Pretreatment of salt-containing hydrolysates, especially for use in fermentation processes

Abstract

The present invention relates to a method for reducing the salt content of a salt-containing hydrolysate. The present invention further relates to a method for 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, with 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 salt-containing hydrolysate. The present invention further relates to a method for 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, with a growth medium comprising the reduced salt hydrolysate. [Background technology]

[0002] Single-cell oil (SCO) produced by oleaginous microorganisms can be used in the production of advanced biofuels as an alternative source of edible vegetable oil. In contrast to crop cultivation for oil production, microbial cultivation is not seasonal, and biotechnology plants allow for efficient land use and vertical scale-up. However, a major weakness of ecological SCO production is the selection of cost-effective feedstocks. Given this background, the use of industrial waste streams has great potential to solve waste treatment and feedstock issues for advanced bioprocesses. However, most industrial waste streams contain compounds that inhibit microbial growth and prevent the efficient production of target products such as microbial oils.

[0003] The pulp and paper industry is one of the major sources of waste containing high concentrations of biodegradable carbon. One of the main processes for cellulosic fiber production, besides alkaline kraft pulping, is acid sulfite pulping, which is combined with steam explosion to hydrolyze cells and lignocellulosic compounds and isolate valuable cellulose fibers. The resulting waste stream typically contains a large amount of pentoses, small amounts of hexoses, and uronic acids, in addition to aliphatic carboxylic acids, furans, and phenolic compounds. In most industrial plants, it is used for energy production by anaerobic fermentation to methane or direct combustion. Therefore, value creation for this waste stream is currently limited.

[0004] An advanced example of utilizing lignocellulosic waste for the production of alternative oleochemicals is ethanol production using bacteria or yeast as whole-cell biocatalysts. Furthermore, microbial production of long-chain fatty acids, primarily C13–C21, has been reported in microalgae, bacteria, and oleaginous yeast. However, oleaginous yeast fermentation in a diauxic fermentation mode based on limiting nitrogen or phosphate concentrations results in reduced growth rate, biomass accumulation, and lipid yield. In an example of lipid production in lignocellulosic hydrolysate (LCH) using the fermentation host Rhodosporidium toruloides, a lipid titer of 39.5 g / L was achieved in fed-batch fermentation. However, the given example utilizes hexoses as the carbon source, as opposed to pentoses, which can only be efficiently utilized by C. oleaginosus. Furthermore, lignocellulosic hydrolysates typically contain compounds such as salts that inhibit microbial growth and reduce yield and efficiency.

[0005] Hydrolysates of side, waste, or residual streams of lignocellulosic biomass contain high salt concentrations and high contents of soluble oligo- and monomeric lignins, furans, and compounds that inhibit microbial growth, such as heavy metals, which interfere with any fermentation process that uses such side, waste, and / or residual streams. To date, intensive concentration followed by combustion is the only possible and applied use of such side, waste, or residual stream hydrolysates.

[0006] Thus, there remains a need to convert waste streams, such as lignocellulosic hydrolysates, into suitable growth substrates. Additionally, there remains a need to provide methods for reducing the salt content of hydrolysates used as or in growth media for culturing microorganisms. There also remains a need to convert hydrolysates, such as lignocellulosic hydrolysates from acid pulping, into suitable growth media or growth media supplements. There also remains a need for efficient methods for producing target products, such as microbial lipids. There also remains a need for environmentally friendly, cost-effective methods for producing target products, such as SCO. Summary of the Invention

[0007] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to produce further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The specification should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments, or combining one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0008] In a first aspect, the present invention provides a method for reducing the salt content of a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, comprising the steps of: a) providing a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, more preferably a salt-containing lignocellulosic hydrolysate; b) optionally neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate; optionally, the neutralized hydrolysate has a pH 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 hydrolysate of step a) or the neutralized hydrolysate of step b) to obtain a hydrolysate containing precipitated salts; optionally, the hydrolysate containing precipitated salts has a pH in the range of about pH 4 to about pH 8.5; d) adding a chelating agent to the hydrolysate of step c); e) optionally adjusting the pH of the hydrolysate 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 even more preferably about pH 6 to about pH 7; f) obtaining a hydrolysate with reduced salt content; The present invention relates to a method comprising:

[0009] In one embodiment, the hydrolysate provided in step a) is a hydrolysate from paper production, such as a hydrolysate from pulp production, a forestry hydrolysate, an agricultural hydrolysate, a food hydrolysate, a food waste hydrolysate, a biofuel waste hydrolysate, a textile hydrolysate, an animal tissue hydrolysate, a plant tissue hydrolysate, a microbial biomass hydrolysate, an industrial waste hydrolysate, a municipal waste hydrolysate, or any combination thereof; Preferably, the hydrolysate is a lignocellulosic hydrolysate, preferably a pulping waste liquor hydrolysate and / or a pulping-derived hydrolysate, more preferably an acid pulping-derived hydrolysate.

[0010] In one embodiment, the hydrolysate provided in step a) is a hydrolysate obtained by physical, chemical, enzymatic and / or biological treatment of a substrate, preferably biomass; Preferably, the physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and combinations thereof; The chemical treatment is selected from alkaline treatment, acid treatment, and treatment at neutral pH; preferably, the chemical treatment is treatment with any of salt, acid, peroxide, and any combination thereof, preferably treatment with sulfide, sulfite, and / or bisulfite; the enzymatic treatment is a treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylases, peptidases, such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, ligninolytic enzymes, and any combination thereof; The biological treatment is a treatment with a microorganism, preferably a treatment with a microorganism selected from bacteria, yeasts, and fungi.

[0011] In one embodiment, the hydrolysate provided in step a) comprises salt in an amount ranging 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 It contains carbon in an amount ranging from about 0.1% to about 65% by weight.

[0012] In one embodiment, the hydrolysate provided in step a) comprises salts selected from sulfates, sulfides, sulfites, nitrates, nitrites, chlorides, and any combination thereof; preferably, the salts comprise sulfates, sulfides, and / or sulfites.

[0013] In one embodiment, the hydrolysate provided in step a) comprises lignols, lignans, organic acids, and / or sugars; optionally, the sugars comprise xylose, glucose, mannose, and / or galactose; preferably, the sugars comprise monosaccharides, preferably xylose; preferably, the organic acid comprises acetic acid.

[0014] 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; Preferably, the chelating agent is selected from Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, Ca(H2PO4), CaHPO4, Ca3(PO4), (NH4)(H2PO4), and Na3PO4; More preferably, the chelating agent is KH2PO4.

[0015] In one embodiment, the method comprises a step e) of adjusting the pH of the hydrolysate by adding NaOH, KOH, CH3COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid, or any combination thereof, preferably NaOH and / or KOH, to the hydrolysate of step c) and / or step d).

[0016] In one embodiment, the method further comprises sterilizing the reduced salt content hydrolysate preferably obtained in step f) to obtain a sterilized reduced salt content hydrolysate; Preferably, the sterilization comprises heat sterilization, ultra-high temperature treatment, and / or sterile filtration.

[0017] In a further aspect, the present invention provides a method for producing a target product, preferably a microbial oil, comprising the steps of: i) providing a hydrolysate having reduced salt content by carrying out a method for reducing the salt content of a salt-containing hydrolysate, as defined herein; ii) culturing a microorganism, preferably an oleaginous microorganism, with a growth medium comprising or consisting of the hydrolysate provided in step i), thereby causing the microorganism to produce a target product; preferably causing the oleaginous microorganism to produce a microbial oil; iii) optionally, enzymatically treating the microorganism, preferably the oleaginous microorganism; optionally, the enzymatically treating comprises enzymatically treating the microorganism without any solvent-based extraction or chemical-based demulsification; iv) obtaining the target product, preferably a microbial oil; The present invention relates to a method comprising:

[0018] In one embodiment, the microorganism is an oleaginous microorganism, preferably an oleaginous yeast, more preferably a species of the genus Cutaneotrichosporon, even more preferably Cutaneotrichosporon oleaginosus.

[0019] In one embodiment, the target product is selected from microbial oils, glycerol, free fatty acids, mono-, di- 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 a microbial oil.

[0020] In one embodiment, the reduced salt hydrolysate provided in step i) comprises acetic acid and / or xylose; Preferably, the hydrolysate comprises or consists of a lignocellulosic hydrolysate.

[0021] In one embodiment, the culturing in step ii) comprises adding acetate and / or a carbon source other than acetate to the growth medium; Preferably, the acetic acid is added in the form of a feed comprising or consisting of acetic acid, 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 acetate.

[0022] In one embodiment, the growth medium comprises a sugar, such as xylose, in an amount ranging from 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 ranging from about 0.01 g / L to about 100 g / L, preferably from about 1 g / L to about 50 g / L, and more preferably from about 5 g / L to about 10 g / L.

[0023] In a further aspect, the present invention relates to a hydrolysate with reduced salt content obtained and / or obtainable by a process for reducing the salt content of a salt-containing hydrolysate, as defined herein.

[0024] In a further aspect, the present invention relates to a composition obtained and / or obtainable by a method for producing a target product as defined herein.

[0025] In one embodiment, the composition comprises or consists of a target product obtained and / or obtainable by a method of producing a target product, as defined herein. [Brief explanation of the drawings]

[0026] The invention is further described by reference to the following drawings. [Figure 1] Figure 1 shows a comparison of growth behavior under different fermentation conditions (LCH - lignocellulosic hydrolysate, N-limited - nitrogen-limited fermentation, cb - feeding - consumption-based feeding, co-feeding - a combination of consumption-based and continuous feeding of LCH). Biomass accumulation after inoculation of fermentations with various starting sugars and acetate consumption-based feeding in all cases except the nitrogen-limited condition. Curve fitting with the Gompertz function. Error bars indicate two standard deviations. [Figure 2] Figure 2 shows lipid analysis for the five most important fermentation conditions. Starting carbon sources are abbreviated as follows: Glu - glucose, LCH - lignocellulosic hydrolysate, LCHco - feed - lignocellulosic hydrolysate as starting carbon and constant feed, 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 main fatty acids quantified by GC-FID. Error bars indicate two standard deviations. [Figure 3] Figure 3 shows a comparison of feeding strategies at a 0.25 L scale in the DASbox® system (LCH-lignocellulosic hydrolysate). Biomass accumulation and substrate consumption for the control (50% acetic acid, consumption-based feeding) and the best two operating modes (50:50 acetic acid:LCH co-feeding and 1 mL / h LCH continuous feeding). Total lipid titer and LCH share of total carbon uptake achieved after 65 hours. Error bars indicate two times the standard deviation. [Figure 4] Figure 4 shows confocal microscopy images of cells fermenting lignocellulosic hydrolysate with an acetic acid-based feed after 24 hours (a) and 71 hours (b), and with a continuous feed of LCH combined with an acetic acid feed after 24 hours (c) and 71 hours (d). [Figure 5] Figure 5 shows the annual production costs of yeast oil in $ / Mt for three fermentation strategies analyzed using TEA: LCHcb-feed - LCH with acetate consumption-based feed, glucosecb-feed - glucose with acetate consumption-based feed, LCHco-feed - continuous feed of LCH, and acetate consumption-based feed. The feed rates for each were set to produce yeast oil at a rate of 0.81 Mt / h (LCHcb-feed: 1 Mt / h, glucosecb-feed: 0.151 Mt / h, LCHco-feed: 2.1 Mt / h). [Figure 6] FIG. 6 illustrates an exemplary embodiment of the method of the present invention. [Figure 7] Figure 7 shows the average sediment after each step of pretreatment. [Figure 8] Figure 8 shows a comparison of pellets obtained from KH2PO4 titration of CaCO3 treated hydrolysates. [Figure 9]Figure 9 shows photographs of samples after autoclaving. The media were prepared as described in Table 2, neutralized, autoclaved, and centrifuged. The supernatant (SN) and pellet for each condition are shown. It can be seen that the pellet is much more abundant in the condition with only CaCO3 added. Furthermore, in the conditions without KH2PO4 added, the color of the supernatant is much darker, except for condition 3, which was supplemented with complete medium. However, autoclaving the media with complete medium results in the formation of HMF and furfural, which may inhibit microbial growth. [Figure 10] Figure 10 shows growth curves based on optical density at 600 nm measured in a photometer cuvette. Conditions 2, 4, and 5 behaved similarly to control condition 1. Only condition 3, in which the hydrolysate was charcoal treated, showed significantly lower growth. [Figure 11] Figure 11 shows a 100x magnification micrograph of a cell culture grown in a culture medium containing the five different carbon sources listed in Table 3. After 24 hours, no large lipid droplets have yet formed in any of the conditions. After 120 hours, lipid droplets are indicated by a halo reflection. In conditions 1 and 2, an average of two lipid droplets are formed per cell. In condition 3, not all cells have lipid droplets; those that do have two to three. In conditions 4 and 5, one lipid droplet is formed per cell, indicating the highest possible lipid content. In condition 2, insoluble matter is clearly visible. All methods referred to in the following figure descriptions were carried out as described in detail in the Examples. Reference is made below to the Examples for the purpose of illustrating, but not limiting, the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention aims to provide a hydrolysate with reduced salt content that can be used as a growth medium. Advantageously, a method for reducing the salt content of a hydrolysate can increase the value of products and / or waste products, such as biomass, so that these products become suitable growth media and / or suitable additives for growth media. Advantageously, waste products, such as spent pulp liquor or other hydrolysates, that contain salt(s) that inhibit microbial growth can be processed into valuable hydrolysates with reduced salt content that can be used as or in growth media for microbial growth. The method for reducing the salt content of a hydrolysate effectively upcycles waste products, such as biogenic waste.

[0028] The inventors have surprisingly found that a target product can be efficiently produced using a microorganism cultured using a hydrolysate with reduced salt content, such as a lignocellulosic hydrolysate with reduced salt content, as a growth substrate. The inventors have unexpectedly found that the target product production method of the present invention can produce the target product in high yield. Furthermore, the inventors have found that reducing the salt content can increase the value of waste hydrolysates, such as spent pulp liquor, as a growth substrate for microbial cultivation. Methods for reducing the salt content of hydrolysates are highly effective in producing hydrolysates that can be used as and / or in growth media. The present invention makes it possible, for the first time, to utilize side streams, waste streams, and / or residual streams from lignocellulosic biomass for microbial cultivation after reducing the salt content.

[0029] Interestingly, the inventors have found that microorganisms exhibit a unique ability to grow perfectly well on reduced-salt hydrolysates that exhibit high contents of soluble oligo- and monomeric lignins, as well as high tolerance and uptake capacity for furans. In current fermentation systems, microorganisms actually exhibit much higher growth rates on reduced-salt lignocellulosic hydrolysates compared to model lignocellulosic hydrolysates.

[0030] The present invention provides a method for reducing the salt content of a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, comprising the steps of: a) providing a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, more preferably a salt-containing lignocellulosic hydrolysate; b) optionally neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate; optionally, the neutralized hydrolysate has a pH 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 hydrolysate of step a) or the neutralized hydrolysate of step b) to obtain a hydrolysate containing precipitated salts; optionally, the hydrolysate containing precipitated salts has a pH in the range of about pH 4 to about pH 8.5; d) adding a chelating agent to the hydrolysate of step c); e) optionally adjusting the pH of the hydrolysate 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 even more preferably about pH 6 to about pH 7; f) obtaining a hydrolysate with reduced salt content; The present invention relates to a method comprising:

[0031] In one embodiment, the method reduces the salt content, such as the sulfate content, of a salt-containing hydrolysate by at least 1%, preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, and even more preferably at least 80%. In one embodiment, the reduction in salt content comprises a significant reduction in salt content, preferably by at least 1%, preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, and even more preferably at least 80%.

[0032] In one embodiment, the term "reducing the salt content" as used herein relates to reducing the total salt content of a hydrolysate and / or reducing the amount of a specific salt in the hydrolysate, particularly reducing the amount of at least one salt, for example, reducing the sulfate content of a hydrolysate. In one embodiment, a method for reducing the salt content comprises reducing the sulfate content and, optionally, further comprising reducing the calcium content of the hydrolysate. In one embodiment, the salt(s) composition of the hydrolysate is changed independently of the total salt content. For example, the method of the present invention can reduce the amount of sulfate present in the hydrolysate (thus changing the total salt(s) composition of the hydrolysate), while maintaining or changing the total salt content of the hydrolysate. In one embodiment, the terms "content," "amount," and "concentration" are used interchangeably. In one embodiment, a method for reducing the salt content of a hydrolysate 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 a hydrolysate comprises reducing the salt concentration of at least one salt present in the hydrolysate, for example, reducing the sulfate concentration; optionally, the total salt content of the hydrolysate is reduced. In one embodiment, the salt reduced in the method for reducing the salt content of a hydrolysate is sulfate. In one embodiment, reducing the salt content comprises or consists of reducing the sulfate content. In one embodiment, the method for reducing the salt content of a salt-containing hydrolysate is a method for reducing the sulfate content of a sulfate-containing hydrolysate. In one embodiment, the method for reducing the salt content of a salt-containing hydrolysate further comprises, for example, reducing the calcium concentration of the hydrolysate. In one embodiment, the hydrolysate with reduced salt content obtained in step f) of the method for reducing the salt content is a hydrolysate with reduced sulfate content, optionally with reduced calcium content.In one embodiment, the hydrolysate with reduced salt content obtained in step f) of the method for reducing the salt content has a salt composition that differs from the salt composition of the hydrolysate provided in step a); preferably, the concentration of at least one salt, such as the sulfate concentration, is reduced in the hydrolysate obtained in step f) compared to the concentration of said at least one salt in the hydrolysate provided in step a); optionally, the total salt content of the hydrolysate obtained in step f) is the same as or different from the total salt content of the hydrolysate provided in step a).

[0033] The term "hydrolysate," as used herein in the context of the method for reducing the salt content of a hydrolysate according to the present invention, relates to any product of hydrolysis, particularly the product of hydrolysis of biomass, such as lignocellulosic biomass. For example, the hydrolysate may be derived from solid paper waste, pulp, spent pulp 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 hydrolysate comprises sulfate. The hydrolysate may comprise carbohydrates, particularly sugars, sugar degradation products, and / or lignin degradation products. In one embodiment, the hydrolysate comprises sugars, particularly xylose, and / or acetic acid. In one embodiment, the hydrolysate is derived from and / or prepared from lignocellulosic biomass. In one embodiment, the terms "derived from" and "prepared from" or "derived from" and "prepared from" are used interchangeably. In one embodiment, the term "derived from" as used herein in the context of a hydrolysate refers to a hydrolysate prepared or prepared, particularly obtained, from a starting material such as biomass by any method known to those skilled in the art, preferably by physical, chemical, enzymatic, and / or biological treatment of the starting material, particularly the substrate, preferably biomass. In one embodiment, the starting material, particularly the substrate, comprises or consists of: paper production products such as pulp, spent pulp liquor, bleaching waste, paper, and / or paper waste; forest products such as wood and / or sawdust; agricultural products such as straw; food products or food waste such as bread; biofuel production waste such as microbial biomass; hydrogen production waste such as microbial biomass; textile products such as wool, cotton, and / or hemp, optionally biodegradable textile products; 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 hydrolysate, optionally biodegradable industrial waste; 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, the production of biofuels may be associated with the production of hydrogen. In one embodiment, the biofuel waste hydrolysate is a hydrogen-producing waste hydrolysate.

[0034] For example, the biomass may relate to the biodegradable fraction of products, waste, and / or residues of biological origin, e.g., from agriculture, forestry, and / or related industries, including fishing and aquaculture. For example, the biomass may relate to the biodegradable portion of industrial and domestic waste. In one embodiment, the industrial waste is selected from the group consisting of side streams and / or waste streams from food processing, pulp production, paper production, agricultural industry, biofuels, and / or forestry. In one embodiment, the microbial biomass is selected from bacterial biomass, fungal biomass, yeast biomass, microalgae biomass, and combinations thereof; preferably, the microbial biomass is fungal biomass, particularly yeast biomass. In one embodiment, the fungal biomass comprises or consists of biomass of a microbial organism selected from the group consisting of Aspergillus, Fusarium, Trichoderma, Ascobolus, Rhizopus, and combinations thereof. For example, the fungal biomass can comprise biomass of Trichoderma reesei and / or Aspergillus niger. In one embodiment, the yeast biomass comprises or consists of biomass of a microorganism selected from the group consisting of Saccharomyces, Yarrowia, Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, Rhodotorula, Candida, Cutaneotrichosporon, and any combination of the foregoing; preferably, the yeast biomass comprises biomass of Cutaneotrichosporon oleaginosus and / or Saccharomyces cerevisiae.

[0035] The term "lignocellulosic biomass" is meant to refer to biomass comprising cellulose, hemicellulose, lignin, and combinations thereof. In one embodiment, the hydrolysate is derived from paper production products such as pulp, spent pulp liquor, paper, and / or paper waste; forest products such as wood and / or sawdust; agricultural products such as straw; food products or food waste such as bread; biofuel production waste such as microbial biomass; hydrogen production waste such as microbial biomass; textile products such as wool, cotton, and / or hemp, optionally biodegradable textile products; animal tissue 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 hydrolysate, optionally biodegradable industrial waste; municipal waste; or any combination thereof.

[0036] In one embodiment, the hydrolysate is a hydrolysate derived from paper production, such as pulp or pulp hydrolysate, spent pulp liquor, paper hydrolysate, and / or paper waste hydrolysate; a hydrolysate derived from forestry, such as wood hydrolysate and / or sawdust hydrolysate; an agricultural hydrolysate, such as straw hydrolysate; a food hydrolysate, such as fruit peel hydrolysate; a food waste hydrolysate, such as bread residue hydrolysate; a biofuel production waste hydrolysate, such as microbial biomass hydrolysate; a hydrogen production waste, such as microbial biomass hydrolysate; a textile hydrolysate, such as wool hydrolysate, cotton hydrolysate, and / or hemp hydrolysate; an animal tissue hydrolysate, such as meat hydrolysate; a plant tissue hydrolysate, such as a crop hydrolysate; a microbial biomass hydrolysate, such as fungal biomass hydrolysate, bacterial biomass hydrolysate, and / or yeast biomass hydrolysate; an industrial waste hydrolysate, such as hemp hydrolysate; a municipal waste hydrolysate; or any combination thereof.

[0037] The term "spent pulp liquor" as used herein refers to any spent pulp liquor known to those skilled in the art, particularly the liquid effluent from the cooking of wood during pulping. Spent pulp liquor typically contains wood components such as lignin and further contains cooking agents such as saprophytic agents, sulfites, or sulfates. The term "pulp" as used herein refers to pulp from a paper production process, particularly a lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibers from raw materials such as wood, fiber crops, waste paper, or rags. Pulp is the primary raw material used in the industrial production of paper and other paper products. Pulp production can include mechanical pulping, thermomechanical pulping, chemi-thermomechanical pulping, chemical pulping, and organic solvent pulping. Chemical pulping can include the Kraft process, the sulfite process, and / or the soda pulping process. In one embodiment, the hydrolysate from pulp production is a hydrolysate from chemical pulping, preferably acid pulping. In one embodiment, the hydrolysate is or is derived from spent pulp liquor.

[0038] In a preferred embodiment, the hydrolysate is derived from biomass, for example, lignocellulosic biomass. In one embodiment, the hydrolysate is a hydrolysate from paper production, such as a hydrolysate from pulp production, a forestry-derived hydrolysate, an agricultural hydrolysate, a food hydrolysate, a food waste hydrolysate, a biofuel waste hydrolysate, a textile hydrolysate, an animal tissue hydrolysate, a plant tissue hydrolysate, a microbial biomass hydrolysate, an industrial waste hydrolysate, a municipal waste hydrolysate, or any combination thereof. In one embodiment, the hydrolysate is a biogenic hydrolysate. In one embodiment, the term "biogenic hydrolysate" as used herein relates to a hydrolysate of a biogenic product, such as a biogenic waste. A biogenic product is a product made by or in a living organism. In one embodiment, the biogenic hydrolysate comprises or consists of a lignocellulosic hydrolysate. In a preferred embodiment, the hydrolysate is or is derived from acidic pulping, particularly a waste stream and / or residual stream from acidic pulping. Advantageously, the methods of the present invention increase the value of pentose sugars in waste streams and / or residual streams from acid pulping as feedstock for culturing microorganisms, such as Cutaneotrichosporon sp. Additionally, the methods of the present invention advantageously reduce the salt content of growth-inhibiting salts, such as sulfate. By reducing the salt content of the hydrolysate, e.g., by reducing the sulfate content of the hydrolysate, the hydrolysate becomes useful, for example, as feedstock for culturing microorganisms.

[0039] In one embodiment, the reduced-salt hydrolysate provided in step i) comprises a volatile organic acid, xylose, and / or glucose; preferably, the hydrolysate comprises or consists of a lignocellulosic hydrolysate. For example, the volatile organic acid may comprise acetic acid. In one embodiment, the culturing in step ii) comprises adding a volatile organic acid, such as acetic acid, a carbon source other than the volatile organic acid, and / or additional nutrients to the growth medium; preferably, the volatile organic acid is added in the form of a feed comprising or consisting of the volatile organic acid; 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; optionally, the feed further comprises a carbon source other than the volatile organic acid, such as a carbon source other than acetic acid.

[0040] In one embodiment, the hydrolysate is or is derived from a waste stream of paper production, preferably a pentose sugar fraction of a waste stream of paper production. In one embodiment, the salt-containing hydrolysate, for example a pentose sugar fraction of a waste stream of paper production, - hexoses in an amount between 0.5% and 5%, pentoses in an amount between 1% and 10%, oligosaccharides in an amount between 0.3% and 1%, volatile fatty acids in an amount between 0.1% and 3%, and soluble lignin in an amount between 0.2% and 1%; or - hexoses in an amount between 5% and 25%, pentoses in an amount between 30% and 65%, oligosaccharides in an amount between 2% and 15%, volatile fatty acids in an amount between 0.5% and 25%, and soluble lignin in an amount between 1% and 10%; or - hexoses in an amount of 15% to 35%, pentoses in an amount of 1% to 25%, oligosaccharides in an amount of 10% to 40%, volatile fatty acids in an amount of 3% to 25%, and soluble lignin in an amount of 5% to 25%. Includes:

[0041] In one embodiment, the hydrolysate is a hydrolysate obtained by physical, chemical, enzymatic, and / or biological treatment of a substrate, preferably biomass; optionally, the substrate is selected from paper production products such as pulp, pulp liquor, paper, and / or paper waste; forest products such as wood and / or sawdust; agricultural products such as straw; food products or food waste such as bread; biofuel production waste such as microbial biomass; hydrogen production waste such as microbial biomass; textile products 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 hydrolysate; municipal waste; and any combination thereof. In one embodiment, the hydrolysate is provided by carrying out physical, chemical, enzymatic, and / or biological treatment of a substrate, preferably biomass. In one embodiment, the substrate is selected from paper production products such as pulp, spent pulp liquor, paper, and / or paper waste; forest products such as wood and / or sawdust; agricultural products such as straw; food products or food waste such as bread; biofuel production waste such as microbial biomass; hydrogen production waste such as microbial biomass; textile products such as wool, cotton, and / or hemp; animal tissue 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 hydrolysate; municipal waste; and any combination thereof. In one embodiment, the substrate is lignocellulosic biomass. In one embodiment, the hydrolysate is a lignocellulosic hydrolysate derived from wood, preferably hardwood.

[0042] In one embodiment, the physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and combinations thereof. In one embodiment, the chemical treatment is selected from alkaline treatment, acidic treatment, and treatment at neutral pH; preferably, the chemical treatment is treatment with any of salt, acid, peroxide, and any combination thereof, preferably treatment with sulfide, sulfite, and / or bisulfite. In one embodiment, the enzymatic treatment is treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylases, peptidases, such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, ligninolytic enzymes, and any combination thereof. In one embodiment, the biological treatment is treatment with microorganisms, preferably treatment with microorganisms selected from bacteria, yeast, and fungi. For example, hydrolysates can be produced by chemical and physical treatments, such as acid or alkaline treatment followed by steam explosion. For example, the enzymatic treatment can be carried out by any of LiP (EC 1.11.1.14), MnP (EC 1.11.1.13), laccase (EC 1.10.3.2), bO-4 ether cleaving enzymes such as b-etherase, bO-4 aryl-ether cleaving enzymes, O-demethylases, HO-generating oxidases, aryl-alcohol oxidases (EC 1.1.3.7), quinone reductases (EC 1.6.5.5), cellobiose dehydrogenases (EC 1.1.99.18), catechol 2,3-dioxygenases (EC 1.13.11.2), perhydrolases, lipases (EC 3.1.1.3), and / or any combination thereof.

[0043] In one embodiment, the salt-containing hydrolysate comprises a salt in an amount ranging 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 a salt selected from sulfate, sulfide, sulfite, nitrate, nitrite, chloride, and any combination thereof. In one embodiment, the salt-containing hydrolysate comprises sulfate in an amount ranging from about 0.0005 mol / L to about 8 mol / L, optionally from about 0.05 mol / L to about 0.4 mol / L, e.g., about 0.2 mol / L. In one embodiment, the salt-containing hydrolysate comprises sulfate and optionally further salts.

[0044] In one embodiment, the salt-containing hydrolysate comprises a salt selected from sulfate, sulfide, sulfite, nitrate, nitrite, chloride, and any combination thereof, preferably sulfate. In a preferred embodiment, the salt-containing hydrolysate comprises a salt selected from sulfate, sulfide, sulfite, and any combination thereof. The hydrolysate may contain salt, particularly a large amount of salt, such as 8 mol / L, as a result of chemical processing, such as acid pulping, to prepare the hydrolysate.

[0045] In one embodiment, the salt-containing hydrolysate comprises carbon in an amount ranging from about 0.1% to about 65% by weight, preferably from about 0.1% to about 35% by weight, for example about 25% by weight; preferably, "% by weight" refers to the dry weight of the hydrolysate. In a preferred embodiment, the carbon is present in the hydrolysate in the form of biodegradable carbon sources, such as sugars and organic acids. In one embodiment, the carbon is present in the hydrolysate in the form of sugars, such as xylose, glucose, mannose, and / or galactose, and / or in the form of organic acids, such as acetic acid. In one embodiment, the hydrolysate comprises sugars, such as xylose, glucose, mannose, and / or galactose, and / or comprises acetic acid. In one embodiment, the hydrolysate provided in step a) comprises lignols, lignans, organic acids, and / or sugars; optionally, the sugars comprise or consist of xylose, glucose, mannose, and / or galactose; preferably, the sugars comprise monosaccharides, preferably xylose; and preferably, the organic acid comprises acetic acid. In one embodiment, the hydrolysate provided in step a) comprises lignols, lignans, organic acids, and / or sugars; optionally, the sugars comprise or consist of xylose, glucose, mannose, and / or galactose; preferably, the sugars comprise monosaccharides, preferably xylose; preferably, the organic acid comprises acetic acid.

[0046] In one embodiment, the hydrolysate comprises sugars in an amount ranging from about 10 g / L to about 250 g / L, e.g., about 115 g / L. In one embodiment, the hydrolysate comprises xylose in an amount ranging from about 30 g / L to about 100 g / L, e.g., about 77 g / L. In one embodiment, the hydrolysate comprises glucose in an amount ranging from about 0 g / L to about 20 g / L, e.g., about 0.05 g / L to about 20 g / L, e.g., about 12 g / L. In one embodiment, the hydrolysate comprises acetic acid in an amount ranging from about 0 g / L to about 20 g / L, e.g., about 0.05 g / L to about 20 g / L, e.g., about 12 g / L. In one embodiment, the hydrolysate comprises furan in an amount ranging from about 0 g / L to about 10 g / L, e.g., about 0.05 g / L to about 10 g / L, e.g., about 5 g / L. In one embodiment, the hydrolysate comprises lignin-derived compounds in an amount ranging from about 0 g / L to about 200 g / L, such as from about 0.05 g / L to about 200 g / L, for example about 90 g / L.

[0047] In one embodiment, the terms "hydrolysate", "salt-containing hydrolysate", and "hydrolysate provided in step a)" are used interchangeably. In one embodiment, the reduced-salt hydrolysate obtained in step f) differs from the hydrolysate provided in step a) only in its salt content, and optionally in pH and / or sterilization. The reduced-salt hydrolysate obtained in step f) can have the characteristics described for the hydrolysate provided in step a).

[0048] In one embodiment, the method for reducing salt content comprises neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate. In one embodiment, the neutralized hydrolysate has a pH in the range of about pH 4 to about pH 8, preferably in the range of about pH 6 to about pH 8, more preferably in the range of about pH 6.5 to about pH 7.5. In one embodiment, the neutralized hydrolysate obtained in step b) has 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, about pH 7.0, or about pH 7.5. In one embodiment, neutralizing the pH of the hydrolysate comprises increasing the pH of the hydrolysate to a maximum of pH 7.5 or pH 8. In one embodiment, the neutralization comprises neutralizing the hydrolysate of step a) by increasing the pH of the hydrolysate to obtain a neutralized hydrolysate, e.g., to obtain a hydrolysate of a maximum of pH 7.5 or a maximum of pH 8. In one embodiment, the neutralized hydrolysate has a pH of about pH 7.

[0049] The method for reducing the salt content comprises adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, and / or Mg(OH)2, preferably CaCO3 and / or Ca(OH)2, to the hydrolysate of step a) or the neutralized hydrolysate of step b) to obtain a hydrolysate containing precipitated salts. The inventors have found that adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, and / or Mg(OH)2 can provide a hydrolysate suitable for use as a growth substrate for microorganisms. In particular, the addition of CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2, or a combination thereof sufficiently removes growth-inhibiting substances, particularly salts, and establishes the suitability of the hydrolysate for use as a growth substrate. Advantageously, CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2, or combinations thereof, function as an overliming agent to promote precipitation of salts present in the hydrolysate. In one embodiment, the hydrolysate including precipitated salts obtained in step c) has a pH in the range of about pH 4 to about pH 8.5, such as 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, adding CaCO3, Ca(OH)2, CaO, MgO, MgCO3, Mg(OH)2, or a combination thereof to the hydrolysate of step a) or the neutralized hydrolysate 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 in the range of 1 g / L to 150 g / L. In one embodiment, the adding 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%, even more preferably at least 50%, and even more preferably at least 80% of the salt content of the hydrolysate of step a) or the neutralized hydrolysate of step b) precipitates.

[0050] In one embodiment, the adding comprises mixing, preferably thoroughly mixing, the CaCO3, Ca(OH), CaO, MgO, MgCO3, and / or Mg(OH)2 with the hydrolysate to promote precipitation of the salt(s). In one embodiment, the method comprises mixing the CaCO3, Ca(OH), CaO, MgO, MgCO3, and / or Mg(OH)2 with the hydrolysate, such as by stirring, shaking, and / or vortexing. The inventors have found that adding CaCO3, Ca(OH), CaO, MgO, MgCO3, Mg(OH)2, or combinations thereof to a salt-containing hydrolysate efficiently precipitates the salts of the hydrolysate, providing a hydrolysate useful as a growth substrate.

[0051] The method of the present invention includes adding a chelating agent to the hydrolysate of step c), which may 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 alternative counterion.

[0052] In one embodiment, the chelating agent is selected from MPO, MHPO, MHPO, MHPO, MPO, (NH)(HPO), and any combination thereof, where M is a metal. Advantageously, when the chelating agent is selected from MPO, MHPO, MHPO, MHPO, MPO, (NH)(HPO), and any combination thereof, the resulting reduced salt hydrolysate allows for efficient growth of microorganisms, such as production of a target product. The inventors have found that when a reduced salt hydrolysate used as a growth substrate is prepared using a chelating agent selected from MPO, MHPO, MHPO, MHPO, MPO, (NH)(HPO), and any combination thereof, where M is a metal, the cultivation of microorganisms, and in particular production of a target product, becomes highly efficient. The inventors have found that the hydrolysate is particularly useful for subsequent use as a growth substrate when the chelating agent is selected from M3PO4, M2HPO4, MH2PO4, MHPO4, MPO4, (NH4)(H2PO4), and any combination thereof, as other chelating agents such as EDTA, EGTA, and EHPG may inhibit microbial growth.

[0053] 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, 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 hydrolysate with reduced salt content is particularly advantageous for subsequent use as a growth medium. In particular, the inventors have found that when such chelating agents are used to produce hydrolysates with reduced salt content, the resulting reduced salt hydrolysates enable particularly efficient production of the target product by microorganisms grown using such hydrolysates as a growth substrate. In one embodiment, adding a chelating agent to the hydrolysate in step c) comprises adding the chelating agent at a concentration in the range of 0.001 g / L to 500 g / L, preferably in the range of 1 g / L to 150 g / L.

[0054] In one embodiment, the method comprises adjusting the pH of the hydrolysate of step c) and / or the pH of the hydrolysate of step d) to a pH in the range of about pH 2 to about pH 10. In one embodiment, the method comprises adjusting the pH of the hydrolysate of step c) and / or the pH of the hydrolysate of step d) to a pH in the range of about pH 3 to about pH 8.5, such as a range of about pH 5 to about pH 8.5 or a range of about pH 5 to about pH 8; preferably a range of about pH 3.5 to about pH 7.5; more preferably a range of about pH 5 to about pH 7; even more preferably a range of about pH 6 to about pH 7.

[0055] In one embodiment, adjusting the pH comprises adding an acid or a base. In one embodiment, the method comprises a step e) of adjusting the pH of the hydrolysate, which is carried out by adding NaOH, KOH, CH3COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid, or any combination thereof, preferably NaOH and / or KOH, to the hydrolysate of step c) and / or step d). The inventors have found that when the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, or any combination thereof, the hydrolysate is particularly advantageous for subsequent cultivation of microorganisms. In one embodiment, the adjustment is carried out by adding NaOH, KOH, CH3COOH, HCl, or any combination thereof.

[0056] In one embodiment, for example, when the hydrolysate having a reduced salt content is used for culturing a microorganism, adjusting the pH comprises adjusting the pH to about pH 3 to about pH 8.5, e.g., about pH 5 to about pH 8.5, or about pH 5 to about pH 8. In one embodiment, for example, when the hydrolysate having a reduced salt content is used for culturing an acidophilic microorganism and / or an alkaliphilic microorganism, adjusting the pH comprises adjusting the pH to about pH 2 to about pH 10. In one embodiment, for example, when the hydrolysate having a reduced salt content is used for culturing an oleaginous microorganism, such as a strain of Cutaneotrichosporon, adjusting the pH comprises adjusting the pH to about pH 6 to about pH 7, e.g., about pH 6.5. Advantageously, adjusting the pH of the hydrolysate provides the hydrolysate with a pH suitable for culturing a microorganism. For example, when the hydrolysate is used for highly efficient fermentation of a microorganism, such as an oleaginous yeast, a pH of about 6.5 may be provided. In particular, the pH of the hydrolysate may be adjusted to suit the requirements of the microorganism of interest, for example, the microorganism being cultured in the method of producing the target product of the present invention.

[0057] In one embodiment, the method preferably comprises sterilizing the reduced-salt hydrolysate obtained in step f) to obtain a sterilized reduced-salt hydrolysate. In one embodiment, the method comprises sterilizing the salt-containing hydrolysate provided in step a), the neutralized hydrolysate obtained in step b), the precipitated salt-containing hydrolysate obtained in step c), the mixture obtained in step d), the mixture obtained in step e), and / or the reduced-salt hydrolysate obtained in step f). In one embodiment, the method comprises one or more sterilization steps. In one embodiment, the sterilization comprises heat sterilization, ultra-high temperature treatment, and / or sterile filtration. In one embodiment, the sterilization step comprises heat sterilization, ultra-high temperature treatment, and / or sterile filtration. Advantageously, sterilizing the hydrolysate makes the hydrolysate suitable as a growth medium for culturing microorganisms. In particular, sterilizing the hydrolysate prevents contamination of the growth medium for culturing microorganisms. In one embodiment, the reduced-salt hydrolysate obtained in step f) is filtered through activated carbon. The inventors have found that filtering the hydrolysate through activated carbon effectively reduces the content of toxic phenols and furans.

[0058] In one embodiment, obtaining a hydrolysate with reduced salt content comprises obtaining a hydrolysate having a salt content reduced by at least 1%, preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, and even more preferably at least 80% compared to the salt-containing hydrolysate provided in step a). In one embodiment, the reduced-salt hydrolysate obtained in step f) is sterilized. In one embodiment, the reduced-salt hydrolysate obtained in step f) is suitable as a growth medium. In one embodiment, the reduced-salt hydrolysate obtained in step f) is for use in a method of producing a target product of the invention. In one embodiment, the reduced-salt hydrolysate obtained in step f) comprises sugars such as xylose, glucose, mannose, and / or galactose, and / or organic acids such as acetic acid. In one embodiment, the reduced-salt hydrolysate obtained in step f) comprises an amount of carbon in the range of about 0.1% to about 65% by weight; preferably, "weight" refers to the dry weight of the hydrolysate. In one embodiment, the reduced salt hydrolysate obtained in step f) is used in a method for producing a target product of the invention. In one embodiment, the reduced salt hydrolysate obtained in step f) is provided in the form of a growth medium. In one embodiment, the reduced salt hydrolysate obtained in step f) is a hydrolysate intended for use in a microbial culture, such as a method for producing a target product of the invention.

[0059] The present invention also provides a method for producing a target product, preferably a microbial oil, comprising the steps of: i) providing a hydrolysate having reduced salt content by carrying out a method for reducing the salt content of a salt-containing hydrolysate, as defined herein; ii) culturing a microorganism, preferably an oleaginous microorganism, with a growth medium comprising or consisting of the hydrolysate provided in step i), thereby causing the microorganism to produce the target product; preferably causing the oleaginous microorganism to produce a microbial oil; iii) optionally, enzymatically treating the microorganism, preferably an oleaginous microorganism; optionally, said enzymatically treating comprises enzymatically treating the microorganism without any solvent-based extraction or chemical-based demulsification; iv) obtaining the target product, preferably a microbial oil; The present invention relates to a method comprising:

[0060] The term "target product" as used herein refers to any product of interest, particularly any product that can be produced by a microorganism, such as microbial oil, glycerol, free fatty acids, mono-, di-, and triglycerides, phospholipids, sphingolipids, polyols, alcohols such as ethanol, organic acids, biodiesel, hydrogen, methane, biopolymers, carotenoids, cellulose, squalene, sterols, vitamins, phenolic compounds, pigments, proteins such as peptides and enzymes, DNA, RNA, or other products of interest. The target product may be any product of interest to a person skilled in the art, such as microbial oil, microbial oil containing compounds of interest, and / or compounds obtained from microbial oil. For example, microbial oil may contain compounds such as antioxidants and other lignin-derived compounds.

[0061] 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 includes any compound derived from lignin. In one embodiment, the other products of interest and / or the other lignin-derived compounds may be any compound derived from lignin, preferably ethyl pyrocatechuate, 2,3-dihydroxybenzoic acid, coniferyl aldehyde, syringaldehyde, methyl vanillate, 3,4-dihydroxybenzaldehyde, gentisic 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, ferulaldehyde, 3-formylphenol, 1-hydroperoxy-4-methoxybenzene, coumarin, vanillin, methylphthalic anhydride, ayapanin, indole, benzeneacetonin, benzophenone ... Tolyl, 5-aminopentanal, syringic 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-tetramethoxynaphthalene, 1,3,6,8-tetramethoxynaphthalene, dimethyl 2-[(2-methoxyphenyl) phenyl)methylidene]butanedioate, 5,10-dimethoxy-2,2-dimethylpyrano[3,2-g]chromen-8-one, Sweetenocoumarin 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-trimethoxyphenyl)propan-1-one, syringaresinol, lenampicillina, β-D-glucopyranoside, tubocurarine, psymberin, mesaconitine, aschantin, phenylpropanolamine, monomethyl phenylphosphonate, hernialin, 4-methylumbelliferone, chromone, 2-benzofurancarboxaldehyde, glycocoumarin, imbricaric acid, phenethylrutinoside, fluticasone 17β-carboxylic acid, gentisic acid, protocatechuic acid, 7-azaindolizine, and syringaresinol.

[0062] In one embodiment, the phenolic compound is selected from the group consisting of tocopherols, such as α-, β-, γ-, and δ-tocopherol; tocotrienols, such as α-, β-, γ-, and δ-tocotrienol; tocomonoenols, such as α- and β-tocomonoenol; phytoestrogens; chalcones, such as arbutin, phloretin, phloridzin, and chalconaringenin; flavonoids, such as flavonols, flavones, flavanones, flavanols, flavan-3-ols, anthocyanidins, isoflavones, and condensed tannins; and non-flavonoids, such as coumarins and phenolic acids. In one embodiment, the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 and vitamin K.

[0063] As used herein, the terms "microbial lipid" and "microbial oil" refer to lipids produced by oleaginous microorganisms, such as yeast oil, bacterial oil, and / or fungal oil. In one embodiment, the term "microbial lipid" is used interchangeably with "single-cell oil" or "microbial oil." Microbial lipids are typically rich in unsaturated fatty acids. In one embodiment, the microbial lipid is an edible microbial lipid. Such microbial lipids can be used to prepare food products containing the microbial lipid. 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.

[0064] In one embodiment, culturing a microorganism, preferably an oleaginous microorganism, in step ii) comprises using a growth medium comprising or consisting of the hydrolysate provided in step i) as a 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 can comprise the hydrolysate as a growth substrate, in particular to provide carbon, nitrogen, and / or phosphate to the microorganism. In one embodiment, the term "enabling the microorganism to produce a target product" as used herein relates to providing suitable growth conditions for the microorganism. In one embodiment, the microorganism produces the target product as a by-product of microbial growth, such as a by-product of fermentation. In one embodiment, the microorganism produces the target product during cultivation.

[0065] In one embodiment, the growth medium comprises the hydrolysate and further comprises other components, such as buffers, trace elements, vitamins, and / or additional carbon, nitrogen, and / or phosphate sources.

[0066] In one embodiment, the microorganism is selected from yeast, fungi, bacteria, and microalgae. In one embodiment, the microorganism is an oleaginous microorganism, preferably an oleaginous yeast. In one embodiment, the microorganism is selected from Rhodosporidium, Yarrowia, Rhodotorula, Candida, Lipomyces, Cutaneotrichosporon, or Trichosporon, preferably Cutaneotrichosporon, more preferably Cutaneotrichosporon oleaginosus, e.g., Cutaneotrichosporon oleaginosus (ATCC 20509). In one embodiment, the microorganism is an oleaginous microorganism, preferably an oleaginous yeast, more preferably Cutaneotrichosporon, even more preferably Cutaneotrichosporon oleaginosus. In one embodiment, the microorganism is an oleaginous microorganism, preferably an oleaginous yeast, more preferably Cutaneotrichosporon, even more preferably Cutaneotrichosporon oleaginosus. In one embodiment, the microorganism is an oleaginous microorganism, and the target product comprises or consists of microbial oil. In one embodiment, the target product comprises or consists of a microbial oil, which optionally comprises squalene, sterols, vitamins, and / or phenolic compounds.

[0067] In one embodiment, the microorganism may be a wild-type or genetically modified microorganism. In one embodiment, the genetic modification may be in the form of accelerated evolution, directed evolution, random mutagenesis, and / or targeted engineering. In one embodiment, accelerated evolution, directed evolution, and / or random mutagenesis include genome modification by UV treatment, chemical treatment, genetic breeding, error-prone PCR, or other PCR-based methods such as gene shuffling, Agrobacterium-mediated transformation, selection, and / or screening. In one embodiment, targeted engineering includes genome integration, modification, knockout, CRISPR-Cas-mediated gene knockdown, Agrobacterium-mediated transformation, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), targeted mutagenesis, site-directed mutagenesis with promoter modification, RNAi, siRNA, and / or combinations thereof. In one embodiment, the purpose of the genetic modification is to improve the tolerance of the microorganism to reduced-salt hydrolysates, to improve the tolerance of the microorganism to salt content in the culture conditions, to improve the yield of the target product, and / or to alter the chemical and / or physical properties of the target product and / or its composition.

[0068] In one embodiment, the reduced-salt hydrolysate provided in step i) comprises sugars such as acetic acid and / or xylose. In one embodiment, the hydrolysate comprises xylose, acetic acid, and optionally glucose. In one embodiment, the reduced-salt hydrolysate provided in step i) comprises or consists of a lignocellulosic hydrolysate. In one embodiment, the reduced-salt hydrolysate provided in step i) is a hydrolysate obtained in step f) of the method for reducing salt content of the present invention. In one embodiment, the reduced-salt hydrolysate provided in step i) is obtained by the method for reducing salt content of the present invention.

[0069] In one embodiment, culturing a microorganism, preferably an oleaginous microorganism, with a growth medium comprising or consisting of the hydrolysate provided in step i) comprises a fermentation culture of the microorganism, wherein the target product is produced as a by-product of the fermentation culture. In one embodiment, culturing a microorganism with a growth medium comprising or consisting of the hydrolysate provided in step i) comprises: a period of 1 to 7 days, preferably 2 to 4 days; 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; 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; in a medium selected from a minimal nitrogen medium, a minimal phosphate medium, a minimal sulfate medium, and an acetate-rich medium, containing the hydrolysate provided in step i); and / or The method includes culturing the microorganisms at a dissolved oxygen (pO2) in the range of 10% to 90%, preferably 20% to 80%, and more preferably 30% to 65%.

[0070] Advantageously, the cultivation causes the microorganism to produce the target product.

[0071] The term "growth medium" as used herein preferably refers to a cell culture medium comprising a carbon source, a nitrogen source, and / or a phosphate source. In one embodiment, the terms "culture medium" and "growth medium" are used interchangeably. In one embodiment, the growth medium comprises or consists of the hydrolysate provided in step i), the hydrolysate providing the carbon source, the nitrogen source, and / or the 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 of organic phosphate compounds, such as parathion, malathion, phospholipids, ATP, ADP, AMP, and organic phosphate compounds; and inorganic phosphates, such as H3PO4, M2HPO4, MH2PO4, MHPO4, and MPO4 (wherein M is a metal ion).

[0072] In one embodiment, the growth medium includes a carbon source and, optionally, a buffer, salts, trace elements, a nitrogen source, peptone, and / or yeast extract. For example, the growth medium can include sugar in an amount ranging from about 10 g / L to about 100 g / L, e.g., about 30 g / L, and / or acetic acid in an amount ranging from about 10 g / L to about 50 g / L, e.g., about 30 g / L. For example, the growth medium can include any of NaHPO, KHPO, CHCOO·Na, MgSO·7H0, CaCl·2H0, ZnSO·7H0, MnCl·6H0, CuSO·5H0, CHO·Fe·HN, urea, peptone, yeast extract, and combinations thereof.

[0073] In one embodiment, the growth medium has a carbon to nitrogen weight ratio (C:N) of <200, more preferably ≦100, even more preferably in the range of 5 to 80. In one embodiment, the growth medium has a carbon to phosphate weight ratio (C:P) of <500, more preferably ≦150, even more preferably in the range of 25 to 100. The inventors have found that a medium having a carbon to nitrogen weight ratio (C:N) of <100, more preferably ≦80, even more preferably in the range of 10 to 80 and / or a carbon to phosphate weight ratio (C:P) of <500, more preferably ≦150, even more preferably in the range of 10 to 100 allows for high yields of the target product(s) to be achieved. In one embodiment, the culturing in step ii) comprises or consists of nitrogen-limited fermentation, for example using a growth medium having a carbon to nitrogen weight ratio (C:N) in the range of 50 to 200.

[0074] In one embodiment, culturing the microorganism comprises subjecting the microorganism to suitable growth conditions. In one embodiment, the terms "cultivating" and "growing" are used interchangeably. In one embodiment, culturing a microorganism, preferably an oleaginous microorganism, using a growth medium comprising or consisting of the hydrolysate provided in step i) comprises fermentation culturing of the microorganism. In one embodiment, the term "fermentation culturing" as used herein refers to growing a microorganism, such as yeast, under fermentation conditions. For example, fermentation refers to culturing a microorganism, particularly a heterotrophic microorganism, using a feedstock, such as in aerobic and / or anaerobic culture. For example, fermentation conditions may include fermentation at a temperature in the range of 10°C to 45°C, preferably in the range of 15°C to 40°C, and more preferably in the range of 20°C to 33°C; at a pH in the range of 4 to 9, preferably in the range of 5 to 8, and more preferably in the range of 5.5 to 7.5; in a medium selected from minimal nitrogen medium, minimal phosphate medium, minimal sulfate medium, and acetate-rich medium; and / or at a dissolved oxygen (pO2) in the range of 5% to 90%, preferably 20% to 80%, and more preferably 30% to 60%. In one embodiment, step ii) of culturing the microorganism comprises a fermentation culture. In one embodiment, step ii) of culturing the microorganism is carried out in a fed-batch mode, a semi-continuous mode, or a continuous mode, preferably in a continuous mode.

[0075] In one embodiment, the step ii) of culturing a microorganism in a growth medium comprises using the hydrolysate as a substrate, and optionally additional feeds. In one embodiment, the hydrolysate used as a substrate for growing the microorganism comprises a carbon source, a nitrogen source, and / or a phosphate source. In one embodiment, the hydrolysate provided in step i) is used as a substrate for growing the microorganism in step ii).

[0076] In one embodiment, the growth medium in step ii) comprises a carbon source, a nitrogen source, a phosphate source, an organic acid, a trace metal, and / or a vitamin. In one embodiment, the growth medium comprises any of: a monosaccharide, preferably a pentose or hexose, more preferably glucose, xylose, mannitol, arabinose, fructose, mannose, sorbitol, lactose, or sucrose; an oligosaccharide; an amino acid; a fatty acid; an organic acid, preferably acetic acid; a mineral; a vitamin; a trace element; and combinations thereof. In one embodiment, the trace metal is selected from Mo, Cu, Zn, Mn, Ni, and Fe. In one embodiment, the vitamin is 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 peptidic hydrolysates (preferably, the peptidic hydrolysates include components of animal tissue, plant tissue, microbial biomass, and / or the yeast); N-acetylglucosamine; and urea; preferably, 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 compounds; and inorganic phosphates such as H3PO4, M2HPO4, MH2PO4, MHPO4, and MPO4 (wherein M is a metal ion).

[0077] In one embodiment, the organic acid is 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, the respective salt(s) of such acids, and combinations thereof. Preferably, the organic acid is acetic acid. It should be noted that the term "organic acid" as used herein is intended to encompass the respective organic acids regardless of their degree of protonation, i.e., in their protonated state(s) and deprotonated state(s), e.g., when in aqueous solution at a pH at which they are protonated or deprotonated, respectively, according to their respective pKa value(s). The term "organic acid" as used herein is also intended to encompass the salt(s) of the organic acid, e.g., the respective metal salt(s) of such organic acid. Examples of such metal salts are alkali or earth alkali salts of the respective organic acids. The salt may be in dissociated or undissociated form. Without wishing to be bound by any theory, the inventors believe that the presence of an organic acid, such as acetic acid, allows for further productivity enhancement of the process for producing the target product of the invention, while the presence of a carbon source allows for an increase in total biomass.

[0078] In one embodiment, the culturing in step ii) comprises adding to the growth medium an additional amount of a lignocellulosic hydrolysate, such as the hydrolysate provided in step i), a lignocellulosic hydrolysate other than the hydrolysate provided in step i), a carbon source other than the lignocellulosic hydrolysate, and / or additional nutrients; Preferably, the further amount of hydrolysate provided in step i), the lignocellulosic hydrolysate, the carbon source, and / or the additional nutrients are added in the form of a feed comprising or consisting of volatile organic acids, 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, and 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 1.75 mol / L to 15.75 mol / L; Optionally, the feed further comprises a carbon source other than acetate.

[0079] In one embodiment, the culturing in step ii) comprises adding one or more carbon sources and / or additional nutrients in the form of a feed; Optionally, the feed is provided continuously, semi-continuously, on a consumption basis, a pH basis, a dissolved oxygen basis, an off-gas CO2 concentration basis, a time-staggered basis, and / or combinations thereof; Preferably, the feed is provided in a continuous and / or consumption-based manner. In one embodiment, the terms "providing a feed" and "adding a feed" as used herein relate to adding a feed to a growth medium, especially during cultivation, e.g., in a continuous and / or consumption-based manner.

[0080] In one embodiment, the growth medium comprises the hydrolysate as a carbon source. In one embodiment, the culturing in step ii) comprises adding a carbon source other than acetate, such as acetate and / or sugars, to the growth medium. For example, the growth medium may be supplemented with the hydrolysate and, optionally, additional acetate and / or additional carbon sources other than acetate. For example, the acetate may be added to the growth medium in the form of a feed comprising or consisting of acetate. In one embodiment, the term "feed" refers to a liquid feed, e.g., an input stream and / or a solid feed. In one embodiment, the feed comprises the hydrolysate and, optionally, acetic acid. For example, the feed, particularly a liquid feed, may comprise liquid acetic acid and / or a liquid comprising acetic acid. In one embodiment, the feed has an acetate concentration in the range of 1 mol / L to 20 mol / L, preferably 1.75 mol / L to 15.75 mol / L. In one embodiment, the feed comprises the hydrolysate with reduced salt content, and optionally comprises a buffer. In one embodiment, the feed comprises the reduced salt hydrolysate, optionally in diluted form, and further comprises acetic acid, hi one embodiment, the feed further comprises a carbon source other than acetic acid.

[0081] In one embodiment, the growth medium is configured to contain biodegradable carbon in an amount of 10% (w / v) or less, preferably 7% (w / v) or less, more preferably 5% (w / v) or less, for example about 3% (w / v), for example by adjusting the amount of each of the hydrolysates present in the growth medium. In one embodiment, the growth medium contains carbon, particularly sugars and organic acids, in an amount of 10% (w / v) or less, preferably 7% (w / v) or less, more preferably 5% (w / v) or less, for example about 3% (w / v).

[0082] Biodegradable carbon typically refers to carbon that can be decomposed and / or used by microorganisms and / or enzymes, for example, biodegradable carbon is carbon that can be metabolized by microorganisms. In one embodiment, the biodegradable carbon comprises sugar(s) and / or organic acid(s), such as acetic acid. In one embodiment, the culture comprises a consumption-based feed, such as a consumption-based acetic acid feed and / or a consumption-based hydrolysate feed. The advantage of using hydrolysates as feedstocks is that they are very cost-effective and environmentally friendly. Compared to hydrolysates with no reduced salt content, hydrolysates with reduced salt content have the advantage of removing growth-inhibiting salts and significantly improving culture efficiency.

[0083] 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 of 50% (v / v) acetic acid. In one embodiment, the feed comprises a mixture of the acetic acid solution and the reduced salt hydrolysate; optionally, the acetic acid solution and the hydrolysate are mixed in a ratio of about 1:1 to about 5:1.

[0084] In one embodiment, the culturing in step ii) comprises providing a growth medium comprising or consisting of a hydrolysate, and further comprises providing a feed, preferably a feed stream, comprising, for example, acetic acid and / or hydrolysate. In one embodiment, the terms "feed stream," "feed medium," and "substrate feed" are used interchangeably. For example, the growth medium may comprise an initial amount of the hydrolysate and optionally acetic acid, and the growth medium may be supplemented during culturing with a feed comprising an additional amount of the hydrolysate provided in step i). The inventors have found that a feed comprising acetic acid and hydrolysate maximizes hydrolysate consumption and conversion. In one embodiment, the culturing in step ii) comprises providing a growth medium comprising or consisting of an initial amount of the 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 culturing. In one embodiment, the addition of the additional amount of hydrolysate and / or the addition of the acetic acid is performed in a consumption-based or continuous manner. For example, the amount of carbon source, such as acetate and / or sugar, present in the growth medium may be measured, and if necessary, additional carbon sources, e.g., additional amounts of hydrolysate and / or acetate, may be added to provide suitable growth conditions for the microorganism. In one embodiment, the culturing in step ii) includes measuring the carbon content of the growth medium, preferably by HPLC, e.g., using an Agilent 1260 Infinity II LC system equipped with a diode array (DA) detector and a refractive index (RI) detector. For separation, a Phenomenex column, Rezex ROA-organic H+8%, with a mobile phase of 5 mM H2SO4 may be used. An isocratic flow of 0.5 mL / min may be applied for 60 minutes at an oven temperature of 70°C. Detection by RID may be performed at 40°C. In one embodiment, the growth medium comprises a sugar; preferably a sugar selected from xylose, glucose, fructose, arabinose, mannose, galactose, and combinations thereof; preferably, the growth medium comprises the sugar in an amount ranging from about 0.1 g / L to about 100 g / L, preferably <50 g / L.In one embodiment, the growth medium contains an organic acid, preferably acetic acid, in an amount ranging 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. The present inventors have found that when the growth medium contains the respective concentrations of sugar and / or organic acid, culture becomes highly efficient.

[0085] In one embodiment, the culturing in step ii) comprises a continuous feed and / or a consumption-based feed, hi one embodiment, the culturing in step ii) comprises a continuous feed comprising an amount of the hydrolysate provided in step i) and / or a consumption-based feed comprising an organic acid, in particular acetic acid, and / or a carbon source other than the organic acid.

[0086] In one embodiment, the culturing in step ii) includes measuring the sugar concentration of the growth medium, preferably by HPLC, for example, using an Agilent 1260 Infinity II LC system equipped with a diode array (DA) detector and a refractive index (RI) detector. Separation can be performed using a Phenomenex column, Rezex ROA-organic H+8%, with a mobile phase of 5 mM H2SO4. An isocratic flow rate of 0.5 mL / min can be applied for 60 minutes at an oven temperature of 70°C. RID detection can be performed at 40°C. In one embodiment, the culturing in step ii) includes measuring the organic acid concentration, for example, acetic acid concentration, of the growth medium, preferably by HPLC, for example, using an Agilent 1260 Infinity II LC system equipped with a diode array (DA) detector and a refractive index (RI) detector. Separation can be performed using a Phenomenex column, Rezex ROA-organic H+8%, with a mobile phase of 5 mM H2SO4. A uniform concentration flow of 0.5 mL / min may be applied for 60 minutes at an oven temperature of 70°C. Detection by RID is performed at 40°C. In one embodiment, if the measured carbon concentration, sugar concentration, and / or acetic acid concentration are too low to provide suitable growth conditions for the microorganism, for example, if the sugar concentration is less than 0.1 g / L and / or the organic acid concentration is less than 0.01 g / L, the culture includes adding additional amounts of hydrolysate, sugar, and / or acetic acid. In one embodiment, the culture includes adding a feed, preferably a feed stream, to the growth medium; the feed, preferably the feed stream, includes hydrolysate and optionally additional acetic acid. For example, the feed may include about 500 mL of acetic acid per feed and about 500 mL of hydrolysate per feed. In one embodiment, the feed comprises about 10% (v / v) acetic acid to about 100% (v / v) acetic acid, preferably about 10% (v / v) acetic acid to about 90% (v / v) acetic acid. In one embodiment, the feed comprises about 10% (v / v) to about 100% (v / v) of the hydrolysate, preferably a lignocellulosic hydrolysate. In one embodiment, the feed comprises acetic acid and the hydrolysate in a ratio of 10:1 to 1:10.In one embodiment, the carbon content, sugar content, and / or organic acid content is measured using HPLC.

[0087] In a preferred embodiment, the hydrolysate comprises an organic acid such as acetic acid. In one embodiment, the growth medium comprises the hydrolysate and optionally an additional organic acid, such as acetic acid. For example, if the microorganism requires a large amount of organic acid, additional organic acids may be added to the growth medium, particularly in addition to the organic acids contained in the hydrolysate. For example, the feed and / or growth medium may comprise the organic acids contained in the hydrolysate and may further comprise additional organic acids. In one embodiment, the growth medium and / or feed comprises the hydrolysate and is further supplemented with an organic acid and / or a carbon source. In one embodiment, the term "hydrolysate" as used herein in the context of a method for producing a target product relates to a hydrolysate with a reduced salt content, in particular a hydrolysate with a reduced salt content provided in step i) of the method for producing a target product according to the present invention and / or a hydrolysate with a reduced salt content obtained in step f) of the method for reducing the salt content of a hydrolysate according to the present invention.

[0088] In one embodiment, the method for producing the target product comprises, particularly after step ii) of culturing the microorganism, a step of lysing the microorganism. For example, lysing the microorganism can facilitate obtaining the target product. In one embodiment, the step of lysing the microorganism comprises any of enzymatic hydrolysis, temperature shock, chemical treatment, high-pressure homogenization, ultrasonic homogenization, and any combination thereof.

[0089] In one embodiment, the method includes enzymatically treating the microorganisms by enzymatically treating the microorganisms without any solvent-based extraction or chemical-based demulsification, preferably by purely enzymatically treating the microorganisms without any solvent-based extraction or chemical-based demulsification. In one embodiment, the term "purely enzymatic treatment of the microorganisms without any solvent-based extraction or chemical-based demulsification" refers to enzymatic treatment of the microorganisms a) without extraction using one or more solvents, or b) without demulsification using one or more (suitable) chemical reagents, or c) without both a) and b). Preferably, such term refers to enzymatic treatment without any exposure to extraction solvents and without any exposure to demulsifying chemical reagents. This term also excludes any other pretreatment of the grown microorganisms, e.g., oleaginous microorganisms. It should be noted that in an embodiment of the present invention, "purely enzymatic treatment" excludes the performance of any pretreatment of the grown microorganisms, which may be chemical (using one or several chemical reagents to which the grown microorganisms are exposed) or physical (changing physical conditions, such as temperature, pressure, exposure to ultrasound and / or light, irradiation with electromagnetic radiation, etc.). In one embodiment, the purely enzymatic treatment of the microorganisms is treatment of the microorganisms with a hydrolase alone or with a combination of a hydrolase and a protease / protease followed by a hydrolase. In one embodiment, the hydrolase is obtained from a fungus, preferably a filamentous fungus, more preferably a fungus of the genera Trichoderma, Aspergillus, Penicillium, Aureobasilium, and Fusarium. In one embodiment, the hydrolase is obtained from a fungus cultured in the presence of an induction system, preferably an induction system being a component of the microorganism cultured in step ii).

[0090] In one embodiment, obtaining the target product comprises collecting the target product by density-based separation, drying, flotation, solvent-based extraction, chromatography, distillation, dissociation, supercritical fluid extraction, enfleurage, press extraction, demulsification, decantation, and / or aspiration, preferably by density-based separation. In one embodiment, obtaining the target product comprises collecting the target product from the culture medium, for example, from the unsaponifiable matter of the produced microbial oil and / or from cell debris, preferably from the unsaponifiable matter of the produced microbial oil, optionally after an oil extraction step. In one embodiment, the terms "obtaining" and "collecting" are used interchangeably. In one embodiment, the target product is obtained and / or collected using any 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, semi-continuous, or continuous mode, preferably in a continuous mode.

[0091] 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 an inert or noble gas, and / or deodorization.

[0092] Hydrolysates, particularly lignocellulosic hydrolysates, typically contain organic acids such as acetic acid and sugars, making them highly advantageous for fermentation by microorganisms, such as oleaginous microorganisms. The sugars present in the hydrolysate allow for an efficient initial growth phase for the oleaginous microorganisms, while the organic acids, such as acetic acid, allow for an efficient oil-producing phase. The inventors have found that culture is particularly efficient when the hydrolysate contains xylose, acetic acid, and optionally glucose.

[0093] As used herein, terms such as "of the present invention," "in accordance with the present invention," and "in accordance with the present invention" are intended to refer to all aspects and embodiments of the present invention as described and / or claimed herein. As used herein, the term "comprising" is interpreted as encompassing both "including" and "consisting of," each of which refers to an embodiment specifically contemplated according to the present invention and, therefore, separately disclosed. As used herein, "and / or" should be interpreted as meaning that each of the two specified features or components is specifically disclosed, regardless of the presence or absence of the other feature or component. In one embodiment, the terms "at least one" and "one or more" are used interchangeably. For example, "A and / or B" should be interpreted as meaning that (i) A, (ii) B, and (iii) each of A and B are specifically disclosed, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the specified numerical value. As one skilled in the art will appreciate, the specific such deviations in the numerical values ​​for a given technical effect will depend on the nature of that technical effect. For example, natural or biological technical effects may generally exhibit greater such deviations than man-made or engineered technical effects. When an indefinite or definite article, e.g., "a," "an," or "the," is used when referring to a singular noun, the plural of that noun is also included unless otherwise specified.

[0094] Co-feeding sugars and acetate during cultivation has been shown to achieve high lipid yields, avoid fermentation-limiting problems, and achieve high lipid titers. The inventors have demonstrated that the method of the present invention can efficiently produce target products when using oleaginous microorganisms such as C. oleaginosus for SCO production. Advantageously, C. oleaginous can use phenolic compounds such as coumarate and resorcinol as carbon sources, but these are the main components of depolymerized lignin and are often harmful to microbial growth. Because C. oleaginosus is a yeast that metabolizes a variety of different sugars, including xylose, glucose, and the derivative N-acetylglucosamine, it can efficiently utilize not only hemicellulose but also monomers derived from cellulose, chitin, and lignin, the most abundant biopolymers on Earth. In addition to its metabolic flexibility, inhibitory compounds such as hydroxymethylfurfural (HMF) have little effect on C. oleaginosus's lipid productivity. The present inventors have found that microorganisms such as C. oleaginosus can advantageously use such hydrolysates efficiently if the salt content of biogenic waste streams, particularly hydrolysates from, for example, the pulp and paper industry, is reduced before using the hydrolysates for culturing the microorganisms.

[0095] Advantageously, the method for reducing the salt content of a salt-containing hydrolysate enables a hydrolysate, such as a lignocellulosic hydrolysate, containing an amount of salt that inhibits microbial growth to be converted into a hydrolysate with reduced salt content that can be used as a growth substrate for microbial growth. For example, such a reduced-salt hydrolysate, e.g., a reduced-salt spent pulp liquor, can be used as a carbon source for microbial production of a target product, such as microbial oil. Advantageously, the method for producing a target product of the present invention using a hydrolysate as a growth substrate is much more cost-effective and environmentally friendly than a method for producing a target product using glucose as a growth substrate for microbial growth. Thus, economic performance is improved.

[0096] In one embodiment, the method for producing a target product, preferably a microbial oil, comprises: i) a) providing a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, more preferably a salt-containing lignocellulosic hydrolysate; b) optionally neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate; optionally, the neutralized hydrolysate has a pH 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 hydrolysate of step a) or the neutralized hydrolysate of step b) to obtain a hydrolysate comprising precipitated salts; optionally, the hydrolysate comprising precipitated salts has a pH in the range of about pH 4 to about pH 8.5; d) adding a chelating agent to the hydrolysate of step c); e) optionally adjusting the pH of the hydrolysate 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, even more preferably about pH 6 to about pH 7; and f) Obtaining a hydrolysate with reduced salt content providing a hydrolysate having a reduced salt content by carrying out ii) culturing a microorganism, preferably an oleaginous microorganism, with a growth medium comprising or consisting of the hydrolysate provided in step i), in particular the hydrolysate obtained in step f), thereby causing the microorganism to produce the target product; preferably causing the oleaginous microorganism to produce a microbial oil; iii) optionally, enzymatically treating the microorganism, preferably an oleaginous microorganism; optionally, said enzymatically treating comprises enzymatically treating the microorganism without any solvent-based extraction or chemical-based demulsification; iv) obtaining the target product, preferably a microbial oil; Includes:

[0097] Advantageously, the hydrolysate provided in step i) results in similar or even more efficient growth characteristics compared to growth characteristics using only xylose, glucose, and / or acetate as carbon sources and is much more cost-effective. [Example]

[0098] Example 1: Materials and Methods sugar analysis Sugars and short 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 (DA) and refractive index (RI) detector was used. Separation was performed using a Phenomenex column, Rezex ROA-organic H+ 8%, with a mobile phase of 5 mM H2SO4. An isocratic flow rate of 0.5 mL / min was applied for 60 min at an oven temperature of 70 °C. RID detection was performed at 40 °C.

[0099] elemental analysis Elemental analyses were performed using a Euro EA CHNS elemental analyzer (HEKAtech Ltd.) by dynamic spontaneous combustion in a Sn boat at approximately 1800 °C, followed by gas chromatographic separation and detection using a thermal conductivity detector (TCD).

[0100] ash 2 to 4 g of the neutralized and freeze-dried lignocellulose hydrolysate was burned at 1000°C for 3 hours to produce incinerated material. After cooling overnight in a desiccator, the amount was determined by weight measurement.

[0101] dry weight The dry weights of substrate solutions and biomass samples were determined gravimetrically. To measure dry cell weight, 4 mL of fermentation broth was transferred to a pre-weighed tube, centrifuged (4500 rcf, 20 min), and washed twice with an equal volume of water or, in the case of lipid-rich cells, 50% EtOH. Alternatively, 0.5 mL of lipid-rich broth was filtered through a pre-weighed 0.2 μm filter and washed three times with 2 mL of water. Samples were frozen and lyophilized. At least technical duplicates were measured for each biological replicate.

[0102] Sulfate determination The determination of sulfate in the hydrolysate was carried out chemically by treatment with CaCO3 and BaCl2. The resulting BaSO4 precipitate was quantified gravimetrically.

[0103] lipid content For lipid content analysis, cells from fermentations were centrifuged, washed twice with 50% EtOH, and dissolved in water. Cells were mechanically disrupted using a Maximator HPL6 high-pressure homogenizer. Triplicate 7 mL aliquots of disrupted cell solution were frozen and lyophilized. Lipid extraction was performed using chloroform and methanol following the modified Bligh and Dyer method. Immediately, 100–200 mg of biomass was weighed into a glass tube and 4 mL of Cl3CH:MeOH (2:1) and 1 mL of HO (0.58% NaOH) were added. After shaking at 120 rpm for 60 minutes, the tube was centrifuged at 2000 rcf for 10 minutes, and the bottom layer was transferred to a new glass tube. An additional 3 mL of Cl3CH:MeOH (2:1) was added to the upper phase of the first tube and immediately mixed. After centrifugation, the bottom layer was transferred to the tube containing the Firth extract, and 2 mL of HO (0.58% NaOH):MeOH (1:1) was added. After mixing and centrifugation, the bottom phase was transferred to a new pre-weighed glass tube, and the solvent was evaporated under a stream of nitrogen. The lipid content was determined gravimetrically.

[0104] Fatty Acid Profile Unwashed and freeze-dried samples obtained from the fermentation process were subjected to fatty acid methyl esterification (FAME) followed by gas chromatography (GC) analysis. Known amounts of 3–10 mg were weighed into glass vials, and all subsequent steps were automated using a Gerstel Multi-Purpose Sampler MPS robotic. For quantification, a 10 g / L C19 TAG internal standard in toluene was used. First, 490 μL of toluene and 10 μL of the internal standard were added and mixed at 1000 rpm for 1 minute. Then, 1 mL of 0.5 M sodium methoxide in methanol 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 5% hydrochloric acid in methanol was added, and the mixture was heated to 80 °C with shaking at 800 rpm for 20 minutes, after which it was cooled to 5 °C. After adding 400 μL of ddH2O and mixing at 1000 rpm for 30 seconds, 1 mL of hexane was added and extracted by shaking three times at 2000 rpm for 20 seconds in a quickMix device. The sample was centrifuged at 1000 rpm for 3 minutes and cooled at 5°C. A 200 μL sample of the organic phase was transferred to a 1.5 mL vial for chromatography. Fatty acids were quantified using gas chromatography with flame ionization detection (GC-FID). GC-MS was performed to identify acids using a Thermo Scientific TRACE™ Ultra gas chromatograph coupled to a Thermo DSQ™ II mass spectrometer and a Triplus™ Autosampler injector in positive ion mode. A Stabilwax® fused silica capillary column (30 m x 0.25 mm, 0.25 μm film thickness) was used for separation. The temperature profile for the analysis was set at an initial column temperature of 50°C and increased at a rate of 4°C / min to a final temperature of 250°C. Hydrogen was used as the carrier gas at a constant flow rate of 35 mL / min. Standardization was performed using the FAMEs Marine Oil Standard (20 components from C14:0 to C24:1).

[0105] Pretreatment of LCH To precipitate sulfate in the hydrolysate, 20 g of CaCO3 was added to 1 L of LCH in a 5 L beaker with constant stirring. The hydrolysate was then left to completely release CO2. The LCH was then frozen overnight at -20°C. After thawing, the LCH was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. 20 g of KH2PO4 was added to the remaining liquid to remove excess calcium, and the pH was adjusted to 7 with 3 M NaOH. The LCH was centrifuged at 10,000 rpm for 10 minutes, sterile filtered, and used in the culture experiments.

[0106] OD measurement for process monitoring The optical density at 600 nm (OD ) of the culture samples 600 Cell growth was monitored by measuring the OD 600 The culture medium was diluted to a concentration of 0.1 to 1.

[0107] Strains and precultures 50 mL of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) in an Erlenmeyer flask containing antibiotics (0.05 g / L kanamycin, 0.1 g / L ampicillin) was inoculated with a single colony of C. oleaginosus (ATCC20509) from a YPD plate. The flask was incubated at 28°C with constant shaking at 120 rpm for 2 days. These yeast precultures were used as inoculum in various fermentation setups.

[0108] Fermentation medium Different media were used for nitrogen limitation or co-fermentation with acetate. 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, and 0.000025 g / L Calcium.

[0109] Techno-economic analysis A technoeconomic analysis (TEA) was conducted to estimate the total capital investment and operating costs for oil production from LCH using C. oleaginosus. The process was designed using an in silico consequential approach according to previous laboratory results, available literature data, and mathematical functions built into SuperPro Designer (SPD) version 10 (Intelligen, Inc., Scotch Plains, NJ, USA). A production plant with a lipid production capacity of 0.81 metric tons per hour (t / h) was created for the process simulation. Feedstock requirements and chemicals required for LCH pretreatment were estimated based on the medium composition used in this study. Equations and parameters for simulating yeast biomass production, lipid formation, and enzymatic hydrolysis were based on results obtained in this study or previous experiments. To calculate energy balances, equipment dimensions, and purchase prices, values ​​from the SPD database were supplemented with current published prices and data from the literature. This in silico plant featured several operations for LCH pretreatment, fermentation, and downstream processing, including single-cell oil recovery and recycling of waste streams and by-products. A list of the modules used in the SPD for different fermentation conditions (co-feeding of glucose and LCH with acetate, combination of continuous and consumption-based feeding) is included in the Appendix.

[0110] Of all process parameters, lipid productivity has the greatest impact on Cost 1. Lipid productivity was estimated from the lipid titer obtained from fermentations performed in a 1 L DASGIP® system after 71 hours. Lipid productivity [g / L / h] = lipid titer [g / L] / hour [h]

[0111] Lipid productivity was calculated for three conditions: glucose and acetate co-feeding, LCH and acetate co-feeding, and a combination of continuous feed and consumption-based acetate co-feeding. Equipment acquisition costs (PC) were calculated using an internal SPD function based on the equipment size. Installation cost factors for PC were included with values ​​ranging from 1.1 to 1.3. Cost factors for storage piping and site development were set at 4%, 9%, and 5%, respectively.

[0112] Capital interest was included as 6% of the total investment. Utility and labor costs, as well as waste disposal costs, were estimated based on current German prices. The costs of consumables and raw materials were set according to current market prices. Maintenance costs were 3% and insurance costs were 0.7% and were included as costs dependent on ability to pay.

[0113] Example 2: Results and Discussion Analysis of lignocellulosic hydrolysates To develop pretreatment strategies, a comprehensive analysis of lignocellulosic hydrolysate (LCH) obtained from spent pulp liquor from a pulping process was performed to gain a deeper understanding of its composition. The dark brown solution contained a small amount of insoluble particles. The pH was 1.7, and the dry mass was 247.7 ± 13.9 g / L. A high sulfate content of 19.4 ± 2.0 g / L was determined. HPLC analysis was used to quantify sugars, organic acids, and furans. HPLC analysis showed that the major sugar in the hydrolysate was xylose at 77.05 ± 2.46 g / L, with small amounts of other sugars present, 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), resulting in a total sugar content of 115.60 ± 4.99 g / L. The total organic acid concentration was 16.83 ± 1.43 g / L, with acetic acid being the predominant organic acid at 12.34 ± 1.20 g / L. Significant amounts of hydroxymethylfurfural (HMF) at 4.53 ± 0.59 g / L and furfural at 0.68 ± 0.04 g / L were detected. Total ash was 0.70 ± 0.01 g / L, with low concentrations of phosphorus (0.035 g / L) and undetectable nitrogen. The remainder of the material was derived from lignols and lignans, as well as other plant metabolites, totaling 89.95 ± 22.97 g / L. The hydrolysate is a waste stream from the industrial production of cellulose fiber using hardwood as a raw material and an acid pulping process. Chemical hydrolysis of lignocellulose revealed the presence of sugar monomers, organic acids, lignols, and lignans. The presence of phenolic compounds and furans can be problematic for fermentation processes due to their generally inhibitory effect on microbial growth. The high sulfate content and acidic pH result from chemical hydrolysis and therefore must be neutralized before LCH can be used in yeast fermentation. However, the high content of xylose and other sugars makes LCH an ideal carbon source. The very low amounts of nitrogen, phosphorus, and other elements such as sulfur, magnesium, or calcium require additional nutrient supplementation before using the hydrolysate in a fermentation medium.

[0114] Pretreatment of lignocellulosic hydrolysate To convert waste stream LCH into a carbon source suitable for yeast fermentation, the challenges of low pH and a lack of essential nutrients needed to be addressed with a suitable pretreatment strategy. In the first step, LCH was neutralized and sterilized using different methodologies. It was then mixed with essential salts, buffer compounds, nitrogen sources, nutrients, and trace elements. The resulting medium was tested at a small scale for culturing C. oleaginosus in 24-well deep plates. Neutralization with NaOH from pH 1.7 to pH 7 resulted in high salt concentrations and consequently reduced growth. To reduce the amount of sulfate, CaCO3 was added and allowed to react to form partially insoluble CaSO4. Addition of 20 g / L CaCO3 proved to be slightly excessive, but was only able to raise the pH to 5.6. Therefore, NaOH was used for titration to neutrality, resulting in a solution with lower salt content and better growth behavior. Various thermal methods were tested for sterilization, but all resulted in the formation of insoluble particles. Therefore, filtration was used for sterilization. Nevertheless, insoluble particles formed again after the addition of other medium compounds such as buffer salts, vitamins, nitrogen, and trace elements, as well as during cultivation. In another approach, LCH was filtered through activated carbon to reduce the content of toxic phenols and furans. Unfortunately, this strategy also reduced the amount of available carbon source, resulting in a dramatic decrease in growth.

[0115] The formation of insoluble particles was observed with the fermentation time, which was due to the high [Ca +2 The calcium phosphate derived from the fermentation of LCH is one of the contributing factors. Therefore, LCH was titrated with KH2PO4 to remove excess calcium in the form of insoluble Ca3(PO4)2 before fermentation. Because this process resulted in an acidic solution, a final neutralization with NaOH was performed after KH2PO4 treatment. This pretreated LCH resulted in growth similar to that of the control containing xylose, glucose, and acetic acid (XGA). Details of the pretreatment experiment are provided in the Supplementary Data.

[0116] In this way, the lignocellulosic hydrolysate was neutralized without increasing the salt concentration or forming insoluble materials even when buffer salts or trace elements were added. The sugar content was maintained, making it suitable as a carbon source for C. oleaginosus. This optimized pretreatment strategy was applied to all LCHs used in the fermentation in this study.

[0117] Nitrogen-limited fermentation Fermentation under nutrient limitations, such as phosphate or nitrogen stress, is a common strategy for inducing lipid accumulation in oleaginous yeast. Therefore, nitrogen-limited fermentation was performed using LCH as the carbon source. To understand the effects of lignocellulosic compounds on yeast metabolism, a control medium was designed. The control contained only the major sugars of LCH in the respective ratios: xylose (8.28%), glucose (1.03%), and acetic acid (0.69%), abbreviated as XGA. Fed-batch fermentation was performed in a DASGIP® system at a maximum volume of 1 L. A 500 mL starting medium was used with a 3% sugar content from either LCH or XGA. The nitrogen content was adjusted to a C / N ratio of 120. Pretreated pure LCH or XGA was fed at a rate of 10 mL / h starting after 12 h and continuously at 5 mL / h from 36 to 60 h. Fermentation results showed that the biomass formed in LCH and XGA was 7.02±0.88g / L and 16.65±0.24g / L, respectively, indicating better growth performance in XGA medium. Visual observation under a microscope revealed the formation of several lipid droplets within each cell.

[0118] The inhibition of growth on lignocellulosic hydrolysate compared to growth on XGA can probably be explained by the higher concentrations of inhibitory furans and other phenolic compounds. In general, nitrogen limitation induces lipid formation but also limits biomass formation, since nitrogen is required for protein synthesis and other metabolic processes.

[0119] Acetic acid-based fermentation using sugar as a carbon source As previously described, enhanced growth and increased lipid accumulation in C. oleaginosus have been demonstrated by combining a sugar-containing starting medium with a consumption-based acetate feed. Glucose, xylose, and a mixed control of xylose, glucose, and acetate (XGA) were tested as starting sugars with a consumption-based acetate feed. Because of the consumption-based feed, the acetate concentration remained constant, and sugars were consumed within the first 40 h after inoculation. After complete sugar consumption, biomass continued to increase, reaching 35.11 ± 1.11 g / L, 39.95 ± 4.59 g / L, and 39.00 ± 0.76 g / L for glucose, xylose, and XGA by 71 h. 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 2a). The same was true for the carbon conversion rates from substrate carbon to lipid carbon, which were 20.4 ± 2.7%, 23.0 ± 1.4%, and 23.5 ± 0.7% for glucose, xylose, and XGA (Figure 2b). The fatty acid profile of the oil is shown in Figure 2c. The most abundant fatty acid was C18:1 at approximately 54%, followed by C16:0 (~24%), 18:0 (~15%), and C18:2 (~6%), with trace amounts of C18:3, C16:1, and C22:0 each at less than 0.3%.

[0120] Fermentation using glucose and xylose as sole carbon sources showed similar growth behavior to that using XGA, indicating good simultaneous uptake of both glucose and xylose. This is a significant advantage for C. oleaginosus as a fermenting strain, as it allows the full potential of biomass as a carbon source to be exploited. Furthermore, acetate can be efficiently diverted to lipid synthesis metabolism via acetyl-CoA synthase. This results in a more efficient metabolic pathway than starting from sugar molecules, making it an effective method for increasing lipid biosynthesis in C. oleaginosus.

[0121] Acetic acid-based fermentation of lignocellulosic hydrolysates It has been shown that single-cell oil can be successfully produced from refined sugars by acetic acid-based fermentation. However, to make the process more commercially attractive, the waste stream LCH mentioned above was applied as the carbon source instead of expensive sugars. Lignocellulosic hydrolysate is the spent pulp liquor from acidic hardwood pulping, and beneficially, this process also produces acetic acid by cleaving the acetyl group from the xylose of the hardwood. Therefore, the second carbon group in this process, acetic acid, can be produced in situ.

[0122] Natural products, such as lignocellulosic hydrolysates, contain furans and phenols that can have inhibitory effects on organisms. To determine the optimal balance between carbon supply and the toxicity level of inhibitory compounds, fermentations were performed using three different starting concentrations of LCH. Hydrolysate amounts were used to achieve bioavailable carbon concentrations of 3%, 5%, and 7% in the starting medium. Fermentations at the highest LCH concentration, corresponding to 7% sugars, resulted in a longer lag period and slower overall growth, with a phagocytosis rate of 1.21 g / L / h compared to 1.33 g / L / h at 3%. Dry biomass formation was 58.90 ± 1.05 g / L at 3%, followed by 39.64 ± 6.30 g / L and 51.92 ± 0.18 g / L at 7% and 5%. Therefore, increasing the concentration to 5% resulted in a slightly longer lag period and a slight decrease in biomass after 3 days of fermentation. Lipid titers were similar in the two lower concentrations (25.59±1.79 g / L and 25.75±2.02 g / L) but lower in the 7% LCH (19.83±1.38 g / L).

[0123] A starting carbon source concentration of 3% resulted in the shortest lag period, the fastest growth rate, and the best overall biomass formation. This explains the longer lag period and slower growth rate at higher concentrations due to the higher concentrations of inhibitory furans and phenols. Therefore, a starting concentration of 3% was used for all subsequent experiments.

[0124] Acetic acid-based fermentation of lignocellulosic hydrolysates under optimized conditions Optimized conditions for the fermentation of C. oleaginosus in LCH were evaluated by fermentation of three biological replicates. A starting carbon content of the hydrolysate equivalent to 3% sugars and acetic acid were used. After 71 h, a high biomass formation of 55.73 ± 5.20 g / L was achieved, a 7-fold increase compared to nitrogen-limited fermentation of LCH. Furthermore, biomass formation was 50% higher with LCH than with glucose or xylose under comparable reaction conditions (Figure 1). However, sugar consumption showed a similar profile to the fermentation process using a single sugar. The lipid titer after 71 h was twice as high (42.1 ± 1.7 g / L) as glucose fermentation (18.5 ± 3.6 g / L) (Figure 2a). Carbon conversion was 33.6% from substrate carbon to lipid (Figure 2b), with lipid per dry biomass being 76.05 ± 9.04%. The fatty acid profile did not show any significant changes compared to the lipid profile of fermentations with single sugars or XGA (Fig. 2c).

[0125] The optimized fermentation strategy for C. oleaginosus in LCH with a consumption-based supply of acetic acid showed the best performance in terms of biomass accumulation, lipid titer, and lipid yield, with only minor changes in lipid profile. The improved performance when LCH was used as a substrate may be due in part to the presence of lignols and lignans, which C. oleaginosus can partially metabolize. Taken together, this waste stream combined with oleaginous yeast is a promising new sustainable single-cell oil production process. It combines the application of a waste stream rich in xylose, lignols, and lignans with a fermentation strategy that results in high lipid yields.

[0126] Screening of various strategies for supplying LCH Consumption-based acetate feeding results in lipid productivity as high as over 85%. Nevertheless, compared to acetate, LCH is a waste product and a cheap feedstock with limited value-creation options. To increase LCH uptake by oleaginous yeast relative to acetate consumption, different fermentation modes were compared. Five fermentation conditions were compared simultaneously in duplicate using a DASbox® 12 parallel bioreactor system. All conditions utilized consumption-based feeding with 50% (v / v) acetate.

[0127] As the first fermentation mode, we tested a combined feeding of a mixture of LCH and acetic acid. LCH was added to 50% (v / v) acetic acid to achieve final concentrations of either 10% (v / v) or 50% (v / v). The biomass of cells fed with the 50:10 and 50:50 acetic acid:LCH mixtures (51.5 ± 3.7 g / L and 59.6 ± 1.2 g / L) exceeded that of cells fed with acetic acid alone (50.8 ± 0.1 g / L) after 65 h, with the 50:50 co-feeding being the highest (Figure 3). The lipid titer after 65 h was measured at 28.4 ± 0.4 g / L and 30.4 ± 1.4 g / L for the 50:10 and 50:50 feeds, respectively, compared with 25.2 ± 3.1 g / L in the control setup (Figure 3).

[0128] As an additional feeding strategy, pure pretreated LCH was continuously fed into the reactor throughout the fermentation process at two feed rates: 0.5 mL / h or 1 mL / h starting 12 h after inoculation, in addition to a consumption-based feed of 50% (v / v) acetic acid. Dry biomass values ​​reached after 65 h were 50.4 ± 2.2 g / L and 50.4 ± 4.6 g / L at 0.5 mL / h and 1 mL / h, respectively. Maximum lipid titers were 30.5 ± 2.4 g / L at 0.5 mL / h and 26.3 ± 2.7 g / L at 1 mL / h, exceeding the control (25.2 ± 3.1 g / L).

[0129] To select the setting with the highest LCH turnover, the percentage of LCH consumed as a percentage of total carbon consumed was calculated. The strategy with 1 mL / h continuous feeding yielded the highest percentage of consumed LCH (21.3 ± 2.9%), followed by 50:50 acetate:LCH (12.5%) and 0.5 mL / h continuous feeding (10.4 ± 1.2%). The difference between the replicates with continuous LCH feeding can be explained by the different amounts of acetate consumed. Because the LCH occupancy rate with 50:50 acetate:LCH remained constant throughout the fermentation, it was not possible to calculate a standard deviation.

[0130] Additionally, the carbon conversion rate from fed carbon to lipids was calculated for all conditions. In comparison, the setup using continuous feeding yielded the highest carbon conversion rates: 19.8 ± 2.0% at 0.5 mL / h and 18.8 ± 0.2% at 1 mL / h. The control showed a conversion rate of 16.4 ± 0.2%, while co-feeding with an acetate:LCH mixture resulted in a conversion rate of 16.7 ± 0.1% at a 50:10 ratio and 16.7 ± 0.5% at a 50:50 ratio. The control and the two best feeding strategies are visualized in Figure 3.

[0131] The overall evaluation of the feeding strategies focused on the percentage of LCH consumed and lipid titer. In this regard, the two best feeding strategies were 50:50 acetate:LCH cb-feeding and co-feeding with a continuous feed of 1 mL / h LCH. By providing more LCH during the fermentation process, these strategies were able to significantly increase the percentage of LCH. At the same time, the starting concentration of inhibitory compounds was not increased, preventing the prolonged induction period observed with high LCH concentrations in the DASGIP® system (3.5).

[0132] Co-fermentation with continuous feeding of lignocellulosic hydrolysate Using a 1 mL / h continuous LCH feed in the DASbox® configuration resulted in the highest percentage of consumed LCH relative to the total carbon source and the second-highest carbon conversion rate of all conditions tested. Therefore, this condition was selected for the DASGIP® system. The 1 L scale also allowed for a more direct comparison of fermentation production with other conditions tested in the DASGIP® system. When a consumption-based feed of 90% acetic acid was used in conjunction with a continuous LCH feed of 3.3 mL / h, biomass accumulation reached 29.0 ± 5.87 g / L after 71 hours, and the lipid titer was 22.7 ± 1.95 g / L. HPLC analysis showed that carbon substrates were consumed evenly throughout the fermentation, with the exception of xylose, which accumulated slightly, from 15.5 ± 1.11 g / L after 65 hours to 17.44 ± 1.85 g / L after 71 hours. The accumulation of xylose was likely due to a slightly too fast feed rate, resulting in an excess sugar supply and an increase in the concentration of inhibitory compounds. The proportion of LCH in total consumed carbon was increased from 10.9 ± 31.4% to 37.6 ± 2.3%. This increased the relative consumption of LCH, a more cost-effective feedstock than acetate, by C. oleaginosus. At the same time, the substrate carbon converted to lipid carbon remained at a similar level (32.0 ± 1.5%) compared to 33.5 ± 2.1% in the fermentation using only acetate as a consumption-based feed. As detected by Nile Red-stained cells shown in Figure 4, the cell phenotype, growth behavior, and lipid droplet formation were similar for both fermentation strategies. After 24 h of fermentation, cells had already begun to form several lipid droplets, exhibiting an oval shape. After 71 h of fermentation, cells primarily consisted of one or up to two lipid droplets filling the majority of the cell, and the cells had a ring-shaped morphology. Overall, when using the continuous feeding operation mode, the substrate to lipid conversion of C. oleaginosus was comparable to that of a consumption-based feed with acetate. To further optimize the fermentation mode, the acetate to sugar feed ratio must be adapted to avoid sugar accumulation, as indicated by the xylose concentration after 71 h.

[0133] Techno-economic analysis Aside from the cumulative results of this study, we conducted a consequential techno-economic analysis (TEA) to assess the economic feasibility of a commercial-scale production plant using LCH as an industrially relevant feedstock. Two different modes of LCH fermentation from 1 L-scale DASGIP® experiments were implemented and compared for potential cost savings and maximization of LCH utilization: acetate consumption-based feeding (LCHcb-feeding) using LCH as the starting carbon source and a combination of LCH consumption and continuous feeding (LCHco-feeding). Both fermentation modes were compared with an operating configuration using glucose as the starting carbon source for acetate consumption-based feeding (glucosecb-feeding). Simulations showed that LCHcb-feeding had the lowest CAPEX at $24.4 million, compared with $34.5 million for LCHcb-feeding and $33.9 million for LCHco-feeding. The increased capital costs were due to the different fermenter volumes and vessel volumes in each model. To simulate individual lipid productivity, different fermenter combinations had to be used for the fermentation strategies due to the different residence times in the fermentation conditions. Capital costs remained the same, except for the vessel quantity and volume. Depreciation was calculated at 10% over 10 years, resulting in $2.4 million, $3.4 million, and $3.4 million for the LCHcb-fed, glucosecb-fed, and LCHco-fed systems, respectively. The quantity and volume of vessels also resulted in differences in operating costs, particularly electricity costs (LCHcb-supply: $2.3 million, glucosecb-supply: $4.8 million, LCHco-supply: $4.0 million), cooling water costs (LCHcb-supply: $0.5 million, glucosecb-supply: $1.0 million, LCHco-supply: $0.9 million), labor costs (LCHcb-supply: $1.5 million, glucosecb-supply: $2.2 million, LCHco-supply: $1.8 million), maintenance costs (LCHcb-supply: $0.6 million, glucosecb-supply: $0.8 million, LCHco-supply: $0.8 million), and raw material costs (LCHcb-supply: $12.4 million, glucosecb-supply: $13.2 million, LCHco-supply: $10.9 million). The final costs of yeast oil calculated from the in silico model were $3,500 / ton, $4,500 / ton, and $4,000 / ton, respectively (Fig. 5a).All calculated pricing structures are within the current price range for organic palm oil ($2,500–$3,000 / ton). Further optimization of the fermentation process and the resulting lipid conversion rate could potentially lower the retail price of the LCH process in the future. However, the consequential TEA applied in this study focuses more on the relative changes in input and output conditions. Therefore, the calculated price per ton should be considered as a range. Prices of other vegetable oils used in biofuel production, such as canola oil, are well below $1,000 / ton. However, utilizing industrial waste streams avoids competition with edible oils, thereby enhancing the sustainability of the LCH-based oil production process presented in this study.

[0134] Furthermore, because acetic acid accounts for the majority of raw material costs, we conducted a sensitivity analysis considering different acetic acid prices (LCHcb-fed: 94%, glucosecb-fed: 90%, LCHco-fed: 86%). Because acetic acid prices in Western Europe have fluctuated between $600 / ton and $1,500 / ton from 2012 to 2021, the cost of yeast oil per ton is highly dependent on the current acetic acid price. In the sensitivity analysis, acetic acid costs were assumed to be $600 / ton and $1,500 / ton, compared with the value of $1,000 / ton used in the TEA. As shown in Figure 5b, the LCHcb-fed model responded most strongly to price changes. A price decrease to $600 / ton reduced production costs by 19.3% ($3,100 / ton), while an increase in acetic acid prices to $1,500 / ton increased production costs by 24.1% ($4,800 / ton). For LCHco-supply, total production costs decreased by 13.5% ($3,800 / ton) and increased by 16.9% ($5,100 / ton) for each acetic acid price. The LCHco-supply model was less sensitive to changes in acetic acid price due to the larger proportion of LCH in yeast-available carbon. However, overall, LCHcb-supply exhibited the lowest production costs relative to acetic acid price declines or increases, despite experiencing the highest percentage change in acetic acid price. The primary reason for acetic acid price volatility is that the United States and China supply 17% and 55% of the world's acetic acid, respectively. Problems or disruptions in these supply chains can significantly reduce availability, significantly impacting pricing. To avoid such dependencies, it would be best to produce and supply acetic acid locally, even locally.

[0135] In summary, our lab-scale experiments for biotechnological valorization of industrial waste streams using C. oleaginosus were validated in an in silico fermentation setting. Compared to glucose, LCH was demonstrated to be cost-effective for the production of single-cell oil at a biorefinery scale. Such a resource-efficient approach may lead to a more self-sustaining, circular, and sustainable economy in the future.

[0136] Example 3: Comparison of pretreatment methods Various pretreatment conditions were tested, and the hydrolysate was used to formulate a fermentation medium. 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 g / L 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. The medium was used to cultivate the oleaginous yeast, Cutaneotrichosporon oleaginosus, in a 24-well deep plate with shaking incubation. After 72 hours of culture, the cell density was measured as OD600 using a plate reader EnSpire 2. Cell morphology and the formation of insoluble particles were observed using a microscope at 100x magnification.

[0137] In the absence of significant addition of phosphate-containing compounds, cultures showed the formation of insoluble particles during incubation. In many cases, these particles were not present before incubation because the samples were sterile filtered. Growth behavior could be improved by several pretreatment strategies, including the addition of phosphate-containing compounds.

[0138] [Table 1-1]

[0139] [Table 1-2]

[0140] Example 4: Elimination of salts and insoluble materials in a novel pretreatment Acidic (pH 1-2) lignocellulosic hydrolysate was first treated with CaCO3 (20 g / L), and the insoluble particles were separated by centrifugation and weighed. The same procedure was repeated after adding KH2PO4 (20 g / L) and neutralizing with NaOH (18 g / L). The amount of material removed as precipitate is shown for each step in Figure 7. After this pretreatment, the solution became lighter in color, while the precipitate was dark, suggesting that some of the lignin compounds were removed from the solution by this treatment. The results of this example are shown in Figure 7.

[0141] Example 5: Titration of lignocellulosic hydrolysate after CaCO treatment The CaCO3-treated lignocellulosic hydrolysate was titrated with KH2PO4 and pelleted. 50 mg of KH2PO4 was added in increments, and after each addition, the solution was centrifuged in a new tube. As can be seen in Figure 8, a dark precipitate formed at each step. The color indicates that this treatment separated material from the hydrolysate.

[0142] Example 6: Comparison of pretreatments on appearance The hydrolysate and media preparations were compared: one with CaCO3 only, and the other with CaCO3 and KH2PO4, followed by neutralization with NaOH. Because the media salts contained phosphate, three different conditions were added for each treatment. One condition contained no media; the next condition contained trace elements, vitamins, and yeast extract but no salts; and the final condition contained complete media. After media formulation, all solutions were autoclaved (20 min, 121°C). Samples were centrifuged and observed for the formation of insoluble material.

[0143] [Table 2]

[0144] The results are shown in Figure 9.

[0145] Example 7: Comparison of growth and cell morphology associated with lipid production Lignocellulosic hydrolysates were subjected to various pretreatments, and their cell morphology and growth behavior were compared. Cell morphology, based on the size and number of lipid bodies contained, provides an indication of potential lipid accumulation. Various pretreatment conditions were compared with a model substrate containing xylose, glucose, and acetate in various amounts of actual hydrolysate. The pretreated hydrolysates and the model substrate were incorporated into a nitrogen-limited (C / N ratio 50) culture medium and cultured in shake flasks at 28°C for 120 hours.

[0146] [Table 3]

[0147] The growth curves obtained under different pretreatment conditions are shown in FIG. 10, and the cell morphology is shown in FIG.

[0148] conclusion Here, we present a novel strategy for producing microbial lipids, e.g., by the oleaginous yeast C. oleaginosus, using lignocellulosic hydrolysate and acetic acid as the primary carbon source. To this end, fermentations were performed at 0.25 L and 1 L scales, achieving a maximum lipid titer of 42.1 ± 1.7 g / L under optimized conditions. LCH consumption increased up to 37.6 ± 2.3% of total carbon consumed. Furthermore, a techno-economic analysis demonstrated the economic benefits of using LCH over glucose. Finally, we present a sustainable and economical strategy for generating single-cell oil as an alternative platform for producing biofuels and oleochemicals.

[0149] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. 1. A method for reducing the salt content of a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, comprising: a) providing a salt-containing hydrolysate, preferably a salt-containing biogenic hydrolysate, more preferably a salt-containing lignocellulosic hydrolysate; b) optionally neutralizing the pH of the hydrolysate of step a) to obtain a neutralized hydrolysate; optionally, said neutralized hydrolysate has a pH in the range of about pH 4 to about pH 8; c) CaCO ３ , Ca(OH) ２ , CaO, MgO, MgCO ３ , and / or Mg(OH) ２ , preferably CaCO ３ and / or Ca(OH) ２ to the hydrolysate of step a) or the neutralized hydrolysate of step b) to obtain a hydrolysate comprising precipitated salts; optionally, said hydrolysate comprising precipitated salts has a pH in the range of from about pH 4 to about pH 8.5; d) adding a chelating agent to the hydrolysate of step c); e) optionally adjusting the pH of the hydrolysate 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, even more preferably about pH 6 to about pH 7; f) obtaining a hydrolysate with a reduced salt content; A method comprising:

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

3. the hydrolysate provided in step a) is a hydrolysate obtained by physical, chemical, enzymatic and / or biological treatment of a substrate, preferably biomass; Preferably, the physical treatment is selected from mechanical treatment, pressure treatment, heat treatment, steam explosion, combustion, and combinations thereof; the chemical treatment is selected from alkaline treatment, acidic treatment, and treatment at neutral pH; preferably, the chemical treatment is treatment with any of salt, acid, peroxide, and any combination thereof, preferably treatment with sulfide, sulfite, and / or bisulfite; the enzymatic treatment is treatment with one or more enzymes selected from hydrolases, preferably endo- and exo-glycoside hydrolases, glycosylases, peptidases, such as endo- and exo-peptidases, proteases, amylases, dehydrogenases, peroxidases, ligninolytic enzymes, and any combination thereof; 3. The method according to claim 1 or 2, wherein the biological treatment is a treatment with a microorganism, preferably a treatment with a microorganism selected from bacteria, yeasts and fungi.

4. the hydrolysate provided in step a) contains salt in an amount ranging 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 10. The method of any one of the preceding claims, comprising carbon in an amount ranging from about 0.1% to about 65% by weight.

5. 10. The method of any one of the preceding claims, wherein the hydrolysate provided in step a) comprises salts selected from sulfates, sulfides, sulfites, nitrates, nitrites, chlorides, and any combination thereof; preferably, said salts comprise sulfates, sulfides, and / or sulfites.

6. 10. The method of any one of the preceding claims, wherein the hydrolysate provided in step a) comprises lignols, lignans, organic acids, and / or sugars; optionally, the sugars comprise xylose, glucose, mannose, and / or galactose; preferably, the sugars comprise monosaccharides, preferably xylose; and preferably, the organic acids comprise acetic acid.

7. The chelating agent is M ３ P.O. ４ , M ２ HPO ４ , M.H. ２ P.O. ４ , M.H.P.O. ４ , MPO ４ , (NH ４ ) (H ２ P.O. ４ ), and any combination thereof, wherein M is a metal; Preferably, the chelating agent is Na ３ P.O. ４ , Na ２ HPO ４ , NaH ２ P.O. ４、 K ３ P.O. ４ , K. ２ HPO ４ , K.H. ２ P.O. ４ , Ca(H ２ P.O. ４ ) ２ , CaHPO ４ , Ca ３ (P.O. ４ ) ２ , (NH ４ ) (H ２ P.O. ４ ), and Na ３ P.O. ４ Selected from: More preferably, the chelating agent is KH ２ P.O. ４ 10. The method of any one of the preceding claims, wherein:

8. e) adjusting the pH of the hydrolysate, wherein the adjustment is carried out with a pH adjusting agent such as NaOH, KOH, CH ３ 10. The method according to any one of the preceding claims, carried out by adding COOH, HCl, KCl, sulfuric acid, phosphoric acid, acetic acid, hydrocyanic acid, carbonic acid, or any combination thereof, preferably NaOH and / or KOH, to the hydrolysate of step c) and / or step d).

9. Preferably, the method further comprises sterilizing the reduced salt content hydrolysate obtained in step f) to obtain a sterilized reduced salt content hydrolysate; 10. The method according to any one of the preceding claims, wherein the sterilization preferably comprises heat sterilization, ultra-high temperature treatment, and / or sterile filtration.

10. 1. A method for producing a target product, preferably a microbial oil, comprising: i) providing a hydrolysate with reduced salt content by carrying out a method according to any one of the preceding claims; ii) culturing a microorganism, preferably an oleaginous microorganism, with a growth medium comprising or consisting of the hydrolysate provided in step i), thereby causing said microorganism to produce a target product; preferably causing said oleaginous microorganism to produce a microbial oil; iii) optionally, enzymatically treating said microorganisms, preferably said oleaginous microorganisms; optionally, said enzymatically treating comprises enzymatically treating said microorganisms without any solvent-based extraction or chemical-based demulsification; iv) obtaining the target product, preferably a microbial oil; A method comprising:

11. 11. The method of claim 10, wherein the microorganism is an oleaginous microorganism, preferably an oleaginous yeast, more preferably a species of the genus Cutaneotrichosporon, even more preferably Cutaneotrichosporon oleaginosus.

12. the target product is selected from microbial oils, glycerol, free fatty acids, mono-, di- 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; 12. The method of claim 10 or 11, wherein the target product preferably comprises a microbial oil.

13. the reduced salt hydrolysate provided in step i) comprises acetic acid and / or xylose; A method according to any one of claims 10 to 12, wherein the hydrolysate preferably comprises or consists of a lignocellulosic hydrolysate.

14. The culturing in step ii) comprises adding acetate and / or a carbon source other than acetate to the growth medium; Preferably, the acetic acid is added in the form of a feed comprising or consisting of acetic acid, 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; 14. The method of any one of claims 10 to 13, wherein the feed optionally further comprises a carbon source other than acetate.

15. the growth medium comprises a sugar, such as xylose, in an amount ranging from about 0.1 g / L to about 250 g / L, preferably <100 g / L; and / or 15. The method of any one of claims 10 to 14, wherein the growth medium comprises acetic acid in an amount ranging from about 0.01 g / L to about 100 g / L, preferably from about 1 g / L to about 50 g / L, and more preferably from about 5 g / L to about 10 g / L.

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