Method for reducing emulsion formation during the fermentation process

Abstract

A method for producing farnesene oil, comprising: fermenting yeast using a supply of raw materials in a fermentation vessel to produce whole cell broth (WCB); and adding a surfactant to the fermentation vessel to reduce emulsification inside the fermentation vessel.

Description

Detailed description of the invention

[0001] [background] [1. Field]

[0001] The present disclosure relates to an improved method for the recovery of terpinoids, lipophilic isoprenoids, lipids and biomass from fermentation broth by reducing emulsion formation, comprising inhibiting emulsion formation in the fermentation vessel during the fermentation process and / or simultaneously deemulsifying the emulsion, and further comprising adding a surfactant to the fermentation vessel during the fermentation process. The method also affects the surface chemistry of the cells and their interaction with the fermentation medium, thereby simplifying the separation of biomass.

[0002] [2. Explanation of related technologies]

[0002] Terpenoids, lipids, and biomass can be produced by microorganisms such as yeast and microalgae via fermentation, and the cells produce oil and biomass containing intracellular or extracellular products that are converted into emulsions inside the fermenter. To recover the desired products, the fermented broth is centrifuged and separated into a heavy phase (HP) and a light phase (LP). HP mainly contains cellular biomass and a portion of the aqueous fermentation medium. LP contains the products in the form of oil, emulsion, and a portion of the fermentation medium. In certain processes, the recovery of products from the light phase is carried out by adding a surfactant while mixing and heating to break down the emulsion. The combination of the light phase (LP) and the surfactant is sent to a centrifuge or other separation mechanism to recover the products.

[0003]

[0003] A challenge in the fermentation production of organic compounds is the difficulty in recovering them from the fermentation mixture. Separation of organic compounds from the fermentation mixture often relies on the use of surfactants to break down emulsions containing organic compounds. However, the addition of surfactants increases costs, and the surfactants become impurities that must be removed by downstream processes (DSP). Furthermore, some emulsions are not broken down by the addition of surfactants, while others are only partially broken down by the addition of surfactants. Further steps or additional conditions required to break down the emulsions, such as the addition of heat, carry the risk of generating further impurities. There is a need for a method to recover fermentation products from the culture medium in high yield while reducing the amount of surfactants used.

[0004] [Brief Overview]

[0004] In one embodiment, a method for reducing emulsion formation inside the fermenter during the production of organic compounds may include fermenting microorganisms using a supply material in the fermentation vessel to produce whole cell broth (WCB) containing organic compounds, adding a surfactant to the supply material, whole cell broth, a portion of the whole cell broth, or a combination thereof in the fermentation vessel, optionally removing a portion of the whole cell broth, and heating the fermentation vessel.

[0005]

[0005] In one embodiment, the microorganism is a bacterium. The bacterium may be Escherichia, optionally E. coli, Bacillus, or Lactobacillus species.

[0006]

[0006] In one embodiment, the microorganism is a fungus, optionally a yeast. The yeast may be a Saccharomyces, optionally S. cerevisiae, or Pichia species.

[0007]

[0007] In one embodiment, the microorganism is an alga, optionally a Chlorella species.

[0008]

[0008] In one embodiment, the microorganism is a recombinant microorganism.

[0009]

[0009] In one embodiment, the surfactant is selected from anionic surfactants, nonionic surfactants, or a combination thereof.

[0010]

[0010] In one embodiment, the surfactant is an anionic surfactant selected from sulfates and sulfonates, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate, disulfonates, sodium dodecylnaphthalene sulfate, dialkylbenzenealkyl sulfates and sulfonates, acids, optionally abitic acid, alkyldiphenyl oxide disulfonates containing two C6-C16 hydrocarbon chains branched to each of the sulfonated aromatic rings, and combinations thereof.

[0011]

[0011] In one embodiment, the surfactant is a copolymer of propylene oxide (PO) and ethylene oxide (EO), which includes a nonionic surfactant selected from polyether polyols, and optionally, one made by capping polypropylene glycol with ethylene oxide. The surfactant may be a nonionic surfactant which is a polyether glycol having a molecular weight of at least 2400 g / mol, less than 2800 g / mol, or less than 3000 g / mol. The anionic surfactant may be selected from sulfates and sulfonates.

[0012]

[0012] In one embodiment, the nonionic surfactant is a polyether polyol. The surfactant may be L-62 (polyether polyol). The surfactant may be L-81 (polyether polyol).

[0013]

[0013] In one embodiment, the surfactant is added in an amount of about 0.001% v / v (volume / volume) to 1% v / v (volume / volume). The surfactant may be added in an amount of about 0.01 to 1% v / v. The surfactant may be added in an amount of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 1% v / v. The surfactant may be added in an amount of about 0.02 to 0.06% v / v, 0.03 to 0.05% v / v or 0.03 to 0.04% v / v.

[0014]

[0014] In one embodiment, the surfactant is added in solid form, as needed, in crystalline, amorphous, pelletized, granular, or a combination thereof. The surfactant may be added in pure form, solution, optionally in diluted solution, saturated solution, or supersaturated solution form.

[0015]

[0015] In one embodiment, fermentation lasts for about 1 to 14 days. Fermentation lasts for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0016]

[0016] In one embodiment, a portion of the whole cell broth is removed after 1 day. A portion of the whole cell broth may be removed after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

[0017]

[0017] In one embodiment, step (b) is approximately 24 hours.

[0018]

[0018] In one embodiment, the method further includes isolating organic compounds from whole cell broth.

[0019]

[0019] In one embodiment, the organic compound includes an isoprenoid (terpenoid).

[0020]

[0020] In one embodiment, the method further includes isolating the isoprenoid.

[0021]

[0021] In one embodiment, the isoprenoid is selected from the group consisting of (a) hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes, or (b) an isoprenoid that is not a carotenoid, or (c) a C5-C 20 isoprenoid, or (d) selected from the group consisting of abietadiene, amorphadiene, caryophyllene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene. The isoprenoid is a C5-C 20 isoprenoid. The isoprenoid can be a hemiterpene, optionally isoprene. The isoprenoid can be a monoterpene. The monoterpene can be selected from caryophyllene, geraniol, linalool, limonene, myrcene, ocimene, β-pinene, sabinene, γ-terpinene, terpinolene, or a combination thereof. The isoprenoid can be a sesquiterpene isoprenoid. The sesquiterpene isoprenoid can be selected from abietadiene, amorphadiene, farnesene, optionally α-farnesene, β-farnesene, or a combination thereof, farnesol, nerolidol, patchoulol, valencene, and a combination thereof. The isoprenoid can be farnesene. The isoprenoid can be α-farnesene, β-farnesene, or a mixture thereof. The isoprenoid can be a diterpene, optionally geranylgeraniol.

[0022]

[0022] In one embodiment, the method can further include separating the whole cell broth into an organic phase containing the isoprenoid compound and a heavy phase containing the solid and liquid fermentation medium.

[0023]

[0023] In one embodiment, the method can further include separating the whole cell broth into a liquid phase containing the isoprenoid compound and a solid phase containing the microorganism and cell debris.

[0024]

[0024] In one embodiment, the method may further include separating a liquid phase containing an isoprenoid compound into a light phase containing the isoprenoid compound and a heavy phase containing a dead cell layer, cell debris, and a fermentation aqueous phase.

[0025]

[0025] In one embodiment, the method may further include further purifying the isoprenoid.

[0026]

[0026] In one embodiment, the method includes an air supply to the fermenter at about 3 L / min to 10 L / min.

[0027]

[0027] In one embodiment, the dissolved oxygen in the fermenter can be maintained above about 15%.

[0028]

[0028] In one embodiment, the dissolved oxygen in the fermenter can be maintained at 0%.

[0029]

[0029] In one embodiment, the surfactant reduces emulsion formation. The surfactant can reduce emulsion formation in the range of about 1% to 100% compared to emulsion formation without the addition of the surfactant. The surfactant can reduce emulsion formation between about 10%, 20%, 30%, 40%, 50%, 60%, 80%, or 90% compared to emulsion formation in the fermenter without the addition of the surfactant.

[0030]

[0030] In one embodiment, the surfactant reduces the amount of dead cells (dead cell layer) in the whole cell broth. The surfactant can reduce the amount of dead cells (dead cell layer) by about 1% to 50%.

[0031]

[0031] In one embodiment, the surfactant prevents emulsion formation.

[0032]

[0032] This patent or application file includes at least one color drawing. Copies of this patent or patent application with color drawings are provided by the Patent Office upon request and payment of the required fees.

Brief Description of the Drawings

[0033] [Figure 1] The following shows a 14 mL sample taken from a 10 L fermentation tank for the eighth time and centrifuged at 11,000 × g for 3 minutes (1st photo = sample from a control fermentation tank sprayed at a rate of 3 L / min, 2nd photo = sample from a second control fermentation tank, but sprayed with air at a rate of 6 L / min, 3rd photo = sample in which L62 was added once inside the fermentation tank (added once between each sampling), 4th photo = sample in which L62 was added inside the fermentation tank (L62 was added every 2 hours until sampling). The samples were centrifuged at 11,000 × g for 3 minutes. Figure 2-1: Centrifuged sample from fermenter NB5 without added surfactant (control). Figure 2-2: Centrifuged sample from fermenter NB6, where 25% was collected instead of the usual 30-40% from other fermenters. Figure 2-3: Centrifuged sample from fermenter NB7, to which L-62 was added once between samplings. Figure 2-4: Centrifuged sample from fermenter NB8, to which L-62 was added semi-continuously between samplings. [Figure 2] This figure shows the %v / v of biomass for samples from fermentation tank NB5 (control), fermentation tank NB7 (L62 added once between samplings), and fermentation tank NB8 (L62 added semi-continuously). This figure also shows the %v / v of oil in fermentation tank NB7 (L62 added once between samplings) and fermentation tank NB8 (L62 added semi-continuously). The figure also shows the amount of surfactant added in grams inside NB7 (L62 added once between samplings) and inside fermentation tank NB8 (L62 added semi-continuously). [Figure 3] This figure shows the estimated emulsion amount at each sampling time in control fermenter NB5, the estimated crude oil amount at each sampling time in fermenter NB7 (L62 added once between samplings), and the estimated crude oil amount at each sampling time in fermenter NB8 (L62 added semi-continuously). This figure also shows the surfactant (in grams) added to the inside of fermenter NB7 (L62 added once between samplings) and the surfactant (in grams) added to the inside of fermenter NB8 (L62 added semi-continuously to the inside of the fermenter). [Figure 4]The four main phases after demulsification and centrifugation are shown. The top layer represents crude oil containing farnesene. The second layer from the top represents the dead cell layer (DCL). The third layer from the top represents the aqueous phase, mainly containing aqueous media derived from fermentation. The fourth layer from the top is mainly solid matter derived from biomass and the fermentation process.

[0034] [Detailed explanation]

[0037] Before further describing the subject matter, it should be understood that this disclosure is not limited to the specific embodiments of this disclosure described below, as variations of certain embodiments may be made and remain within the scope of the attached claims. It should also be understood that the terms used are intended to describe, and not to limit, certain embodiments. Rather, the scope of this disclosure is established by the attached claims.

[0035]

[0038] In this specification and the appended claims, the singular forms “a,” “an,” and “it” encompass multiple subjects unless otherwise explicitly indicated. Unless otherwise specified, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs.

[0036] [Definition]

[0039] As used herein, “organic compound,” “bioorganic compound,” or “microbial organic compound” broadly refers to organic compounds produced by microbial cells, including recombinant microbial cells and naturally occurring microbial cells.

[0037]

[0040] As used herein, “cells” broadly refers to microorganisms that can grow in liquid growth media.

[0038]

[0041] As used herein, “dry weight” or “dry substance” broadly refers to the weight determined in the relative absence of water. For example, a reference to a cell as containing a certain percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all of the water has been removed (to a certain weight).

[0039]

[0042] As used herein, “dry cell weight,” “dry cell material,” or “total suspension solid” broadly refers to the weight measured in the relative absence of water after washing the sample for removal of insoluble solids.

[0040]

[0043] As used herein, “isoprenoid compound” broadly refers to compounds that can be derived from isopentenyl diphosphate (IPP).

[0041]

[0044] "Microbial cells" refer to organisms such as algae, bacteria, fungi, mites, and combinations thereof, including single-celled organisms.

[0042]

[0045] As used herein in relation to fermentation products, “cell-related” broadly refers to fermentation products related to host cells or host cell debris.

[0043]

[0046] As used herein, “emulsion” broadly refers to a mixture of two immiscible liquids, such as water and oil. As used herein, it particularly refers to a mixture of an organic compound as envisioned herein and water.

[0044]

[0047] As used herein, “host cell” broadly refers to a microbial cell used for the production of an organic compound. A host cell may be a recombinant cell, meaning that it has been genetically modified to induce or increase the production of an organic compound. A host cell may contain foreign DNA and / or have one or more genetic modifications that affect the production of an organic compound compared to a wild-type organism. However, a host cell can also be considered a microbial cell that naturally produces the organic compound of interest.

[0045]

[0048] As used herein, "surfactant" broadly refers to a compound that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid.

[0046] [Farnesene production process]

[0049] The current farnesene process involves fermentation by genetically modified yeast microorganisms to produce farnesene oil, which forms an emulsion in a fermentation broth. The emulsion in the fermenter is harvested partially or whole in the form of whole cell broth (WCB), and in downstream processing (DSP), the biomass is separated from the emulsion in a first-stage solid-liquid (S / L) centrifugation step, and the clarified cell broth (CCB), which contains the emulsion, liquid fermentation broth, proteinaceous material, dead cells, and cell debris, is advanced to the next step. In the next step, the CCB is heated to 75-80°C, and L-62 surfactant is added inline and mixed in a tank. The mixture of this surfactant and CCB is centrifuged to separate the crude oil. The crude oil contains organic compounds, such as isoprenoids (e.g., farnesene oil). Because the process is continuous, the composition of the emulsion varies with each harvest, making it difficult to add the surfactant in a uniform proportional amount. Due to variability in the deemulsification process and the presence of oil in dead cells, approximately 8–10% product loss is observed in the liquid-liquid (L / L) biphase.

[0047]

[0050] The resulting crude oil contains surfactant residue, so it is also necessary to distill the crude oil to separate the farnesene oil from the surfactant. This resulted in additional process costs. The evaporator also became a bottleneck in expanding production capacity. The inventors attempted to prevent emulsion formation in the fermenter or complete demulsification within the fermenter by adding a surfactant inside the fermenter during the fermentation process.

[0048] [Reduction of emulsion formation]

[0051] The method described herein aims to eliminate or reduce emulsion formation in the source and / or demulsify any emulsions that do form by adding a surfactant to the fermentation vessel during the fermentation process. The addition of the surfactant may be a single addition between harvests, or a specified amount of surfactant added at harvest or during the fermentation process (e.g., regular additions of different amounts of surfactant at regular intervals). The method described herein prevents emulsion formation in the fermentation vessel and / or achieves demulsification. The method described herein reduces the amount of surfactant consumed for farnesene oil production by up to 91% compared to current processes. The method described herein provides improved downstream biomass recovery. Furthermore, the inventors unexpectedly found that supplying a sufficient amount of dissolved oxygen more than doubled the fermentation productivity of farnesene oil. The inventors also found a reduction in the amount of dead cells in the harvested material or the harvested whole cell broth, indicating that the surfactant reduced the shear effect on cells during fermentation.

[0049]

[0052] The methods described herein facilitate the separation and recovery of terpenoids, lipid oils, biomass, and any other products that form emulsions or similar compositions during the fermentation process. The recovered terpenoids and other oils can be used as precursors for the production of other products, including vitamins, pharmaceuticals, fragrances, cosmetics, animal feed, and fuel oils. The methods described herein can also be used for the separation and purification of intracellular products, or for the concentration and recovery of microorganisms when the microorganisms are used in products such as animal feed. The methods described herein reduce chemical consumption, waste, and costs, and improve product quality.

[0050]

[0053] The methods described herein solve problems associated with demulsification or biomass separation processes by preventing emulsion formation or performing demulsification simultaneously. The methods described herein reduce the problems of long fermentation processes by increasing productivity, solve the time-consuming problems of biomass and emulsion separation, mixing, heating, and multi-stage centrifugation, and reduce material transport costs. The methods described herein significantly reduce the consumption of chemicals associated with the demulsification of fermentation products. The methods described herein eliminate or reduce the processes required to separate surfactants from the final product. The methods described herein also reduce the environmental footprint by eliminating the need for evaporators, reducing the need for wastewater treatment, and reducing or eliminating other environmental problems associated with chemicals and salts currently used in conventional methods.

[0051]

[0054] The advantages of the method described herein are that emulsion formation is eliminated or minimized, and if emulsion formation occurs, it is demulsified in the fermenter. This eliminates the need for heating and the addition of chemicals to the fermentation broth for demulsification in downstream processes, thus significantly reducing chemical consumption. Another advantage of the method described herein is that surfactant consumption is reduced by up to 91%. Other advantages of the method described herein include higher quality products and the elimination of additional separation and purification steps such as evaporation or distillation. The method described herein reduces the environmental footprint by reducing the disposal of chemicals into the environment. The method described herein reduces the disposal of products in waste streams and improves productivity and recovery.

[0052] [Addition of L-62 to the inside of the fermentation tank during fermentation]

[0055] The method described herein involves adding a surfactant (L-62) during fermentation, thereby reducing emulsion formation, including complete demulsification within the fermenter. Using the method described herein, a reduction of up to 91% in surfactant consumption can be achieved compared to a comparative method in which the surfactant is added in a downstream process to demulsify the emulsion.

[0053]

[0056] The addition of L-62 during fermentation resulted in a 2-4% improvement in overall recovery compared to the current process, and a 6-8% improvement compared to adding L-62 to the harvest tank. As a result of adding L-62 to the fermenter, two-stage centrifugation can be reduced to single-stage centrifugation. Based on the crude oil properties determined by solid-liquid (S / L) centrifugation, L / L centrifugation can be used in the refining process. The successful addition of L-62 to the fermenter eliminates the need to heat the CCB to, for example, 75-80°C. Product loss to the dead cell layer (DCL) in the L / L heavy phase stream separated by L / L centrifugation was reduced from 8-10% in the current process to approximately 3-6%. Adding surfactants to the inside of the fermenter reduced cell death by reducing the shear effect on the biomass.

[0054] [Production of Farnese oil]

[0057] The method described herein provides a process for recovering isoprenoid compounds produced by fermentation from a fermentation medium, comprising: (a) fermenting isoprenoid-producing microorganisms; (b) (after the fermentation period) demulsifying the fermentation medium or a fraction thereof in a fermenter in the presence of a surfactant to produce a stream having an organic phase containing isoprenoid compounds and a phase heavier than the organic phase; and (c) separating the stream obtained in step (b) into an organic phase containing isoprenoid compounds ("light phase"), a phase heavier than the organic phase, and a solid phase ("heavy phase") optionally containing host cells and cell debris.

[0055]

[0058] The pH range of the fermentation medium or its fraction is 4 to 7. The pH range of the fermentation medium or its fraction is 4.8 to 5.2, preferably 5.0.

[0056]

[0059] The surfactant is selected from anionic surfactants, nonionic surfactants, or a combination thereof. The anionic surfactant is selected from sulfates and sulfonates, and the nonionic surfactant is a polyether polyol. Both are preferably water-soluble at room temperature (20°C). Preferably, the surfactant is L-62, also known as TERGITOL®, and 99.0% or more is polyalkylene glycol (CAS 9003-11-6).

[0057]

[0060] The surfactant concentration in the fermentation medium or its fraction in emulsion reduction step (a) is approximately 0.01 to 1.0% v / v. For example, the surfactant concentration in the fermentation medium or its fraction in emulsion reduction step (a) may be approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 1.0% v / v. The concentration may be approximately 0.03, 0.04, or 0.05% v / v. The concentration may be approximately 0.01 to 0.05 v / v% or 0.02 to 0.06 v / v%.

[0058]

[0061] The separation step (b) may consist of solid-liquid separation followed by a liquid-liquid purification step.

[0059]

[0062] The method described herein is for the recovery of isoprenoids as fermentation products from fermentation media, (a) Adding a surfactant to the fermentation vessel and allowing isoprenoid-producing microorganisms to ferment in the vessel, (b) Heat the fermentation vessel to approximately 25°C to 35°C, (c) Harvesting all or part of the fermented broth, (d) Selectively separating by centrifugation a heavy phase containing solids including microorganisms, cell debris and cellular material and liquid fermentation medium from a light phase mainly containing organic phase mainly containing isoprenoid compounds (and selectively further purifying the crude oil mainly containing isoprenoid compounds by distillation into pure isoprenoid compounds and small amounts of impurities), (e) Optionally, further purify the light phase by centrifugation into a liquid organic phase mainly containing isoprenoids and a small amount of heavy phase containing solids and liquid fermentation medium. (f) Optionally, further purify the crude oil, which mainly contains isoprenoids, by distillation to pure isoprenoid compounds and small amounts of impurities. This may include the recovery of [the product / service].

[0060] [Microbial fermentation]

[0063] Isoprenoids can be produced by microbial fermentation. For example, farnesene, a sesquiterpene isoprenoid, can be produced by microbial fermentation. Typically, microorganisms are cultured under conditions suitable for the production of organic compounds by microbial host cells. Suitable conditions include many parameters such as temperature range, aeration and / or oxygen supply level, pH, and culture medium composition. Each of these conditions, individually and in combination, is typically modified and / or optimized to allow the microbial host cells to grow and / or ensure the optimal production of the desired organic compound. Exemplary culture media include broth or gel. Host cells can be grown in a culture medium containing a carbon source used for host cell growth. Exemplary carbon sources include carbohydrates such as glucose, fructose, and cellulose, which can be directly metabolized by host cells. Furthermore, enzymes can be added to the culture medium to promote the recruitment of carbon sources (e.g., depolymerization of starch or cellulose into fermentable sugars) and subsequent metabolism. The culture medium may optionally contain additional nutrients required by specific microbial strains, such as inorganic nitrogen sources and minerals, for example, ammonia or ammonium salts. Other growth conditions, such as temperature and cell density, are generally selected to provide an economical process. Temperatures during the growth and growth phases, respectively, can range from above the freezing point of the culture medium to approximately 50°C. Fermentation can be carried out aerobic, anaerobic, or substantially anaerobic. Simply put, anaerobic conditions refer to an environment lacking oxygen. Substantially anaerobic conditions include, for example, culture, batch fermentation, or continuous fermentation where the dissolved oxygen concentration in the culture medium remains at 0-10% of the saturation level. Substantially anaerobic conditions also include cell growth or standing in liquid medium or on solid agar in a sealed chamber maintained in an atmosphere of less than 1% oxygen. The oxygen ratio can be maintained, for example, by spraying the culture medium with an N2 / CO2 mixture or one or more other suitable non-oxygen gases. Fermentation can be carried out continuously, in batches, or a combination thereof. Conventional fermentation bioreactors, shaking flasks, test tubes, microtiter dishes, and Petri plates can be used.

[0061]

[0064] In a preferred embodiment, the present invention is a method for producing an organic compound, (a) Fermenting microorganisms using supplied raw materials in a fermentation vessel to produce whole cell broth (WCB) containing organic compounds, (b) Adding a surfactant to the feedstock, whole cell broth, a portion of the whole cell broth, or a combination thereof in the fermentation vessel, (c) Selectively removing a portion of the whole cell broth, (d) Heating the fermentation vessel and The present invention provides a method comprising supplying air or oxygen to a fermenter, wherein the organic compound includes isoprenoids. As shown in the examples, preferably the supply of air or oxygen to the fermenter is approximately 3 L / min to 10 L / min. Preferably, the air or oxygen is supplied simultaneously with or after the addition of the surfactant. Preferably, the supply of air or oxygen is applied continuously. More preferably, the supply of air or oxygen is applied continuously for at least 50% of the time, more preferably at least 70% of the time, even more preferably at least 90% of the time, even more preferably at least 95% of the time, and most preferably at least 100% of the time, from the start of surfactant addition to the end of fermentation. The supply of air or oxygen is preferably supplied via a separate supply line from the surfactant supply line. Therefore, the supply of air or oxygen is preferably supplied in parallel with the supply of surfactant. Preferably, the amount of air or oxygen supplied is increased over time. The supply of air or oxygen may be increased gradually or in stages. Most preferably, the supply of air or oxygen is started simultaneously with or after the addition of the surfactant and subsequently increased over time. More preferably, the amount of oxygen added increases over time. Preferably, the method includes supplying pure oxygen to the fermenter following the supply of air to the fermenter. More preferably, the method may include supplying pure oxygen to the fermenter following the supply of air to the fermenter, preferably about 3 L / min to 10 L / min. Pure oxygen is understood herein to mean a gas containing 99% by volume or more oxygen, more preferably 99.6% by volume or more oxygen. The selection of microorganisms, surfactants and isoprenoids is as described above and below in this specification. More preferably, the surfactant is a polyether polyol, even more preferably a polyether polyol containing propylene oxide (PO) and / or ethylene oxide (EO), most preferably a copolymer of propylene oxide (PO) and ethylene oxide (EO). Most preferably, the isoprenoid is farnesene.

[0062]

[0065] In a preferred embodiment, the present invention is a method for producing an organic compound, (a) Fermenting microorganisms using supplied raw materials in a fermentation vessel to produce whole cell broth (WCB) containing organic compounds, (b) Adding a surfactant to the feedstock, whole cell broth, a portion of the whole cell broth, or a combination thereof in the fermentation vessel, (c) Selectively removing a portion of the whole cell broth, (d) Heating the fermentation vessel and The present invention also provides a method comprising an organic compound, comprising an isoprenoid, and wherein the dissolved oxygen in the fermenter is maintained at more than about 15%. Aeration of the culture medium can be adequately carried out by the aforementioned air or oxygen supply. More preferably, the method comprises supplying air or oxygen to the fermenter, after which the dissolved oxygen in the fermenter is maintained at more than about 15%. The selection of microorganisms, surfactants and isoprenoids is as described above and below in this specification. More preferably, the surfactant is a polyether polyol, even more preferably a polyether polyol comprising propylene oxide (PO) and / or ethylene oxide (EO), most preferably a copolymer of propylene oxide (PO) and ethylene oxide (EO). Most preferably, the isoprenoid is farnesene. As shown in the examples, aeration of the fermentation medium advantageously allowed for increased biomass and oil formation. As a result, the above method according to the present invention may preferably be a method that does not involve a demulsification step.

[0063] [Microbial cells]

[0066] Microorganisms suitable for fermentation are well known in the art. Suitable microorganisms include, but are not limited to, bacteria such as Escherichia (e.g., Escherichia coli), Bacillus or Lactobacillus species, fungi, particularly Saccharomyces (e.g., S. cerevisiae) or Pichia, or algae such as Chlorella. The microbial host cell may be a fungus, preferably a yeast. The microorganism may be able to produce the desired organic compound naturally, or it may be genetically modified to ensure the production of the desired organic compound, such as farnesene (e.g., recombinant microorganisms).

[0064]

[0067] Microorganisms suitable for use in the methods described herein are capable of producing farnesene, and in particular, microorganisms capable of secreting farnesene. Non-limiting examples of microorganisms suitable for the present invention are the genetically modified host cells described in International Publication Nos. 2013 / 071172, 2014 / 144135, 2008 / 039499, 2007 / 140339, and 2006 / 014837.

[0065] [Addition of surfactants]

[0068] The method described herein provides a reduction in emulsion formation during fermentation (including complete demulsification within the fermenter) and eliminates the need for a demulsification step in downstream processes. The method may be carried out by adding the surfactant at the start of the fermentation process, at the start of the inoculation step, or at 24 hours into the logarithmic growth phase. The method may be carried out by adding the surfactant once between partial samplings of the fermentation medium, or by adding the surfactant at time intervals, for example, every hour, every two hours, every three to five hours, every four to ten hours, or every 24 hours or every 48 hours.

[0066] [Harvesting and recovery of isoprenoid compounds]

[0069] This method can also be carried out by taking a fraction of the fermentation medium or by harvesting the entire fermentation medium. The harvested fermentation broth can be separated into a fermentation medium fraction using a solid-liquid centrifuge. The fermentation medium fraction may be a liquid stream obtained by extracting isoprenoid compounds from the solid-liquid biphase obtained by solid-liquid separation of the fermentation medium into a solid-liquid biphase and a solid-liquid light phase. Some organic compounds are actually related to the cells and / or host cell debris contained in this solid-liquid biphase (e.g., located within the internal cell compartment or adsorbed onto the cell wall).

[0067]

[0070] The fermentation medium fraction may be a liquid stream obtained by extracting isoprenoid compounds from the liquid-liquid biphase. This liquid-liquid biphase is obtained by liquid-liquid separation of the solid-liquid light phase into a liquid-liquid biphase and a liquid-liquid light phase. The solid-liquid light phase is obtained from the fermentation medium by optional solid-liquid separation into a solid-liquid biphase and a solid-liquid light phase, as previously described. This liquid-liquid biphase can be subjected to a second solid-liquid separation to separate it into a second solid-liquid light phase and a second solid-liquid biphase. Subsequently, isoprenoid compounds are extracted from the second solid-liquid light phase.

[0068]

[0071] The fermentation medium fraction may be a liquid stream obtained by extracting isoprenoid compounds from a phase heavier than the organic phase or from a solid phase containing host cell debris and cells.

[0069]

[0072] Fermentation media or fractions thereof may contain up to 35% v / v of cells (live or dead) and / or host cell debris. The %v / v of cells and / or debris can be measured by conventional methods, such as volume ratio measurements obtained by centrifugation in a capillary tube. For example, fermentation media may contain up to 35% v / v or up to 30% v / v of cells. Generally, about 1–5% v / v are cells that still contain isoprenoid compounds.

[0070] [Surfactants]

[0073] Surfactants are typically amphiphilic organic compounds, meaning they contain both hydrophobic and hydrophilic groups. Anionic surfactants contain anionic functional groups such as sulfates, sulfonates, phosphates, and carboxylates. Suitable surfactants include, but are not limited to, nonionic surfactants, anionic surfactants, and combinations thereof, particularly water-soluble anionic surfactants.

[0071]

[0074] Examples of anionic surfactants include, but are not limited to, sulfates and sulfonates, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate, sodium dodecylnaphthalene sulfate, dialkylbenzenealkyl sulfates and sulfonates, acids such as abitic acid, and combinations thereof. Other suitable anionic surfactants include, for example, alkyldiphenyl oxide disulfonates containing two branched C6-C16 hydrocarbon chains on each of the sulfonated aromatic rings, such as Dow Chemical Company's DOWFAX® 2A1.

[0072]

[0075] The surfactant is a sulfonate, particularly a disulfonate. Advantageously, this surfactant is water-soluble.

[0073]

[0076] Examples of nonionic surfactants include copolymers of propylene oxide (PO) and ethylene oxide (EO), such as polyether polyols, particularly those produced by capping polypropylene glycol with ethylene oxide, like the one commercially available from Dow Chemical Company as TERGITOL® L.

[0074]

[0077] The nonionic surfactant is a polyether glycol having a molecular weight of at least 2400 g / mol. In particular, the molecular weight is less than 3000 g / mol, preferably less than 2800 g / mol. The molecular weight may have a value within the range defined by any combination of these limit values.

[0075]

[0078] The nonionic surfactant is a polyether glycol having a molecular weight of at least 2400 g / mol. Advantageously, this surfactant is water-soluble.

[0076]

[0079] Combinations of these surfactants with any of the aforementioned surfactants may be included in the methods described herein.

[0077]

[0080] Surfactants can be added in amounts of approximately 0.01–1% v / v. For example, surfactants can be added in amounts of approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 1% v / v. Surfactants can also be added in amounts of approximately 0.02–0.06% v / v, 0.03–0.05% vv, or 0.03–0.04% vv.

[0078]

[0081] Surfactants can be introduced as solids (e.g., crystalline, amorphous, pelletized, and / or granular forms) and / or as solutions containing, for example, water, alcohol, or combinations thereof (e.g., diluted solutions, saturated solutions, or supersaturated solutions).

[0079]

[0082] The pH of the fermentation medium or its fraction is in the range of 2 to 7.

[0080]

[0083] At the end of the demulsification step (a), a stream is obtained having an organic phase containing isoprenoid compounds, a phase heavier than the organic phase, and a solid phase optionally containing host cell debris and cells.

[0081]

[0084] This stream may more particularly contain an oil organic phase, also referred to as the "organic phase", which contains isoprenoid compounds, a final residual emulsion phase, a heavy phase, also referred to as the "phase heavier than the organic phase", which mainly contains water, and finally a deposit at the bottom, which may contain debris and dead cells if present.

[0082] [Organic compound]

[0085] The organic compounds contemplated herein can be isoprenoid compounds, particularly terpenes or polyterpenes, such as farnesene.

[0083]

[0086] Terpenes are a large class of hydrocarbons produced by many organisms. They are derived by linking isoprene (C5H8) units and are classified by the number of isoprene units present. Hemiterpenes consist of a single isoprene unit. Isoprene itself is considered the only hemiterpene. Monoterpenes are made from two isoprene units and have the molecular formula C 10 H 16 Examples of monoterpenes are myrcene, geraniol, limonene and terpineol. Sesquiterpenes are composed of three isoprene units and have the molecular formula C 15 H 24 Examples of sesquiterpenes are farnesene, farnesol, amorph-4,11-diene and patchoulol. Diterpenes are made from four isoprene units and have the molecular formula C 20 H 32 Examples of diterpenes are cafestol, kaurene, cembrene and taxadiene. Sesterterpenes are made from five isoprene units and have the molecular formula C 25 H 40 An example of a sesterterpene is geranyl farnesol. Triterpenes consist of six isoprene units and have the molecular formula C 30 H 48 Sesterterpenes consist of eight isoprene units and have the molecular formula C 40 H 64It possesses the following characteristics. Biologically important tetraterpenes include acyclic lycopene, monocyclic γ-carotene, and bicyclic α- and β-carotene. Polyterpenes consist of long chains of many isoprene units. Natural rubber consists of polyisoprene in which the double bonds are in the cis configuration.

[0084]

[0087] When terpenes are chemically modified (for example, through oxidation or rearrangement of their carbon skeleton), the resulting compounds are generally called terpenoids, also known as isoprenoids.

[0085]

[0088] Isoprenoid compounds are typically composed of repeating 5-carbon isopentenyl diphosphate (IPP) units, but irregular isoprenoids and polyterpenes have also been reported.

[0086]

[0089] The isoprenoid compounds produced by the method described herein are (a) selected from the group consisting of hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes and polyterpenes, or (b) isoprenoids that are not carotenoids, or (c)C5~C 20 isoprenoid, or (d) Selected from the group consisting of abietadiene, amorphousdiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

[0087]

[0090] Isoprenoid compounds are C5-C 20 It can be an isoprenoid, and in particular, it does not have to be a carotenoid.

[0088]

[0091] The isoprenoid compound may be a hemiterpene, such as isoprene.

[0089]

[0092] The isoprenoid compounds are monoterpenes and may be selected from carene, geraniol, linalool, limonene, myrcene, ocimene, β-pinene, sabinene, γ-terpinene, terpinolene, or combinations thereof.

[0090]

[0093] The isoprenoid compounds are sesquiterpenes and can be selected from abietadiene, amorphousdiene, farnesene (α-farnesene, β-farnesene), farnesol, nerolidol, patchulol, valencene, and combinations thereof.

[0091]

[0094] The isoprenoid compound is a diterpene and may be geranylgeraniol.

[0092]

[0095] The isoprenoid compound may be a sesquiterpene. The isoprenoid compound may be farnesene. The isoprenoid compound may be α-farnesene, β-farnesene, or a mixture thereof.

[0093] [Overall recovery process]

[0096] The methods described herein involve the production and recovery of organic compounds, particularly isoprenoid compounds, from a fermentation mixture. The fermentation mixture (also referred herein as fermenter broth or whole cell broth (WCB)) typically includes microorganisms, a culture medium, and fermentation products or organic compounds produced by the microorganisms once the culture is initiated. These organic compounds are preferably released or secreted as organic compounds by the microorganisms in the culture medium, from which these organic compounds can be recovered.

[0094]

[0097] The recovery method described herein involves separating the cell fraction from the remaining fermentation medium. This may be done by centrifugation, which generates a microbial pellet that is discarded, while the organic phase, generally the supernatant, is used for further extraction of organic compounds. Some of the organic compounds of interest remain bound, at least partially, to the microbial host cells during the standard recovery process (referred to herein as cell-associated organic compounds). These are typically lost along with the microbial pellet in the current microbial production process.

[0095]

[0098] The fraction from which the cell fraction has been removed from the fermentation medium may be further processed for the recovery of organic compounds using conventional recovery processes, including but not limited to liquid-liquid separation.

[0096]

[0099] Another cause of organic compound loss is emulsion formation, which is inherent in microbial production systems. In fact, emulsion formation can be promoted in fermentation media by mechanical energy from fermentation (e.g., from a stirrer or fermentation gases produced by microbial host cells) or by microbial host cells or various biomolecules within them.

[0097]

[0100] This specification provides a recovery process for organic compounds from a fermentation mixture, characterized by comprising the recovery of the organic compounds contained in an emulsion, in this specification an emulsion of organic compounds in water.

[0098]

[0101] The method described herein provides an improved recovery of organic compounds from a fermentation mixture, the method comprising recovering organic compounds present in the fermentation medium in an emulsion in the presence of a salt and a surfactant.

[0099]

[0100] The method described herein may include the steps of providing a fermentation mixture comprising a microorganism, a culture medium, and a fermentation product or organic compound produced by the microorganism, and adding a surfactant to the fermentation vessel to reduce emulsion formation.

[0100] [Solid-liquid separation process]

[0101] The method described herein may include a solid-liquid separation step. The solid-liquid separation step separates microorganisms from the fermentation mixture. The stream containing microorganisms is also referred herein as “microbial pellet” or “solid-liquid biphase”. This stream contains microorganisms and cell-associated organic compounds and may further contain host cell debris, culture medium and organic compounds. The solid-liquid biphase mainly contains biomass. The supernatant or light phase obtained by solid-liquid separation of the fermentation mixture is also referred herein as “solid-liquid light phase” or “concentrated clarified broth (CCB)”, and contains culture medium, free organic compounds (organic phase) and organic compounds contained in oil, and may further contain host cell debris. There is also an option to recover isoprenoid compounds from the light phase in this solid-liquid separation step.

[0101]

[0102] Solid-liquid separation of fermentation mixtures can be achieved by techniques known to those skilled in the art, including but not limited to centrifugation, filtration, and decantation, preferably centrifugation. Centrifugal separators can separate fermentation mixtures in batch or continuous flow. Preferably, the methods described herein use continuous flow centrifugation. Non-limiting examples of centrifuges suitable for solid-liquid separation of fermentation mixtures taught herein are disc centrifuges, such as disc centrifuges with nozzles. Centrifugal separation conditions can preferably be determined by those skilled in the art to achieve the desired solid-liquid separation.

[0102] [Separation process]

[0103] In this step, the organic phase obtained in step (a) can be separated. The organic phase containing isoprenoid compounds is separated by liquid-liquid separation such as centrifugation. In the liquid-liquid separation step, organic compounds are separated from the phase heavier than the organic phase. The obtained light phase containing organic compounds is also referred to herein as “crude raw material,” “organic phase,” or “liquid-liquid light phase.” This stream contains organic compounds produced by fermentation and may further contain some cells, generally dead cells. The recovered heavy phase, also referred to herein as “phase heavier than the organic phase” or “liquid-liquid heavy phase,” contains culture medium, cells, and may further contain host cell debris, free organic compounds, and organic compounds contained in the emulsion. A further solid phase can be obtained, also referred to herein as “waste phase” or “waste composition,” which contains culture medium, host cell debris, cells, and may further contain free organic compounds and organic compounds contained in the emulsion.

[0103]

[0104] Liquid-liquid separation can be achieved by known techniques, including but not limited to centrifugation, filtration, and decantation, preferably by centrifugation.

[0104]

[0105] Centrifugal separators can separate liquid-liquid light phases in batch or continuous flow configurations. Preferably, the methods described herein use continuous flow centrifugation. A non-limiting example of a centrifuge suitable for liquid-liquid separation of fermentation mixtures taught herein is a disk centrifuge. Centrifugal separation conditions can preferably be determined by those skilled in the art to achieve the desired liquid-liquid separation.

[0105] [Extraction of relevant isoprenoid compounds from within cells]

[0106] The solid phase obtained in step (b), which is heavier than the organic phase or contains host cell debris and cells, the aforementioned solid-liquid bilayer phase, or liquid-liquid bilayer phase, all contain cells (dead cells or living cells depending on the fraction) and / or host cell debris, and all of these contain some isoprenoid compound.

[0106]

[0107] Isoprenoids in one or more of these phases of cells and / or host cell debris are extracted to obtain a liquid stream containing isoprenoid compounds. This extraction can be obtained by any suitable technique known in the art for extracting / releasing organic compounds from cells and / or host cell debris. These techniques include, but are not limited to, mechanical, chemical, enzymatic, physical, or combinations thereof. Mechanical techniques include, but are not limited to, cell homogenization, sonication of cells, cooling and compression of cells, grinding of cells, or combinations thereof. In preferred embodiments, grinding is used to extract organic compounds from cells and / or host cell debris.

[0107]

[0108] Examples of mechanical cell processing include, but are not limited to, the use of French press cell disruptors, ultrasonic generators, homogenizers, ball mills, rod mills, pebble mills, bead mills, high-pressure grinding rolls, vertical shaft impact devices, industrial blenders, high-shear mixers, paddle mixers, and Polytron homogenizers, as well as combinations thereof.

[0108]

[0109] Physical treatments include, but are not limited to, heating, depressurizing, and osmotic shock of cells. Examples of cell heating include, but are not limited to, resistance heating, convection heating, steam heating, heating in a fluid bath, heating by solar energy, and heating by concentrated solar energy. Any of these can be carried out in a tank, pool, tube, conduit, flask, or other containment device.

[0109]

[0110] Chemical treatments include, but are not limited to, contacting cells with chemicals such as antibiotics, chelating agents, solvents, detergents, chaotropic agents, or combinations thereof.

[0110]

[0111] Enzymatic lysis refers to the dissolution of a cell wall or cell membrane by contacting the cell with one or more enzymes. Enzymatic methods include lysis by enzymes or autolysis.

[0111] [Examples] [Example 1] [Addition of L-62 in 2 x 10L fermentation tanks] [Main steps]

[0112] One 10L fermenter was used as a control (NB5-F-164), a second 10L fermenter was used for adding a specific amount of L-62 immediately after each sampling and at 24-hour intervals (NB7-F-164), and a third 10L fermenter was used for continuous addition of L-62 during fermentation (NB8-F-164). However, continuous addition was unsuitable due to the pump stroke size, so the addition of L-62 was changed to a semi-continuous method (addition was started with a specific amount added at 1-hour intervals, and then changed to 2-hour intervals for convenience). The added L-62 was diluted to 25% with water and filtered sterilized. Dilution was necessary to facilitate pumping and small-volume addition. To ensure there were no problems caused by cell inoculation into the 10L fermenters, L-62 was not added until 24 hours of LH. At LH24, L-62 was added in an amount equivalent to that required for the current process based on the amount harvested. The amount added decreased with each harvest until LH240, when L-62 was no longer added. At NB7, L-62 was added again at the next harvest, and at NB8, it was added periodically at 2-hour intervals until fermentation was complete or harvesting.

[0112]

[0113] Aeration in each of the three fermenters was maintained at an equivalent air supply of 3 L / min to each fermenter. Upon initiation of L-62 addition, biomass and cellular oxygen demand increased significantly in NB7 and NB8, and dissolved oxygen (DO) levels were lower compared to the control. DO levels began to decrease, reaching zero in NB7 and NB8 at approximately LH96. Air supply was increased to 6 L / min in NB7 and NB8. No L-62 was added between the 8th and 9th samplings. After LH120, air supply was increased to 6 L / min to 10 L / min (maximum for the 10 L fermenter) in NB7 and NB8, while 3 L / min was maintained in the control. This increased biomass in the NB7 and NB8 fermenters, and at approximately LH216, the dissolved oxygen levels in NB7 and NB8 began to decline towards zero again. At LH288, 2-3 L / min of oxygen was supplied to NB7 and NB8 to increase the dissolved oxygen concentration.

[0113]

[0114] The effects of biomass volume and emulsion or crude oil were analyzed by capillary centrifugation of samples at each sampling and harvesting stage.

[0114] [Effects of L-62 addition on biomass and oxygen demand]

[0115] In all three fermenters, cell viability and biomass were similar in LH24 (before L-62 addition). Upon initiation of L-62 addition, biomass and oxygen requirements increased in NB7 and NB8. After L-62 addition, oil was detected in the total cell broth in subsequent samples, and direct centrifugation was sufficient to recover the crude oil (e.g., DSP demulsification was unnecessary). Increasing the aeration rate from 3 L / min to 10 L / min was insufficient to maintain DO above 0% due to the significant increase in cell proliferation rate after L-62 addition. Continuous supply of approximately 3 L / min of pure oxygen helped maintain dissolved oxygen above 15%.

[0115] [Effects of L-62 addition on crude oil]

[0116] After adding L-62, NB7 and NB8 contained oil in the samples, while the control contained an emulsion as expected. This indicates that emulsion formation in the fermenter is inhibited during fermentation, or that complete demulsification is possible.

[0116]

[0117] The amount of L-62 added decreased daily from LH24 to LH240, and no L-62 was added between the 8th and 9th samplings. No oil was detected in the 9th sample (all were emulsions). L-62 addition was resumed, and oil was observed again in the next (10th) sampling. This supports the effect of L-62 addition on the oil obtained from whole cell broth.

[0117] [Effects of L-62 addition on sample yield]

[0118] Extraction from NB7 and NB8 increased proportionally to biomass and dissolved oxygen percentage, but this was also affected by L-62 addition. Extraction increased from LH24 to LH96 and decreased until aeration increased. The maximum extraction obtained after aeration supply increased to a maximum of 10 L / min. Extraction began to decrease until pure oxygen supplementation was introduced one day before harvest. The minimum amount of L-62 required to prevent emulsification was used as the minimum requirement base and line to help estimate the reduction in L-62 consumption.

[0118] [Effects of L-62 addition on total crude oil volume]

[0119] As the amount extracted increases, the total amount of crude oil extracted also increases. In most cases, the graph of total crude oil is similar to the graph of total biomass. When L-62 addition was stopped at LH240, no crude oil was observed in the whole cell broth.

[0119] [Effects of L-62 addition on crude oil concentration]

[0120] The addition of L-62 increased biomass, resulting in an increased yield. This led to an increase in the total crude oil yield, but a slight decrease in crude oil (equivalent to concentration) per unit weight of yield. In most samples, the concentration appeared to be similar to that of the control.

[0120] [The effect of L-62 addition on crude oil quality]

[0121] The crude oil obtained in the laboratory appears lighter in color compared to the control. GC data showed that the purity of the crude oil with L-62 added was superior to that of the control.

[0121] [Effects of adding L-62 to fermentation tanks on surfactant consumption]

[0122] The amount of L-62 added started at approximately 1.5 g per kg of sample, gradually decreased to zero, and then increased again by up to 0.5 g per sample. Based on the observed consumption rate, the total consumption of L-62 based on whole cell broth decreased by 83% in NB7 and 82% in NB8. Based on laboratory demulsification of the control sample, the total consumption of L-62 decreased by 88% in both NB7 and NB8.

[0122] [Effects of L-62 addition on sugar yield]

[0123] Recalculation and fermentation data showed approximately 2% yield loss for surfactants added to the fermenter compared to the control group. Semi-continuous addition of L-62 was slightly superior in terms of productivity and recovery compared to single addition. L-62 addition significantly increased the amount of whole cell broth (WCB). Crude oil recovery increased by 24% in NB7 and 32% in NB8.

[0123] [Example 2] [Adding L-62 to a 10L fermentation tank] [Main steps]

[0124] The aeration supply rate for the control fermenter remained the same as in Example 1 at 3 L / min. Aeration for fermenters NB7 (L-62 added once between samplings) and NB8 (30% of the required amount of L-62 added once, with the remainder added in a semi-continuous mode) was set to 10 L / min, and pure oxygen was supplied from the start of the test. L-62 addition started after LH24. During the 12-day test period, approximately 30% of the required amount of L-62 was added at once, and the remaining amount was then added every two hours until the next sampling to maintain a constant level. NB7 was maintained under the same conditions as NB8.

[0124] [Effects of L-62 addition on biomass]

[0125] A slight difference in biomass at LH24 was observed between NB8 and the control (before L-62 addition). When L-62 addition was initiated, biomass increased significantly in NB8. Based on capillary tube measurements, the biomass of NB7, which had the same aeration as NB8 (10 L / min + pure oxygen supplementation), was slightly less than that of NB8. Dissolved oxygen concentration remained high at approximately 50% in NB7, while dissolved oxygen in NB8 was approximately 25% for most of the test.

[0125] [Effects of L-62 addition on crude oil]

[0126] In fermentation tanks to which L-62 was added, crude oil was obtained from the whole cell broth throughout the experiment. The inventors observed that the v / v ratio of the crude oil obtained in the samples collected increased until day 5 (LH96), and then remained constant until harvest.

[0126] [Effect of L-62 addition on sample yield]

[0127] The addition of L-62 significantly increased the amount collected (NB8). Aeration with pure oxygen supplementation resulted in a similar amount of collected material (NB7).

[0127] [Example 3] [Effect of L-62 addition on DSP recovery rate] [Main procedure (equivalent to adding L-62 to the harvest tank)]

[0128] Approximately 420g samples were taken from the fifth sampling from fermentation tanks NB5 and NB6, heated to 75°C, and 0.4% w / w L-62 was added for demulsification. The samples were mixed for 2 hours and centrifuged. The four phases (oil, dead cell layer (DCL), aqueous phase, and S / L HP layer) were decanted, collected, and weighed. Samples from each phase, including the starting WCB, were analyzed by GC. The sample from the fifth sampling of fermentation tank NB8 was centrifuged as is, and the four phases were separated in the same manner. The farnesene oil ("fene oil") distribution in each phase was analyzed by gas chromatography (GC).

[0128] [Results and Observations]

[0129] The overall recovery rates for NB5 (control) and NB6 were lower than the current recovery rates (recovery rate approximately 85%, total loss approximately 15%, current recovery rate approximately 88%). The loss distribution for the control was low in L / L HP and high in S / L HP, but the opposite was true in the current process. L-62 addition resulted in relatively low losses in both S / L HP and L / L HP. Compared to adding L-62 to the harvest tank, L-62 addition (NB8) reduced total losses by approximately 45-50%. Compared to the current process, L-62 addition reduced total losses by approximately 25%. The overall recovery rate when L-62 was added to the harvest tank was approximately 82-85%, while when L-62 was added inside the fermenter, the recovery rate was approximately 88-91%. In contrast, the current recovery rate is approximately 88%.

[0129] [Table 1]

[0130] [Example 4] [Effect of L-62 addition on downstream process (DSP) recovery rates] [procedure]

[0130] For NB5, samples and harvested material from batch 165 were mixed in a tank. Approximately 420 grams were sampled and 0.8% L-62 was added in the laboratory at 75°C to disemulsify, followed by centrifugation. For NB8, samples from batch 165 were centrifuged in the laboratory. The four phases were separated, weighed, and analyzed by GC.

[0131] [result]

[0131] In NB5, the recovery rate of 85.9% was lower than that of the current process, but the total loss rate was also higher at 17.2%. For NB5, the loss distribution was similar to that of the current process, with the L / L HP loss rate being approximately 8.7%, compared to approximately 8-10% in the current process. In NB5, the required amount of L-62 was twice the current consumption. In NB8, the overall recovery rate based on crude oil was high, but the total loss rate was close to the total loss rate obtained from extraction. The DCL loss distribution of NB8 was much smaller than that of NB5 or the current process.

[0132] [Table 2]

[0133] [Example 5] [The effect of "heating" fermentation broth harvested from L-62 added to the inside of a fermentation tank on DSP recovery rate] [Main steps]

[0132] For NB5, samples and harvested materials from batch 165 were mixed in a tank using a mini pilot. Approximately 400 grams of WCB were sampled, and after demulsification with 0.8% L-62 at 75°C in the laboratory, the sample was centrifuged. For NB8, the WCB sample from batch 165 was heated to 75°C in the laboratory and centrifuged. Two phases were separated from each sample, weighed, and analyzed by GC based on the purity of the crude oil.

[0134] [Results and Observations]

[0133] For NB5, the overall recovery rate was similar to other laboratory-scale tests and lower than the current process. For NB8, the overall recovery rate was 88%, which was equivalent to the current process. Crude oil with L-62 added showed higher purity than the control.

[0135] [Table 3]

[0136] [Example 6] [Effects of heat sterilization on the efficacy of surfactants] [Main steps]

[0134] WCB obtained from a 10L fermenter (NB5-F-1774) used as a control for strain development. The test of this fermenter was extended for two days, and L-62 that had been autoclaved was tested.

[0137] [result]

[0135] When WCB was centrifuged as is, the S / L HP loss rate was equivalent to that of the current process. When WCB was used to perform demulsification equivalent to that when L-62 was added to the harvest tank, the S / L HP loss rate was significantly higher than that of the current process. When CCB was separated and demulsified in the same way as the current process, the total loss rate was equivalent to that of the current process: Table 5. WCB with added L-62 had a loss rate approximately 2.5% lower compared to the current process: Table 5.

[0138] [Table 4]

[0139] [Table 5]

[0140] [Example 7]

[0136] L-62 (used in the first batch of 10L fermentation) was diluted to 25% with water, filtered, and autoclaved. Two-phase separation was observed during cooling. Both phases were mixed, and 450g of the sample heated to 75°C was set aside for bench-scale demulsification testing (Table 4). The remaining autoclaved L-62 was tested by adding it to a 10L fermenter. Strain development work was carried out, and at the end of the experiment, the efficacy of autoclaved L-62 was tested for 2 days using a control (NB5-F-1774). Table 5.

[0141] [Results and Observations]

[0137] L-62 diluted to 25% with water and autoclaved showed phase separation upon cooling. After mixing, phase separation was not observed for 24 hours. After several days, phase separation was observed again. To demulsify on a bench scale, the same amount of autoclaved L-62 as regular L-62 was required. For demulsification in a fermenter, the same amount of autoclaved L-62 as filter-sterilized L-62 was required.

[0142] [Conclusion]

[0138] The inventors have found that by adding L-62 in the fermentation tank, emulsion formation can be effectively prevented and / or emulsification and separation can be completed in the fermentation tank.

[0143]

[0139] Adding L-62 to the fermentation tank reduced surfactant consumption by 91%. This represents a significant cost reduction compared to the processes currently used in this industry.

[0144]

[0140] By adding L-62 to the fermentation tank, the two-stage centrifugal separation can be reduced to a single-stage centrifugal separation. Based on the crude oil characteristics from the S / L centrifugal separator, an L / L centrifugal separator can be used in the refining process.

[0145]

[0141] Adding L-62 to the fermentation tank eliminates the need to heat the CCB to, for example, 75-80°C.

[0146]

[0142] Adding L-62 to the 10L fermentation tank improved the overall recovery rate in DSP by 2-4%. This represents a significant cost reduction compared to the process currently used in this industry.

[0147]

[0143] Adding L-62 to the fermenter significantly increased productivity in DSP (in optimized tests, productivity increased by up to 70% with maximum airflow and oxygen supply, while DO was maintained at approximately 30%).

[0148]

[0144] In an optimized test with a 10L flow rate (DO>30%), adding L-62 to the fermenter may slightly reduce the farnesene oil yield relative to sugar.

[0149] [Example 8] [10L test]

[0145] Two 10L fermenters were prepared to test the addition of surfactants inside the fermenter under conditions equivalent to current production. NB2-F-177 was used as a control, and NB5-F-177 as the experimental fermenter. The airflow rate was set to 3L / min, and L-62 addition was started with LH24. Approximately 30% of the estimated required amount of L-62 was added with LH24, and the remaining 70% was added in equal amounts every two hours until two hours before the next harvest. These procedures were repeated after each harvest until the final harvest.

[0150]

[0146] Each sample and the final harvest were collected and processed. The control whole cell broth was centrifuged to recover the CCB. The CCB sample was brought into the laboratory and thawed by heating at 75°C and adding L-62 in the same manner as the current production process. The WCB with L-62 added was centrifuged to recover the high-moisture oil. This oil sample was recentrifuged in the laboratory to recover the crude oil.

[0151]

[0147] Data from laboratory and pilot processes showed a recovery rate of 90% for farnesene oil and a loss rate of 7.9% in DCL in the control case, and a recovery rate of 88% when L-62 was added to the inside of the fermenter. Laboratory data for WCB demulsification showed a recovery rate of 79%, a loss rate of 6% in DCL, and a loss rate of 7.8% in S / L HP. As shown in previous experiments, the addition of L-62 to WCB (equivalent to the addition of L-62 to the harvest tank) resulted in a decrease in product recovery. On the other hand, when WCB with L-62 added to the inside of the fermenter was centrifuged in the laboratory, the product recovery rate was 88%, the loss rate in DCL was 3.5%, and the loss in S / L HP was 6.3%.

[0152]

[0148] All references cited herein are incorporated by reference in such a manner as is specifically and individually indicated, each reference being incorporated by reference. Any reference is made to its disclosure prior to the filing date and should not be construed as an acknowledgment that this disclosure does not have prior rights to such reference by prior invention.

[0153]

[0149] It is understood that each of the above elements, or two or more of them, may find useful applications together in other types of ways different from those described above. Without further analysis, the foregoing is sufficient to illustrate the intent of this disclosure, and others, by applying their current knowledge, can easily adapt it to various applications without omitting features that, from the perspective of the prior art, appropriately constitute the essential features of the general or specific aspects of this disclosure described in the appended claims. The embodiments described above are presented merely as examples, and the scope of this disclosure should be limited only by the following claims.

Claims

1. A method for reducing emulsion formation inside a fermenter during the production of organic compounds, (a) Fermenting microorganisms using supplied raw materials in a fermentation vessel to produce whole cell broth (WCB) containing organic compounds, (b) Adding a surfactant to the feed material, the whole cell broth, a portion of the whole cell broth, or a combination thereof in the fermentation vessel, (c) Selectively removing a portion of the whole cell broth, (d) Heating the fermentation vessel and A method that includes this.

2. The aforementioned microorganisms are bacteria, fungi, or algae. The bacteria are preferably Escherichia coli (E. coli), Bacillus, or Lactobacillus species. The fungus is preferably a yeast such as Saccharomyces or Pichia species, and The method according to claim 1, wherein the algae is preferably of the Chlorella species.

3. The method according to claim 1 or 2, wherein the microorganism is a recombinant microorganism.

4. The surfactant is selected from anionic surfactants, nonionic surfactants, or combinations thereof. The anionic surfactant is selected from one or more of the following: sulfates and sulfonates, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate, disulfonates, sodium dodecylnaphthalene sulfate, dialkylbenzenealkyl sulfates and sulfonates, acids, optionally avitic acid, alkyldiphenyl oxide disulfonates containing two C6-C16 hydrocarbon chains branched to each of the sulfonated aromatic rings, and combinations thereof, and The method according to any one of claims 1 to 3, wherein the nonionic surfactant is selected from copolymers of propylene oxide (PO) and ethylene oxide (EO), which optionally include polyether polyols and those produced by capping polypropylene glycol with ethylene oxide.

5. The method according to claim 4, wherein the nonionic surfactant is a polyether glycol having a molecular weight of at least 2400 g / mol, less than 2800 g / mol, or less than 3000 g / mol, and / or the anionic surfactant is selected from sulfates and sulfonates.

6. The method according to any one of claims 1 to 5, wherein the surfactant is L-62 (polyether polyol) or L-81 (polyether polyol).

7. The method according to any one of claims 1 to 6, wherein the surfactant is added in an amount of about 0.001% v / v (volume / volume) to 1% v / v (volume / volume), more preferably about 0.01 to 1% v / v, more preferably about 0.02 to 0.06% v / v or 0.03 to 0.05% v / v.

8. The method according to any one of claims 1 to 7, wherein the fermentation is continued for approximately 1 to 14 days, and a portion of the whole cell broth is removed after 1 day.

9. The method according to any one of claims 1 to 8, wherein step (b) is continued for approximately 24 hours, and the method further comprises isolating an organic compound from the whole cell broth.

10. The organic compound comprises an isoprenoid, and the isoprenoid is (a) Selected from the group consisting of hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes and polyterpenes, (b) Is an isoprenoid that is not a carotenoid, (c) C ５ ~C ２０ isoprenoid, or (d) The method according to any one of claims 1 to 9, selected from the group consisting of abietadiene, amorphousdiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

11. The method according to any one of claims 1 to 10, wherein the organic compound comprises, consists of, or is essentially composed of farnesene.

12. The whole cell broth is separated into an organic phase containing the isoprenoid compound and a biphase containing solid and liquid fermentation media. Purifying the isoprenoid compound and The method according to any one of claims 1 to 11, further comprising:

13. The method according to any one of claims 1 to 12, comprising supplying air to a fermentation tank at a rate of approximately 3 L / min to 10 L / min, and / or maintaining the dissolved oxygen in the fermentation tank at a rate of more than approximately 15%.

14. The method according to any one of claims 1 to 13, wherein the dissolved oxygen in the fermentation tank is maintained at 0%.

15. The method according to any one of claims 1 to 14, wherein the surfactant reduces emulsion formation by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, or 90% compared to emulsion formation in a fermenter without the addition of the surfactant.

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