INTEGRATED PROCESS FOR PRODUCTION OF BIOFUELS

专利摘要:
integrated process for the production of biofuels the present invention refers to an integrated process, which comprises a first biotechnical process, which produces biofuel ei or starting material for biofuels and uses a microorganism capable of producing enzymes, and a second biotechnical process, which produces biofuel ei or starting material for biofuel. the process comprising said microorganisms is grown and biofuels and i or starting material for biofuel and enzymes are produced. the culture of microorganisms, the supernatant or an enriched protein fraction or a dilution of the supernatant comprises a catalytically active enzyme (s) are introduced into the first i or the second biotechnical process, or as raw material for said process (s) treated. the invention also relates to the use of enzymes produced in the biofuel production process or in other applications, as an enzyme preparation or as an enzyme source. the invention also relates to the use of the produced lipids and alcohols as biofuels, as a biofuel component or as a starting material for the production of biofuels.
公开号:BR112013016198B1
申请号:R112013016198-1
申请日:2011-12-19
公开日:2020-03-03
发明作者:Perttu Koskinen；Reijo Tanner
申请人:Neste Oil Oyj；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: “PROCESS
INTEGRATED FOR THE PRODUCTION OF BIOFUELS.
FIELD OF THE INVENTION [001] The present invention relates to an integrated process for the production of biofuels from lignocellulosic materials.
BACKGROUND OF THE INVENTION [002] Lignocellulose is the most abundant biopolymer on Earth. Lignocellulose is the main structural component of woody and non-woody plants such as grass. Lignocellulosic biomass refers to plant biomass, which is composed of cellulose, hemicellulose and lignin. Large quantities of lignocellulosic waste are produced through forestry, wood and paper and cellulose industries and agricultural practices (straw, firewood, sugarcane bagasse, bran) and many agro-industries. Municipal waste also contains fractions that can be considered as lignocellulosic waste, such as paper or cardboard, garden waste or construction wood waste. Due to the high abundance and low price, lignocellulosic residues are preferred materials for the production of biofuels. In addition, dedicated medeira or herbaceous energy crops with biomass productivity have gained interest as the use of biofuels.
[003] The production of biofuels, especially,
2/99 ethanol, from lignocellulosic materials by microbial fermentation, has been studied extensively. The biggest challenge for the use of lignocellulose for the microbiological production of biofuels biofuel raw materials poses the complexity of the lignocellulosic material and its resistance to biodegradation. In lignocellulose, cellulose fibers (20 - 50% dry matter weight) are covalently incorporated into the matrix found in hemicellulose (20 - 40%), pectin (2 - 20%) and lignin (10 - 20%) forming a structure very resistant to biodegradation. In addition, hemicellulose sugar residues contain a variable mixture of hexoses (eg, glucose, mannose and galactose) and pentoses (eg, arabinose and xylose), depending on the biomass.
[004] The pretreatment of lignocellulosic material with a high yield of sugars that are usable by microorganisms represents one of the biggest challenges. Significant cost reductions are required in the costs of the enzymes required in the hydrolysis of sugar polymers to sugar monomers that are usable by desired microorganisms. In addition, the economically viable production of biofuels from lignocellulosic materials requires efficient conversion of all major carbohydrate components from complex material to biofuels. The production of cellulosic ethanol includes two challenges
3/99 main: traditional ethanol producing organisms, such as beer yeast (Saccharomyces) or Zymomonas mobilis (bacteria) are not able to use pentoses, which are sources of carbon and / or energy for the production of ethanol. This leads to an inefficient use of total sugars in lignocelluloses to ethanol. Wild beer yeast strains (Saccharomyces) or Zymomonas mobilis cannot use polymeric sugars in lignocellulose as carbon and / or energy sources for the production of ethanol. Enzymes for the hydrolysis of sugar polymers to monomers need to be purchased, but the enzyme costs are too high. Brewer's yeast or genetically modified Zymomonas mobilis strains capable of using xylose have been developed, but have not been proven to be robust enough for large-scale long-term operations. The same applies to genetically modified beer yeast capable of using cellulose. Ethanol-producing bacteria using Pentoses or other yeasts in addition to Saccharomyces exist, such as Pachysolen tannophilus, Pichia stipitis, and Candida shehate, however their low ethanol tolerance, low robustness and high sensitivity to inhibitors have prevented their commercial use.
[005] Enzymatic hydrolysis is generally carried out in a separate step from the production process of
4/99 biofuels by commercial enzymes purchased and produced outside the actual biofuel production process.
[006] Lignocellulose hydrolysates were also used for the production of single cell oils. Hydrolysis of lignocellulose was typically performed by pretreating the lignocellulosic material to monomeric sugars before feeding to the bioprocess.
[007] US patent application publication 2009217569 describes the production of single cell oil from various lignocellulosic and other hydrolyzed materials, such as straw, wood, pulp and paper industry, recyclable fiber waste, solid urban waste , algae biomass. For the manufacture of biofuel it comprises the treatment of source material with water, acid or alkali and contacting the filtrate or precipitate with a lipid-producing microorganism. US patent application publication 2009064567 describes the production of single cell oil from hydrolysates of cellulose materials for biodiesel and jet biofuel production by Stramenopiles. US 20090011480 describes the production of single cell oil by heterotrophically cultivated algae and fungi from depolymerized lignocellulosic materials, such as straw, wood, pulp mill residues, switchgrass. CN 101148630 describes the production of single-cell oil from
5/99 hydrolyzed wheat, corn or rice straw hemicellulose obtained by steam explosion by bacteria or fungi.
[008] Furthermore, in the state of the art, the production of polymeric lipids directly from sugars in lignocellulose, such as xylan, has been described by Fall et al. (1984), or cellulose by Lin et al. (2010). US 2010028484 describes the production of single cell oil co-products, such as vinasse or DDGS, from the production of ethanol from corn as a raw material.
[009] WO 2010042842 describes the production of single cell oil from hydrolyzed lignocellulose by mixed culture of microorganisms (s) capable of degrading polymeric sugars into lignocellulose and at least one species of seaweed. The crop is grown in successive aerobic and anaerobic cultures, where fatty acids are produced from sugars and anaerobic fermentation products. However, the process leads to a low efficiency in the production of oil from lignocellulose since fermentation products (alcohols, etc.) are used as carbon sources for the production of lipids.
[0010] WO 2010006228 describes sequential production of biofuels from lignocelluloses. In the first stage, the anaerobic fermentation of organisms capable of producing from polymeric sugar alcohols in lignocellulose hydrolysates, in the second stage, the
6/99 spent culture, possibly containing at least one fermentation product, is treated with algae in order to accumulate oils from single-celled organisms.
SUMMARY OF THE INVENTION [0011] It is an object of the present invention to provide a solution to the problems encountered in the prior art. Specifically, the present invention aims to provide a beneficial technical solution to the problems encountered in biofuel production processes.
[0012] It is another objective of the present invention to provide a beneficial technical solution to the problems encountered in the large-scale production of biofuels. In particular, it is an object of the invention to provide a solution to the problems encountered in the large-scale production of biofuels by microbiological processes, such as fermentation of alcohols or aerobic fermentation of oil from a single cell.
[0013] The third objective of the present invention is to provide a beneficial technical solution to the problems encountered in the large-scale production of ethanol or other alcohols or mixtures of alcohols.
[0014] It is yet another objective of the present invention to provide a solution, which allows to update the economy of biofuel production.
7/99 [0015] It is yet another objective of the present invention to provide a solution, which allows to reduce the environmental load.
[0016] The present invention aims, in particular, to solve the problems related to the manufacture of transportation of biofuels, such as alcohols, biodiesel and / or diesel, gasoline or renewable jet fuel.
[0017] To achieve these objectives, the invention is characterized by the characteristics that are listed in the independent claims. Other claims represent preferred embodiments of the invention.
[0018] The present invention is based on the finding that certain lipid-producing microorganisms efficiently produce lipids from polymeric sugars directly in the lignocellulose material. It has surprisingly been discovered that, in a single cell oil production process, a significant amount of exoenzymes is produced. It was also discovered that these exoenzymes remain active and can be collected from the spent culture medium.
[0019] In addition, the invention is based on the finding that certain organisms produce exoenzymes that have activity towards different polysaccharides.
[0020] In one aspect, the present invention provides an integrated process, which comprises a first process
8/99 biotechnological, which produces a starting material for the component or biofuel and uses a microorganism capable of producing enzymes, and a second biotechnological process, which produces a starting material or component for biofuels. The process comprises that microorganisms can produce components or starting material for the biofuel and enzymes, or component or starting material for the biofuel.
[0021] Enzymes can be recovered from culture of microorganisms, the spent culture medium or supernatant.
[0022] The cells of the supernatant and microorganisms are optionally separated from the culture of microorganisms. Biofuel (s) are recovered from the culture of microorganisms and / or from cells of the microorganism. Typically, the supernatant or a fraction of protein enriched from the supernatant or a dilution of the supernatant comprising catalytically active enzyme (s) is introduced into the first and / or second biotechnological process, or as feedstock for the process (s) that is treated.
[0023] In one embodiment of the invention, a process produces lipids from polysaccharides (polymeric sugars) and simultaneously produces extracellular enzymes capable of depolymerizing sugars. Enzymes are
9/99 reused in another biofuel or raw material production process by organisms typically unable to use polysaccharides.
[0024] The present invention offers the following advantages / solutions:
- More complete use of lignocellulosic materials for the production of biofuels.
- The efficient use of the flow of hemicellulose for the production of biofuels. Currently used ethanol producers are not able to use pentoses efficiently.
- Production of valuable products, suitable for the production of biofuels, together with the production of enzymes.
- Cost savings in enzyme costs. Production of necessary enzymes in ethanol, single cell oil or on-site butanol process. Reduces the need for enzymatic treatment before use, such as stabilization.
- Bioprocess for the production of consolidated lipids (enzymatic digestion and fermentation) reduces the costs of decreasing or eliminating the need for an enzyme produced in a separate refinery.
- The flow of enzyme (liquid culture) needs minimal processing, since anaerobic /
10/99 aerobic reduces the risk of contamination.
[0025] In addition, the production of enzymes for the hydrolysis of cellulose and / or hemicellulose at the site is advantageous for several reasons and improves the economy of biofuel production:
- Reduction of downstream processing costs, including water and enzyme stabilization,
- Decreased transport and packaging costs,
- Reduction of losses through direct transfer of enzymes to the second biofuel production process,
- Decrease in capital costs versus dedicated (remote) facilities,
- The use of the same raw material or raw material from the same source for the production of the enzyme and the production of biofuels in direct induction and adaptation of enzymes to the raw material,
- Control of simple processes and output tuning and opportunities for improvement directly within the biorefinery in the production of biofuels and production of the enzyme.
BRIEF DESCRIPTION OF THE FIGURES [0026] Figs. 1 to 6 show the process diagrams;
[0027] Fig. 7 shows Xylose released in the hydrolysis test by volume of culture broth. As a substrate,
11/99 200 mg of birch wood xylan was used.
[0028] Fig. 8 shows Xylose released in the protein hydrolysis test. As substrate, 200 mg of birch wood xylan was used.
[0029] Fig. 9 Glucose in model hydrolysis tests by volume of culture broth. As a substrate, 1 g of cellulose was used.
[0030] Fig. 10 Glucose released in the protein hydrolysis test. As a substrate, 1 g of cellulose was used.
[0031] Fig. 11 shows Xylose released in the hydrolysis test by volume of culture broth. As substrate, 200 mg of birch wood xylan was used.
[0032] Fig. 12 shows Xylose released in the protein hydrolysis test. As substrate, 200 mg of birch wood xylan was used.
[0033] Fig. 13 shows glucose in the hydrolysis assay model by volume of culture broth. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
[0034] Fig. 14 shows glucose released in the protein hydrolysis test. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
12/99 [0035] Fig. 15 shows Xylose released in the hydrolysis test by volume of culture broth. As a sub-strate, 200 mg xylan birch wood was used.
[0036] Fig. 16 shows Xylose released in the protein hydrolysis test. As substrate, 200 mg of birch wood xylan was used.
[0037] Fig. 17 shows glucose in the model of hydrolysis assays by volume of culture broth. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
[0038] Fig. 18 shows glucose released in the protein hydrolysis test. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
DETAILED DESCRIPTION OF THE INVENTION
Definitions [0039] Single cell oil production process refers here to a process, which comprises the stages of formation or allows the formation of a lipid synthesis microorganism and allowing the mass thus obtained from the organism to produce and / or store (accumulate) lipids, recovering cells from the liquid phase, and extracting or recovering lipids from cells. In certain cases, single cell oil can also be extracellular, such as excreted or released from cells in
13/99 culture during or after cultivation.
[0040] As described herein, the present invention preferably uses microorganisms capable of producing lipids and enzymes. A microorganism refers, in some embodiments of the invention, to two or more microorganisms. In some embodiments, enzymes are produced by one microorganism and lipids by another microorganism. In some embodiments, more than one of different strains of microorganisms are used for the production of lipid and / or the production of enzymes.
[0041] The term lipid refers to a grease substance, the molecule of which usually contains, as a part, an aliphatic hydrocarbon chain, which dissolves in non-polar organic solvents, but is poorly soluble in water. Lipids are an essential group of large molecules in living cells. Lipids are, for example, fats, oils, waxes, wax esters, sterols, terpenes, isoprenoids, carotenoids, polyhydroxyalkanoates, nucleic acids, fatty acids, fatty alcohols, fatty aldehydes, fatty acid esters, phospholipids, glycolipids, sphingolipids and acylglycerols, such as triglycerides, diacylglycerols, or monoacylglycerols.
[0042] Preferred lipids in the present invention are fats, oils, waxes, acylglycerols and fatty acids and their derivatives, in particular, wax esters and
14/99 triacylglycerols.
[0043] Within the scope of the present invention, single cell oil is used as a synonym for lipids and fat.
[0044] Lipid recovery refers to a process, in which the lipid (intracellular lipid) is recovered by mechanical, chemical, thermomechanical or autocatalytic methods, or by a combination of these methods, the microorganism cells.
[0045] Residual cell mass means, from a fraction of flowing semi-solid or solid material, which contains microorganisms treated for the recovery of intracellular lipids [0046] By the term alcohol is meant that any organic compound in which a functional group hydroxyl (-OH) is attached to a carbon atom. Alcohol here refers generally to an organic compound containing a hydroxyl group that is produced * - by microorganisms. Typical alcohols produced by microorganisms include, but are not limited to, ethanol, n-butanol, iso-butanol, propanol and / or iso-propanol. Alcohols are typically produced by anaerobic fermentation. Alcohols can be produced together with aldehydes, such as acetone, or organic acids, such as acetic acid and / or butyric acid, and gaseous products, such as C02 and / or H2.
[0047] The term integration process or process
Integrated 15/99 is a combination of at least two unit operations that exploit interactions between different units in order to employ resources effectively, improve energy efficiency, improve material balance, maximize profit and / or minimize costs . At least one of the two unit operations in the integrated process receives the material and / or energy, and can be dose-dependent, from another operation unit. The integration of processes taking into account the interactions between different unit operations, from the beginning, instead of optimizing them separately. Integration process is not limited to the design of new plants, but also covers the retrofit design (for example, new units to be installed in an old factory) and the operation of existing systems. Preferably, the unit operations are located in situ. However, this is not necessary and, in some embodiments of the invention, unit operations are located separately.
[0048] In one embodiment of the invention, the production of the enzyme is initiated and / or maintained through the addition of an enzyme inducer for the culture of microorganisms. This usually results in an increase in the amount of enzymes produced. In particular, in continuous cultivation it is important to keep the amount of inducors at a sufficient level to maintain the production of extracellular enzymes.
16/99 [0049] A culture medium here refers to a medium used for the cultivation of microorganisms. The culture medium here comprises typically polymeric sugars. The culture medium can be supplemented with minerals, micronutrients, macronutrients, growth factors and buffering agents.
[0050] The present invention provides a process to improve the efficiency and economy of production of biofuels from lignocellulosic materials. The invention also provides a process for decreasing the entry of externally produced enzymes for the hydrolysis of sugar polymers using microorganisms that produce biofuels that are capable of degrading polymeric sugars by exoenzymes. In addition, the invention provides a process for the production of valuable compounds suitable as biofuels, or as a raw material for the production of biofuels, together with the production of enzymes. The enzymes are preferably, at least in part, used on the spot and / or sold outside the integrated process.
[0051] The present invention relates to the use of lignocellulosic material efficiently for the production of lipids from biofuels. The invention reveals a process for the production of biofuels, such as lipids, ethanol and butanol, from cellulose and / or fractions of
17/99 hemicellulose by microbiological processes of an integrated process. More specifically, a process is provided for the use of hemicellulose or cellulose fraction as a raw material for the production of lipid by microorganisms, which are capable of using polymeric sugars by exoenzymes.
[0052] In one aspect, the present invention provides a process for the production of biofuels, such as ethanol, lipids and / or butanol, from lignocellulose materials or fractions thereof, such as from cellulose and / or fractions hemicellulose, by microbiological processes of an integrated process.
[0053] In addition, the invention improves the overall efficiency of the use of carbohydrates, using different microorganisms in the production of biofuels from different fractions of lignocellulose (for example, cellulose and hemicellulose or their fractions): The organisms used for the production of biofuels from cellulose and hemicellulose are optimized in terms of their efficiency in using sugars and the production of biofuels from that fraction.
[0054] In one embodiment of the invention, the present invention provides a process for using lignocellulose fractions, in particular hemicellulose and / or cellulose fractions as a raw material for the production of
18/99 lipid by microorganisms, which are capable of using polymeric sugars for exoenzymes.
[0055] In the preferred embodiment of the invention, the lignocellulose hemicellulose fraction containing polymeric sugars is used for the production of lipid using oleaginous organisms, which are capable of using polymeric hemicellulose producing exoenzymes. The enzymes recovered and / or enriched from the spent culture medium of hemicellulose lipid production also have cellulose degradation activity and can be used for cellulose hydrolysis in another process.
[0056] Another embodiment of the invention uses alcohol-producing microorganisms for the use of lignocellulose fractions, in particular hemicellulose and / or cellulose fractions as raw materials, which are capable of using polymeric sugars for exoenzymes. The exoenzymes that result from the use of polymeric sugars are reused in the production of biofuels or the production of raw material for biofuels in a first or before a second biofuel production process. Polymeric sugar-degrading exoenzymes produced by microorganisms (eg cellulases, hemicellulases, glucosidases, xylanases, arabinases, galactosidases, mannases) that produce biofuels (eg lipid, ethanol, butanol, EBA = acetone-butanol-ethanol) from
19/99 hemicellulose and / or cellulose in first bioprocesses (bioprocess 1) can be reused in the production of biofuel or biofuel raw material (for example, ethanol, butanol, ABE, lipid) from cellulose and / or hemicellulose.
[0057] In a specific embodiment of the invention, enzymes are partly recycled in the first biofuel production process using organisms capable of producing enzymes and biofuels.
[0058] In one embodiment of the invention, the process includes, in any case, a biofuel production process, preferably a lipid production process, using sugar polymers from lignocellulosic material (cellulose or hemicellulose) and organisms preferably lipid-producing organisms, which have the capacity to use these polymer sugars. Another embodiment of the invention uses alcohol-producing microorganisms for the use of lignocellulose fractions, in particular hemicellulose and / or cellulose fractions as a raw material, which are capable of using polymeric sugars for exoenzymes. In addition, exoenzymes capable of hydrolyzing sugars are reused in another bioprocess for the production of biofuels. Enzymes can be reused, for example, to saccharify polymeric sugars before the second production process of
20/99 biofuels or (during) the second biofuel production process. The second biofuel production process can be a single cell oil production process, ethanol, butanol or ABE. The processes are preferably integrated (biorefineries) or enzymes produced in the first biofuel production process can be collected, purified and sold out to be used in the production of biofuels from lignocellulosic materials.
[0059] In one embodiment of the invention, the first biofuel production process (process 1) uses raw material that contains polymeric sugars. Process 1 uses microorganisms that are capable of using polymeric sugars for exoenzymes and capable of producing fuels in the same process. Process 1 produces ethanol, butanol, acetone, butanol, ethanol, or lipids. Process 1 is preferably an aerobic or aerated lipid production process. Process 1 preferably uses microorganisms that are capable of using both hemicellulose and cellulose by producing exoenzymes.
[0060] Exoenzymes produced in the process are recovered and re-used in the hydrolysis of polymeric sugars for the production of biofuels in the second process (process 2) or process 2.
[0061]
In one embodiment, process 2 of the invention
21/99 uses microorganisms that are capable of producing biofuels from monomeric sugars, but are not capable of producing exoenzymes using polymeric sugars. Process 2 produces ethanol, butanol, acetone, butanol, ethanol, or lipids. Process 2 is preferably an anaerobic process for the production of ethanol, butanol, or acetone-ethanol-butanol.
[0062] Typical process options and raw materials, raw materials can contain other components of lignocellulose, such as lignin and / or pectin, in addition to hemicellulose and / or cellulose:
[0063] Case l: Process 1 uses hemicellulose; process 2 uses cellulose;
[0064] Case 2: Process 1 uses cellulose; process 2 uses hemicellulose;
[0065] Case 3: Process 1 uses cellulose; Process 2 uses cellulose;
[0066] Case 4: Process 1 uses hemicellulose; Process 2 uses hemicellulose;
[0067] Case 5: Process 1: uses a mixture of hemicellulose and cellulose (any combination); Process 2: uses a mixture of hemicellulose and cellulose (any combination);
[0068] Case 6: Process 1: uses a mixture of hemicellulose and cellulose (any combination); Process 2 uses
22/99 cellulose;
[0069] Case 7: Process 1: uses a mixture of hemicellulose and cellulose (any combination); Process 2 uses hemicellulose.
[0070] In a preferred embodiment of the invention, enzymes capable of hydrolyzing polymeric sugars are produced in an aerobic or aerated bioprocess that also produces biofuels or raw materials for biofuels, preferably lipids. The aerobic bioprocess allows efficient production of enzymes.
[0071] According to a preferred embodiment of the invention, hydrolysis and biofuels or production of biofuel raw material are carried out in a single step using microorganisms that are capable of both enzymes that are capable of hydrolysis of oligomeric sugars and production of biofuels. This type of approach with cellulase (and / or hemicellulose) production, cellulose (and / or hemicellulose) hydrolysis and one-step fermentation is often called consolidated bioprocessing. Consolidated bioprocessing offers the potential for cost reduction and greater efficiency than the processes that characterize production dedicated to cellulase (and / or hemicellulase). This results in avoided costs of capital, substrate and other raw materials and utilities associated with cellulase production. In addition, it offers
23/99 possibility of obtaining higher hydrolysis rates and, consequently, reduced reactor volume and capital investment, using consolidated bioprocessing. Consolidated bioprocessing reduces significant costs, eliminating, or at least decreasing, the need for an enzyme produced in a separate bioprocess.
[0072] In one embodiment of the invention, enzyme production and single cell oil production occur simultaneously or sequentially in any order. Typically, production of the enzyme starts earlier. The enzyme produced degrades the polymer biomass in the culture medium, thus producing components for the growth of the microorganism.
[0073] In one embodiment of the invention, cellulose or hemicellulose fractions are divided into two parts. A part of cellulose or hemicellulose can be used to grow biofuel-producing organisms that have enzymatic capabilities for the degradation of polymeric sugars. The enzymes can be recovered from the metabolized culture medium, or spent culture medium comprising enzymes can be reused for the production of biofuels from the second cellulose part or the hemicellulose. In a preferred embodiment of the invention, lipids and alcohols are produced by microbiological processes, such as microorganisms using
24/99 polymeric sugars capable of accumulating lipids are used for a part of cellulose or hemicellulose. Alcohols are produced from another part of cellulose or hemicellulose treated by enzymes recovered from the spent culture medium of the first process using lipid producers capable of using polymeric sugars. In one embodiment of the invention, lipids and ethanol, butanol or lipids and lipid and acetone-butanol-ethanol (ABE) are produced. Similarly, ABE and lipids, ABE and ethanol, ethanol and ABE or ethanol and lipids can be produced.
[0074] The process, as described here, is not limited to the use in the production of only biofuels. It can be combined with any type of aerobic or anaerobic fermentation starting using polysaccharides as carbon and / or energy sources.
[0075] In one embodiment of the invention, a mixture of cellulose and hemicellulose is used for the production of biofuels by microorganisms that have enzymatic capabilities to degrade polymeric sugars. Enzymes can be recovered from the metabolized culture medium, or spent culture medium, including the enzymes can be reused to hydrolyze cellulose or hemicellulose for the production of biofuels using organisms that are unable to use polymeric sugars. In a preferred embodiment of the invention, lipids and ethanol
25/99 are produced by microbiological processes, such as microorganisms using polymeric sugar capable of accumulating lipids are used for a part of cellulose or hemicellulose. Ethanol is produced from another part of the cellulose or by hemicellulose treated by enzymes recovered from the spent culture medium of the first process using lipid producers capable of using polymeric sugars. Likewise, in yet another embodiment of the invention, lipids and butanol or lipids and ABE are produced. Likewise, ABE and lipids, ABE and ethanol, ethanol and ABE or ethanol and lipids can be produced.
[0076] In one embodiment of the invention, the raw material for the process contains polymeric sugars from both cellulose and hemicellulose (any combination) and uses microorganisms capable of producing exoenzymes for the hydrolysis of sugar polymers and biofuels (Process 1). The exoenzymes that are in the spent culture medium are recovered and reused in the saccharification of polymeric sugars from both hemicellulose and cellulose before or in another bioprocess (Process 2) to produce biofuels that use organisms that are not capable of using polymeric sugars. As an example, hydrolysates containing polymeric sugars of hemicellulose and cellulose are divided into two fractions, a fraction used for Process 1 and another fraction for Process 2.
26/99 [0077] In one embodiment of the invention, the sugars used for Process 1 consist mainly of hemicellulose, but also some cellulosic sugars in polymeric form, usually 0.5 to 20% (w / w), usually 0, 5 to 10% (w / w). For example, the flow containing hemicellulose polymeric sugars is completed with a flow containing polymeric cellulose sugars. In yet another embodiment of the invention, the sugars used for Process 1 consist mainly of cellulose, but also some hemicellulosic sugars in the polymeric form, generally 0.5 to 30% (w / w), usually from 1 to 20% ( p / p). For example, the stream containing the polymeric cellulose sugars is supplemented with a stream containing the polymeric hemicellulose sugars.
[0078] In another embodiment of the invention, microorganisms using polymeric sugar produce sets of enzymes that have the ability to use both cellulose and hemicellulose.
[0079] In one embodiment of the invention, the organism capable of using sugars and the production of polymeric biofuels or material for another organ, and biofuels capable of producing biofuels or starting material for the starting biofuels, but is not able to use , polymeric sugars are added in the same reactor (mixed culture).
27/99 [0080] In one embodiment of the invention, cells from the biofuel production process using polymeric sugars are removed, and the spent culture medium, as such, including enzymes capable of hydrolyzing the sugar polymers, they are fed to another biofuel production process with organisms that are not capable of using polymeric sugars.
[0081] The supernatant and the cells do not need to be completely separated. In some embodiments, the supernatant comprises 1% to 30% of the culture cells of the original microorganism. In some embodiments, the supernatant comprises 2 to 15%, in some embodiments from 3 to 10%, in some embodiments 5 - 8% of the culture cells of the original microorganism.
[0082] In one embodiment of the invention, the microorganism is even used in the production of ethanol from lipids and lignocellulosic material. The production of lipids is obtained in an aerated process (aerobic cultivation), which is the production of ethanol is obtained in anaerobic or microaerobic cultivation.
[0083] By integrating aerobic and anaerobic bioprocesses, significant cost reductions and increases in raw material and chemicals for the total productivity of biofuels can be obtained in comparison with the operational units independently.
28/99 [0084] By combining the aerobic production of lipids as a first bioprocess (Process 1), and alcohols as a second bioprocess (Process 2), it is possible to transform most of the lignocellulosic material into compounds suitable for biofuel applications ( alcohols, short-chain solvents C and lipids). Integration of a lipid and bioprocesses that produce enzyme for a fuel distillery producing ethanol, butanol, or ABE can increase the total alcohol production capacity, along with the lipid production capacity.
[0085] In one embodiment of the invention, the aerobic process supplies process water to an anaerobic process and vice versa. The recirculation of process water between aerobic and anaerobic bioprocesses decreases the risk of microbial contamination, since oxygen is very toxic for anaerobic microbial strains, and on the other hand, aerobic microorganisms do not grow well under anaerobic conditions.
[0086] After anaerobic process (process 1 or 2), solvents are traditionally separated from the water fraction by distillation and suspended solids are separated after distillation from water by decantation. In one embodiment of the invention, the biomass cells and residual cellulosic polymers (and proteins containing protein if the raw material was used as the raw material) circulate in the
29/99 aerobic process. The spent culture medium, possibly containing remaining sugars, such as unbroken oligomers, pentoses and cellobiosis, diarabinose and xylobiosis, can be recirculated for aerobic processing as well. If the anaerobic process uses Saccharomyces wild-type yeasts to produce ethanol, these sugars are not used. In ABE's Clostridium-based production, the bacteria can use a pentose as organisms normally produce lipids in the aerobic process.
[0087] According to one embodiment of the invention, biofuels are produced in an integrated bioprocess, in which the lignocellulosic material is divided into two fractions, one containing cellulose and the other containing hemicellulose. The fractionation of lignocellulose to the fraction of cellulose and hemicellulose can be done by any suitable method. The hemicellulose and / or cellulose fraction may contain lignin or lignin and / or pectin residues. In one embodiment of the invention, biofuels (lipid, ethanol, butanol or ABE) are produced from polymeric hemicellulose using organisms that have enzymatic capabilities for the degradation of sugar polymers. The enzymes can be recovered from the metabolized culture medium, or spent culture medium, the enzymes can be reused in the hydrolysis of polymeric cellulose to produce second biofuel bioprocessing (ethanol,
30/99 butanol, ABE, lipids). Alternatively, in another embodiment of the invention, biofuels are produced from polymeric cellulose through the use of organisms that have enzymatic capacities for the degradation of sugar polymers. The enzymes can be recovered from the metabolized culture medium, or spent culture medium, the enzymes can be reused in the hydrolysis of polymeric hemicellulose for the production of biofuels in a second bioprocess.
[0088] In practice, the efficiency of separating hemicelluloses from cellulose is not 100% and the cellulose fraction contains some hemicellulose. The hemicellulose that remains in the cellulose fraction can be hydrolyzed by enzymes recovered from the culture medium used from the first bioprocess (Process 1). In addition, in practice, the hemicellulose fraction may contain some polymeric cellulose, and if organisms that can use both hemicellulose and polymeric cellulose are used, hemicellulase and cellulase enzymes can be recovered from the spent culture medium and re -used in the second biofuel production process (Process 2).
[0089] In addition, cellulose and hemicellulose degradation may involve the same enzymes, such as, but not limited to cellobiases. Therefore, useful enzymes from spent culture medium in polymeric hemicellulose
31/99 can be recovered and reused for the hydrolysis of polymeric cellulose.
[0090] Figure 1 describes an embodiment of the invention, in which the production of aerobic and anaerobic lipids of alcohol production is integrated. In this concept of lignocellulosic raw material for both bioprocesses, it can be pre-treated to form cellulose and hemicellulose fractions by any technology, such as, but not limited to, hot water extraction or organosolv methods. Aerobic lipid production process uses hemicellulose as raw material, while alcohol production uses cellulose as raw material. For the aerobic lipid production process (process 1), lipid-producing microorganism (s) is chosen, which produce at least hemicellulases, preferably both hemicellulases and cellulases.
[0091] According to the embodiment of the invention described in Figure 1, aerobic lipid production and anaerobic alcohol production are integrated. The second bioprocess (Process 2) comprises the anaerobic fermentation of alcohol is faster, if the enzyme celobiase is added. The enzyme celobiase is obtained, preferably at least partially, from spent culture medium of the aerobic process of lipid production, or, alternatively, or in addition to commercial celobiase can be used.
32/99 [0092] In the second bioprocess comprising alcohol production bioprocess (process 2), production strains that are not capable of using lignocellulosic pentoses or disaccharides, such as Saccharomyces yeast, can be used. Organisms capable of using pentoses and / or polymeric sugars can also be used, such as Clostridia for the production of ethanol or ABE. Potentially, the spent culture medium and / or Process 2, after product recovery, can be recycled to produce bioprocess lipid. Vinasse, that is, a solid fraction of alcoholic fermentation, an anaerobic process can potentially be treated in the pretreatment process or hydrolysis in the same lignocellulose process before feeding the aerobic production of lipids. In addition, vinasse and / or spent culture liquid (liquid phase) from the anaerobic process may contain cellulose remains, such as cellulose oligomers, which can act as a cellulase production inducer in the lipid production process using organisms that have activity cellulase. In addition, the spent culture liquid from the anaerobic process (process 2) may contain other organic compounds, such as organic acids, alcohols, glycerol, which can be converted to lipids in anaerobic bioprocesses.
[0093] The spent culture medium from the aerobic lipid production process (Process 1) can be recovered, or
33/99 concentrated for the enrichment of enzymes, or used as such, as dilution water from the hydrolyzate inlet to process 2.
[0094] Figure 2 describes an embodiment of the invention, in which aerobic lipid production and anaerobic alcohol production is integrated. The aerobic lipid production process uses cellulose as raw material, while alcohol production uses hemicellulose as raw material. The lipid-producing aerobic process (process 1), lipid-producing microorganisms (s) are chosen which produce both at least cellulases, preferably both hemicellulases and cellulases. The hemicellulase and / or cellulase enzymes are recovered and reused for the hydrolysis of cellulose in other bioprocesses that contain the anaerobic fermentation of alcohols, such as ethanol, butanol or ABE, preferably the production of ethanol or butanol.
[0095] In one embodiment of the invention, cellulose or hemicellulose fractions are divided into two parts. A part of cellulose or hemicellulose can be used to grow biofuel-producing organisms that have enzymatic capabilities for the degradation of sugar polymers. Enzymes can be recovered from the metabolized culture medium, or spent culture medium, from enzymes can be reused for the production of biofuels from
34/99 second part of cellulose or hemicellulose. In a preferred embodiment of the invention, lipids and ethanol are produced by microbiological processes, such as microorganisms using polymeric sugars capable of accumulating lipids are used for a part of cellulose or hemicellulose. Ethanol is produced from another part of cellulose or by hemicellulose treated by enzymes recovered from the spent culture medium of the first process using lipid producers capable of using polymeric sugars. Likewise, however, in another embodiment of the invention, lipids and butanol or lipid and acetone-butanol-ethanol are produced. Likewise, acetone-butanol-ethanol and ethanol can be produced.
[0096] Figure 3 describes an example, of the production of biofuels from cellulose, by an integration of two bioprocesses. The first bioprocess comprises the aerobic production of lipids, which also produces exoenzymes capable of degrading polymeric cellulose. The enzymes are re-used for the hydrolysis of cellulose in another bioprocess using cellulose, which comprises the anaerobic fermentation of alcohols, such as ethanol, butanol or ABE, preferably the production of ethanol or butanol. Figure 4 describes a similar bioprocess to integrate bioproduction of biofuels, but the process uses
35/99 raw material hemicellulose. Preferably, the first bioprocess is the production of lipids and the second Bioprocess is the production of butanol or ABE.
[0097] Therefore, any combination: lipid + ethanol; lipid + butanol; lipid + ABE, butanol + ethanol; ABE + ethanol; + ethanol ethanol, etc., in any order can be used in various embodiments of the invention.
[0098] According to one embodiment of the invention, raw materials containing cellulose or hemicellulose are divided into two fractions.
[0099] The process, as described here, is not limited to the use in the production of only biofuels. It can be combined with any type of aerobic or anaerobic fermentation starting using polysaccharides as sources of carbon and / or energy.
[00100] In one embodiment of the invention, a mixture of cellulose and hemicellulose is used for the production of biofuels by microorganisms that have enzymatic capacities to degrade polymeric sugars. Cellulose and hemicellulose can be derived from the same material or are fractions of different raw materials. The mixture may also contain some or remains of lignin or legnin and / or pectin degradation products. In a bioprocess, such organisms are preferably used, which produce biofuels and also hemicellulases and cellulases.
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Enzymes can be recovered from the metabolized culture medium, or spent culture medium, including enzymes can be reused to hydrolyze cellulose or hemicellulose for the production of biofuels using organisms that are unable to use polymeric sugars. In a preferred embodiment of the invention, lipids and ethanol are produced by microbiological processes, such as microorganisms using polymeric sugars capable of accumulating lipids are used for a part of the cellulose or hemicellulose. Alcohols, such as ethanol, butanol or ABE, are produced from another part of cellulose or hemicellulose treated by enzymes recovered from the spent culture medium of the first process using lipid producers capable of using polymeric sugars. Likewise, however, in another embodiment of the invention, lipids and butanol or lipid and acetone-butanol-ethanol are produced. Likewise, acetone-butanol-ethanol and ethanol, ABE and lipids, ethanol and ABE, ethanol or lipids can be produced.
[00101] In one embodiment of the invention, the raw material for the process comprises polymeric sugars from both hemicellulose and cellulose and uses microorganisms capable of producing exoenzymes for the hydrolysis of sugar polymers and biofuels (Process 1). Exoenzymes in the
37/99 spent culture medium is recovered and reused in saccharification of polymeric sugars from both hemicellulose and cellulose before or in another biofuel production process (Process 2) that use organisms that may not be able to use polymeric sugars. As an example, hydrolysates containing polymeric sugars of hemicellulose and cellulose are divided into two fractions, a fraction used for Process 1, and another fraction used for Process 2.
[00102] A specific embodiment of the invention, using a mixture of cellulose and hemicellulose of the lignocellulosic raw material is shown in Figure 5. In this embodiment, in the aerobic process of lipid production of the invention including organisms capable of using polymeric sugars in cellulose and hemicellulose is integrated with the anaerobic alcohol production process, such as ethanol, butanol or ABE production process (Process 2). Vinasse and / or spent culture liquid can be recycled from the alcohol production process with the lipid production process, which may contain polymeric sugars that can act as inducers for the production of hemicellulases and cellulases in the production process of lipids. In addition, the spent culture liquid from the anaerobic process (Process 2) may contain other organic compounds, such as organic acids, alcohols, glycerol, which can be converted to
38/99 lipids in anaerobic bioprocesses. Furthermore, the spent culture medium or vinasse from process 2 may contain enzymes that are beneficial to process 1.
[00103] In one embodiment of the invention, the sugars used for the first bioprocess (Process 1) comprise mainly hemicellulose polymeric sugars, but also some cellulosic sugars in polymeric form. For example, the flow containing hemicellulose polymeric sugars is completed with a flow containing polymeric cellulose sugars. The First Bioprocess uses microorganisms capable of producing exoenzymes for the hydrolysis of polymeric sugars and biofuels. The second bioprocess (Process 2) uses raw material that comprises cellulose and microorganisms that are capable of producing cellulose biofuels in sugars, but which are not capable of using polymeric sugars. The spent Process 1 exoenzymes of culture medium used are recovered and reused in the saccharification of polymeric sugars from both hemicellulose and cellulose before or in the second bioprocess (bioprocess 2). Figure 6 describes an example of bioprocesses, such integration, where the first bioprocess is an aerobic, microbial lipid production process and the second bioprocess is an anaerobic process, alcoholic fermentation. More preferably, the second bioprocess is an ethanol or butanol fermentation process.
[00104] In yet another embodiment of the invention, the sugars used for the first bioprocess (Process 1) consist mainly of polymeric cellulose sugars, but also some hemicellulosic sugars in polymeric form. Flow, for example, containing polymeric cellulose sugars is completed with a flow containing polymeric hemicellulose sugars. The first bioprocess uses microorganisms capable of producing exoenzymes for the hydrolysis of polymeric sugars and biofuels. The second bioprocess (Process 2) uses raw material that comprises hemicellulose and microorganisms that are capable of producing biofuels from sugars in the hemicellulose, but that are not capable of using polymeric sugars. Exoenzymes of spent culture medium from Process 1 are recovered and reused in the saccharification of polymeric sugars from both hemicellulose and cellulose before or in the second bioprocess (bioprocess 2). Figure 7 describes an example of a bioprocess, as integrated, where the first bioprocess is an aerobic, microbial lipid production process and the second bioprocess is an anaerobic, alcoholic fermentation process. More preferably, the second bioprocess is a butanol and / or ABE fermentation production process.
[00105] In another embodiment of the invention, microorganisms using polymeric sugar produce
40/99 sets of enzymes that have the ability to use both cellulose and hemicellulose.
[00106] In one embodiment of the invention, the organism capable of using polymeric sugars and producing biofuel components and another organism capable of producing biofuel components, but is not capable of using polymeric sugars, which are added in the same reactor (mixed culture ).
[00107] In one embodiment of the invention, cells from the biofuel production process with polymeric sugars are removed, and the spent culture medium, as such, including enzymes capable of hydrolyzing the sugar polymers, are fed to another production process of biofuels with organisms that are not capable of using polymeric sugars.
[00108] More specifically, in one embodiment of the invention microorganisms are used, such as those that are capable of accumulating lipids or alcohol production, such as ethanol, butanol or ABE, which can use pentoses, thus increasing the efficiency of use of lignocellulose. In addition, organisms, such as lipid-accumulating organisms or alcohol-producing organisms, are also able to use polymeric sugars in cellulose and / or hemicellulose in exoenzymes. Enzymes can be recovered from the spent culture medium and used for
41/99 hydrolysis of sugar polymers in biofuel production processes, where microorganisms are unable to use polymeric sugars. In a specific embodiment of the invention, both bioprocesses use microorganisms that are capable of using polymeric sugars capable of producing biofuels or starting material for biofuels.
[00109] In one embodiment of the invention, the microorganism is even used in the production of lipid and alcohol, in particular, ethanol from lignocellulosic material. The production of lipids is obtained in an airy process (aerobic cultivation), whereas alcohol, in particular, ethanol production is obtained in anaerobic or microaerobic cultivation.
[00110] In a specific embodiment of the invention it includes an integrated process of one of the bioprocesses in an integrated process that is a mesophilic process producing biofuels and, possibly, with enzymes, the operating temperature below 45 ° C, preferably below 40 ° C, while the other bioprocess is a thermophilic process for the production of biofuels and, possibly, enzymes, with an operating temperature above 45 ° C, preferably above 55 ° C. An example of such an integrated process is a mesophilic lipid production aerobics with anaerobic thermophilic alcohol (s), such as ethanol, production process. THE
42/99 combination of mesophilic and thermophilic processes can be beneficial for the recycling and reuse of enzymes between bioprocesses. As an example, the enzyme-containing supernatant, also likely to comprise some cells, produced in the medium in the mesophilic process are reused in a thermophilic process. Process enzymes can be thermostable mesophilic to tolerate thermophilic process temperatures, while the residual cells present in the supernatant are inactivated. And therefore, it is not able to grow in the thermophilic process. Alternatively, vice versa, organisms grown in the thermophilic temperature range, do not grow well in the mesophilic temperature range in the next bioprocess and do not contaminate the process.
Recycling of Effluents and Biomass [00111] The invention allows the recycling of effluents from one bioprocess to another bioprocess. In the preferred embodiment of the invention, the ingrate uses at least one aerobic and one anaerobic bioprocess for the production of biofuels. This reduces the risk of contamination when recycling waste from the aerobic process to the anaerobic process. The lipids produced by aerobic bioprocesses can use effluent compounds from anaerobic bioprocesses as a source of carbon and nutrients. Anaerobic bioprocesses can be, for example, ethanol fermentation, butanol acetone fermentation or fermentation
43/99 butanol-ethanol (ABE-fermentation). These bioprocesses typically result in organic acids, such as acetic acid, butyric acid or acetaldehyde in fermentation of the effluent.
[00112] In addition to enzymes, recycling of biomass and / or culture medium spent between bioprocesses provides nutrients, minerals and / or growth factors, such as proteins, amino acids, vitamins, coenzymes, metabolites, which decrease the need for anabolism in microorganisms and therefore increase the production of microbial biofuels, in particular, the production of lipids, which is a step in the aerobic process. In addition, microbial cells from the anaerobic process contain lipids, such as membrane lipids, which can be used by lipid-producing organisms, for example, incorporated or transformed into lipid triacylglycerol producers. Organisms producing lipids can use residual alcohols from the fermentation broth of the alcohol production process, after product recovery. In a specific embodiment of the invention, fermentation alcohols are not recovered, and the fermentation broth, which contains alcohols and possible active enzymes, is fed into the aerobic process of lipid production, where they are converted to lipids.
[00113] The supernatant of lipid production in
44/99 bioprocesses or aerobic alcohol production in anaerobic bioprocesses can be collected at different times, in order to optimize the amount of enzymes for reuse in other bioprocesses. In one embodiment of the invention, the biomass and / or supernatant is removed, in part, from the aerobic bioprocess during fermentation, in order to optimize the activity of enzymes for reuse, and the lipid content of the biomass.
[00114] In one embodiment of the invention, the cell residue and other solid waste from lipid production is after recovery of lipid or biomass from anaerobic alcohol production can be (thermo) mechanically, chemically or enzymatically treated before recycling back to the lipid production process or anaerobic alcohol production bioprocesses. If a cascade system is used, biomass can be recycled to one or all of the reactors in a cascade system. It is also possible to recycle biomass and / or fermentation broth between cascade fermenters, with or without the treatment of cells in the medium. This can shorten the fermentation time, increasing the amount of biomass or active microorganisms and / or enzymes.
[00115] In one embodiment of the invention, solid and biomass residues, spent and / or recovered culture medium or enzymes from culture medium
45/99 enriched are partially recycled in the same bioprocess. This can improve the production of enzymes and / or biofuels in the bioprocess.
[00116] The recycling of metabolized biomass and / or culture medium can result in the accumulation of minerals, inert materials and other compounds, as well as lead to inhibitons. Therefore, the amount of recirculation is optimized, and a certain amount of spent biomass and / or culture medium is removed periodically.
Pretreatment of liqnocellulose before fermentation [00117] Pretreatment of lignocellulose in order to improve the digestibility of polymeric sugar hydrolyzing enzymes can be carried out by any known method. Pretreatment can include the fractionation (separation) of possible cellulose and hemicellulose and lignin by any known method. The choice of the correct pretreatment method is largely dependent on the type of lignocellulosic raw material to be used in the process. There are several methods / technologies that are only suitable for a particular type of raw material. Preferably, the separation of hemicelluloses and / or cellulose is done with a method that produces hydrolysates that do not inhibit the growth of lipid-producing microorganisms. The hemicellulose and cellulose fractions may contain sugars, mainly, or at least in part, in the polymeric form.
46/99
One embodiment of the invention is the use of hot water extraction to extract hemicellulose. In addition, hemicellulose, hot water extraction can remove minerals from lignocellulosic materials that are preferable in fermentation, which reduces the need for mineral additions in culture medium. In another embodiment, pretreatment with organic acid is carried out, such as treatment with acetic acid, formic acid, ethyl acetate, lactic acid or malic acid, or any combinations thereof. In yet another embodiment of the invention, pretreatment with acid, such as with sulfuric acid, is performed. Also steam explosion with or without an acid catalyst is used. Also methods, such as organosolv pretreatment, such as methanol treatment such as using ethanol, acetone or any mixture thereof, possibly supplemented with an acid catalyst, such as sulfuric acid or sulfur dioxide (SO2) can be used. Also other methods, such as ammonia assisted pretreatment, ammonia fiber expansion, percolation of recycled ammonia or limestone pretreatments can be used. The lignocellulose material can be (thermo) mechanically treated, for example, the reduced particle size with any methods, such as, but not limited to, crushing or grinding, before or in the pre-treatment medium.
47/99 [00118] Purification and / or separation of fractions of cellulose, hemicellulose and lignin may not be necessary before feeding biomass for a process that produces exoenzymes capable of hydrolyzing polymeric sugars in lignocellulosic materials and produces biofuels, such as lipids .
Biomass Recycling [00119] Microbial biomass (cells), or biomass residues, such as biomass after lipid recovery, can be recycled from the first bioprocess to the second bioprocess. In addition, or alternatively, biomass or microbial biomass residues can be recycled from the second bioprocess to the first bioprocess. Microbial biomass can potentially be recycled with the supernatant. Microbial biomass can be treated (thermo) mechanically, enzymatically and / or chemically before feeding bioprocesses. In one embodiment of the invention, the microbial biomass for recycling is treated from the same operating unit, where the lignocellulosic biomass is treated. In one embodiment of the invention, the microbial biomass to be recycled goes through the same treatment as lignocellulosic biomass before being fed to a bioprocess, that is, the feed for the microbial production of biofuels or raw material for the production of
48/99 biofuels. The recycled microbial biomass contains nutrients that can be beneficial for the bioprocess to be fed. The first bioprocess can be lipid or alcohol production, while the second can be bioprocess for alcohol or lipid production.
[00120] Microbial biomass, biomass residues, such as biomass, after oil recovery, and bioprocess supernatants can be recycled between bioprocesses. The supernatant and the bioprocess cells contain nutrients and / or enzymes that can be recycled between the bioprocesses and are advantageous for the bioprocess. Biomass and / or recycling supernatants can improve the overall yield of the product in bioprocesses and decrease the need to purchase minerals or nutrients outside and can thus improve the economics of biofuel processes.
[00121] In one embodiment of the invention, at least a part of the supernatant and at least a part of the microbial biomass, or biomass residues, from a bioprocess is fed into bioprocess 2.
[00122] In one embodiment of the invention, at least part of the supernatant, and / or at least part of the cells and / or cell residue from bioprocess 2 can be recycled back to bioprocess 1.
[00123] If a bioprocess is an aerobic process, such as a microbial one, lipid production can use waste
49/99 organic, such as organic acids, alcohols, aldehydes or in the supernatant from an alcohol production process.
[00124] In one embodiment of the invention, process water or part of it, from the second biotechnological process after separation of the biofuel is recycled to the dilution water of the raw material of the first biotechnological process and / or second biotechnological process, from preferably the first biotechnological process.
[00125] In one embodiment of the invention, the supernatant or effluents from or after the recovery of alcohol from the ethanol or butanol production process is recycled to a lipid production process. The lipid production process can use remains of ethanol or butanol in the supernatant or effluent for microbial growth and / or lipid production. Therefore, the recovery of ethanol or butanol does not need to be complete, since the remaining ethanol or butanol can be used for the production of lipids in the subsequent biofuel production process by recirculating the effluent. This is advantageous since the very high (yield) product removal efficiency (ethanol or butanol) typically results in increased capital or operating costs. Enabling slightly lower product recovery yields can reduce operating or
50/99 capital.
[00126] In another embodiment of the invention, the supernatant or effluents from or after recovery from the fermentation process from ABE is recycled to a lipid production process. The lipid production process can use remains of ABE, in the supernatant or effluent for microbial growth and / or lipid production. Therefore, the recovery of ethanol does not need to be complete, since the remaining ABE can be used for the production of lipids in the subsequent biofuel production process, by recycling the effluent. Raw Materials [00127] The method can be applied to any lignocellulosic materials, including woody or non-woody plants, herbaceous or other materials containing cellulose and / or hemicellulose: The materials can be agricultural waste (such as wheat straw, rice straw , straw, bark, corn straw, cane bagasse), energy crops (such as grasses, Miscanthus, yellow grass, willow, water hyacinth), wood materials or waste (including sawmill and cellulose and / or waste from mills) paper or fractions, such as hemicellulose, passed liquor sulfite, waste fiber and / or primary sludge), peat moss or, microorganisms or municipal paper waste. Also low-lignin materials, materials such as macroalgae or biomass from
51/99 microalgae can be used. In addition, the materials can also be fractions of hemicellulose or cellulose from industrial practices. The invention can use any type of cellulose fraction. The invention can use any type of hemicellulose fractions, containing, for example, but not limited to galactoglucomannan, xylan or arabinoxylan as main fractions. Raw materials or certain fractions of hemicellulose, and / or cellulose, as raw materials of different origin, plant species, or industrial processes can be mixed together and used as raw materials for biological processes according to the invention.
[00128] The fraction of hemicelluloses and / or cellulose, containing polymeric sugars can be fed to the bioprocess that produces exoenzymes capable of hydrolyzing polymeric sugars in lignocellulosic materials and producing biofuels, such as lipids or alcohols, in any form, that is, solid or dissolved form, or as partially solid form and partially dissolved form.
[00129] In one embodiment of the invention, lignocellulosic biomass is added as a solid in the form of bioprocess to produce exoenzymes capable of hydrolyzing polymeric sugars in lignocellulosic materials and producing biofuels, such as lipids. In one embodiment of the invention, the solid lignocellulosic material may have been
52/99 mechanically treated to obtain smaller particle size, for example, by grinding or grinding, but has not been pre-treated for separate cellulose, hemicellulose and lignin fractions, before fermentation. In yet another embodiment of the invention, the solid fraction of the lignocellulose is, in addition to the mechanical treatment for the reduction of the size of the particles, treated with the methods that were opened or released from the lignocellulose structure, before feeding for the production of exoenzymes of bioprocessing capable of degrading polymeric sugars and biofuels (Process 1). This solid fraction of lignocellulose may contain cellulose and / or hemicellulose and lignin in polymeric form.
[00130] The fraction of lignin, if fractionated from lignocellulose, can be used for any known purposes, such as, but not limited to energy and heat production, for the production of biochemicals (bioplastics, resins), structural biomaterials, pyrolysis of hydro-deoxygenation or gasification and Fischer-Tropsch synthesis of compounds that can be used as chemicals, biofuels and / or lubricants.
[00131] The advantage of the invention is that the process produces the necessary exoenzymes by itself (in situ) without the need to reduce or add other enzymes that are not produced by the strains used in the described process. Exoenzymes produced in a lipid production process
53/99 cellulose and / or hemicellulose are recovered from the culture medium and used in the integrated process for the production of biofuels or biofuel raw material with organisms that are not capable of using polymeric sugars. In another embodiment of the invention, the culture medium, from the production of lipids from polymeric sugars containing exoenzymes capable of degrading polymeric sugars, after cell recovery is concentrated in enzymes or used without enrichment as a culture medium for biofuel or raw material for biofuel production of sugars by polymeric organisms that are not capable of using polymeric sugars.
[00132] The raw materials for the production of biofuels according to the invention include those that preferably contain at least some polymeric sugars.
[00133] In the most preferred embodiment of the invention, the raw material of biomass is lignocellulosic or any of its fractions.
[00134] In another embodiment of the invention, the raw material is starch or contains starch. Some examples of materials that contain starch include, but are not limited to corn, cereals such as wheat and barley, tapioca, cassava, rice, potatoes, sweet potatoes, yams and
54/99 microalgae.
[00135] According to the invention, the first bioprocess uses raw materials containing starch, using microorganisms that are capable of using polymeric sugars in starch and the production of biofuels. The starch hydrolysis of enzymes in the supernatant of the first bioprocess are fed to the second bioprocess for the microbial production of biofuels that hydrolyze the starch from polymeric sugars. The second bioprocess uses microorganisms that are unable to use polymeric sugars in starch, or alternatively, use microorganisms that are capable of using polymeric sugars in starch. The introduction of enzymes with starch hydrolysis activity to increase starch hydrolysis for the second bioprocess.
Liqnocellulose Hydrolysis [00136] Cellulose typically does not dissolve in fresh water. The hydrolysis of solid cellulose normally requires three different types of enzymes: endoglucanases, exoglucanases and β-glucosidases endoglycanases (EC 3.2.1 0.4), operated mainly on the amorphous part of cellulose, attacking randomly in internal tanks of the cellulose Macromolecule. Exoglucanases or cellobiohydrolases (EC 3.2.1 .91) attack the end of the cellulose chain, mainly hydrolyzing one cellobiose unit at a time.
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Exoglucanases are also capable of hydrolyzing crystalline cellulose polymer. Finally, the hydrolysis of cellobiose to glucose monomers is done by β-glucosidase (EC 3.2.1 .21).
[00137] Cellulose hydrolysis generally needs the cooperation of many different cellulases. The amount of different glycosylhydrolases analyzed is very high, more than 90 different enzymes are already accounted for (even more under study) in 14 different families as an example, cellobiohydrolase domains (CBH I, II), endoglucanese domains (EG I,
II. Ill, IV, V) and beta-glucosidase domains (BGL I, II).
[00138] For the total enzymatic hydrolysis of hemicellulose (xylans, arabinoxylans and glucomannans) several different enzymes are required, which must be activated at the same time. First attack is typically carried out by enzymes, such as endoxylanases (1,4-D-xylan xylanohydrolases), endoarabinases and endomannanases (1,4-β-Dmanane mananohydrolases). For example, Trichoderma reesei has at least four different endoxylanases and one endomananase.
[00139] Enzymes capable of catalyzing the hydrolysis of hemicellulose oligomers after the operation of endohemicellulases are, for example, β-xylosidase, βarabinosidase, β-mannosidase and β-glucosidase (EC 33.2.1 .21). (3.2.1 CE. 0.22) for breaking for side connections
56/99 residuals included in the oligomers' -glucuronidase (EC 3.2.1 0.139), '-arabinodase (EC 3.2.1 0.55) and' -D-galactosidase are required. The removal of acetyl components requires the operation of the esterases (EC 3.2.1.72).
[00140] Furthermore, enzymatic hydrolysis of lignin requires activity of oxidative enzymes, such as lignin peroxidase (LiP CE 1.1 1.1.14), manganese-dependent peroxidase (MnP CE 1.11.1.13) and laccase (Ec 1 .10.3 0.2 ). Modification of lignin needs the cooperation of many enzymes, coenzymes and electron transport system between donors and final recipients. The chemical structure and binding of lignin to cellulose and hemicellulose is more important than the amount of lignin.
[00141] ABE fermentation or ABE production refers to a process in which a mixture of acetone, butanol (nbutanol) and ethanol is produced by bacterial fermentation. In some cases, iso-propanol is produced instead of acetone, depending on the bacterial strain.
[00142] A process for the production of alcohols, typically comprises the cultivation of anaerobic microorganisms in a bio-reactor, typically in a fermenter. The microorganism is allowed to produce alcohol. The alcohol is collected from the fermentation broth, typically by distillation. Alcohol, such as ethanol and / or butanol, can be recovered and used as a biofuel. Ethanol
57/99 typically needs to be dehydrated to a concentration of 99.5% before being used as biofuels, for example, as mixtures of ethanol and gasoline in vehicles. Microorganisms [00143] For the bioprocess (Process 1) containing animal feed, including polymeric sugars suitable microorganisms can be any microorganisms that are capable of using sugars and polymers capable of producing the appropriate compounds for biofuel purposes. In the preferred embodiment of the invention, the lipid production of organisms used in the invention can be any organisms that can use polymeric sugars in hemicellulose and / or cellulose.
[00144] These organisms include, but are not limited to bacteria, such as Streptomyces, Bacillus or filamentous fungi, such as Aspergillus, Cephalosporium, Fusarium, Humicola, Microsphaeropsis, Nigrospora, Penicillium, Phanerochaete, Phomopsis, Rhizopus, Sclerocystis, Trichoderma or Trichoderma such as A. niger, A. terreus, A. oryzae, A. nidulans, F. oxysporum, Phanerochaete chrysosporium, R. oryzae or Trichoderma reesei, yeasts, such as Cryptococcus or Trichosporon, such as Cryptococcus albidus or Trichosproron cutaneum. Oilseed microorganisms that are genetically modified to be able to use polymeric sugars in cellulose and / or hemicellulose are also part
58/99 of the invention. In addition, organisms capable of using polymeric sugars in cellulose and / or hemicellulose which are genetically modified for improved lipid production, are also included in the present invention.
[00145] Microorganism capable of producing both enzymes and lipids is preferably a fungus, yeast or bacterium, preferably belonging to a genus selected from the group of Aspergillus, Humicola, Rhizopus and Trichoderma, or a yeast belonging to the Cryptococcus genus or a bacterium belonging to Streptomyces.
[00146] In the most preferred embodiment of the invention, lipid-producing microorganisms are used that can use polymeric sugars from both hemicellulose and cellulose, i.e., they have both hemicellulose and cellulase activity. These organisms include, but are not limited to, filamentous fungi, such as Aspergillus, such as Aspergillus terreus, and bacteria, such as Streptomyces.
[00147] For the use of the second bioprocess (Process 2), in which the polymeric sugars have been enzymatically digested, producing lipid organisms that are not capable of using polymeric sugars can be used. However, the process can also use organisms that are capable of using polymeric sugars. Lipid-producing organisms are selected from the group of bacteria, cyanobacteria, fungi, such as
59/99 yeasts and filamentous fungi (molds), archaebacteria or microalgae. Microorganisms can easily accumulate lipids or have been genetically modified to accumulate lipids or to improve lipid accumulation. Organisms producing lipids include, but are not limited to, the following organisms:
[00148] Species of microalgae belonging to the genera including Dunaliella, Chlorella, Botryococcus, Brachiomonas, Chlorococcum, Crypthecodinium, Euglena, hematococci, Chlamydomas, Isochrysis, Pleurochrysis, Pavlova, Prototheca, Phaeodocochone, Phaeodochochone, Phaeodachochone, Nitzschia, Nannochloropsis, Navicula, Nannochloris, Scihizochytrium, Sceletonema, Thraustochytrium, Ulkenia, Tetraselmis and Synechocystse.
[00149] Species of filamentous fungi belonging to the following genera Aspergillus, Mortierella, Chaetomium, Claviceps, Cladosporidium, Cunninghamella, Emericella, Fusarium, Glomus, Mucor, Paecilomyces, Penicillium, Pythium, Rhizopus, Trichoderma, Clichoderma, Zygorhchychurchium, Zygorhchy Ustilago.
[00150] Yeasts belonging to the following genera clavispora, Deparyomyces, Pachysolen, Kluyveromyces, Galactomyces, Hansenula, Saccharomyces, Waltomyces, Endomycopsis, Cryptococcus, such as Cryptococcus curvatus,
60/99
Rhodosporidium, such as Rohodosporidium toruloides, Rhodotorula, such as Rhodotorula glutinis, Yarrowia, such as Yarrowia lipolytica, Pichia, such as Pichia stipitis, Candida, such as Candida curvata, Lipomyces, such as Lipomyces starkeyi, Trichosporon, such as Trichosporon cutum pullulans.
[00151] Bacteria belonging to the following genus Acinetobacter, Actinobacter, Alcanivorax, Aerogenes, Anabaena, Arthrobacter, Bacillus, Clostridium, Dietzia, Gordonia, Escherichia, Flexibacterium, Micrococcus, Mycobacterium, Nocardia, Nostoc, Oscillatoria, Rhodococcus, Rhodocephaly Rhodopseudomonas, Shewanella, Shigella, Vibrio and Streptomyces. More preferably, bacteria comprise Rhodococcus opacus, Acinetobacter, Nocardia or Streptomyces.
[00152] The organisms used for the production of ethanol can be selected from a group of bacteria, cyanobacteria, fungi, such as yeasts and filamentous fungi (molds), and microalgae, more preferably, bacteria, filamentous fungi and yeasts. Microorganisms can easily produce ethanol or have been genetically modified to accumulate lipids or to improve lipid accumulation. Ethanol producers include organisms that are capable of using monomeric or polymeric sugars with materials
61/99 lignocellulosic. Ethanol producing organisms include, but are not limited to, the following organisms:
[00153] Fungi, such as yeasts belonging to the following Saccharomyces genera, such as S. cerevisiae or S. uvarum, Candida, such as C. shehatae, Pachysolen, such as P. tannophilus, Pichia, such as P. stipitis and Schizosaccharomyces, like S. pombe.
[00154] Filamentous fungi belonging to the following genera such as Aurobasidium, such as A. pullulans and Fusarium, such as F. avenaceum or F. oxysporum.
[00155] Bacteria belonging to the following genera such as Bacteroides, Geobacillus, Clostridium such as C. thermocellum or C. saccharolyticum, Erwinia, such as E. chrysanthemi, Escherichia, such as E. coli, Klebsiella, such as K. oxytoca, Sarcina, Raoultella , ruminococcus, Spirochaeta, Thermoanaerobacter, such as T. ethanolicus, T. mathranii, T. thermohydrosulfuricus, T. thermoanaerobacterium, T. aciditolerans, T. aotearoense, T. polysaccharolyticum, T. thermosaccharolyticum, T.zeae, Thermobrachium, such as T. cekere , and Zymomonas, such as Z. mobilis.
[00156] The organisms used for the production of butanol, or acetone-butanol-ethanol, or iso-butanol-ethanolacetone can be selected from a group of bacteria, cyanobacteria, fungi, such as yeasts and filamentous fungi (molds) microalgae more of
62/99 preferably bacteria, filamentous fungi and yeasts, more preferably, bacteria. Butanol, or acetone-butanoletanol includes organisms that produce it, which are capable of using monomeric or polymeric sugars with lignocellulosic materials. Butanol or acetone-butanol-ethanol includes the production of organisms that include, but are not limited to, the following organisms:
[00157] Bacteria belonging to the following genera such as Clostridium, such as C. acetobutylicum, C.beijerinckii, C. butyricum, C. aurantibutyricum, C. saccharoperbutylacetonicum and Escherichia, such as E. coli.
[00158] “Oleaginous microorganism refers here as a microorganism that accumulates at least 15% (w / w) of its biomass as lipid when grown under suitable or efficient conditions for the production of lipids.
[00159] Mass of a single cell containing lipids means an autotrophic, heterotrophically and / or myxotrophically formed mass of a single cell and cell mycelium with a lipid content of at least 3%, preferably at least 10%, preferably at least less than 15% (w / w) or more of the microorganism's dry matter. Enzymes [00160] Enzymes that are part of the present invention include especially those that are capable of converting sugars into one usable from microorganisms.
63/99
Typically, such enzymes are hydrolytic enzymes, such as those that are capable of converting sugar polymers to sugar monomers. This is usually not done by a single enzyme, but by a group of enzymes. Alternatively, enzymes capable of converting sugars into usable form for microorganisms include isomerases.
[00161] Cellulase or cellulolytic enzyme refers to a group of enzymes produced by fungi, mainly, such as filamentous fungi or yeasts, bacteria, plants, by the animals that catalyze the cellulose hydrolysis, also called cellulolysis. The EC number for cellulase enzymes is EC 3.2.1 0.4. Several different types of cellulases are known, which differ structurally and mechanically. In general, cellulases include, based on the type of catalyzed reaction, endo-cellulases, exo-cellulases, cellobiases or beta-glycosidases, oxidative cellulases and cellulose phosphorylases.
[00162] Hemicellulase refers to a group of enzymes produced mainly by fungi, such as filamentous fungi or yeasts, bacteria, plants, of animals by which they catalyze the hydrolysis of hemicellulose. For example, enzymes involved in xylan hydrolysis include endoxylanases, acetyl-xylanesterases, α-D-glucuronidases, '-Larabinofuranosidases, ferulic acid esterase and βxilosidases. In addition, the enzymes involved in hydrolysis
64/99 of galactoglucomannan include endo-mannanases, acetylmannanesterases, β-galactosidase, β-glucosidases, βManosidases. In addition, enzymes involved in the hydrolysis of arabinogalac-tan include β-galactosidase and Endo-aLarabinanase. These enzymes can be found in the following EC numbers: EC 3.2.1.8, EC 3.2.1 .37, EC 3.2.1 .55, EC 3.2.1 .99, EC 3.2.1 .139, EC 3.2.1 .78, EC 3.2.1 .25, EC 3.2.1 .22, EC 3.2.1 .21, EC 3.2.1 .89, EC 3.1 .1 .72, EC 3.1 .1 .6, EC 3.1 .1 .73.
[00163] Hemicellulose refers to a group of complex carbohydrates found in a lignocellulosic material that, with other carbohydrates (for example, pectins), surround the cellulose fibers of plant cells. The composition of hemicelluloses depends on the type of plant. Most common types of hemicelluloses include xylan, glucoronoxylan, glucomannan, galactoglucomannan, arabinoxylan, xyloglucan and arabinogalactan.
[00164] Lignocellulosic material or lignocellulosic biomass refers to biomass, which is composed of cellulose, hemicellulose, lignin and or any of its fractions.
[00165] Saccharification refers to the hydrolysis of polymeric sugars to sugar monomers. Saccharification is typically carried out using enzymes
65/99 capable of hydrolyzing polymeric sugars.
Bioprocess [00166] Microbial production of lipids can be carried out with any known method or a method developed in the future. Typically, the microbial process of lipid production includes the cultivation of microorganisms in aerated bioreactors in submerged culture. Microorganisms are grown in a liquid culture medium that comprises a carbon and energy source, such as hemicellulose and / or cellulose sugars, and macro and micronutrients. Cultivation can be carried out, for example, as batch cultivation, batch cultivation, continuous cultivation. The culture can also be carried out in a cascade process. In cultivation, microorganisms are left to grow and accumulate intracellular lipids. Some microorganisms may also be able to excrete lipids into the culture medium.
[00167] The microbial lipid production process can also be carried out in reactors, where the amount of free water is low, or when the production is carried out on a solid or semi-solid surface. The cell mass or other biomass does not dissolve in water, it can be extracted with aqueous solutions, in order to obtain the enzymes in soluble form.
[00168] Microbial production of ethanol, butanol, or
6/99 acetone-ethanol-butanol is carried out with any known method or a method developed in the future. Typically, microorganisms are grown in a submerged fermenter. Microorganisms are grown in a liquid culture medium that consists of carbon and energy sources, such as hemicellulose and / or cellulose sugars, and macro and micronutrients. Cultivation can be carried out, for example, as batch cultivation, batch cultivation, continuous cultivation. The culture can also be carried out in a cascade process.
Recovery of enzymes from spent culture medium [00169] Enzymes can be recovered from cultured microorganisms, spent culture medium, supernatant and cells of the microorganism by any known and suitable method, or by any suitable method developed in the future. The same also applies to methods by which enzymes can be separated into fractions with the desired enzyme activities.
[00170] A preferred method for the recovery of enzymes is a method by which the culture of microorganisms, the supernatant, or any combination of them can be treated by a technician skilled in the subject to carry out the recovery of enzymes, maintaining their catalytic activity .
[00171] The supernatant cells and / or microorganisms can be separated from the culture of
67/99 microorganisms and used as an enzyme preparation or as an enzyme source. The supernatant represents a substantially cell-free fraction, which comprises the spent culture medium. The supernatant can also be called a fermentation liquid, a liquid phase or both cultures or culture broth.
[00172] The separation of the supernatant and the cells can be done by any suitable method to maintain the catalytic activity of the enzymes.
[00173] A method by which the culture of the microorganism or supernatant or protein enriched fraction comprising catalytically active enzyme (s) are recovered can be based on their molecular size, ionic behavior, water solubility, the solubility of different solutes or solubility in a mixture of solutes containing a factor or factors with surface activity or an active surface compound or a buffering salt.
[00174] Enzymes can be recovered from the culture medium by various procedures, including, but not limited to, procedures such as centrifugation, filtration, extraction, spray drying, evaporation or precipitation.
[00175] If necessary, enzymes can be purified or isolated by various procedures, including but not limited to chromatography, procedures
68/99 electrophoretic, differential solubility, SDS-PAGE, or extraction.
[00176] Enzymes can be stabilized, for example, by salt, sugar or glycerol.
[00177] In addition, enzymes can be formulated for the desired application.
[00178] Extracellular enzymes are enzymes excreted into the culture medium or released by lysis of the cells from the cells into the culture medium. Extracellular enzymes can be recovered from the supernatant.
[00179] In one embodiment of the invention, the protein fraction is enriched with the supernatant. Enrichment can be performed simply, for example, by concentrating the supernatant.
[00180] In some embodiments, the protein fraction is enriched by at least 10%, typically at least 20%, in various embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, compared to the original liquid phase. Examples of suitable methods are methods based on the ionic properties of proteins, molecular size, solubility, active surface properties or hydrophobic interactions. Preferably, the recovery of the enzyme fraction is carried out under conditions, where the temperature is 70 ° C or lower.
[00181] In one embodiment of the invention, the protein fraction in the supernatant is enriched at least once (1 x), typically at least twice (2x), preferably at least three times (3x). In some embodiments, the protein fraction in the aqueous phase of the microorganism culture or in the supernatant is enriched at least 5 times, in some embodiments, at least 10 X, or 20 X, or 30 X, or 40 x, or 50 x , or 60 x, or 70 x, or 80 x, or 90 x or 100 x, or calculated as enzyme activity per unit volume and / or per total protein.
[00182] In addition, in some embodiments, the supernatant can be diluted before use in the integrated process.
Production of biofuels from lipids [00183] Biofuels refers to solid, liquid or gaseous fuel derived mainly from biomass or bio-waste and is different from fossil fuels, which are derived from organic plant waste and prehistoric animals.
[00184] According to the European Directive 2003/30 / EU biodiesel refers to a methyl ester produced from vegetable oil or oil of animal origin, with diesel quality to be used as biofuel. More broadly, biodiesel refers to long chain alkyl esters, such as methyl, ethyl or propyl esters, from
70/99 diesel quality vegetable or animal oil. Biodiesel can be produced from lipids, where the lipids of microorganisms can originate from bacteria, a fungus (a yeast or a mold), algae or other microorganism.
[00185] Renewable diesel refers to a fuel that is produced by a hydrogen treatment of lipids from an animal, plant or microorganism, or mixtures thereof, by which lipid microorganism may originate from a bacterium, a fungus (a yeast or mold), algae or other microorganism. Renewable diesel can also be produced from waxes derived from biomass by gasification and Fischer-Tropsch synthesis. Optionally, in addition to the hydrogen treatment, isomerization or other processing steps can be performed. Renewable diesel process can also be used to produce jet fuels and / or gasoline. The production of renewable diesel has been described in patent publications EP 1396531, EP 1398364, EP 1741767 and EP 1741768.
[00186] Biodiesel or renewable diesel can be mixed with fossil fuels. Suitable additives, such as preservatives and antioxidants, can be added to the fuel product.
[00187] Lubricant refers to a substance, such as fat, lipid or oil, which reduces friction when
71/99 applied as a surface coating for moving parts. Two other main functions of a lubricant are to remove heat and to dissolve impurities. Lubricant applications include, but are not limited to use in internal combustion engines, such as engine oils, fuel additives, in oil driven devices, such as pumps and hydraulic equipment, or in different types of bearings. Normally lubricants contain 75 - 100% base oil and the rest are additives. Suitable additives are, for example, detergents, storage stabilizers, antioxidants, corrosion inhibitors, dehazers, demulsifiers, defoaming agents, cosolvents and lubricating additives (see for example, US 7,691,792). Lubricating base oil may originate from mineral oil, vegetable oil, animal oil or from bacteria, fungi (a yeast or a mold), algae or other microorganism. Base oil can also originate from waxes derived from biomass by gasification and Fischer-Tropsch synthesis. The viscosity index is used to characterize the base oil. Typically a high viscosity index is preferred.
[00188] The lipids produced according to the method described in the present invention can be used as raw material for the production of biodiesel, renewable diesel, jet fuel or gasoline. Biodiesel is made up of
72/99 by fatty acid methyl esters, and is typically produced by transesterification. In transesterification, acylglycerols are converted to alkyl esters of long-chain fatty acid (methyl, ethyl or propyl). Renewable diesel refers to the fuel that is produced by treating hydrogen (hydrogen deoxygenation, hydrogenation or hydroprocessing) of lipids, in the treatment of hydrogen, acylglycerols are converted into corresponding alkanes (paraffins). Alkanes (paraffins) can be further modified by isomerization or other alternative processes. Renewable diesel process can also be used to produce jet fuels and / or gasoline. In addition, lipid cracking can be performed for the production of biofuels. In addition, lipids can be used as biofuels directly in certain applications.
[00189] The lipids produced with the method can be used as a base for lubricating oils (lubricating oils) or as a starting material for the production of base oils for lubricants [00190] The term lipid refers to a grease substance , whose molecule generally contains, as a part, an aliphatic hydrocarbon chain, which dissolves in non-polar organic solvents, but is poorly soluble in water. Lipids are an essential group of large molecules
73/99 in living cells. Lipids are, for example, fats, oils, waxes, wax esters, sterols, terpenes, isoprenoids, carotenoids, polyhydroxyalkanoates, nucleic acids, fatty acids, fatty alcohols, fatty acid esters, phospholipids, glycolipids, sphingolipids and acylglycerols.
[00191] The term aciglycerol refers to an ester of glycerol and fatty acids. Acylglycerols occur naturally as fats and fatty oils. Examples of acylglycerols include triacylglycerols (TAG, triglycerides), diacylglycerols (diglycerides) and monoacylglycerols (monoglycerides).
Oil Recovery [00192] Microorganisms containing lipids can be separated from the culture medium by any known methods, such as using filtration or decanting techniques. Alternatively, centrifugation with commercial centrifuges on an industrial scale with a large volume capacity can be used to separate the desired products.
[00193] In various embodiments of the invention, the oil, or oil precursors, can be recovered from cell biomass or culture medium by any method known in the art or developed in the future. Such methods include, but are not limited to, solvent extraction
74/99 organic. In various embodiments of the invention, cells of the microorganism can be disrupted to facilitate the separation of oil and other components. Any known method for cell disaggregation can be used, such as ultrasound, osmotic shock, mechanical shear force, cold pressing, thermal shock, enzyme-catalyzed or self-directed autolysis.
[00194] The residue from the extracted oil cells can be used for energy production, for example, combustion or treated with the anaerobic digestion process, or used as animal feed. Waste extracted from oil cells, or a fraction of waste from the cell can also be recycled back into bioprocesses to be used as a source of nutrients.
Alcohol recovery (ethanol, butanol, ABE) [00195] The recovery of ethanol from the fermentation broth can be done by any method. Traditionally, distillation is used. When distillation is used as a technology for separating alcohol products, savings can be achieved by combining product recovery unit processes, such as distillation for the alcohol production process and oil extraction for the alcohol production process. lipids including recovery of oil extraction solvent, energetically together. Heat recovery processes can regenerate
75/99 process that can be used in another production process of the integration unit, such as enrichment, or concentration of hydrolysates or for other purposes.
[00196] In one embodiment of the invention, alcohols from the anaerobic process of alcohol production are used as extraction solvent, possibly together with other lipid extraction solvents from microbial cells. In a specific embodiment of the invention, from the anaerobic fermentation process, ethanol is used in conjunction with a non-polar solvent, such as hexane, to extract lipids from oil-rich cells formed in the lipid-producing aerobic process .
[00197] In the recovery of alcohols, instead of distillation, which destroys the activity of most enzymes, even though high vacuum is during distillation, other methods, such as pervaporation or membrane technology can be used. Alcohol recovery can be carried out with fermentation broth methods containing biomass and active enzymes during the last stages of the fermentation cascade or after batch fermentation. This allows the maintenance of the enzyme activity in a spent culture medium or those bound in sugar oligomers, in order to recover, enrich them for reuse, such as recycling them for the bioprocess for the hydrolysis of
76/99 lignocellulosic biomass. With membrane technology, it is possible to control the molecular size of molecules, such as enzymes, for recovery, enrichment and reuse. Enzyme recovery and reuse can improve the efficiency and productivity of products in an entire integrated system for the production of biofuels.
[00198] For process steps using high temperature, such as distillation, modification steps such as catalytic modification of process water and ammonium or weak acid hydrolysis steps can be added to separate biomass (increase in yield, acceleration of fermentation).
Separation of butanol or ABE [00199] The recovery of butanol and / or an acetone-butanol-ethanol mixture from the fermentation broth can be done by any known method or a method developed in the future. Butanol has traditionally been recovered from the ABE fermentation broth by distillation, which is energy intensive. Alternative methods include, but are not limited to, freezing by fermentation broth, gaseous crystallization, pervaporation, membrane extraction, reverse osmosis, adsorption or liquid-liquid extraction.
[00200] In the recovery of the product from the fermentation process, alcohol, the technologies that allow the
77/99 maintenance of non-inhibitory product concentration and high productivity and high cell density (concentration of biomass cells) can be applied. Such as alcohol recovery can be done during fermentation or from a fermentation recycling stream, or by any other method.
[00201] According to the invention, product recovery methods are preferred, which do not destroy the hydrolytic activity of the enzymes in the culture broth, and thus allow the reuse of enzymes.
[00202] In summary, various embodiments of the invention are described below with the help of the following clauses numbered 1 to 29:
CLAUSES [00203] 1. An integrated process comprising
- A first biotechnological process, which produces biofuel and / or starting material for biofuel and uses a microorganism capable of producing enzymes and
- A second biotechnological process, which produces biofuel and / or raw material for biofuels, in which the process comprises the steps of cultivating the said microorganisms and production of biofuels and / or starting material for the biofuel and enzymes, or biofuel or material in
78/99 starting for biofuels,
- optionally, separate the supernatant and the microorganism cells from the culture of microorganisms,
- separate biofuel or starting material for the biofuel (s) from culture of microorganisms or cells of microorganisms,
- introducing the microorganism culture, the supernatant or a fraction of enriched protein from the supernatant or a dilution of the supernatant comprising lithically active catalytic enzyme (s) for the first and / or the second biotechnological process, or treating the raw material for ( s) process (es).
[00204] 2. The process, according to clause 1, in which the product of the first process comprises alcohol (s) or lipids, preferably lipids.
[00205] 3. The process, according to clause 1 or 2, in which the product of the second process comprises alcohol (s) or lipids, preferably alcohol (s).
[00206] 4. The process, according to any one of clauses 1 to 3, in which the product of the process is recovered by means of a method of preserving the catalytic activity of the enzymes, preferably hydrolytic enzymes, in the supernatant.
[00207] 5. The process, according to any of clauses 1 to 4, in which the alcohol comprises ethanol, butanol,
79/99 isopropanol, butanol, ethanol and / or acetone-butanol-ethanol.
[00208] 6. The process, according to any one of clauses 1 to 5, in which the organic material fed to the lipid production process comprises at least 50% of lignocellulose or a fraction of lignocellulose, which preferably comprises at least least 10% polymeric sugars from the sugar fraction.
[00209] 7. The process, according to clause 6, in which the lignocellulosic biomass or a fraction thereof, has agricultural residues, such as straw, bagasse or stem, energy crops, such as grasses, Miscanthus, willow, water hyacinth or reed canary grass, micro or macro algae, wood or forest waste, fractions of cellulose and the paper industry or waste, paper waste or urban waste containing lignocellulose.
[00210] 8. The process, according to any one of clauses 1 to 7, in which at least part of the organic material fed to the lipid production process comprises starch.
[00211] 9. The integrated process, according to any one of clauses 1 to 8, in which the microorganism, in the first process uses hemicellulose, cellulose or, both or a mixture of hemicellulose and cellulose or its fractions.
[00212] 10. The integrated process, according to any one of clauses 1 to 9, in which the microorganism, in the
80/99 second process uses hemicellulose, cellulose or, both or a mixture of cellulose and hemicellulose fractions.
[00213] 11. The process, according to clause 1 to 10, in which the microorganism capable of producing both lipids and enzymes is a fungus, yeast or bacterium, preferably belonging to a genus selected from the group Aspergillus, Humicola, Rhizopus and Trichoderma, or yeasts belonging to the genus Criptococcus or a bacterium belonging to Streptomyces.
[00214] 12. The method according to any one of clauses 1 to 11, in which the ethanol-producing microorganism is a yeast or a bacterium, preferably a yeast that belongs to a genus selected from the group of Saccharomyces, Pichia, and Candida, or a bacterium, preferably, belonging to a genus selected from the group of Zymomonas, Clostridia, Escherichia and Thermoanaerobacter.
[00215] 13. The process, according to any of clauses 1 to 12, in which acetone, butanol, ethanol or iso-propanol-butanol-ethanol-producing organisms belonging to the genus Clostridium.
[00216] 14. The process, according to any one of clauses 1 to 13, in which the enzymes comprise exoenzymes, preferably enzymes associated with the hydrolysis of hemicellulose and / or cellulose.
81/99 [00217] 15. The process, according to any one of clauses 1 to 14, in which the enzymes comprise hemicellulases, xylanases, mannanases, arabinases, galactosidases, glucosidases, mannosidases, xilosidases, arabinofuranosidase, esterases, cellulases, endo -cellulases, exo-cellulases, or cellobiases beta-glycosidases, cellulases, oxidatives or cellulose phosphorylase or any mixtures thereof.
[00218] 16. The process, according to any of clauses 1 to 15, in which the need for certain enzymes obtained outside the integrated process is reduced by at least 5%, preferably at least 30% of the enzymes produced in the integrated process [00219] 17. Process, according to any of clauses 1 to 16, in which part of the enzymes are used outside the integrated process.
[00220] 18. The process, according to any of clauses 1 to 17, in which the microorganisms in the first biotechnological process are capable of producing both hemicellulases and / or cellulases.
[00221] 19. The process, according to any of clauses 1 to 18, in which the biomass from the first biotechnological process or part of it is recycled to the second biotechnological process.
[00222] 20. The process, according to any of the
82/99 clauses 1 to 19, in which the biomass or part of it, the supernatant and / or enzymes from the first biotechnological process or part of it / is / are recycled to the first biotechnological process.
[00223] 21. The process, according to any of clauses 1 to 20, in which the biomass, supernatant and / or enzymes from the second biotechnological process or part of it / is / are recycled back to the first biotechnological process .
[00224] 22. The process, according to any of clauses 1 to 21, in which the process water or part of it, the second separation process after biofuel is recycled to the dilution water of the raw material of the first biotechnological process and / or second biotechnological process, preferably to the first biotechnological process.
[00225] 23. Use of lipids produced according to the process as defined by any one of clauses 1 to 22 as biofuels, as a biofuel component or as a starting material for the production of biofuels.
[00226] 24. The use, according to clause 23, in which the biofuel is biodiesel or renewable diesel, gasoline and / or aviation fuel.
[00227] 25. Use of alcohols (s) produced according to
83/99 with the process as defined by any one of clauses 1 to 22 as biofuels, as a biofuel component or as a starting material for the production of biofuels.
[00228] 26. An enzyme preparation obtained by the process as defined by any of clauses 1 to 22.
[00229] 27. Use of the produced enzyme, according to the process as defined by any of clauses 1 22 or the enzyme preparation as defined by clause 26, in a biofuel production process, preferably in a process of alcohol production, or in another application, as an enzyme preparation or as an enzyme source.
[00230] 28. An integrated process system for the production of lipids and alcohol production, which comprises the processes that use lignocellulosic material, or its fractions, as raw material for the production of lipids and alcohol, and one or both producing enzymes for the alcohol production process.
[00231] 29. An integrated process system for the production of lipids and the production of alcohol, comprising the processes that use lignocellulosic material, or its fractions, as raw material for the production of lipids and alcohol, and a lipid production process producing
84/99 enzymes for the alcohol production process, preferably ethanol production process.
[00232] It is an objective of the following examples to illustrate the invention and should not be construed as limiting the invention in any other way.
EXAMPLES [00233] Enzyme activities in spent culture broth from filamentous fungi that produce fat were determined by hydrolysis test with pure xylan cellulose as substrates.
Methods
Definition of Sugar:
[00234] In order to define the sugar content of a solution, the solution was made in an appropriate dilution that was filtered through 0.2 pm, before HPLC analysis.
[00235] The column used in the definition of sugar was the ion exchanger Shodex Açúcar SP 0810 in the form of lead (in the stationary phase). The column dimensions were 8.0 mm (ID) x 300 mm. The eluent was water (flow rate 0.6 ml / min) and the column temperature was 60 ° C. The detector was Rl Shimatzu 10A RID and the pump was A6 and the self-sampling was Shimatzu SIL 20A. The results were processed using the Class-VP software.
Fatty acid analysis:
[00236] The fatty acid composition of the samples was
85/99 determined as in the method described by Suutari et al. (nineteen ninety). Lipids in the samples were hydrolyzed first to free fatty acids, which were saponified in their sodium salts and, subsequently, methylated in methyl esters. Fatty acid methyl esters were analyzed using a gas chromatograph.
Protein concentration analysis:
[00237] The protein concentration of the culture broths was analyzed after filtering the broth through Whatman3 filter paper. The protein concentration was analyzed according to the Bio-Rad Protein assay (based on the Bradford method).
Hydrolysis tests:
[00238] The culture broth was filtered through Whatman3 filter paper prior to the hydrolysis test.
[00239] The xylanase activity was determined as follows. A 100 ml Erlenmeyer flask was used as a reaction vessel. It was filled with 20 ml of 1% solution of birch wood xylan (Sigma) in phosphate buffer (0.02 M, pH 5) as substrate, 10 ml of culture broth and 20 ml of phosphate buffer was filtered (0.02 M, pH 5). The hydrolysis reaction was performed on a shaker (140 rpm), 50 ^° C water bath. 1 ml samples were taken from the reaction vessel, immediately after adding the culture broth and after 1, 3, 5, 21/23 hours. The reaction of
86/99 hydrolysis was completed in the 1 ml sample by decreasing the pH by adding 50 pl of 1.33 M sulfuric acid. The released sugars were analyzed by HPLC (see definition of sugar) with mannitol as standard.
[00240] Cellulase activity was determined with 1 g of Whatman cellulose filter paper as a substrate, instead of xylan. The reaction volume was 50 ml, containing 1 g of Whatman filter paper in circles of equal sizes (about 5 mm in diameter), as substrate, 10 ml of culture broth and 40 ml of phosphate buffer were filtered ( 0.02 M, pH 5). The experiment was carried out in another way as with xylan. Strains of microorganisms:
[00241] Lipid-producing microorganisms are generally available to the public from a plurality of collections of recognized microbial cultures, such as ATCC, DSM, etc. Various embodiments of the invention are discussed in the examples that follow, using strains of microorganisms, as follows. Aspergillus oryzae DSM 1861, Aspergillus oryzae DSM 1864 and Aspergillus terreus DSM 1958. Example 1 [00242] This example shows the enzymatic activity formed in the culture broth during the cultivation of Aspergillus oryzae with cellulose-based material for the production of carbon of lipids.
[00243] Aspergillus oryzae was grown for
87/99 production of lipid in different cellulose-based lignocellulose materials. The average base growth contained, per liter of water, 40 g of lignocellulosic material, as a carbon source, 0.5 g of yeast extract, 1 g of MgS04 7H20, 0.5 g of K2HP04, 1 g of KH2P04 and 0, 2 g CACl2 - 2H20 and was supplemented with nitrogen source and traces of metals.
[00244] Experiments 1 - 4 were performed as flask cultures. In experiments 1 - 3, the base medium was supplemented with 3 g NaN03 and 0.02 g of FeS04 7H20 per liter and in experiment 4 the base medium was supplemented with 1 g of (NH4) 2S04 per liter. Parallel cultures were performed in 250 ml Erlenmeyer flasks containing 50 - 100 ml of culture medium. Culture media were inoculated with 1% (v / v) spore suspension of Aspergillus oryzae. The cultures were incubated at 28 ° C in an orbital shaker (160 rpm) for 6 days.
[00245] Experiments 5 - 6 were carried out as bioreactor fermentations.
[00246] In experiment 5, the base medium of the culture was supplemented with 6.5 g of peptone, 0.00015 g of ZnS04 7H20, 0.0001 g of CuCl-2H20 and 0.00625 g MNCl2 4H20 per liter of growth base medium. The carbon source was cellulose, which was added to the culture to give a final concentration of 55 g / l. For the inoculation of spore suspension, it was prepared by applying a
88/99 total of 24 ml of sterilized water in two culture spores in A. oryzae PDA petri dishes. The spores were suspended with a spreader and 1 L of culture medium was inoculated with the suspension. The fermentation was carried out at a temperature of 28 ° C, with 0.6 l / min of aeration and 350 450 rpm of agitation. The pH culture was 5.7 and was adjusted with 3 M NaOH during cultivation. Enzymatic activities were determined after 233 hours of incubation.
[00247] In experiment 6, the base growth medium was supplemented with 0.46 g peptone, 0.00015 g ZnS04 7H20, 0.0001 g CuCl-2H20 and 0.00625 g MNCl2 -4H20 per liter. The carbon source was cellulose, which was added to the culture to give a final concentration of 50 g / l. The culture medium was inoculated with 50 ml of 48 h grown in suspension of Aspergillus oryzae. The fermentation was carried out in a volume of culture medium L at 28 ° C of temperature, with 0.8 l / min of aeration and 350 - 450 rpm of agitation. The pH culture was 5.7 and was adjusted with 3 M NaOH during cultivation. Enzymatic activities were determined after 188 hours of incubation.
[00248] The culture broths were separated and the protein concentration and the tested xylanase and cellulase activity as described above.
Table 1. The source of carbon and nitrogen, culture volume, as well as determined protein concentration.
89/99
Exp Carbon source Nitrogen source Culture volume(ml) Conc. inProtein(mg / ml) 1 Manual ¹ fabric, grown with a Fritsch grinder sprayer NaNO3 50 0.19 2 SolkaFloc(cellulosepurified) NaNO3 100 0.11 3 Cellulose ² , treated by crushing, 0.3-Alpine sieve, 0.2-Alpine sieve NaNO3 100 0.06 4 Birch flour (grown with Gorgens turbo-motor) (NH4) 2SO4 100 0.18
90/99
5 Cellulose ² , same treatment as in exp. 3 Peptone 1000 0.49 ³ 6 Cellulose ² , same treatment as in exp. 3 Peptone / (NH ₄ ) 2SO ₄ 1000 0.11
¹ professional manual Lotus tissue pattern, fiber raw material: recycled fiber, Georgia-Pacific Nordic.
² UPM, Wisabetula, Bleached birch of Sulfate fiber 790.388 4/15/2008 Wisapulp. Hemicellulose ca. 15%.
³ broth concentrated three times by ultrafiltration (10,000 Da in an Amicon Ultra 8200 filter stirred by Millipore cell ultrafiltration)
[00249] At the experiment 6, the content of lipids was determined how being 4%. [00250] O sugar model during the tests in
hydrolysis as milligrams per milliliter of culture broth and milligram per milligram of protein as a function of time is shown in Figures 7 to 10. Figure 7 shows the xylose released in the hydrolysis assay by volume of culture broth. As substrate, 200 mg of birch wood xylan was used. Figure 8 shows the xylose model in the protein hydrolysis test. As substrate, 200 mg of birch wood xylan was used. Figure 9 shows the glucose in the model in volume hydrolysis assays
91/99 of culture broth. As a substrate, 1 g of cellulose was used. Figure 10 shows glucose in the protein hydrolysis test model. As a substrate, 1 g of cellulose was used.
[00251] All six culture broths tested from cultures showed significant xylanase activity. Only three of the six culture broths showed signs of cellulase activity, indicating a weak ability to produce these enzymes under specific conditions.
[00252] This example shows that Aspergillus oryzae can produce lignocellulolytic enzymes in the culture broth. The example shows that A. oryzae can have as much xylan degradation activity as cellulose.
[00253] The enzyme production was selectively degrading xylan, in most cases, three of the cultures showed poor cellulase activity. The reuse of hydrolytic enzymes produced by A. oryzae in the production of lipid can reduce the amount of commercial enzymes required in lignocellulose hydrolysis. or their fractions.
Example 2 [00254] This example shows the enzymatic activity formed in the culture broth during the cultivation of Aspergillus terreus with hemicellulose-based material, as a carbon source for the production of lipids.
[00255] Aspergillus terreus was grown for
92/99 production of lipid in a hemicellulose wheat straw as a carbon substrate in a volume of 2 liters in a bioreactor. The culture medium consisting of 50 ml of Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco) 10 x stock solution suspended in 2 L of water and supplemented with per liter: 10.0 g of yeast extract, 1 g of (NHéUSQé, 1 g of MgS04 7H20, 0.5 g of K2HP04, 1 g of KH2P04, 0.2 g CACl2 2H20 and 2 g of cellulose. The culture medium was inoculated with 150 ml of a pre-culture of A. terreus 24 h The fermentation was carried out at 35 ° C, with 3.0 l / min of aeration and 200 - 430 rpm of agitation.The pH of the Culture was 5.7 and was adjusted with NaOH 3 M, during cultivation, during cultivation, hemicellulose solution was fed to the fermenter.The enzymatic activities were determined after 165 hours of incubation.
The culture broth was separated and was partially concentrated by ultrafiltration in an Amicon ultrafiltration cell stirred with a 10,000 Da filter (Millipore). The concentration of proteins and lipids and the activity of xylanase and cellulase were tested as described above.
[00257] The lipid content in the biomass containing mycelium of the fungus, hemicellulose and residual cellulose was 15% by dry weight. The protein concentration was 0.72 mg / ml in the non-concentrated culture broth and 2.15 mg / ml in the broth
93/99 concentrated.
[00258] The sugar model during hydrolysis tests as milligrams per milliliter of culture broth and milligram per milligram of protein as a function of time is shown in Figures 11 to 14. Figure 11 shows the xylose released in the hydrolysis test by volume of culture broth. As substrate, 200 mg of birch wood xylan was used. Figure 12 shows the xylose released in the test by protein hydrolysis. As substrate, 200 mg of birch wood xylan was used. Figure 13 shows the glucose model in hydrolysis assays by volume of culture broth. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used. Figure 14 shows glucose in the protein hydrolysis test model. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
[00259] This example indicates that Aspergillus terreus can produce both intracellular lipids and hydrolytic enzymes in the culture broth. The example shows that A. terreus produces enzymes, and excretes them into a growth medium, which has both xylan and cellulose degradation activity. This enzyme can be separated, concentrated and used in the hydrolysis of lignocellulosic material, such as
94/99 material containing both cellulose and / or polymeric hemicellulose. The re-use of enzymes produced by A. terreus in the production of lipid can reduce the amount of commercial enzymes required in the hydrolysis of lignocellulose or its fractions.
Example 3 [00260] The example shows the integration of ethanol production with lipid production. Ethanol is produced from cellulose hydrolyzed by enzymes in the metabolized culture medium obtained from the production of lipids by mold.
[00261] In Example 3 it was found that the culture medium spent from the culture and the lipid production of Aspergillus terreus in wheat straw hemicellulose supplemented with cellulose contains enzymes, xylanases and cellulases susceptible to hydrolysis of lignocellulosic materials.
[00262] The spent culture medium is used in this culture to hydrolyze cellulose. Cellulose or pure cellulose wheat straw is added to the spent culture medium, treated by ultrafiltration in an Amicon ultrafiltration cell under agitation with a 10,000 Da filter (Milliporos), from A. lipid cultivation and production. terreus in hemicellulose of wheat straw supplemented with cellulose. The solution is incubated at 30
C for 16
200 h for
95/99 saccharification of cellulose. After saccharification, nutrients (NH4) 2HP04 (0.5 g / L), MgS04-7H20 (0.025 g / L) and yeast extract (10.0 g / L) are added to the solution and the solution is inoculated with yeast Saccharomyces cerevisiae.
S. cerevisiae is grown for 48 - 120 h at 36 ° C maintaining the pH between 5.0 and 6.5 anaerobically. After culture (fermentation), the cells are removed from the culture medium by filtration through a 0.2 pm or 0.45 pm filter, and / or by centrifugation at 5000xg for 5 min. The ethanol concentration of the culture medium can be measured by gas chromatography or by liquid chromatography, such as by HPLC.
Example 4 [00263] The example shows the integration of acetonaethanol-butanol (ABE) and the lipid production processes. Hemicellulose ABE is produced from wheat straw and / or cellulose hydrolyzed by enzymes in the metabolized culture medium obtained from the production of lipids by mold.
[00264] Hemicellulose and / or the fraction of wheat straw is divided into two fractions. One fraction is used to produce lipids by A. terréus mold, as described in example 3. The other fraction is used to produce ABE by Clostridium acetobutylicum bacteria.
[00265] In Example 3 it was found that the medium of
6/99 culture spent from culture and lipid production of Aspergillus terreus in hemicellulose wheat straw supplemented with cellulose contain enzymes, xylanases and cellulases susceptible to hydrolysis of lignocellulosic material.
[00266] The culture medium used in this cultivation is used to hydrolyze wheat hemicellulose. Wheat straw hemicellulose is added to the spent culture medium, treated by ultrafiltration in an Amicon ultrafiltration cell under agitation with a 10,000 Da filter (Milli-pores), from A. terreus lipid cultivation and production in wheat straw hemicellulose supplemented with cellulose. The solution is incubated at 30 - 70 ° C for 16 - 200h for saccharification of hemicellulose. After saccharification, yeast extract (10.0 g / L) is added to the solution and the solution is sterilized at 121 ° C for 15 min. After sterilization and cooling to room temperature, minerals, buffer, and vitamins are added to the medium, for example, according to the medium P2, which is described in Baer et al. (1987). The medium is placed in an anaerobic flask or flask and inoculated with the bacteria Clostridium acetobutylicum. The culture is incubated 34 - 90 h at 35 ° C maintaining the pH between 5.0 and 5.5. After culture (fermentation), cells are removed from the culture medium by filtration through a 0.45 pm or 0.2 pm filter, and / or by centrifugation at 5000xg for
97/99 min. The concentrations of acetone, butanol and ethanol in the culture medium can be measured by gas chromatography or by liquid chromatography, such as by HPLC.
Example 5 [00267] This example shows the enzymatic activity formed in the culture broth during the cultivation of Aspergillus oryzae with hemicellulose-based material, as a carbon source for the production of lipids.
[00268] Aspergillus oryzae was grown for the production of lipid in a wheat straw hemicellulose as a carbon substrate in a volume of 2 liters in a bioreactor. The culture medium consisting of 50 ml of Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco) 10 x stock solution suspended in 2 L of water and supplemented with per liter: 10.0 g of yeast extract, 1 g of (NH4) 2S04, 1 g of MgS04 7H20, 0.5 g of K2HP04, 1 g of KH2P04 and 0.2 g CACl2 2H20.
[00269] The culture medium was inoculated with 200 ml of 72 h pre-culture of A. oryzae. Fermentation was carried out in 2 L of medium volume cultivation at 30 ° C of temperature with 3.0 l / min of aeration and 200 - 410 rpm agitation. The pH of the culture was 5.7 and was adjusted with 3 M NaOH, during cultivation. During cultivation, hemicellulose solution was fed to the fermenter. Enzymatic activities were determined after 144 hours of incubation.
98/99 [00270] The culture broth was separated and the protein concentration and xylanase and cellulase activity tested as described above. The concentration of biomass lipids contained mycelium of the fungus and residual hemicellulose of 21% by dry weight. The protein concentration was 0.61 mg / ml in non-concentrated culture broth and 1 0.65 mg / ml in the concentrated broth.
[00271] The sugar released during hydrolysis tests as milligrams per milliliter of culture broth and milligram per milligram of protein as a function of time is shown in Figures 12 to 15. Figure 12 shows the xylose released in the volume hydrolysis assay of culture broth. As substrate, 200 mg of birch wood xylan was used. Figure 13 shows the xylose released in the test by protein hydrolysis. As substrate, 200 mg of birch wood xylan was used. Figure 14 shows the glucose model in hydrolysis assays by volume of culture broth. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used. Figure 15 shows glucose in the protein hydrolysis test model. As a substrate, 1 g of cellulose was used. Some xyloses were released from hemicellulose from the culture broth used.
[00272] This example shows that Aspergillus oryzae
9/99 can produce lipid and as a by-product of a culture broth with hydrolytic activities that can be reused in the hydrolysis of lignocellulosic material. REFERENCES [00273] Baer SH, Blaschek HP, Smith TL. 1987. Effect of Butanol Challenge and Temperature on Lipid Composition and Membrane Fluidity of Butanol-Tolerant Clostridium acetobutylicum. Applied and Environmental Microbiology 53: 2854 - 2861.
[00274] Fall R, Phelps P, Spindler D. 1984. Bioconversion of xylan to triglycerides by oil-rich yeasts. Applied and Environmental Microbiology. 47: 1130 - 1134.
[00275] Lin H, Chang W, Ding H-T, Chen X-J, Zhou Q-F, Zhao Y-Hu. 2010. Direct microbial conversion of wheat straw into lipid by a cellulolytic fungus of Aspergillus oryzae A-4 in solid-state fermentation. Bioresource Technology 101: 7556 - 7562.
[00276] Lynd LR, van Zyl WH, McBride JE, Laser M. 2005. Consolidated bioprocessing of cellulosic biomass: an update. Current Opinion in Biotechnology 16: 577 - 583.
[00277] Suutari M, Liukkonen K, Laakso S. 1990. Temperature adaptation in yeasts: the role of fatty acids. Journal of General Microbiology 136: 1469 - 1474.

权利要求:
Claims (12)
[1]
1. Integrated process for the production of single cell oil and alcohol production characterized by the fact that it comprises the steps of:
(a) cultivating a fungus belonging to the genus Aspergillus in a culture medium in a process for the production of single cell oil, in which the culture medium comprises hemicellulose and the fungus produces single cell oil and xylanase;
(b) cultivating a microorganism in a culture medium in an alcohol production process, in which the microorganism produces alcohol and the culture medium comprises:
(i) lignocellulosic material, or its fractions, and the xylanase produced in step (a) or a fraction of the microorganism culture from step (a) that comprises xylanase; or (ii) lignocellulosic material, or its fractions;
in that the material treated lignocellulosic, or your fractions were treated with the produced xylanase at stage (a), or in that the material treated lignocellulosic, or your fractions were treated with a fraction of the culture of
microorganisms from step (a) comprising xylanase.
[2]
2. Process according to claim 1, characterized by the fact that the alcohol is ethanol, butanol, isopropanol, or mixtures thereof.
[3]
3. Process according to claim 1, characterized by the fact that it further comprises separating the supernatant and the fungal cells from the culture of the process for producing single cell oil from step (a), and
Petition 870190071912, of 7/26/2019, p. 31/41
2/4 introduce the separated supernatant or the separated fungal cells into the culture medium of the process for producing alcohol from step (b).
[4]
4. Process, according to claim 1, characterized by the fact that it also comprises separating the supernatant and fungal cells from the culture of the process for producing single cell oil from step (a), treating the lignocellulosic material or its fractions with a separate supernatant or with separate fungal cells, and introduce the treated lignocellulosic material or its fractions into the culture medium of the process for producing alcohol from step (b).
[5]
5. Process, according to claim 1, characterized by the fact that it also comprises recovering the xylanase produced in the process for producing single cell oil from step (a) and introducing the recovered xylanase in the culture medium of the process for producing alcohol from step (b).
[6]
6. Process, according to claim 1, characterized by the fact that it also comprises recovering the xylanase produced in the single cell oil production process of step (a), treating the lignocellulosic material or its fractions with the recovered xylanase, and introducing the treated lignocellulosic material or its fractions in the culture medium of the process for producing alcohol from step (b).
[7]
7. Process according to claim 1, characterized by the fact that the fungal culture supernatant of step (a) comprises xylanase, and the
Petition 870190071912, of 7/26/2019, p. 32/41
3/4
culture gives stage (b) comprises the supernatant, a dilution of same or a fraction of even enriched with protein. 8. Process, according with claim 1,
characterized by the fact that it also comprises recovering the single cell oil from the process for producing single cell oil from step (a) using a recovery method that preserves the catalytic activity of the xylanase produced in the process for producing single cell oil from step (a).
[8]
9. Process according to claim 1, characterized by the fact that the culture medium in the process for producing alcohol from step (b) further comprises starch.
[9]
10. Process according to claim 1, characterized by the fact that the fungi of the process for producing single cell oil from step (a) still produce the enzymes selected from the group consisting of hemicellulases, cellulases, mannanases, arabinases, galactosidases, glycosidases, mannosidases, xilosidases, arabinofuranosidase, esterases, endo-cellulases, exocellulases, cellobiases or beta-glucosidases, oxidative cellulases or cellulose phosphorylase or any mixtures thereof.
[10]
11. Process, according to claim 1, characterized by the fact that it also comprises recovering the xylanase from the process for producing single cell oil from step (a), and recycling the recovered xylanase to the
Petition 870190071912, of 7/26/2019, p. 33/41
4/4 culture medium of the single cell oil production process.
[11]
12. Process, according to claim 1, characterized by the fact that it also comprises recovering the lipids from the process for producing single cell oil from step (a) and reacting the lipids as alcohol in a transesterification reaction for biodiesel production .
[12]
13. Process, according to claim 1, characterized by the fact that it also comprises recovering the alcohol from the process for producing alcohol from step (b) and reacting the alcohol with lipids in a transesterification reaction to produce biodiesel.

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US10479716B2|2016-12-01|2019-11-19|University Of Louisiana At Lafayette|Biorefinery method and system for isolated environments|

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-03-03| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-04-07| B25D| Requested change of name of applicant approved|Owner name: NESTE OYJ (FI) |

优先权:

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申请号 | 申请日 | 专利标题

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US201061459963P| true| 2010-12-22|2010-12-22|

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US61/459,963|2010-12-22|

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EP10196556.4|2010-12-22|

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EP10196556A|EP2468875A1|2010-12-22|2010-12-22|An integrated process for producing biofuels|

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PCT/FI2011/051129|WO2012085340A1|2010-12-22|2011-12-19|An integrated process for producing biofuels|

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