

The present invention relates to the preamble (preamble) according to claim 1 is produced lipid or lipid mixt from organic raw material method.The present invention relates to the lipid produced by described method or lipid mixt equally as according to the biofuel of claim 17 and according to the purposes of the biofuel of claim 18.The present invention relates to the method according to claim 19 purifying city water drain equally.

Technical background

Be well known that, be very large-scale from the purposes of the traffic fuel of fossil feedstock manufacturing, and consume just in sustainable growth.Therefore, quite by rights emerged in large numbers about abundance, environmental effect and the Sustainable development aspect of fossil energy resource and be important global challenge.In this reference system, the renewable alternative materials of traffic fuel has been emerged in large numbers the target for cumulative interest.

Towards a step based on the fuel production of renewable natural resources is to attempt, and at least in part, replaces fossil feedstock with organic materials.Even adopt this method, we still can imagine the problem that is very difficult to solve.Proportional with the consumption that fossil feedstock is present, the amount of desired organic raw material is very large, even only some fossil feedstock is replaced.In several connections, observed the unidirectional of organic natural resources and consume on a large scale or the regeneration that is used for the cultivated area of this purpose may cause multifarious consequence of natural biology and grain primary production equilibrated consequence, these will be insoluble.Organic materials changes into the form that can be used for production traffic fuel in the mode of Energy Efficient and has proposed technological challenge equally.

For traffic fuel, particularly advantageous raw material sources will be organic fatties, particularly more be higher than the triacylglycerol of corresponding for example carbohydrate or alcohols considerably as the triacylglycerol of its intrinsic energy.In addition, it is known best, and it can be converted into the component of traffic fuel via suitable effective chemical method, such as diesel oil, biochemical diesel oil or renewable diesel.Yet the shortage of natural fat raw material reserves has proposed limiting factor.Based on present fatty resource, the biofuel of no more than marginal industrial output is feasible, at the most.Therefore, increase fatty reserves requirement oil plant and cultivate appreciable increase.This big variation of the turnout sector of oil plant cultivation conversely, has intense influence to the balance of world market grain economy.This needs still are in the supposition level, self have been revealed as a large amount of risings of grain and feedstuff raw material price.

The total amount of the organic substance of natural reproducible is sizable; Calculate with the carbon amount, considerably more greater than annual consumption as the fossil-carbon of traffic fuel.Yet, the major part of these renewable materials, about 60%, form by compound, also contain a large amount of oxygen, so their fuel value is quite low.

On the formerly known technical foundation, the known carbohydrate of relative lower energy content that in theory can utilize is transformed into the more macroergic compound that contains how reducible hydrocarbon chain, particularly use as known chemistry, such as gasification technology (gasification technique), method (the R.Agrawal that a kind of energy is extremely intensive, N.R.Singh, F.H.Ribeiro and W.N.Delgass2007. " Sustainahle fuel for the transportation sector " PNAS104:4828-4833 and WO 2006/117317), wherein the total efficiency between charging and the output still is low.Corresponding underlying issue is relevant with biotechnological means according to known technology equally, and the hexose that utilizes these biotechnological meanss will be contained in the carbohydrate changes into more high-octane compound.This example is the production of alcohols, particularly ethanol, and this is described in other specification sheetss US2002/0185447, US 5637502 and WO 03/038067.

Patent specification US 2004/0231661 has also described by water and acid extraction and by hydrolysis and has handled lignocellulose-containing material, so that form the wood sugar and the glucose that can be used to ethanol preparation.Patent specification US 5,221,357 has described to handle by acid hydrolysis and has contained hemicellulose and cellulosic material, and mechanically and by acid hydrolysis handles solid phase to produce the monose that can be used to ethanol preparation, such as pentose and hexose.Patent specification US 4,752,579 has described by acid and/or alkali and enzymic hydrolysis and has handled the shell of cereal seed to separate monose.

Some specification sheetss have been described the lipid production of carrying out from different organic materialss by microbial fermentation.Carbohydrate is used in patent specification US 4,368,056 suggestion in butylic fermentation, it exists with low content in trade waste; And suggestion use microorganism glyceryl ester in the production of biochemical diesel oil, it generates in fermentation.People (Dai such as publication 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.Vol 6 (18), pp.2130-2134,19 September 2007) described be present in vegetable material straw such as the corn stem, the extraction of the carbohydrate in leaf and the paddy by grinding and acid treatment, and utilizes the filtrate that obtains from described processing and washing water as the raw material of producing lipid by the Rhodotorula yeast.Be used to the production of biochemical diesel oil from the lipid of microorganism acquisition.People such as Angerbauer (Angerbauser, C., Sierbenhofer, M.Mittelbach, M. and Guebtz, G.M.Conversion of sludge into lipids by Lipomyces starkeyi for biodiesel production.Bioresource Technology 99 (2008) 3051-3056), conversely, described by alkali and acid wastewater sludge is handled, and it is used as the raw material of biochemical diesel oil in belong to the lipid production that yeast carries out by saccharomyces oleaginosus.

The problem that aforesaid method ran into comprises such fact, and the productive rate of the sugar that can be utilized by microorganism still is low, and perhaps the method that the needs of scale operation are had less importance is applicable to and uses biomaterial as source material.Be apparent that the quantitative objective collection of biofuel manufacturing can not reach by method described herein and used source material.

Therefore, still be starved of new technical scheme, particularly such technical scheme promptly can be used for having the more scheme of the compound of high energy content than the preceding more renewable organic carbon hydrate reserves of the earth being changed into, no matter composition its chemistry or structure.It can change into effectively that to have more high-octane compound (such as fat) will be particularly important with being present in carbohydrate in the various organic materialss to how solving, and its general can be adapted to traffic fuel purposes more or be adapted to the raw material of this purposes.

Summary of the invention

The purpose of this invention is to provide at how the organic-biological material is converted into and have more new departure of the problem of macroergic compound.

Particularly, the purpose of this invention is to provide at how will be converted into the scheme of the problem that is applicable to the lipid of producing biochemical diesel oil from the obtainable carbohydrate ingredient of organic-biological material.

More accurate, the method according to this invention is characterized in that what has been stated at the characteristic of claim 1.

Conversely, purposes according to the present invention is characterized in that what has been stated in claim 17, and the biofuel of being stated in the claim 18.

Method according to purifying city water drain of the present invention is characterized in that, is stated in the claim 19.

The present invention is based on such observation, and promptly when handling biological substance with diverse ways so that recycled fiber is plain and the timesharing of hemicellulose level, Mierocrystalline cellulose and hemicellulose fraction are further degraded, and then microbial population occurs in described fraction quickly.Surprisingly, find in above-mentioned carbohydrate fraction that the microorganism that contains ATP citrate-lyase is also in growth (EC 2.3.3.8, former EC 4.1.3.8), microorganism is with lipid whereby, and particularly triacylglycerol is collected in the cell.Based on these observations, the present invention has developed and has handled biomaterial so that wherein contained Mierocrystalline cellulose and hemicellulose separate with all the other biomaterials, be hydrolyzed then so that hydrolysate is suitable for collecting the microorganism growth of lipid, and be suitable for the production of lipid, and be fit to utilize the lipid that so forms as the raw material of making biochemical diesel oil.

According to specification sheets of the present invention, can be fit to be synthesized the carbohydrate that the microorganism of lipid utilizes from the organic raw material production that comes from multiple source, and/or can be from producing the carbohydrate fraction by the lipid of microorganisms producing.According to the present invention, especially, can produce described carbohydrate from the biomaterial that contains hemicellulose, Mierocrystalline cellulose, starch or non-starch polysaccharide.The suitable carbohydrate that is utilized by microorganism is monose and oligosaccharides particularly, and this comprises hexose and pentose two classes.Described carbohydrate also can the polymeric form, produces the microorganism of lipid so that it can utilize carbohydrate polymer if selected.

Timber with its various ways, comprises the recyclable organism material of the maximum reserves that can be recovered at present.The purposes of timber, particularly its machinery or thermomechanical processing or from the manufacturing or the production of the mechanical substance of timber, be large-scale, and as a kind of method, produce many carbohydrate containing than rill.These side cuts for the timber highly industry have had been found that very little economic value added; And in many cases, they cause cost, because must eliminate the environmental load that is caused by them.As technological challenge, be problematic especially as these effluent.Described effluent is big on volume, but their carbohydrate content is normally low.As rare carbohydrate solutions based on water, they not too are applicable to is intended to utilize in the technology of the carbohydrate in the solution by chemical mode.

Correspondingly, purpose of the present invention also provides the scheme at problem how to utilize the large-scale industry effluent that contains biomaterial, it requires expensive cleansing operation at present or still can not be utilized fully by present method, but it still has the possibility as energy derive.

In the method according to the invention, organic source material is handled, described organic source material contains Mierocrystalline cellulose, hemicellulose, starch, all these, its some mixtures or its degraded product, or alternatively, starch or non-starch carbohydrate former state or be connected in Mierocrystalline cellulose or hemicellulosic materials.Described source material can be carried out mechanically, heat mechanically, physically, chemically, the pre-treatment of biology ground, or carry out pre-treatment by the combination of these processing, perhaps it may be fit to former state and uses.When containing the carbohydrate of polymerized form, preferably adopt water or acid or alkali or its combination that it is handled.After these pre-treatment according to this specification sheets, mixture is divided into filtrate and solid matter, promptly precipitation (Fig. 1) reclaims filtrate or two kinds of fractions.Preferably will adopt the processing regeneration of the source material that water or acid or alkali or their combination carry out, and the filtrate after the precipitation separation will be merged mutually.Preferably the filtrate that will obtain in containing alkaline purification after former state or the above-mentioned recycling, is transported to mixture, and the acid treatment of carrying out source material therein is to increase the amount of solvable monose.Filtrate that will in these are handled, produce or any or source material former state in the precipitation, perhaps source material and filtrate or precipitation or multiple filtrate or multiple sedimentary combination, after possible pre-treatment such as neutralization, decolouring and filtering, be used for the manufacture order cytolipin.Also these filtrates can be merged, dilution or concentrate to reach the microorganism that suitable contents of monosaccharides and composition are used for the manufacture order cytolipin.

From each alternative processing of source material, produced the precipitation of different compositions, depend on whether processing is employing water or carries out in the presence of acid or alkali.In order to increase sugar yield, preferably precipitation is transported to machinery and grinds, former state or in the presence of water, acid or alkali has obtained filtrate and precipitation once more from this processing.Filtrate or precipitation or its some compositions are used for the manufacture order cytolipin, or preferably will precipitate when needing and use strong acid treatment.As acid-treated result, produced filtrate and precipitation once more, therefrom can be with filtrate or precipitation or the extremely production of unicellular lipid of these combination carry.Combine by grinding with handling, can be deposited in addition higher acid concentration in handle.Can be with as the result of this processing and filtrate that produces or precipitation or its composition are used for the production of unicellular lipid.Also precipitation can be removed and burn or be used for production or its precursor of biofuel via additive method.Can or be used for the production of unicellular lipid as multiple composition with each filtrate fraction or precipitation or source material former state.Can with the precipitation that from above-mentioned processing step, obtains by provide in this specification sheets the preceding or after processing step handle once more.In order to increase sugar yield, therefore preferred the employing than stronger acid or the alkali of processing before handled precipitation.

In addition, can carry out enzyme between different processing steps handles or microbial fermentation.

The present invention relates to a kind of method equally, and wherein used alkali and acid are circulated in technology once more.

The present invention relates to a kind of method equally, and the unicellular organism matter that wherein is used for present method is used as the desired biomaterial of the present invention and recirculation, when having reclaimed the lipid of being produced.

Can from the precipitation that processing obtained of carrying out, reclaim developable economically component according to present method.Conversely, also filtrate can be used for other microbiological processes except that lipid production.

Provide scheme according to the method for this specification sheets at problem, described problem little by little is divided into the less fraction that contains relatively large monomer sugar unit for the hexose and the pentose monosaccharides that how will be contained in the biomass carbohydrate, and the microorganism of synthetic lipid can more effectively utilize these fractions to produce lipid.When combining with any processing step, therefrom insoluble substance is promptly precipitated with soluble substance and separate, therefore therefrom produced filtrate, can will be contained in hexose and the pentose former state in the filtrate or after the pre-treatment of necessity, be used for the production of unicellular lipid as the filtrate mixture that is used for unicellular lipid production.Except that filtrate, also precipitation or filtrate and sedimentary composition can be used for the production of unicellular lipid.

Overall merit of the present invention is, it can be used to use simple technology and unit operation, these have used in industry, be used to produce the chemical compound that is rich in energy with Energy Efficient with mode environment, lipid, from the compound of the biogenetic derivation that contains less energy, such as hexose and pentose monosaccharides or by these oligopolymer that forms, and these mixture.

In the method according to the invention, particularly preferably be, by machinery or thermomechanical grinding, new surface is exposed in the organic materials usually, this can adopt the acid of water or varying strength or alkali to handle once more, and therefore can provide solution and precipitation to distribute, therefrom, particularly adopt solution, can obtain the sugar that can be used for the microorganism lipid production more.

When merging solution and sedimentary plural number and plant educt, compare with the single-phase water solution, for machinery or thermomechanical grinding, obtained the quite big hexose that has improved and the productive rate of pentose.

In the method according to the invention, by the filtrate of merging acquisition from bronsted lowry acids and bases bronsted lowry is handled, or by merging filtrate and precipitation, the stream that obtains from multiple processing neutralizes mutually.Therefore, filtrate or precipitation or its composition are the former state available, and under the situation of not regulating acidity, or it is less to regulate the demand of acidity at least.Plural number planted different biomass and also be favourable from the possibility that the biomass of different sources merge mutually.

The exploitability of present method becomes and stresses, so that the wherein formed sugar that can be used for lipid production can be used for microbiology naturally, former state or partly is so that also produce other compounds, such as alcohols.

Special benefits of the present invention is, except in and the effluent or main flow of the machinery of timber and hot mechanical treatment, it is equally applicable to neutralize and discharges the other biological material of the carbohydrate that is used to produce lipid.

The invention further relates to the mixture that produces from the recycle once more by fiber according to present method and form the method for lipid or lipid mixt, described fiber is at hot mechanical treatment, is adopting in the processing of alkali or acid and produce.

Be difficult to other organic raw material of extensive neutral, according to this specification sheets, can be processed to be hexose and pentose or become the oligopolymer that forms by these, and forming lipid from these by microorganism, these organic raw material are recycled fibers that the recycle of the beet pulp that for example obtains from newspaper, from beet and cereal such as avenaceous shell and straw obtains; And other similar devaluation parts of field cultivated plant, sawdust, purified ground wood pulp, straw and peat (the particularly slight peat that decomposes).Up to the present almost unemployed other organic materialss are (submerged) marsh or under water biomass, biomass from the catchment of Mierocrystalline cellulose factory, and become biomass, or become other organic Municipal wastes that dump zone or incineration from the active sludge plant of municipal sewage system.Also can handle these organic materialss, so that make the carbohydrate that is contained in these materials can be used for microorganism with manufacture order cellular biomass and lipid by the numerous embodiments that is used alternatingly present method.For example, can handle Municipal waste water by present method, whereby, will have such advantage among other advantages, promptly harmful microorganism is die in processing.

Fundamental point of the present invention is with the biomaterial of carbohydrate containing, no matter what organic materials it derives from, to handle so that obtain to be suitable for monomer hexose and/or the pentose or their oligopolymer of unicellular lipid production from carbohydrate.Preferably handle by the combination of two or more processing.

Use the method according to this invention, be suitable for the yardstick of the scale operation needs of biofuel, can plant different biomass from plural number and produce source material simultaneously, these source materials can be used for producing microbial cell group's the production of lipid and the production of lipid.Can produce the component that is suitable for the microorganism lipid production, no matter and the composition of biomass, operability and structure.Significant advantage of the present invention also is that by the method according to this invention, but high productivity is produced the available carbohydrate, and can reduce and regulate the needed chemical cost of acidity.

Compare with the existence of prior art, invention described here provides the breakthrough technology, and it combines contains the conversion that the two biomaterial of Mierocrystalline cellulose and hemicellulose becomes hexose and pentose.By identical embodiment, the present invention also makes the monosaccharide unit can utilize the starch that is contained in these biomaterials and non-starch polysaccharide become the production available carbohydrate of unicellular lipid.The present invention is enforceable especially, so that it can be applied to the technical scale of material, described material source is in the reproducible natural resources that is produced by industry or community or the effluent of their devaluation.Can use the method according to this invention to handle the material of cellulose and hemicellulose in a controlled manner, so that therefrom form the precursor of unicellular lipid, described precursor can be utilized in the production of unicellular lipid by the microorganism of safety.

Below, by accompanying drawing and detailed description the present invention has been described more closely.

The accompanying drawing summary

The key step of Fig. 1 Display Realization the method according to this invention.

Fig. 2 shows that hexose and pentose former state or combination are used for the production of cellular material and lipid.

Fig. 3 shows the generation of growth of zymic in the substratum and lipid, has added mixture as carbon source and be used for the production of lipid in substratum, and described mixture is by alkaline purification and the 10% acid hydrolysis generation by the filtrate of acquisition therefrom from husk.

Fig. 4 shows the growth of yeast in substratum, has added the pentose that is purchased as carbon source in substratum.

Method describes in detail

" carbohydrate " refer to comprise aldehyde, acid or ketone groups and, in addition, the organic molecule of a plurality of hydroxyls. Therefore, the scope of carbohydrate comprises by the described compound of term monose, oligosaccharides, polysaccharide, carbohydrate, cellulose, hemicellulose, starch and non-starch carbohydrate.

" cellulose " is the long-chain polysaccharide, and its primary structure is made up of the polymer that the β-1-4 by glucose forms.

" starch " is the long-chain polysaccharide that mainly is made up of α-1-4 and α-1-6 glucose unit.

" available sugar " refers to carbohydrate at this, utilizes described carbohydrate microorganism to breed, and the generation lipid can generate lipid or alcohols with the microorganism that generates alcohols from described carbohydrate.

" hemicellulose " refers to one group of compound being made up of the different hexose of plural number kind and pentose such as galactolipin, mannose, glucose, wood sugar and arabinose.

" monose " is the monomeric unit of carbohydrate, (C-H₂O) n usually is made up of 3-9 carbon atom, and has spatial chemistry difference at one or more carbon atoms. These comprise hexose, and such as glucose, galactolipin, mannose, fructose, they contain 6 carbon atoms, and pentose, and such as wood sugar, ribose and arabinose, they contain 5 carbon atoms.

" oligosaccharides " refers to from the carbohydrate of two or more monose by the formation of O-glycosides key.

" pentose " refers to contain the monose of five carbon atoms.

" hexose " refers to contain the monose of six carbon atom.

" hydrolysis " refers to the fracture under water, acid or alkali impact of carbon-to-carbon, carbon-oxygen, carbon-nitrogen or carbon-sulfide linkage, no matter and whether water participates in reaction. In enzymatic hydrolysis, corresponding reaction is by enzymatic. For example, hydrolysis is a kind of reaction, wherein the peptide bond fission between the amino acid of the O-glycosides key between monose and the carbohydrate or protein.

" adopt water-acid or alkali treatment " in this connection, mean with organic material former state or the product that derives from it extract, mechanically or heat mechanically process, or the combination of these processing is carried out in the presence of water, acid or alkali. According to

-Lowry acid-base theory, acid refer to provide chemical substance, molecule or the ion of hydrogen ion (proton), and alkali refers to accept material, molecule or the ion of hydrogen ion (proton). Acid refers to accept the so-called Lewis acid of duplet equally; Lewis alkali refers to provide the so-called Lewis alkali of base-pair. According to these definition, material is not limited to the aqueous solution as the activity of acid or alkali. According to these definition, in this manual, term " acid " and " alkali " refer to the bronsted lowry acids and bases bronsted lowry catalyst equally. In this manual, acid refers to any sour phase equally, and it can serve as acid therein, such as with gas, solid or liquid form; For example, as the aqueous solution. Correspondingly, alkali refers to any alkali phase, and it serves as alkali therein, such as with gas, solid or liquid form; For example, as the aqueous solution.

" organic source material " in this specification refers to by biogenic any organic substance of living. Organic source material in this specification is also referred to as biomaterial.

Particularly, organic source material refers to comprise the organic material of polysaccharide. " polysaccharide " refers to that it also can contain the compound except monose from the carbohydrate polymer of monose formation. Polysaccharide is, for example, and cellulose, hemicellulose and starch. Polysaccharide comprises equally, among other, and alginates, dextran (glucane), synanthrin and Arabic gum. Outside other polysaccharide, mannosan (mannane) also should be mentioned. Source material can comprise the polysaccharide former state or as mixture, perhaps it can comprise their catabolite.

" SNSP " refers to carbohydrate, and their molecular structure lacks the typical α of starch-1-4 key, or α-1-4 key is rare. SNSP is, for example, and dextran, alginates, synanthrin and Arabic gum. SNSP also comprises hemicellulose and cellulose. Other SNSPs are, for example, and the carbohydrate polymer that in algae, exists.

" cultivated plant " refers to plant, is being for useful purpose with it for planting or sow in the soil of its preparation.

Term " lipid " refers to fatty material, and its molecule contains usually, as a part, and the aliphatic hydrocarbon chain, it is dissolved in organic solvent but is poorly soluble in water.

In the present invention, the lipid that forms in microorganism mainly is three-, two-or list-acetoglyceride, or sterol ester, but other lipids also can form in cell such as phosphatide, free fatty, sterol, dolichol, sphingolipid (sfingolipids), glycolipid and cardiolipin.

The present invention also can be used for the production of biochemical diesel oil or renewable diesel.

According to EU indication 2003/30/EY, " biochemical diesel oil " refers to have the diesel quality that can be used as bio-fuel from the methyl ester of vegetable oil or animal oil production.

Renewable diesel refers to animal, plant or microbe-derived hydrotreated lipid, and whereby, the microorganism lipid can derive from bacterium, yeast, mould, marine alga or other microorganism.

The source material of the method according to this invention can be cellulose, hemicellulose and living beings, be preferably wood pulp, may contain adhesive, it by machinery or thermomechanical method or other physical methods, or chemistry, enzyme or microbiological method or the combination by these methods have produced. In the situation of not revising this method, the vegetable material that contains starch, but such as potato, its seed, maize and the paddy rice rise of part raise crop, respectively, and beet, in addition, comprise the beet pulp that is contained in SNSP wherein, can be equally the source material of the method according to this invention. The part that contains the plant of SNSP such as beta glucan also suits as source material. The method also is fit to use carbohydrate as source material, and described carbohydrate is such as alginates, and it derives from unicellular organism. The composition of above-mentioned source material also can comprise protein and the lipid of variable quantity, and they serve as equally source material for the synthesis of the microbial growth of lipid and are used for lipid production.

The source material of the method according to this invention also can be, for example, from the recycled fibers of the recycling acquisition of newspaper, the shell of beet pulp and cereal, such as oat, sawdust, refining ground wood pulp, peat and straw. Other in the method according to the invention useful source material be, for example, microbiological materials, such as unicellular organism matter, the living beings marsh or under water, comprise algae and microalgae, living beings from the catchment area of cellulose factory, and become living beings from the activated sludge plant of municipal sewage system, or contain biological components and be used at present incineration, compost or be used for other municipal wastes of some additive methods, this carbon that causes being contained in refuse extensively discharges as carbon dioxide.

Method according to preferred embodiment of the present invention comprises at least one step, wherein will be from the filtrate of organic material acquisition according to the present invention or the composition of filtrate, the composition of precipitation or precipitation, organic material former state or any composition in these, be transported to mixture, lipid production takes place therein. Based on being used for growth of microorganism and producing the composition of seviparous filtrate or filtrate or the preferred monose types of compositions of composition of precipitation, the composition that precipitates or filtrate or precipitation, can select the alternate embodiments of the method. Therefore, filtrate or precipitation are selected from the group that produces by processing biomaterial, and wherein said biomaterial preferably is selected from following mass treatment:

I) water,

Ii) acid and

Iii) alkali, and

After this fibrous precipitation is separated with not fibrous filtrate.

Randomly, will precipitate again carry out (i), (ii) or (iii) in the bar any processing once or more times, preferably the gained precipitation is carried out machinery or thermomechanical grinding, and precipitation separation and filtrate.

Depend on and used what biomaterial and expected what monose, with biomaterial water, acid or alkaline purification; Preferred acid or alkali; Usually, the aqueous solution by acid or alkali.When needing, processing can be carried out several times.Can adopt plural number to plant different solution identical biomaterial is handled continuously, and can be added to the water strengthening the separation of carbohydrate and the compound of hydrolysate.

Listed source material among the inventory group I below can be adopted water treatment in first step, or enhancement process adopts the mixture process of water and acid as a result the time if desired.

Biomaterial among the group II is preferred employing acid treatment in first step.

Biomaterial among the group III is preferred employing alkaline purification in first step.When having reclaimed precipitation and filtrate, can adopt acid to handle once more filtrate.

Group I

Derive from timber machinery, thermomechanical, enzyme or microbiology is handled or from these treatment combinations or from the biomaterial of cultivated plant.

Group II

The source material of recycled fibers, beet pulp, husk, straw, wheat bran, cereal-granules, complete cultivated plant, cultivated plant, TMP slurry, MDF slurry or starch-containing or non-starch polysaccharide.

Group III

Sawdust, purified ground wood pulp, husk, straw and xylophyta part, TMP slurry, MDF slurry, beet pulp, cultivated plant, they may contain the starch of variable quantity.

Can by will be for example one or more plant enzymes and join in the treatment soln, preferably join and make in the treatment soln that water makes, come enhancement process.Also can add enzyme between the kinds of processes step handles or microbial fermentation.

In the step of following, any in the microorganism that produces lipid and filtrate or precipitation or their composition contacted in substratum, make microorganism cells production lipid and reclaim lipid.

The fibrous precipitation that also preferably will obtain from above-mentioned biomaterial treatment step uses such method to handle, and wherein fibrous precipitation is mechanically ground, and separates fibrous precipitation and not fibrous filtrate.

In addition, can adopt strong acid treatment to grind the fibrous precipitation of acquisition and separable fibrous precipitation and not fibrous filtrate from machinery.After the acid treatment, also can carry out machinery and grind precipitating once more recycle back, or, as mentioned above, precipitation can be used for utilizing the lipid production of microorganism.

In addition, can with the biomaterial acidifying and mechanically or heat mechanically grind, and separate fibrous precipitation and not fibrous filtrate.

The filtrate that obtains from any above-mentioned processing or precipitation or their composition can be joined the substratum of the microorganism that produces lipid.

Usually, the total amount of carbohydrate is a 0.5-10% weight in the filtrate.In the middle of these, the carbohydrate that can be used for biomass and lipid production generally includes at least 0.5% weight, preferably at least 3% weight, more preferably 4-5% weight.For example,, can from biomaterial, isolate more carbohydrate, like this, the amount of carbohydrate can be increased to more favourable level by biomaterial being ground and extracting again.Yet the amount of carbohydrate preferably is lower than 30% weight in the filtrate, more preferably less than 20% weight.

The microorganism growth that can produce lipid produces lipid then so that they at first produce biomass, or produce simultaneously biomass and lipid the two.

Depend on the source of biomaterial and with treatment process that it carried out (adopting water, acid or alkaline purification), can obtain hexose monose, pentose monosaccharides or with the two of multiple ratio, as shown in Figure 2.From some biomaterials, mainly can obtain hexose, yet from the other biological material, mainly be pentose.Suitable selection along with the microorganism that can produce lipid, can the production that the filtrate of hexose or precipitation or their composition are used for cellular material will be contained mainly, after this, can the production that the filtrate of pentose or precipitation or their composition are used to go deep into the lipid among the cellular material will be contained mainly.Alternatively, lipid can be from hexose production in cellular material.Cellular material and lipid can be from hexose productions.Correspondingly, the suitable selection of microorganism by can producing lipid, can the production that the filtrate of pentose or precipitation or their composition are used for cellular material will be contained mainly, after this, can the production that the filtrate of hexose or precipitation or their composition are used to go deep into the lipid among the cellular material will be contained mainly.Alternatively, lipid can go deep among the cellular material from pentose production, or cellular material and lipid can be from pentose productions.Cellular material and lipid the two also can be from the mixture production of pentose and hexose.

The processing of biomaterial

Below, processing according to the biomaterial of preferred implementation of the present invention has been described.Usually, processing is to carry out with the combination of two or more processing.

Preferably source material is extracted under 90-100 ℃ temperature.The favourable embodiment of acid extraction is to use the mineral acid of 5-10%, such as sulfuric acid, or organic acid, such as citric acid or acetic acid, and in alkaline extraction, the NaOH of preferred 0.5-2.0M.Treatment time is in extensive range; Be preferably 1-10 hour, be generally 2-8 hour, be most suitably 2-4 hour.Also can preferably other be handled, such as the processing of adopting enzyme, microorganism, oxidation or reduction chemical, or the combination of these processing, make up with water extraction.

According to present method, can be preferably be deposited in 100-210 ℃ and be generally under 150-200 ℃ the temperature and mechanically grind what in extraction, produce, preferably carried out 2-20 minute, be generally 5-11 minute.Pressure is preferably 6-8bar.The material that is produced is filtered, use aforesaid method to handle filtrate so that it is suitable for the production of unicellular lipid.Precipitation can be transported to the acid treatment of adopting strong acid, preferred described processing is to adopt 40-72% sulfuric acid, 65-70% vitriolic processing suitably.Normally, the treatment time is 2-8 hour, preferred 2-4 hour.This method can adopt any acid to realize, provides proton catalytic hydrolysis whereby.Suitable acid is, for example, and strong mineral acid, phosphoric acid, sulfuric acid, or the oxygen acid of sulphur, nitrogen, chlorine, bromine and iodine.

The result of hydrolysis can be divided into filtrate and precipitation, filtrate is handled so that be suitable for the production of unicellular lipid, precipitation can be transported to dilute acid soln, the sulphuric acid soln of preferred 5-10%, and can under the pressure of 170-200 ℃ temperature and 6-10bar, carry out 10-20 minute grind.Mixture is divided into filtrate and precipitation, wherein the former is handled so that be suitable for the production of unicellular lipid, and can remove precipitation.

The above-mentioned embodiment of present method is a target with the production that utilizes the carbohydrate that exists in the source material to be used for unicellular lipid fully.Yet, from the mode of extraction source material, also can only realize present method to selected part, for example, when the precipitated fiber material also will be used to other purpose.The method according to this invention, it is characterized in that, preferably in their integral body, comprise above-mentioned steps, but it is unrestricted to the degree that departs from basic skills from the part of carrying out present method, perhaps with unit operation sequence, and in the production of unicellular lipid, use the monose fraction that produces by these operations.According to this specification sheets, also processing step can be joined in present method, wherein will be used for the productions of unicellular organism matter or alcoholic acid from the fraction that contains monose that source material obtains, except that lipid.

In the following description, certain methods according to preferred implementation of the present invention has been described.Identical method can be applied to the starting material except that the starting material that provide in this specification sheets.

I.

According to preferred implementation of the present invention, will comprise that the xylon of ground timber, TMP slurry, sawdust or ground wood pulp is handled according to following composition step:

A. the 100g xylon was extracted preferred 2 hours 2-4 hour at 90-100 ℃ in 1 premium on currency.By filtering separation precipitation and solution, and reclaim solution.The scope of carbohydrate productive rate is 2% to 5% in the solution, depends on the manufacture method of xylon, and the TMP fiber of high treatment temp (above 170 ℃) is generally 4-5%.

B. in order to increase the carbohydrate productive rate, with the fiber fraction in 1 liter about 1 (for example, mineral acid) of 5-10% acid (preferred 5%), pH, under 90-100 ℃ temperature hydrolysis 2-4 hour, preferred 2 hours.Separate remaining precipitation and solution; Reclaim solution.

C. preferably from precipitation, topple over and excessive acid, will be deposited in the defibrator (for example, wing or disc refiner) and grind once more.Preferably temperature of precipitation is increased and enriched material is remained in the mixture by presteaming one minute.Temperature is increased to 150-200 ℃, and preferred 170 ℃, pressure is 6-8bar (wing refining mill).According to timber, in 2 to 15 minutes scope, select to grind the time.By filtering separation precipitation and solution, and reclaim solution.

D. the strong acid with 40-72% (sulfuric acid) joins in the precipitation fraction, makes it room temperature absorption 2-4 hour, preferred 2 hours.Topple over and excessive acid, and will be deposited in the refining mill (for example, wing or disc refiner) and grind once more.Preferably temperature of precipitation is increased, after this do not discharge the water in the enriched material by presteaming one minute.Temperature is increased to 150-200 ℃, and preferred 170 ℃, pressure is 6-8bar (wing refining mill).According to timber, in 2 to 15 minutes scope, select to grind the time.Mixture is filtered precipitation separation and solution.Reclaim solution and will precipitate and be used as fuel.

The solution fraction that any step from form steps A-D obtains further can be handled, measure by decoloring method, pH regulator and other promotion microorganism growth, anhydrate such as removing, and the solution that so obtains can be used for the substratum of microorganism, can make the microorganisms producing lipid.Make up by forming steps A-D, the original wood fiber material of 40-65% can be changed into soluble form from the fiber as source material.The dissolved carbohydrate comprises glucose, semi-lactosi, seminose, wood sugar and pectinose unit, and their total part is typical for used timber type.

II.

According to another preferred implementation of the present invention, xylon, ground timber, recycled fibers, TMP slurry, sawdust or the ground wood pulp of 100g have been used, at 1 liter 4-8% alkaline solution, among the NaOH of preferred 1M, under 90-100 ℃ temperature, extracted 2-4 hour preferred 3 hours.By at room temperature filtering separation precipitation and solution, and reclaim two kinds of fractions.Depend on the processing of source material fiber, the productive rate of carbohydrate falls within the scope of 5-8% in the solution.Preferably this solution is handled by any step in the following composition step:

A. with vat liquor in the above described manner once more former state be used to extract following fibre batch, and reclaim solution.

B. any step among the step B-D of embodiment before the basis adopts sour xylogen and oligosaccharides and the polysaccharide that is hydrolyzed and has been dissolved in wherein to handle with vat liquor.

C. according to the vat liquor of the utilization of the step B among the embodiment I according to steps A recirculation.

In order to increase the monose productive rate, the precipitation of preferably using following method or its combination to reclaim in alkaline purification is handled:

D. according to before embodiment I step B, C, D or according to they institute in steps, precipitation is handled.Mixture is filtered, neutralization solution, and reclaim precipitation and solution the two.

E. will adopt alkali to handle once more from the remaining precipitation of the steps A of embodiment II, preferably under such condition, promptly wherein mixture be ground 2-8 minute, preferred 6 minutes, under the pressure of 4-10bar, preferred 8bar, temperature is 170 ℃.Filter, with solution neutralization and recovery.By this extra grinding, the dissolved quantity of material can be increased to 27% of fiber original vol in the source material.

To form the carbohydrate containing solution (comprising glucose, semi-lactosi, seminose, wood sugar and pectinose unit) that produces among steps A-E at each and be processed into the form that is suitable for the lipid production of being undertaken by microorganism, for example, by decolouring, pH regulator, or anhydrate by from solution, removing, and it is used as the substratum of microorganism, or as its part, and make the microorganisms producing lipid.

III.

According to the 3rd preferred implementation of the present invention, xylon, ground timber, TMP slurry, sawdust, ground wood pulp, recycled fibers (or according to the neutralization of above-mentioned steps B preparation extract) have been used, adopt the acid of 5-10% (preferred 5%) intensity to be hydrolyzed, by treat (for example to add 1 liter of described acid in the hydrolyzed material to every 100g, mineral acid), being about under 1 the pH, use 90-100 ℃ temperature to carry out preferred 2 hours 2-4 hour.By filtering separation precipitation and solution.The amount of fiber is calculated from source material, and solution contains the carbohydrate of 4-14%.Can precipitation as fiber, or can it further be handled to increase the monose productive rate according to the step C in above-mentioned first embodiment or D or the two.From precipitate and separate, neutralization, filtration, and former state is used or with substratum or its part of spissated form as microorganism, make the microorganisms producing lipid with solution.

IV.

According to the 4th preferred implementation of the present invention, use the xylon, ground timber, TMP slurry, sawdust, ground wood pulp of 100g or from the remaining precipitation of steps A of steps A-C or the embodiment II of embodiment I, the acid that adds 5-10%, pH (for example is about 1, mineral acid), make its absorption 2-4 hour, preferred 2 hours.Topple over and excessive acid, and will be deposited in the refining mill (for example, wing or disc refiner) and grind once more.Preferably temperature of precipitation is raise, and enriched material is not flowed out by presteaming one minute.Temperature is increased to 150-200 ℃, and preferred 170 ℃, pressure is 6-8bar (wing refining mill).According to timber, in 2 to 15 minutes scope, select to grind the time.Remove by filtering to precipitate from solution.Can be with residual precipitation as fuel or grind once more in the above described manner to increase the monose productive rate in the solution.With solution neutralization, filter and former state or use, and using in the mode described in the embodiment I-III with spissated form.

V.

According to the 5th preferred implementation of the present invention, use the xylon, ground fiber, TMP slurry, sawdust, ground wood pulp of 100g or according to the residual precipitation of any embodiment among the embodiment I-IV, the strong acid of adding 40-72% (for example, sulfuric acid), make it room temperature absorption 2-4 hour, preferred 2 hours.Topple over and excessive acid, and will be deposited in the refining mill (for example, wing or disc refiner) and grind once more.Preferably temperature of precipitation is increased, and enriched material is not flowed out by presteaming one minute.Temperature is increased to 150-200 ℃, and preferred 170 ℃, pressure is 6-8bar (wing refining mill).According to timber, in 2 to 15 minutes scope, select to grind the time.Carbohydrate mixture is filtered and precipitation separation and solution.From the precipitation as source material, dissolved substances partly falls within the scope of 40-65%.Can maybe it can be ground once more to increase the monose productive rate with residual precipitation as fuel.Solution is neutralized, filters and handles according to any embodiment among the embodiment I-IV.

Alternatively, the strength sulfuric acid that makes 40-72% is adsorbed onto in the precipitation 1-3 hour in room temperature, preferred 1.5 hours.After this, acid is diluted to 5%, and under normal pressure and 100 ℃, boiled 4 hours.Further handle as above.

Utilize the lipid production of microorganism

The invention provides,, be contained in carbohydrate in the source material and hexose and pentose monosaccharides and will be widely used for lipid and unicellular organism matter by microbiological processes via the composition of the filtrate that in a plurality of composition steps, produces.Adopt pre-treatment,, can be used in combination the production that is used for unicellular lipid with the filtrate former state of each recovery or with multiple moisture fraction such as washing, neutralization, decolouring or other processing back operations.Because the processing mode of source material, this is a part of the present invention, and the present invention also is applicable to alcohol production.

With filtrate, promptly moisture fraction, or any combination of filtrate join in the microbiological culture media of having inoculated or inoculated microorganism, make the microorganisms producing lipid.Lipid is reclaimed with the form of microbiological materials, or with lipid and described separating substances, and the therefrom isolated lipid of recovery and microbiological materials the two.Can use known method by from cell, removing lipid or passing through fragmented cell and reclaim lipid.Can with an organic solvent from cracked cell, extract lipid.Can be applicable to lipid recovery method of the present invention, for example, at the publication Yeast of Z.Jacob Lipids:Extraction, Quality Analysis, and Acceptability, Critical Reviews in Biotechnology, 12 (5/6); 463-491 is described in (1992).The preferred method that is used to reclaim lipid is to be separated.The processing that the lipid that will form in microorganism is processed into fatty acid ester also can take place under previous microorganism cells homogenization and the fatty isolating situation subsequently not having.

Preferred implementation of the present invention relates to the method that forms lipid or lipid mixt the processing of organic source material from the carbohydrate mixture that produces, described carbohydrate mixture comprises the hexose and the pentose of monomer and oligopolymer form, according to present method, the mixture of carbohydrate containing is joined in the water-containing medium of having cultivated the microorganism that produces lipid, described culture medium supplemented has the desired nutrient substance of growth, adopt described microorganism to carry out the inoculation of substratum, culturing micro-organisms also makes it to produce lipid, reclaim cellular material, and separate lipid or lipid mixt from cell or fatty cell, or their component is used by former state.

The method according to this invention provides the special elastic at the microbiology lipid production.Containing the two fraction of hexose and pentose is the natural carbon source that is used for the microorganism of many generation lipids.Therefore, this method has microorganism is selected in permission in wide region advantage equally, for example based on the ability of lipid production, the productive rate of biomass, the type or the culture condition of cultivation.Other components of microorganism except that lipid, can be utilized with many different mode energy efficients, therefore improved whole economic performances according to technology of the present invention.The optimal way that utilizes the microbiological materials that does not contain lipid is that hydrolysis and circulation enter the substratum of the microorganism that produces lipid or be used as feed or nutritive substance.Also various ingredients never may be contained in the microbiological materials of lipid and separate, described component is such as (especially) carbohydrate, tinting material, beta-glucan, sterol, sterol ester or protein.

Microorganism is to select from the microorganism of the accumulation fat of natural or hereditary modification, preferably selects from yeast, mould, bacterium and algae, more preferably selects from yeast and mould, most preferably selects from yeast.Importantly, the microorganism that will be utilized can produce lipid from hexose or pentose or from the two.Therefore, the present invention includes the ATP that contains based on them: all microorganisms of the lipid accumulation of Citrate trianion lyase activity (EC 2.3.3.8).

The yeast belong that is applicable to synthetic lipid of the present invention comprises dependent of dead military hero down: Candida, ascomycetous yeast belongs to, saccharomyces oleaginosus belongs to, Rhodotorula and genera cryptococcus, they comprise the bacterial strain with the synthetic lipid of pentose wood sugar, such as candida curvata (D) (Evans, C.T.and Ratledge, 1983.Acomparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture, Lipids 18 623-629), Rhodotorula gracilis (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 red winter spore yeast (Rhodosporidium toruloides), rhodotorula glutinis (Rhodotorula glutinis), herbage rhodotorula bacterium (Rhodotorula graminis), the Si Shi saccharomyces oleaginosus, the produce oil saccharomyces oleaginosus, candida lipolytica, genera cryptococcus, cryptococcus albidus, trichosporon cutaneum and trichosporon pullulans (Fall, R., Phelps, P.and Spindler, D.1984, Bioconversion of Xylan to Triglycerides by Oil-Rich Yeasts.Appl.Environ.Mircobiol.47,1130-1134), and the bacterial strain that the pentose pectinose is synthesized lipid, Si Shi saccharomyces oleaginosus (Naganuma, T., Uzuka, Y.and Tanaka is Factors Affecting Total Cell Number and Lipid Content of the Yeast K.1985.Physiological, Lipomyces starkeyi, J.Gen.Appl.Microbiol.31,29-37).

Correspondingly, be applicable to that the mould Pseudomonas of accumulation fat of the present invention comprises, among other mould Pseudomonas:

-Eurotium

-black wool Pseudomonas

-Clodosporidium

The silver-colored mould genus of-little gram

-Emericella

-Fusarium

-mortierella sp

-mucor

-Penicillium

-rotten mold genus

-Rhizopus

-Trichoderma

Correspondingly, be applicable to that the bacterium genus of accumulation fat of the present invention comprises, among other bacteriums belong to:

-motionless stalk Pseudomonas

-Actinobacter

-Anabaena

-Jie stalk Pseudomonas

-Bacillussubtilis belongs to

-fusobacterium

-Flexibacterium

-micrococcus sp

-branch stalk Pseudomonas

-Nuo Kaer Pseudomonas

-nostocaceae

-Oscillatoria

-Rhodopseudomonas

-Rhod

-Rhodomicrobium

-Rhodopseudomonas

-Si Wani Pseudomonas

-streptomyces

-Vibrio

Correspondingly, be applicable to that little Trentepohlia of accumulation fat of the present invention comprises, among other little Trentepohlias:

-grape Trentepohlia

-one-tenth born of the same parents' Trentepohlias (Brachiomonas)

-clothing Monas

-Chlorella

-Crypthecodinium cohnii belongs to (Crypthecodinium)

-Dunaliella salina belongs to (Dunaliella)

-Euglena

-microballoon Trentepohlia (Nannochloris)

-little Trentepohlia (Nannochloropsis)

-Navicula (Navicula)

-Nitzschia (Nitzschia)

-schizochytrium limacinum belongs to (Schizochytrium)

-rib line Trentepohlia (Sceletonema)

-Scenedesmus

-merge little Trentepohlia (Tetraselmis)

-genus thraustochytrium (Thraustochytrium)

-my Ken Shi Chytridium (Ulkenia)

According to preferred implementation of the present invention, it is synthetic that such microorganism is used to lipid, and they synthesize the cell that enters them with the lipid of fatty acids with the amount that is preferably dry cell weight 12-65% weight.

According to special preferred implementation of the present invention, with the biomass that do not contain lipid that form in the present invention, handle in the mode that is suitable for microorganism as the nutrient substance in the substratum.Except that these components, substratum can be supplemented with the microorganism used therefor preferred ingredients.In order to produce lipid, the common requirement of microorganism is among other, carbon source, it comes from source material in the present invention, nitrogenous source, such as inorganic ammonium salt (for example, and the micro-nutrients source ammonium sulfate) or organic nitrogen source (for example, the cellular material of amino nitrogen, yeast extract or hydrolysis),, such as phosphoric acid salt, vitriol, muriate, microorganism or cationic source (for example, Mg, K, Na, Ca, Fe or Cu ion source), whereby, these components can be joined in the substratum when needing.When using method of the present invention, the lipid concentration of cell is preferably 40% weight, most preferably 65% weight.

The manufacturing of biofuel

The fatty acid ester that is contained in microbe-derived lipid can be handled to be suitable as biofuel by any currently known methods.A kind of preferable methods is to adopt short chain alcohol to carry out transesterify, and particular methanol is to obtain the alcohol ester of lipid acid.

In the manufacturing of biochemical diesel oil and renewable diesel, the impure effluent that produces when when transesterify, taking place, its effluent contains alkylol cpd, such as glycerine or non-esterified fatty acid salt, and it is difficult to develop in the mode of Energy Efficient, it can be utilized the production that is used for unicellular lipid again, but described lipid is former state available or recirculation become biogenetic derivation other contain the material of glyceride.

The advantage of invention

Advantage of the present invention comprises such fact, and promptly present method devices needed is simple, and so long as relates to and making and the correlation technique of operation all is known.The method according to this invention is not limited to any industrial scale, but can easily be amplified or dwindle, according to the amount of the content of carbohydrate and pending source material.Carry out this method and do not require the heating of power consumption, other chemical catalysts that presser unit operation or deacidification, alkali or enzyme catalyst are outer to produce lipid.Present method only requires that use can mix the chemical in the processing of the internal recycle of the method according to this invention or described biomaterial.Present method does not require the dehydration that the available sugar soln is increased expense yet, and former state is used for or be used as microbiological culture media because the carbohydrate solutions of dilution is suitable for.Whole economic performances of present method are to improve by such fact, i.e. biomass that do not contain lipid that produce therein, except that internal recycle, have many different purposes, production such as the individual organic component, as feed or as the raw material of feed, or as the supplemental medium that is used for microorganisms producing.Present method is equally applicable to unicellular organism matter and alcoholic acid production.

Advantage according to biomass graded processing of the present invention is organic materials hydrolysis widely, and therefore, compared with prior art, the biomaterial that is used for unicellular lipid production has better operability.In addition, handle the filtrate that obtains or precipitate the use that neutralizes mutually and reduced the desired chemical that neutralizes from bronsted lowry acids and bases bronsted lowry.

The scheme of prior art has been described the method that contains the microbiology step that is used for alcohol production, and wherein the material of carbohydrate containing is used as raw material, former state or change into monose.Patent application publication US 2002/0185447 and patent US 5637502 have also described the treatment process of carbohydrate, and such as adopting acid or alkaline purification, following is alcoholic fermentation (alcoholic fermentation) after these are handled.Handle about microbiology, two kinds of described methods all are confined to alcohol production, have wherein used the hexose that forms from polysaccharide.In patent application publication US 2003/0096385, used microorganisms producing in prenyl ethanol (alcoholic acid geranyl and farnesyl derivative).Patent application publication WO 03/038067 has described such method, and wherein the genomic modification of fungi microbe can obtain utilizing the biology of pentose.This publication is only at alcohol production.

The invention provides new possible scheme, particularly occur in pulp and paper industry, particularly as the hot mechanical wood working that is used for the traffic fuel feedstocks at the more remarkable moisture effluent exploitation of the mechanical wood working of heat.The present invention consider with being connected of groundwood pulp manufacturing in the biology load of the carbohydrate containing effluent that produces, therefore, the energy expenditure of wastewater treatment remains to be reduced.With concrete form, the present invention is a lipid production technology, it provides the eco-friendly scheme that is used for the raw material of traffic fuel, biochemical diesel oil or renewable diesel at generation, for example from the moisture fraction of rare carbohydrate containing of producing the corresponding mechanical treatment of TMP technology or timber.

Present method also provides the alternatives at other source material exploitations.According to present method, recycled fibers such as printing paper, wrapping material and comparable based on cellulosic material, can be used as source material.

Therefore compared with prior art, the present invention meets principle of sustainable development, and the availability by increasing lipid feedstocks also reduces aggregate demand from organic lipid in other sources.Therefore the present invention has improved the availability based on the biofuel raw material of renewable natural resources, and helps their productive expense is brought to the acceptable terminal user's level of human consumer.

In the place of the full use of considering source material, the present invention not only is confined to the full use of contained carbohydrate.Present method also comprises the stage that the lipid composition that allows can to extract in the wood substance fraction is recovered.When adopting the water treatment source material, filtrate is separated lipid composition, and described lipid composition can reclaim from aqueous solution by the method that those skilled in the art are familiar with.As the acid-treated result of source material, the lipid that can extract in the fraction obtains free fatty acids, described free fatty acids with insoluble alkaline earth salt such as Ca ⁺⁺Salt form separates with filtrate, and after precipitate and separate, they are become alcohol ester by transesterify.Correspondingly, the alkaline extraction of source material produces soap from extractible fraction, and described salt is water miscible, therefore is mixed in the filtrate.In the present invention, described salt is converted to water-insoluble salt such as Ca ⁺⁺Salt form is from aqueous phase separation precipitation and be used to make alcohol fatty acid ester.

The following example intention is illustrated the present invention, and they can not be understood that to limit the scope of the invention by any way.The present invention also is confined to used microorganism strains never in any form.The present invention not only can realize by used bacterial strain, and can be by other bacterial strains mutually of the same race or that belong to, or other microorganisms belong to or the bacterial strain of planting or realize by the microorganism strains of genetic modification.The microorganism of generation lipid is available normally, and they are present in several bacterial strain preservation mechanisms, for example ATCC, DSM or the like.Produce the microorganism of lipid and use the lipid production technology of microorganism (comprising algae) to be described in the literature, for example in works: Single Cell Oils, eds.Z.Cohen and C.Ratledge, AOCS Press, 2005 and Microbial Lipids, eds.C.Ratledge and S.G.Wilkinson, vol.1 and 2, Academic Press, 1988.

Embodiment

Embodiment 1

Xylon, ground timber, TMP slurry, sawdust and each 100g of ground wood pulp were stored in 1 liter of boiling water 90-100 ℃ 2 hours.Solution is filtered out from filamentary material (hereinafter being called precipitation).Reclaim precipitation, and handle precipitation to increase the monose productive rate according to the multiple alternate ways of a kind of use in the alternate ways that provides in embodiment 4 or 5.The carbohydrate productive rate scope of solution is 2% to 6%, depends on source material, when using when high temperature (above 170 ℃) descends ground dragon spruce fiber as source material, is generally 4%.In these solution some are reused under the situation of not evaporating and are used to extract following batch fiber, and some in these solution are evaporated and concentrate until the dry matter content that reaches about 20% weight.The recovery of spissated filtrate is used for the production of unicellular lipid.For the seeds of being extracted, the dissolved carbohydrate is divided into hexose and pentose usually.

Embodiment 2

Xylon, ground timber, recycled fibers, TMP slurry, sawdust and ground wood pulp are joined in 1 liter of 5% alkaline solution (NaOH) separately, and stirred 3 hours at 90-100 ℃.Filter at normal temperature, and precipitation is further handled the production that is used for monose according to embodiment 4.Handle so that the part of each solution is used to handle the fiber of next batch once more extracting solution, a part is neutralized, a part is hydrolyzed according to embodiment 4, has suffered because some xylogen and some oligosaccharides and polysaccharide have been dissolved in alkaline extraction solution.The mean value that never keeps source material weight 5% by described alkaline purification with the solution of source material acquisition.

The precipitation that will obtain in original alkaline purification adopts alkali to handle again, grinds 6 minutes by the alkaline purification before repeating and under the temperature of the pressure of 8bar and 170 ℃.Use thisly further to grind, the amount of material in the alkaline solution of being dissolved in can be increased to the mean value of source material original vol 27%.Solution becomes black, after neutralization, hydrolysis and concentrating, adopts gac and ion-exchanger to handle them and is used for decolouring.Reclaim mixture and use it for the manufacture order cytolipin.

Embodiment 3

With xylon, ground timber, TMP or MDF slurry, sawdust, ground wood pulp, recycled fibers and according to the neutralization of embodiment 2 preparations each 100g of extract in 1 liter of 5% mineral acid (pH 1) 90-100 ℃ of hydrolysis 3 hours.To precipitate and solution separating by filtering.Solution contains the monose of active material amount average 10%.Each solution handled so as a part to be neutralized, to filter and by evaporation concentration until the monose amount that reaches about 20% weight, concentrated solution is reclaimed the production that is used for unicellular lipid.With the part of solution once more former state be used for the hydrolysis of following batch of source material, as described in this embodiment.According to the following examples 5, precipitation is further handled to increase the monose productive rate.

Embodiment 4

With xylon, ground timber, TMP slurry, MDF slurry, sawdust, ground wood pulp, straw, paddy skin with to join concentration according to each 125g of precipitation of embodiment 1-3 be in 10% the sulphuric acid soln, make acid absorption 2 hours.Topple over and excessive acid, and mixture is guided to the wing refining mill, by presteaming 11 minutes temperature is raise, and do not allow enriched material flow out, afterwards, temperature is increased to 160 ℃ and pressure to 8bar, use the wing refining mill to grind.11 minutes the mixture time that grinds is selected at the dragon spruce fiber.After grinding, will precipitate and solution separating.Each precipitation is cut apart so that a part is used to incinerate, and a part is ground according to the following examples 5 once more to increase the monose productive rate.Correspondingly, with solution neutralization, filter and by evaporation concentration until the monosaccharide concentration that reaches about 20%.These solution are reclaimed the production that is used for unicellular lipid.

Embodiment 5

1D. join in 40% sulphuric acid soln with xylon, ground timber, TMP slurry, sawdust, ground wood pulp, straw and corn fibre and according to each 125g of the remaining precipitation of embodiment 1-4, and make it room temperature absorption 2 hours.Topple over and excessive acid, and will be deposited in and grind in the wing defibrator so that temperature of precipitation is raise and do not allow enriched material flow out by presteaming 11 minutes.Then temperature is increased to 170 ℃ and pressure to 8bar.For straw and corn fibre, the optimum time of grinding is 11 minutes.To precipitate and solution separating.The ratio of lysed material on average surpasses 50% of source material.With solution neutralization, filter and by evaporation concentration, and reclaim the production that is used for unicellular lipid.With precipitation is that residual pars fibrosa ground incinerates, and part grinds to increase the monose productive rate once more according to this embodiment.

Equally the vitriol oil (72%) is adsorbed onto in the above-mentioned source material individually, the treatment time is 2 hours, in room temperature.Subsequently, acid is diluted to 5% and under normal pressure and 100 ℃, boiled 4 hours.Further the purposes of the processing and the fraction that produces as above takes place.

Embodiment 6

Can be suitable for the monose of manufacture order cytolipin from husk, straw and the production of paddy skin, direct hydrolysis by using 5% acid or adopt stronger acid dipping and hydrolysis or at first adopt alkaline purification in 5% solution, and can be with hemicellulose and Mierocrystalline cellulose such as top being hydrolyzed individually that embodiment presented.These processing can combine with the dip treating that adopts acid or alkali, repeat to grind to produce heat-ground wood pulp subsequently in wing or disc refiner.Yet, in this embodiment, husk, straw and paddy skin (each 125g) were being carried out presteaming 11 minutes and be processed into heat-ground wood pulp under 170 ℃ temperature in the wing refining mills under the pressure of 8bar.With the hydrolysis 4 hours under 90-100 ℃ and normal pressure in 5% acid (sulfuric acid) of 1 liter of volume of heat-ground wood pulp of being produced.By filtering the solution fraction is separated and neutralization.Subsequently, solution concentration is also reclaimed the production that is used for unicellular lipid.50% of monose productive rate average out to source material.In order to increase the carbohydrate productive rate, will filter the remaining precipitation in back and carry out mechanically processing again of heat, and a part will be incinerated according to embodiment 5.

Embodiment 7

To handle 4 hours at 90-100 ℃ in each comfortable 100 liters of alkaline solution (1.2M NaOH) of husk, straw and paddy skin (each 16kg).Mixture is divided into precipitation and solution.The 49-57% of the average source material dry-matter of raising ends in the solution.Give 5% alkaline solution, as for acid, use sulfuric acid, and they are hydrolyzed as described in example 6 above.Mainly form the mixture of wood sugar and pectinose, also contained a small amount of glucose and semi-lactosi.The contents of monosaccharides of solution becomes the 23-33% of the source material of raising.Before the solution recovery is used for the production of unicellular lipid, reduce by the color of activated carbon treatment with them.

The precipitation that obtains in filtering be impregnated in 5% acid (sulfuric acid), and in the wing refining mill, under the temperature of the pressure of 6bar and 150 ℃, ground 11 minutes.Precipitation is degraded into soluble carbohydrate, mainly is glucose and semi-lactosi, after neutralization and the filtration, its recovery is used for the production of unicellular lipid.Remaining precipitation fraction after the filtration of carrying out after the acid hydrolysis is still ground once more, then according to embodiment 4 hydrolysis in 10% acid.Obtained solution after these are handled, they contain the source material of 55-65% altogether.Solution is reclaimed the production that is used for unicellular lipid.

Embodiment 8

The beet pulp of steam drying is poured in the bucket, and poured enough boiling water into it top so that water has covered beet pulp.By decant with water and solids constituent from before, make the solution cooling.3.6mg/ml dry-matter be dissolved in the water.Moisture level portions is used to handle new batch, and part recovery after concentrating is used for the production of unicellular lipid.With the solid fraction that is obtained, precipitation contains 16.7% dry-matter, and weighing is 748g, is equivalent to do beet pulp as the 125g of dry-matter, and it is adopted 0.4M phosphoric acid (H ₃PO ₄, 500ml) flooded 18 hours in room temperature.Topple over and excessive acid solution, and presteaming 11 minutes before the hot machinery of beginning grinds.Use the wing refining mill that mixture was ground 11 minutes.In attrition process, pressure is 8bar, and temperature is initially 172 ℃, and temperature is 162 ℃ when grinding 10 minutes in the past time.Mixture is removed from refining mill and filtered.The solution fraction, contains 2.75% dry-matter by 2.26 liters.After the filtration, the dry-matter that carries out ground 50% of raising exists in solution.Solution mainly contains monose, glucose, semi-lactosi, pectinose and wood sugar.In addition, having observed in a small amount molecular weight ranges is 500 to 3000 oligosaccharides and high-molecular weight compound more.The solution fraction is neutralized, concentrates and reclaim the production that is used for unicellular lipid.

The solid fiber slurry that will obtain from the above-mentioned processing of beet pulp, precipitation (125g dry-matter) impregnated in 15% hydrochloric acid 4 hours in room temperature.Topple over and excessive acid, and will starch under the same conditions and grind once more.After the filtration stage, observe other 28% monose and entered the solution phase.Discard precipitation.

Embodiment 9

With source material---the dragon spruce fiber carries out heat mechanically to be handled, weighing 46.8g (about 1 liter of volume), and with 0.2N NaOH join it above, to the Na that wherein further adds 5.6g ₂CO ₃MgSO with 1.3g ₄6H ₂O (500ml).With mixture heating up to 50 ℃, in solution, obtain in a small amount glucose, wood sugar, semi-lactosi, pectinose, seminose and other oligopolymer.The pH of solution is about 11, with its filtration and wash with water.Washings and filtrate are merged, and with solution evaporation to 320ml.The amount of dissolved substances is 4.5% (dry-matter) of source material.In order to increase the amount of monose, in solution, add acid, obtain precipitation.To precipitate and solution separating, and the latter is reclaimed and be used for the manufacture order cytolipin.As described in example 3 above, will be deposited in 5% acid and be hydrolyzed into monose.Utilize hydrolysis, produced 1% monose from sedimentable matter, these monose enter solution, and its recovery are used for the production of unicellular lipid after decolouring.

Embodiment 10

The dragon spruce fiber that 45g batch of (about 1 liter) hot mechanical treatment crossed is weighed, add 0.2N NaOH in the above, to the Na that wherein further adds 5.6g ₂CO ₃MgSO with 1.3g ₄6H ₂O (500ml).With mixture heating up to 50 ℃, filter, and wash precipitation with water.Washings and filtrate are merged.In order to increase the amount of monose, add acid in the filtrate after merging, and be hydrolyzed according to embodiment 3.After the neutralization, the hydrolysate that is produced is reclaimed the production that is used for unicellular lipid.Precipitation after the washing is compressed to 300ml, and adds the citrate buffer of 1 liter of pH 4.8, and add cellulase.Oligopolymer and glucose produce in mixture.The mixture recycled as such that enzyme was handled and reclaim, and be used for the manufacture order cytolipin as separated filtrate, solution.

Embodiment 11

125g batch oat husk is provided, and its heat under the pressure of 170 ℃ and 8bar was mechanically ground 2 minutes.After the processing, will precipitate and solution separating by filtering.The solution recovery is used for the manufacture order cytolipin.Use 7g batch precipitation (fiber), to give intensity be 19% sulfuric acid and refluxed 4 hours.Analyze the monose that display process obtains entering with 56% of source material solution, major part is wood sugar, seminose, glucose, semi-lactosi and pectinose.The solution recovery is used for the manufacture order cytolipin.

Embodiment 12

The oat husk of weighing 400g, and add 3 premium on currency and 200g NaOH.Mixture under agitation remained on 90-96 ℃ temperature 2 hours.Cloth and separated fiber are passed in the mixture filtration.With filtrate fraction (50ml) neutralization, and adopt citrate buffer (40ml), add multifect zytase (10ml) its pH regulator to 4.8, and with mixture automatic temperature-control to 50 ℃.Carbohydrate is released into solution, and wherein 25.2% is wood sugar, and 11.8% is pectinose.After 50 hours reaction times, also there is oligopolymer in the solution.Whole mixture recovery is used for the manufacture order cytolipin.

Embodiment 13

Employing 0.4M sulfuric acid floods 125g beet pulp, topples over after 12 hours excessive acid.Slurry is transferred to the wing refining mill, will starches presteaming therein 4 minutes and under the pressure of 150 ℃ and 6bar, ground 11 minutes.Mixture neutralization and adding citric acid are fallen to 4 until pH.Add 10ml polygalacturonase 4450U, i.e. the protein of 178mg/ml (Sigma), and make and be reflected at 25 ℃ and carried out 24 hours.After the reaction, the part of mixture is reclaimed the production that is used for unicellular lipid, and a part is filtered.To return the stage of grinding from filtering the precipitation that obtains, and will adopt the gac of 2g/L to handle from the solution fraction that this processing obtains, guide to anion-exchange column then.To reclaim the production that is used for unicellular lipid from the dry-matter of the obtainable monose of post solution evaporation to 20% concentration.

Embodiment 14

According to embodiment 7 the oat husk is handled, obtained containing the mixture of 23.8g/L glucose, 93.3g/L wood sugar, 37.1g/L pectinose and 9.0g/L semi-lactosi.This mixture is added yeast fungi degradation fat Ye Shi yeast (Yarrowia lipolytica) ATCC 20373 and the rhodotorula glutinis TKK 3031 that is used for synthetic lipid as substratum, former state is as 1: 1 diluent with the corresponding diluent of 11g/L glucose is arranged as a supplement.Culture cycle is 68 hours, and temperature is 28 ℃, jolting under 250rpm, and volume of culture is 50ml.As viewed in Fig. 3, yeast can be grown and do not required synthetic lipid under the situation of adding other nutrient substances.

Embodiment 15

With three primary yeast bacterium rhodotorula glutinises, separate fat Ye Shi yeast and Kluyveromyces marxianus (Kluyveromyces marxianus) (Anam.Candida kefyr) ATCC 42265, adopt wood sugar to cultivate individually as carbon source.Substratum contains 20g/L wood sugar, 10g/L yeast extract and 20g/L peptone.Under the jolting of 200rpm, under the volume of 50ml and 25 ℃, cultivate.Fig. 4 shows that three kinds of all bacterial strains can utilize pentose as carbon source.

Claims (19)

1. method of producing lipid or lipid mixt from the organic source material that comprises polysaccharide, described polysaccharide is selected from the group that comprises Mierocrystalline cellulose, hemicellulose, starch, all these, its any mixture or its degraded product or non-starch polysaccharide, it is characterized in that comprising:

A) source material usefulness is selected from the mass treatment that comprises following group:

I) water,

Ii) acid and

Iii) alkali,

Afterwards, precipitation separation and filtrate, and the precipitation former state that will obtain or in the presence of water, acid or alkali, carry out machinery or thermomechanical grinding, and precipitation separation and filtrate, alternatively, will precipitate and carry out i once more), ii) or iii) any part processing and/or grind once or more times, and

B) microorganism that will produce lipid and the filtrate that so obtains or with plural number kind gained filtrate or with precipitation, perhaps with from any combination of these gained and, randomly,, in substratum, contact with source material, microorganism begins to produce lipid whereby, and

C) reclaim lipid.

2. according to the method for claim 1, it is characterized in that, equally randomly use the method that comprises one or more the following steps to handle precipitation:

D) precipitation that will obtain from a) part is with strong acid treatment and precipitation separation and filtrate, perhaps, and alternatively,

E) will from a) or d) sedimentation acidified that obtains of part and it is carried out mechanically or heat mechanically grinds, precipitation separation and filtrate, perhaps, randomly, use is according to step a), d) or e) in the method for any step will be with optional order from step a), d) or e) the precipitation that obtains of any step handle once more once or more times

F) microorganism that will produce lipid with from a), d) or the e) filtrate or the precipitation that obtain of part, perhaps with from any combination of these acquisitions and, randomly,, in substratum, contact with source material, microorganism cells begins to produce lipid whereby, and

G) reclaim lipid.

3. according to the method for claim 1 or 2, it is characterized in that, described source material come from timber machinery or thermomechanical processing or come from cultivated plant.

4. according to each method among the claim 1-3, it is characterized in that, described source material water or acid treatment.

5. according to the method for claim 1 or 2, it is characterized in that, but described source material comes from the source that is selected from the group that comprises recycled fibers, beet pulp, husk, straw, wheat bran, cereal-granules, complete raise crop, cultivated plant, TMP slurry and MDF slurry.

6. according to claim 1,2 or 5 method, it is characterized in that, described source material acid treatment.

7. according to the method for claim 1 or 2, it is characterized in that described source material comes from the source of the group that is selected from the cultivated plant that comprises sawdust, purified ground wood pulp, husk, straw, TMP slurry, MDF slurry, beet pulp and contain inessential amount starch.

8. according to each method in the claim 1,2 or 7, it is characterized in that, described source material alkaline purification.

9. according to the method for claim 1 or 2, it is characterized in that, described source material come from be selected from comprise biomass microbiological materials, marsh or under water, from the biomass of the catchment of Mierocrystalline cellulose factory, from the biomass of Municipal waste with from the source of the group of the biomass of municipal sewage system.

10. according to each method in the aforementioned claim, it is characterized in that described filtrate contains 0.5-1% weight at least, up to the carbohydrate that can be used for lipid production of 20-30% weight, preferred 4-5% weight.

11. the method according to claim 1 or 2 is characterized in that, step a), d) or e) in the processing of any step carry out once or more times.

12. the method according to claim 1 is characterized in that, a) iii) in the part during, the gained precipitation handled with acid again with the alkaline purification source material, and precipitation separation and filtrate.

13. the method according to claim 1 or 2 is characterized in that, during with the fibrous precipitation of strong acid treatment, gained is precipitated once more mechanically or heat mechanically grinds.

14. according to each method in the aforementioned claim, it is characterized in that, one or more planted enzymes join in the biomaterial treatment soln.

15. according to each method in the aforementioned claim, it is characterized in that, use makes filtrate be more suitable for the method for microorganism growth, such as decoloring method, regulate pH and/or remove and anhydrate or add entry, will further handle from the filtrate that any processing step obtains.

16. according to each method in the aforementioned claim, it is characterized in that, with described source material carry out mechanically, heat mechanically, physically, chemically, the pre-treatment of biology ground, or the combination pre-treatment by these processing.

17. use the lipid produced according to each method among the claim 1-16 or lipid mixt purposes as the raw material of making biofuel.

18. a biofuel is characterized in that lipid is used for its production, wherein said lipid is to use to be produced according to each method among the claim 1-16.

19. the method for a purifying city water drain is characterized in that, uses according to each method among the claim 1-16 and handles municipal sewage system.

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