Pretreatment of salt-containing hydrolysates, in particular for fermentation processes

Abstract

The invention relates to a method for reducing the salt content of salt-containing hydrolysates. The invention also relates to a method of producing a target product, preferably a microbial oil, comprising providing a hydrolysate having a reduced salt content, and culturing a microorganism, preferably an oleaginous microorganism, using a growth medium comprising the hydrolysate having a reduced salt content.

Description

Technical Field

[0001] The present invention relates to a method for reducing the salt content of a hydrolyzate of a brine. The present invention also relates to a method for producing a target product, preferably microbial oil, which comprises providing a hydrolyzate having a reduced salt content, and culturing a microorganism, preferably an oil-producing microorganism, using a growth medium comprising the hydrolyzate having the reduced salt content. Background Art

[0002] Single cell oils (SCOs) produced by oil-producing microorganisms can be used as alternative resources for edible vegetable oils and for the production of advanced biofuels. Different from growing crops for oil production, the cultivation of microorganisms is independent of seasons, and biotech factories enable efficient land use and vertical expansion. However, the main weakness of ecological SCO production is the selection of cost-effective feedstocks. In this context, the use of industrial waste streams has high potential to solve the waste treatment and feedstock problems of advanced bioprocesses. However, most industrial waste streams are defective, for example, they contain components that interfere with the growth of microorganisms, which hinders the efficient production of target products such as microbial oils.

[0003] The pulp and paper industry is one of the main producers of waste streams with high concentrations of biodegradable carbon. In addition to Kraft-pulping, one of the main processes for cellulose fiber production is the combination of acid sulfite pulping with steam explosion to hydrolyze cellulosic and lignocellulosic compounds and to separate high-value cellulose fibers. In addition to aliphatic carboxylic acids, furans, and phenolic compounds, the resulting waste stream usually contains large amounts of pentoses, small amounts of hexoses, and uronic acids. In most industrial plants, it is used for energy production by anaerobic fermentation to methane or direct combustion. Therefore, the value creation of this waste stream is currently limited.

[0004] An advanced example of using lignocellulosic waste for the production of alternative oleochemicals is the production of ethanol using bacteria or yeast as whole-cell biocatalysts. In addition, it has been documented that microalgae, bacteria, and oleaginous yeasts are capable of microbially synthesizing long-chain fatty acids with a main carbon chain length ranging from C13 to C21. However, operating oleaginous yeast fermentation in a secondary fermentation mode (diauxic fermentation mode) by restricting the nitrogen or phosphate concentration results in a decrease in growth rate, biomass accumulation, and lipid production. An example of lipid production on lignocellulosic hydrolysate (LCH) using Rhodosporidium toruloides as a fermentation host showed a fed-batch fermentation reaching a lipid titer of 39.5 g / l. However, the examples cited used hexoses as the carbon source, rather than pentoses, which can only be efficiently utilized by C. oleaginosus. In addition, lignocellulosic hydrolysates typically contain compounds that inhibit microbial growth and reduce yield and efficiency, such as salts.

[0005] The hydrolysates of by-products, waste, or residual material streams of lignocellulosic biomass contain high salt concentrations and high levels of compounds that inhibit microbial growth, such as soluble oligomeric lignin and monomeric lignin, furans, and heavy metals, which impede any fermentation process using such by-products, waste, and / or residual material streams. To date, deep concentration followed by incineration is the only viable and practically applicable utilization method for such hydrolysates of by-products, waste, or residual material streams.

[0006] Therefore, there is an urgent need to convert waste streams such as lignocellulosic hydrolysates into suitable growth substrates. In addition, there is still a need for methods that can reduce the salt content of hydrolysates to make them suitable as growth media for microbial cultivation or for use in growth media for microbial cultivation. There is also a need to convert hydrolysates such as lignocellulosic hydrolysates (e.g., lignocellulosic hydrolysates from acid pulping) into suitable growth media or growth medium supplements. There is also a need for a method for efficiently producing target products such as microbial lipids. In addition, there is a need to provide an environmentally friendly and cost-effective method for producing target products such as SCO. Summary of the Invention

[0007] In the following, the elements of the present invention will be described. These elements are listed by way of specific examples, but it should be understood that they can be combined in any manner and in any number to form more embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to the explicitly described embodiments. It should be understood that this specification supports and encompasses embodiments that combine two or more explicitly described embodiments or that combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, any arrangement and combination of all the described elements in this application should be disclosed by the description of this application, unless the context otherwise indicates.

[0008] In a first aspect, the present invention relates to a method for reducing the salt content of a hydrolyzate, preferably a salt-containing biogenic hydrolyzate, the method comprising:

[0009] a) providing a hydrolyzate, preferably a salt-containing biogenic hydrolyzate, more preferably a salt-containing lignocellulosic hydrolyzate;

[0010] b) optionally, neutralizing the pH of the hydrolyzate of step a) to obtain a neutralized hydrolyzate; wherein, optionally

[0011] the pH of the neutralized hydrolyzate is in the range of about pH 4 to about pH 8;

[0012] c) adding CaCO3,

[0013] Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2 to the hydrolyzate of step a) or the neutralized hydrolyzate of step b), preferably CaCO3 and / or Ca(OH)2, to obtain a hydrolyzate containing precipitated salts; wherein, optionally, the pH of the hydrolyzate containing precipitated salts is in the range of about pH 4 to about

[0014] pH 8.5;

[0015] d) adding a chelating agent to the hydrolyzate of step c);

[0016] e) optionally, adjusting the pH of the hydrolyzate of step c) and / or step d) to a pH in the range of about pH 3 to about pH 8.5, preferably about pH 3.5 to about pH 7.5, more preferably about pH 5 to about pH 7, further preferably about pH

[0017] 6 to about pH 7;

[0018] f) obtaining a hydrolyzate with a reduced salt content.

[0019] In one embodiment, the hydrolysis product provided in step a) is a hydrolysis product derived from the paper industry, such as a hydrolysis product derived from pulp production, a hydrolysis product derived from forestry, a hydrolysis product of agriculture, a hydrolysis product of food, a hydrolysis product of food waste, a hydrolysis product of biofuel waste, a hydrolysis product of textiles, a hydrolysis product of animal tissues, a hydrolysis product of plant tissues, a hydrolysis product of microbial biomass, a hydrolysis product of industrial waste, a hydrolysis product of municipal waste, or any combination thereof;

[0020] Among them, preferably, the hydrolysis product is a lignocellulosic hydrolysis product, preferably a spent liquor hydrolysis product, and / or a hydrolysis product derived from pulping, more preferably a hydrolysis product derived from acid pulping.

[0021] In one embodiment, the hydrolysis product provided in step a) is a hydrolysis product obtained by physically treating, chemically treating, enzymatically treating, and / or biologically treating a substrate (preferably biomass);

[0022] Among them, preferably,

[0023] The physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and any combination thereof;

[0024] The chemical treatment is selected from alkali treatment, acid treatment, and treatment at neutral pH; among them, preferably, the chemical treatment is treatment with salts, acids, peroxides, and any combination thereof, preferably treatment with sulfides, sulfites, and / or bisulfites;

[0025] The enzymatic treatment is treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylating enzymes, peptidases, such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, lignin-degrading enzymes, and any combination thereof; and

[0026] The biological treatment is treatment with microorganisms, preferably treatment with microorganisms selected from bacteria, yeasts, and fungi.

[0027] In one embodiment, the hydrolysis product provided in step a) contains a salt in an amount in the range of about 0.0001 mol / l to about 15 mol / l, preferably about 0.0005 mol / l to about 8 mol / l, and / or

[0028] carbon in a value in the range of about 0.1% to about 65% by weight.

[0029] In one embodiment, the hydrolysis product provided in step a) contains salts selected from sulfates, sulfides, sulfites, nitrates, nitrites, chlorides, and any combination thereof; among them, preferably, the salt contains sulfates, sulfides, and / or sulfites.

[0030] In one embodiment, the hydrolysis product provided in step a) comprises lignophenols, lignans, organic acids, and / or sugars; wherein, optionally, the sugars comprise xylose, glucose, mannose, and / or galactose; wherein, preferably, the sugars comprise monosaccharides, preferably xylose; and wherein, preferably, the organic acids comprise acetic acid.

[0031] In one embodiment, the chelating agent is selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof, where M is a metal;

[0032] wherein, preferably, the chelating agent is selected from Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, (NH4)(H2PO4), and Na3PO4;

[0033] wherein, more preferably, the chelating agent is KH2PO4.

[0034] In one embodiment, the method comprises step e) of adjusting the pH of the hydrolysis product, and the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid, or any combination thereof to the hydrolysis product of step c) and / or step d), preferably by adding NaOH and / or KOH.

[0035] In one embodiment, the method further comprises sterilization, preferably sterilizing the hydrolysis product with reduced salt content obtained in step f) to obtain a sterile hydrolysis product with reduced salt content;

[0036] wherein, preferably, the sterilization comprises heat sterilization, ultra-high temperature treatment, and / or sterile filtration.

[0037] On the other hand, the present invention relates to a method for producing a target product, preferably microbial oil, the method comprising the following steps:

[0038] i) Providing a hydrolysis product with reduced salt content by implementing the method for reducing the salt content of a saline hydrolysis product defined herein

[0039] product;

[0040] ii) Culturing a microorganism (preferably an oil-producing microorganism) using the hydrolysis product provided in step i) or a growth medium composed thereof, thereby enabling the microorganism to produce the target product; preferably enabling the oil-producing microorganism to produce microbial

[0041] Oil and fat;

[0042] iii) Optionally, subject the microorganism (preferably the oil-producing microorganism) to enzymatic treatment; wherein, optionally, the enzymatic treatment comprises subjecting the microorganism to enzymatic treatment without any solvent extraction or chemical demulsification;

[0043] iv) Obtain the target product, preferably microbial oil.

[0044] In one embodiment, the microorganism is an oil-producing microorganism, preferably an oil-producing yeast, more preferably Cutaneotrichosporon sp., and further preferably Cutaneotrichosporon oleaginosus.

[0045] In one embodiment, the target product is selected from microbial oil, glycerol, free fatty acids, monoglycerides, diglycerides and triglycerides, phospholipids, sphingolipids, polyols, alcohols, organic acids, biodiesel, hydrogen, methane, biopolymers, carotenoids, cellulose, squalene, sterols, vitamins, phenolic compounds, pigments, peptides, proteins such as enzymes, DNA, RNA, and any combination thereof;

[0046] Wherein, preferably, the target product comprises microbial oil.

[0047] In one embodiment, the hydrolyzate with reduced salt content provided in step i) comprises acetic acid and / or xylose,

[0048] Wherein, preferably, the hydrolyzate comprises or consists of a lignocellulosic hydrolyzate.

[0049] In one embodiment, the culturing in step ii) comprises adding acetic acid and / or a carbon source other than acetic acid to the growth medium;

[0050] Wherein, preferably,

[0051] the acetic acid is added in the form of a feed comprising acetic acid or consisting of acetic acid, wherein, preferably, the concentration of acetic acid in the feed is in the range of 1 mol / l to 20 mol / l, preferably in the range of 1.75 mol / l to 15.75 mol / l,

[0052] wherein, optionally, the feed further comprises a carbon source other than acetic acid.

[0053] In one embodiment, the growth medium comprises sugar, such as xylose, in an amount in the range of about 0.1 g / l to about 250 g / l, preferably <100 g / l; and / or

[0054] The growth medium contains acetic acid in an amount in the range of from about 0.01 g / l to about 100 g / l, preferably from about 1 g / l to about 50 g / l, more preferably from about 5 g / l to about 10 g / l.

[0055] In another aspect, the present invention relates to a hydrolyzate having a reduced salt content obtainable and / or obtained by a method for reducing the salt content of a saline hydrolyzate as defined herein.

[0056] In another aspect, the present invention relates to a composition obtainable and / or obtained by a method for producing a target product as defined herein.

[0057] In one embodiment, the composition comprises or consists of a target product obtainable and / or obtained by a method for producing a target product as defined herein. Detailed Description

[0058] The present invention aims to provide a hydrolyzate having a reduced salt content that can be used as a growth medium. Advantageously, the method for reducing the salt content of the hydrolyzate enables the value-added utilization of products such as biomass and / or waste, making these products into suitable growth media and / or growth medium additives. Advantageously, saline waste, such as waste liquid or other hydrolyzates, which contain salts that interfere with microbial growth, can be processed into valuable hydrolyzates having a reduced salt content, which can be used as a growth medium for microbial growth or used in a growth medium for microbial growth. The method for reducing the salt content of the hydrolyzate can effectively upgrade and utilize waste products such as biogenic waste.

[0059] The inventors surprisingly found that when using a hydrolyzate having a reduced salt content (such as a lignocellulosic hydrolyzate having a reduced salt content) as a growth substrate to culture microorganisms, a target product can be efficiently produced. The inventors found that, unexpectedly, the method for producing the target product of the present invention enables the high-yield production of the target product. In addition, the inventors found that by reducing the salt content, waste hydrolyzates (such as waste liquid) can be value-added utilized as a growth substrate for microbial culture. The method for reducing the salt content of the hydrolyzate is very effective in producing hydrolyzates that can be used as a growth medium and / or used in a growth medium. The present invention for the first time realizes the utilization of by-products, waste and / or residual streams derived from lignocellulosic biomass after reducing the salt content.

[0060] Interestingly, the inventors found that microorganisms exhibit excellent complete growth ability in the salt-reduced hydrolysis products, showing high tolerance and absorption capacity for high contents of soluble oligomeric lignin and monomeric lignin, as well as furan substances. In the existing fermentation system, compared with the model lignocellulose hydrolysis products, microorganisms exhibit a higher growth rate in the actual salt-reduced lignocellulose hydrolysis products.

[0061] The present invention relates to a method for reducing the salt content of a salt-containing hydrolysis product, preferably a biogenic hydrolysis product containing salt, and the method comprises:

[0062] a) providing a salt-containing hydrolysis product, preferably a biogenic hydrolysis product containing salt, more preferably a lignocellulose hydrolysis product containing salt;

[0063] b) optionally, neutralizing the pH of the hydrolysis product of step a) to obtain a neutralized hydrolysis product; wherein, optionally

[0064] the pH of the neutralized hydrolysis product is in the range of about pH 4 to about pH 8;

[0065] c) adding CaCO3,

[0066] Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2 to the hydrolysis product of step a) or the neutralized hydrolysis product of step b), preferably CaCO3 and / or Ca(OH)2, to obtain a hydrolysis product containing precipitated salt; wherein optionally, the pH of the hydrolysis product containing precipitated salt is in the range of about pH 4 to about pH

[0067] 8.5;

[0068] d) adding a chelating agent to the hydrolysis product of step c);

[0069] e) optionally, adjusting the pH of the hydrolysis product of step c) and / or step d) to a pH in the range of about pH 3 to about pH 8.5, preferably about pH 3.5 to about pH 7.5, more preferably about pH 5 to about pH 7, further preferably about pH

[0070] 6 to about pH 7;

[0071] f) obtaining a hydrolysis product with a reduced salt content.

[0072] In one embodiment, the method reduces the salt content (e.g., sulfate content) of the hydrolyzate of the brine by at least 1%, preferably by at least 10%, more preferably by at least 30%, further preferably by at least 50%, and even more preferably by at least 80%. In one embodiment, the reduction of the salt content comprises a significant reduction of the salt content, preferably by at least 1%, preferably by at least 10%, more preferably by at least 30%, further preferably by at least 50%, and even more preferably by at least 80%.

[0073] In one embodiment, as used herein, the term "reducing salt content" relates to reducing the total salt content and / or the content of a specific salt in the hydrolysis product, in particular reducing the content of at least one salt in the hydrolysis product, for example, relating to reducing the sulfate content in the hydrolysis product. In one embodiment, the method for reducing salt content comprises reducing the sulfate content and optionally further comprises reducing the calcium content of the hydrolysis product. In one embodiment, the salt composition of the hydrolysis product changes independently of the total salt content. For example, the amount of sulfate present in the hydrolysis product can be reduced (thus changing the overall salt composition of the hydrolysis product) by the method of the present invention while maintaining or changing the total salt content of the hydrolysis product. In one embodiment, the terms "content", "amount" and "concentration" are used interchangeably. In one embodiment, the method for reducing the salt content of the hydrolysis product comprises reducing the salt content of at least one salt in the composition, for example reducing the sulfate content. In one embodiment, the method for reducing the salt content of the hydrolysis product comprises reducing the concentration of at least one salt present in the hydrolysis product, for example the sulfate concentration; wherein optionally the total salt content of the hydrolysis product is reduced. In one embodiment, the salt reduced in the method for reducing the salt content of the hydrolysis product is sulfate. In one embodiment, the reducing salt content comprises reducing or consisting of reducing the sulfate content. In one embodiment, the method for reducing the salt content of the saline hydrolysis product is a method for reducing the sulfate content of the hydrolysis product containing sulfate. In one embodiment, the method for reducing the salt content of the saline hydrolysis product further comprises reducing the calcium concentration (e.g., the calcium concentration of the hydrolysis product). In one embodiment, the hydrolysis product with reduced salt content obtained in step f) of the method for reducing salt content is a hydrolysis product with reduced sulfate content, optionally with reduced calcium content. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) of the method for reducing salt content has a different salt composition from the hydrolysis product provided in step a); wherein preferably, compared with the concentration of at least one salt (e.g., sulfate) in the hydrolysis product provided in step a), the concentration of the at least one salt in the hydrolysis product obtained in step f) is reduced; wherein optionally, the total salt content of the hydrolysis product obtained in step f) is the same as or different from the total salt content of the hydrolysis product provided in step a).

[0074] As used herein in the context of a method for reducing the salt content of a hydrolysis product according to the present invention, the term "hydrolysis product" refers to any hydrolysis product, particularly a hydrolysis product of biomass (such as lignocellulosic biomass). For example, the hydrolysis product can be derived from solid paper waste, pulp, spent liquor, wood, sawdust, plants such as crops, straw, food, food waste, biofuel waste, textiles, animal tissue, microbial biomass, municipal waste, and / or industrial waste. In one embodiment, the hydrolysis product contains sulfate. The hydrolysis product can contain carbohydrates, particularly sugars, sugar degradation products, and / or lignin degradation products. In one embodiment, the hydrolysis product contains sugars, particularly xylose, and / or acetic acid. In one embodiment, the hydrolysis product is derived from and / or prepared from lignocellulosic biomass. In one embodiment, the terms "derive from" and "prepare from" or "derived from" and "prepared from" are used interchangeably. In one embodiment, as used herein, in the context of a hydrolysis product, the term "derive from" means that the hydrolysis product is currently or was previously prepared (particularly obtained) from a starting material (such as biomass) by any method known to those skilled in the art, preferably by physically treating, chemically treating, enzymatically treating, and / or biologically treating the starting material (particularly the substrate, preferably biomass). In one embodiment, the starting material (particularly the substrate) comprises or consists of: paper industry products (such as pulp, spent liquor, bleaching waste, paper, and / or paper waste); forest products (such as wood and / or sawdust); agricultural products (such as straw); food or food waste (such as bread); biofuel production waste (such as microbial biomass); hydrogen production waste (such as microbial biomass); textiles (optionally biodegradable textiles, such as wool, cotton, and / or linen); animal tissue (such as meat); plant biomass (such as crops); microbial biomass (such as fungal biomass, bacterial biomass, and / or yeast biomass); industrial waste (optionally biodegradable industrial waste, such as hemp hydrolysis products); municipal waste; or any combination thereof. In one embodiment, the starting material (particularly the substrate) comprises or consists of lignocellulosic biomass. In one embodiment, the biofuel waste comprises or consists of hydrogen production waste. For example, biofuel production can involve hydrogen production. In one embodiment, the biofuel waste is the hydrolysis product of hydrogen production waste.

[0075] For example, the biomass may relate to biodegradable portions of products, wastes, and / or residues of biological origin, such as those derived from agriculture, forestry, and / or related industries, including fisheries and aquaculture. For example, the biomass may relate to biodegradable portions of wastes from industry and households. In one embodiment, the industrial waste is selected from the group consisting of by-products and / or waste streams from food processing, by-products and / or waste streams from pulp production, by-products and / or waste streams from the paper industry, by-products and / or waste streams from agricultural product processing, by-products and / or waste streams from biofuels, and / or by-products and / or waste streams from forestry. In one embodiment, the microbial biomass is selected from bacterial biomass, fungal biomass, yeast biomass, microalgal biomass, and combinations thereof; wherein, preferably, the microbial biomass is fungal biomass, particularly yeast biomass. In one embodiment, the fungal biomass comprises microbial biomass selected from the group consisting of Aspergillus sp., Fusarium sp., Trichoderma sp., Ascoulus sp., Rhizopus sp., and combinations thereof, or consists of the same. For example, the fungal biomass may comprise Trichoderma reesei and / or Aspergillus niger biomass. In one embodiment, the yeast biomass comprises microbial biomass selected from the group consisting of Saccharomyces sp., Yarrowia sp., Rhodosporidium sp., Cryptococcus sp., Trichosporon sp., Lipomyces sp., Rhodotorula sp., Candida sp., Cutaneotrichosporon sp., and combinations of any of the foregoing, or consists of the same; wherein, preferably, the yeast biomass comprises Cutaneotrichosporon oleaginosus and / or Saccharomyces cerevisiae biomass.

[0076] The term "lignocellulosic biomass" refers to biomass containing cellulose, hemicellulose, lignin, and combinations thereof. In one embodiment, the hydrolyzate is derived from the following: paper industry products (such as pulp, spent liquor, paper, and / or paper waste); forest products (such as wood and / or sawdust); agricultural products (such as straw); food or kitchen waste (such as bread); biofuel production waste (such as microbial biomass); hydrogen production waste (such as microbial biomass); textiles (optionally biodegradable textiles, such as wool, cotton, and / or hemp); animal tissues (such as meat); plant biomass (such as crops); microbial biomass (such as fungal biomass, bacterial biomass, and / or yeast biomass); industrial waste (optionally biodegradable industrial waste, such as hemp hydrolyzate); municipal waste; or any combination thereof.

[0077] In one embodiment, the hydrolyzate is a hydrolyzate derived from the paper industry, such as pulp or pulp hydrolyzate, spent liquor, paper hydrolyzate, and / or paper waste hydrolyzate; a hydrolyzate derived from forestry, such as wood hydrolyzate and / or sawdust hydrolyzate; an agricultural hydrolyzate, such as straw hydrolyzate; a food hydrolyzate, such as fruit peel hydrolyzate; a kitchen waste hydrolyzate, such as bread residue hydrolyzate; a biofuel production waste hydrolyzate, such as microbial biomass hydrolyzate; a hydrogen production waste, such as microbial biomass hydrolyzate; a textile hydrolyzate, such as wool hydrolyzate, cotton hydrolyzate, and / or hemp hydrolyzate; an animal tissue hydrolyzate, such as meat hydrolyzate; a plant tissue hydrolyzate, such as crop hydrolyzate; a microbial biomass hydrolyzate, such as fungal biomass hydrolyzate, bacterial biomass hydrolyzate, and / or yeast biomass hydrolyzate; an industrial waste hydrolyzate, such as hemp hydrolyzate; a municipal waste hydrolyzate; or any combination thereof.

[0078] As used herein, the term "spent liquor" refers to any spent liquor known to those skilled in the art, and particularly to the liquid effluent from wood digestion during pulping. Spent liquor typically contains wood components such as lignin and further contains digestants such as caustic, sulfite, or sulfate. As used herein, the term "pulp" refers to the pulp of the papermaking process, particularly the lignocellulosic fiber material prepared by separating cellulose fibers from raw materials (such as wood, fiber crops, waste paper, or scraps) by chemical or mechanical methods. Pulp is the main raw material used in the production of the paper and other paper product industries. Pulp production can include mechanical pulping, thermomechanical pulping, chemi-thermomechanical pulping, chemical pulping, and organosolv pulping. Chemical pulping can include the Kraft process, the sulfite process, and / or the soda pulping process. In one embodiment, the hydrolysis product derived from pulp production refers to the hydrolysis product derived from chemical pulping (preferably acid pulping). In one embodiment, the hydrolysis product is spent liquor or derived from spent liquor.

[0079] In a preferred embodiment, the hydrolysis product is derived from biomass (such as lignocellulosic biomass). In one embodiment, the hydrolysis product is a hydrolysis product derived from the paper industry, such as a hydrolysis product derived from pulp production, a hydrolysis product derived from forestry, agriculture, food, food waste, biofuel waste, textiles, animal tissues, plant tissues, microbial biomass, industrial waste, municipal waste, or any combination thereof; in one embodiment, the hydrolysis product is a biogenic hydrolysis product. In one embodiment, as used herein, the term "biogenic hydrolysis product" refers to the hydrolysis product of a biogenic product (such as biogenic waste). A biogenic product is a product made or composed of living organisms. In one embodiment, the biogenic hydrolysis product contains or consists of lignocellulosic hydrolysis products. In a preferred embodiment, the hydrolysis product is an acid pulping product or derived from an acid pulping process, particularly the waste stream and / or residual stream generated during the acid pulping process or derived from the waste stream and / or residual stream generated during the acid pulping process. Advantageously, by the method of the present invention, pentoses in the waste stream and / or residual stream from acid pulping can be effectively valorized as a raw material for culturing microorganisms (such as Cutaneotrichosporon sp.). In addition, the content of growth-inhibiting salts (such as sulfates) is advantageously reduced by the method of the present invention. By reducing the salt content of the hydrolysis product (such as reducing the sulfate content in the hydrolysis product), the hydrolysis product can be used as a raw material (such as for microbial culture).

[0080] In one embodiment, the hydrolyzate with reduced salt content provided in step i) comprises volatile organic acids, xylose and / or glucose; wherein, preferably, the hydrolyzate comprises or consists of a lignocellulosic hydrolyzate. For example, the volatile organic acid may comprise acetic acid. In one embodiment, the cultivation in step ii) comprises adding volatile organic acids (such as acetic acid), a carbon source other than the volatile organic acid and / or supplementary nutrients to the growth medium; wherein, preferably, the volatile organic acid is added in the form of a feed comprising or consisting of the volatile organic acid; wherein, preferably, the concentration of the volatile organic acid in the feed is in the range of 1 mol / l to 20 mol / l, preferably 1.75 mol / l to 15.75 mol / l; wherein, optionally, the feed further comprises a carbon source other than the volatile organic acid (such as other carbon sources other than acetic acid).

[0081] In one embodiment, the hydrolyzate is a paper production waste stream or is derived from a paper production waste stream, preferably the pentose fraction of a paper production waste stream or the pentose fraction derived from a paper production waste stream. In one embodiment, the saline hydrolyzate (such as the pentose fraction in a paper production waste stream) comprises:

[0082] - hexose in an amount of 0.5% - 5%; pentose in an amount of 1% - 10%; oligosaccharides in an amount of 0.3% - 1%; volatile fatty acids in an amount of 0.1% - 3%; and soluble lignin in an amount of 0.2% - 1%; or

[0083] - hexose in an amount of 5% - 25%; pentose in an amount of 30% - 65%; oligosaccharides in an amount of 2% - 15%; volatile fatty acids in an amount of 0.5% - 25%; and soluble lignin in an amount of 1% - 10%; or

[0084] - hexose in an amount of 15% - 35%; pentose in an amount of 1% - 25%; oligosaccharides in an amount of 10% - 40%; volatile fatty acids in an amount of 3% - 25%; and soluble lignin in an amount of 5% - 25%.

[0085] In one embodiment, the hydrolysis product is a hydrolysis product obtained by subjecting a substrate (preferably biomass) to physical treatment, chemical treatment, enzymatic treatment, and / or biological treatment; wherein optionally, the substrate is selected from: paper industry products (such as pulp, waste liquor, paper, and / or paper waste), forest products (such as wood and / or sawdust), agricultural products (such as straw), food or kitchen waste (such as bread), biofuel production waste (such as microbial biomass), hydrogen production waste (such as microbial biomass), textiles (such as wool, cotton, and / or hemp), animal tissues (such as meat), plant biomass (such as crops), microbial biomass (such as fungal biomass, bacterial biomass, and / or yeast biomass), industrial waste (such as hemp hydrolysis products), municipal waste, and any combination thereof. In one embodiment, the hydrolysis product is provided by subjecting a substrate (preferably biomass) to physical treatment, chemical treatment, enzymatic treatment, and / or biological treatment. In one embodiment, the substrate is selected from: paper industry products (such as pulp, waste liquor, paper, and / or paper waste), forest products (such as wood and / or sawdust), agricultural products (such as straw), food or kitchen waste (such as bread), biofuel production waste (such as microbial biomass), hydrogen production waste (such as microbial biomass), textiles (such as wool, cotton, and / or hemp), animal tissues (such as meat), plant biomass (such as crops), microbial biomass (such as fungal biomass, bacterial biomass, and / or yeast biomass), industrial waste (such as hemp hydrolysis products), municipal waste, and any combination thereof. In one embodiment, the substrate is lignocellulosic biomass. In one embodiment, the hydrolysis product is a lignocellulosic hydrolysis product derived from wood, preferably a lignocellulosic hydrolysis product derived from hardwood.

[0086] In one embodiment, the physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and any combination thereof. The chemical treatment is selected from alkali treatment, acid treatment, and treatment at neutral pH; wherein, preferably, the chemical treatment is treatment with salts, acids, peroxides, and any combination thereof, preferably treatment with sulfides, sulfites, and / or bisulfites. In one embodiment, the enzymatic treatment is treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylating enzymes, peptidases such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, lignin-degrading enzymes, and any combination thereof. In one embodiment, the biological treatment is treatment with microorganisms, preferably treatment with microorganisms selected from bacteria, yeasts, and fungi. For example, the hydrolysate can be obtained by chemical treatment and physical treatment, such as by acid treatment or alkali treatment followed by steam explosion. For example, the enzymatic treatment can be carried out using any of the following enzymes: LiP (EC 1.11.1.14), MnP (EC 1.11.1.13), laccase (EC 1.10.3.2), β-O-4 ether bond cleavage enzyme (such as β-etherase), β-O-4 aryl ether cleavage enzyme, O-demethylase, H2O2-generating oxidase, aryl-alcohol oxidase (EC 1.1.3.7), quinone reductase (EC 1.6.5.5), cellobiose dehydrogenase (EC 1.1.99.18), catechol 2,3-dioxygenase (EC 1.13.11.2), perhydrolase, lipase (EC 3.1.1.3), and / or any combination thereof.

[0087] In one embodiment, the salt-containing hydrolysate contains an amount of salt in the range of from about 0.0001 mol / l to about 15 mol / l, preferably from about 0.0005 mol / l to about 8 mol / l, such as salts selected from sulfates, sulfides, sulfites, nitrates, nitrites, chlorides, and any combination thereof. In one embodiment, the salt-containing hydrolysate contains an amount of sulfate in the range of from about 0.0005 mol / l to about 8 mol / l, optionally in the range of from about 0.05 mol / l to about 0.4 mol / l, such as about 0.2 mol / l. In one embodiment, the salt-containing hydrolysate contains sulfate and optionally also contains other salts.

[0088] In one embodiment, the hydrolyzed product of the brine contains salts selected from the group consisting of sulfates, sulfides, sulfites, nitrates, nitrites, chlorides, and any combination thereof, preferably containing sulfates. In a preferred embodiment, the hydrolyzed product of the brine contains salts selected from the group consisting of sulfates, sulfides, sulfites, and any combination thereof. The hydrolyzed product may contain salts (especially salts at high concentrations, such as 8 mol / L), which are caused by the preparation process of the hydrolyzed product (such as chemical treatment, such as the acid pulping process).

[0089] In one embodiment, the hydrolyzed product of the brine contains carbon in an amount in the range of about 0.1% to about 65% by weight, preferably in the range of 0.1% to about 35% by weight, such as about 25% by weight; wherein, preferably, "by weight" refers to the dry weight of the hydrolyzed product. In a preferred embodiment, the carbon exists in the hydrolyzed product in the form of a biodegradable carbon source (such as sugars and organic acids). In one embodiment, the carbon exists in the hydrolyzed product in the form of sugars (such as xylose, glucose, mannose, and / or galactose) and / or organic acids (such as acetic acid). In one embodiment, the hydrolyzed product contains sugars (such as xylose, glucose, mannose, and / or galactose), and / or contains acetic acid. In one embodiment, the hydrolyzed product provided in step a) contains lignols, lignans, organic acids, and / or sugars; wherein, optionally, the sugars contain xylose, glucose, mannose, and / or galactose, or consist of them; wherein, preferably, the sugars contain monosaccharides, preferably xylose; and wherein, preferably, the organic acids contain acetic acid. In one embodiment, the hydrolyzed product provided in step a) contains lignols, lignans, organic acids, and / or sugars; wherein, optionally, the sugars contain xylose, glucose, mannose, and / or galactose, or consist of them; wherein, preferably, the sugars contain monosaccharides, preferably xylose; and wherein, preferably, the organic acids contain acetic acid.

[0090] In one embodiment, the growth medium comprises sugar in an amount ranging from about 10 g / L to about 250 g / L, such as about 115 g / L. In one embodiment, the growth medium comprises xylose in an amount ranging from about 30 g / L to about 100 g / L, such as about 77 g / L. In one embodiment, the hydrolyzate comprises glucose in an amount ranging from about 0 g / L to about 20 g / L, such as in the range of about 0.05 g / L to about 20 g / L, such as about 12 g / L. In one embodiment, the hydrolyzate comprises acetic acid in an amount ranging from about 0 g / L to about 20 g / L, such as in the range of about 0.05 g / L to about 20 g / L, such as about 12 g / L. In one embodiment, the hydrolyzate comprises furans in an amount ranging from about 0 g / L to about 10 g / L, such as in the range of about 0.05 g / L to about 10 g / L, such as about 5 g / L. In one embodiment, the hydrolyzate comprises lignin-derived compounds in an amount ranging from about 0 g / L to about 200 g / L, such as in the range of about 0.05 g / L to about 200 g / L, such as about 90 g / L.

[0091] In one embodiment, the terms "hydrolyzate", "saline hydrolyzate" and "hydrolyzate provided in step a)" are used interchangeably. In one embodiment, the hydrolyzate with reduced salt content obtained in step f) differs from the hydrolyzate provided in step a) only in salt content, and optionally also in terms of pH value and / or sterilization treatment. The hydrolyzate with reduced salt content obtained in step f) can have any of the characteristics described for the hydrolyzate provided in step a).

[0092] In one embodiment, the method for reducing the salt content comprises neutralizing the pH of the hydrolyzate of step a) to obtain a neutralized hydrolyzate. In one embodiment, the pH value of the neutralized hydrolyzate ranges from about pH 4 to about pH 8, preferably from about pH 6 to about pH 8, more preferably from about pH 6.5 to about pH 7.5. In one embodiment, the pH value of the neutralized hydrolyzate obtained in step b) is about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0 or about pH 7.5. In one embodiment, neutralizing the pH of the hydrolyzate comprises increasing the pH value of the hydrolyzate, for example up to pH 7.5 or pH 8. In one embodiment, the neutralization comprises neutralizing the hydrolyzate of step a) by increasing the pH of the hydrolyzate to obtain a neutralized hydrolyzate, for example to obtain a hydrolyzate with a pH of up to pH 7.5 or up to pH 8. In one embodiment, the neutralized hydrolyzate has a pH of about pH 7.

[0093] The method for reducing the salt content comprises the step of adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2, preferably CaCO3 and / or Ca(OH)2, to the hydrolysis product of step a) or the neutralized hydrolysis product of step b) to obtain a hydrolysis product containing precipitated salts. The inventors have found that adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2 enables a hydrolysis product suitable as a microbial growth substrate to be provided. In particular, by adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2 or a combination thereof, substances interfering with growth, especially salts, are sufficiently removed and the hydrolysis product is made suitable for use as a growth substrate. Advantageously, CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2 or a combination thereof can act as an overliming agent to promote the precipitation of salts present in the hydrolysis product. In one embodiment, the hydrolysis product containing precipitated salts obtained in step c) has a pH in the range of about pH 4 to about pH 8.5, such as a pH of about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5 or about pH 7.0. In one embodiment, the addition of CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2 or a combination thereof to the hydrolysis product of step a) or the neutralized hydrolysis product of step b) comprises adding the CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2 or a combination thereof in an amount in the range of 0.001 g / l to 500 g / l, preferably 1 g / l to 150 g / l. In one embodiment, the addition comprises adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2 or a combination thereof in an amount such that at least 1% (preferably at least 10%, more preferably at least 30%, further preferably at least 50%, most preferably at least 80%) of the salt content in the hydrolysis product of step a) or the neutralized hydrolysis product of step b) is precipitated.

[0094] In one embodiment, the addition comprises mixing (preferably thoroughly mixing) the CaCO3, Ca(OH)2, CaO, MgO, MgCO3, and / or Mg(OH)2 with the hydrolysis product to promote the precipitation of salts. In one embodiment, the method comprises mixing the CaCO3, Ca(OH)2, CaO, MgO, MgCO3, and / or Mg(OH)2 with the hydrolysis product (e.g., by stirring, shaking, and / or vortex mixing). The inventors have found that by adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2, or a combination thereof to the saline hydrolysis product, the salts in the hydrolysis product can be effectively precipitated, thereby providing a hydrolysis product that can be used as a growth substrate.

[0095] The method of the present invention comprises adding a chelating agent to the hydrolysis product of step c). The chelating agent can be any chelating agent known to those skilled in the art, such as a chelating agent selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), EDTA, EGTA, EHPG, and any combination thereof, where M is a metal or a substituted counterion.

[0096] In one embodiment, the chelating agent is selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof, where M is a metal. Advantageously, when the chelating agent is selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof, the obtained hydrolysis product with a reduced salt content enables efficient growth of microorganisms (e.g., for the production of target products). The inventors have found that when a hydrolysis product with a reduced salt content prepared using a chelating agent selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof (where M is a metal) is used as a growth substrate, the efficiency of microbial culture (especially the production of target products) is significantly improved. The inventors have found that when the chelating agent is selected from the M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof, the hydrolysis product is particularly suitable for subsequent use as a growth substrate because other chelating agents (such as EDTA, EGTA, and EHPG) may inhibit microbial growth.

[0097] In a preferred embodiment, the chelating agent is selected from Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, (NH4)(H2PO4), and Na3PO4, where, preferably, the chelating agent is KH2PO4. The inventors have found that when the chelating agent is selected from Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, (NH4)(H2PO4), and Na3PO4, the hydrolyzate with reduced salt content has significant advantages for subsequent use as a growth medium. In particular, the inventors have found that when using such chelating agents to produce a hydrolyzate with reduced salt content, the obtained hydrolyzate with reduced salt content can significantly improve the production efficiency of the microbial target product using the hydrolyzate as a growth substrate. In one embodiment, adding the chelating agent to the hydrolyzate of step c) comprises adding the chelating agent at a concentration in the range of 0.001 g / l - 500 g / l, preferably 1 g / l - 150 g / l.

[0098] In one embodiment, the method comprises adjusting the pH of the hydrolyzate of step c) and / or the hydrolyzate of step d) to a range of about pH 2 to about pH 10. In one embodiment, the method comprises adjusting the pH of the hydrolyzate of step c) and / or the pH of the hydrolyzate of step d) to a range of about pH 3 to about pH 8.5 (e.g., in the range of about pH 5 to about pH 8.5, or in the range of about pH 5 to about pH 8); preferably in the range of about pH 3.5 to about pH 7.5; more preferably in the range of about pH 5 to about pH 7; further preferably in the range of about pH 6 to about pH 7.

[0099] In one embodiment, adjusting the pH comprises adding an acid or a base. In one embodiment, the method comprises step e) of adjusting the pH of the hydrolyzate, and the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid, or any combination thereof to the hydrolyzate of step c) and / or step d), preferably by adding NaOH and / or KOH. The inventors have found that when the pH is adjusted by adding NaOH, KOH, CH3COOH, HCl, or any combination thereof, the hydrolyzate has significant advantages in subsequent microbial culture. In one embodiment, the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, or any combination thereof.

[0100] In one embodiment, for example when the hydrolyzate with reduced salt content is intended for use in culturing microorganisms, the pH adjustment comprises adjusting the pH value to a range of about pH 3 to about pH 8.5 (such as about pH 5 to about pH 8.5, or about pH 5 to about pH 8). In one embodiment, for example when the hydrolyzate product with reduced salt content is intended for use in culturing acidophilic and / or alkaliphilic microorganisms, the pH adjustment comprises adjusting the pH value to a range of about pH 2 to about pH 10. In one embodiment, for example when the hydrolyzate product with reduced salt content is intended for use in culturing oil-producing microorganisms (such as Cutaneotrichosporon sp.), the pH adjustment comprises adjusting the pH value to a range of about pH 6 to about pH 7 (such as about pH 6.5). Advantageously, by adjusting the pH of the hydrolyzate, the hydrolyzate has a pH suitable for culturing microorganisms. For example, when the hydrolyzate is used for high-efficiency fermentation of microorganisms (such as oil-producing yeast), its pH value can be adjusted to about 6.5. In particular, the pH of the hydrolyzate can be adjusted to match the requirements of the target microorganism (such as the microorganism to be cultured in the method for producing the target product of the present invention).

[0101] In one embodiment, the method comprises sterilization, preferably sterilizing the hydrolyzate with reduced salt content obtained in step f) to obtain a sterile hydrolyzate with reduced salt content. In one embodiment, the method comprises hydrolyzing and sterilizing the saline hydrolyzate provided in step a), the neutralized hydrolyzate obtained in step b), the hydrolyzate containing precipitated salts obtained in step c), the mixture obtained in step e) and / or the hydrolyzate with reduced salt content obtained in step f). In one embodiment, the method comprises one or more sterilization steps. In one embodiment, the sterilization comprises thermal sterilization, ultra-high temperature treatment and / or sterile filtration. In one embodiment, the sterilization step comprises thermal sterilization, ultra-high temperature treatment and / or sterile filtration. Advantageously, by sterilizing the hydrolyzate, it can be made suitable as a growth medium for culturing microorganisms. In particular, by sterilizing the hydrolyzate, contamination of the growth medium for culturing microorganisms can be effectively prevented. In one embodiment, the hydrolyzate with reduced salt content obtained in step f) is filtered through activated carbon. The inventors have found that by filtering the hydrolyzate through activated carbon, the content of toxic phenolic and furan compounds is effectively reduced.

[0102] In one embodiment, obtaining the hydrolysis product with reduced salt content comprises obtaining a hydrolysis product having a salt content reduced by at least 1% (preferably at least 10%, more preferably at least 30%, further preferably at least 50%, and most preferably at least 80%) compared to the salt-containing hydrolysis product provided in step a). In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is sterilized. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is suitable as a growth medium. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is used in the method for producing the target product of the present invention. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) comprises sugars (such as xylose, glucose, mannose, and / or galactose), and / or comprises organic acids (such as acetic acid). In one embodiment, the hydrolysis product with reduced salt content obtained in step f) contains carbon in an amount in the range of about 0.1% to about 65% by weight; wherein, preferably, "by weight" refers to the dry weight of the hydrolysis product. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is intended for use in the production method of the target product of the present invention. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is provided in the form of a growth medium. In one embodiment, the hydrolysis product with reduced salt content obtained in step f) is a hydrolysis product intended for use in microbial culture (such as in the production method of the target product of the present invention).

[0103] The present invention also relates to a method for producing a target product (preferably microbial oil), which comprises the following steps:

[0104] i) Providing a hydrolysis product with reduced salt content by implementing the method for reducing the salt content of the salt-containing hydrolysis product defined herein;

[0106] ii) Culturing a microorganism (preferably an oil-producing microorganism) using the growth medium comprising or consisting of the hydrolysis product provided in step i), thereby enabling the microorganism to produce the target product; preferably enabling the oil-producing microorganism to produce microbial oil;

[0108] iii) Optionally, subjecting the microorganism (preferably the oil-producing microorganism) to an enzymatic treatment; wherein, optionally, the enzymatic treatment comprises subjecting the microorganism to the enzymatic treatment without any solvent extraction or chemical demulsification;

[0109] iv) Obtaining the target product, preferably microbial oil.

[0110] As used herein, the term "target product" refers to any target product, particularly microbial oils, glycerol, free fatty acids, monoacylglycerols, diacylglycerols and triacylglycerols, phospholipids, sphingolipids, polyols, alcohols such as ethanol, organic acids, biodiesel, hydrogen, methane, biopolymers, carotenoids, cellulose, squalene, sterols, vitamins, phenolic compounds, pigments, peptides, proteins such as enzymes, DNA, RNA or other products of interest. The target product can be any product of interest to those skilled in the art, such as microbial oils, microbial oils containing the target compound and / or compounds obtained from microbial oils. For example, microbial oils can contain compounds such as antioxidants and other lignin-derived compounds.

[0111] In one embodiment, the target product is selected from microbial oils, polyols, alcohols such as ethanol, organic acids, biodiesel, biopolymers, carotenoids, cellulose, squalene, sterols, vitamins, phenolic compounds, proteins such as enzymes, DNA, RNA, other lignin-derived compounds, proteins such as enzymes, DNA, RNA, and any combination thereof. In one embodiment, the target product comprises any compound derived from lignin. In one embodiment, the other target products and / or the other lignin-derived compounds may be any compound derived from lignin, preferably selected from Pyrocatechuate, 2,3-Dihydroxybenzoic acid, Coniferylaldehyde, Syringaldehyde, Methyl vanillate, 3,4-Dihydroxybenzaldehyde, Gentisate aldehyde, Umbelliferone, 3-Hydroxycoumarin, Asaronic acid, Sinapate, Genipin, 2,4,5-trimethoxybenzoic acid, Vanillactic acid, 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoic acid, 2,4,4'-Trimethoxy-3',6-dihydroxybenzophenone,6-dihydroxybenzophenone, Ferulaldehyde, 3-Formylphenol, 1-Hydroperoxy-4-methoxybenzene, coumarin, vanillin, Methylphthalicanhydride, Ayapanin, Indole, Benzeneacetonitrile, 5-Aminopentanal, Syrinic acid, Ethyl syringate, Pyroglutamylleucine, 4-(2-Methoxyphenyl)furan-2(5H)-one, 2-(4-Hydroxy-1-benzofuran-2-yl)acetic acid, 1-(4-Hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one, 2,3,6,7-tetamethoxynaphthalene, 1,3,6,8-tetramethoxynaphthalene, Dimethyl 2-[(2-methoxyphenyl)methylidene]butanedioate, 5,10-dimethoxy-2,2-dimethylpyrano[3,2-g]chromen-8-one, Swietenocoumarin F, 4-N-[(3,4-dimethoxyphenyl)methylideneamino]-5-nitropyrimidine-4,6-diamine, 3-OH-3”,4”,5,7-tetraMeO Flavone, 1-(2,4,6-trihydroxyphenyl)-3-(3,4,5-(3,4,5-trimethoxyphenyl)propan-1-one (1-(2,4,6-Trihydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)propan-1-one), Syringaresinol, Lenampicilina, beta-D-Glucopyranoside, Tubocurarine, Psymberin, Psymberin, Mesaconitine, aschantin, Phenylpropanolamine, Monomethyl phenylphosphonate, Herniarin, 4-Methylumbelliferone, Chromone, 2-Benzofurancarboxaldehyde, Glycocoumarin, Imbricaric acid, Phenethyl rutinoside, Fluticasone 17beta-carboxylicacid, Gentisic acid, Protocatechuic acid, 7-Azaindolizine, Syringaresinol.,

[0112] In one embodiment, the phenolic compounds are selected from tocopherols such as α-, β-, γ- and δ-tocopherols; tocotrienols such as α-, β-, γ- and δ-tocotrienols; tocomonols such as α- and β-tocomonols; phytoestrogens; chalcones such as arbutin, phloretin, phloridzin and naringin chalcone; flavonoid compounds such as flavonols, flavones, dihydroflavones, flavanols, flavan-3-ols, anthocyanins, isoflavones and condensed tannins; and non-flavonoid compounds such as coumarins and phenolic acids. In one embodiment, the vitamins are selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 and vitamin K.

[0113] As used herein, the terms "microbial lipid" and "microbial oil" refer to lipids produced by oil-producing microorganisms, such as yeast oil, bacterial oil, and / or fungal oil. In one embodiment, the term "microbial lipid" may be used interchangeably with "single cell oil" or "microbial oil". Generally, microbial lipids are rich in unsaturated fatty acids. In one embodiment, the microbial lipid is an edible microbial lipid. Such microbial lipids can be used to prepare foods containing microbial lipids. Advantageously, microbial oils can be used to replace fats with a high saturated fatty acid content and / or environmentally unfriendly fats (such as palm oil).

[0114] In one embodiment, the culturing of the microorganism (preferably an oil-producing microorganism) described in step ii) involves using a growth medium comprising or consisting of the hydrolysate provided in step i) as the growth substrate for the microorganism. In particular, the growth substrate comprises a carbon source, a nitrogen source, and / or a phosphate source. In a preferred embodiment, the hydrolysate provided in step i) comprises a carbon source, a nitrogen source, and / or a phosphate source. For example, the growth medium may comprise the hydrolysate as the growth substrate, particularly to provide carbon, nitrogen, and / or phosphate to the microorganism. In one embodiment, as used herein, the term "causing the microorganism to produce a target product" refers to providing the microorganism with suitable growth conditions. In one embodiment, the microorganism produces the target product as a coproduct (e.g., a fermentation coproduct) of microbial growth. In one embodiment, the microorganism produces the target product during culturing.

[0115] In one embodiment, the growth medium comprises the hydrolysate and further comprises other components, such as buffers, trace elements, vitamins, and / or additional carbon sources, nitrogen sources, and / or phosphate sources.

[0116] In one embodiment, the microorganism is selected from yeast, fungi, bacteria, and microalgae. In one embodiment, the microorganism is an oil-producing microorganism, preferably an oil-producing yeast. In one embodiment, the microorganism is selected from the genus Rhodosporidium, the genus Yarrowia, the genus Rhodotorula, the genus Candida, the genus Lipomyces, Cutaneotrichosporon sp., the genus Trichosporon, preferably selected from Cutaneotrichosporon sp., more preferably Cutaneotrichosporon oleaginosus, such as Cutaneotrichosporon oleaginosus (ATCC 20509). In one embodiment, the microorganism is an oil-producing microorganism, preferably an oil-producing yeast, which is Cutaneotrichosporon sp., and further preferably Cutaneotrichosporon oleaginosus. In one embodiment, the microorganism is an oil-producing microorganism, and the target product comprises or consists of microbial oil. In one embodiment, the target product comprises or consists of microbial oil, wherein the microbial oil optionally comprises squalene, sterols, vitamins, and / or phenolic compounds.

[0117] In one embodiment, the microorganism can be a wild-type or genetically modified microorganism. In one embodiment, the genetic modification can be in the form of directed evolution, random mutagenesis, and / or targeted engineering. In one embodiment, the directed evolution, random mutagenesis, and / or targeted engineering include genome modification by UV treatment, chemical treatment, genetic breeding, error-prone PCR, or other PCR-based methods such as DNA shuffling, Agrobacterium-mediated transformation, selection, and / or screening. In one embodiment, the targeted engineering includes genome integration, modification, knockout, CRISPR-Cas-based gene knockdown, Agrobacterium-mediated transformation, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), targeted mutagenesis, site-directed mutagenesis of promoter modification, RNA interference (RNAi), small interfering RNA (siRNA), and / or combinations thereof. In one embodiment, the purpose of the genetic modification is to improve the tolerance of the microorganism to salts reducing the hydrolyzate, improve the adaptability of the microorganism to the salt content under the culture conditions, increase the yield of the target product, and / or change the chemical and / or physical properties and / or its composition of the target product.

[0118] In one embodiment, the hydrolyzate with reduced salt content provided in step i) comprises acetic acid and / or saccharides (such as xylose). In one embodiment, the hydrolyzate comprises xylose, acetic acid, and optionally glucose. In one embodiment, the hydrolyzate with reduced salt content provided in step i) comprises or consists of a lignocellulosic hydrolyzate. In one embodiment, the hydrolyzate with reduced salt content provided in step i) is the hydrolyzate obtained in step f) of the method for reducing salt content of the present invention. In one embodiment, the hydrolyzate with reduced salt content provided in step i) is obtained by the method for reducing salt content of the present invention.

[0119] In one embodiment, culturing a microorganism (preferably an oil-producing microorganism) using a growth medium comprising or consisting of the hydrolyzate provided in step i) as a growth substrate for the microorganism comprises fermentatively culturing the microorganism, wherein the target product is produced as a by-product of the fermentative culturing. In one embodiment, culturing a microorganism using a growth medium comprising or consisting of the hydrolyzate provided in step i) as a growth substrate for the microorganism comprises culturing the microorganism

[0120] for a duration of 1 to 7 days, preferably 2 to 4 days;

[0121] at a temperature in the range of 10°C to 45°C, preferably in the range of 15°C to 40°C, more preferably in the range of 20°C to 33°C;

[0122] at a pH in the range of pH 4 to pH 9.5, preferably in the range of pH 5 to pH 8.5, more preferably in the range of pH 5.5 to pH 8;

[0123] in a culture medium selected from a low-nitrogen medium, a low-phosphorus medium, a low-sulfur medium, and an acetic acid-rich medium, wherein the culture medium comprises the hydrolyzate provided in step i); and / or

[0124] wherein the dissolved oxygen (pO2) is in the range of 10% - 90%, preferably 20% - 80%, more preferably 30% - 65%.

[0125] Advantageously, through the culturing, the microorganism produces the target product.

[0126] As used herein, the term "growth medium" refers to a cell culture medium, preferably comprising a carbon source, a nitrogen source, and / or a phosphate source. In one embodiment, the terms "medium" and "growth medium" may be used interchangeably. In one embodiment, the growth medium comprises or consists of the hydrolyzate provided in step i), wherein the hydrolyzate provides a carbon source, a nitrogen source, and / or a phosphate source. In one embodiment, the carbon source comprises glucose, xylose, and / or acetic acid. In one embodiment, the nitrogen source comprises ammonium salts, nitrates, amino acids, peptides, N-acetylglucosamine, peptone, yeast extract, and / or urea. In one embodiment, the phosphate source comprises any organic phosphate compound, such as parathion, malathion, phospholipids, ATP, ADP, AMP, and organic phosphate ester compounds; and inorganic phosphates, such as H3PO4, M2HPO4, MH2PO4, MHPO4, and MPO4, where M is a metal ion.

[0127] In one embodiment, the growth medium comprises a carbon source, and optionally a buffer, salts, trace elements, a nitrogen source, peptone, and / or yeast extract. For example, the growth medium may comprise an amount of sugar in the range of about 10 g / l to about 100 g / l (e.g., about 30 g / l), and / or an amount of acetic acid in the range of about 10 g / l to about 50 g / l (e.g., about 30 g / l). For example, the growth medium may comprise any one of Na2HPO4, KH2PO4, CH3COO·Na, MgSO4·7H2O, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·6H2O, Cu SO4·5H2O, C6H8O7·Fe·H3N, urea, peptone, yeast extract, and combinations thereof.

[0128] In one embodiment, the growth medium has a carbon to nitrogen weight ratio (C:N) of <200, more preferably ≤100, and even more preferably 5 to 80. In one embodiment, the growth medium has a carbon to phosphorus weight ratio (C:P) of <500, more preferably ≤150, and even more preferably 25 to 100. The inventors have found that a medium having a carbon to nitrogen weight ratio (C:N) of <100, more preferably ≤80, and even more preferably in the range of 10 to 80, and / or a carbon to phosphorus weight ratio (C:P) of <500, more preferably ≤150, and even more preferably in the range of 10 to 100 can achieve a high yield of the target product. In one embodiment, the culturing process comprises or consists of nitrogen-limited fermentation, such as using a growth medium having a carbon to nitrogen weight ratio (C:N) in the range of 50 to 200.

[0129] In one embodiment, culturing the microorganism comprises maintaining the microorganism under suitable growth conditions. In one embodiment, the terms "culture medium" and "growth medium" are used interchangeably. In one embodiment, culturing the microorganism (preferably an oil-producing microorganism) using the hydrolyzate provided in step i) or a growth medium consisting thereof as a growth substrate for the microorganism comprises fermentatively culturing the microorganism. In one embodiment, as used herein, the term "fermentative cultivation" refers to culturing a microorganism, such as yeast, under fermentation conditions. For example, fermentation involves culturing a microorganism, particularly a heterotrophic microorganism, using a feedstock (e.g., aerobic and / or anaerobic cultivation). For example, the fermentation conditions can comprise fermentation at a temperature in the range of 10°C to 45°C, preferably in the range of 15°C to 40°C, more preferably in the range of 20°C to 33°C; fermentation at a pH in the range of pH 4 to pH 9, preferably in the range of pH 5 to pH 8, more preferably in the range of pH 5.5 to pH 7.5; fermentation in a culture medium selected from a low-nitrogen medium, a low-phosphorus medium, a low-sulfur medium, and an acetic acid-rich medium; and / or cultivation at a dissolved oxygen (pO2) in the range of 5% to 90%, preferably in the range of 20% to 80%, more preferably in the range of 30% to 60%. In one embodiment, the culturing of the microorganism in step ii) comprises fermentative cultivation. In one embodiment, the culturing of the microorganism in step ii) is carried out in a fed-batch manner, a semi-continuous manner, or a continuous manner, preferably in a continuous manner.

[0130] In one embodiment, culturing the microorganism in the growth medium in step ii) comprises using the hydrolyzate as a substrate and optionally adding additional feeds. In one embodiment, the hydrolyzate as a growth substrate for the microorganism comprises a carbon source, a nitrogen source, and / or a phosphate source. In one embodiment, the hydrolyzate provided in step i) is used as a substrate for culturing the microorganism in step ii).

[0131] In one embodiment, the growth medium described in step ii) comprises a carbon source, a nitrogen source, a phosphate source, an organic acid, trace metals, and / or vitamins. In one embodiment, the growth medium comprises any one of the following: monosaccharides, preferably pentoses or hexoses, more preferably glucose, xylose, mannitol, arabinose, fructose, mannose, sorbitol, lactose, sucrose; oligosaccharides; amino acids; fatty acids; organic acids, preferably acetic acid; minerals; vitamins; trace elements; and combinations thereof. In one embodiment, the trace metals are selected from Mo, Cu, Zn, Mn, Ni, and Fe. In one embodiment, the vitamins are selected from vitamin C, vitamin B, vitamin A, and vitamin E. In one embodiment, the nitrogen source is selected from the group consisting of organic nitrogen compounds (such as amines, amides, alkyl nitrates, nitrosamines, nitroarenes, and peroxyacyl nitrates); inorganic nitrogen compounds (such as ammonium salts, nitrates, and nitrites); amino acids; peptides; protein hydrolysates (such as peptone, tryptone, and other peptide hydrolysates, where the peptide hydrolysates preferably comprise animal tissues, plant tissues, microbial biomass, and / or components of the yeast); N-acetylglucosamine; and urea; preferably selected from the group consisting of ammonium salts, amino acids, peptides, N-acetylglucosamine, and urea. In one embodiment, the phosphate source is selected from the group consisting of organic phosphate compounds, such as parathion, malathion, phospholipids, ATP, ADP, AMP, and organic phosphate ester compounds; and inorganic phosphates, such as H3PO4, M2HPO4, MH2PO4, MHPO4, and MPO4, where M is a metal ion.

[0132] In one embodiment, the organic acids are selected from acetic acid, malonic acid, oxalic acid, citric acid, propionic acid, valeric acid, acrylic acid, crotonic acid, butyric acid, isobutyric acid, isovaleric acid, 3-hydroxybutyric acid, 3-hydroxypropionic acid, 2-hydroxybutyric acid, lactic acid, and salts and / or combinations thereof corresponding to these acids. Preferably, the organic acid is acetic acid. It should be noted that as used herein, the term "organic acid" is intended to cover all protonation states of the corresponding organic acid, i.e., including the protonated and deprotonated forms of the acid (e.g., in an aqueous solution, depending on their respective pKa values, they may be in a protonated or deprotonated state under corresponding pH conditions). As used herein, the term "organic acid" is also intended to cover salts of the organic acid, such as metal salts corresponding to the organic acid. Examples of such metal salts are alkali metal salts or alkaline earth metal salts of the corresponding organic acid. The salt may be in its dissociated form or its undissociated form. Without wishing to be bound by any theory, the inventors believe that: the presence of an organic acid (such as acetic acid) can further increase the yield of the method for producing the target product of the present invention, while the presence of a carbon source can increase the total biomass.

[0133] In one embodiment, the cultivation in step ii) comprises adding to the growth medium an additional amount of the hydrolyzate provided in step i), a lignocellulosic hydrolyzate (e.g., a lignocellulosic hydrolyzate different from the hydrolyzate provided in step i), a carbon source of non-lignocellulosic hydrolyzate, and / or additional nutrients;

[0134] wherein, preferably,

[0135] the additional amount of the hydrolyzate provided in step i), the lignocellulosic hydrolyzate, the carbon source, and / or the additional nutrients are added in the form of a feed comprising or consisting of volatile organic acids, wherein, preferably, the pH of the feed is in the range of pH 3 to pH 7, preferably pH 3.5 to pH 6, more preferably pH 4 to pH 5.5, wherein, preferably, the volatile organic acids are present in the feed in an amount in the range of 1 mol / l to 20 mol / l, preferably in the range of 1.75 mol / l to 15.75 mol / l;

[0136] wherein, optionally, the feed further comprises a carbon source other than acetic acid.

[0137] In one embodiment, the cultivation in step ii) comprises adding one or more carbon sources and / or additional nutrients in the form of a feed;

[0138] wherein, optionally, the feed is provided in any one of continuous, semi-continuous, consumption-based, pH-based, dissolved oxygen-based, off-gas CO2 concentration-based, staged, and / or combinations thereof;

[0139] wherein preferably, the feed is provided in a continuous and / or consumption-based manner. In one embodiment, as used herein, the terms "providing a feed" and "adding a feed" refer to adding a feed to the growth medium, particularly during cultivation, for example, in a continuous and / or consumption-based manner.

[0140] In one embodiment, the growth medium comprises the hydrolyzate as a carbon source. In one embodiment, the cultivation in step ii) comprises adding acetic acid and / or a carbon source other than acetic acid (such as sugars) to the growth medium. For example, the growth medium may be supplemented with the hydrolyzate and optionally with additional acetic acid and / or additional carbon sources other than acetic acid. For example, the acetic acid may be added to the growth medium in the form of a feedstock comprising acetic acid or consisting of acetic acid. In one embodiment, the term "feedstock" relates to a liquid feedstock (such as an input stream) and / or a solid feedstock. In one embodiment, the feedstock comprises the hydrolyzate and optionally acetic acid. For example, the feedstock, particularly the liquid feedstock, may comprise liquid acetic acid and / or a liquid containing acetic acid. In one embodiment, the feedstock comprises acetic acid at a concentration in the range of 1 mol / l to 20 mol / l, preferably in the range of 1.75 mol / l to 15.75 mol / l. In one embodiment, the feedstock comprises the hydrolyzate with a reduced salt content and optionally a buffer. In one embodiment, the feedstock comprises the hydrolyzate with a reduced salt content, optionally in a diluted form, and further comprises acetic acid. In one embodiment, the feedstock further comprises a carbon source other than acetic acid.

[0141] In one embodiment, the growth medium is configured to comprise (e.g., by adjusting the content of the hydrolyzate in the medium) an amount of biodegradable carbon of 10% (w / v) or less, preferably 7% (w / v) or less, more preferably 5% (w / v) or less, such as about 3% (w / v). In one embodiment, the growth medium comprises an amount of carbon (particularly sugars and organic acids) of 10% (w / v) or less, preferably 7% (w / v) or less, more preferably 5% (w / v) or less, such as about 3% (w / v).

[0142] Biodegradable carbon generally refers to carbon that can be degraded and / or utilized by microorganisms and / or enzymes; for example, biodegradable carbon refers to carbon that can be metabolized by microorganisms. In one embodiment, the biodegradable carbon comprises sugars and / or organic acids (such as acetic acid). In one embodiment, the cultivation comprises a feedstock mode based on consumption, such as a feedstock based on acetic acid consumption and / or a feedstock based on hydrolyzate consumption. The advantage of using the hydrolyzate as a raw material is its cost-effectiveness and environmental friendliness. Compared with the hydrolyzate without a reduced salt content, the hydrolyzate with a reduced salt content has the advantage of removing growth-inhibiting salts and greatly improving the efficiency of cultivation.

[0143] In one embodiment, the feed comprises about 50% (v / v) acetic acid. In one embodiment, the feed comprises an acetic acid solution, preferably an acetic acid solution with an acetic acid content of 50% (v / v). In one embodiment, the feed comprises a mixture of the acetic acid solution and the hydrolyzate with a reduced salt content; wherein optionally, the acetic acid solution and the hydrolyzate are mixed at a ratio of about 1:1 to about 5:1.

[0144] In one embodiment, the cultivation in step ii) comprises providing a growth medium comprising or consisting of a hydrolysate, and further comprises providing a feed, preferably a feed stream, such as comprising acetic acid and / or a hydrolysate. In one embodiment, the terms "feed stream", "feed medium" and "substrate feed" can be used interchangeably. For example, the growth medium may comprise an initial amount of the hydrolysate and optionally acetic acid, and additional amounts of the hydrolysate provided in step i) may be supplemented during the cultivation by the feed. The inventors have found that a feed comprising acetic acid and a hydrolysate maximizes hydrolysate consumption and conversion. In one embodiment, the cultivation in step ii) comprises providing a growth medium comprising or consisting of an initial amount of the hydrolyzed hydrolysate provided in step i), and optionally further comprises adding an additional amount of the hydrolysate provided in step i) and / or adding acetic acid to the growth medium during the cultivation. In one embodiment, the addition of the additional amount of the hydrolysate and / or the addition of acetic acid is carried out in a consumption-based or continuous manner. For example, the content of carbon sources (such as acetic acid and / or sugars) in the growth medium can be measured, and if it is necessary to provide suitable growth conditions for the microorganism, carbon sources (such as an additional amount of the hydrolysate and / or acetic acid) can be added. In one embodiment, the cultivation in step ii) comprises measuring the carbon content of the growth medium, preferably by high performance liquid chromatography (HPLC), for example using an Agilent 1260 Infinity II liquid chromatography (LC) system equipped with a diode array detector (DA) and a refractive index detector (RI). To achieve separation, a Rezex ROA-organic H+ 8% column produced by Phenomenex can be used, and 5 mM H2SO4 can be used as the mobile phase. An isocratic elution flow rate of 0.5 mL / min can be used to run for 60 minutes, and the oven temperature is set at 70 °C. Detection can be carried out in a refractive index detector (RID) at 40 °C. In one embodiment, the growth medium comprises sugars; preferably sugars selected from xylose, glucose, fructose, arabinose, mannose, galactose and combinations thereof; wherein, preferably, the growth medium comprises the sugars in an amount in the range of about 0.1 g / L to about 100 g / L, preferably <50 g / L. In one embodiment, the growth medium comprises an amount of organic acid (preferably acetic acid) in the range of about 0.01 g / l to about 100 g / l, preferably in the range of about 1 g / l to about 50 g / l, more preferably in the range of about 5 g / l to about 10 g / l. The inventors have found that when the growth medium contains specific concentrations of sugars and / or organic acids, the cultivation process has a significant synergistic effect.

[0145] In one embodiment, the culturing process described in step ii) includes continuous feeding and / or consumption-based feeding methods. In one embodiment, the culturing in step ii) includes: continuous feeding of a certain amount of the hydrolyzate provided in step i), and / or consumption-based feeding including organic acids (especially acetic acid) and / or including other carbon sources in addition to the organic acids.

[0146] In one embodiment, the cultivation of step ii) comprises determining the sugar concentration of the growth medium, preferably by high performance liquid chromatography (HPLC), for example using an Agilent 1260 Infinity II LC system equipped with a diode array detector (DA) and a refractive index detector (RI). For separation, a Rezex ROA-organic H+ 8% column produced by Phenomenex can be used, and 5 mM H2SO4 can be used as the mobile phase. An isocratic elution flow rate of 0.5 mL / min can be used for 60 minutes, and the oven temperature is set at 70 °C. Detection can be carried out in a refractive index detector (RID) at 40 °C. In one embodiment, the cultivation of step ii) comprises determining the concentration of organic acids (such as acetic acid concentration) in the growth medium, preferably by high performance liquid chromatography (HPLC), for example using an Agilent 1260 Infinity II LC system equipped with a diode array detector (DA) and a refractive index detector (RI). For separation, a Rezex ROA-organic H+ 8% column produced by Phenomenex can be used, and 5 mM H2SO4 can be used as the mobile phase. An isocratic elution flow rate of 0.5 mL / min can be used for 60 minutes, and the oven temperature is set at 70 °C. Detection is carried out in a refractive index detector (RID) at 40 °C. In one embodiment, if the detected carbon concentration, sugar concentration and / or acetic acid concentration is too low (such as sugar concentration below 0.1 g / L and / or organic acid concentration below 0.01 g / L) to provide suitable growth conditions for the microorganism, the cultivation comprises: adding an additional amount of hydrolyzate, adding sugar and / or adding acetic acid. In one embodiment, the cultivation comprises adding a feed (preferably a feed stream) to the growth medium; wherein the feed (preferably the feed stream) comprises hydrolyzate and optionally additional acetic acid. For example, the feed can comprise about 500 ml of acetic acid per liter of feed and about 500 ml of hydrolyzate per liter of feed. In one embodiment, the feed comprises about 10% (v / v) to about 100% (v / v) acetic acid, preferably about 10% (v / v) to about 90% (v / v) acetic acid. In one embodiment, the feed comprises about 10% (v / v) to about 100% (v / v) of the hydrolyzate (preferably lignocellulosic hydrolyzate). In one embodiment, the feed comprises acetic acid and the hydrolyzate in a ratio of 10:1 - 1:10. In one embodiment, high performance liquid chromatography (HPLC) can be used to determine the carbon content, sugar content and / or organic acid content.

[0147] In a preferred embodiment, the hydrolysis product comprises an organic acid (such as acetic acid). In one embodiment, the growth medium comprises the hydrolysis product and optionally an additional organic acid (such as acetic acid). For example, if the microorganism requires a large amount of organic acid, an additional organic acid can be added to the growth medium, especially in addition to the organic acid contained in the hydrolysis product. For example, the feed and / or growth medium can contain the organic acid contained in the hydrolysis product and can be additionally supplemented with other organic acids. In one embodiment, the growth medium and / or feed contains the hydrolysis product and is additionally supplemented with an organic acid and / or a carbon source. In one embodiment, the term "hydrolysis product" as used herein in the context of the method for producing a target product specifically refers to a hydrolysis product having a reduced salt content, especially the hydrolysis product having a reduced salt content provided in step i) of the method for producing a target product according to the present invention, and / or the hydrolysis product having a reduced salt content obtained according to step f) of the method for reducing the salt content of the hydrolysis product according to the present invention.

[0148] In one embodiment, the method for producing a target product comprises the step of lysing the microorganism, especially after culturing the microorganism in step ii). For example, by lysing the microorganism, the acquisition of the target product can be facilitated. In one embodiment, the step of lysing the microorganism comprises any one of enzymatic hydrolysis, temperature shock, chemical treatment, high-pressure homogenization, ultrasonic homogenization, and any combination thereof.

[0149] In one embodiment, the method comprises the step of subjecting the microorganism to enzymatic treatment without any solvent extraction or chemical demulsification involved, preferably a pure enzymatic treatment of the microorganism that does not involve any solvent extraction or chemical demulsification at all. In one embodiment, the term "pure enzymatic treatment of the microorganism that does not involve any solvent extraction or chemical demulsification at all" refers to the enzymatic treatment of the microorganism in the following situations: a) one or more solvents are not used for extraction; b) one or more (applicable) chemical reagents are not used for demulsification; or c) neither a) nor b) is the case simultaneously. Preferably, the term specifically refers to an enzymatic treatment method that completely eliminates the situation of contacting extraction solvents and also completely eliminates the situation of contacting demulsification chemical reagents. The term also aims to exclude the implementation of any other pretreatment on the cultured microorganism (such as oil-producing microorganisms). It should be noted that in the embodiments of the present invention, "pure enzymatic treatment" excludes any pretreatment on the cultured microorganism, and the pretreatment can be chemical pretreatment (using one or more chemical reagents that the cultured microorganism will contact) or physical pretreatment (such as changing physical conditions, including temperature, pressure, ultrasonic and / or light treatment, electromagnetic radiation irradiation, etc.). In one embodiment, the pure enzymatic treatment of the microorganism means treating the microorganism with a hydrolase alone or treating it jointly / sequentially with a protease. In one embodiment, the hydrolase is derived from a fungus, preferably a filamentous fungus, more preferably a fungus selected from the genus Trichoderma, Aspergillus, Penicillium, Aureobasidium, and Fusarium. In one embodiment, the hydrolase is obtained from a fungus cultured in the presence of an induction system; wherein, preferably, the induction system is a component of the microorganism cultured in step ii).

[0150] In one embodiment, said obtaining the target product comprises harvesting the target product by density-based separation, drying, floating, solvent-based extraction, chromatography, distillation, maceration, supercritical fluid extraction, enfleurage, press extraction, demulsification, decantation, and / or aspiration, preferably by density-based separation. In one embodiment, said obtaining the target product comprises harvesting from the culture medium, from the produced microbial oil (e.g., from the unsaponifiables of the produced microbial oil), and / or from cell residues (preferably from the unsaponifiables of the produced microbial oil), optionally after an oil extraction step. In one embodiment, the terms "obtaining" and "harvesting" are used interchangeably. In one embodiment, the target product is obtained and / or harvested by any one of centrifugation, filtration, distillation, organophilic pervaporation, solid-phase microextraction, and combinations thereof. In one embodiment, the method for producing the target product is carried out in a fed-batch mode, a semi-continuous mode, or a continuous mode, preferably in a continuous mode.

[0151] In one embodiment, the method for producing the target product further comprises purifying the target product obtained in step iv), preferably using a separation method comprising chromatography, affinity-based separation, organic solvent extraction, ionic-liquid extraction, supercritical fluid extraction, liquid-liquid extraction, solid phase extraction, flash extraction, steam extraction, vacuum distillation, distillation under inactive or noble gases, and / or deodorization.

[0152] Hydrolysis products, especially lignocellulosic hydrolysis products, are extremely favorable for the fermentation of microorganisms (such as oil-producing microorganisms) because these hydrolysis products usually contain organic acids (such as acetic acid) and sugars. The sugars in the hydrolysis products enable the oil-producing microorganisms to achieve an efficient first growth stage, while the organic acids (such as acetic acid) enable an efficient oil production stage. The inventors have found that the efficiency of the cultivation method is particularly high when the hydrolysis products contain xylose, acetic acid and optionally glucose.

[0153] As used herein, the terms "of the present invention", "according to the present invention", "in accordance with the present invention" and similar expressions are intended to refer to all aspects and embodiments of the invention described and / or claimed herein. As used herein, the term "comprising" shall be construed to cover both the meanings of "including" and "consisting of", both of which are specifically indicated, and thus each embodiment separately disclosed according to the present invention is also within this scope. When used herein, "and / or" shall be understood to specifically disclose each of the two specified features or components with or without the other. In one embodiment, the terms "at least one" and "one or more" may be used interchangeably. For example, "A and / or B" shall be understood to specifically disclose each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that can be understood by those skilled in the art and still ensure the relevant technical effects of the features. This term generally indicates a deviation of ±20%, ±15%, ±10% from the indicated value, for example, a deviation of ±5%. As understood by those of ordinary skill in the art, the specific deviation range of a numerical value for a particular technical effect will depend on the nature of that technical effect. For example, natural or biotechnological effects may generally have a greater such deviation range than artificial or engineering effects. Where an indefinite or definite article is used when referring to a singular noun, for example, "a", "an" or "the", unless otherwise expressly stated, the expression includes the plural form of that noun.

[0154] Studies have shown that high lipid yields can be obtained by simultaneously feeding sugar and acetic acid during cultivation, which can not only avoid the problem of fermentation limitation but also achieve high lipid titers. The inventors have shown that the target product can be efficiently produced by using the method of the present invention (for example, using oleaginous microorganisms such as C. oleaginosus to produce single-cell oils). Advantageously, C. oleaginosus can utilize phenolic compounds (such as coumaric acid and resorcinol) as carbon sources, which are the main components of depolymerized lignin and are usually harmful to microbial growth. C. oleaginosus can efficiently utilize the monomers of the most abundant biopolymers on earth (cellulose, chitin, lignin, and hemicellulose) because this yeast can metabolize a variety of different sugars, including xylose, glucose, and its derivative N-acetylglucosamine. In addition to its metabolic flexibility, inhibitory compounds (such as hydroxymethylfurfural (HMF)) have little effect on the lipid productivity of C. oleaginosus. The present inventors have found that, advantageously, microorganisms such as C. oleaginosus can efficiently utilize biomass waste streams (especially hydrolysates, such as those from the pulp and paper industry) if the salt content of such hydrolysates has been reduced before using them for culturing microorganisms.

[0155] Advantageously, the method for reducing the salt content of the saline hydrolysate can convert a hydrolysate containing an amount of salt that inhibits microbial growth (such as a lignocellulosic hydrolysate) into a hydrolysate with a reduced salt content, which can be used as a culture substrate for microbial growth. For example, such hydrolysates with a reduced salt content (such as waste liquids with a reduced salt content) can be used as a carbon source for the production of target products (such as microbial oils) by microorganisms. Advantageously, the method of using a hydrolysate as a growth substrate to produce a target product according to the present invention has significant cost-effectiveness and environmental friendliness advantages compared to the method of using glucose as a microbial growth substrate to produce a target product. Therefore, the economic performance is improved.

[0156] In one embodiment, the method for producing a target product (preferably microbial oil) comprises the following steps:

[0157] i) Providing a hydrolysate with a reduced salt content by performing the following steps:

[0158] a) Providing a saline hydrolysate, preferably a bio-based saline hydrolysate, more preferably a lignocellulosic saline hydrolysate;

[0159] b) Optionally, neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate; wherein, optionally

[0160] the pH of the neutralized hydrolysate is in the range of about pH 4 to about pH 8;

[0161] c) Add CaCO3,

[0162] Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2 to the hydrolysis product of step a) or the neutralized hydrolysis product of step b), preferably CaCO3 and / or Ca(OH)2, to obtain a hydrolysis product containing precipitated salts; wherein optionally, the pH of the hydrolysis product containing precipitated salts is in the range of about pH 4 to about pH

[0163] 8.5;

[0164] d) Add a chelating agent to the hydrolysis product of step c);

[0165] e) Optionally, adjust the pH of the hydrolysis product of step c) and / or step d) to a pH in the range of about pH 3 to about pH 8.5, preferably about pH 3.5 to about pH 7.5, more preferably about pH 5 to about pH 7, and further preferably about pH

[0166] 6 to about pH 7; and

[0167] f) Obtain a hydrolysis product with a reduced salt content.

[0168] ii) Cultivate a microorganism (preferably an oil-producing microorganism) using the hydrolysis product provided in step i) (especially the hydrolysis product obtained in step f) or consisting thereof as a growth medium, so that the microorganism produces a target product; preferably, the oil-producing microorganism produces microbial oil;

[0169] iii) Optionally, perform an enzymatic treatment on the microorganism (preferably the oil-producing microorganism); wherein, optionally, the enzymatic treatment of the microorganism is carried out without any solvent extraction or chemical demulsification;

[0170] iv) Obtain the target product, preferably microbial oil.

[0171] Advantageously, compared with the growth characteristics using only xylose, glucose and / or acetic acid as carbon sources, the hydrolysis product provided in step i) exhibits similar or even more efficient growth characteristics and is more cost-effective.

[0172] All methods mentioned in the following figure captions are carried out according to the details described in the examples. The following will be described with reference to the examples, which are only for illustration and not for limiting the present invention. Brief Description of the Drawings

[0173] The present invention will now be further described with reference to the following drawings.

[0174] Figure 1Shows a comparison of the growth behavior under different fermentation conditions (LCH - lignocellulosic hydrolysate, N - limited - nitrogen - limited fermentation condition, cb - feed - consumption - based feeding, co - feed - combination of consumption - based feeding and continuous feeding of LCH with LCH). Biomass accumulation after fermentation inoculation with consumption - based feeding of different starting sugars and acetic acid was curve - fitted with the Gompertz function in all cases except the nitrogen - limited condition. Error bars show two - fold standard deviation.

[0175] Figure 2 Shows the results of lipid analysis for five of the most important fermentation conditions. The abbreviations for the starting carbon sources are as follows: Glu - glucose, LCH - lignocellulosic hydrolysate, LCH co - feed - lignocellulosic hydrolysate as the starting carbon source with continuous feeding, Xyl - xylose. (a) Lipid titer after 71 h of fermentation at 1 L scale. (b) Carbon conversion from substrate carbon to lipid carbon. (c) Fatty acid profile of the major fatty acids quantified by GC - FID. Error bars show two - fold standard deviation.

[0176] Figure 3 Shows Comparison of feeding strategies in the system at 0.25 L scale (LCH - lignocellulosic hydrolysate). Biomass accumulation and substrate consumption of the control group (50% acetic acid, consumption - based feeding) and two optimal operation modes (acetic acid:LCH = 50:50 co - feeding and 1 mL / h continuous feeding of LCH). Total lipid titer reached after 65 h and the proportion of LCH in total carbon uptake. Error bars show two - fold standard deviation.

[0177] Figure 4 Shows confocal microscopy images of lignocellulosic hydrolysate fermentation: (a) after 24 h of acetic - acid - based feeding, (b) after 71 h of acetic - acid - based feeding; and cell images after (c) 24 h and (d) 71 h of the combination of continuous LCH feeding and acetic - acid feeding.

[0178] Figure 5Shows the annual production cost of yeast oil ($ / Mt) analyzed by TEA for three fermentation strategies. LCH cb-feed - lignocellulosic hydrolysate (LCH) and acetic acid based on fed-batch consumption, Glucose cb-feed - glucose and acetic acid based on fed-batch consumption, LCH co-feed - continuous feeding of LCH combined with acetic acid based on fed-batch consumption. The feeding rate of each raw material is set to produce yeast oil at a rate of 0.81 Mt / h (LCH cb-feed: 1 Mt / h, Glucose cb-feed: 0.151 Mt / h, LCH co-feed: 2.1 Mt / h).

[0179] Figure 6 Shows an exemplary embodiment of the method of the present invention.

[0180] Figure 7 Shows the average precipitate after each pretreatment step.

[0181] Figure 8 Shows the comparison of the precipitate obtained by titrating the hydrolysate treated with CaCO3 with KH2PO4.

[0182] Figure 9 Shows the images of the samples after autoclaving. The medium was prepared and neutralized according to the method described in Table 2, and then autoclaved and centrifuged. The supernatant (SN) and precipitate under each treatment condition are shown in the figure. It can be seen that significantly more precipitate is produced under the treatment condition with only CaCO3 added. In addition, the color of the supernatant is significantly darker under the condition without KH2PO4 added (except for condition 3, because this condition added full medium). However, when autoclaving the medium with full medium, HMF and furfural are formed, which can inhibit the growth of microorganisms.

[0183] Figure 10 Shows the growth curve based on the optical density at 600 nm, which was obtained by measuring in a cuvette in a spectrophotometer. The performance of conditions 2, 4, and 5 is similar to the control condition 1. Only condition 3 (the hydrolysate treated with activated carbon) shows a significantly lower growth level.

[0184] Figure 11Shows micrographs of cell cultures grown in culture media containing the five different carbon sources described in Table 3 at 100-fold magnification. After 24 hours, no obvious large lipid droplets were formed under all conditions. After 120 hours, the lipid droplets were visualized by light halo reflection. Under Conditions 1 and 2, an average of two lipid droplets were formed per cell. Under Condition 3, not all cells contained lipid droplets; and the cells that contained lipid droplets had 2 to 3. Under Conditions 4 and 5, only one lipid droplet was formed per cell, which indicated the highest lipid content that could be achieved. Under Condition 2, insoluble particles were clearly visible.

[0185] Example

[0186] Example 1: Materials and Methods

[0187] Sugar Analysis

[0188] Carbohydrates and short-chain organic acids were analyzed by high-performance liquid chromatography (HPLC). All samples were filtered through a 10 kDa filter. An Agilent 1260 Infinity II LC system equipped with a diode array detector (DA) and a refractive index detector (RI) was used. To achieve separation, a Rezex ROA-organic H+ 8% chromatographic column produced by Phenomenex was employed, and 5 mM H2SO4 was used as the mobile phase. An isocratic elution flow rate of 0.5 ml / min was run for 60 minutes, and the oven temperature was set at 70 °C. Detection was carried out at 40 °C in the refractive index detector (RID).

[0189] Elemental Analysis

[0190] Elemental analysis was completed using a Euro EA CHNS elemental analyzer (HEKAtech Ltd.). Dynamic spontaneous combustion was carried out at approximately 1800 °C in a tin boat, followed by gas chromatography separation and detection using a thermal conductivity detector (TCD).

[0191] Ash

[0192] 2 g to 4 g of neutralized and lyophilized lignocellulosic hydrolysis products were incinerated at 1000 °C for 3 hours to ash. After the samples were cooled overnight in a desiccator, their mass was determined by gravimetry.

[0193] Dry Weight

[0194] The dry weights of both the substrate solution and the biomass samples were determined by gravimetry. To determine the dry cell weight, 4 mL of the fermentation broth was transferred to a pre-weighed centrifuge tube, centrifuged (4500 rcf, 20 minutes), and washed twice with an equal volume of water or 50% ethanol (for lipid-rich cells). Alternatively, 0.5 mL of lipid-rich culture broth was filtered through a pre-weighed 0.2-μm filter paper and washed three times with 2 mL of water. The samples were frozen and lyophilized. Each biological replicate sample was measured with at least technical replicates.

[0195] Quantification of Sulfate

[0196] Quantification of sulfate in the hydrolysate was accomplished chemically, using CaCO3 and BaCl2 treatment. The resulting BaSO4 precipitate was quantified by gravimetry.

[0197] Lipid Content

[0198] For lipid content analysis, the fermented cells were centrifuged and washed twice with 50% EtOH, then dissolved in water. The cells were mechanically disrupted by a high-pressure homogenizer type HPL6 from Maximator. Three 7-mL aliquots of the disrupted cell solution were frozen and lyophilized. Chloroform-methanol lipid extraction was performed using a modified Bligh and Dyer method. The brief procedure was as follows: 100 - 200 mg of biomass was weighed into a glass tube, and 4 mL of Cl3CH:MeOH (2:1) and 1 mL of H2O (containing 0.58% NaOH) were added. After shaking at 120 rpm for 60 minutes, it was centrifuged at 2000 rcf for 10 minutes, and the lower layer liquid was transferred to a new glass tube. An additional 3 mL of Cl3CH:MeOH (2:1) was added to the upper phase in the first tube and briefly mixed. After centrifugation, the lower layer liquid was transferred to the tube containing the first extract, and 2 mL of a water (0.58% NaOH):MeOH (1:1) mixture was added. After mixing and centrifugation, the lower phase was transferred to a pre-weighed new glass tube, and the solvent was evaporated under a nitrogen stream. The lipid content was determined by gravimetry.

[0199] Fatty Acid Profile

[0200] After the fatty acids of the unwashed and lyophilized samples from the fermentation process were methyl esterified (FAME), the fatty acid profiles were determined by gas chromatography. Samples with a known weight of 3 mg to 10 mg were weighed into glass vials, and all subsequent steps were completed using the Multi Purpose Sampler MPS robotic of Gerstel. C19 TAG dissolved in toluene at 10 g / L was used as an internal standard for quantification. First, 490 μl of toluene and 10 μl of the internal standard were added, and the mixture was mixed at 1000 rpm for 1 minute; then 1 mL of a methanol solution of 0.5 M sodium methoxide was added, and the solution was heated to 80 °C and shaken at 750 rpm for 20 minutes. After cooling to 5 °C, 1 ml of a methanol solution of 5% hydrochloric acid was added, and the mixture was heated to 80 °C and reacted with shaking at 800 rpm for 20 minutes, and then cooled to 5 °C again. 400 μl of ddH2O was added, and the mixture was mixed at 1000 rpm for 30 seconds, then 1 ml of n-hexane was added, and the mixture was extracted three times by shaking at 2000 rpm in a quickMix device for 20 seconds each time. After the sample was centrifuged at 1000 rpm for 3 minutes and cooled to 5 °C, 200 μl of the organic phase was transferred to a 1.5 mL chromatographic injection vial. Fatty acids were quantitatively analyzed using gas chromatography-flame ionization detector (GC-FID). Fatty acid identification was completed by GC-MS: using the TRACE TM Ultra gas chromatograph of Thermo Scientific (TRACE TM Ultra Gas Chromatograph), equipped with Thermo DSQ TM II mass spectrometer (Thermo DSQ TM II mass spectrometer) and Triplus TM Autosampler injector (Triplus TM Autosampler injector), and analysis was carried out in the positive ion mode. Using fused silica capillary column ( fused silica capillary column, 30 m × 0.25 mm, film thickness 0.25 μm) for separation. The analytical temperature program was set as follows: the initial column temperature was 50 °C, and it was heated to the final temperature of 250 °C at a rate of 4 °C / min. Hydrogen was used as the carrier gas with a constant flow rate of 35 ml / min. Standardization was carried out using FAMEs Marine Oil Standard (containing 20 components from C14:0 to C24:1).

[0201] LCH pretreatment

[0202] To precipitate sulfates in the hydrolysis product, 20 g of CaCO3 was added to 1 L of LCH in a 5 L beaker with continuous stirring. Subsequently, the hydrolysis product was left to stand until the complete release of CO2. Then, LCH was frozen at -20 °C overnight. After thawing, LCH was centrifuged at 10000 rpm for 10 minutes and the supernatant was collected. 20 g of KH2PO4 was added to the remaining liquid to remove excess calcium ions, and the pH was adjusted to 7 with 3M NaOH. LCH was centrifuged at 10000 rpm for 10 minutes and used for the culture experiment after sterile filtration.

[0203] OD measurement was used for process monitoring

[0204] Cell growth was monitored by measuring the optical density (OD 600 ) of the culture samples at 600 nm. The samples were diluted with the corresponding medium to an OD 600 value between 0.1 and 1.

[0205] Strains and precultures

[0206] 50 mL of YPD medium (containing 10 g / l yeast extract, 20 g / l peptone, and 20 g / l glucose) was placed in an Erlenmeyer flask, to which antibiotics (0.05 g / l kanamycin, 0.1 g / l ampicillin) were added. A single colony of C. oleaginosus (ATCC 20509) was picked from the YPD plate and inoculated into this medium. The Erlenmeyer flask was cultured at 28 °C and 120 rpm with constant shaking for 2 days. These yeast precultures were used as inoculum for different fermentation experiments.

[0207] Fermentation medium

[0208] For experiments on nitrogen limitation or co-fermentation with acetic acid, different media were used respectively. The basal medium consisted of 0.9 g / l Na2HPO4, 2.4 g / l KH2PO4, 2 g / l MgSO4·7H2O, 0.5 CaCl2·2H2O, 0.00000055 g / l ZnSO4·7H2O, 0.000024 g / l MnCl2·6H2O, 0.000025 g / l.

[0209] Techno-economic analysis

[0210] To estimate the total capital investment and operating costs for lipid production from C. oleaginosus using LCH, a techno-economic analysis (TEA) was performed. The method was designed to use a recursive modeling approach, with computer (in silico) simulations based on previous laboratory results, available data from the literature, and mathematical functions integrated in SuperPro Designer (SPD) version 10 (Intelligen, Inc., Scotch Plains, NJ, USA). In the process simulation, a lipid production plant with a capacity of 0.81 metric tons per hour (t / h) was constructed. The raw material requirements and the estimation of the chemicals required for LCH pretreatment were based on the medium composition used in this study. The mathematical equations and simulation parameters for yeast biomass generation, lipid formation, and enzymatic hydrolysis were based on the results obtained in this study or previous experiments. To calculate the energy balance, equipment sizing, and purchase prices, SPD database data were used, supplemented with current open market prices and literature data. The computer-simulated plant included multiple processes such as LCH pretreatment, fermentation, and downstream processing, covering single-cell oil recovery, waste stream, and by-product recycling. The list of modules used in SPD for different fermentation conditions (glucose and co-feeding of LCH and acetic acid, continuous feeding, and a combination of consumption-based feeding) is provided in the appendix.

[0211] Among all the process parameters, lipid productivity had the most significant impact on cost 1. Lipid productivity was calculated based on the lipid concentration after 71 h of fermentation in a 1 L system.

[0212] Lipid productivity [g / l / h] = Lipid titer [g / l] / Time [h]

[0213] The lipid productivities were calculated for three conditions: co-feeding of glucose and acetic acid, co-feeding of LCH and acetic acid, and a combination of continuous feeding and consumption-based co-feeding of acetic acid. The equipment purchase cost (PC) was calculated based on the equipment size using the built-in function in SPD. The installation cost factor ranged from 1.1 to 1.3 relative to the PC. The cost factors for storage tanks, pipelines, and site development were set at 4%, 9%, and 5%, respectively.

[0214] The capital interest was calculated at 6% of the total investment. The utility costs, labor costs, and waste treatment costs were estimated based on current German prices. The costs of consumables and raw materials were set according to current market prices. Maintenance and insurance, as costs related to the facility, were calculated at 3% and 0.7%, respectively.

[0215] Example 2: Results and Discussion

[0216] Analysis of Lignocellulosic Hydrolysates

[0217] A comprehensive analysis of the lignocellulosic hydrolysis products (LCH) in the pulping process waste liquor was carried out to gain an in-depth understanding of its composition and thus formulate a pretreatment strategy. The dark brown solution contains a small amount of insoluble particles. It has a pH of 1.7 and a dry mass of 247.7 ± 13.9 g / l. The sulfate content was detected to be as high as 19.4 ± 2.0 g / l. HPLC was used to quantitatively analyze sugars, organic acids and furan compounds. The results showed that the main sugar in the hydrolysis products is xylose, with 77.05 ± 2.46 g / l, and small amounts of other sugars such as glucose (11.51 ± 0.39 g / l), mannose (8.21 ± 0.84 g / l), galactose (6.47 ± 0.21 g / l) and others (12.37 ± 1.10 g / l), and the total sugar content is 115.60 ± 4.99 g / l. The organic acids have a total concentration of 16.83 ± 1.43 g / l, with acetic acid as the main component, having a content of 12.34 ± 1.20 g / l. Considerable amounts of hydroxymethylfurfural (HMF) (4.53 ± 0.59 g / l) and furfural (0.68 ± 0.04 g / l) were detected. The total ash content was measured to be 0.70 ± 0.01 g / l, with a low phosphorus concentration (0.035 g / l) and no nitrogen detected. The remaining substances are derived from lignophenols, lignans and other plant metabolites, with a total content of 89.95 ± 22.97 g / l.

[0218] The hydrolysis products are a waste stream generated during the industrial production of cellulose fibers from hardwood by an acidic pulping process. The chemical hydrolysis of lignocellulose produced the detected monosaccharides, organic acids, lignophenols and lignans. The phenolic compounds and furan substances contained may have an adverse effect on the fermentation process because these compounds usually inhibit the growth of microorganisms. The high sulfate content and acidic pH value result from the chemical hydrolysis itself. Therefore, LCH must be neutralized before being used in yeast fermentation. Despite the above problems, the high content of xylose and other sugars makes it an ideal carbon source. However, due to the extremely low content of nitrogen, phosphorus and other elements such as sulfur, magnesium and calcium, additional nutrients need to be supplemented before using the hydrolysis products in the fermentation medium.

[0219] Pretreatment of Lignocellulosic Hydrolysis Products

[0220] To convert the spent liquor LCH into a carbon source suitable for yeast fermentation, it is necessary to address the problems of its too low pH value and lack of essential nutrients through appropriate pretreatment strategies. In the first step of pretreatment, different methods are used to neutralize and sterilize LCH. Then it is mixed with essential salts, buffer compounds, nitrogen sources, nutrients, and trace elements. The prepared medium is tested on a small scale in 24-well deep-well plates for culturing C. oleaginosus. Neutralizing the pH from 1.7 to 7.0 using NaOH results in too high a salt concentration, which inhibits growth. To reduce the sulfate content, CaCO3 is added to react with it to form insoluble CaSO4. Experiments show that adding 20 g / L CaCO3 is slightly excessive, but it can only raise the pH to 5.6. Therefore, NaOH is finally titrated to neutrality, and the resulting solution has a lower salt content and is more conducive to growth. For sterilization, different heat methods are tested, but they all result in the formation of insoluble particles. Therefore, the filtration sterilization method is selected. However, insoluble particles will form again after adding culture medium components such as buffer salts, vitamins, nitrogen sources, and trace elements, as well as during the culturing process. Among different methods, LCH is filtered through activated carbon to reduce the content of toxic phenols and furans. Unfortunately, this strategy also reduces the content of effective carbon sources, resulting in significantly limited growth.

[0221] The continuous formation of insoluble particles was observed throughout the fermentation process, mainly calcium phosphate precipitation caused by a high calcium ion concentration ([Ca 2+ ). Therefore, before fermentation, KH2PO4 is titrated to LCH to remove excess calcium ions by forming insoluble Ca3(PO4)2. Since this process causes the solution to acidify, NaOH is used for final neutralization after KH2PO4 treatment. The LCH pretreated in this way enables the growth to reach a level comparable to that of the xylose-glucose-acetic acid (XGA) control group. The specific pretreatment experimental data can be seen in the supplementary materials.

[0222] In this way, the lignocellulose hydrolysate is neutralized without a high salt concentration and without forming insoluble materials when adding buffer salts or trace elements. The sugar content is maintained so that it can be used as a carbon source for C. oleaginosus. All LCH used in the fermentation in this study adopts this optimized pretreatment scheme.

[0223] Nitrogen-limited fermentation

[0224] Fermentation under nutrient limitation (such as phosphate or nitrogen stress) is a common strategy to trigger lipid accumulation in oleaginous yeasts. Therefore, nitrogen-limited fermentation was carried out using LCH as the carbon source. To explore the effects of lignocellulose-derived components on yeast metabolism, a control medium was set up. The control contained only the main sugars from LCH, with their respective components and proportions being: xylose (8.28%), glucose (1.03%), and acetic acid (0.69%), thus abbreviated as XGA. In the system, fermentation was carried out in fed-batch mode with a maximum volume of 1 L. The sugar content (from LCH or XGA) in the initial 500 ml of medium was set at 3%. The nitrogen content was adjusted to achieve a C / N ratio of 120. The pretreated pure LCH or XGA medium was continuously fed at a rate of 10 ml / h starting from 12 h and decreased to 5 ml / h during 36 - 60 h. Fermentation produced biomass formation of 7.02 ± 0.88 g / l and 16.65 ± 0.24 g / l for LCH and XGA respectively, indicating better growth performance on the XGA medium. Microscopic observation showed the formation of multiple lipid droplets inside the cells.

[0225] It is speculated that compared with growth on XGA, high concentrations of inhibitory furan and phenolic compounds can explain the growth inhibition on lignocellulose hydrolysates. Generally, although nitrogen limitation can induce lipid synthesis, it will simultaneously limit biomass formation (because nitrogen is an essential element for metabolic processes such as protein synthesis).

[0226] Acetic acid-based fermentation with sugars as the carbon source

[0227] As previously discussed, by combining an initial sugar-containing medium with a feeding strategy based on consumed acetic acid, the growth rate and lipid accumulation of C. oleaginosus can be simultaneously increased. Glucose, xylose, and the xylose-glucose-acetic acid mixed control group (XGA) were used as the starting sugar sources, and a feeding strategy based on consumed acetic acid was tested. The concentration of acetic acid was kept constant by feeding based on consumption; the sugars were completely consumed within 40 h after inoculation. After the sugars were completely consumed, the biomass further increased and reached 35.11 ± 1.11 g / l, 39.95 ± 4.59 g / l, and 39.00 ± 0.76 g / l for glucose, xylose, and XGA respectively at 71 h, and the lipid titers were 18.5 ± 3.6 g / l, 23.6 ± 0.5 g / l, and 21.6 ± 2.8 g / l for glucose, xylose, and XGA respectively ( Figure 2 a). This also applies to the carbon conversion from substrate carbon to lipid carbon, i.e., 20.4 ± 2.7%, 23.0 ± 1.4%, and 23.5 ± 0.7% for glucose, xylose, and XGA respectively ( Figure 2 b). The fatty acid composition of the oil is as Figure 2As shown in c, the most abundant is C18:1 (about 54%), followed by C16:0 (about 24%), C18:0 (about 15%) and C18:2 (about 6%), as well as trace amounts (each less than 0.3%) of C18:3, C16:1 and C22:0.

[0228] Fermentation experiments using glucose and xylose as sole carbon sources showed growth characteristics similar to those of the fermentation process using XGA. This indicates that both glucose and xylose can be efficiently and simultaneously absorbed and utilized. This is a significant advantage for C. oleaginosus as a fermentation strain, as it can fully utilize the potential of biomass as a carbon source. In addition, acetic acid can be efficiently imported into the lipid synthesis metabolic pathway through acetyl-CoA synthetase. This forms a more efficient metabolic pathway compared to the metabolic pathway starting from sugar molecules, and thus becomes an effective method for improving lipid biosynthesis in C. oleaginosus.

[0229] Fermentation of lignocellulosic hydrolysates based on acetic acid

[0230] It was shown that single cell oil can be successfully produced from refined sugars using acetic acid fermentation. However, to achieve a more commercially attractive process, we used the aforementioned waste stream LCH as a carbon source instead of expensive sugars. The lignocellulosic hydrolysate is derived from acidic hardwood pulping waste liquor. Advantageously, this method also produces acetic acid by cleaving acetyl groups from hardwood xylose. Thus, acetic acid, the second carbon source required for the process, can be produced on-site.

[0231] Natural products such as lignocellulosic hydrolysates contain furan and phenolic compounds that may inhibit microbial growth. To determine the optimal balance between carbon source supply and inhibitor toxicity levels, fermentations with three different initial concentrations of LCH were conducted. The amount of hydrolysate was used for concentrations of 3%, 5% and 7% bioavailable carbon in the initial medium. Fermentation experiments using the highest concentration of LCH (equivalent to 7% sugar content) showed a longer lag phase and a slower overall growth rate, with a growth rate of 1.21 g / l / h, while the growth rate of the 3% concentration group was 1.33 g / l / h. The dry biomass formation of the 7% and 5% concentration groups was 39.64 ± 6.30 g / l and 51.92 ± 0.18 g / l, respectively, while the 3% concentration group reached 58.90 ± 1.05 g / l. Therefore, when the concentration was increased to 5%, a slight extension of the lag phase and a slight decrease in biomass were observed after three days of fermentation. The lipid titers of the two lower concentration groups were similar (25.59 ± 1.79 g / l and 25.75 ± 2.02 g / l), but the yield of the 7% LCH group was lower (19.83 ± 1.38 g / l).

[0232] An initial carbon source concentration of 3% achieved the shortest lag phase, the best growth rate, and the highest total biomass. Thus, higher concentrations led to increased concentrations of inhibitory furan and phenolic compounds, which explained the extended lag phase and decreased growth rate. Therefore, an initial concentration of 3% was used in all subsequent experiments.

[0233] Optimized conditions fermentation of lignocellulosic hydrolysates based on acetic acid

[0234] The optimized conditions for the fermentation of C. oleaginosus on LCH were evaluated through three sets of biological replicate experiments. Hydrolysates with an initial carbon content equivalent to 3% sugar and acetic acid content were used. High biomass formation of 55.73 ± 5.20 g / l was achieved after 71 h, which was seven-fold higher than the nitrogen-limited fermentation of LCH. Additionally, under equivalent reaction conditions, biomass formation using LCH as the carbon source was 50% higher than that of glucose or xylose ( Figure 1 ). However, the sugar consumption pattern was similar to the fermentation process using a single sugar source. The lipid droplet titer (42.1 ± 1.7 g / l) after 71 h was twice that of glucose fermentation (18.5 ± 3.6 g / l) ( Figure 2 a). The carbon conversion rate was 33.6% of the substrate carbon being converted to carbon lipids ( Figure 2 b), and the lipid content in the dry biomass reached 76.05 ± 9.04%. The fatty acid composition did not show significant changes compared to the lipids from single sugar sources or XGA fermentation ( Figure 2 c).

[0235] Using an optimized fermentation strategy based on acetic acid consumption feeding, C. oleaginosus showed the best performance in terms of biomass accumulation, lipid droplet titer, and lipid production on LCH, with minor changes in lipid distribution. The improved performance of LCH as a substrate may be due to the fact that the lignophenols and lignin compounds it contains can be partially metabolized by C. oleaginosus. In summary, this combination of waste stream and oil-producing yeast provides a promising new process for sustainable single-cell oil production. This process combines the application of waste streams rich in xylose, lignophenols, and lignin with fermentation strategies, resulting in high lipid yields.

[0236] Screening of LCH feeding strategies

[0237] Feeding based on consumed acetic acid can achieve a high lipid yield of over 85%. However, compared to acetic acid, LCH, as a waste material with limited value creation pathways, is a cheaper raw material. To increase the uptake of LCH by oil-producing yeast relative to acetic acid, different fermentation modes were compared. In this context, the use of The twelve - parallel bioreactor system simultaneously compared five fermentation conditions (each with a double replicate). All experimental conditions used consumption - based feeding of 50% (v / v) acetic acid.

[0238] As the first fermentation mode, the mixed feeding of LCH and acetic acid was tested. LCH was added to 50% (v / v) acetic acid to final concentrations of 10% (v / v) and 50% (v / v). After 65 hours, the biomass in the acetic acid:LCH mixed - feeding groups of 50:10 and 50:50 (51.5 ± 3.7 g / l and 59.6 ± 1.2 g / l) exceeded that of the pure - acetic - acid control group (50.8 ± 0.1 g / l), with the 50:50 group being the highest ( Figure 3 ). After 65 hours, the lipid titers in the acetic acid:LCH mixed - feeding groups of 50:10 and 50:50 were 28.4 ± 0.4 g / l and 30.4 ± 1.4 g / l, respectively, while that in the control group was measured to be 25.2 ± 3.1 g / l ( Figure 3 ).

[0239] As an additional feeding strategy, in addition to the consumption - based feeding of 50% (v / v) acetic acid, starting from 12 h after inoculation, pure pre - treated LCH was continuously fed into the reactor at 0.5 ml / h or 1 ml / h throughout the fermentation process. After 65 hours, the biomass was 50.4 ± 2.2 g / l (0.5 ml / h group) and 50.4 ± 4.6 g / l (1 ml / h group). The maximum lipid titers were 30.5 ± 2.4 g / l for 0.5 ml / h and 26.3 ± 2.7 g / l for 1 ml / h, thus both being higher than the control (25.2 ± 3.1 g / l).

[0240] To screen the scheme with the highest LCH conversion rate, the proportion of LCH in the total carbon - source consumption was calculated. The LCH consumption proportion in the 1 - mL / h continuous - feeding group was the highest (21.3 ± 2.9%), followed by the 50:50 acetic - acid:LCH group (12.5%) and the 0.5 - mL / h continuous - feeding group (10.4 ± 1.2%). The differences between replicates in the continuous - feeding groups were due to different acetic - acid consumption amounts. Since the LCH proportion in the 50:50 acetic - acid:LCH group remained constant during fermentation, the standard deviation could not be calculated.

[0241] In addition, the carbon conversion rates of the feed carbon source and lipids were calculated for all conditions. The carbon conversion rate was the highest in the continuous feeding group, 19.8 ± 2.0% for the 0.5 mL / h group and 18.8 ± 0.2% for the 1 mL / h group. The carbon conversion rate of the control group was 16.4 ± 0.2%, while those of the acetic acid:LCH mixed co-feeding groups were 16.7 ± 0.1% (50:10) and 16.7 ± 0.5% (50:50), respectively. The comparison of the effects between the control group and the two optimal feeding strategies is shown in Figure 3 .

[0242] For the comprehensive evaluation of the feeding strategies, two indicators, namely the proportion of LCH consumption and the lipid titer, were focused on. Accordingly, the two optimal feeding strategies were the consumption-based feeding of acetic acid:LCH at 50:50 and the co-feeding with continuous feeding of 1 mL / h LCH. These strategies significantly increased the proportion of LCH consumption by increasing the supply of LCH during the fermentation process. At the same time, the initial concentration of inhibitors was not increased, avoiding the phenomenon of extended lag phase caused by high concentration of LCH in the system (3.5.).

[0243] Continuous co-fermentation with lignocellulose hydrolysate feed

[0244] In the system, continuous feeding of 1 mL / h LCH was adopted, achieving the highest proportion of LCH carbon source (21.3 ± 2.9%) and the second highest carbon conversion rate (18.8 ± 0.2%). Therefore, this condition was selected for system verification. The 1 L-scale experiment could also more directly compare the fermentation output with other conditions tested in the system. Using the combination of 90% acetic acid consumption-based feeding and continuous feeding of 3.3 mL / h LCH, after 71 hours, the biomass accumulation reached 29.0 ± 5.87 g / l and the lipid titer was 22.7 ± 1.95 g / l. HPLC analysis showed that the carbon source was consumed evenly throughout the fermentation process, and only xylose accumulated slightly from 15.5 ± 1.11 g / l at 65 hours to 17.44 ± 1.85 g / l at 71 hours. The accumulation of xylose was presumably due to a slightly higher feeding rate, resulting in an excessive supply of sugar and an increase in the concentration of inhibitory compounds. The proportion of LCH in the total consumed carbon source increased from 10.9 ± 31.4% to 37.6 ± 2.3%. Therefore, the relative consumption of LCH by C. oleaginosus was increased, and LCH is a more cost-effective raw material compared to acetic acid. At the same time, the conversion rate of substrate carbon to lipid carbon remained stable (32.0 ± 1.5%), comparable to 33.5 ± 2.1% in the fermentation with pure acetic acid consumption-based feeding. The two fermentation strategies showed similar performance in terms of cell phenotype, growth behavior, and lipid droplet formation, as shown in Figure 4As shown in the Nile red stained cells. After 24 h of fermentation, the cells had started to form multiple lipid droplets but were oval in shape. By 71 h, the cells mainly contained 1-2 lipid droplets that occupied most of the intracellular space and presented a round morphology. Overall, when using the operation mode of additional continuous feeding, the substrate-lipid conversion rate of C. oleaginosus was comparable to that of acetic acid-based feeding based on consumption. To further optimize the fermentation mode, the feeding ratio of acetic acid to sugar should be adjusted according to the xylose concentration after 71 h to avoid sugar accumulation.

[0245] Techno-economic analysis

[0246] In addition to the cumulative results of this study, we conducted a related techno-economic analysis (TEA) to evaluate the economic feasibility of a commercial production plant using LCH as an industrial raw material. Based on a 1 L scale Experimental data were used to compare the implementation of two LCH fermentation modes to optimize cost and feedstock utilization: acetic acid-based feeding on consumed LCH (LCHcb-feed), and a combination of LCH consumption and continuous feeding (LCH co-feed). The two fermentation modes were compared with an operating setup where glucose was used as the starting carbon source with acetic acid-based feeding (Glucose cb-feed). According to the simulation, the capital expenditure (CAPEX) of LCH cb-feed was the lowest ($24.4M). In contrast, the CAPEX of LCH cb-feed was $34.5M, and the CAPEX of LCH co-feed was $33.9M. The difference in capital costs was due to the different numbers and volume configurations of fermenters in each model. Due to differences in residence time under fermentation conditions, different combinations of fermenters were used for fermentation strategies to simulate individual lipid productivity. Except for the vessel quantity and volume, the equipment costs were the same. Calculated at a 10% depreciation rate over 10 years, the annual depreciation amounts were: LCH cb-feed $2.4M, Glucose cb-feed $3.4M, LCH co-feed $3.4M. The number and volume of the tanks also led to differences in operating costs, especially for electricity (LCH cb-feed: $2.3M, Glucose cb-feed: $4.8M, LCH co-feed: $4.0M), cooling water (LCH cb-feed: $0.5M, Glucose cb-feed: $1.0M, LCH co-feed: $0.9M), labor costs (LCH cb-feed: $1.5M, Glucose cb-feed: $2.2M, LCH co-feed: $1.8M), maintenance (LCH cb-feed: $0.6M, Glucose cb-feed: $0.8M, LCH co-feed: $0.8M), and raw materials (LCH cb-feed: $12.4M, Glucose cb-feed: $13.2M, LCH co-feed: $10.9M). The final costs of yeast oil calculated from the computer simulation model were $3,500 / t, $4,500 / t, and $4,000 / t respectively ( Figure 5a). All price ranges are within the current price range of organic palm oil ($2500 - 3000 / t). Looking ahead, by further optimizing the fermentation process and increasing the lipid conversion rate, the production cost of the LCH process is expected to continue to decrease. However, as applied in this study, subsequent TEA focuses more on the relative changes in input and output conditions. Therefore, the calculated price per ton should only be regarded as a reference range. The prices of other vegetable oils for biofuels (such as canola oil) are generally lower than $1000 / t. However, using industrial waste streams as raw materials can avoid competition with edible oils, thus enhancing the sustainability advantage of the LCH-based oil production process.

[0247] In addition, the sensitivity analysis focuses on the impact of acetic acid price, which accounts for the majority of raw material costs (LCH cb-feed: 94%; Glucose cb-feed: 90%; LCH co-feed: 86%). The price of acetic acid in Western Europe fluctuated between $600 - $1500 / t from 2012 to 2021, resulting in a significant dependence of yeast oil cost on the real-time acetic acid price. For the sensitivity analysis, it is assumed that the acetic acid cost is $600 / t and $1500 / t, and compared with $1000 / t used in the TEA. The model using LCH cb-feed responds most significantly to price changes, as Figure 5 shown in b. A price reduction to $600 / t leads to a 19.3% decrease in production cost ($3100 / t), and a high acetic acid price of $1500 / t leads to a 24.1% increase ($4800 / t). For LCH co-feed, for the corresponding acetic acid prices, the total production cost decreases by 13.5% ($3800 / t) and increases by 16.9% ($5100 / t). Due to the higher proportion of LCH carbon sources available for yeast, the LCH co-feed model responds less sensitively to changes in acetic acid price. Although the LCH cb-feed model has the largest percentage response to acetic acid price changes, it still maintains the lowest production cost in both scenarios of price increase or decrease. The fluctuation of acetic acid price is mainly because China and the US respectively account for 55% and 17% of the global supply. Its supply chain problems can lead to a sharp price reduction by significantly affecting market supply. Localizing or even on-site production of acetic acid would be the best solution to avoid such supply chain dependence.

[0248] In summary, we verified the laboratory-scale experiment of biotechnologically valorizing industrial waste streams using C. oleaginosus through computer-simulated fermentation. Compared with glucose, it was demonstrated that LCH shows better cost-effectiveness in the production of single-cell oils at the biorefinery scale. Such methods of efficient resource utilization are expected to promote a more independent, circular, and sustainable economic development in the future.

[0249] Example 3: Comparison of Pretreatment Methods

[0250] Different pretreatment conditions were tested, and the hydrolysates were used to formulate fermentation media. The basal medium consisted of a carbon source (equivalent to 10 g / L glucose), as well as 0.9 g / L Na2HPO4, 2.4 g / L KH2PO4, 4.5 g / L CH3COO·Na, 2 g / L MgSO4·7H2O, 0.5 CaCl2·2H2O, 0.00055 mg / L ZnSO4·7H2O, 0.024 mg / L MnCl2·6H2O, 0.025 mg / L CuSO4·5H2O, 0.027 mg / L C6H8O7·Fe·H3N, 0.25 g / L urea, and 1 g / L yeast extract. In a 24-well deep-well plate shaking culture, the oleaginous yeast Cutaneotrichosporon oleaginosus was cultured using this medium. After 72 h of culture, the OD600 value was measured using an EnSpire2 microplate reader as an indicator of cell density. The cell morphology and the formation of insoluble particles were observed using a microscope at a magnification of 100x.

[0251] Without adding a large amount of phosphate-containing compounds, the culture showed the formation of insoluble particles during cultivation. These particles did not exist before cultivation because, in many cases, the samples were sterile filtered. The growth conditions can be improved by some pretreatment strategies, including adding phosphate-containing compounds.

[0252] Table 1: Lists various tested pretreatment conditions, including the maximum cell density and microscopic observation results after 72 h of culture

[0253]

[0254]

[0255]

[0256] Example 4: Removal of salts and insolubles in a novel pretreatment

[0257] First, acidic (pH 1 - 2) lignocellulose hydrolysates were treated with CaCO3 (20 g / L), and the insoluble particles were separated by centrifugation and weighed. The same repeated operation was carried out after adding KH2PO4 (20 g / L) and neutralizing with NaOH (18 g / L). The mass of the precipitate removed in each step is as Figure 7 shown. Since the color of the solution became lighter and the precipitate was dark after pretreatment, it can be speculated that some lignin compounds were removed by this treatment. The results of this example are shown in Figure 7 .

[0258] Example 5: Titration experiment of lignocellulose hydrolysates treated with CaCO3

[0259] The hydrolysate of lignocellulose treated with CaCO3 was titrated with KH2PO4 and centrifuged to precipitate. KH2PO4 was added stepwise at 50 mg per time, and after each addition, the solution was transferred to a new centrifuge tube for centrifugation. In each step, a dark precipitate was formed, as Figure 8 shown. The change in the precipitate color indicated that this treatment effectively separated the components in the hydrolysate.

[0260] Example 6: Comparison of the Appearance of Pretreatment

[0261] In one case, only CaCO3 was used. In another case, the hydrolysate and the culture medium were pretreated with CaCO3 and KH2PO4 and then neutralized with NaOH. Since the salts in the culture medium contained phosphates, the addition in each treatment varied under three different conditions. One group did not add the culture medium; another group added trace elements, vitamins, and yeast extract (but no salts); the last group added the complete culture medium. After the culture medium was prepared, all samples were autoclaved (20 min, 121 °C). The samples were centrifuged and the formation of insoluble matter was observed.

[0262] Table 2: Overview of the Formulations of Six Test Culture Media

[0263]

[0264] The results are shown in Figure 9 ...

[0265] Example 7: Correlation Analysis of Growth Status, Cell Morphology, and Lipid Yield

[0266] The lignocellulose hydrolysate was pretreated differently, and its effects on cell morphology and growth behavior were compared. The morphology of the cells gave an indication of the potential amount of lipids accumulated, the size and number of lipid bodies included. All pretreatment conditions were compared with a model substrate (containing xylose, glucose, and acetic acid in the corresponding amounts of the actual hydrolysate). The pretreated hydrolysate and the model substrate were formulated in a medium with nitrogen limitation (C / N ratio of 50) and cultured in a shake flask at 28 °C for 120 hours.

[0267] Table 3: Summary of the Pretreatment and Control Conditions Tested in This Experiment

[0268]

[0269] The growth curves corresponding to different pretreatment conditions are as Figure 10 shown, and the cell morphology is shown in Figure 11 ...

[0270] Conclusion

[0271] This paper presents a new strategy for producing microbial oils using lignocellulosic hydrolysis products (LCH) and acetic acid as the main carbon sources by oleaginous yeasts (such as C. oleaginosus). To this end, fermentations were carried out at 0.25 l and 1 l scales, and a maximum lipid titre of 42.1 ± 1.7 g / l was achieved under optimized conditions. The proportion of LCH consumption increased to 37.6 ± 2.3% of the total carbon source consumption. In addition, techno-economic analysis further confirmed that using LCH has significant economic advantages compared to glucose. In summary, we have developed a sustainable and cost-effective single-cell oil production process, providing an alternative platform for the production of biofuels and oil-based chemicals.

[0272] The features of the present invention disclosed in the description, claims and / or drawings can be implemented separately and in any combination thereof to achieve the spirit of the present invention in various forms.

Claims

1. A method for reducing the salt content of a salt-containing hydrolysis product (preferably a salt-containing biogenic hydrolysis product), which comprises: a) providing a salt-containing hydrolysis product, preferably a salt-containing biogenic hydrolysis product, more preferably a salt-containing lignocellulosic hydrolysis product; b) Optionally, neutralize the pH of the hydrolysis product of step a) to obtain a neutralized hydrolysis product; wherein, Optionally, the pH of the neutralized hydrolysis product is in the range of about pH 4 to about pH 8; c) adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3 and / or Mg(OH)2, preferably CaCO3 and / or Ca(OH)2, to the hydrolysis product of step a) or the neutralized hydrolysis product of step b) to obtain a hydrolysis product containing precipitated salts; wherein, optionally, the pH of the hydrolysis product containing precipitated salts is in the range of about pH 4 to about pH 8.5; d) adding a chelating agent to the hydrolysis product of step c); e) optionally, adjusting the pH of the hydrolysis product of step c) and / or step d) to a pH in the range of about pH 3 to about pH 8.5, preferably about pH 3.5 to about pH 7.5, more preferably about pH 5 to about pH 7, and further preferably about pH 6 to about pH 7; f) obtaining a hydrolysis product with a reduced salt content.

2. The method according to claim 1, wherein The hydrolysis product provided in step a) is a hydrolysis product derived from the paper industry, such as a hydrolysis product derived from pulp production, a hydrolysis product derived from forestry, an agricultural hydrolysis product, a food hydrolysis product, a food waste hydrolysis product, a biofuel waste hydrolysis product, a textile hydrolysis product, an animal tissue hydrolysis product, a plant tissue hydrolysis product, a microbial biomass hydrolysis product, an industrial waste hydrolysis product, a municipal waste hydrolysis product, or any combination thereof; wherein, preferably, the hydrolysis product is a lignocellulosic hydrolysis product, preferably a waste liquid hydrolysis product, and / or a hydrolysis product derived from pulping, more preferably a hydrolysis product derived from acid pulping.

3. The method according to claim 1 or 2, wherein The hydrolysis product provided in step a) is a hydrolysis product obtained by physically treating, chemically treating, enzymatically treating, and / or biologically treating a substrate (preferably biomass); wherein, preferably, the physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and any combination thereof; the chemical treatment is selected from alkali treatment, acid treatment, and treatment at neutral pH; wherein, preferably, the chemical treatment is treatment with salts, acids, peroxides, and any combination thereof, preferably treatment with sulfides, sulfites, and / or bisulfites; the enzymatic treatment is treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylating enzymes, peptidases, such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, lignin-degrading enzymes, and any combination thereof; and the biological treatment is treatment with microorganisms, preferably treatment with microorganisms selected from bacteria, yeasts, and fungi.

4. The method according to any one of the preceding claims, wherein, The hydrolyzate provided in step a) contains salts in an amount in the range of from about 0.0001 mol / l to about 15 mol / l, preferably from about 0.0005 mol / l to about 8 mol / l, and / or carbon in a value in the range of from about 0.1% by weight to about 65% by weight.

5. The method according to any one of the preceding claims, wherein, The hydrolyzate provided in step a) contains salts selected from sulfates, sulfides, sulfites, nitrates, nitrites, chlorides and any combination thereof; wherein, preferably, the salts contain sulfates, sulfides and / or sulfites.

6. The method according to any one of the preceding claims, wherein, The hydrolyzate provided in step a) contains lignophenols, lignans, organic acids and / or sugars; wherein, optionally, the sugars contain xylose, glucose, mannose and / or galactose; wherein, preferably, the sugars contain monosaccharides, preferably xylose; and wherein, preferably, the organic acids contain acetic acid.

7. The method according to any one of the preceding claims, wherein, The chelating agent is selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4) and any combination thereof, where M is a metal; wherein, preferably, the chelating agent is selected from Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, (NH4)(H2PO4) and Na3PO4; wherein, more preferably, the chelating agent is KH2PO4.

8. The method according to any one of the preceding claims, wherein, The method comprises step e) of adjusting the pH of the hydrolyzate, and, wherein, the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid or any combination thereof to the hydrolyzate of step c) and / or step d), preferably by adding NaOH and / or KOH.

9. The method according to any one of the preceding claims, wherein, The method further comprises sterilization, preferably sterilizing the hydrolyzate with a reduced salt content obtained in step f) to obtain a sterile hydrolyzate with a reduced salt content; wherein, preferably, the sterilization comprises thermal sterilization, ultra-high temperature treatment and / or sterile filtration.

10. A method for producing a target product (preferably microbial oil), comprising the following steps: i) providing a hydrolyzate with a reduced salt content by implementing the method according to any one of the preceding claims; ii) culturing a microorganism (preferably an oil-producing microorganism) using the hydrolyzate provided in step i) or a growth medium consisting thereof, so that the microorganism produces the target product; preferably causing the oil-producing microorganism to produce microbial oil; iii) Optionally, subject the microorganism (preferably the oil-producing microorganism) to enzymatic treatment; wherein, Optionally, the enzyme treatment comprises carrying out the enzyme treatment of the microorganism without any solvent extraction or chemical demulsification; iv) obtaining the target product, preferably microbial oil.

11. The method according to claim 10, wherein, The microorganism is an oil-producing microorganism, preferably an oil-producing yeast, more preferably Cutaneotrichosporon sp., and further preferably Cutaneotrichosporon oleaginosus.

12. The method according to claim 10 or 11, wherein The target product is selected from microbial oils, glycerol, free fatty acids, monoglycerides, diglycerides and triglycerides, phospholipids, sphingolipids, polyols, alcohols, organic acids, biodiesel, hydrogen, methane, biopolymers, carotenoids, cellulose, squalene, sterols, vitamins, phenolic compounds, pigments, peptides, proteins such as enzymes, DNA, RNA, and any combination thereof; Preferably, the target product comprises microbial oil.

13. The method according to any one of claims 10 to 12, wherein, The hydrolyzate with reduced salt content provided in step i) contains acetic acid and / or xylose, Preferably, the hydrolyzate comprises or consists of a lignocellulosic hydrolyzate.

14. The method according to any one of claims 10 to 13, wherein, The cultivation in step ii) comprises adding acetic acid and / or a carbon source other than acetic acid to the growth medium; Preferably, The acetic acid is added in a feed form comprising or consisting of acetic acid, and preferably, the concentration of acetic acid in the feed is in the range of 1 mol / l to 20 mol / l, preferably in the range of 1.75 mol / l to 15.75 mol / l. Optionally, the feed further comprises a carbon source other than acetic acid.

15. The method according to any one of claims 10 to 14, wherein, The growth medium contains sugars such as xylose in an amount in the range of about 0.1 g / l to about 250 g / l, preferably <100 g / l; and / or The growth medium contains acetic acid in an amount in the range of about 0.01 g / l to about 100 g / l, preferably about 1 g / l to about 50 g / l, more preferably about 5 g / l to about 10 g / l.

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