Method for producing a microbial oil
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- OMV DOWNSTREAM GMBH
- Filing Date
- 2026-02-03
- Publication Date
- 2026-08-06
Smart Images
Figure EP2026052728_06082026_PF_FP_ABST
Abstract
Description
[0001] Method for producing a microbial oil
[0002] The present invention relates to a method for producing a microbial oil from biomass . In addition, the invention relates to methods for producing olefins, methods for producing biodiesel as well as methods for producing sustainable aviation fuel (SAF) .
[0003] The quest for sustainable alternatives to fossil fuels has intensified in recent years due to the escalating concerns over climate change and the depletion of non-renewable resources . In this context, microbial oil derived from oleaginous microorganisms is gaining more and more attention due to its potential to serve as a renewable source for the production of biodiesel and sustainable aviation fuel (SAF) as well as for other industrial applications .
[0004] Microbial oil is typically produced from biomass such as agricultural residues like wheat straw, rice straw, corn stover, or sugarcane bagasse, which are rich in carbohydrates . As a first step, the biomass may be pretreated to hydrolyze complex carbohydrates into simpler sugars, which the microorganisms can consume as a carbon source . Pretreatment may be achieved by a wide range of different processes, including steam explosion, dilute acid hydrolysis, enzymatic pretreatment, hot water treatment, or standard pulp industry processes . The pretreated biomass then serves as a fermentation substrate for cultivating oleaginous microorganisms, wherein the sugars contained in the pretreated biomass are metabolically converted into lipids . Oleaginous microorganisms, such as certain species of yeast, algae or bacteria, can accumulate a significant proportion of their cell mass as lipids, typically at least 20 % (w / w) . After fermentation, the cells are typically lysed using mechanical, chemical, or biological processes and the lipids are extracted to obtain a microbial oil . The microbial oil can then be further processed, e . g. , in a transesterification step to produce biodiesel, or in a hydroprocessing step to produce SAF. Processes for the production of microbial oils from biomass are known, e . g. , from WO 2008 / 151149 A2, WO 2011 / 130573 Al,
[0005] WO 2015 / 086780 Al, WO 2011 / 073781 A2 , WO 2016 / 108185 Al,EP 3 009 515 Al, and CN 112 941 120 B .
[0006] However, despite the obvious environmental benefits, the commercial viability of microbial oil production still faces challenges, primarily related to the efficiency and overall cost-effectiveness of existing processes . Therefore, there remains a big need to improve the efficiency of such processes to enable their large-scale deployment in the interest of replacing fossil fuels .
[0007] It is an obj ect of the present invention to address this need, in particular, to provide new and improved methods for producing a microbial oil from biomass .
[0008] Therefore, the present invention provides a method for producing a microbial oil from biomass, the method comprising the following steps :
[0009] - Step A: Providing the biomass;
[0010] - Step B : Pretreating the biomass to obtain a fermentation substrate, wherein the pretreating preferably comprises a solid-liquid separation step for removing insoluble components;
[0011] - Step C : Culturing an oleaginous microorganism in a fermentation broth comprising the fermentation substrate, wherein lipids are produced within cells of the oleaginous microorganism;
[0012] - Step D: Contacting the cells from the fermentation broth with a lysis composition under lysis conditions, wherein the lysis composition comprises a fermentable acid, thereby obtaining a lysis mixture, wherein at least a part of the produced lipids is released from the cells;
[0013] - Step E : Isolating at least a part of the released lipids from the lysis mixture to obtain the microbial oil and a lipid-depleted lysis mixture;
[0014] wherein at least a part of the lipid-depleted lysis mixture from Step E is withdrawn as a recycle stream, and wherein at least a part of the recycle stream comprising residual fermentable acid from the lysis composition is included in the fermentation broth in Step C .In the context of the invention, it was found that the efficiency of the overall process can be significantly improved when a fermentable acid is used for lysis (Step D) and when a part of said fermentable acid is recycled as part of a recycle stream to the fermentation broth (Step C) . Thus, the recycle stream withdrawn after the lipids have been isolated from the lysis mixture comprises a part of the fermentable acid that was added to the cells in Step D as part of the lysis composition. In this way, the acid that was used for cell lysis in Step D can be used as a carbon source for the oleaginous microorganisms in Step C and be metabolically converted into lipids . This significantly reduces waste and increases the overall efficiency, as at least a part of the acid used for lysis is consumed in the process rather than going to waste . As used herein, the recycle stream or part thereof "comprising residual fermentable acid from the lysis composition" may also be referred to as the respective stream comprising at least a part of the fermentable acid that was introduced as part of the lysis composition in Step D.
[0015] The acid comprised in the lysis composition is a fermentable acid. As used herein, "fermentable" means that the acid can be used as a carbon source by the oleaginous microorganisms and be metabolically converted into lipids . When a fermentable acid is used for cell lysis in the inventive process, and when said fermentable acid is recycled as part of the recycle stream to the fermentation broth (Step C) , it can be consumed by the oleaginous microorganisms to produce more lipids .
[0016] Preferably, the fermentable acid is an organic acid. Organic acids can typically be fermented by oleaginous microorganisms and are therefore advantageous for the reasons described above . In a preferred embodiment, the fermentable acid is a Ci-Ce organic acid. In this context, a "Ci" organic acid refers to an organic acid having i carbon atoms . Small organic acids, in particular Ci-Ce organic acids, can typically be particularly well metabolized by oleaginous microorganisms and are therefore especially preferred. Even more preferably, the fermentable acid is a C1-C5 organic acid, even more preferably a C1-C4 organic acid.
[0017] Preferably, the fermentable acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-butyric acid,iso-valeric acid and mixtures thereof . These acids are particularly well suited as carbon sources for the oleaginous microorganisms .
[0018] It has turned out to be particularly advantageous, when the lysis composition comprises mixtures of fermentable acids . It was unexpectedly observed that this can further increase lysis efficiency. Without wishing to be bound to a particular theory, the inventors believe that when a single type of acid is used for cell lysis, and when the cells were already exposed to this same acid during the fermentation step, the cells can develop a resistance against lysis by this type of acid. Using mixtures of different acids can reduce the likelihood of such resistances developing and can therefore increase lysis efficiency.
[0019] Thus, in a preferred embodiment, the lysis composition comprises at least two distinct fermentable acids . Each of the distinct fermentable acids is preferably as defined above . Thus, each of the two distinct fermentable acids preferably is a Ci-Ce organic acid, more preferred a C1-C5 organic acid, even more preferred a C1-C4 organic acid. Preferably, each of the two distinct fermentable acids is selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-butyric acid and iso-va-leric acid.
[0020] The at least a part of the recycle stream comprising residual fermentable acid from the lysis composition is preferably added to the fermentation broth during the culturing in Step C . However, the recycle stream may also be included in the fermentation broth indirectly, by adding it to the biomass in Step B . In this case, fermentable acid from the recycle stream can be included in the fermentation broth as part of the fermentation substrate . Thus, in a preferred embodiment, the pretreating in Step B comprises mixing at least a part of the recycle stream with the biomass . This embodiment is particularly advantageous, since the acid contained in the recycle stream can act as a pretreatment agent, leading to acid hydrolysis of complex carbohydrates contained in the biomass into simpler sugars, which the oleaginous microorganisms can use as fermentation substrate . At the same time, at least a part of the fermentable acid that was added to the biomass as part of the recycle stream can becarried over to the fermentation broth in Step C, providing an additional carbon source for the microorganisms . Therefore, the acid can fulfil three different roles in this embodiment : it is used as lysis agent to lyse the cells of the oleaginous microorganism; it is used as a pretreatment agent to hydrolyze complex carbohydrates contained in the biomass to fermentable sugars; and it is used itself as a carbon source for the oleaginous microorganism to produce more lipids . This significantly increases efficiency of the overall process and reduces waste .
[0021] In a further preferred embodiment, a first part of the recycle stream is added to the biomass in Step B and a further part of the recycle stream is added to the fermentation broth in Step C . In this case, the acid contained in the recycle stream can act as a pretreatment agent, as detailed above, and additional amounts of the recycle stream are added to the fermentation broth in Step C, providing even more carbon source for the oleaginous microorganism. This can further increase the efficiency of the overall process .
[0022] Preferably, at least a part of the inventive process is operated as a continuous process . It is particularly preferred that the at least a part of the lipid-depleted lysis mixture from Step E is continuously withdrawn as a recycle stream, and that the at least a part of the recycle stream comprising residual fermentable acid from the lysis composition is continuously fed to the fermentation broth during the culturing in Step C or to the biomass in Step B; in particular to the fermentation broth during the culturing in Step C . Preferably, at least Steps C, D, and E of the inventive process are operated as a continuous process; preferably the inventive process is a continuous process . Operating the process continuously can significantly enhance the overall efficiency.
[0023] In a preferred embodiment of the inventive method, the pretreating in Step B comprises adding a further fermentable acid to the biomass . It is particularly preferred, that the pretreating in Step B comprises both mixing at least a part of the recycle stream (which comprises the fermentable acid from the lysis Step D) with the biomass and adding a further fermentable acid to the biomass . In this case, an even more effective pretreatment can be achieved. For the further fermentable acid, the sameembodiments are preferred as for the fermentable acid used in Step D of the inventive process . The further fermentable acid can also be used as a carbon source for the oleaginous microorganisms, resulting in a higher efficiency of the overall process and less waste . Preferably, the further fermentable acid is an organic acid, preferably a Ci-Ce organic acid, more preferred a C1-C5 organic acid, even more preferred a C1-C4 organic acid.
[0024] Preferably, the further fermentable acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-bu-tyric acid, iso-valeric acid and mixtures thereof . It is particularly preferred, that the further fermentable acid is identical to the fermentable acid contained in the lysis composition.
[0025] In the context of the inventive method, any suitable type of biomass can be used, in particular any type of biomass that is suitable for the production of C5 and / or Ce sugars e . g. by hydrolysis . The biomass preferably comprises cellulose, hemicellulose and / or lignin. The biomass can be derived from plants, animals and / or microorganisms . Preferably, the biomass includes agricultural waste, industrial waste, forestry waste, municipal waste and / or energy crops .
[0026] Preferably, the biomass is lignocellulosic biomass . Lignocellulosic biomass typically refers to plant biomass that comprises cellulose, hemicellulose, and lignin. Lignocellulosic biomass is available in high abundance and at low costs, e . g. , from forestry, timber and pulp and paper industries, as well as from agriculture . In addition, also municipal solid waste can comprise lignocellulosic biomass, e . g. , paper or cardboard waste and garden waste .
[0027] In a preferred embodiment, the biomass is selected from wheat straw, rice straw, corn stover and / or sugarcane bagasse . These biomass sources are typically available in high amounts and at low costs and are therefore particularly preferred.
[0028] During Step B of the inventive method, the biomass is pretreated to obtain a fermentation substrate . As used herein, the term "pretreating" preferably refers to the transformation of biomass into a form that can be utilized as a carbon source by the oleaginous microorganisms . Preferably, pretreatmentcomprises the production of fermentable sugars from complex carbohydrates . Alternatively, or in addition, pretreatment may comprise the production of fermentable acids, especially volatile fatty acids, from the biomass, e . g. through anaerobic digestion. Preferably, pretreatment comprises converting polysaccharides contained in the biomass to monosaccharides, in particular Ce and / or C5 sugars, which can be used as a carbon source by the oleaginous microorganisms . Preferably the biomass comprises cellulose and the pretreatment involves the hydrolysis of cellulose to obtain glucose . It is also preferred that the biomass comprises hemicellulose and the pretreatment involves the hydrolysis of hemicellulose to obtain monosaccharides such as xylose, mannose, galactose, rhamnose and / or arabinose .
[0029] Therefore, the fermentation substrate preferably comprises monosaccharides, preferably Ce and / or C5 sugars, especially selected from the group consisting of glucose, xylose, mannose, galactose, rhamnose and / or arabinose . In the prior art, pretreatment is typically achieved by processes such as steam explosion, dilute acid hydrolysis, enzymatic pretreatment, hot water treatment, or standard pulp industry processes . Such methods are also suitable in the context of the present invention and are described, e . g. , in EP 3 009 515 Al and in
[0030] WO 2015 / 086780 Al .
[0031] In a preferred embodiment, the pretreating in Step B comprises steam explosion, preferably wherein the biomass is treated with saturated steam at a temperature of 160-260°C .
[0032] In another preferred embodiment, the pretreating in Step B comprises hot water treatment . Hot water treatment preferably comprises mixing the biomass with water to a dry matter content between 5-40 % (w / w) and heating the mixture to a temperature between 140 °C and 240 °C for a time period of 1-120 minutes .
[0033] In another preferred embodiment, the pretreating in Step B comprises anaerobic digestion. For instance, the biomass may undergo degradation by microorganisms in an anaerobic fermenter, wherein the biomass is converted into fermentable substrates such as fermentable acids, in particular volatile fatty acids .
[0034] In the context of the inventive method, it is particularly preferred that the pretreating comprises acid hydrolysis . Asdescribed above, in a preferred embodiment the pretreating in Step B comprises mixing at least a part of the recycle stream with the biomass, forming a pretreatment mixture . Since the pretreatment mixture contains fermentable acid from the recycle stream, which was originally introduced into the process as a part of the lysis composition, acid hydrolysis of the biomass can occur . Thus, the fermentable acid contained in the recycle stream causes acid hydrolysis of the biomass, wherein polysaccharides such as cellulose are converted into monosaccharides such as glucose, as described above .
[0035] When the pretreating in Step B comprises adding a further fermentable acid to the biomass, as described above, an even more efficient hydrolysis can be achieved. In this embodiment, the pretreatment mixture contains both the fermentable acid from the recycle stream and the further fermentable acid added to the biomass .
[0036] Prior to the mixing with the at least a part of the recycle stream (and optionally the further fermentable acid) , the biomass may be milled. Milling allows reducing the particle size of the biomass, which in turn facilitates acid hydrolysis .
[0037] Preferably, the pretreating in Step B comprises heating the pretreatment mixture formed by the mixing of the at least a part of the recycle stream (and optionally the further fermentable acid) with the biomass to a temperature between 20 °C and 300 °C, preferably between 25 °C and 260 °C, more preferred between 30 °C and 220 °C . Preferably, the pretreatment mixture is heated to the indicated temperature for a time period between 5 and 360 minutes, more preferred between 10 and 240 minutes, more preferred between 15 and 120 minutes . Heating the pretreatment mixture in this way allows for particularly efficient hydrolysis .
[0038] Preferably, the pretreating as described above is carried out in a continuous reactor .
[0039] Preferably, the pretreating comprises a solid-liquid separation step for separating insoluble components . In a preferred embodiment, the pretreating comprises a filtration step . Such a solid-liquid separation step, in particular a filtration step, allows to separate and remove lignin or other insoluble components from the hydrolyzed cellulose and / or hemicellulose . Theremoval of insoluble components prior to fermentation is advantageous, since the presence of lignin and particulate material may adversely affect fermentation performance and may cause fouling or mechanical stress in downstream equipment and damage sensors in the bioreactor . Thus, preferably, the pretreating in Step B comprises a first step of converting the biomass into a mixture comprising fermentable compounds and insoluble components, followed by a solid-liquid separation step for separating said insoluble components from a liquid phase comprising the fermentable compounds, thereby obtaining the liquid phase as the fermentation substrate . In this context, the term "converting the biomass" encompasses, for example, any of the previously described methods such as acid hydrolysis, steam explosion, hot water treatment, or anaerobic digestion.
[0040] The solid-liquid separation step may be carried out by any suitable method known in the art, such as filtration, centrifugation ( for example using a decanter or disc-stack centrifuge) or other suitable solid-liquid separation techniques . Preferably, at least a portion of the insoluble components is removed, more preferably a majority by weight of the insoluble components present prior to the solid-liquid separation step, more preferably substantially all insoluble components are removed.
[0041] The amount of insoluble components in the fermentation substrate may be quantified as total suspended solids (TSS) , preferably in accordance with ASTM D5907-18. In preferred embodiments, at least 80 wt% of TSS, more preferred at least 90 wt%, more preferred at least 95 wt%, more preferred at least 99 wt%, more preferred substantially all TSS present prior to the solidliquid separation step are removed. In preferred embodiments, the fermentation substrate after the solid-liquid separation step comprises less than 20, 000 mg / L, preferably less than 1, 000 mg / L total suspended solids (TSS) . It is particularly preferred that the fermentation substrate is essentially free of insoluble components . For the purposes of the inventive method, any suitable type of oleaginous microorganism known in the art can be used. As used herein, the term "oleaginous microorganism" preferably refers to a microorganism that is able to produce high levels of lipids, in particular that it is able to accumulate more than 20 % (w / w) , preferably more than 30 % (w / w) , morepreferred more than 40 % (w / w) , more preferred more than 50 % (w / w) , more preferred more than 60 % (w / w) of its dry cell weight as lipids, preferably as triglyceride oil .
[0042] Thus, preferably, the oleaginous microorganism comprises lipids, preferably triglyceride oil, in an amount of at least 20 % (w / w) by dry cell weight, preferably at least 30 % (w / w) , more preferred at least 40 % (w / w) , more preferred at least 50 % (w / w) , more preferred at least 60 % (w / w) .
[0043] The oleaginous microorganism is preferably selected from the group consisting of filamentous fungi, yeast, bacteria and / or algae . Suitable species of oleaginous microorganisms are described, e . g. , in WO 2015 / 086780 Al . For instance, preferred ( filamentous) fungal strains are from species from genera Aspergillus such as Aspergillus oryzae, Giberella , such as Giberella Fujikori Mortierella such as Mortierella isabellina, Chaeto-mium, Claviceps , Cladospori di um, Cunninghamella, Emericella, Fusarium, Glomus, Mucor, Pseudozyma, Pythium, Rhizopus , such as Rhizopus oryzae, Tremella, Zygorhynchus , Humicola, Cladosporium, Malbranchea, Umbelopsis such as Umbel opsis isabellina and Usti-lago. Particularly preferred fungal species are from genera Aspergillus and / or Mortierella . Preferred yeast strains are those belonging to species from genera Geotrichum, Deparyomyces , Pachysolen, Galactomyces , Hansenula, Leucosporidium, Sporobolo-myces, Sporidiobolus , Waltomyces , Cryptococcus , such as Cryptococcus curvatus , Rhodosporidium, such as Rhodospori di um toruloides or Rhodospori di um fluviale, Rhodotorula, such as Rhodotorula glutinis, Yarrowia, such as Yarrowia lipolytica, Candida such as Candida curvata, Lipomyces such as Lipomyces starkeyi and Trichosporon such as Trichosporon cutaneum or Trichosporon pullulans . Particularly preferred yeasts are from genera Lipomyces , Rhodosporidium and Cryptococcus . Preferred bacteria are those belonging to the species from genera Rhodococcus , Gordania , Rhodobacter , Acinetobacter and Streptomyces . Particularly preferred algae are microalgae, such as microalgae species from genera comprising Brachiomonas , Crypthecodinium, Chlorella, Dunaliella, Hantzschia, Nannochloris, Nannochloropsis , Nitzschia, Prototheca, Scenedesmus , Schizochytrium, Traustro-chytrium and Ulkenia .
[0044] In a preferred embodiment, the oleaginous microorganism isan oleaginous yeast, especially Cryptococcus oleaginosus . It was found that Cryptococcus sp. especially Cryptococcus oleaginosus, are able to utilize fermentable acids particularly effectively as carbon source and are particularly well suited for the production of microbial oils in the context of the inventive method .
[0045] Other particularly preferred oleaginous microorganisms include those selected from the group consisting of Cryptococcus oleaginosus , Yarrowia lipolytica, Rhodotorula toruloides , Rhodosporidium toruloides , Rhodotorula graminis , Pichia kudria-vezii, Candida lypomcyes , Lipomyces starkeyi , Trichosporon po-rosum, Debaryomyces hansenii , Kluyveromyces marxianus , Ka-zachstania unispora, and / or Zygotorulaspora florentina . These species were found to exhibit high productivity for producing microbial oils from substrates in the context of the inventive method .
[0046] In Step C of the inventive method, the oleaginous microorganism is cultured in a fermentation broth comprising the fermentation substrate . Preferably, the culturing comprises inoculating the fermentation substrate with the oleaginous microorganism to obtain a fermentation broth and incubating the fermentation broth under conditions that allow for growth of the oleaginous microorganism. During the culturing, the sugars and organic acids contained in fermentation substrate can be metaboli-cally converted into lipids . The lipids typically accumulate as intracellular lipids within the cells of the oleaginous microorganism, however the lipids may also be secreted or at least partly secreted to the fermentation broth.
[0047] The skilled person is familiar with suitable methods for culturing the oleaginous microorganism. For instance, the culturing may be done by batch, fed batch or continuous cultivation. Preferably, the oleaginous microorganisms are cultured in a bioreactor, especially a continuous stirred tank bioreactor .
[0048] The skilled person is also familiar with suitable conditions that allow for growth of the oleaginous microorganisms and for accumulation of lipids . Preferably, the culturing is carried out under aerobic conditions . Preferably, the culturing is carried out at atmospheric pressure . Preferably, the culturing iscarried out at a temperature between 30 and 37 °C . Preferably, the culturing is carried out for a time period of at least 24 hours, preferably at least 48 hours, more preferred at least 72 hours, more preferred at least 96 hours, more preferred at least 120 hours . Preferably, the culturing is carried out until at least 20 % (w / w) , preferably at least 30 % (w / w) , more preferred at least 40 % (w / w) , more preferred at least 50 % (w / w) , more preferred at least 60 % (w / w) , more preferred at least 70 % (w / w) of the dry cell weight of the oleaginous microorganism consists of lipids .
[0049] Thus, preferably, the culturing comprises inoculating the fermentation substrate with the oleaginous microorganism to obtain a fermentation broth and incubating the fermentation broth at a temperature between 30 and 37 °C; preferably under aerobic conditions; preferably at a pressure of between 1-4 bar . Preferably the fermentation broth is incubated for a time period of at least 24 hours, preferably at least 48 hours, more preferred at least 72 hours, more preferred at least 96 hours, more preferred at least 120 hours; preferably at least until the lipid proportion of the dry cell weight of the oleaginous microorganism reaches the above-mentioned values .
[0050] During the culturing, the cells of the oleaginous microorganisms preferably consume at least a part of the fermentable acid that was added to the fermentation broth as part of the recycle stream. Preferably, at least 80 wt%, more preferred at least 90 wt%, more preferred at least 95 wt%, more preferred at least 99 wt%, more preferred 100 wt% of the fermentable acid is consumed by the cells of the oleaginous microorganisms during the culturing in Step C .
[0051] Preferably, during the culturing in Step C, the fermentation broth comprises fermentable acids in a comparatively high concentration. Preferably, the total concentration of fermentable acids in the fermentation broth during culturing is at least 10 g / L, more preferred at least 15 g / L, more preferred at least 20 g / L, more preferred at least 25 g / L, more preferred at least 30 g / L, based on the liquid phase of the fermentation broth. In preferred embodiments, the total concentration of fermentable acids in the fermentation broth is in the range of from 10 to 120 g / L, more preferred from 15 to 100 g / L, more preferred from20 to 80 g / L, more preferred from 25 to 70 g / L, more preferred from 30 to 60 g / L . These ranges are also preferred for all embodiments of the fermentable acid specified herein. For instance, in the preferred embodiment, in which the fermentable acid is a Ci-Ce organic acid, it is preferred that the total concentration of Ci-Ce organic acids in the fermentation broth during culturing is as defined above . In the preferred embodiment, in which the fermentable acid is acetic acid, it is preferred that the total concentration of acetic acid in the fermentation broth during culturing is as defined above; i . e . , it is preferred that the concentration of acetic acid in the fermentation broth during culturing is at least 10 g / L, more preferred at least 15 g / L, more preferred at least 20 g / L, more preferred at least 25 g / L, more preferred at least 30 g / L, based on the liquid phase of the fermentation broth. In preferred embodiments, the concentration of acetic acid in the fermentation broth is in the range of from 10 to 120 g / L, more preferred from 15 to 100 g / L, more preferred from 20 to 80 g / L, more preferred from 25 to 70 g / L, more preferred from 30 to 60 g / L .
[0052] In a preferred embodiment of the inventive method, during the culturing in Step C a further fermentable acid is added to the fermentation broth. Adding a further fermentable acid to the fermentation broth allows providing an additional carbon source for the oleaginous microorganisms, which can result in more lipid production. In the context of this embodiment, the same embodiments are preferred for the further fermentable acid as described above for the fermentable acid used in Step D of the inventive process . Thus, preferably the further fermentable acid is an organic acid, preferably a Ci-Ce organic acid, more preferred a C1-C5 organic acid, even more preferred a C1-C4 organic acid. Preferably, the further fermentable acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, isobutyric acid, iso-valeric acid and mixtures thereof . It is particularly preferred, that the further fermentable acid is identical to the fermentable acid contained in the lysis composition .
[0053] In Step D of the inventive method, the cells of the oleaginous microorganism that are present in the fermentation brothcan be lysed to produce a lysate comprising the produced lipids . For this purpose, the cells of the oleaginous microorganism are contacted with a lysis composition comprising a fermentable acid to obtain a lysis mixture . The cells of the oleaginous microorganism are subj ected to lysis conditions . As used herein the term "lysis conditions" preferably refers to conditions allowing for at least partial cell disruption, under which at least a part of cellular components, especially lipids, are released from the cells . Thus, in Step D of the inventive method, preferably at least a part of the cells of the oleaginous microorganism are lysed and at least a part of the lipids is released from the cells . The skilled person is familiar with conditions suitable for cell lysis . Preferably, at least 70 % of the cells, more preferred at least 80 %, more preferred at least 90 %, more preferred 100 %, are lysed. As used herein, percentages referring to cell lysis are based on the number of cells (i . e . , number lysed / total number) . The percentage of cells that have been lysed can preferably be determined by Trypan blue staining followed by microscopic counting.
[0054] In a preferred embodiment, the cells of the oleaginous microorganisms are harvested from the fermentation broth before cell lysis . Thus, preferably, Step D of the inventive method comprises harvesting the cells of the oleaginous microorganisms from the fermentation broth and adding a lysis composition comprising a fermentable acid to the harvested cells to obtain a lysis mixture . Preferably, the mass ratio between the harvested cells and the lysis composition in the lysis mixture is between 2 : 1 and 1 : 20, preferably between 1 : 1 and 1 : 15, more preferred between 1 : 1 and 1 : 10 (harvested cells : lysis composition) . The mass of the harvested cells is preferably determined as the biomass dry weight . The skilled person is familiar with suitable methods to harvest the cells . For instance, the harvesting can be achieved by centrifugation.
[0055] In an alternative embodiment, which is also preferred, the lysis composition in Step D is added to the fermentation broth to obtain the lysis mixture, preferably without prior harvesting of the cells .
[0056] As described above, the fermentable acid comprised in the lysis composition preferably is an organic acid, preferably a Ci-Ce organic acid, more preferred a C1-C5 organic acid, even more preferred a C1-C4 organic acid, especially selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-bu-tyric acid, iso-valeric acid and mixtures thereof .
[0057] Preferably, the concentration of the fermentable acid in the lysis composition is at least 20 % (w / w) , preferably at least 40 % (w / w) , more preferred at least 60 % (w / w) , more preferred at least 70 % (w / w) , more preferred at least 80 % (w / w) , more preferred at least 85 % (w / w) , more preferred at least 90 % (w / w) , more preferred at least 95 % (w / w) . Providing a larger concentration of acid in the lysis composition allows for more efficient lysis .
[0058] Preferably, the lysis mixture has a pH below 5, preferably below 4, more preferred below 3.5. Preferably, the pH is between 1 and 5, preferably between 1 and 4, more preferred between 2 and 3.5. In the course of the invention it has turned out that with the indicated pH values, a particularly efficient lysis can be achieved.
[0059] Preferably, in Step D of the inventive method the lysis mixture is heated to a temperature of at least 50 °C, more preferred at least 100 °C, more preferred at least 120 °C, more preferred at least 140 °C, more preferred at least 150 °C . Preferably, it is heated to a temperature between 50 and 250 °C, preferably between 100 and 240 °C, more preferred between 120 and 220 °C, more preferred between 140 and 200 °C, more preferred between 150 and 180 °C . Heating the lysis mixture also allows enhancing lysis efficiency.
[0060] Without wishing to be bound to any particular theory, the inventors believe based on observations during experiments that at lower temperatures the proteins only denature but typically do not hydrolyze . It is believed that the denatured proteins can form strong emulsions in the presence of phospholipids leading to less efficient oil recovery and slower separation of phases . A large layer or matrix can appear between the polar and non-polar fraction of the liquid due to this phenomenon. At higher temperatures, in particular above 100 °C, and especially above 120 °C, proteins as well as other components of the cell wallhydrolyze, leading to even higher recovery rates of the oil and faster separation of the layers .
[0061] Preferably, the lysis mixture is incubated, preferably at the above-mentioned temperatures, until at least 70 % of the cells, preferably at least 80 %, more preferred at least 90 %, more preferred 100 %, are lysed. As used herein, percentages referring to cell lysis are based on the number of cells (i . e . , number lysed / total number) . The percentage of cells that have been lysed can preferably be determined by Trypan blue staining followed by microscopic counting.
[0062] In Step E of the inventive method, at least a part of the released lipids is isolated from the lysis mixture to obtain the microbial oil, leaving behind a lipid-depleted lysis mixture . The skilled person is familiar with suitable methods for separating lipids from cell lysates . Suitable methods are described, e . g. , in WO 2011 / 130573 Al and EP 3 009 515 Al and include, among others, extraction with a hydrophobic solvent, extraction using liquefaction, oil liquefaction, and supercritical CO2 extraction .
[0063] In the context of the inventive method, it is preferred that Step E comprises an extraction with a non-polar solvent . Preferably, the non-polar solvent is an organic solvent, preferably a water-immiscible organic solvent . Preferably, the non-polar solvent is selected from the group consisting of n-hexane, acetone, petroleum ether, ethyl ether and combinations thereof ; especially n-hexane .
[0064] Extraction with non-polar solvents such as n-hexane are well known in the art . Preferably, a lysate obtained from Step D of the inventive method is mixed with the non-polar solvent, the resulting mixture is allowed to separate into an aqueous phase and an organic phase, and the organic phase comprising at least a part of the released lipids is isolated. The lipids may be isolated from the organic phase and / or purified to obtain the microbial oil . In this case, the aqueous phase contains at least a part of the fermentable acid from the lysis composition and the recycle stream may be obtained by withdrawing at least a part of the aqueous phase .
[0065] In a preferred embodiment, after the extraction the solventis recovered from the lipids by evaporation and preferably reused in the process . This further increases the overall efficiency of the process and reduces waste .
[0066] After the released lipids are isolated from the lysis mixture, at least a part of the lipid-depleted lysis mixture is recycled as a recycle stream, as detailed above . The lipid-depleted lysis mixture contains various components that can be used as a carbon source by the oleaginous microorganism when they are recycled. This concerns not only the fermentable acid but also cell debris resulting from cell lysis . Thus, preferably, the recycle stream also contains cell debris .
[0067] It is preferred that a large part of the lysis mixture of Step D is recycled after separation of the released lipids and included in the fermentation broth in Step C . Preferably, at least 10 wt%, more preferred at least 20 wt%, more preferred at least 30 wt%, more preferred at least 50 wt%, more preferred at least 70 wt% of the lysis mixture is recycled as part of the recycle stream and included in the fermentation broth in Step C .
[0068] In a preferred embodiment of the inventive method, a further fermentable acid is added to the recycle stream before it is mixed with the biomass and / or added to the fermentation broth. Adding a further fermentable acid in this away allows for additional lysis to occur in case the recycle stream contains cells of the oleaginous microorganism that have not been fully lysed. In the context of this embodiment, the same embodiments are preferred for the further fermentable acid as described above for the fermentable acid used in Step D of the inventive process . Thus, preferably the further fermentable acid is an organic acid, preferably a Ci-Ce organic acid, more preferred a C1-C5 organic acid, even more preferred a C1-C4 organic acid. Preferably, the further fermentable acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-butyric acid, isovaleric acid and mixtures thereof . It is particularly preferred, that the further fermentable acid is identical to the fermentable acid comprised in the lysis composition.
[0069] The recycle stream may contain toxic and / or inhibitory compounds . In order to prevent accumulation of such compounds inthe recycle stream, part of the recycle stream may be purged. Thus, in a preferred embodiment of the inventive method, a purge stream is removed from the recycle stream before it is mixed with the biomass and / or added to the fermentation broth. The purge stream may comprise compounds that are toxic and / or inhibitory to the cells of the oleaginous microorganism. By removing a purge stream, such toxic and / or inhibitory compounds can be removed and thus prevented from accumulating in the process . In this way, a more robust cell growth of the oleaginous microorganism can be ensured.
[0070] Preferably, the purge stream comprises cell debris . On the other hand, it is also preferred that at least a part of the cell debris contained in the lipid-depleted lysis mixture from Step E is included in the part of the recycle stream that is included in the fermentation broth in Step C . Also cell debris or other components that may not be easily consumable by cells of the oleaginous microorganism in Step C may become consumable as they undergo hydrolysis by acid treatment in the lysis Step D. Thus, they may become consumable as they are continued to be recycled in the process .
[0071] In a preferred embodiment, the inventive method further comprising the step of hydroprocessing the microbial oil . Hydroprocessing the microbial oil can advantageously remove impurities, reduce the oxygen content, and / or increase the degree of saturation of fatty acid components in the oil .
[0072] The microbial oil obtained by the inventive method can be used in a broad range of applications . For instance, it may be converted to olefins, to biodiesel or to SAF. Suitable methods for further processing of microbial oil into such products are known in the art and described, e . g. , in WO 2011 / 130573 Al and in EP 3 009 515 Al .
[0073] Therefore, in another aspect, the present invention provides a method for producing olefins, comprising the following steps :
[0074] - Producing a microbial oil using the method according to the invention; and
[0075] - Converting the microbial oil into olefins .
[0076] The microbial oil can be converted into olefins usingstandard processes known in the art . Preferably the microbial oil is converted into olefins by cracking, especially steam cracking. Preferably, the microbial oil is hydroprocessed followed by cracking, especially steam cracking. Preferably the olefins comprise ethylene and / or propylene .
[0077] Therefore, in another aspect, the present invention provides a method for producing biodiesel, comprising the following steps :
[0078] - Producing a microbial oil using the method according to the invention; and
[0079] - Converting the microbial oil into biodiesel .
[0080] Typically, biodiesel is essentially made up of long-chain fatty acid mono-alkyl esters, in particular methyl, ethyl or propyl esters . Preferably, the biodiesel comprises one or several of the following fatty acid alkyl esters : fatty acid methyl esters (FAME) , fatty acid ethyl esters (FAEE) , fatty acid butyl esters (FABE) . In particular, the biodiesel can contain one or several fatty acids selected from myristate, palmitate, stearate, oleate, linolenate, arachidate and behenate . Preferably the biodiesel is biodiesel according to European standard EN 14214 : 2008 .
[0081] Converting the microbial oil into biodiesel is preferably achieved by transesterification. Transesterification refers to the reaction taking place between a fatty acid and an alcohol to form an ester . Thus, free fatty acids contained in the microbial oil may be converted into the above-mentioned esters by transesterification. Suitable transesterification methods are well known to the skilled person and described, e . g. , in
[0082] EP 1 682 466 Al, EP 1 795 576 Al, EP 1 681 337 Al and
[0083] EP 1 640 437 Al .
[0084] In a further aspect, the present invention provides a method for producing sustainable aviation fuel (SAF) , comprising the following steps :
[0085] - Producing a microbial oil using the method according to any one of the preceding claims; and
[0086] - Converting the microbial oil into SAF.
[0087] Preferably the SAF is aviation fuel according to thestandard ASTM D1655-22.
[0088] Methods for converting microbial oil into aviation fuel are known to the skilled person and described, e . g. , in
[0089] WO 2011 / 130573 Al . Preferably, the SAP is produced from the microbial oil by fluid catalytic cracking or by hydroprocessing, preferably by hydrodeoxygenation.
[0090] Unless specified otherwise, all parameters as used herein correspond to parameters at IUPAC SATP-conditions ( „Standard Ambient Temperature and Pressure" ) , in particular a temperature of 25 °C and a pressure of 101.300 Pa .
[0091] Percentages (indicated as "%", "wt%" and the like) as used herein correspond to weight per weight (w / w) unless specified otherwise . Similarly, ratios used herein correspond to weight ratios (w / w) unless specified otherwise .
[0092] Pressures given in "bar" indicate absolute pressures ("bara") , unless specified otherwise .
[0093] As used herein, "dry cell weight" (DCW) preferably refers to the weight of microbial cells obtained after separating the cells from the culture broth, preferably by centrifugation, and drying at 105 °C under atmospheric pressure until no further weight loss is observed.
[0094] As used herein, the phrase "at least a part of" or similar phrases refer to any subset of a specified material, component, or stream, which may include part or all of the specified entity. This definition is intended to encompass not only partial quantities but also the entirety of the material, component, or stream in question. Consequently, whenever "at least a part of" a stream or other entity is mentioned, it is also preferred to use the entire stream or entity. For instance, when it is mentioned that at least a part of the recycle stream is included in the fermentation broth, it is also preferred that the recycle stream (as a whole) is included in the fermentation broth.
[0095] The present invention is further illustrated by the following figures, without being limited thereto .
[0096] Figure 1 shows a process flow diagram of an embodiment of the method for producing a microbial oil from biomass .
[0097] Figure 2 shows a process diagram of an implementation of themethod for producing a microbial oil from biomass, including calculated mass flow rates (kg / h) , as described in Example 2.
[0098] In the embodiment shown in Figure 1, biomass 1, such as solid agricultural residue, is pretreated by forming an acidic pretreatment mixture 2, which contains a fermentable acid such as acetic acid. The pretreatment mixture 2 is heated to a temperature between 30 °C and 220 °C under atmospheric pressure for a time period between 15 and 120 minutes . The acid present in the pretreatment mixture allows for acid hydrolysis of polysaccharides such as cellulose and hemicellulose contained in the biomass to occur, whereby C5 and Ce sugars are formed. The pretreatment mixture 2 is preferably filtered to remove lignin and other insoluble components and a fermentation substrate 3 comprising fermentable monosaccharides as well as the fermentable acid that was used for pretreatment is obtained as the filtrate . The fermentation substrate 3 is inoculated with an oleaginous microorganism, preferably oleaginous yeast such as Cryptococcus oleaginosus, from seed train 4 to form a fermentation broth 5. The fermentation broth is mixed and aerated in a continuous stirred tank bioreactor at 30 to 37 °C und atmospheric pressure . Under these conditions, the microorganisms can metabolize both the monosaccharides and the fermentable acid that were contained in the fermentation substrate 3, leading to growth as well as to the production of lipids within cells of the oleaginous microorganisms . The cells are grown until a large proportion, preferably 60-80 % (w / w) of the dry cell weight, consists of lipids . Next, the cells are harvested by centrifugation and the harvested cells 6 are conveyed to a tank, in which a lysis composition 7 is added at a mass ratio of 1 : 1 to 1 : 15 (harvested cells : lysis composition) to form a lysis mixture 8. The lysis composition 7 contains a fermentable acid, such as acetic acid, preferably at a concentration of 80 to 95 % (w / w) . The tank is pressurized to a pressure between 1 and 10 bar, agitated, and the lysis mixture 8 is heated to a temperature between 50 and 250 °C, whereby cell lysis occurs and at least a part of the produced lipids is released from the cells . The lipids are then isolated from the resulting lysate 9. In the embodiment shown in Figure 1, this is achieved by extraction with a water-immiscible organic solvent 10 such as hexane . The water-immiscible organicsolvent 10 is mixed with the lysate 9 in a separator 11, which may e . g. be a centrifuge or a mixer settler . From the separator 11, an organic phase comprising at least a part of the released lipids is obtained. Optionally, at least a part of the water-immiscible organic solvent 10 may be separated and recovered by evaporation. The remaining composition comprising at least a part of the produced lipids is obtained as microbial oil 12. In addition to the organic phase, an aqueous phase is obtained from the separator 11 as a lipid-depleted lysis mixture 13. The li-pid-depleted lysis mixture 13 forms a recycle stream 14, which comprises at least a part of the fermentable acid that was introduced into the process through the lysis composition 7. A further separator 15 is used to periodically remove a purge stream 16 from the recycle stream 14. A part of the purified recycle stream 17 is then conveyed to the pretreatment step, where it is mixed with the biomass 1 to form the pretreatment mixture 2. In this way, the fermentable acid that was used for cell lysis is recycled and used for acid hydrolysis of polysaccharides contained in the biomass 1. In addition, another part of the purified recycle stream 17 is conveyed to the bioreactor and added to the fermentation broth 5. In this way, the fermentable acid that was used for cell lysis is recycled and can be consumed as a carbon source by the oleaginous microorganisms . By recycling the acid in this way, the overall efficiency and cost-effectiveness of the process can significantly be increased and waste can be reduced.
[0099] Example 1 : Production of microbial oil using fermentable acids for cell lysis .
[0100] An oleaginous yeast was cultured in a 1 liter bioreactor in a fermentation broth containing glucose as carbon source, and ammonium sulfate as nitrogen source . The culturing was carried out for 3 days with controlled parameters of temperature 30 °C, pH of 7 and dissolved oxygen content of 50% . The resulting biomass contained between 20-30 wt% lipids . After culturing, the fermentation broth was centrifuged to obtain a total water content between 20 and 30 wt% .
[0101] For cell lysis, a lysis composition consisting of 95 wt%acetic acid in water was used. The centrifuged broth and lysis composition were poured into a closed tubular reactor that was rotated for mixing and heated up to a temperature of 150 °C . The mass ratio of dry cell weight (DCW) to lysis composition was 1 : 10. The rotating heating mixture had a pH of around 2, 5 and samples were taken at different time intervals . Samples were taken after 15, 60 and 120 minutes and the lysis efficiency was determined by the total extraction efficiency (wt% of oil recovered from the total cells) . Utilizing hexane as solvent the following extraction efficiencies were observed:
[0102]
[0103] After extraction with the non-polar solvent, the aqueous and non-polar phases were separated. The non-polar phase contained the microbial oil as well as the solvent, while the aqueous phase contained the residual lysis composition and cell debris . This aqueous phase can be recycled to the bioreactor, where its contents can be used as a carbon source for the oleaginous microorganisms and be metabolically converted into lipids .
[0104] The obtained results indicate that cell lysis using fermentable organic acids can be utilized with a high efficiency, while having the added benefit that the aqueous phase can be recycled into the fermentation broth, decreasing the need for acid recovery and purification while providing additional carbon source to the microorganisms for lipid production, thereby increasing the overall process efficiency.
[0105] Example 2 : Process scheme modeling of the method for producing a microbial oil .
[0106] To evaluate a concrete implementation of the inventive method, a process scheme was modeled, and the mass flow rates of various streams within the process were calculated, as shown in Figure 2. The process depicted in Figure 2 involves the following components :- FERMENT : Fermentation bioreactor for culturing the oleaginous microorganism.
[0107] - LYSTANK: Lysis tank for lysing the cells of the oleaginous microorganism.
[0108] - SEP1 : Separator for extracting released lipids from the lysis mixture .
[0109] - SEP2 : Separator for recovering solvent used for extraction. - Bl, B2, B3, B5 : Mixers .
[0110] Additionally, the process shown in Figure 2 involves the following streams :
[0111] - F-MEDIUM: Fresh fermentation substrate added to the bioreactor .
[0112] - BROTH: Fermentation broth obtained from the bioreactor .
[0113] - NEWACID: Lysis composition comprising a fermentable acid. - MIX1 : Lysis mixture obtained from mixing the lysis composition (NEWACID) , the fermentation broth (BROTH) and a part of the recycle stream containing fermentable acid (RECLOOP) .
[0114] - OFFGAS : Gas released from the lysis tank.
[0115] - LYSED: Lysate obtained after cell lysis .
[0116] - SOLV: Solvent for extracting released lipids .
[0117] - MIX3 : Mixture obtained after mixing the lysate (LYSED) with the solvent (SOLV) .
[0118] - EXT : Organic phase obtained from the separator (SEP1 ) , containing the lipids released from the cells of the oleaginous microorganism.
[0119] - OIL : Microbial oil isolated from the organic phase (EXT) after removal of the solvent (SOLV) used for extraction.
[0120] - RAFF: Lipid-depleted lysis mixture obtained from separator (SEP1 ) after separation of the organic phase (EXT) containing the lipids released from the cells of the oleaginous microorganism.
[0121] - SOLIDS : Cell debris obtained from separator (SEP1 ) after separation of the organic phase (EXT) containing the lipidsreleased from the cells of the oleaginous microorganism. - RECFERM: Part of the lipid-depleted lysis mixture (RAFF) recycled to the fermentation bioreactor (FERM) .
[0122] - RECTOT : Mixture of lipid-depleted lysis mixture (RAFF) and cell debris (SOLIDS) recycled to the fermentation bioreactor (FERM) .
[0123] - RECLOOP : Part of the lipid-depleted lysis mixture (RAFF) recycled to be included in the lysis mixture (MIX1 ) .
[0124] For each of these streams, mass flow rates were calculated. These are indicated next to the respective streams in Figure 2, expressed in kilograms per hour (kg / h) .
[0125] As can be seen from Figure 2, the recycling of the lipid-depleted lysis mixture allows keeping the fermentable acid of the lysis composition (NEWACID) in the process, and using it as a carbon source for the oleaginous microorganisms in the fermentation bioreactor (FERM) . The mass flows indicated in Figure 2 represent one example for implementing the inventive method and may be adjusted as desired, e . g. , depending on the plant and bioreactor size . Additionally, the total amount recycled (the RAFF and, optionally, SOLIDS stream) , as well as the relative amounts of recycled lipid-depleted lysis mixture (RAFF) conveyed to the bioreactor (RECTOT) and to the lysis tank (RECLOOP) may also be adjusted as desired.
[0126] Example 3 : Comparison of processes for producing a microbial oil with and without recycling streams .
[0127] To further assess the impact of recycling fermentable acid from the cell lysis to the fermentation broth, two different experiments were carried out . For both experiments, fermentation of oleaginous yeast was conducted in a 1-liter bioreactor, at 30 °C and at pH 7. The agitation was controlled based on the dissolved oxygen level in the reactor . The following experiments were conducted.
[0128] No . 1 Batch fermentation without recycling stream : A control experiment using a fermentation medium having a C / N ratio of 50, and comprising 100 g / L glucose and 30 g / L acetic acid was used. The fermentation was conducted in batch mode without any feed.The fermentation was run until glucose and acetic acid were completely consumed. After fermentation, the cells were collected by centrifugation, lysed by addition of glacial acetic acid and incubation at 150°C for 1 hour, and lipids were recovered by extraction with n-hexane .
[0129] No . 2 Acid recycling to fermentation: Fermentation was carried out essentially as described in experiment No . l, using a fermentation medium having a C / N ratio of 50, and comprising 100 g / L glucose . However, acetic acid was added through a recycling stream rather than being present in the base medium. Initially, the concentration of acetic acid in the fermenter was adjusted to 20 g / L . In addition, a recycling stream comprising 500 g / L acetic and lysed cell debris was fed to the fermenter at a rate of 1.5 g / L / h. Cell lysis and lipid extraction were carried out essentially as in experiment No . l .
[0130]
[0131] As shown in the table above, the addition of a recycling stream substantially increased both growth yield and lipid yield .
Claims
Claims :1 . A method for producing a microbial oil ( 12 ) from biomass ( 1 ) , the method comprising the following steps :- Step A: Providing the biomass ( 1 ) ;- Step B : Pretreating the biomass ( 1 ) to obtain a fermentation substrate ( 3 ) , wherein the pretreating comprises a solidliquid separation step for removing insoluble components ; - Step C : Culturing an oleaginous microorganism in a fermentation broth ( 5 ) comprising the fermentation substrate ( 3 ) , wherein lipids are produced within cells of the oleaginous microorganism;- Step D : Contacting the cells from the fermentation broth ( 5 ) with a lysis composition ( 7 ) under lysis conditions , wherein the lysis composition ( 7 ) comprises a fermentable acid, thereby obtaining a lysis mixture ( 8 ) , wherein at least a part of the produced lipids is released from the cells ;- Step E : I solating at least a part of the released lipids from the lysis mixture ( 8 ) to obtain the microbial oil ( 12 ) and a lipid-depleted lysis mixture ( 13 ) ;wherein at least a part of the lipid-depleted lysis mixture ( 13 ) from Step E is withdrawn as a recycle stream ( 14 ) , and wherein at least a part of the recycle stream ( 14 ) comprising residual fermentable acid from the lysis composition ( 7 ) is included in the fermentation broth ( 5 ) in Step C .2 . The method according to claim 1 , wherein the fermentable acid is a Ci-Ce organic acid, preferably selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-butyric acid, iso-valeric acid and mixtures thereof .3 . The method according to claim 1 or 2 , wherein the lysis composition ( 7 ) comprises at least two distinct fermentable acids .
4. The method according to claim 3, wherein each of the two distinct fermentable acids is a Ci-Ce organic acid, preferably selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, formic acid, valeric acid, caproic acid, iso-butyric acid and iso-valeric acid.
5. The method according to any one claims 1 to 4, wherein the pretreating in Step B comprises mixing at least a part of the recycle stream ( 14 ) with the biomass ( 1 ) .
6. The method according to any one claims 1 to 5, wherein a first part of the recycle stream ( 14 ) is added to the biomass ( 1 ) in Step B and a further part of the recycle stream ( 14 ) is added to the fermentation broth (5) in Step C .
7. The method according to any one of claims 1 to 6, wherein the pretreating in Step B comprises adding a further fermentable acid to the biomass ( 1 ) , preferably wherein the further fermentable acid is identical to the fermentable acid contained in the lysis composition (7 ) .
8. The method according to any one of claims 1 to 7, wherein the biomass ( 1 ) is lignocellulosic biomass, preferably selected from wheat straw, rice straw, corn stover and / or sugarcane bagasse .
9. The method according to any one of claims 1 to 8, wherein the oleaginous microorganism is an oleaginous yeast .
10. The method according to any one of claims 1 to 9, wherein during the culturing in Step C a further fermentable acid is added to the fermentation broth (5) , preferably wherein the further fermentable acid is identical to the fermentable acid contained in the lysis composition (7 ) .11 . The method according to any one of claims 1 to 12 , wherein a purge stream ( 16 ) is removed from the recycle stream ( 14 ) before it is mixed with the biomass ( 1 ) and / or added to the fermentation broth ( 5 ) .12 . The method according to any one of claims 1 to 11 , further comprising the step of hydroprocessing the microbial oil ( 12 ) .13 . A method for producing olefins , comprising the following steps :- Producing a microbial oil ( 12 ) using the method according to any one of claims 1 to 12 ; and- Converting the microbial oil ( 12 ) into olefins .14 . A method for producing biodiesel , comprising the following steps :- Producing a microbial oil ( 12 ) using the method according to any one of claims 1 to 12 ; and- Converting the microbial oil ( 12 ) into biodiesel .15 . A method for producing sustainable aviation fuel ( SAP) , comprising the following steps :- Producing a microbial oil ( 12 ) using the method according to any one of claims 1 to 12 ; and- Converting the microbial oil ( 12 ) into SAF .